Evaluation of 64 Cu-Labeled Bifunctional ChelateBombesin Conjugates

Several bifunctional chelates (BFCs)were investigated as carriers of 64 Cu for PET imaging. The most widely used chelator for 64 Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N 0 , N 00 ,N 000 -tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelatorpeptide conjugates labeled with 64 Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and com- pared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclo- nonane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo(9.3.1)penta- deca-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 ,βAla 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 humanbreastcancer cells.A convenientsynthetic approach forthe attachmentof aniline-BFC to peptides on solidsupport 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 cleavageandpurification,thepeptide-conjugateswerelabeledwith 64 Cuusing( 64 Cu)Cu(OAc)2in0.1Mammoniumacetatebufferat 100 � C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 � Ct o achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC- BBNcomplexesretainedhighaffinityfortheGRPR.AllBFC-BBN 64 Cu-conjugateswerestableforover20hwhenincubatedat37 � C in mouse plasma samples. However, in vivo, only 37% of the 64 Cu/Oxo-DO3A complex remained intact after 20 h while the 64 Cu/ DOTA-BBN complex was completely demetalated. In contrast, both 64 Cu/NOTA- and 64 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 64 Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.