title: Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates.
abstract: 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',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 (64)Cu, 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 H(2)N-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 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-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-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 (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling 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 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °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.
authors.affiliation: Centre d'imagerie moléculaire de Sherbrooke, Department of Nuclear Medicine and Radiobiology, Université de Sherbrooke, 3001, 12th North Avenue Sherbrooke, Qubec, Canada, J1H 5N4.
authors.forename: Samia
authors.initials: S
authors.lastname: Ait-Mohand
authors.forename: Patrick
authors.initials: P
authors.lastname: Fournier
authors.forename: Véronique
authors.initials: V
authors.lastname: Dumulon-Perreault
authors.forename: Garry E
authors.initials: GE
authors.lastname: Kiefer
authors.forename: Paul
authors.initials: P
authors.lastname: Jurek
authors.forename: Cara L
authors.initials: CL
authors.lastname: Ferreira
authors.forename: François
authors.initials: F
authors.lastname: Bénard
authors.forename: Brigitte
authors.initials: B
authors.lastname: Guérin
mesh_terms: D001667:Binding, Competitive; D001839:Bombesin / Q000737:chemistry*; D001943:Breast Neoplasms / Q000000981:diagnostic imaging*; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry*; D003301:Copper Radioisotopes; D004355:Drug Stability; D005260:Female; D006573:Heterocyclic Compounds, 1-Ring; D006801:Humans; D010455:Peptides; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals; D018004:Receptors, Bombesin / Q000032:analysis / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D002614:Chelating Agents; D003301:Copper Radioisotopes; D006573:Heterocyclic Compounds, 1-Ring; D010455:Peptides; D019275:Radiopharmaceuticals; D018004:Receptors, Bombesin; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D001839:Bombesin