Thi,Nle]BBN(6-14) monomer and dimers for prostate cancer PET imaging

Background: Gastrin-releasing peptide receptors [GRPR] are highly over-expressed in multiple cancers and have been studied as a diagnostic target. Multimeric gastrin-releasing peptides are expected to have enhanced tumor uptake and affinity for GRPR. In this study, a Cu-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA]monomer and two NOTA-dimers of [D-Tyr,bAla, Thi, Nle]bombesin(6-14) ] [BBN(6-14)] were compared. Methods: Monomeric and dimeric peptides were synthesized on solid phase support and radiolabeled with Cu. NOTA-dimer 1 consists of asymmetrically linked BBN(6-14), while NOTA-dimer 2 has similar spacer between the two BBN(6-14) ligands and the chelator. In vitro GRPR-binding affinities were determined with competitive binding assays on PC3 human prostate cancer cells. In vivo stability and biodistribution of radiolabeled compounds were assessed in Balb/c mice. Cellular uptake and efflux were measured with radiolabeled NOTA-monomer and NOTAdimer 2 on PC3 cells for up to 4 h. In vivo biodistribution kinetics were measured in PC3 tumor-bearing Balb/c nude mice by μ-positron emission tomography [μPET] imaging and confirmed by dissection and counting. Results: NOTA-monomer, NOTA-dimers 1 and 2 were prepared with purity of 99%. The inhibition constants of the three BBN peptides were comparable and in the low nanomolar range. All Cu-labeled peptides were stable up to 24 h in mouse plasma and 1 h in vivo. Cu/NOTA-dimer 2 featuring a longer spacer between the two BBN(6-14) ligands is a more potent GRPR-targeting probe than Cu/NOTA-dimer 1. PC3 tumor uptake profiles are slightly different for Cu/NOTA-monomer and Cu/NOTA-dimer 2; the monomeric BBN-peptide tracer exhibited higher tumor uptake during the first 0.5 h and a fast renal clearance resulting in higher tumor-to-muscle ratio when compared to Cu/NOTA-dimer 2. The latter exhibited higher tumor-to-blood ratio and was retained longer at the tumor site when compared to Cu/NOTA-monomer. Lower ratios of tumor-to-blood and tumor-to-muscle in blocking experiments showed GRPR-dependant tumor uptake for both tracers. Conclusion: Both Cu/NOTA-monomer and Cu/NOTA-dimer 2 are suitable for detecting GRPR-positive prostate cancer in vivo by PET. Tumor retention was improved in vivo with Cu/NOTA-dimer 2 by applying polyvalency effect and/or statistical rebinding.