Abstract
BackgroundGastrin-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 64Cu-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA]-monomer and two NOTA-dimers of [D-Tyr6,βAla11, Thi13, Nle14]bombesin(6-14) ] [BBN(6-14)] were compared.MethodsMonomeric and dimeric peptides were synthesized on solid phase support and radiolabeled with 64Cu. 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 NOTA-dimer 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.ResultsNOTA-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 64Cu-labeled peptides were stable up to 24 h in mouse plasma and 1 h in vivo. 64Cu/NOTA-dimer 2 featuring a longer spacer between the two BBN(6-14) ligands is a more potent GRPR-targeting probe than 64Cu/NOTA-dimer 1. PC3 tumor uptake profiles are slightly different for 64Cu/NOTA-monomer and 64Cu/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 64Cu/NOTA-dimer 2. The latter exhibited higher tumor-to-blood ratio and was retained longer at the tumor site when compared to 64Cu/NOTA-monomer. Lower ratios of tumor-to-blood and tumor-to-muscle in blocking experiments showed GRPR-dependant tumor uptake for both tracers.ConclusionBoth 64Cu/NOTA-monomer and 64Cu/NOTA-dimer 2 are suitable for detecting GRPR-positive prostate cancer in vivo by PET. Tumor retention was improved in vivo with 64Cu/NOTA-dimer 2 by applying polyvalency effect and/or statistical rebinding.
Highlights
Gastrin-releasing peptide receptors [gastrin-releasing peptide receptor (GRPR)] are highly over-expressed in multiple cancers and have been studied as a diagnostic target
BBN is involved in regulating exocrine secretion, smooth muscle contraction and gastrointestinal hormone release [18], and it is widely expressed in the central nervous system [19]. [D-Tyr6,bAla11, Thi13, Nle14]BBN(6-14) [BBN(6-14)] is a potent modified GRPR agonist peptide that binds to GRPR with high affinity [20]
We reported a convenient synthetic approach for the preparation of two NOTA-BBN homodimers and their labeling with 64Cu (Figure 2). micro-positron emission tomography (μPET) imaging on PC3 human prostate carcinomas xenografted in Balb/c nude mice was performed to compare the diagnostic properties of 64Cu/NOTA-BBN homodimers homodimers to those of the 64Cu/NOTA-BBN monomer
Summary
Gastrin-releasing peptide receptors [GRPR] are highly over-expressed in multiple cancers and have been studied as a diagnostic target. A 64Cu-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA]monomer and two NOTA-dimers of [D-Tyr6,bAla, Thi, Nle14]bombesin(6-14) ] [BBN(6-14)] were compared. GRPR is overexpressed on 84% of all human prostate cancers according to a study by Markwalder and Reubi [5]. These receptors represent an interesting molecular target for radiolabeled BBN analogs as diagnostic or radiotherapeutic applications for prostate cancer. Smith et al successfully labeled modified BBN(7-14) analogs with 64Cu for potential use in diagnostic imaging using 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA] or NO2A as chelating agents and obtained stable compounds [23,24]
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