Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

Bombesin receptor 2 (BB2) and integrin αvß3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB2- and integrin αvß3-targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD2-BBN heterotrimer, composed of (7-14)BBN-NH2 peptide (BBN) linked to the E[ c(RGDyK)]2 dimer peptide (RGD2), bearing the new linker type [Pro-Gly]12. The heterodimer E[c(RGDyK)]2-PEG3-Glu-(Pro-Gly)12-BBN(7-14)-NH2 (RGD2-PG12-BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good αvß3 affinities and high BB2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( Ki = 24 nM). 64Cu-DOTA and 64Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB2 and integrin αvß3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide.

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [18F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.

A Four-Arm Star-Shaped Poly(ethylene glycol) (StarPEG) Platform for Bombesin Peptide Delivery to Gastrin-Releasing Peptide Receptors in Prostate Cancer.

We present the design, synthesis, and characterization of a novel cancer biomarker delivery platform, the star-shaped four-arm poly(ethylene glycol) (StarPEG). Using the multidisplay platform we were able to synthesize a bombesin (BBN) positron emission tomography (PET) probe featuring four copies of 8-Aoc-BBN peptides (where 8-Aoc is 8-aminooctanic acid), which we named StarPEG-BBN. Cell binding studies showed that StarPEG-BBN had a good binding affinity to PC3 cells (IC50 = 65.3 ± 3.4 nM). Cell uptake studies showed that the binding was specific (blocking vs no-blocking, P < 0.05). Mice were then implanted with PC3 cells and divided into two groups, one injected with 64Cu-StarPEG-BBN and the other 250 µg of unlabeled 8-Aoc-BBN along with 64Cu-StarPEG-BBN. In vivo images revealed that StarPEG-BBN had good tumor uptake (4.2 ± 0.4% ID/g at 4 h post-injection (p.i.)) and was significantly blocked by coinjection of unlabeled 8-Aoc-BBN at 4 h p.i. (P = 0.003). The small animal PET quantification was further verified by the biodistribution study at 24 h p.i. Our study demonstrated that the novel four-arm PEG platform StarPEG as a cancer biomarker multimerization/delivery platform conserves binding specificity, improves drug loading, is capable of achieving good tumor uptake, and has great potential in cancer treatment and molecular imaging.