Multivalent Bifunctional Chelator Scaffolds for Gallium-68 Based Positron Emission Tomography Imaging Probe Design: Signal Amplification via Multivalency

The role of the multivalent effect has been well recognized in the design of molecular imaging probes toward the desired imaging signal amplification. Recently, we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds (Bu-t(3)-1-COOH, Bu-t(3)-2-(COOH)(2), and Bu-t(3)-3-(COOH)(3)) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for Ga-68-based PET probe design and signal amplification via the multivalent effect. For proof of principle, a known integrin alpha(v)beta(3) specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H(3)1, H(3)2, and H(3)3), which present 1-3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin alpha(v)beta(3), binding affinities (IC50 values), enhanced specific binding was observed for multivalent conjugates (H32: 43.9 +/- 16.1 nM; H(3)3: 14.7 +/- 5.0 nM) as compared to their monovalent counterpart (H(3)1: 171 +/- 60 nM) and the intact c(RGDyK) peptide (204 +/- 76 nM). The obtained conjugates were efficiently labeled with Ga-68(3+) within 30 min at room temperature in high radiochemical yields (>95%). The in vivo evaluation of the labeled conjugates, Ga-68-1, Ga-68-2, and Ga-68-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin alpha(v)beta(3) positive PC-3 tumor xenografts (n = 3). All Ga-68-labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h postinjection (p.i.). The PET signal amplification in PC-3 tumor by the multivalent effect was dearly displayed by the tumor uptake of the 68Ga-labeled conjugates (Ga-68-3: 2.55 +/- 0.50%ID/g; Ga-68-2: 1.90 +/- 0.10%ID/g; Ga-68-1: 1.66 +/- 0.15%ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications as diagnostic gallium radiopharmaceuticals.