[68Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold-synthesis and evaluation

Over the last years Gallium-68 (Ga-68) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). Ga-68 labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for Ga-68 resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based Ga-68 labeled radiopharmaceuticals. As proof of principle we report on the synthesis and Ga-68-radiolabeling of a trimeric FSC-RGD conjugate, [Ga-68]FSC-(RGD)(3), targeting alpha(v)beta(3) integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)(3) with Ga-68. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, alpha(v)beta(3) integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies alpha(v)beta(3) positive human melanoma M21 and alpha(v)beta(3) negative M21-L cells were used. [Ga-68]FSC-(RGD)(3) was prepared with high radiochemical yield (>98%). Distribution coefficient was - 3.6 revealing a hydrophilic character, and an IC50 value of 1.8 +/- 0.6 nM was determined indicating a high binding affinity for alpha(v)beta(3) integrin. [Ga-68]FSC-(RGD)(3) was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 degrees C for 2 hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the alpha(v)beta(3) positive tumor was 4.3% ID/g 60 min p.i. which was 3-fold higher than the monomeric [Ga-68]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [Ga-68]FSC-(RGD)(3) serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for alpha(v)beta(3) integrin in vivo for imaging tumorinduced neovascularization. (C) 2014 Elsevier Inc. All rights reserved.