Fusarinine C (FSC), a siderophore‐based chelator coupled with the model peptide c(RGDfK) (FSC(succ‐RGD) 3 ), revealed excellent targeting properties in vivo using positron emission tomography (PET). Here, we report the details of radiolabeling conditions and specific activity as well as selectivity for 68 Ga. 68 Ga labeling of FSC(succ‐RGD) 3 was optimized regarding peptide concentration, pH, temperature, reaction time, and buffer system. Specific activity (SA) of [ 68 Ga]FSC(succ‐RGD) 3 was compared with 68 Ga‐1,4,7‐triazacyclononane, 1‐glutaric acid‐4,7 acetic acid RGD ([ 68 Ga]NODAGA‐RGD). Stability was evaluated in 1000‐fold ethylenediaminetetraacetic acid (EDTA) solution (pH 7) and phosphate‐buffered saline (PBS). Metal competition tests (Fe, Cu, Zn, Al, and Ni) were carried out using [ 68 Ga]‐triacetylfusarinine C. High radiochemical yield was achieved within 5 min at room temperature, in particular allowing labeling with 68 Ga up to pH 8 with excellent stability in 1000‐fold EDTA solution and PBS. The 10‐fold to 20‐fold lower concentrations of FSC(succ‐RGD) 3 led to the same radiochemical yield compared with [ 68 Ga]NODAGA‐RGD with SA up to 1.8 TBq/µmol. Metal competition tests showed high selective binding of 68 Ga to FSC. FSC is a multivalent siderophore‐based bifunctional chelator allowing fast and highly selective labeling with 68 Ga in a wide pH range and results in stable complexes with high SA. Thus it is exceptionally well suited for the development of new 68 Ga‐tracers for in vivo molecular imaging with PET.

Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga 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 (68)Ga 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 (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvβ3 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 (68)Ga. 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, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [(68)Ga]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 αvβ3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60min p.i. which was 3-fold higher than the monomeric [(68)Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [(68)Ga]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 αvβ3 integrin in vivo for imaging tumor-induced neovascularization.

Introduction: Ga-68-labeled RGD peptides in combination with PET allow non-invasive determination of alpha(v)beta(3) integrin expression which is highly increased during tumor-induced angiogenesis. The aim of this study was to synthesize and evaluate two RGD peptides containing alternative chelating systems, namely [Ga-68]NS3-RGD and [Ga-68]Oxo-DO3A-RGD and to compare their in vitro and in vivo properties with [Ga-68]DOTA- and [Ga-68]NODAGA-RGD.Methods: Syntheses of both radiotracers followed standard SPPS protocols. For in vitro characterization distribution coefficients, protein binding abilities, serum stabilities, and alpha(v)beta(3) integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay alpha(v)beta(3) positive human melanoma M21 and alpha(v)beta(3) negative M21-L cells were used.Results: Ga-68-labeling of NS3-RGD resulted in good radiochemical purity, whereas HPLC analysis showed two peaks with a ratio of 1:6 for [Ga-68]Oxo-DO3A-RGD. Distribution coefficients were -3.4 for [Ga-68]Oxo-DO3A-RGD and -2.9 for [Ga-68]NS3-RGD. Both radiotracers were stable in PBS solution at 37 degrees C for 2h but lack stability in human serum. Protein binding was approximately 40% of the total activity for [Ga-68]NS3-RGD and 70% for [Ga-68]Oxo-DO3A-RGD, respectively, resulting in high blood pool activities. Biodistribution assays confirmed these findings and showed an additional high uptake in liver and kidneys, especially for (Ga-68]NS3-RGD. Furthermore, [Ga-68]Oxo-DO3A-RGD showed nearly the same activity concentrations in alpha(v)beta(3) positive and alpha(v)beta(3) negative tumors.Conclusions: [Ga-68]Oxo-DO3A-RGD and [Ga-68]NS3-RGD have inferior characteristics compared to already existing Ga-68-labeled RGD peptides and thus, both are not suited to image alpha(v)beta(3) integrin expression. Of all our tested RGD peptides, [Ga-68]NODAGA-RGD still possesses the most favorable imaging properties. Moreover this study shows that the use of appropriate chelators to achieve good targeting properties of Ga-68-labeled biomolecules and careful in vitro and in vivo evaluation including comparative studies of different strategies are essential components in designing an effective imaging agent for PET. (C) 2013 Elsevier Inc. All rights reserved.

A molecular target involved in the angiogenic process is the αvβ3 integrin. It has been demonstrated in preclinical as well as in clinical studies that radiolabelled RGD peptides and positron emission tomography (PET) allow noninvasive monitoring of αvβ3 expression. Here we introduce a 68Ga-labelled NOTA-conjugated RGD peptide ([68Ga]NODAGA-RGD) and compare its imaging properties with [68Ga]DOTA-RGD using small animal PET.