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

Abstract

Over the last years Gallium-68 (68Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). 68Ga 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 68Ga 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 68Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and 68Ga-radiolabeling of a trimeric FSC-RGD conjugate, [68Ga]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 68Ga. 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.[68Ga]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. [68Ga]FSC-(RGD)3 was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 °C for 2 hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60 min p.i. which was 3-fold higher than the monomeric [68Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [68Ga]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.

Radiolabeled Cyclic RGD Peptide Bioconjugates as Radiotracers Targeting Multiple Integrins

“…23,24,34–50 In the literature, a few 68 Ga-labeled trimeric RGD peptides were used as PET radiotracers. 11,14 It would be interesting to compare them with their dimeric and tetrameric analogs with respect to the tumor uptake and T/B ratios of 99m Tc, 111 In, 64 Cu and 68 Ga radiotracers in the same model, and determine the optimal multiplicity for development of α v β 3 -targeted cyclic RGD peptides.…”

Comparison of biological properties of 99m Tc-labeled cyclic RGD Peptide trimer and dimer useful as SPECT radiotracers for tumor imaging

“…In the literature, only a few radiolabeled cyclic RGD trimers were reported [41,42]. There was no comparison in biodistribution properties between the radiolabeled cyclic RGD dimers and trimers.…”

Evaluation of two novel 64Cu-labeled RGD peptide radiotracers for enhanced PET imaging of tumor integrin αvβ3

“…For example, it is now known that increasing the hydrophilic character of RGD peptides improves its circulation half-lives, whereas cyclization reduces sensitivity toward enzymatic degradation [18, 19], and that multimerization significantly increases the integrin α v β 3 binding affinity of these antagonists due to a polyvalence effect [20, 21]. A clear tendency towards focusing on increasing the absolute tumor uptake of RGD-based tracers, through the synthesis of dimeric, trimeric, tetrameric, and even octameric versions of the cyclic RGD can be identified in recent literature [22–27]. However, these strategies result in an increased non-specific uptake of the peptides on off-target tissues.…”