title: [(68)Ga]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 ((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.
authors.affiliation: Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Knetsch
authors.affiliation: Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Chuangyan
authors.initials: C
authors.lastname: Zhai
authors.affiliation: Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Rangger
authors.affiliation: Division of Molecular Biology, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Blatzer
authors.affiliation: Division of Molecular Biology, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Hubertus
authors.initials: H
authors.lastname: Haas
authors.affiliation: Ministry of Science and Technology (MOST), Thailand Institute of Nuclear Technology (TINT), Bangkok, Thailand.
authors.forename: Piriya
authors.initials: P
authors.lastname: Kaeopookum
authors.affiliation: Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Haubner
authors.affiliation: Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria. Electronic address: clemens.decristoforo@uki.at.
authors.forename: Clemens
authors.initials: C
authors.lastname: Decristoforo
mesh_terms: D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry*; D060326:Chemistry Techniques, Synthetic; D005240:Feasibility Studies; D005290:Ferric Compounds / Q000737:chemistry*; D005710:Gallium Radioisotopes; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry*; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007553:Isotope Labeling; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D049268:Positron-Emission Tomography / Q000379:methods*; D017262:Siderophores / Q000737:chemistry*; D014018:Tissue Distribution
chemical_list: D002614:Chelating Agents; D005290:Ferric Compounds; D005710:Gallium Radioisotopes; D006877:Hydroxamic Acids; D039302:Integrin alphaVbeta3; D010456:Peptides, Cyclic; D017262:Siderophores; C507169:cyclic arginine-glycine-aspartic acid peptide; C010966:fusigen
keywords: 68Ga; Bifunctional chelator; RGD; Siderophors; αvβ3 integrin