[(68)Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvß3 integrin expression based on a novel siderophore derived chelating scaffold-synthesis and evaluation.

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 αvß3 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, αvß3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvß3 positive human melanoma M21 and αvß3 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 αvß3 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 αvß3 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 αvß3 integrin in vivo for imaging tumor-induced neovascularization.

[68Ga]NS3-RGD and [68Ga] Oxo-DO3A-RGD for imaging α(v)ß3 integrin expression: synthesis, evaluation, and comparison.

INTRODUCTION: 68Ga-labeled RGD peptides in combination with PET allow non-invasive determination of α(v)ß3 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 [68Ga]NS3-RGD-RGD and [68Ga]Oxo-DO3A-RGD and to compare their in vitro and in vivo properties with [68Ga]DOTA- and [68Ga]NODAGA-RGD. METHODS: Syntheses of both radiotracers followed standard SPPS protocols. For in vitro characterization distribution coefficients, protein binding abilities, serum stabilities, and α(v)ß3 integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay α(v)ß3 positive human melanoma M21 and α(v)ß3 negative M21-L cells were used. RESULTS: 68Ga-labeling of NS3-RGD resulted in good radiochemical purity, whereas HPLC analysis showed two peaks with a ratio of 1:6 for [68Ga]Oxo-DO3A-RGD. Distribution coefficients were -3.4 for [68Ga]Oxo-DO3A-RGD and -2.9 for [68Ga]NS3-RGD. Both radiotracers were stable in PBS solution at 37°C for 2 h but lack stability in human serum. Protein binding was approximately 40% of the total activity for [68Ga]NS3-RGD and 70% for [68Ga]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 [68Ga]NS3-RGD. Furthermore, [68Ga]Oxo-DO3A-RGD showed nearly the same activity concentrations in α(v)ß3 positive and α(v)ß3 negative tumors. CONCLUSIONS: [68Ga]Oxo-DO3A-RGD and [68Ga]NS3-RGD have inferior characteristics compared to already existing 68Ga-labeled RGD peptides and thus, both are not suited to image α(v)ß3 integrin expression. Of all our tested RGD peptides, [68Ga]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 68Ga-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.

Radiolabelling of peptides for PET, SPECT and therapeutic applications using a fully automated disposable cassette system.

OBJECTIVES: Radiolabelled somatostatin analogues have found wide clinical use in nuclear medicine for both diagnostic and therapeutic applications. Here, we describe the development of a fully automated synthesis system allowing radiolabelling of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-derivatized peptides with 68Ga/¹¹¹In/¹77Lu and 9°Y, meeting radiation safety and pharmaceutical requirements. MATERIALS AND METHODS: The system consists of a syringe pump, a holder for insertion of a single use multivalve cassette, a heater and a removable radiation shielding. 68Ga labelling was performed in acetate buffer and ¹77Lu, 9°Y and ¹¹¹In labelling in ascorbate buffer, respectively, followed by purification on a C18 cartridge and final sterile filtration. Cross-contamination was prevented by using disposable cassettes and also by ensuring pharmaceutical standards. Radiochemical purity (RCP) was determined by instant thin-layer chromatography on silica gel impregnated glass fibres and reversed-phase high performance liquid chromatography. RESULTS: 68Ga-DOTA-peptides were prepared with high RCP (>91%) and radiochemical yields (RCY>80% decay corrected) and 68Ge content was less than 0.0001% in all cases. Synthesis time did not exceed 30 min. ¹¹¹In, ¹77Lu and 9°Y labelling of DOTA-peptides resulted again in high yields (approximately 90%) and RCP (approximately 95%) and total synthesis time of less than 45 min. Radiation dose to fingers was considerably reduced when compared with manual labelling procedures. CONCLUSION: The described system allows fully automated, aseptic preparation of DOTA-peptides radiolabelled with different radionuclides in high radiochemical yields and pharmaceutical quality suitable for clinical application.

[68Ga]NODAGA-RGD for imaging αvß3 integrin expression.

PURPOSE: A molecular target involved in the angiogenic process is the α(v)ß(3) 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 α(v)ß(3) expression. Here we introduce a (68)Ga-labelled NOTA-conjugated RGD peptide ([(68)Ga]NODAGA-RGD) and compare its imaging properties with [(68)Ga]DOTA-RGD using small animal PET. METHODS: Synthesis of c(RGDfK(NODAGA)) was based on solid phase peptide synthesis protocols using the Fmoc strategy. The (68)Ga labelling protocol was optimized concerning temperature, peptide concentration and reaction time. For in vitro characterization, partition coefficient, protein binding properties, serum stability, α(v)ß(3) binding affinity and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET imaging were carried out. For both in vitro and in vivo evaluation, α(v)ß(3)-positive human melanoma M21 and α(v)ß(3)-negative M21-L cells were used. RESULTS: [(68)Ga]NODAGA-RGD can be produced within 5 min at room temperature with high radiochemical yield and purity (> 96%). In vitro evaluation showed high α(v)ß(3) binding affinity (IC(50) = 4.7 ± 1.6 nM) and receptor-specific uptake. The radiotracer was stable in phosphate-buffered saline, pH 7.4, FeCl(3) solution, and human serum. Protein-bound activity after 180 min incubation was found to be 12-fold lower than for [(68)Ga]DOTA-RGD. Biodistribution data 60 min post-injection confirmed receptor-specific tumour accumulation. The activity concentration of [(68)Ga]NODAGA-RGD was lower than [(68)Ga]DOTA-RGD in all organs and tissues investigated, leading to an improved tumour to blood ratio ([(68)Ga]NODAGA-RGD: 11, [(68)Ga]DOTA-RGD: 4). MicroPET imaging confirmed the improved imaging properties of [(68)Ga]NODAGA-RGD compared to [(68)Ga]DOTA-RGD. CONCLUSION: The introduced [(68)Ga]NODAGA-RGD combines easy accessibility with high stability and good imaging properties making it an interesting alternative to the (18)F-labelled RGD peptides currently used for imaging α(v)ß(3) expression.