[⁶⁸Ga]NS₃-RGD and [⁶⁸Ga] Oxo-DO3A-RGD for imaging α_vβ₃ integrin expression: synthesis, evaluation, and comparison

Introduction:

⁶⁸Ga-labeled RGD peptides in combination with PET allow non-invasive determination of α_vβ₃integrin 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 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_vβ₃ integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay α_vβ₃ positive human melanoma M21 and α_vβ₃ 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-DO3ARGD and −2.9 for [⁶⁸Ga]NS₃-RGD. Both radiotracers were stable in PBS solution at 37°C for 2h 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_vβ₃ positive and α_vβ₃ 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_vβ₃ 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.