Be spoilt for choice with radiolabelled RGD peptides: Preclinical evaluation of ⁶⁸Ga-TRAP(RGD)₃

Gallium-68 is rapidly gaining importance, as this generator-produced PET isotope is available independent of on-site cyclotrons, enabling radiopharmaceutical production with comparably simple techniques at low cost. The recently introduced TRAP chelator combines the advantage of straightforward design of multimeric ⁶⁸Ga-radiopharmaceuticals with very fast and efficient ⁶⁸Ga-labeling. We synthesized a series of five cyclo(RGDfK) peptide trimers and determined their α_vβ₃ integrin affinities in competition assays on α_vβ₃-expressing M21 human melanoma cells against ¹²⁵I-echistatin. The compound with highest IC₅₀, Ga-TRAP(RGD)₃, showed more than 7-fold higher affinity compared to the monomers F-Galacto-RGD and Ga-NODAGA-c(RGDyK). TRAP(RGD)₃ was radiolabeled with ⁶⁸Ga in a fully automated GMP compliant manner. CD-1 athymic nude mice bearing M21/M21L human melanoma xenografts were used for biodistribution studies, blockade experiments, metabolite studies and PET imaging. ⁶⁸Ga-TRAP(RGD)₃ exhibited high M21 tumor uptake (6.08±0.63% ID/g, 60min p.i.), was found to be fully stable in vivo, and showed a fast renal clearance. Blockade studies showed that uptake in the tumor, as well as in all other tissues, is highly integrin specific. A comparison of biodistribution and PET data of ⁶⁸Ga-TRAP(RGD)₃ with those of ⁶⁸Ga-NODAGA-c(RGDyK) and ¹⁸F-Galacto-RGD showed that the higher affinity of the trimer effects a larger dynamic response of tracer uptake to integrin expression, i.e., enhanced integrin-specific uptake in all tissues. We conclude that ⁶⁸Ga-TRAP(RGD)₃ could allow for imaging of low-level integrin expression in tissues which are not visible with the two competitors. Overall, the study constitutes proof of concept for the favourable in vivo properties of TRAP-based ⁶⁸Ga radiopharmaceuticals.