In vivo biokinetic and metabolic characterization of the 68Ga-labelled α5β1-selective peptidomimetic FR366

Purpose Integrins are transmembrane receptors responsible for cell-cell adhesion and cell-extracellular matrix binding and play an important role in angiogenesis and tumour metastasis. For this reason, integrins are increasingly used as targets for molecular imaging. Up to now interest has mostly been focused on the integrin subtype alpha v beta 3. However, targeting of other subtypes such as the integrin alpha 5 beta 1 is also of high interest due to its central role in colonization of metastatic cells, resistance of tumour cells to chemotherapy and ionizing radiation, and tumour aggressiveness. Recently, a highly active antagonist ligand (2,2'-(7-(1-carboxy-4-((6-((3-(4-(((S)-1-carboxy-2-(2-(3-guanidinobenzamido)acetamido)ethyl)carbamoyl)-3,5-dimethylphenoxy)propyl)amino)-6-oxohexyl)amino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid, FR366) for the integrin subtype alpha 5 beta 1 with high selectivity versus alpha v beta 3, has been developed and tested successfully in preliminary in vitro and in vivo experiments. Here, we present our results of an investigation of the use of Ga-68-labelled alpha 5 beta 1 ligand in PET imaging. Methods The free alpha 5 beta 1 peptidomimetic ligand was functionalized with a spacer (6-aminohexanoic acid) and the bifunctional chelator 1-((1,3-dicarboxy)propyl)-4,7-(carboxymethyl)-1,4,7-triazacyclononane (NODAGA) to yield FR366 and labelled with Ga-68. To confirm selective in vivo targeting of alpha 5 beta 1, female BALB/c nude mice xenografted with alpha 5 beta 1-expressing RKO cells in the right shoulder and alpha 5 beta 1/alpha v beta 3-expressing M21 cells in the left shoulder were subjected to PET/CT scans and biodistribution experiments. Specificity of tracer uptake was proven by blocking studies. Metabolic stability of the injected tracer was measured in urine and in plasma. Results MicroPET/CT scans with radiolabelled FR366 showed a good tumour-to-normal tissue ratio with low uptake in the liver (0.32 +/- 0.14 %ID/g) and good retention of Ga-68-NODAGA-FR366 in the tumour (0.71 +/- 0.20 %ID/g and 0.40 +/- 0.12 %ID/g for RKO and M21 tumours, respectively, at 90 min after injection). Biodistribution experiments showed uptake in the alpha 5 beta 1-expressing RKO tumour of 1.05 +/- 0.23 %ID/g at 90 min after injection. Specificity of tracer uptake was demonstrated by injection of 5 mg/kg unlabelled ligand 10 min prior to tracer injection, resulting in a 67 % reduction in uptake in the RKO tumour. The tracer was found to be metabolically stable in urine and plasma 30 min after injection. Conclusion Our results show that PET imaging of alpha 5 beta 1 expression with the Ga-68-labelled alpha 5 beta 1-specific ligand is feasible with good image quality. Thus, FR366 is a promising new tool for investigating the role of alpha 5 beta 1 in angiogenesis and the influence of this integrin subtype on cancer aggressiveness and metastatic potential.