In vivo biokinetic and metabolic characterization of the super(68)Ga-labelled alpha 5 beta 1-selective peptidomimetic FR366

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-ca rboxy-2-(2-(3-guanidinobenzamido)acetamido)ethyl)carbamoyl)-3,5-di m e thylphenoxy)propyl)amino)-6-oxohexyl)amino)-4-oxobutyl)-1,4,7-tria z o nane-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 super(68)Ga-labelled alpha 5 beta 1 ligand in PET imaging. 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-triazacyclonon a ne (NODAGA) to yield FR366 and labelled with super(68)Ga. 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. MicroPET/CT scans with radiolabelled FR366 showed a good tumour-to-normal tissue ratio with low uptake in the liver (0.32 plus or minus 0.14 %ID/g) and good retention of super(68)Ga-NODAGA-FR366 in the tumour (0.71 plus or minus 0.20 %ID/g and 0.40 plus or minus 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 plus or minus 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