In vivo biokinetic and metabolic characterization of the 68Ga-labelled α5β1-selective peptidomimetic FR366
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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 αvβ3. However, targeting of other subtypes such as the integrin α5β1 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 α5β1 with high selectivity versus αvβ3, 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 68Ga-labelled α5β1 ligand in PET imaging.
The free α5β1 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 68Ga. To confirm selective in vivo targeting of α5β1, female BALB/c nude mice xenografted with α5β1-expressing RKO cells in the right shoulder and α5β1/αvβ3-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 ± 0.14 %ID/g) and good retention of 68Ga-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 α5β1-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.
Our results show that PET imaging of α5β1 expression with the 68Ga-labelled α5β1-specific ligand is feasible with good image quality. Thus, FR366 is a promising new tool for investigating the role of α5β1 in angiogenesis and the influence of this integrin subtype on cancer aggressiveness and metastatic potential.
Keywordsα5β1 Integrin antagonist Peptidomimetics PET imaging
We thank Christina Lesti, Rosel Oos and Marianna Kallinger for technical assistance (cell culture and animal handling), Martina Anton and Edelburga Hammerschmid for help with the FACS analysis and Sybille Reder, Markus Mittelhäuser and Marco Lehmann for performing the animal PET/CT experiments.
C.D. optimized the FACS analysis protocol and measurements, elaborated and prepared the PET/CT images, performed the data analysis, and wrote the manuscript. K.P. carried out 68Ga-labelling, performed cell binding studies, the FACS analysis and in vivo experiments (biodistribution, metabolic stability, PET/CT analysis). S.N. and F.R. synthesized and characterized the integrin antagonist. J.N. and H.J.W. proof-read the manuscript and provided advice on 68Ga labelling. A.J.B. and M.S. were responsible for the molecular imaging experiments. A.J.B., M.S. and H.K. initiated and supervised the project. All authors contributed to writing and editing the manuscript.
Compliance with ethical standards
This work was carried out within the MOBITUM project P3 “Improved ligands for quantitative monitoring of integrin expression” (MOBITEC/MOBITUM 01EZ0826) at TU Munich and was supported partially by ERC grant MUMI, ERC-2011-ADG_20110310, by the SFB 824 project C5 “Evaluation of imaging of αVβ3 expression with PET for response assessment of antiangiogenic therapies” and the Center of Integrated Protein Science Munich (CIPSM). H.K. is grateful for financial support from the Center of Integrated Protein Science Munich (CIPSM) and from King Abdulaziz University KAU (grant no. HiCi/25-3-1432).
Conflicts of interest
Ethical approval for animal studies
All animal experiments were approved by the “Regierung von Oberbayern” (animal permission no. 55.2-1-45-2531-94-10) and were performed in accordance with current animal welfare regulations in Germany.
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