Biological characterization of [¹⁸F]-radiolabeled anilinopyrimidine: a potential radiotracer for EphB4 receptor imaging

Background:

The receptor tyrosine kinase EphB4 and its ligand ephrinB2 play a critical role in tumor angiogenesis. Therefore, EphB4/ephrinB2 system is a potential target for tumor diagnosis and therapy, and different approaches including tyrosine kinase inhibitors are under investigation. Our study aimed the development and biological characterization of a novel radiotracer targeted to EphB4.
Material and Methods:
Based on a known EphB4 kinase inhibitor (lead compound 1) containing a bis anilinopyrimidine motif a methylsulfonyl residue was replaced by 3 fluoropropylsulfonyl moiety (2) followed by 18F-radiolabeling ([18F]2). Human melanoma cells A375 transfected with human EphB4 full length protein (A375 EphB4) or mock plasmid (A375 pIRES) were analyzed for cell viability and EphB4 tyrosine phosphorylation after incubation with compounds 1 and 2. Cellular uptake of [18F]2 was investigated using specific inhibitors. [18F]2 was analyzed in biodistribution experiments and dynamic positron emission tomography (PET) studies using nude mice bearing A375 EphB4 or A375 pIRES tumors.
Results:
Overexpression of EphB4 in A375 EphB4 cells was confirmed by mRNA and protein expression. Both compound 1 and 2 reduced EphB4 tyrosine phosphorylation and influenced cell morphology and viability. [18F]2 showed internalization by A375 and transfected A375 cells. Partial inhibitory effects of different specific inhibitors provided information on possible uptake mechanisms, which further have to be characterized. Biodistribution and PET studies revealed high stability of [18F]2 in vivo with fast hepatobiliary excretion. No differences between A375 EphB4 and A375 pIRES tumors could be observed in uptake of [18F]2.
Conclusion:
The novel compound 2 substantially inhibited EphB4 tyrosine phosphorylation, when radiolabelded with 18F represents a potential PET tracer. Since [18F]2 was internalized by A375 melanoma cells in vitro the use of [18F]2 for imaging of EphB4 in vivo by PET is supported. However, further studies will deal with optimization of its pharmacokinetic properties.