Characterization of 11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation

The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with C-11. We report the initial physical and biologic characterization of this novel ligand. Methods: C-11-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline-or lipopolysaccharide-treated mice at 15, 30, and 60 min after radio-tracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity C-11-GSK1482160, receptor density and Kd were 1.15 +/- 0.12 nM and 3.03 +/- 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant k(on), dissociation constant k(off), and binding potential (k(on)/k(off)) in HEK293-hP2X7R cells were 0.2312 +/- 0.01542 min(-1).nM(-1), 0.2547 +/- 0.0155 min(-1), and 1.0277 +/- 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline-and lipopolysaccharide-treated brain sections showed a respective 1.8-and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of C-11-GSK1482160 in saline-and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic C-11-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of (11)CGSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.