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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 11 C. We report the initial physical and biologic characterization of this novel ligand. Methods:  11 C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) 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 radiotracer 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 11 C-GSK1482160, receptor density and K d 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 11 C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11 C-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 30 min. 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 C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

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

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