title: Characterization of11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation.
abstract: 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 with11C. We report the initial physical and biologic characterization of this novel ligand.Methods:11C-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-activity11C-GSK1482160, receptor density and Kdwere 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constantkon, dissociation constantkoff, and binding potential (kon/koff) 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 of11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic11C-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 of11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana pterrito@iupui.edu.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Territo
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Jill A
authors.initials: JA
authors.lastname: Meyer
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Peters
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Riley
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Brian P
authors.initials: BP
authors.lastname: McCarthy
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Mingzhang
authors.initials: M
authors.lastname: Gao
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Green
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Qi-Huang
authors.initials: QH
authors.lastname: Zheng
authors.affiliation: Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, Indiana.
authors.forename: Gary D
authors.initials: GD
authors.lastname: Hutchins
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000493:pharmacokinetics; D004660:Encephalitis / Q000000981:diagnostic imaging* / Q000378:metabolism*; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D057054:Molecular Imaging / Q000379:methods; D049268:Positron-Emission Tomography / Q000379:methods*; D011761:Pyrrolidonecarboxylic Acid / Q000493:pharmacokinetics*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D002250:Carbon Radioisotopes; D018836:Inflammation Mediators; C000604998:N-(2-chloro-3-(trifluoromethyl)benzyl)-N-methyl-5-oxopyrrolidine-2-carboxamide; D019275:Radiopharmaceuticals; D058486:Receptors, Purinergic P2X7; D011761:Pyrrolidonecarboxylic Acid
keywords: 11C-GSK1482160; P2X7 receptor; neuroinflammation; purinergic; receptors