Characterization of 11C-GSK1482160 for Targeting the P2X7 receptor as a biomarker for neuroinflammation

Paul Territo, Jill A. Meyer, Jonathan S. Peters, Amanda A. Riley, Brian P. McCarthy, Mingzhang Gao, Wang Min, Mark Green, Qi-Huang Zheng, Gary Hutchins

Research output: Contribution to journalArticle

46 Citations (Scopus)

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 with 11C. 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 1 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 11C-GSK1482160, receptor density and Kd were 1.15 6 0.12 nM and 3.03 6 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 6 0.01542 min21nM21, 0.2547 6 0.0155 min21, and 1.0277 6 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 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide 1 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 11CGSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

Original languageEnglish (US)
Pages (from-to)458-465
Number of pages8
JournalJournal of Nuclear Medicine
Volume58
Issue number3
DOIs
StatePublished - Mar 1 2017

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Purinergic P2X7 Receptors
Lipopolysaccharides
Biomarkers
Western Blotting
Brain
Cell Count
Immunohistochemistry
N-(2-chloro-3-(trifluoromethyl)benzyl)-N-methyl-5-oxopyrrolidine-2-carboxamide
Purinergic Receptors
Molecular Imaging
HEK293 Cells
Blood-Brain Barrier
Fluorescent Antibody Technique
Hippocampus
Ligands
Kidney

Keywords

  • 11C-GSK1482160
  • Neuroinflammation
  • P2X7 receptor
  • Purinergic
  • Receptors

ASJC Scopus subject areas

  • Radiology Nuclear Medicine and imaging

Cite this

Characterization of 11C-GSK1482160 for Targeting the P2X7 receptor as a biomarker for neuroinflammation. / Territo, Paul; Meyer, Jill A.; Peters, Jonathan S.; Riley, Amanda A.; McCarthy, Brian P.; Gao, Mingzhang; Min, Wang; Green, Mark; Zheng, Qi-Huang; Hutchins, Gary.

In: Journal of Nuclear Medicine, Vol. 58, No. 3, 01.03.2017, p. 458-465.

Research output: Contribution to journalArticle

Territo, Paul ; Meyer, Jill A. ; Peters, Jonathan S. ; Riley, Amanda A. ; McCarthy, Brian P. ; Gao, Mingzhang ; Min, Wang ; Green, Mark ; Zheng, Qi-Huang ; Hutchins, Gary. / Characterization of 11C-GSK1482160 for Targeting the P2X7 receptor as a biomarker for neuroinflammation. In: Journal of Nuclear Medicine. 2017 ; Vol. 58, No. 3. pp. 458-465.
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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 with 11C. 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 1 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 11C-GSK1482160, receptor density and Kd were 1.15 6 0.12 nM and 3.03 6 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 6 0.01542 min21nM21, 0.2547 6 0.0155 min21, and 1.0277 6 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 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide 1 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 11CGSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.",
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author = "Paul Territo and Meyer, {Jill A.} and Peters, {Jonathan S.} and Riley, {Amanda A.} and McCarthy, {Brian P.} and Mingzhang Gao and Wang Min and Mark Green and Qi-Huang Zheng and Gary Hutchins",
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TY - JOUR

T1 - Characterization of 11C-GSK1482160 for Targeting the P2X7 receptor as a biomarker for neuroinflammation

AU - Territo, Paul

AU - Meyer, Jill A.

AU - Peters, Jonathan S.

AU - Riley, Amanda A.

AU - McCarthy, Brian P.

AU - Gao, Mingzhang

AU - Min, Wang

AU - Green, Mark

AU - Zheng, Qi-Huang

AU - Hutchins, Gary

PY - 2017/3/1

Y1 - 2017/3/1

N2 - 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 11C. 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 1 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 11C-GSK1482160, receptor density and Kd were 1.15 6 0.12 nM and 3.03 6 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 6 0.01542 min21nM21, 0.2547 6 0.0155 min21, and 1.0277 6 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 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide 1 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 11CGSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

AB - 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 11C. 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 1 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 11C-GSK1482160, receptor density and Kd were 1.15 6 0.12 nM and 3.03 6 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 6 0.01542 min21nM21, 0.2547 6 0.0155 min21, and 1.0277 6 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 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide 1 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 11CGSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.

KW - 11C-GSK1482160

KW - Neuroinflammation

KW - P2X7 receptor

KW - Purinergic

KW - Receptors

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