title: Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat.
abstract: OBJECTIVE
  The P2X7 receptor is implicated in inflammation and pain and is therefore a potential target for therapeutic intervention. Here, the development of a native tissue radioligand binding, localization and ex vivo occupancy assay for centrally penetrant P2X7 receptor antagonists is described.
  
  
  METHODS
  Autoradiography studies using the P2X7 antagonist radioligand [³H]-A-804598 were carried out in rat brain and spinal cord. Subsequent in vitro binding and ex vivo occupancy assays were performed using rat cortex homogenate.
  
  
  RESULTS
  P2X7 expression was shown to be widespread throughout the rat brain, and in the grey matter of the spinal cord. In binding assays in rat cortex homogenate, ∼60% specific binding was achieved at equilibrium. In kinetic binding assays, k(on) and k(off) values of 0.0021·min⁻¹·nM⁻¹ and 0.0070·min⁻¹ were determined, and the K(d) derived from kinetic measurements was consistent with that derived from saturation analysis. Novel P2X7 antagonists inhibited the binding of [³H]-A-804598 to rat cortex P2X7 receptors with K(i) values of <40 nM. In an ex vivo occupancy assay, a P2X7 antagonist dosed orally to rats caused a concentration-dependent inhibition of the specific binding of [³H]-A-804598 to rat cortex.
  
  
  CONCLUSIONS
  The present study describes the development of an assay that allows localization of P2X7 receptors, the measurement of the binding affinity of P2X7 receptor antagonists in native tissue, and provides a means of determining central P2X7 receptor occupancy. These assays could form an important part of a P2X7 drug discovery programme.
authors.affiliation: Pfizer Global Research and Development, Sandwich, UK. sarah.able@pfizer.com
authors.forename: S L
authors.initials: SL
authors.lastname: Able
authors.forename: R L
authors.initials: RL
authors.lastname: Fish
authors.forename: H
authors.initials: H
authors.lastname: Bye
authors.forename: L
authors.initials: L
authors.lastname: Booth
authors.forename: Y R
authors.initials: YR
authors.lastname: Logan
authors.forename: C
authors.initials: C
authors.lastname: Nathaniel
authors.forename: P
authors.initials: P
authors.lastname: Hayter
authors.forename: S D
authors.initials: SD
authors.lastname: Katugampola
mesh_terms: D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D055808:Drug Discovery; D006146:Guanidines / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D058990:Molecular Targeted Therapy; D011485:Protein Binding; D058920:Purinergic P2X Receptor Antagonists / Q000378:metabolism* / Q000494:pharmacology; D011804:Quinolines / Q000097:blood / Q000378:metabolism* / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism*
chemical_list: C539459:2-cyano-1-((1S)-1-phenylethyl)-3-quinolin-5-ylguanidine; D006146:Guanidines; D058920:Purinergic P2X Receptor Antagonists; D011804:Quinolines; D058486:Receptors, Purinergic P2X7