Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY 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 AND IMPLICATIONS: 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.