[³H]A-804598 ([³H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors

Abstract

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC₅₀ = 10 nM), mouse (IC₅₀ = 9 nM) and human (IC₅₀ = 11 nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([³H]A-804598; 8.1 Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [³H]A-804598 labeled a single class of high affinity binding sites (K_d = 2.4 nM and apparent B_max = 0.56 pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [³H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r = 0.95, P < 0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [³H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.

Introduction

P2X7 receptors belong to the family of ATP-activated ionotropic P2X receptors, which include seven homomeric receptor subtypes (P2X1–P2X7) (North, 2002). The P2X7 receptor, previously termed the P2_Z receptor (Di Virgilio et al., 2001), was initially cloned from rat (Surprenant et al., 1996) and human brain (Collo et al., 1997). P2X7 receptors are selectively expressed on cells of hematopoietic lineage including mast cells, lymphocytes, erythrocytes, fibroblasts, and peripheral macrophages (Surprenant et al., 1996). Within the CNS, functional P2X7 receptors are localized on microglia and Schwann cells, as well as on astrocytes (Ferrari et al., 1996, Collo et al., 1997, Sim et al., 2004).

The distribution of P2X7 receptors and the fact that high concentrations of ATP are required to activate the receptor support the hypothesis that the P2X7 receptor functions as a “danger” sensor associated with tissue inflammation or damage (Ferrari et al., 2006). Data from studies of P2X7 receptor knockout mice (Labasi et al., 2002, Chessell et al., 2005) support this hypothesis since P2X7 knockout mice show less chronic inflammation and nerve injury induced pain relative to wild-type controls. The role of P2X7 receptors in mediating some forms of persistent pain in experimental models of nerve injury is further supported by data showing that selective P2X7 receptor antagonists reduce pain sensitivity in these models (Nelson et al., 2006, Honore et al., 2006; Lappin et al., 2005; McGaraughty et al., 2007).

A number of P2X7 receptor antagonists including KN-62, PPADS, oATP and Brilliant Blue G (BBG) have been identified in recent years (Donnelly-Roberts and Jarvis, 2007). A common feature of these antagonists is their differential affinity for human versus rodent P2X7 receptors (Donnelly-Roberts and Jarvis, 2007, Chessell et al., 1998; Michel et al., 2007). For example, KN-62 (Humphreys et al., 1998) potently blocks human P2X7 receptors, but lacks activity at the rat receptor. More recently disclosed adamantine-based antagonists including AZ116453743 (Stokes et al., 2006) and compound 17 (Michel et al., 2007) also show a significant reduction in potency at the rat receptor relative to human P2X7 receptors. Other antagonists like oATP and PPADS show only very weak affinity for blocking P2X7 receptors and are nonselective (Donnelly-Roberts and Jarvis, 2007). Additionally, many of these antagonists have slow association rates and do not competitively block agonist activation of P2X7 receptors (Michel et al., 2007). The inability of these antagonists to block rodent P2X7 receptors is an obvious impediment to determining the physiological roles of P2X7 modulation in experimental models. More recently, several structurally distinct and competitive P2X7 receptor antagonists (e.g. A-740003 and A-438079) have been described that show less human versus rat species differences and have sufficient pharmacokinetic properties to enable their use for in vivo studies (Honore et al., 2006, McGaraughty et al., 2007). However, a limitation of these newer P2X7 antagonists for certain types of in vitro studies (e.g. radioligand binding studies) is their moderate nanomolar affinity (pIC₅₀ = 6.5–7.5) at rat P2X7 receptors.

This report describes the pharmacology of A-804598 (Fig. 1), a structurally novel competitive antagonist of P2X7 receptors that has high affinity for blocking mouse, rat, and human P2X7 receptors. A-804598 was radiolabeled ([³H]A-804598; 8.1 Ci/mmol, Fig. 1) and shown to be a useful radioligand for the study of rat recombinant P2X7 receptors.