Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX 2 receptor

Abstract

Background and purpose: The OX 2 receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX 2 receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino] -N-pyridin-3-ylmethyl-acetamide (EMPA). Experimental approach: The affinity of [ 3H]EMPA was assessed in membranes from HEK293-hOX 2-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A- or orexin-B-induced accumulation of [ 3H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX 2 receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala 11,D-Leu 15]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats. Key results: [ 3H]EMPA bound to human and rat OX 2-HEK293 membranes with K D values of 1.1 and 1.4 nmol·L -1 respectively. EMPA competitively antagonized orexin-A- and orexin-B-evoked accumulation of [ 3H]IP at hOX 2 receptors with pA 2 values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [ 3H]EMPA for OX 2 receptors. EMPA significantly reversed [Ala 11,D-Leu 15]orexin-B- induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure. Conclusions and implications: EMPA is a high-affinity, reversible and selective OX 2 receptor antagonist, active in vivo, which should prove useful for analysis of OX 2 receptor function. © 2009 The British Pharmacological Society.