Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX2 receptor

The OX2 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 OX2 receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). The affinity of [H-3]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 [H-3]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX2 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. [H-3]EMPA bound to human and rat OX2-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 [H-3]IP at hOX(2) receptors with pA(2) values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [H-3]EMPA for OX2 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. EMPA is a high-affinity, reversible and selective OX2 receptor antagonist, active in vivo, which should prove useful for analysis of OX2 receptor function.