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Br J Pharmacol. 2009 Apr;156(8):1326-41. doi: 10.1111/j.1476-5381.2009.00127.x.

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

Author information

1
Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland. parichehr.malherbe@roche.com

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 [(3)H]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 [(3)H]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:

[(3)H]EMPA bound to human and rat OX(2)-HEK293 membranes with K(D) values of 1.1 and 1.4 nmol x L(-1) respectively. EMPA competitively antagonized orexin-A- and orexin-B-evoked accumulation of [(3)H]IP at hOX(2) receptors with pA(2) values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [(3)H]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.

PMID:
19751316
PMCID:
PMC2697736
DOI:
10.1111/j.1476-5381.2009.00127.x
[Indexed for MEDLINE]
Free PMC Article

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