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

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


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.

MeSH terms

  • Administration, Oral
  • Aminopyridines / administration & dosage
  • Aminopyridines / metabolism
  • Aminopyridines / pharmacokinetics
  • Aminopyridines / pharmacology*
  • Animals
  • Autoradiography
  • Behavior, Animal / drug effects*
  • Binding Sites
  • Binding, Competitive
  • Brain / drug effects*
  • Brain / metabolism
  • CHO Cells
  • Calcium Signaling / drug effects
  • Cell Membrane / drug effects*
  • Cell Membrane / metabolism
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Humans
  • Injections, Intraperitoneal
  • Inositol Phosphates / metabolism
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Kinetics
  • Male
  • Mice
  • Motor Activity / drug effects*
  • Neuropeptides / metabolism
  • Orexin Receptors
  • Orexins
  • Radioligand Assay
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, G-Protein-Coupled / antagonists & inhibitors*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, Neuropeptide / antagonists & inhibitors*
  • Receptors, Neuropeptide / genetics
  • Receptors, Neuropeptide / metabolism
  • Sulfonamides / administration & dosage
  • Sulfonamides / metabolism
  • Sulfonamides / pharmacokinetics
  • Sulfonamides / pharmacology*
  • Transfection


  • Aminopyridines
  • HCRT protein, human
  • Inositol Phosphates
  • Intracellular Signaling Peptides and Proteins
  • N-ethyl-2-((6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)amino)-N-pyridin-3-ylmethyl-acetamide
  • Neuropeptides
  • Orexin Receptors
  • Orexins
  • Receptors, G-Protein-Coupled
  • Receptors, Neuropeptide
  • Sulfonamides