title: Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX(2) receptor.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland. parichehr.malherbe@roche.com
authors.forename: P
authors.initials: P
authors.lastname: Malherbe
authors.forename: E
authors.initials: E
authors.lastname: Borroni
authors.forename: L
authors.initials: L
authors.lastname: Gobbi
authors.forename: H
authors.initials: H
authors.lastname: Knust
authors.forename: M
authors.initials: M
authors.lastname: Nettekoven
authors.forename: E
authors.initials: E
authors.lastname: Pinard
authors.forename: O
authors.initials: O
authors.lastname: Roche
authors.forename: M
authors.initials: M
authors.lastname: Rogers-Evans
authors.forename: J G
authors.initials: JG
authors.lastname: Wettstein
authors.forename: J-L
authors.initials: JL
authors.lastname: Moreau
mesh_terms: D000284:Administration, Oral; D000631:Aminopyridines / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D001522:Behavior, Animal / Q000187:drug effects*; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007274:Injections, Intraperitoneal; D007295:Inositol Phosphates / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D009043:Motor Activity / Q000187:drug effects*; D009479:Neuropeptides / Q000378:metabolism; D064446:Orexin Receptors; D000068797:Orexins; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018013:Receptors, Neuropeptide / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000008:administration & dosage / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D014162:Transfection
chemical_list: D000631:Aminopyridines; C000596229:HCRT protein, human; D007295:Inositol Phosphates; D047908:Intracellular Signaling Peptides and Proteins; C546023:N-ethyl-2-((6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)amino)-N-pyridin-3-ylmethyl-acetamide; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D013449:Sulfonamides