Biochemical and Electrophysiological Characterization of Almorexant, a Dual Orexin 1 Receptor (OX1)/Orexin 2 Receptor (OX2) Antagonist: Comparison with Selective OX1 and OX2 Antagonists

  1. Pari Malherbe,
  2. Edilio Borroni,
  3. Emmanuel Pinard,
  4. Joseph G. Wettstein and
  5. Frédéric Knoflach
  1. CNS Research (P.M., E.B., J.G.W., F.K.) and Chemistry Discovery (E.P.), F. Hoffmann-La Roche Ltd., Basel, Switzerland
  1. Address correspondence to:
    Dr. Pari Malherbe, F. Hoffmann-La Roche Ltd., Bldg. 69/333, CH-4070 Basel, Switzerland. E-mail: parichehr.malherbe{at}roche.com

Abstract

Recent preclinical and clinical research has shown that almorexant promotes sleep in animals and humans without disrupting the sleep architecture. Here, the pharmacology and kinetics of [3H]almorexant binding to human orexin 1 receptor (OX1)- and human orexin 2 receptor (OX2)-human embryonic kidney 293 membranes were characterized and compared with those of selective OX1 and OX2 antagonists, including 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042), 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea (SB-408124), and N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA). The effect of these antagonists was also examined in vitro on the spontaneous activity of rat ventral tegmental area (VTA) dopaminergic neurons. [3H]Almorexant bound to a single saturable site on hOX1 and hOX2 with high affinity (Kd of 1.3 and 0.17 nM, respectively). In Schild analyses using the [3H]inositol phosphates assay, almorexant acted as a competitive antagonist at hOX1 and as a noncompetitive-like antagonist at hOX2. In binding kinetic analyses, [3H]almorexant had fast association and dissociation rates at hOX1, whereas it had a fast association rate and a remarkably slow dissociation rate at hOX2. In the VTA, orexin-A potentiated the basal firing frequency to 175 ± 17% of control in approximately half of the neurons tested. In the presence of 1 μM SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 μM EMPA or 1 μM almorexant, the effect of orexin-A was completely antagonized. In conclusion, almorexant exhibited a noncompetitive and long-lasting pseudo-irreversible mode of antagonism as a result of its very slow rate of dissociation from OX2. The electrophysiology data suggest that OX2 might be more important than OX1 in mediating the effect of orexin-A on slow-firing of VTA dopaminergic neurons.

Footnotes

  • A preliminary account of some of the results has been published in abstract form [Knoflach F, Borroni E, Kratzeisen C, Marcuz A, Humbel U, Weber M, Zenner MT, Pinard E, Wettstein JG, and Malherbe P (2008) Biochemical and electrophysiological characterization of a dual OX1R/OX2R antagonist, almorexant and its comparison with the selective OX1R and OX2R antagonists SB-674042 and EMPA. Soc Neurosci Abstr 34:826.817].

  • ABBREVIATIONS: OX1, orexin 1 receptor; OX2, orexin 2 receptor; ACT-078573, almorexant, (2R)-2-{(1S)-6,7-dimethoxy-1-[2-(4-trifluoromethylphenyl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide; CHO, Chinese hamster ovary; REM, rapid eye movement; IP, inositol phosphates; HEK, human embryonic kidney; FLIPR, fluorometric imaging plate reader; DA, dopamine; EMPA, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide; SB-674042, 1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone; Cp-2, 1-(9-oxo-8-trifluoromethyl-1,2,3,9-tetrahydro-pyrrolo[2,1-b]quinazolin-3-yl)-1-((S)-1-phenyl-ethyl)-3-(2-trifluoromethoxy-phenyl)-urea; Cp-3, 2-methyl-5-phenyl-thiazole-4-carboxylic acid cyclobutyl-[3-(4-fluoro-phenoxy)-propyl]-amide; Cp-4, 2-[[4-chloro-2-(hydroxy-phenyl-methyl)-phenyl]-(3,4-dimethoxybenzenesulfonyl)-amino]-N-methyl-acetamide; Cp-5, ((S)-1-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-3,3-dimethyl-2-[(pyridin-4-ylmethyl)-amino]-butan-1-one; SB-334867, 1-(2-methyl-benzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride; SB-408124, 1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea; JNJ-10397049, 1-(2,4-dibromophenyl)-3-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]urea; VTA, ventral tegmental area; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; ACSF, artificial cerebrospinal fluid; CRC, concentration-response curve; NSB/TB%, nonspecific binding/total bound radioactivity; Cp-1, (2R)-2-{(1S)-6,7-dimethoxy-1-[2-(6-trifluoromethyl-pyridin-3-yl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide.

    • Accepted June 17, 2009.
    • Received January 29, 2009.
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  1. Published online before print June 19, 2009, doi: 10.1124/mol.109.055152 Molecular Pharmacology September 2009 vol. 76 no. 3 618-631
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