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

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 [ 3 H]almorexant binding to human orexin 1 receptor (OX 1 )-and human orexin 2 receptor (OX 2 )-human embryonic kidney 293 membranes were characterized and compared with those of selective OX 1 and OX 2 antagonists, including 1- -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. [3 H]Almorexant bound to a single saturable site on hOX 1 and hOX 2 with high affinity (K d of 1.3 and 0.17 nM, respectively). In Schild analyses using the [ 3 H]inositol phosphates assay, almorexant acted as a competitive antagonist at hOX 1 and as a noncompetitive-like antagonist at hOX 2 . In binding kinetic analyses, [ 3 H]almorexant had fast association and dissociation rates at hOX 1 , whereas it had a fast association rate and a remarkably slow dissociation rate at hOX 2 . In the VTA, orexin-A potentiated the basal firing frequency to 175 Ϯ 17% of control in approximately half of the neurons tested. In the presence of 1 M SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 M EMPA or 1 M 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 OX 2 . The electrophysiology data suggest that OX 2 might be more important than OX 1 in mediating the effect of orexin-A on slow-firing of VTA dopaminergic neurons.Orexins (also called hypocretins) belong to a family of neuropeptides that are exclusively synthesized in the neuronal cell bodies of the lateral hypothalamic areas. Orexin-A/hypocretin-1 (33 amino acids) and orexin-B/hypocretin-2 (28 amino acids) are derived from the proteolytic processing of 130 amino acid polypeptide prepro-orexin (de Lecea et al., Article, publication date, and citation information can be found at

Role of Orexin-1 Receptor Mechanisms on Compulsive Food Consumption in a Model of Binge Eating in Female Rats

“…On the contrary, the OX 1 R antagonist GSK1059865 showed a very poor hypnotic ability to induce and maintain sleep. These findings are in keeping with other reports suggesting that OX 2 R might be more important than OX 1 R in mediating the OX effect on sleep (Sakurai, 2007;Brisbare-Roch et al, 2007;Malherbe et al, 2009;Dugovic et al, 2009;Di Fabio et al, 2011).…”

Mapping the Binding Pocket of Dual Antagonist Almorexant to Human Orexin 1 and Orexin 2 Receptors: Comparison with the Selective OX1 Antagonist SB-674042 and the Selective OX2 Antagonist N-Ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA)

“…Forty-eight hours after transfection, the cells were loaded for 1 h at 37°C with 4 M Fluo-4 acetoxymethyl ester (Invitrogen, Carlsbad, CA) in loading buffer (1ϫ Hanks' balanced salt solution and 20 mM HEPES). The cells were washed five times with loading buffer to remove excess dye, and [Ca 2ϩ ] i was measured using a FLIPR (Molecular Devices, Sunnyvale, CA) as described previously (Malherbe et al, 2009b). Orexin-A (50 mM stock solution in dimethyl sulfoxide) was diluted in FLIPR buffer plus 0.1% BSA.…”

“…at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org hOX 2 (Malherbe et al, 2009b). The biochemical characterization of an OX 2 antagonist, EMPA, with 900-fold selectivity in binding for OX 2 over OX 1 has been reported (Malherbe et al, 2009a (Malherbe et al, 2009b).…”

“…Furthermore, the biochemical characterization of almorexant (a high-affinity dual OX 1 /OX 2 ) has demonstrated that it had an apparent noncompetitive and longlasting pseudo-irreversible mode of antagonism that was due to its very slow rate of dissociation from OX 2 , whereas it displayed a competitive mode of antagonism at OX 1 (Malherbe et al, 2009b). 1-(5-(2-Fluoro-phenyl)-2-methylthiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone (SB-674042), which is a highaffinity and selective OX 1 antagonist (Langmead et al, 2004), has been also shown to behave in an apparent noncompetitive manner at hOX 1 similar to that of almorexant at hOX 2 (Malherbe et al, 2009b).…”

Novos sedativos hipnóticos

“…Os resultados destes primeiros estudos demonstraram que o almorexant reduziu o alerta e aumentou o tempo de sono em modelos animais e humanos, quando administrado durante a fase ativa do ciclo circadiano 40,41 .…”