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 (K_d 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.