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

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-(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. [(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 +/- 17% of control in approximately half of the neurons tested. In the presence of 1 mu M SB-674042 or SB-408124, the effect of orexin-A was only partially antagonized. However, in the presence of 1 mu M EMPA or 1 mu 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.