Kinetic properties of 'dual' orexin receptor antagonists at OX1 and Ox2 receptors

We have studied the interactions between human OX1 and OX2 receptors and various “dual” orexin receptor antagonists using radioligand binding with [3H] BBAC (S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). [3H] BBAC binds rapidly and reversibly to both hOX1 and hOX2 receptors. Competition experiments were performed at different time points (15 – 240 min) with [3H]BBAC and SB-649868, almorexant, suvorexant (MK4305) and filorexant (MK6096) in comparison to BBAC and IPSU (2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) a selective OX2 receptor antagonist, presented in the companion paper. Further, we have extended these studies to determine the kinetic features of the “dual” orexin receptor antagonists on calcium signaling using FLIPR® in cells expressing human, rat or mouse OX1 and OX2 receptors. The calcium signaling studies confirm what was observed in radioligand binding: almorexant reaches equilibrium very slowly at OX2 receptors, whereas SB-649868, suvorexant and filorexant may take hours to reach steady state at OX1 and/or OX2 receptors. On the other hand, compounds such as BBAC or IPSU bind rapidly and do reach equilibrium very quickly in both binding and / or functional assays. In summary, the “dual” antagonists tested here reach equilibrium very slowly; whereas they all tend to be rather unselective under non-equilibrium conditions, after equilibrium is reached, each ligand demonstrates a distinct selectivity profile. The data also suggest that since equilibrium will be reached slowly in vitro and probably in vivo, receptor occupancy may be longer lasting than predicted by pharmacokinetic studies measuring plasma or brain levels of these antagonists.