Department of Pharmacology and Therapeutics, Faculty of Medicine, Dentistry and Health Sciences, School of Medicine, The University of Melbourne Parkville, VIC, Australia ; The Florey Institute of Neuroscience and Mental Health, The University of Melbourne Parkville, VIC, Australia.
Department of Neuroscience, Novartis Institutes for Biomedical Research Basel, Switzerland.
Front Neurosci. 2013 Dec 3;7:230. doi: 10.3389/fnins.2013.00230. eCollection 2013.
Orexin receptor antagonists represent attractive targets for the development of drugs for the treatment of insomnia. Both efficacy and safety are crucial in clinical settings and thorough investigations of pharmacokinetics and pharmacodynamics can predict contributing factors such as duration of action and undesirable effects. To this end, we studied the interactions between various "dual" orexin receptor antagonists and the orexin receptors, OX1R and OX2R, over time using saturation and competition radioligand binding with [(3)H]-BBAC ((S)-N-([1,1'-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide). In addition, the kinetics of these compounds were investigated in cells expressing human, mouse and rat OX1R and OX2R using FLIPR® assays for calcium accumulation. We demonstrate that almorexant reaches equilibrium very slowly at OX2R, whereas SB-649868, suvorexant, and filorexant may take hours to reach steady state at both orexin receptors. By contrast, compounds such as BBAC or the selective OX2R antagonist IPSU ((2-((1H-Indol-3-yl)methyl)-9-(4-methoxypyrimidin-2-yl)-2,9-diazaspiro[5.5]undecan-1-one) bind rapidly and reach equilibrium very quickly in binding and/or functional assays. Overall, the "dual" antagonists tested here tend to be rather unselective under non-equilibrium conditions and reach equilibrium very slowly. Once equilibrium is reached, each ligand demonstrates a selectivity profile that is however, distinct from the non-equilibrium condition. The slow kinetics of the "dual" antagonists tested suggest that in vitro receptor occupancy may be longer lasting than would be predicted. This raises questions as to whether pharmacokinetic studies measuring plasma or brain levels of these antagonists are accurate reflections of receptor occupancy in vivo.
食欲素受体拮抗剂代表了开发治疗失眠症药物的有吸引力的靶点。在临床环境中，疗效和安全性都至关重要，对药代动力学和药效动力学的深入研究可以预测作用持续时间和不良作用等影响因素。为此，我们使用 [(3)H]-BBAC((S)-N-([1,1'-联苯]-2-基)-1-(2-((1-甲基-1H-苯并[d]咪唑-2-基)硫代)乙酰基)吡咯烷-2-甲酰胺)，通过饱和和竞争放射性配体结合研究了各种“双重”食欲素受体拮抗剂与食欲素受体 OX1R 和 OX2R 随时间的相互作用。此外，还使用 FLIPR®测定法研究了这些化合物在表达人、鼠和大鼠 OX1R 和 OX2R 的细胞中的动力学。我们证明，阿莫雷克斯坦在 OX2R 上达到平衡非常缓慢，而 SB-649868、苏沃雷生和菲洛雷生可能需要数小时才能在两个食欲素受体上达到稳定状态。相比之下，像 BBAC 或选择性 OX2R 拮抗剂 IPSU((2-((1H-吲哚-3-基)甲基)-9-(4-甲氧基嘧啶-2-基)-2,9-二氮杂螺[5.5]十一烷-1-酮)这样的化合物结合和/或功能测定中快速结合并迅速达到平衡。总的来说，这里测试的“双重”拮抗剂在非平衡条件下往往相当不具有选择性，并且达到平衡非常缓慢。一旦达到平衡，每种配体都表现出一种选择性特征，但与非平衡条件不同。测试的“双重”拮抗剂的缓慢动力学表明，体外受体占有率可能比预测的持续时间更长。这引发了一个问题，即测量这些拮抗剂的血浆或脑水平的药代动力学研究是否准确反映了体内受体占有率。
