Kinetic properties of “dual” orexin receptor antagonists at OX1R and OX2R orexin receptors

Abstract

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 [3H]-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.

Orexin/Hypocretin Based Pharmacotherapies for the Treatment of Addiction: DORA or SORA?

“…fluorescence 9.0 8.0 [71] pK b -Orexin-A induced IP1 accumulation 9.1 9.8 [197] pK b -Orexin-A induced Ca 2? fluorescence b 9.1-8.9 9.4-9.8 [212] Suvorexant (MK-4305) pK i -Radioligand-displacement binding assay 9.3 9.5 [77] IC 50 -Orexin-A induced Ca 2? fluorescence 50 nM 56 nM [77] pK b -Orexin-A induced Ca 2?…”

“…fluorescence 6.9 6.9 [71] pK b -Orexin-A induced Ca 2? fluorescence b 7.8 8.3-9.1 [212] SB-649868 pK i -Radioligand-displacement binding assay 9.5 9.4 [66] pK b -Orexin-A induced IP1 accumulation 9.7 9.6 [197] pK b -Orexin-A induced Ca 2? fluorescence b 9.0-9.4 9.5-9.8 [212] Filorexant (MK-6096) pK i -Radioligand-displacement binding assay 8.6 9.5 [71] pK b -Orexin-A induced Ca 2?…”

“…fluorescence b 7.8 8.3-9.1 [212] SB-649868 pK i -Radioligand-displacement binding assay 9.5 9.4 [66] pK b -Orexin-A induced IP1 accumulation 9.7 9.6 [197] pK b -Orexin-A induced Ca 2? fluorescence b 9.0-9.4 9.5-9.8 [212] Filorexant (MK-6096) pK i -Radioligand-displacement binding assay 8.6 9.5 [71] pK b -Orexin-A induced Ca 2? fluorescence 9.0 8.0 [71] pK b -Orexin-A induced IP1 accumulation 9.1 9.8 [197] pK b -Orexin-A induced Ca 2?…”

Role of orexin receptor subtypes in the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from dorsal root ganglion of carrageenan-treated rats

“…Doses of KCl and ionomycin indicate the concentration (mM) in the analytical chamber. Doses used were determined by a series of pilot experiments based on the outcome of previously reported studies (13)(14)(15)(16)(17). The vehicle for selected doses of orexin-A (20 pmol), MK-4305 (200 pmol) and SB 334867 (2 pmol) in the present study did not alter the baseline fluorescence intensity.…”

Synthesis of [11C]MK-1064 as a new PET radioligand for imaging of orexin-2 receptor

“…Conversely, OX2R is expressed in the tuberomammillary nucleus. 5 The available experimental evidences strongly support that OX2R plays a key role in motivation, arousal and sleep-wake regulation, and is associated with sleep disorders, irregularities in central vestibular motor control, feeding and gastrointestinal disorders and drug abuse. 6 Although OX1R is thought to regulate sleep-wakefulness and energy homeostasis, particularly food intake, the therapeutic potential of selective antagonism of OX1R is still under evaluation.…”