Edited by: Christopher J. Winrow, Merck, USA
Reviewed by: Anthony J. Hannan, University of Melbourne, Australia; Zoë Bichler, National Neuroscience Institute, Singapore
*Correspondence: Daniel Hoyer, Department of Pharmacology and Therapeutics, Faculty of Medicine, Dentistry and Health Sciences, School of Medicine, The University of Melbourne, Parkville, VIC 3010, Australia e-mail:
This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience.
†Present address: Markus Fendt, Institute for Pharmacology and Toxicology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany; Laura H. Jacobson, The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Australia
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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-((1
The orexin receptors, OX1R and OX2R, were deorphanised in 1998, when two independent teams identified the peptides orexin A and orexin B (de Lecea et al.,
Orexin is exclusively expressed by orexin producing neurons within the perifornical nucleus, the dorsomedial hypothalamic nucleus, and the dorsal and lateral hypothalamic areas (Peyron et al.,
The orexin receptors are widely distributed in the brain in a pattern consistent with orexin neuron projections (Trivedi et al.,
Although orexin was originally named for its role in feeding behavior (Sakurai et al.,
Narcolepsy with cataplexy is associated with severe daytime sleepiness (Tafti et al.,
Not surprisingly, the orexin system has attracted substantial attention for the development of drugs for the treatment of insomnia. Dual orexin receptor antagonists or possibly selective OX2R antagonists are likely to be effective without some of the undesirable side effects of currently available treatments. Benzodiazepines and sedative hypnotics are commonly prescribed and inhibit arousal through activation or positive allosteric modulation of the GABAA receptor. However, reported side effects include morning sedation, anxiety, anterograde amnesia, impaired balance and sleep behaviors such as sleep walking and eating (Buysse,
A number of orexin receptor antagonists have been developed that are expected to have advantages over classic sleep promoting drugs (see Uslaner et al.,
During the characterization of orexin receptor antagonists, we and others (Malherbe et al.,
[3H]-BBAC ((S)-N-([1,1′-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide, Specific activity 73.76Ci/mmol) was synthesized at Novartis Pharma AG Basel (Isotope Laboratories). BBAC, SB-649868, suvorexant, filorexant, and IPSU (2-((1
Chinese Hamster Ovary (CHO) cells stably transfected with the cDNA encoding the human OX1R (CHO-hOX1) or OX2R (CHO-hOX2) were used (kindly provided by T. Cremer and Dr. S. Geisse, NIBR Basel, Switzerland). For measurements of calcium accumulation using FLIPR® (Fluorescent Imaging Plate Reader) assay, CHO or Human Embryonic Kidney (HEK) cells stably expressing mouse, rat or human OX1R or OX2R (kindly provided by Dr. A. Chen, GNF, San Diego, CA, USA) were used. All cells were cultured in 1:1 Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 Nutrients Mixture (F12) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 μ/ml (100 g/ml)/streptomycin (100 μg/ml), Fungizone (250 μg/ml), and Geneticin (G418, 50 mg/ml). Cells were maintained in a humidified incubator at 37°C in 5% CO2. For crude cell membrane preparations, cells were washed and harvested in 10 mM HEPES (pH 7.5), and centrifuged at 4°C for 5 min at 2500 g. The cell pellet was either stored at −80°C or used directly.
Cell membranes were resuspended in binding assay buffer at 4°C (10 mM HEPES, pH 7.5, 0.5% (w/v) bovine serum albumin (BSA), 5 mM MgCl2, 1 mM CaCl2, and 0.05% Tween 20) and homogenized with a Polytron homogenizer at 50 Hz for 20 s. Cells were incubated with [3H]-BBAC in binding assay buffer in 96-deep well plates (Fisher Scientific). Aliquots of [3H]-BBAC were measured using liquid scintillation spectrometry on a LS 6500 scintillation counter (Beckman Coulter) to determine the amount radioactivity added to each well. Non-specific Binding (NSB) was determined in the presence of 1 μM almorexant. After the indicated incubation time, bound and free radioligand were separated by vacuum filtration using a Filtermate™ Cell Harvester (Perkin Elmer) and filtered onto 96-well deep GF/b filter plates (Millipore) which had been pre-treated with 0.5% (w/v) polyethyleneimine. Filter plates were rapidly washed three times with wash buffer (10 mM Tris-HCl, 154 mM NaCl, pH 7.4) at 4°C, dried and 25 μl of Microscint™ (Perkin Elmer) was added to each well. Radioactivity was quantified using a TopCount™ microplate counter (Perkin Elmer).
Binding was performed with eight concentrations of [3H]-BBAC (50 μl, 1–20 nM) to construct saturation curves. CHO-hOX1 or CHO-hOX2 cell membranes (150 μl/well) were incubated for 60 min in 96-deep well plates at room temperature with radioligand in binding assay buffer (50 μl) in the presence or absence of almorexant (1 μM, 50 μl), in a final volume of 250 μl. [3H]-BBAC binding was measured in triplicate in at least three independent experiments. Data in the figures is representative of the mean ± s.e.m. of a single experiment.
Competition experiments were performed with a single concentration of radioligand and six concentrations of competitor (unlabeled ligands; BBAC, almorexant, SB-649868, suvorexant, filorexant or IPSU). 4.6 nM [3H]-BBAC (chosen from saturation experiments to provide 80–90% specific binding, 50 μl) was added simultaneously with various concentrations of unlabeled ligand (0.1 nM–10 μ M) to membranes (150 μl/well) in 50 μl/well of assay buffer with a total volume of 250 μl/well. The amount of [3H]-BBAC bound to receptors was determined at room temperature at different time points (ranging from 15 min to 4 h) and terminated by rapid vacuum filtration and liquid scintillation counting. Binding at a given concentration of competitor at a given time was measured in triplicate in at least three independent experiments. Data in figures is representative of the mean ± s.e.m. of a single experiment.
All data was analyzed using GraphPad Prism 4.0 (GraphPad, San Diego, USA). The saturation data was fit to a non-linear regression model for saturation binding with consideration for one site binding. In addition, saturation binding data was also analyzed according to Scatchard (Scatchard,
Determination of orexin A-stimulated calcium accumulation was performed over 2 days using FLIPR® (Fluorescent imaging plate reader from Molecular Devices-FLIPR384). Cells expressing either human, rat or mouse OX1R or OX2R were seeded at 8,000 cells/well in black 384 well clear bottom plates and incubated overnight at 37°C. The following day, medium was discarded and cells loaded with 50 μ l of 1 mM Fluo-4 AM (Invitrogen F14202) in dimethyl sulfoxide in working buffer (Hanks' balanced salt solution, 10 mM HEPES) and incubated for 60 min at 37°C. The loading buffer was removed and cells were washed with 100 μ l working buffer containing 200 mM CaCl2, 0.1% BSA, and 2.5 mM Probenecid (pH 7.4) to remove the excess Fluo-4 AM. Working buffer was added and plates were incubated 10–15 min at room temperature. The assay plate was then transferred to the Molecular Devices-FLIPR384. The baseline calcium signal was recorded for 10 s, then the antagonist of interest was injected (10 μl at 3 times the final concentration) and the calcium signal recorded every second for 1 min, then every 2 s 40 times. Plates were then incubated at room temperature for 30 min, 1, 2, or 4 h. Calcium signals were again measured as above, this time orexin A (15 μl) was injected at 3 times the final concentration. For each experiment, full orexin A concentration response curves were generated on each plate: they served to calculate the EC50 for that plate and to adapt the EC80 values in the subsequent experiments, which vary according to cell line and passage number.
The concentration response curves were analyzed according to the law of mass action, for both orexin A (EC50), and antagonists (IC50) with slope factors and maximal/minimal effects; the antagonist data was transformed according to Cheng and Prusoff (
[3H]-BBAC bound both OX1R and OX2R with high affinity and KD values of about 7 nM and 1 nM, respectively (Figure
Competition experiments were performed with the various antagonists at 15, 30, 45 min, 1, 2, or 4 h and the graphs illustrate the competition curves at the different times. As expected from the saturation experiments described above, as well as in further kinetic experiments to be reported elsewhere, BBAC reached equilibrium quickly at both OX1R and OX2R (15–30 min), and there was no significant difference in IC50 values measured between 30 min and 4 h, as illustrated by superimposable competition curves at both orexin receptors (Figure
In contrast to BBAC, the competition curves for almorexant shifted to the left with time moderately at OX1R and substantially at OX2R (Figure
The SB-649868 competition curves on OX1R shifted to the left over time up to 4 h, whereas at OX2R binding appeared to be rather stable (Figure
Similarly, the suvorexant competition curves for both OX1R and OX2R shifted to the left over time, although the effect on OX2R was somewhat less pronounced (Figure
The filorexant competition curves at OX1R were rather insensitive to incubation time, whereas OX2R curves shifted to the left over time, even up to 4 h (Figure
The IPSU competition curves at OX1R and OX2R do not show time-dependency (Figure
In the calcium accumulation assays performed at mouse, rat and human OX1R and OX2R, we first confirmed that orexin A produces stable results and that the apparent potency is largely comparable when the effects of antagonists are measured following incubation times of between 30 min and 4 h. Indeed, pEC50 values for orexin were largely time-independent at both OX1R and OX2R. This suggests the cells and receptors used were stable and would allow incubation times of up to 4 h in the subsequent experiments (Tables
Orexin A | 9.52 | 0.10 | 3 | 9.48 | 0.01 | 541 | 9.51 | 0.07 | 3 | 9.63 | 0.10 | 3 |
Almorexant | 7.84 | 0.05 | 6 | 7.73 | 0.06 | 26 | 7.75 | 0.07 | 6 | 7.75 | 0.07 | 6 |
Filorexant | 8.65 | 0.09 | 6 | 8.79 | 0.08 | 12 | 8.78 | 0.11 | 6 | 8.55 | 0.06 | 6 |
Suvorexant | 8.39 | 0.09 | 6 | 8.74 | 0.10 | 8 | 8.75 | 0.12 | 6 | 8.73 | 0.15 | 6 |
SB-649868 | 9.03 | 0.05 | 6 | 8.87 | 0.09 | 12 | 9.29 | 0.08 | 6 | 9.38 | 0.10 | 6 |
IPSU | 6.32 | 0.08 | 6 | 6.29 | 0.05 | 6 | 6.28 | 0.09 | 6 | 6.14 | 0.08 | 6 |
Orexin A | 8.92 | 0.08 | 3 | 9.01 | 0.09 | 3 | 9.01 | 0.05 | 3 | 8.86 | 0.09 | 3 |
Almorexant | 7.90 | 0.06 | 6 | 7.90 | 0.07 | 6 | 7.95 | 0.08 | 6 | 7.76 | 0.08 | 6 |
Filorexant | 9.12 | 0.13 | 6 | 9.21 | 0.18 | 6 | 9.30 | 0.17 | 6 | 8.92 | 0.18 | 6 |
Suvorexant | 8.82 | 0.20 | 6 | 9.40 | 0.36 | 6 | 9.37 | 0.19 | 6 | 9.24 | 0.17 | 6 |
SB-649868 | 9.59 | 0.11 | 6 | 9.90 | 0.19 | 6 | 10.17 | 0.10 | 6 | 10.14 | 0.06 | 6 |
IPSU | 6.65 | 0.05 | 6 | 6.62 | 0.06 | 6 | 6.56 | 0.13 | 6 | 6.25 | 0.06 | 6 |
Orexin A | 9.17 | 0.14 | 3 | 9.18 | 0.04 | 31 | 9.36 | 0.15 | 3 | 9.25 | 0.14 | 3 |
Almorexant | 7.73 | 0.05 | 6 | 7.63 | 0.10 | 10 | 7.47 | 0.07 | 6 | 7.41 | 0.11 | 4 |
Filorexant | 8.28 | 0.10 | 6 | 8.39 | 0.14 | 9 | 8.18 | 0.08 | 6 | 8.23 | 0.08 | 6 |
Suvorexant | 8.90 | 0.14 | 6 | 8.98 | 0.18 | 4 | 9.03 | 0.08 | 6 | 8.99 | 0.08 | 6 |
SB-649868 | 9.80 | 0.12 | 6 | 9.37 | 0.14 | 7 | 10.39 | 0.16 | 6 | 10.33 | 0.17 | 6 |
IPSU | 6.48 | 0.04 | 6 | 6.31 | 0.12 | 4 | 6.54 | 0.06 | 6 | 6.49 | 0.06 | 6 |
Orexin A | 8.76 | 0.05 | 3 | 8.79 | 0.01 | 548 | 8.61 | 0.07 | 3 | 8.70 | 0.02 | 3 |
Almorexant | 8.33 | 0.05 | 6 | 8.82 | 0.06 | 29 | 8.80 | 0.15 | 6 | 9.09 | 0.22 | 6 |
Filorexant | 9.45 | 0.09 | 6 | 9.65 | 0.06 | 11 | 9.73 | 0.10 | 6 | 9.77 | 0.09 | 6 |
Suvorexant | 9.00 | 0.14 | 6 | 9.48 | 0.14 | 8 | 9.46 | 0.19 | 6 | 9.53 | 0.20 | 6 |
SB-649868 | 9.52 | 0.05 | 6 | 9.43 | 0.09 | 15 | 9.77 | 0.03 | 6 | 9.82 | 0.05 | 6 |
IPSU | 8.00 | 0.10 | 6 | 7.97 | 0.07 | 6 | 7.82 | 0.08 | 6 | 7.68 | 0.11 | 6 |
Orexin A | 8.40 | 0.01 | 3 | 8.34 | 0.07 | 6 | 8.48 | 0.06 | 3 | 8.58 | 0.03 | 3 |
Almorexant | 8.25 | 0.08 | 6 | 8.65 | 0.06 | 6 | 8.99 | 0.19 | 6 | 9.18 | 0.10 | 6 |
Filorexant | 9.13 | 0.11 | 6 | 9.38 | 0.08 | 6 | 9.60 | 0.11 | 6 | 9.75 | 0.10 | 6 |
Suvorexant | 8.71 | 0.19 | 6 | 9.06 | 0.18 | 6 | 9.22 | 0.20 | 6 | 9.37 | 0.22 | 6 |
SB-649868 | 9.34 | 0.06 | 6 | 9.50 | 0.05 | 6 | 9.81 | 0.07 | 6 | 9.85 | 0.05 | 6 |
IPSU | 7.63 | 0.14 | 6 | 7.55 | 0.11 | 6 | 7.62 | 0.12 | 6 | 7.55 | 0.11 | 6 |
Orexin A | 8.78 | 0.07 | 3 | 9.05 | 0.02 | 53 | 8.94 | 0.01 | 3 | 9.18 | 0.02 | 3 |
Almorexant | 7.72 | 0.06 | 5 | 8.03 | 0.05 | 14 | 8.09 | 0.05 | 6 | 8.38 | 0.05 | 6 |
Filorexant | 8.67 | 0.13 | 6 | 8.68 | 0.06 | 9 | 8.84 | 0.10 | 6 | 8.89 | 0.11 | 6 |
Suvorexant | 7.99 | 0.11 | 6 | 8.17 | 0.14 | 4 | 8.24 | 0.11 | 6 | 8.35 | 0.08 | 6 |
SB-649868 | 8.74 | 0.07 | 6 | 8.55 | 0.08 | 7 | 8.93 | 0.06 | 6 | 9.04 | 0.04 | 6 |
IPSU | 7.15 | 0.04 | 6 | 7.10 | 0.09 | 4 | 7.26 | 0.06 | 6 | 7.22 | 0.07 | 6 |
A thorough exploration of the pharmacokinetics and pharmacodynamics of drug candidates is important in drug development. Ideal sleep-enabling compounds have distinct profiles: rapid absorption and induction of sleep, low blood drug concentrations 8 h after dosing and efficacy in the absence of side effects (Wilson et al.,
With this in mind we sought to characterize the kinetic features of various “dual” orexin receptor antagonists at OX1R and OX2R. We selected antagonists that have either been used clinically or are currently under development for the treatment of insomnia and sleep disorders, including almorexant, SB-649868, suvorexant, and filorexant. We compared the kinetic features of these compounds with those of BBAC (a fast binding dual orexin receptor antagonist that was also used as a radioligand in the present studies) and IPSU, an OX2R antagonist (see Betschart et al.,
We observed that the radioligand [3H]-BBAC binds with high affinity, rapidly and reversibly to both OX1R and OX2R. In competition assays, unlabeled BBAC was a fast dual receptor binder, as illustrated by competition curves which are virtually superimposable irrespective of receptor type or incubation time. The slight shift to the right as time increased indicates the concentration dependence of the association rate, since the concentrations of unlabeled ligand used in the competition experiments (Figure
For the dual orexin receptor antagonists tested, time-dependent changes in the apparent affinities for the receptors were found. The affinity of SB-649868 at hOX1R increased markedly between 15 min and 4 h, whilst time had little effect on the affinity at hOX2R (Figure
The time-dependent binding translated into differences in the more functional FLIPR® calcium assay in whole cells expressing human, rat, or mouse OX1 and OX2 receptors. Almorexant acted as a pseudo-irreversible or very slowly equilibrating antagonist at human, rat or mouse OX2R, whereas, at OX1R for all three species, almorexant behaved as a fast equilibrating antagonist. This data suggests that although originally described as a dual antagonist with very similar affinity for both receptors, almorexant is in fact a slowly equilibrating and somewhat selective OX2R antagonist, if sufficient time is given for the ligand to reach equilibrium. Similar findings were made in the calcium experiments with suvorexant, SB-649868 and filorexant, indicating that all display slow equilibration at one and/or the other orexin receptor (see Tables
The differences in binding kinetics between the orexin receptor antagonists demonstrated here are likely to have implications for pharmacodynamics. Suvorexant is a pertinent example: studies of pharmacokinetics revealed a long dose-dependent apparent terminal half-life (between 9 and 12 h, Merck Sharp and Dohme Corporation,
The Food and Drug Administration (FDA, USA) have concluded that although suvorexant is efficacious, it is not considered safe at doses higher than 20 mg (Farkas,
The individual contribution of orexin receptors to sleep architecture is a matter of debate since, to our knowledge, no selective OX1R or OX2R antagonist has been tested in patients with insomnia. However, rodent models are rather good predictors of the effects of orexin receptor antagonists on sleep. In rodents, OX2R antagonism appears sufficient to induce sleep: almorexant is effective in the OX1R KO whereas it has no effect on the sleep wake cycle in OX2R or in double receptor KO mice (Mang et al.,
In addition to almorexant, SB-649868 and suvorexant have reached phase II clinical trials for the treatment of insomnia. Clinical data suggests that the main effect on total sleep time is largely due to an increase in REM sleep and decreased latency to REM, with modest effects on non-REM or slow wave sleep, if at all (Bettica et al.,
Overall, the clinical data appears to confirm the preclinical data collected in mice or rats which demonstrates dual orexin receptor antagonists or dual receptor KOs induce sleep with a very strong REM component, whereas OX2R KO or antagonism has more balanced sleep phenotypes (Willie et al.,
Still, kinetics are of primary importance in sleep and an appropriate balance must be reached for therapeutic efficacy and safety. If target occupancy is too short, the patient will wake up in the middle of the night as happened with early formulations of Z drugs such as zolpidem and zaleplon (Besset et al.,
Gabrielle E. Callander prepared the data for publication and wrote the manuscript. Morenike Olorunda, Dominique Monna, Edi Schuepbach, and Daniel Langenegger have carried out the experiments, contributed to the development of the assays and performed some of the data analysis. Claudia Betschart and Samuel Hintermann have synthesized a number of compounds and led the chemistry efforts and contributed to writing. Emmanuelle Briard contributed to the synthesis of a number of radioligands used in these studies. Dirk Behnke, Simona Cotesta, Grit Laue, and Silvio Ofner have synthesized other compounds and/or have performed a number of analyses in relation to pharmacokinetics. Christine E. Gee, Markus Fendt, and Laura H. Jacobson have performed
With the exception of Gabrielle E. Callander, the authors are either past or present Novartis employees, or have been supported by Novartis.
The authors would like to thank T. Cremer and Dr. S. Geisse, (NIBR Basel, Switzerland) and Dr. A. Chen, (GNF, San Diego, CA, USA) for providing cells and cell membranes expressing orexin receptors.
serotonin
(S)-N-([1,1′-biphenyl]-2-yl)-1-(2-((1-methyl-1H-benzo[d]imidazol-2-yl)thio)acetyl)pyrrolidine-2-carboxamide
Bovine Serum Albumin
cyclic AMP
Chinese Hamster Ovary
Dulbecco's Modified Eagle's Medium
Ham's F12 nutrients mixture
Federal Drug Administration of the United States Department of Health and Human Services
FLuorescent Imaging Plate Reader
γ-aminobutyric acid
Human Embryonic Kidney
2-((1
Knock Out
Non-specific Binding
orexin receptor 1
orexin receptor 2
Rapid Eye Movement (sleep state).