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Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [43]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [117]. Orexin signaling has been associated with regulation of sleep and wakefulness, reward and addiction, appetite and feeding, pain gating, stress response, anxiety and depression. Currently the orexin receptor ligands in clinical use are the dual orexin receptor antagonists suvorexant and lemborexant and daridorexant, which are used as hypnotics, and several dual and OX2-selective antagonists are under development. Multiple orexin agonists are in development for the treatment of narcolepsy and other sleep disorders. Orexin receptor 3D structures have been solved [146, 144, 55, 126, 47, 109, 7, 145].

Background Inflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO–sGC–cGMP) signaling plays an essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO–sGC–cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models. Methods Target engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling [phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB)]. In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia [ionized calcium binding adaptor molecule 1 (Iba1)] and astrocytes [glial fibrillary acidic protein (GFAP)] was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex. Results In vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood–brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle. Conclusions These data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.

Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [42]) are activated by the endogenous polypeptides orexin-A and orexin-B (also known as hypocretin-1 and -2; 33 and 28 aa) derived from a common precursor, preproorexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [109]. Currently the only orexin receptor ligands in clinical use are suvorexant and lemborexant, which are used as hypnotics. Orexin receptor crystal structures have been solved [134, 133, 54, 117, 46].

Effective treatments for neurodegenerative diseases remain elusive and are critically needed since the burden of these diseases increases across an aging global population. Nitric oxide (NO) is a gasotransmitter that binds to soluble guanylate cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Impairment of this pathway has been demonstrated in neurodegenerative diseases. Normalizing deficient NO-cGMP signaling could address multiple pathophysiological features of neurodegenerative diseases. sGC stimulators are small molecules that synergize with NO, activate sGC, and increase cGMP production. Many systemic sGC stimulators have been characterized and advanced into clinical development for a variety of non-central nervous system (CNS) pathologies. Here, we disclose the discovery of CY6463, the first brain-penetrant sGC stimulator in clinical development for the treatment of neurodegenerative diseases, and demonstrate its ability to improve neuronal activity, mediate neuroprotection, and increase cognitive performance in preclinical models. In several cellular assays, CY6463 was demonstrated to be a potent stimulator of sGC. In agreement with the known effects of sGC stimulation in the vasculature, CY6463 elicits decreases in blood pressure in both rats and mice. Relative to a non-CNS penetrant sGC stimulator, rodents treated with CY6463 had higher cGMP levels in cerebrospinal fluid (CSF), functional-magnetic-resonance-imaging-blood-oxygen-level-dependent (fMRI-BOLD) signals, and cortical electroencephalographic (EEG) gamma-band oscillatory power. Additionally, CY6463 improved cognitive performance in a model of cognitive disruption induced by the administration of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. In models of neurodegeneration, CY6463 treatment increased long-term potentiation (LTP) in hippocampal slices from a Huntington’s disease mouse model and decreased the loss of dendritic spines in aged and Alzheimer’s disease mouse models. In a model of diet-induced obesity, CY6463 reduced markers of inflammation in the plasma. Furthermore, CY6463 elicited an additive increase in cortical gamma-band oscillatory power when co-administered with donepezil: the standard of care in Alzheimer’s disease. Together, these data support the clinical development of CY6463 as a novel treatment for neurodegenerative disorders.

Historically, pharmacological therapies have used mechanisms such as γ-aminobutyric acid A (GABAA) receptor potentiation to drive sleep through broad suppression of central nervous system activity. With the discovery of orexin signaling loss as the etiology underlying narcolepsy, a disorder associated with hypersomnolence, orexin antagonism emerged as an alternative approach to attenuate orexin-induced wakefulness more selectively. Dual orexin receptor antagonists (DORAs) block the activity of orexin 1 and 2 receptors to both reduce the threshold to transition into sleep and attenuate orexin-mediated arousal. Among DORAs evaluated clinically, suvorexant has pharmacokinetic properties engineered for a plasma half-life appropriate for rapid sleep onset and maintenance at low to moderate doses. Unlike GABAA receptor modulators, DORAs promote both non-rapid eye movement (NREM) and REM sleep, do not disrupt sleep stage–specific quantitative electroencephalogram spectral profiles, and allow somnolence indistinct from normal sleep. The preservation of cognitive performance and the ability to arouse to salient stimuli after DORA administration suggest further advantages over historical therapies.

The orexin (also known as hypocretin) G protein-coupled receptors (GPCRs) regulate sleep and other behavioral functions in mammals, and are therapeutic targets for sleep and wake disorders. The human receptors hOX1R and hOX2R, which are 64% identical in sequence, have overlapping but distinct physiological functions and potential therapeutic profiles. We determined structures of hOX1R bound to the OX1R-selective antagonist SB-674042 and the dual antagonist suvorexant at 2.8-Å and 2.75-Å resolution, respectively, and used molecular modeling to illuminate mechanisms of antagonist subtype selectivity between hOX1R and hOX2R. The hOX1R structures also reveal a conserved amphipathic α-helix, in the extracellular N-terminal region, that interacts with orexin-A and is essential for high-potency neuropeptide activation at both receptors. The orexin-receptor crystal structures are valuable tools for the design and development of selective orexin-receptor antagonists and agonists.

Orexins are neuropeptides synthesized in the lateral hypothalamus that influence arousal, feeding, reward pathways, and the response to stress. However, the role of orexins in repeated stress is not fully characterized. Here, we examined how orexins and their receptors contribute to the coping response during repeated social defeat and subsequent anxiety-like and memory-related behaviors. Specifically, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to stimulate orexins prior to each of five consecutive days of social defeat stress in adult male rats. Additionally, we determined the role of the orexin 2 receptor in these behaviors by using a selective orexin 2 receptor antagonist (MK-1064) administered prior to each social defeat. Following the 5 day social defeat conditioning period, rats were evaluated in social interaction and novel object recognition paradigms to assess anxiety-like behavior and recognition memory, respectively. Activation of orexin neurons by DREADDs prior to each social defeat decreased the average latency to become defeated across 5 days, indicative of a passive coping strategy that we have previously linked to a stress vulnerable phenotype. Moreover, stimulation of orexin signaling during defeat conditioning decreased subsequent social interaction and performance in the novel object recognition test indicating increased subsequent anxiety-like behavior and reduced recognition memory. Blocking the orexin 2 receptor during repeated defeat did not alter these effects. Together, our results suggest that orexin neuron activation produces a passive coping phenotype during social defeat leading to subsequent anxiety-like behaviors and memory deficits.

Orexin signaling, known to modulate arousal and vigilance, is also involved in nociception as orexin neurons project to regions of the brain and spinal cord involved in pain processing, and the administration of orexin peptides can alter pain response in a wide range of preclinical models. Pharmacological treatment with the potent, selective and structurally distinct dual orexin receptor antagonists (ORAs) DORA-12 and DORA-2 significantly reduced pain responses during both phases I and II of the mouse formalin pain model and significantly reversed hyperalgesia in the rat complete Freund's adjuvant pain model, respectively. Significant antinociceptive effects of DORA-12 in the formalin model were also observed in orexin 1 receptor (OX1R) knockout mice, but not orexin 2 receptor (OX2R) or OX1R/OX2R double knockout mice. Mechanical hypersensitivity was significantly reduced with a series of structurally distinct, potent and highly selective ORAs (DORA-2, DORA-12 and DORA-22) in the rat spinal nerve ligation (SNL) injury model of neuropathic pain. Selective pharmacological targeting of OX2R with 2-SORA-7 also reduced pain responses in acute inflammatory (complete Freund's adjuvant) and neuropathic (SNL) rat pain models. Performance on the rotarod test of psychomotor performance and baseline thermal sensitivity were not affected in OX1R/OX2R knockout mice or ORA-treated mice, indicating that the observed pain-reducing effects were not due to sedation or motor deficits. These findings indicate that ORAs have pain-reducing effects across a number of acute and chronic neuropathic preclinical mouse and rat pain models. Further studies on the potential pain-relieving effects of orexin receptor antagonism are warranted.

Background Insomnia is increasingly recognized to be comorbid with one or more medical conditions. This study used an online research platform to characterize insomnia across different mental and physical conditions. Methods A custom cross-sectional survey was fielded online to 31,208 users of the patient community PatientsLikeMe. The survey queried members on National Sleep Foundation-defined insomnia risk ( waking up feeling unrefreshed , difficulty falling asleep , waking in the middle of the night , or waking too early ). Results Complete results were obtained from 5256 patients with 11 comorbid conditions. Seventy-six percent of US-based respondents were at risk for insomnia. Patients who reported difficulty falling asleep were found to have nearly twice the odds of self-reporting insomnia (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.5–2.1) when compared to those who do not have difficulty falling asleep, whereas those who reported waking during the night or waking up unrefreshed were no more likely (OR: 1.025 and 1.032, respectively) to report that they suffered from insomnia than those who did not experience these issues. Although insomnia was self-reported as severe or very severe across most conditions, few respondents had actually been diagnosed with insomnia by a physician. After adjustment for age and gender, there was an independent and strong effect of primary condition severity on insomnia risk, and those with severe epilepsy (0.93), depressive disorders (0.92), and fibromyalgia (0.92) occupied the highest risk probabilities. Conclusions The high rate of severity and frequency of insomnia across a multitude of mental and physical conditions reveals an opportunity for better disease management through enhanced insomnia awareness.

Background The neuropeptides orexin A and B play a role in reward and feeding and are critical for arousal. However, it was not initially appreciated that most prepro-orexin synthesizing neurons are almost exclusively concentrated in the perifornical hypothalamus, which when stimulated elicits panic-associated behavior and cardiovascular responses in rodents and self-reported “panic attacks” and “fear of dying” in humans. More recent studies support a role for the orexin system in coordinating an integrative stress response. For instance, orexin neurons are highly reactive to anxiogenic stimuli, are hyperactive in anxiety pathology, and have strong projections to anxiety and panic-associated circuitry. Although the two cognate orexin receptors are colocalized in many brain regions, the orexin 2 receptor (OX2R) most robustly maps to the histaminergic wake-promoting region, while the orexin 1 receptor (OX1R) distribution is more exclusive and dense in anxiety and panic circuitry regions, such as the locus ceruleus. Overall, this suggests that OX1Rs play a critical role in mobilizing anxiety and panic responses. Methods Here, we used a CO2-panic provocation model to screen a dual OX1/2R antagonist (DORA-12) to globally inhibit orexin activity, then a highly selective OX1R antagonist (SORA1, Compound 56) or OX2R antagonist (SORA2, JnJ10397049) to assess OX1R and OX2R involvement. Results All compounds except the SORA2 attenuated CO2-induced anxiety-like behaviors, and all but the SORA2 and DORA attenuated CO2-induced cardiovascular responses. Conclusions SORA1s may represent a novel method of treating anxiety disorders, with no apparent sedative effects that were present with a benzodiazepine.

Antagonism of orexin receptors has shown clinical efficacy as a novel paradigm for the treatment of insomnia and related disorders. Herein, molecules related to the dual orexin receptor antagonist filorexant were transformed into compounds that were selective for the OX2R subtype. Judicious selection of the substituents on the pyridine ring and benzamide groups led to 6b; which was highly potent, OX2R selective, and exhibited excellent development properties.

Objective To examine the effect of the orexinergic blockade with a dual orexin receptor antagonist (DORA) on experimental models of peripheral and central trigeminal as well as cortical activation relevant to migraine and migraine aura. Methods In this study we used a precursor of suvorexant, a dual orexin receptor antagonist #12 (DORA-12) in established experimental in vivo models of dural trigeminovascular nociception in rat. Neurogenic dural vasodilation and electrophysiological recordings of second order trigeminocervical neurons were used to study trigeminal nociceptive mechanisms directly. KCl-evoked cortical spreading depression was also used as a surrogate for migraine aura. Results Neurogenically-induced vasodilation of the middle meningeal artery, caused by nociceptive activation of peripheral afferent projections of the trigeminal nerve, was attenuated by intravenous DORA-12 (1 mg kg − 1 ). Second-order trigeminocervical complex neuronal activity was significantly inhibited by intravenous DORA-12 (1 mg kg − 1 ). DORA-12 significantly reduced susceptibility to KCl-evoked cortical spreading depression. Conclusion The study provides the first direct evidence, that simultaneous antagonism on both orexin receptors is able to attenuate trigeminal nociceptive activity as well as to induce an elevation of the threshold for the induction of a cortical spreading depression (CSD). In the clinical context, these data imply that targeting the hypothalamic orexinergic system may offer an entirely novel mechanism for the preventive treatment of migraine with and without aura.

We identified gene networks linking sleep and affect, highlighting those oppositely perturbed by sleep loss and depression., To understand the transcriptomic organization underlying sleep and affective function, we studied a population of (C57BL/6J × 129S1/SvImJ) F2 mice by measuring 283 affective and sleep phenotypes and profiling gene expression across four brain regions. We identified converging molecular bases for sleep and affective phenotypes at both the single-gene and gene-network levels. Using publicly available transcriptomic datasets collected from sleep-deprived mice and patients with major depressive disorder (MDD), we identified three cortical gene networks altered by the sleep/wake state and depression. The network-level actions of sleep loss and depression were opposite to each other, providing a mechanistic basis for the sleep disruptions commonly observed in depression, as well as the reported acute antidepressant effects of sleep deprivation. We highlight one particular network composed of circadian rhythm regulators and neuronal activity–dependent immediate-early genes. The key upstream driver of this network, Arc, may act as a nexus linking sleep and depression. Our data provide mechanistic insights into the role of sleep in affective function and MDD.

Study objective To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day. Methods Tg(ChAT-eGFP)86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7:00 am (lights on) and 7:00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on. Results There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7:00 am and 7:00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the cold-inducible RNA-binding protein. Conclusions Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined.

Orexin receptor antagonists have demonstrated clinical utility for the treatment of insomnia. The majority of clinical efforts to date have focused on the development of dual orexin receptor antagonists (DORAs), small molecules that antagonize both the orexin 1 and orexin 2 receptors. Our group has recently disclosed medicinal chemistry efforts to identify highly potent, orally bioavailable selective orexin 2 receptor antagonists (2-SORAs) that possess acceptable profiles for clinical development. Herein we report additional SAR studies within the 'triaryl' amide 2-SORA series focused on improvements in compound stability in acidic media and time-dependent inhibition of CYP3A4. These studies resulted in the discovery of 2,5-disubstituted isonicotinamide 2-SORAs such as compound 24 that demonstrated improved stability and TDI profiles as well as excellent sleep efficacy across species.

Analogs of the dual orexin receptor antagonist filorexant were prepared. Replacement of the ether linkage proved highly sensitive toward modification with an acetylene linkage providing compounds with the best in vitro and in vivo potency profiles.

Insomnia persistently affects the quality and quantity of sleep. Currently approved treatments for insomnia primarily target γ-aminobutyric acid-A (GABA-A) receptor signalling and include benzodiazepines and GABA-A receptor modulators. These drugs are used to address this sleep disorder, but have the potential for side effects such as tolerance and dependence, making them less attractive as maintenance therapy. Forward and reverse genetic approaches in animals have implicated orexin signalling (also referred to as hypocretin signalling) in the control of vigilance and sleep/wake states. Screening for orexin receptor antagonists using in vitro and in vivo methods in animals has identified compounds that block one or other of the orexin receptors (single or dual orexin receptor antagonists [SORAs and DORAs], respectively) in animals and humans. SORAs have primarily been used as probes to further elucidate the roles of the individual orexin receptors, while a number of DORAs have progressed to clinical development as pharmaceutical candidates for insomnia. The DORA almorexant demonstrated significant improvements in a number of clinically relevant sleep parameters in animal models and in patients with insomnia but its development was halted. SB-649868 and suvorexant have demonstrated efficacy and tolerability in Phase II and III trials respectively. Furthermore, suvorexant is currently under review by the Food and Drug Administration for the treatment of insomnia. Based on the publication of recent non-clinical and clinical data, orexin receptor antagonists potentially represent a targeted, effective and well-tolerated new class of medications for insomnia. Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA)A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABAA receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABAA modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABAA modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep.

While a correlation between blockade of the orexin 2 receptor (OX2R) with either a dual orexin receptor antagonist (DORA) or a selective orexin 2 receptor antagonist (2-SORA) and a decrease of wakefulness is well established, less is known about selective blockade of the orexin 1 receptor (OX1R). Therefore, a highly selective orexin 1 antagonist (1-SORA) with suitable properties to allow in vivo interrogation of OX1R specific pharmacology in preclinical species remains an attractive target. Herein, we describe the discovery of an optimized 1-SORA series in the piperidine ether class. Notably, a 4,4-difluoropiperidine core coupled with a 2-quinoline ether linkage provides OX1R selective compounds. The combination with an azabenzimidazole or imidazopyridine amide substituent leads to analogs 47 and 51 with >625-fold functional selectivity for OX1R over OX2R in rat. Compounds 47 and 51 possess clean off-target profiles and the required pharmacokinetic and physical properties to be useful as 1-SORA tool compounds.

Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the predominant mediator of arousal promotion. The potential for single OX2R antagonism to effectively promote sleep has yet to be demonstrated in humans. MK-1064 is an OX2R-single antagonist. Preclinically, MK-1064 promotes sleep and increases both rapid eye movement (REM) and non-REM (NREM) sleep in rats at OX2R occupancies higher than the range observed for dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I studies in healthy human subjects evaluated safety, tolerability, pharmacokinetics and sleep-promoting effects of MK-1064, and demonstrated dose-dependent increases in subjective somnolence (via Karolinska Sleepiness Scale and Visual Analogue Scale measures) and sleep (via polysomnography), including increased REM and NREM sleep. Thus, selective OX2R antagonism is sufficient to promote REM and NREM sleep across species, similarly to that seen with dual orexin receptor antagonism.

Study objectives In addition to enhancing sleep onset and maintenance, a desirable insomnia therapeutic agent would preserve healthy sleep's ability to wake and respond to salient situations while maintaining sleep during irrelevant noise. Dual orexin receptor antagonists (DORAs) promote sleep by selectively inhibiting wake-promoting neuropeptide signaling, unlike global inhibition of central nervous system excitation by gamma-aminobutyric acid (GABA)-A receptor (GABAaR) modulators. We evaluated the effect of DORA versus GABAaR modulators on underlying sleep architecture, ability to waken to emotionally relevant stimuli versus neutral auditory cues, and performance on a sleepiness-sensitive cognitive task upon awakening. Methods DORA-22 and GABAaR modulators (eszopiclone, diazepam) were evaluated in adult male rhesus monkeys (n = 34) with continuous polysomnography recordings in crossover studies of sleep architecture, arousability to a classically conditioned salient versus neutral acoustical stimulus, and psychomotor vigilance task (PVT) performance if awakened. Results All compounds decreased wakefulness, but only DORA-22 sleep resembled unmedicated sleep in terms of underlying sleep architecture, preserved ability to awaken to salient-conditioned acoustic stimuli while maintaining sleep during neutral acoustic stimuli, and no congnitive impairment in PVT performance. Although GABAaR modulators induced lighter sleep, monkeys rarely woke to salient stimuli and PVT performance was impaired if monkeys were awakened. Conclusions In nonhuman primates, DORAs' targeted mechanism for promoting sleep protects the ability to selectively arouse to salient stimuli and perform attentional tasks unimpaired, suggesting meaningful differentiation between a hypnotic agent that works through antagonizing orexin wake signaling versus the sedative hypnotic effects of the GABAaR modulator mechanism of action.

Orexin (hypocretin) neuropeptides promote wakefulness by signaling through two G-protein coupled receptors, Orexin 1 Receptor (OX(1)R) and Orexin 2 Receptor (OX(2)R). MK-6096 is an orally bioavailable potent and selective reversible antagonist of OX(1)R and OX(2)R currently in clinical development for insomnia. In radioligand binding and functional cell based assays MK-6096 demonstrated potent binding and antagonism of both human OX(1)R and OX(2)R (3 nM in binding, 11 nM in FLIPR), with no significant off-target activities against a panel of170 receptors and enzymes. MK-6096 occupies 90% of human OX(2)Rs expressed in transgenic rats at a plasma concentration of 142 nM, and dose-dependently reduced locomotor activity and significantly increased sleep in rats (3-30 mg/kg) and dogs (0.25 and 0.5 mg/kg). DORA-22, an analog of MK-6096, exhibits similar sleep promoting properties that are absent OX(1/2)R double knockouts, demonstrating the mechanism of action and specificity of these effects. These findings with a novel, structurally distinct class of OxR antagonists provide further validation of the orexin pathway as an effective target to promote normal sleep. Comparative analysis of the biochemical and pharmacokinetic properties of these compounds relative to other OXR antagonists provides a basis for understanding the attributes critical for in vivo efficacy. This mechanism is distinct from current standard of care such that MK-6096 represents a novel and selective therapeutic for the treatment of insomnia. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Orexins are neuropeptides that regulate wakefulness and arousal. Small molecule antagonists of orexin receptors may provide a novel therapy for the treatment of insomnia and other sleep disorders. In this Letter we describe the design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as orexin receptor antagonists. The design of these constrained analogs was guided by an understanding of the preferred solution and solid state conformation of the diazepane central ring.

Silent Night: Antagonism of the orexin (or hypocretin) system has recently been identified as a novel mechanism for the treatment of insomnia. Herein, we describe discovery of a dual (OX(1)R/OX(2)R) orexin receptor antagonist featuring a 1,4-diazepane central constraint that blocks orexin signaling in vivo. In telemetry-implanted rats, oral administration of this antagonist produced a decrease in wakefulness, while increasing REM and non-REM sleep.

NMR spectroscopy, X-ray crystallography, and molecular modeling studies indicate that N,N-disubstituted-1,4-diazepane orexin receptor antagonists exist in an unexpected low-energy conformation that is characterized by an intramolecular pi-stacking interaction and a twist-boat ring conformation. Synthesis and evaluation of a macrocycle that enforces a similar conformation suggest that this geometry mimics the bioactive conformation.

A series of OX2R/OX1R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.

Neuropathy target esterase (NTE) is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants, and plasticizers, leading to OP-induced delayed neuropathy in people (>30,000 cases of human paralysis) and hens (the best animal model for this demyelinating disease). The active site region of NTE as a recombinant protein preferentially hydrolyzes lysolecithin, suggesting that this enzyme may be a type of lysophospholipase (LysoPLA) with lysolecithin as its physiological substrate. This hypothesis is tested here in mouse brain by replacing the phenyl valerate substrate of the standard NTE assay with lysolecithin for an “NTE-LysoPLA” assay with four important findings. First, NTE-LysoPLA activity, as the NTE activity, is 41–45% lower in Nte -haploinsufficient transgenic mice than in their wild-type littermates. Second, the potency of six delayed neurotoxicants or toxicants as in vitro inhibitors varies from IC 50 0.02 to 13,000 nM and is essentially the same for NTE-LysoPLA and NTE ( r 2 = 0.98). Third, the same six delayed toxicants administered i.p. to mice at multiple doses inhibit brain NTE-LysoPLA and NTE to the same extent ( r 2 = 0.90). Finally, their in vivo inhibition of brain NTE-LysoPLA generally correlates with delayed toxicity. Therefore, OP-induced delayed toxicity in mice, and possibly the hyperactivity associated with NTE deficiency, may be due to NTE-LysoPLA inhibition, leading to localized accumulation of lysolecithin, a known demyelinating agent and receptor-mediated signal transducer. This mouse model has some features in common with OP-induced delayed neuropathy in hens and people but differs in the neuropathological signs and apparently the requirement for NTE aging.

Dual orexin receptor antagonists (DORAs), or orexin 1 (OX1) and orexin 2 (OX2) receptor antagonists, have demonstrated clinical utility for the treatment of insomnia. Medicinal chemistry efforts focused on the reduction of bioactivation potential of diazepane amide 1 through the modification of the Western heterocycle resulted in the discovery of suvorexant, a DORA recently approved by the FDA for the treatment of insomnia. A second strategy towards reducing bioactivation risk is presented herein through the exploration of monocyclic quinazoline isosteres, namely substituted pyrimidines. These studies afforded potent DORAs with significantly reduced bioactivation risk and efficacy in rodent sleep models. Surprisingly, side products from the chemistry used to produce these DORAs yielded isomeric pyrimidine-containing diazepane amides possessing selective OX2R antagonist (2-SORA) profiles. Additional exploration of these isomeric pyrimidines uncovered potent 2-SORA diazepane amides with sleep efficacy in mouse EEG studies.

Background The current standard of care for insomnia includes gamma-aminobutyric acid receptor A (GABAA) activators, which promote sleep as well as general central nervous system depression. Dual orexin receptor antagonists (DORAs) represent an alternative mechanism for insomnia treatment that induces somnolence by blocking the wake-promoting effects of orexin neuropeptides. The current study compares the role and interdependence of these two mechanisms on their ability to influence sleep architecture and quantitative electroencephalography (qEEG) spectral profiles across preclinical species. Results Active-phase dosing of DORA-22 induced consistent effects on sleep architecture in mice, rats, dogs, and rhesus monkeys; attenuation of active wake was accompanied by increases in both non─rapid eye movement (NREM) and rapid eye movement (REM) sleep. Eszopiclone, a representative GABAA receptor modulator, promoted sleep in rats and rhesus monkeys that was marked by REM sleep suppression, but had inconsistent effects in mice and paradoxically promoted wakefulness in dogs. Active-phase treatment of rats with DORA-12 similarly promoted NREM and REM sleep to magnitudes nearly identical to those seen during normal resting-phase sleep following vehicle treatment, whereas eszopiclone suppressed REM even to levels below those seen during the active phase. The qEEG changes induced by DORA-12 in rats also resembled normal resting-phase patterns, whereas eszopiclone induced changes distinct from normal active- or inactive-phase spectra. Co-dosing experiments, as well as studies in transgenic rats lacking orexin neurons, indicated partial overlap in the mechanism of sleep promotion by orexin and GABA modulation with the exception of the REM suppression exclusive to GABAA receptor modulation. Following REM deprivation in mice, eszopiclone further suppressed REM sleep while DORA-22 facilitated recovery including increased REM sleep. Conclusion DORAs promote NREM and importantly REM sleep that is similar in proportion and magnitude to that seen during the normal resting phase across mammalian animal models. While limited overlap exists between therapeutic mechanisms, orexin signaling does not appear involved in the REM suppression exhibited by GABAA receptor modulators. The ability of DORAs to promote proportional NREM and REM sleep following sleep deprivation suggests that this mechanism may be effective in alleviating recovery from sleep disturbance. Electronic supplementary material The online version of this article (doi:10.1186/1471-2202-15-109) contains supplementary material, which is available to authorized users.

The current special topics issue of Frontiers in Neuroscience “Insomnia and Beyond—Exploring the Therapeutic Potential of Orexin Receptor Antagonists” comprises 20 papers from leaders in the orexin/hypocretin field presenting their latest findings and thorough reviews, and will undoubtedly serve as an important reference for others engaged in this field of study. Since the independent identification of the orexin/hypocretin system in 1998 by two teams investigating hypothalamic signaling (de Lecea et al., 1998; Sakurai et al., 1998), the field has rapidly expanded to include potential roles of orexin in processes as diverse as sleep/wake, addiction, feeding, pain and anxiety. Orexin neuropeptides (OX-A, OX-B) are cleaved from a common precursor and secreted from orexinergic neurons localized in the lateral hypothalamus. Orexins (also passionately referred to as hypocretins by many investigators) bind and activate two G-protein coupled receptors (OX1R and OX2R), with OX-A binding equally, and OX-B having ~10 fold selectivity for OX2R. Although orexinergic neurons are localized to less than 100,000 hypothalamic neurons in the human brain, orexin receptors exhibit overlapping and distinct expression throughout the CNS, with significant expression in brain regions associated with arousal and vigilance state, as well as reward and stress processing. Following their seminal discovery of this novel neuropeptide system 16 years ago, authors of the foundational Proc. Natl. Acad. Sci. and Cell papers joined by other exceptional researchers provide their latest insights into the function of orexin signaling and its therapeutic potential. Drs. de Lecea and Huerta (2014) explain their current view on the interaction of orexin neurons with other neuromodulatory systems based on recent optogenetic experiments. A paper from Kohlmeier et al. (2013) presents an original investigation on the interaction of the orexin system with cholinergic and monoaminergic neurons using OX1 and OX2 receptor knockout mice. Two separate papers from Etori et al. (2014) and Tortorella et al. (2013) provide insight into potential synaptic interactions between the hypothalamus, locus coeruleus and oral pontine reticular nucleus, suggesting that orexin signaling within this circuit modulates stage succession during sleep-wakefulness cycles. Expanding upon the first observations of genetic disruptions of orexin receptors in dogs, Thompson et al. (2014) provide a pharmacogenetic overview of orexin peptides and their receptors, and describe the potential impact of polymorphisms observed in human genetic studies. Finally, work described in Flores et al. (2013) illustrates cannabinoid-hypocretin cross-talk within the CNS, an emerging area of interest for the field. The identification of a neuropeptide system with restricted expression, key functions in fundamental CNS processes and approachability of discrete GPCR subtypes has made orexin receptors valuable targets for the next generation of CNS therapeutics, with multiple teams actively characterizing small molecules to understand the pharmacology and potential therapeutic applications of targeting the orexin system. Equihua et al. (2013) takes the reader on a journey comparing the merits and challenges of orexin receptor antagonists as potential sleep medications relative to currently used hypnotics. A paper from Callander et al. (2013) representing a team formerly at Novartis, provides substantial in vitro characterization and describes differential kinetic properties of dual orexin receptor antagonists (DORAs) that have undergone clinical evaluation. Two pharmacological papers from Morairty et al. (2014) (work by groups UCSF and SRI), and Ramirez et al. (2013) (Merck Research Laboratories), demonstrate that DORAs lack the impairment on motor and cognitive function in rodents exhibited by positive GABA-A receptor modulators. Etori et al. (2014) (Kanazawa University), Dugovic et al. (2014) (Janssen), and Hoyer et al. (2013) (team formerly at Novartis) explore the potential differential impact of DORAs as well as antagonists selective for OX1R and OX2R on non-REM and REM sleep architecture. With regard to other potential indications for OXR antagonists beyond insomnia, two manuscripts from teams at Lilly Research Laboratories and MUSC (Anderson et al., 2014; Fitch et al., 2014) describe a novel OX2R selective antagonist, and its effects on rodent models of ethanol addiction and depression relative to a DORA and OX1R selective antagonist. In addition, (Steiner et al., 2013) (Actelion), investigated the impact of chronic pharmacological OX1R blockade in a rat model of diet-induced obesity. A further area of orexin research which is being actively investigated focuses on the impact of orexin signaling on sympathetic autonomous nervous system activation, both during sleep and wake stages, as well as during heightened arousal and stress. Two excellent reviews from leaders in the field (Carrive, 2013; Li and Nattie, 2014) provide an overview of the current perspectives regarding cardiovascular and respiratory control by orexins. (Colas et al., 2014) present evidence supporting the existence of an orexin circuit between central and peripheral nervous system, where orexin signaling potentially modulates sensory processing at dorsal root ganglia. Together with the increasing availability of pharmacological tools, investigations of the role of orexin in limbic emotional processing have also intensified during the last 3–4 years. Two detailed reviews shed light on this topic, from both academic and industry perspectives (Yeoh et al., 2014 from the University of Newcastle and the Hunter Medical Research Institute; Merlo Pich and Melotto, 2014, formerly of Glaxo Smith Kline). These two papers outline recent findings from rodent studies identifying involvement of orexins in mediating compulsive behavior, as well as panic, depression and anxiety-like reactions during withdrawal from drugs of abuse. Importantly, this special issue highlights the exquisite foundational and translational work being conducted by teams at leading academic and biopharmaceutical laboratories, focused on investigating a range of therapeutic opportunities. Although it has only been 16 years since the identification of the orexin/hypocretin system, the wealth of knowledge obtained thus far has set the stage for the next decade of orexin research. It is with great appreciation to the contributing authors and with much enthusiasm that we present this special topics issue of Frontiers in Neuroscience.

The ability to awaken from sleep in response to important stimuli is a critical feature of normal sleep, as is maintaining sleep continuity in the presence of irrelevant background noise. Dual orexin receptor antagonists (DORAs) effectively promote sleep across species by targeting the evolutionarily conserved wake-promoting orexin signaling pathway. This study in dogs investigated whether DORA-induced sleep preserved the ability to awaken appropriately to salient acoustic stimuli but remain asleep when exposed to irrelevant stimuli. Sleep and wake in response to DORAs, vehicle, GABA-A receptor modulators (diazepam, eszopiclone and zolpidem) and antihistamine (diphenhydramine) administration were evaluated in telemetry-implanted adult dogs with continuous electrocorticogram, electromyogram (EMG), electrooculogram (EOG), and activity recordings. DORAs induced sleep, but GABA-A modulators and antihistamine induced paradoxical hyperarousal. Thus, salience gating studies were conducted during DORA-22 (0.3, 1, and 5 mg/kg; day and night) and vehicle nighttime sleep. The acoustic stimuli were either classically conditioned using food reward and positive attention (salient stimulus) or presented randomly (neutral stimulus). Once conditioned, the tones were presented at sleep times corresponding to maximal DORA-22 exposure. In response to the salient stimuli, dogs woke completely from vehicle and orexin-antagonized sleep across all sleep stages but rarely awoke to neutral stimuli. Notably, acute pharmacological antagonism of orexin receptors paired with emotionally salient anticipation produced wake, not cataplexy, in a species where genetic (chronic) loss of orexin receptor signaling leads to narcolepsy/cataplexy. DORA-induced sleep in the dog thereby retains the desired capacity to awaken to emotionally salient acoustic stimuli while preserving uninterrupted sleep in response to irrelevant stimuli.

The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched (13)C6(15)N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically "heavy" proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Recent clinical studies have demonstrated that dual orexin receptor antagonists (OX1R and OX2R antagonists or DORAs) represent a novel treatment option for insomnia patients. Previously we have disclosed several compounds in the diazepane amide DORA series with excellent potency and both preclinical and clinical sleep efficacy. Additional SAR studies in this series were enabled by the expansion of the acetonitrile-assisted, diphosgene-mediated 2,4-dichloropyrimidine synthesis to novel substrates providing an array of Western heterocycles. These heterocycles were utilized to synthesize analogs in short order with high levels of potency on orexin 1 and orexin 2 receptors as well as in vivo sleep efficacy in the rat.

Ataxia telangiectasia (A-T) is an autosomal recessive disease characterized by normal brain development followed by progressive neurodegeneration. The gene mutated in A-T (ATM) is a serine protein kinase implicated in cell cycle regulation and DNA repair. The role of ATM in the brain and the consequences of its loss on neuronal survival remain unclear. We studied the role of ATM in adult neural progenitor cells in vivo and in vitro to define the role of ATM in dividing and postmitotic neural cells from Atm-deficient (Atm−/−) mice in a physiologic context. We demonstrate that ATM is an abundant protein in dividing neural progenitor cells but is markedly down-regulated as cells differentiate. In the absence of ATM, neural progenitor cells of the dentate gyrus show abnormally high rates of proliferation and genomic instability.Atm−/− cells in vivo, and in cell culture, show a blunted response to environmental stimuli that promote neural progenitor cell proliferation, survival, and differentiation along a neuronal lineage. This study defines a role for ATM during the process of neurogenesis, demonstrates that ATM is required for normal cell fate determination and neuronal survival both in vitro and in vivo, and points to a mechanism for neuronal cell loss in progressive neurodegenerative diseases.

The genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain upstream cis-acting regulatory regions termed peroxisome proliferator response elements (PPRE). Transcription of these genes is mediated through the binding of peroxisome proliferator-activated receptor alpha (PPARalpha), which binds to a PPRE as a heterodimer with the 9-cis-retinoic acid receptor (RXRalpha). Here we demonstrate that the HD-PPRE is also a target for the constitutive androstane receptor beta (CARbeta). In vitro binding analysis showed that CARbeta bound the HD-PPRE, but not the AOx-PPRE, as a heterodimer with RXRalpha. Binding of CARbeta/RXRalpha to the HD-PPRE occurred via determinants that overlap partially with those required for PPARalpha/RXRalpha binding. In vivo, CARbeta/RXRalpha activated transcription from an HD-PPRE luciferase reporter construct. Interestingly, CARbeta was shown to also modulate PPARalpha/RXRalpha-mediated transactivation in a response element-specific manner. In the presence of the peroxisome proliferator, Wy-14,643, CARbeta had no effect on PPARalpha/RXRalpha-mediated transactivation from the HD-PPRE but antagonized transactivation from the AOx-PPRE in both the presence and the absence of proliferator. Our results illustrate that transcription of the AOx and HD genes is differentially regulated by CARbeta and that the HD gene is a specific target for regulation by CARbeta. Overall, this study proposes a novel role for CARbeta in the regulation of peroxisomal beta-oxidation.

The orexinergic system has been implicated in a number of behaviors, including reward and incentive motivation. Orexin 1 receptor antagonism has been reported to reduce drug self-administration, conditioned place preference, and reinstatement in rodents, but the role of the orexin 2 receptor is unclear. Here we evaluated the impact of the novel and selective orexin 2 receptor antagonist, 2-SORA 18, on motivation for nicotine as measured by responding on a progressive ratio schedule, as well as cue-induced reinstatement of a response previously associated with nicotine reward, and nicotine-induced reinstatement. 2-SORA 18 demonstrated selective effects on these behaviors. Specifically, doses up to 60 mg/kg 2-SORA 18 were without significant effect on nicotine self-administration or nicotine-induced reinstatement, but doses as low as 15 mg/kg 2-SORA 18 completely blocked cue-induced reinstatement. These findings indicate that orexin 2 receptor antagonism might have utility for attenuating relapse, particularly for patients sensitive to environmental stimuli associated with drug taking.