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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'.

A novel phenyl acetamide series of short-acting T-type calcium channel antagonists has been identified and evaluated using in vitro and in vivo assays. Heterocycle substitutions of the 4-position of the phenyl acetamides afforded potent and selective antagonists that exhibited desired short plasma half-lives across preclinical species. Lead compound TTA-A8 emerged as a compound with excellent in vivo efficacy as indicated by its significant modulation of rat sleep architecture in an EEG telemetry model, favorable pharmacokinetic properties, and excellent preclinical safety. TTA-A8 recently progressed into human clinical trials, and in line with our predictions, preliminary studies (n = 12) with a 20 mg oral dose afforded a high C max of 1.82 ± 0.274 μM with an apparent terminal half-life of 3.0 ± 1.1 h.

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinson's disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Ca(v)3.1, 3.2, 3.3) voltage dependently and with high potency (IC(50) ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Ca(v)3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Ca(v)3.1 and Ca(v)3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.

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.

People with developmental disabilities sleep less and experience higher incidence of clinical sleep disorders than the general population. Exploring the neurophysiology linking sleep with daytime performance in patients with developmental disabilities is now possible using minimally sufficient sleep and sleep-sensitive behavioral assays. Although frequent sampling represents the primary difficulty, it is required to untangle coincident effects of sleep quality amidst circadian variation. Recent evidence finds high quality sleep promotes brain plasticity, improves health measures, and enriches quality of life. Sleep treatments for apnea, insomnia, restless limbs, and conditioned sleep-aversion are available, although not readily provided, for people with developmental disabilities. This population would gain both clinical and behavioral benefits as improved sleep-monitoring, behavioral testing, and sleep-treatment technology is adapted to their needs.

Objective : A concentric spheres model was used in an earlier paper to estimate the effects of volume conduction, reference electrode and spatial filtering on different EEG coherence measures. EEG data are used here to verify theoretical predictions. Methods : Three EEG data sets were: (1) 64 channel, recorded during 7 alternating periods of resting and mental calculation. (2) 128 channel, for comparison of eyes open versus eyes closed coherence. (3) 128 channel, recorded during deep sleep (stages 3 and 4) and REM. Results : The directions of large scale (lobeal) coherency changes between brain states are relatively independent of coherence measure. However, coherence between specific electrode pairs is sensitive to method and frequency. Average reference and digitally linked mastoids provide reasonable semi-quantitative estimates of large-scale neocortical source coherence. Close bipolar, Laplacian, and dura image methods remove most reference electrode and volume conduction distortion, but may underestimate coherence by spatial filtering. Conclusion : Each EEG coherence method has its own potential sources of error and provides coherence estimates for different neural population sizes located in different locations. Thus, studies of coherence and brain state should include several different kinds of estimates to take full advantage of information in recorded signals.

Insomnia is a common disorder that can be comorbid with other physical and psychological illnesses. Traditional management of insomnia relies on general central nervous system (CNS) suppression using GABA modulators. Many of these agents fail to meet patient needs with respect to sleep onset, maintenance, and next-day residual effects and have issues related to tolerance, memory disturbances, and balance. Orexin neuropeptides are central regulators of wakefulness, and orexin antagonism has been identified as a novel mechanism for treating insomnia with clinical proof of concept. Herein we describe the discovery of a series of α-methylpiperidine carboxamide dual orexin 1 and orexin 2 receptor (OX(1) R/OX(2) R) antagonists (DORAs). The design of these molecules was inspired by earlier work from this laboratory in understanding preferred conformational properties for potent orexin receptor binding. Minimization of 1,3-allylic strain interactions was used as a design principle to synthesize 2,5-disubstituted piperidine carboxamides with axially oriented substituents including DORA 28. DORA 28 (MK-6096) has exceptional in vivo activity in preclinical sleep models, and has advanced into phase II clinical trials for the treatment of insomnia.

Modulation of TASK-3 (Kcnk9) potassium channels affect neurotransmitter release in thalamocortical centers and other sleep-related nuclei having the capacity to regulate arousal cycles and REM sleep changes associated with mood disorders and antidepressant action. Circumstantial evidence from this and previous studies suggest the potential for TASK-3 to be a novel antidepressant therapeutic target; TASK-3 knock-out mice display augmented circadian amplitude and exhibit sleep architecture characterized by suppressed REM activity. Detailed analysis of locomotor activity indicates that the amplitudes of activity bout duration and bout number are augmented in TASK-3 mutants well beyond that seen in wildtypes, findings substantiated by amplitude increases in body temperature and EEG recordings of sleep stage bouts. Polysomnographic analysis of TASK-3 mutants reveals increases in nocturnal active wake and suppressed REM sleep time while increased slow wave sleep typifies the inactive phase, findings that have implications for the cognitive impact of reduced TASK-3 activity. In direct measures of their resistance to despair behavior, TASK-3 knock-outs displayed significant decreases in immobility relative to wildtype controls in both tail suspension and forced swim tests. Treatment of wildtype animals with the antidepressant Fluoxetine markedly reduced REM sleep, while leaving active wake and slow wave sleep relatively intact. Remarkably, these effects were absent in TASK-3 mutants indicating that TASK-3 is either directly involved in the mechanism of this drug's action, or participates in parallel pathways that achieve the same effect. Together, these results support the TASK-3 channel to act as a therapeutic target for antidepressant action.

Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX(1)R and OX(2)R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX(2)R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 μM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.

The discovery and synthesis of 4,4-disubstituted quinazolinones as T-type calcium channel antagonists is reported. Based on lead compounds 2 and 3, a focused SAR campaign driven by the optimization of potency, metabolic stability, and pharmacokinetic profile identified 45 as a potent T-type Ca2+ channel antagonist with minimized PXR activation. In vivo, 45 suppressed seizure frequency in a rat model of absence epilepsy and showed significant alterations of sleep architecture after oral dosing to rats as measured by EEG.

Orexins are excitatory neuropeptides that regulate arousal and sleep. Orexin receptor antagonists promote sleep and offer potential as a new therapy for the treatment of insomnia. In this Letter, we describe the synthesis of constrained diazepanes having a 3,9 diazabicyclo[4.2.1]nonane bicyclic core with good oral bioavailability and sleep-promoting activity in a rat EEG model.

A novel series of quinazolinone T-type calcium channel antagonists have been prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 by modifications of the 3- and 4-positions of the quinazolinone ring afforded potent and selective antagonists that displayed in vivo central nervous system efficacy in epilepsy and tremor models, as well as significant effects on rat active wake as measured by electrocorticogram.

The forebrain cholinergic system promotes higher brain function in part by signaling through the M 1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M 1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M 1 mAChR. BQCA reduces the concentration of ACh required to activate M 1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M 1 −/− mice demonstrates that BQCA requires M 1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M 1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M 1 , suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M 1 receptor and represents a promising therapeutic strategy for cognitive disorders.

Orexin is a key neurotransmitter of central arousal and reward circuits in the CNS. Two receptors respond to orexin signaling, Orexin 1 Receptor (OX1R) and Orexin 2 Receptor (OX2R) with partially overlapping brain distributions. Genetic and pharmacological studies suggest orexin receptor antagonists could provide therapeutic benefit for insomnia and other disorders in which sleep/wake cycles are disrupted. Preclinical data has also emerged showing that the orexin system is involved in the behavioral and neurological effects of drugs of abuse (Aston-Jones et al., 2009; Harris et al., 2005). Here we report sleep promoting effects of a recently described small molecule dual orexin receptor OX1R and OX2R antagonist. This dual orexin receptor antagonist (DORA) also inhibits the ability of subchronic amphetamine to produce behavioral sensitization measured 10 days following pre-treatment. Transcriptional profiling of isolated reward and arousal circuits from brains of behaviorally sensitized animals showed that the DORA blocked the significant alteration of gene expression levels in response to amphetamine exposure, particularly those associated with synaptic plasticity in the VTA. Further, DORA attenuates the ability of nicotine to induce reinstatement of extinguished responding for a reinforcer, demonstrating selectivity of the effect to reward pathways and not to food intake. In summary, these data demonstrate efficacy of a dual orexin receptor antagonist for promotion of sleep and suggest that pharmacological inhibition of the orexin system may play a role in both prevention of drug-induced plasticity and drug-relapse.

The epidemics of obesity and metabolic disorders have well-recognized health and economic burdens. Pharmacologic treatments for these diseases remain unsatisfactory with respect to both efficacy and side-effect profiles. Here, we have identified a potential central role for T-type calcium channels in regulating body weight maintenance and sleep. Previously, it was shown that mice lacking CaV3.1 T-type calcium channels have altered sleep/wake activity. We found that these mice were also resistant to high-fat diet–induced weight gain, without changes in food intake or sensitivity to high-fat diet–induced disruptions of diurnal rhythm. Administration of a potent and selective antagonist of T-type calcium channels, TTA-A2, to normal-weight animals prior to the inactive phase acutely increased sleep, decreased body core temperature, and prevented high-fat diet–induced weight gain. Administration of TTA-A2 to obese rodents reduced body weight and fat mass while concurrently increasing lean muscle mass. These effects likely result from better alignment of diurnal feeding patterns with daily changes in circadian physiology and potentially an increased metabolic rate during the active phase. Together, these studies reveal what we believe to be a previously unknown role for T-type calcium channels in the regulation of sleep and weight maintenance and suggest the potential for a novel therapeutic approach to treating obesity.

DATA PRESENTED IN OUR PAPER1 (1) ADDRESSED COMPARISONS OF FULLY AUTOMATED AND SEMI-AUTOMATED APPROACHES TO EVALUATE SLEEP measures from a single clinical trial of an established insomnia treatment; (2) examined the extent to which human supervision was effective in enhancing quality of semi-automated scoring; and (3) provided comparison between two methods of automated and semi-automated systems on the same clinical data set. This work is relevant to understanding efficacy of human effort to improve accuracy of fully automated scoring. In his Letter to the Editor, Zammit incorrectly contends that, in our paper, automated scoring techniques were evaluated using data “processed under controlled conditions in a core PSG laboratory.”2 In fact, what we used were copies of actual recordings made at the sites of data collection. They were from all patients completing the clinical study protocol and were sent to the automated and semi-automated scoring labs free of preprocessing. Variability of manual scoring is an issue that has been addressed in a plethora of work, some of which was referenced in our paper [references 3–4,6–7,11–12]1. We provided comparison of our results with the previous work to the extent possible. We also focused on comparisons of scoring methods with respect to the derived values of clinical endpoints. Cohen's Kappa values 0.79 – 0.87 from page 124 of Silber et al.3 cited by Zammit are for the intra-rater not inter-rater variability. Danker-Hopke et al. [reference 26, in Silber et al.3], reported a Kappa value of 0.68 in a study using 5 sleep stages. Our results were for 6 sleep stages and showed that agreement between automated scoring performed at various sites, with differing automated scoring systems and partial review procedures was Kappa 0.67. When the same automated scoring approaches were used, but reviews of the scores were by different scorers with differing protocols, Kappa increases to 0.74 (c.f. Table 1 1). Zammit points to a low agreement of automated scoring in Stage 1, presumably in comparison with strict manual scoring. However, in Silber et al., one finds that: “the inter-rater variability of Stage 1 sleep is in the fair range of Cohen Kappa, 0.21–0.4” and that due to this low agreement the rules for its scoring “needed reassessment”. We thank Zammit for reminding readers that the Standards4 require “automatically scored tracings be reviewed on an epoch-by-epoch basis,”2 and we do not disagree with this. We disagree that this requirement “renders automated scoring unnecessary.”2 Our results suggest that epoch-by-epoch review of automated scores done by different reviewers, renders a higher reproducibility compared with epoch-by-epoch manual scoring and may thus provide an opportunity for an improvement in technique for the larger field of sleep medicine to consider as it evolves. Reluctance to investigate techniques that may facilitate/improve scoring seems counter-intuitive to the advancement of this field. We hope that our work is a step to evaluate technologies that can more rapidly and objectively examine clinical EEG data than human scoring alone. We endorse that more work is needed to better understand the benefits of changing current approaches, and this is a small step in that endeavor. We thank Zammit for sharing his opinions and look forward to further exchange of ideas as the field progresses.

Publisher Summary Sleep disorders are an international health concern and pose elevated risk factors for the development of other medical and psychiatric disorders including depression, obesity, and cardiovascular disease. They have enormous socio-economic impact based on the combined cost of the three most prevalent sleep disorders: insomnia, sleep-related breathing disorders, and Excessive Daytime Sleepiness (EDS). Understanding the neural control of sleep and wakefulness has largely paralleled the history of neuroscience discovery as a whole. As new tools and methodologies became available to research, sleep scientists have been quick to take advantage of those techniques and apply them to common, replicable experimental paradigms related to sleep. This chapter provides an overview of the classical techniques used to measure sleep and describes progress from relatively crude methods of brain transection and lesion studies to more complicated methods, including gene cloning and the production of transgenic models. Animal models of sleep, including changes in sleep due to age and disease, are a versatile set of laboratory tools to help uncover both the neurophysiology of arousal state control and the daytime complaints arising from a variety of medical disorders. Sleep naturally crosses all disciplines of physiology and neuroscience. Such ubiquity makes animal models of sleep easily accessible and discernible scientific methods for improving human health.

Objective To evaluate the performance of 2 automated systems, Morpheus and Somnolyzer24X7, with various levels of human review/editing, in scoring polysomnographic (PSG) recordings from a clinical trial using zolpidem in a model of transient insomnia. Methods 164 all-night PSG recordings from 82 subjects collected during 2 nights of sleep, one under placebo and one under zolpidem (10 mg) treatment were used. For each recording, 6 different methods were used to provide sleep stage scores based on Rechtschaffen & Kales criteria: 1) full manual scoring, 2) automated scoring by Morpheus 3) automated scoring by Somnolyzer24X7, 4) automated scoring by Morpheus with full manual review, 5) automated scoring by Morpheus with partial manual review, 6) automated scoring by Somnolyzer24X7 with partial manual review. Ten traditional clinical efficacy measures of sleep initiation, maintenance, and architecture were calculated. Results Pair-wise epoch-by-epoch agreements between fully automated and manual scores were in the range of intersite manual scoring agreements reported in the literature (70%-72%). Pair-wise epoch-by-epoch agreements between automated scores manually reviewed were higher (73%-76%). The direction and statistical significance of treatment effect sizes using traditional efficacy endpoints were essentially the same whichever method was used. As the degree of manual review increased, the magnitude of the effect size approached those estimated with fully manual scoring. Conclusion Automated or semi-automated sleep PSG scoring offers valuable alternatives to costly, time consuming, and intrasite and intersite variable manual scoring, especially in large multicenter clinical trials. Reduction in scoring variability may also reduce the sample size of a clinical trial.