Orexin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Orexin receptors [79]) 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, prepro-orexin or orexin precursor, by proteolytic cleavage and some typical peptide modifications [120, 79]. 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, lemborexant and daridorexant, which are used as hypnotics, and several dual, as well as OX1- and OX2-selective antagonists are under development for different indications. Multiple orexin agonists are in development for the treatment of narcolepsy and other sleep disorders. Orexin receptor 3D structures have been solved [150, 148, 55, 130, 47, 113, 7, 149].

Aim Dysfunction of nitric oxide (NO) – soluble guanylate cyclase (sGC) – cyclic guanosine monophosphate (cGMP) signalling is implicated in the pathophysiology of cognitive impairment and dementia. Zagociguat is a central nervous system-(CNS-) penetrant sGC stimulator designed to amplify NO-cGMP signalling in the CNS. This article reports on a phase 1b study evaluating the safety and pharmacodynamic effects of zagociguat. Methods In this randomized crossover study, 24 healthy participants ≥65 years of age were planned to receive 15 mg zagociguat or placebo once daily for two 15-day periods separated by a 27-day washout. Adverse events, vital signs, electrocardiograms, and laboratory tests to assess safety. Pharmacokinetics of zagociguat were evaluated in blood and CSF. Pharmacodynamic assessments included evaluation of cerebral blood flow, CNS tests, pharmaco-electroencephalography, passive leg movement, and biomarkers in blood, cerebrospinal fluid, and brain. Results Twenty-four participants were enrolled and 12 participants completed both treatment periods, while 12 participants completed only one treatment period. Zagociguat was well tolerated and penetrated the blood-brain barrier. Zagociguat induced modest decreases in blood pressure. No consistent effects of zagociguat on other pharmacodynamic parameters were detected. Conclusion Zagociguat was well tolerated and induced modest systemic blood pressure reductions consistent with other sGC stimulators. No clear pharmacodynamic effects of zagociguat were detected, perhaps due to optimal CNS function in healthy participants. Studies in participants with proven reduced cerebral blood flow or CNS function may be an avenue for further evaluation of the compound.

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].

Soluble guanylate cyclase (sGC) and its product, cyclic guanosine monophosphate, play a role in learning and memory formation. Zagociguat (CY6463) is a novel stimulator of sGC being developed for the treatment of neurodegenerative disease. Single zagociguat doses of 0.3, 1, 3, 10, 20, 30, and 50 mg were administered once to healthy participants in a single-ascending-dose phase; then zagociguat 2, 5, 10, and 15 mg was administered q.d. for 14 days in a multiple-ascending-dose phase; and, finally, zagociguat 10 mg was administered once in both fed and fasted state in a food-interaction phase. Safety of zagociguat was evaluated by monitoring treatment-emergent adverse events, suicide risk, vital signs, electrocardiography, and laboratory tests. Pharmacokinetics of zagociguat were assessed through blood, urine, and cerebrospinal fluid sampling. Pharmacodynamic effects of zagociguat were evaluated with central nervous system (CNS) tests and pharmaco-electroencephalography. Zagociguat was well-tolerated across all doses evaluated. Zagociguat exposures increased in a dose-proportional manner. Median time to maximum concentration ranged from 0.8 to 5 h and mean terminal half-life from 52.8 to 67.1 h. CNS penetration of the compound was confirmed by cerebrospinal fluid sampling. Zagociguat induced up to 6.1 mmHg reduction in mean systolic and up to 7.5 mmHg reduction in mean diastolic blood pressure. No consistent pharmacodynamic (PD) effects on neurocognitive function were observed. Zagociguat was well-tolerated, CNS-penetrant, and demonstrated PD activity consistent with other sGC stimulators. The results of this study support further development of zagociguat.

Phelan-McDermid syndrome (PMS) was initially called the 22q13 deletion syndrome based on its etiology as a deletion of the distal long arm of chromosome 22. These included terminal and interstitial deletions, as well as other structural rearrangements. Later, pathogenetic variants and deletions of the SHANK3 gene were found to result in a phenotype consistent with PMS. The association between SHANK3 and PMS led investigators to consider disruption/deletion of SHANK3 to be a prerequisite for diagnosing PMS. This narrow definition of PMS based on the involvement of SHANK3 has the adverse effect of causing patients with interstitial deletions of chromosome 22 to “lose” their diagnosis. It also results in underreporting of individuals with interstitial deletions of 22q13 that preserve SHANK3. To reduce the confusion for families, clinicians, researchers, and pharma, a simple classification for PMS has been devised. PMS and will be further classified as PMS- SHANK3 related or PMS- SHANK3 unrelated. PMS can still be used as a general term, but this classification system is inclusive. It allows researchers, regulatory agencies, and other stakeholders to define SHANK3 alterations or interstitial deletions not affecting the SHANK3 coding region.

Abstract 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.

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.

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.

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.

The timing and propensity of sleep are regulated by two interactive processes: circadian rhythmicity and sleep homeostasis. It has been demonstrated that the two processes are likely to converge at molecular levels, involving networks of genes including the molecular circadian clock machinery. However, since the molecular pathways involved in sleep homeostasis are still elusive, it is unclear how the circadian and homeostatic signals integrate to regulate sleep beyond a handful of clock genes. In ~200 (C57BL/6J x 129S1/SvImJ) F2 mice, we collected a comprehensive dataset containing: 1) phenotypic data of affective behaviors and sleep (including baseline, recovery after 6-h sleep deprivation, and sleep after restraint stress), 2) genotypic data across the genome, and 3) microarray data in the prefrontal cortex, hippocampus, midbrain-thalamus, and hypothalamus. We reconstructed gene networks in each of these brain regions. We also integrated this dataset with multiple circadian and sleep genomics datasets that are publicly available, in order to identify convergent networks that link both circadian and sleep homeostatic processes. Using our mouse dataset, we uncovered brain-region-conserved, as well as brain-region-specific, gene networks that are associated with sleep phenotypes. These sleep gene networks are cell-type specific and can be functionally annotated with specific cellular processes, revealing molecular pathways key to sleep function and regulation. Via Integrated analysis with publicly available datasets, we identified a number of gene networks that are enriched with differentially expressed genes responding to sleep loss and cycling genes peaking at particular times of the day. Particularly, our analysis in the prefrontal cortex highlights a Clock-driven network and a sphingolipid-metabolism network, whose overall network gene expression are cycling with opposite phases and are affected by sleep deprivation in opposite directions. This analysis of molecular networks important to sleep regulation reveals novel insights into convergent pathways that integrate circadian timing and homeostatic signals in the regulation of sleep. This work was supported by a research grant from Merck Research Laboratories, by the Defense Advanced Research Projects Agency and the U.S. Army Research Office (W911NF101006), and by the National Institute of Mental Health of the NIH (J.R.S.; F30MH106293).

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 coldinducible 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.

One of the main approaches for a disease-modifying therapy in Alzheimer's disease (AD) today, is therapeutic intervention in the amyloidogenic APP processing pathway, either via reduction in the production of Aβ monomeric precursors (secretase inhibition) or via facilitation of Aβ peptide clearance/neutralization (immunotherapy). The novel BACE inhibitor, verubecestat, was recently shown to produce a dose-dependent reduction of several APP pathway components—Aβ40, Aβ42 and sAPPβ—in healthy volunteer and AD patient CSF (Kennedy, 2016 Sci. Transl. Med. 8:363). As Aβ oligomers (AβO) embody a neurotoxic intermediary APP derivative en route to the deposition of plaques in AD brain, the present study examined whether acute inhibition of BACE would also alter AβO pool in humans. Specifically, we evaluated the pharmacodynamic (PD) response of toxic AβO, and their relationship to the response of Aβ40, in healthy volunteers after acute administration of an investigational BACE inhibitor molecule. Serial CSF samples were collected in 21 healthy males following single dose administrations of placebo, 10, 60 or 800 mg of an investigational BACE inhibitor. The PD effects in the subjects were evaluated by quantifying the levels of AβO and Aβ40 over 36 hours post-acute administration. Immunoassay-based analysis of AβO was performed as previously described (Savage, 2014 J. Neurosci. 34(8):2884–97). Robust time- and dose-dependent reductions of AβO, with similar concomitant drop of Aβ40 in the CSF of BACE inhibitor-treated subjects, were observed, reaching ∼90% relative to the baseline. These were significant for all summary measure responses tested: time-weighted averages, minimum values, concentration at three different time points, as well as time to reach minimum values. Finally, the kinetics and magnitude of response of oligomers compared with those of monomers, suggesting a great level of cooperativity between Aβ species. Together, these findings in healthy volunteers demonstrate that acute BACE inhibition reduced AβO with a similar rate and extent as monomer, suggesting potential further utility of this therapeutic approach. Future studies will examine the correlation of the magnitude and kinetics of CSF Aβ oligomers with monomeric precursors in an AD cohort after administration of secretase therapy.

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.

Orexin receptor (OX1R, OX2R) antagonism induces sleep architecture characterized by increases in both NREM and REM reminiscent of unmedicated sleep. REM sleep is thought to be controlled in part by noradrenergic neurons of the locus coeruleus (LC), a site of selective OX1R expression. This work utilizes selective DORA and 2-SORA antagonists in combination with prazosin (alpha1-noradrenergic blocker) in genetic models to determine specific roles of OX1R and OX2R in sleep architecture changes induced by DORAs and 2-SORAs. Sleep architecture was evaluated by polysomnography in preclinical animals (mice, rats, dogs, and rhesus monkeys) via radio-telemetry implants in across 3–5 days of drug administration. Stages of sleep were quantified with automated software modules evaluating ECoG, EMG, EOG (dog, monkey) data. Drug-induced sleep stage changes were correlated with receptor occupancy in transgenic rats expressing hOX2R at Cmax. A double-blind, randomized, 4-period crossover Phase I polysomnography study evaluated responses to single doses of 2-SORA, MK-1064, in twenty healthy male subjects. In rats, NREM and REM sleep induced by DORAs is statistically no different from un-medicated inactive phase sleep. Mouse genetic models indicate that OX2R primarily modulates orexin-induced arousal, while OX1R appears involved in vigilance state gating. Prazosin similarly augmented REM sleep induced by DORAs and 2-SORAs, however, indicating that noradrenergic signaling from the LC is not maximally inhibited by either treatment. In rats, sleep was induced by lower receptor occupancies of DORAs relative to 2-SORAs. Across mammals including humans, REM sleep was induced by MK-1064, a 2-SORA exhibiting 3000x binding selectivity for OX2R over OX1R. DORAs promote sleep by attenuating arousal and reducing sleep stage threshold through mechanisms beyond alpha1-noradrenergic signaling of the LC. 2-SORAs promote REM sleep across mammals in a manner similar to DORAs and do not appear to measurably differentiate from DORAs in sleep architecture. This work was funded by Merck & Co., Inc.