Orexin Receptor Research Articles

In Silico Screening of Rauwolfia Serpentina Phytochemicals as Orexin Receptor-2 Agonists for the Management of Narcolepsy

The neuropeptide orexin/hypocretin plays a crucial role in numerous physiological processes such as regulation of sleep/wakefulness, appetite and emotions. Dysregulation of orexin signaling has been implicated in hypersomnia, especially in narcolepsy, characterized by excessive daytime sleepiness (EDS), sudden loss of muscle tone while awake (cataplexy), sleep paralysis, and hallucinations. Lack of orexins contributes to the development of narcolepsy, thus small-molecule orexin receptor agonists are promising therapeutics for narcolepsy. In this study, we employed several bioinformatics tools to screen and identify effective phytochemicals of Rauvolfia serpentina that may act as Orexin 2 receptor (OX2R) agonists. In silico methods such as protein‒ligand interaction analysis, drug‒likeness evaluation, ADMET analysis, molecular docking, molecular dynamics simulation, and biological activity prediction, have been employed extensively. Among all screened phytochemicals from R. serpentina, both tetraphylline and yohimbine were identified as promising drug candidates due to their favorable ADMET profile, high docking scores, permeability of the blood‒brain barrier, and significant RMSD, RMSF, Rg, and SASA values. However, the study is solely based on in silico study, therefore in vitro and in vivo experimental studies are suggested to validate the potency and efficacy of these medications.

Role of OX/OXR cascade in insomnia and sleep deprivation link Alzheimer's disease and Parkinson's disease: Therapeutic avenue of Dual OXR Antagonist (DORA).

Sleep plays a role in the elimination of neurotoxic metabolites that are accumulated in the waking brain as a result of neuronal activity. Long-term insomnia and sleep deprivation are associated with oxidative stress, neuroinflammation, amyloid beta (Aβ) deposition, and Lewy body formation, which are known to increase the risk of mild cognitive impairment (MCI) and dementia. Orexin A (OXA) and orexin B (OXB), two neuropeptides produced in the lateral hypothalamus, are known to influence the sleep-wake cycle and the stress responses through their interactions with OX receptor 1 (OX1R) and OX receptor 2 (OX2R), respectively. OX/OXR cascade demonstrates intricate neuroprotective and anti-inflammatory effects by inhibiting nuclear factor-kappa B (NF-kB) and PLC/Ca2+ pathway activation. OX1R binds OXA more strongly than OXB by one-order ratio, whereas OX2R binds both OXA and OXB with equal strengths. Overexpression of OXs in individuals experiences sleep deprivation, circadian rhythm disturbances, insomnia-associated MCI, Parkinson's disease (PD), and Alzheimer's disease (AD). Many dual OXR antagonists (DORAs) have been effective in their clinical studies, with suvorexant and daridorexant receiving FDA clearance for insomnia therapy in 2014 and 2022 respectively. The results of clinical studies suggested that there is a new pharmaceutical option for treating insomnia and the sleep deprivation-AD/PD relationship by targeting the OXR system. DORAs treatment reduces Aβ deposition in the brain and improves synaptic plasticity and circadian expression. This review indicates the link between sleep disorders and MCI, DORAs are an appropriate medication category for treating insomnia, and sleep deprivation links AD and PD.

The role of the hypothalamus-gut microbiota in the pathogenesis of periparturient fatty liver disease in dairy cows.

During the periparturient period, dairy cows experience negative energy balance due to reduced feed intake, leading to adipose tissue breakdown, liver damage, and fat accumulation. This study examined the gut-liver-brain axis to explore the link between fatty liver disease, changes in hypothalamic appetite-related neurons, and microbiome shifts in dairy cows. Thirty cows were monitored, with daily DMI recordings and blood sampling. Postpartum brain, liver, and ileal contents were collected from 10 selected cows, divided into two groups: H-DMI (slight DMI decrease) and L-DMI (severe DMI decrease). The L-DMI group of cows exhibited higher plasma NEFA, BHBA, ALT, and AST levels, along with severe hepatic steatosis and lipid accumulation. Transcriptome sequencing of the hypothalamic arcuate nucleus (ARC) revealed decreased expression of Hypocretin Neuropeptide Precursor (HCRT), orexin-A (OX-A), Orexin Receptor Type 1 (OX1R), and Cannabinoid Receptor 1 (CB1) in the L-DMI group, while Pro-opiomelanocortin (POMC) and Melanocortin 4 Receptor (MC4R) expression increased. Metagenomic analysis of ileal contents showed reduced abundance of Ruminococcus spp. in the L-DMI group, which may be associated with fatty liver disease (FL). Integrated omics analysis showed that increased MC4R expression was correlated with the elevated abundance of bacteria such as Akkermansia glycaniphila, and reduced abundance of species such as Methanobrevubacter thaueri and Ruminococcus spp. Decreased HCRT expression was also linked to Akkermansia glycaniphila. In conclusion, these changes may affect DMI through the OX-A/POMC pathway, with neurological and gut microbiome alterations potentially leading to appetite suppression, negative energy balance, and the development of fatty liver disease.

Pharmacodynamic, Safety and Pharmacokinetic Effects of Co-Administration of the Selective Orexin-1 Receptor Antagonist INDV-2000 and Buprenorphine in Treatment Seeking Individuals With Opioid Use Disorder

Pharmacodynamic, Safety and Pharmacokinetic Effects of Co-Administration of the Selective Orexin-1 Receptor Antagonist INDV-2000 and Buprenorphine in Treatment Seeking Individuals With Opioid Use Disorder

Orexin Receptor Mechanisms Involved in Methamphetamine-Induced Sleep Disruption in Female Rhesus Monkeys

Orexin Receptor Mechanisms Involved in Methamphetamine-Induced Sleep Disruption in Female Rhesus Monkeys

Exploring the Silent Connection Between Sleep Disorders (i.e., OSA, Insomnia) and Cardiovascular Diseases: Pathophysiology and Insights

Sleep is a complex physiological phenomenon crucial for health. Despite this, millions suffer from sleep disorders, contributing to a range of health issues, particularly cardiovascular diseases (CVD). The pathophysiological mechanisms linking sleep disorders, such as insomnia and obstructive sleep apnea (OSA), to cardiovascular risk factors include disruptions in inflammatory, autonomic, and metabolic pathways. Increased sympathetic nervous system activity, chronic inflammation, and metabolic dysregulation stemming from poor sleep can lead to conditions like hypertension, obesity, and insulin resistance, significantly elevating the risk for CVD. This article reviews the connection between sleep quality and cardiovascular disease risks. Sleep disorders (i.e., insomnia and OSA) have been found to impact cardiovascular risk factors adversely. Studies have found an association between abnormal sleep and increased cardiovascular morbidity and mortality by higher risks of hypertension, diabetes, obesity, and dyslipidemia. The review also discusses non-pharmacological interventions, such as relaxation training, Cognitive behavioral therapy for insomnia (CBT-I), and red-light therapy, which have shown efficacy in improving sleep quality and reducing cardiovascular risks. Dual orexin receptor antagonists and Ashwagandha promise to enhance sleep quality and cardiovascular health, but further research is needed. Addressing sleep disorders and promoting healthy sleep practices are essential for mitigating the global burden of cardiovascular diseases, underscoring the need for continued research and effective public health interventions.

Orexin Receptor Antagonists for Insomnia in Patients with Serious Illness #496.

Orexin Receptor Antagonists for Insomnia in Patients with Serious Illness #496.

Utilization of Potentially Inappropriate Sedative-Hypnotic and Atypical Antipsychotic Medications among Elderly Individuals with Insomnia and Alzheimer's Disease.

This study assessed the utilization of potentially inappropriate medications (PIM) including oral sedative-hypnotic and atypical antipsychotic (OSHAA), healthcare resource utilization (HCRU), and costs among elderly individuals with insomnia and in the subpopulation with Alzheimer's Disease (AD) who also had a diagnosis of insomnia. Using claims database containing International Classification of Diseases, 10th Revision (ICD-10) codes, the cohort included individuals aged ≥ 65 with incident insomnia (EI, N=152,969) and AD insomnia subpopulation (ADI, N=4,888). Proportion of patients utilizing atypical antipsychotics or oral sedative-hypnotic medications, namely z-drugs, benzodiazepines, doxepin, Dual Orexin Receptor Antagonists (DORAs), and melatonin agonists, were assessed. Inappropriate OSHAA utilization was defined as per the American Geriatrics Society (AGS) Beers criteria. Multivariable models were utilized to compare HCRU and costs between PIM-OSHAA and no PIM-OSHAA groups. Among the EI cohort, z-drugs (13.39%) were the most commonly utilized OSHAA, and in the ADI cohort, it was AAPs (29.97%). PIM-OSHAA was utilized by 20% of the EI and 35% of the ADI cohorts. Patients with PIM-OSHAA use among the EI cohort had a higher annualized adjusted mean HCRU (pharmacy visits: 31.21 vs. 23.68; ambulatory & outpatient visits: 18.55 vs. 16.85) and costs, primarily due to medical costs (mean total cost: $36,676.08 vs. $31,346.54) compared to those without. Substantial utilization of PIM-OSHAA was observed in EI and ADI cohorts. PIM-OSHAA use was associated with higher HCRU and costs. These findings underscore the importance of appropriate treatment strategies for insomnia in the elderly population especially in those with concurrent AD.

The involvement of orexin-1 receptors in modulation of feeding and anxiety-like behavior in rats with complete Freund's adjuvant-induced temporomandibular joint disorder.

Orexin-A (OXA), a neuropeptide produced in the hypothalamus, is recognized for its role in modulating orofacial nociception and regulating feeding behaviors, as well as its impact on psychophysiological responses. This study investigated the role of orexin-1 receptors (OX1R) in modulating nociceptive behaviors induced by noxious stimulation of the temporomandibular joint (TMJ) and the associated changes in mood and feeding behaviors in rats with complete Freund's adjuvant (CFA)-induced temporomandibular disorders (TMDs). Bilateral cannulation of the lateral ventricles was performed in rats. To induce nociception, CFA was injected unilaterally into the left TMJ of the rats. Nociceptive behaviors were assessed using the hot plate and tail flick tests, while anxiety-like behavior and food intake were evaluated using an elevated plus maze (EPM) and a food preference device, respectively. The results demonstrated a significant increase in nociceptive scores and anxiety-like behaviors, along with reductions in water and food consumption following CFA injection. However, post-treatment with OXA at concentrations of 50 and 100pM/rat significantly decreased thermal nociceptive scores, alleviated anxiety-like behavior, and increased water and food intake. These beneficial effects were reversed when OXA was co-administered with SB-334867 (40nM/rat), an OX1R antagonist. Collectively, our findings suggest that OX1R signaling plays a role in the modulation of anxiety-like behavior and abnormalities in food intake in CFA-treated rats. Understanding the involvement of OXA and its receptors in CFA-induced TMJ nociception and behavioral changes may pave the way for potential therapeutic interventions targeting OX1R signaling in the management of TMD-associated symptoms.

Deficiency of orexin receptor type 1 in dopaminergic neurons increases novelty-induced locomotion and exploration

Orexin signaling in the ventral tegmental area and substantia nigra promotes locomotion and reward processing, but it is not clear whether dopaminergic neurons directly mediate these effects. We show that dopaminergic neurons in these areas mainly express orexin receptor subtype 1 (Ox1R). In contrast, only a minor population in the medial ventral tegmental area express orexin receptor subtype 2 (Ox2R). To analyze the functional role of Ox1R signaling in dopaminergic neurons, we deleted Ox1R specifically in dopamine transporter-expressing neurons of mice and investigated the functional consequences. Deletion of Ox1R increased locomotor activity and exploration during exposure to novel environments or when intracerebroventricularely injected with orexin A. Spontaneous activity in home cages, anxiety, reward processing, and energy metabolism did not change. Positron emission tomography imaging revealed that Ox1R signaling in dopaminergic neurons affected distinct neural circuits depending on the stimulation mode. In line with an increase of neural activity in the lateral paragigantocellular nucleus (LPGi) of Ox1RΔDAT mice, we found that dopaminergic projections innervate the LPGi in regions where the inhibitory dopamine receptor subtype D2 but not the excitatory D1 subtype resides. These data suggest a crucial regulatory role of Ox1R signaling in dopaminergic neurons in novelty-induced locomotion and exploration.

Deficiency of orexin receptor type 1 in dopaminergic neurons increases novelty-induced locomotion and exploration

Orexin signaling in the ventral tegmental area and substantia nigra promotes locomotion and reward processing, but it is not clear whether dopaminergic neurons directly mediate these effects. We show that dopaminergic neurons in these areas mainly express orexin receptor subtype 1 (Ox1R). In contrast, only a minor population in the medial ventral tegmental area express orexin receptor subtype 2 (Ox2R). To analyze the functional role of Ox1R signaling in dopaminergic neurons, we deleted Ox1R specifically in dopamine transporter-expressing neurons of mice and investigated the functional consequences. Deletion of Ox1R increased locomotor activity and exploration during exposure to novel environments or when intracerebroventricularely injected with orexin A. Spontaneous activity in home cages, anxiety, reward processing, and energy metabolism did not change. Positron emission tomography imaging revealed that Ox1R signaling in dopaminergic neurons affected distinct neural circuits depending on the stimulation mode. In line with an increase of neural activity in the lateral paragigantocellular nucleus (LPGi) of Ox1RΔDAT mice, we found that dopaminergic projections innervate the LPGi in regions where the inhibitory dopamine receptor subtype D2 but not the excitatory D1 subtype resides. These data suggest a crucial regulatory role of Ox1R signaling in dopaminergic neurons in novelty-induced locomotion and exploration.

TAK-925 (danavorexton), an Orexin Receptor 2 Agonist, Reduces Opioid-Induced Respiratory Depression and Sedation Without Affecting Analgesia in Healthy Adult Males.

Orexin neuropeptides help regulate sleep/wake states, respiration, and pain. However, their potential role in regulating breathing, particularly in perioperative settings, is not well understood. TAK-925 (danavorexton), a novel, orexin receptor 2-selective agonist, directly activates neurons associated with respiratory control in the brain and improves respiratory parameters in rodents undergoing fentanyl-induced sedation. This study assessed the safety and effect of danavorexton on ventilation in healthy men in an established remifentanil-induced respiratory depression model. This single-center, double-blind, placebo-controlled, two-way crossover, phase 1 trial randomized (1:1) 13 healthy men to danavorexton (11mg [low-dose] then 19mg [high-dose]) or placebo, under remifentanil infusion, on two occasions separated by a ≥36-hour washout period. Remifentanil infusion was titrated under isohypercapnic conditions to achieve ~30% to 40% decrease in minute ventilation (from ~20 to ~14 L/minute) before danavorexton/placebo administration. Assessments included safety, ventilation measurements, sedation, and pain tolerance. 4 (30.8%) danavorexton-treated participants and 1 (8.3%) placebo-treated participant experienced treatment-emergent adverse events (all mild in severity). Insomnia, lasting 1 day, occurred in 1 participant, and was considered related to danavorexton. Compared with placebo, low- and high-dose danavorexton significantly increased ventilation variables (observed mean [95% confidence interval] change, sensitivity analysis model-based p-values) including minute volume (8.2[5.0, 11.4] and 13.0[9.4, 16.5] L/min), tidal volume (312[180, 443] and 483[309, 657] mL), and respiratory rate (3.8[1.9, 5.7] and 5.2[2.7, 7.7] breaths/min) (all P<0.001). High-dose danavorexton significantly decreased sedation on visual analog scale (-29.7[-54.1, -5.3] mm, P<0.001) and Richmond Agitation Sedation Scale (0.4[0.0, 0.7], P<0.001), compared with placebo. Improvements in respiratory variables continued beyond completion of danavorexton infusion. No significant differences in pain tolerance were observed between danavorexton doses or between danavorexton and placebo (~13% increase from baseline; low-dose:P=0.491; high-dose:P=0.140). Danavorexton has effects on respiration and wakefulness in an opioid-induced respiratory depression setting without reversing opioid analgesia.

TAK-994 Mechanistic Investigation into Drug-Induced Liver Injury.

The frequency of drug-induced liver injury (DILI) in clinical trials remains a challenge for drug developers despite advances in human hepatotoxicity models and improvements in reducing liver-related attrition in preclinical species. TAK-994, an oral orexin receptor 2 agonist, was withdrawn from phase II clinical trials due to the appearance of severe DILI. Here, we investigate the likely mechanism of TAK-994 DILI in hepatic cell culture systems examined cytotoxicity, mitochondrial toxicity, impact on drug transporter proteins, and covalent binding. Hepatic liabilities were absent in rat and non-human primate safety studies, however, murine studies initiated during clinical trials revealed hepatic single-cell necrosis following cytochrome P450 induction at clinically relevant doses. Hepatic cell culture experiments uncovered wide margins to known mechanisms of intrinsic DILI, including cytotoxicity (>100× Cmax/IC50), mitochondrial toxicity (>100× Cmax/IC50), and bile salt efflux pump inhibition (>20× Css, avg/IC50). A potential covalent binding liability was uncovered with TAK-994 following hepatic metabolism consistent with idiosyncratic DILI and the delayed-onset clinical toxicity. Although idiosyncratic DILI is challenging to detect preclinically, reductions in total daily dose and covalent binding can reduce the covalent body binding burden and, subsequently, the clinical incidence of idiosyncratic DILI.

Circadian aspects in nonpharmacologic and pharmacologic treatment of insomnia.

Insomnia disorder is a frequent sleep disorder leading to significant health and economic consequences. It has been proposed that individuals with insomnia may experience compromised deactivation systems of arousal, leading to a chronic state of hyperactivation of arousal known as hyperarousal, along with instability in the flip-flop system. Such disruptions may have a primarily impact on the sleep homeostatic drive process. Insomnia may indeed be associated with a disruption in the body's internal clock, known as chronodisruption. Despite the differentiation established in diagnostic nosology between insomnia disorder and circadian rhythm disorders, there is a significant body of evidence suggesting a complex interplay and frequent co-occurrence between these two conditions. In particular, circadian factors can predispose individuals to insomnia disorders, as well as precipitate and perpetuate their symptoms. Accordingly numerous pieces of evidence suggest that both pharmacologic and nonpharmacologic options for treating insomnia can have a resynchronization effect on circadian rhythms. The first-line treatment for chronic insomnia, according to current guidelines, is cognitive behavioral therapy for insomnia while pharmacologic interventions comprise of benzodiazepine receptor agonists also known as Z-drugs and short- to medium-acting benzodiazepines, melatonergic agonists such as ramelteon and melatonin 2mg prolonged release, and dual orexin receptor antagonists such as daridorexant, suvorexant, and lemborexant. At the same time, certain therapies recommended for circadian rhythm disorders can be utilized as adjunctive treatments for insomnia. Therefore, this chapter will discuss the circadian aspects of insomnia disorder and of its therapeutic approach. Furthermore, the effects of chronobiologic interventions, recommended for the treatment of circadian rhythm sleep-wake disorders, will be examined in individuals afflicted with chronic insomnia.

Association Between Hypnotics, Accidents, and Injuries: A Study Based on the Adverse Drug Event Reporting Database in Japan.

The use of hypnotic drugs can lead to accidents and injuries. However, few reports have shown their association with these events after adjusting for many concomitant medications. This study aimed to determine whether the use of hypnotic drugs was associated with accidents and injuries. Using the Japanese Adverse Event Reporting Database, 772,387 reports published between September 2023 and April 2004 were analyzed. Reporting odds ratios (RORs) and 95% confidence intervals (CIs) for accidents and injuries associated with each hypnotic drug were calculated after adjusting for potential confounders. Of the total, 12,484 reports indicated association of hypnotic drugs with accidents and injuries. The use of each hypnotic drug was associated with accidents, injuries, and other adverse events. However, a multivariate analysis adjusted for age, sex, reporting year, and concomitant medications showed a considerable decrease in ROR for melatonin receptor agonists (adjusted ROR=1.26; 95%CI=1.03-1.55) and dual orexin receptor antagonists (DORAs) (adjusted ROR=1.04; 95%CI=0.86-1.25). Particularly in DORAs, a loss of signal for accidents and injuries was observed. The risk of accidents and injuries may vary with hypnotic drug use; however, DORAs may be less frequently associated with these events. The results of this study provide useful information for the selection of hypnotic drugs.

Learned phenotypes emerge during social stress modifying hippocampal orexin receptor gene expression

Psychological distress, including anxiety or mood disorders, emanates from the onset of chronic/unpredictable stressful events. Symptoms in the form of maladaptive behaviors are learned and difficult to treat. While the origin of stress-induced disorders seems to be where learning and stress intersect, this relationship and molecular pathways involved remain largely unresolved. The hippocampus, studied for its role in learning, is divided into regions that designate the passage of neuronal signaling during memory formation, including dentate gyrus (DG), CA3, CA2, and CA1. Inputs into these hippocampal subregions, like those from hypothalamic orexinergic neurons, may modify learning outcomes. We have previously shown the orexin system to balance stress states, where receptor subtypes prompt opposing actions on behavior. Here, we explore the connection between hippocampal orexin receptors and learning during stress. In a social stress/learning paradigm separating mice into stress resilient and vulnerable populations, hippocampal Orx1R and Orx2R transcription is regulated in a phenotype-dependent fashion. We further identified Orx1R as highly expressed in the hilus of DG, while Orx2R is abundant in CA2. Finally, we designed an experiment where mice were provided prior exposure to a stressful environment, which ultimately modified behavior, as well as transcription of hippocampal orexin receptors.

Effectiveness and Safety of Suvorexant in Preventing Delirium: A Systematic Review and Meta-Analysis

Introduction: Delirium is a common and severe complication in hospitalized patients, particularly among the elderly and those in intensive care units or post-surgery. Suvorexant, a dual orexin receptor antagonist, has been proposed as a potential preventive treatment for delirium, but its safety and effectiveness have not been comprehensively analyzed. Methods: We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) assessing Suvorexant for adult delirium prevention, identified through four database searches. The primary outcome was the incidence of delirium, and the secondary outcome was the incidence of adverse events. A random-effects model was used for data synthesis. Results: Three studies, all conducted in Japan, involved 307 patients and were included in the analysis. The pooled results demonstrated that Suvorexant significantly reduced the incidence of delirium, with an odds ratio (OR) of 0.43 [95% confidence interval (CI) 0.21–0.87, p = 0.02; I2 = 16%]. A seven-day administration of Suvorexant showed a statistically significant protective effect (OR 0.50, 95% CI 0.28–0.90, p = 0.02; I2 = 0%), whereas three-day administration did not reach statistical significance (OR 0.56, 95% CI 0.24–1.27, p = 0.16; I2 = 19%). Additionally, there was no significant increase in adverse events (OR 0.91, 95%CI 0.50–1.64, p = 0.75; I2 = 0%). Conclusion: Suvorexant is an effective and safe option for preventing delirium, particularly with seven-day administration periods. However, further research is required to determine the optimal dosing and duration for maximum effectiveness.

Function of orexin-1 receptor signaling in the olfactory tubercle in odor-guided attraction and aversion

While olfactory behaviors are influenced by neuromodulatory signals, the underlying mechanism remains unknown. The olfactory tubercle (OT), a component of the olfactory cortex and ventral striatum, consists of anteromedial (am) and lateral (l) domains regulating odor-guided attractive and aversive behaviors, respectively, in which the amOT highly expresses various receptors for feeding-regulated neuromodulators. Here we show functions of appetite-stimulating orexin-1 receptor (OxR1) signaling in the amOT. When odor-food reward associated mice underwent OxR1 antagonist injection in the amOT, their odor-attractive behavior was suppressed and odor-aversive behavior was conversely induced. Although odor-attractive mice showed activation of attraction-promoting dopamine receptor type 1-expressing D1 cells in the amOT, the antagonist injection increased activation of aversion-promoting D2 cells in the amOT and D1 cells in the lOT. The results highlight the amOT as the crucial structure integrating OxR1 signaling and odor information, thereby controlling metabolic status-dependent olfactory behavior through the cell type- and domain-specific activation.

Urinary excretion profiles of the orexin receptor antagonist suvorexant and its metabolites.

Suvorexant is an orexin receptor antagonist used in the treatment of insomnia. In this study, we investigated the urinary excretion profiles of suvorexant and its major metabolites, including conjugates, to obtain fundamental information for proving exposure to suvorexant in criminal cases. Urine specimens were collected from three subjects for maximum 168h after a single oral ingestion of suvorexant (10mg), and suvorexant and its metabolites in urine were determined using liquid chromatography-tandem mass spectrometry with a C18 semi-micro column. The carboxylic and hydroxy metabolites (M4 and M9) were identified with authentic standards synthesized in our laboratory, and their glucuronides and other hydroxy metabolites (M8 and M10) were tentatively detected based on measured exact masses and product ion spectra of them. Suvorexant, M4 and M9 would be detectable for 20-34h, 6-7days and 42-61h after intake, respectively. The quantitative results demonstrated that the molar ratios of accumulated amounts of M4 and M9 including their glucuronides excreted in urine to dose ranged about 2.6-6.2% and 0.37-0.51%, respectively, while that of the unchanged parent was much lower (0.011-0.013%). The ratios of the amount of glucuronide to the total amount of M4 and M9 excreted in urine was less than 10% and approximately 90%, respectively. The urinary excretion profiles indicated that M4 and M9 would be effective indicators for proving suvorexant intake, and M4 could be detected until one week after intake even without enzymatic hydrolysis (limit of detection: 0.05ng/mL).

Pharmacodynamics of the orexin type 1 (OX1) receptor in colon cancer cell models: A two-sided nature of antagonistic ligands resulting from partial dissociation of Gq.

Orexins have important biological effects on the central and peripheral nervous systems. Their primary ability is to regulate the sleep-wake cycle. Orexins and their antagonists, via OX1 receptor have been shown to have proapoptotic and antitumor effects on various digestive cancers cell models. We investigated, (1) the ability of orexin-A and its antagonists to regulate OX1 receptor expression at the cell surface and (2), how OX1 antagonists induced proapoptotic effect in cancer cells models. The OX1 receptor internalisation is determined by imaging flow cytometry in colon cancer cell models and the OX1 receptor coupling to G proteins via bioluminescence resonance energy transfer and molecular dynamic simulation. Orexin-A induced rapid receptor internalisation within 15 min via β-arrestin 2 recruitment, whereas antagonists had no effect. Furthermore, Gq is critical for receptor internalisation and signalling pathways, and no other G proteins appear to be recruited. Surprisingly, antagonists induced recruitment and conformational changes in Gq protein. Simulated molecular dynamics of agonists/orexin receptor/Gq complexes show that antagonists exhibits a similar binding mode, stable at the binding site and show conformational changes of ECL2, similar to that of the agonists. OX1 receptor activation induced orexin/β-arrestin-dependent internalisation, which was independent of the apoptotic pathway induced by orexins and antagonists. In addition, antagonists activate the Gq protein, suggesting its putative partial dissociation. These results suggest that the development of OX1 receptor targeting molecules, including orexin antagonists with antitumor properties, may pave the way for innovative cancer therapies.