Orexin Knockout Research Articles

0039 Investigating the role of striatal dopamine in sleep and narcolepsy-cataplexy

Abstract Introduction Dopamine release in the striatum is known to govern reward, motor, and sleep behaviors. There also exists a link between pharmacological manipulation of dopamine receptors and cataplexy occurrence, yet the precise spatial and temporal dynamics of dopamine release in relation to cataplexy onset are not understood. In the present study, we evaluate dopamine release dynamics in orexin knockout (OX KO) mice during cataplexy and physiological behaviors. Methods OX KO mice were implanted with EEG/EMG electrodes, and dLight1.1 was expressed in the nucleus accumbens core (NAcc), shell (NAcSh), and dorsolateral striatum (DLS) to perform in vivo fiber photometry of dopamine release in freely-moving animals. In NAcc and NAcSh mice, ChrimsonR was retrogradely expressed in the ventral tegmental area (VTA), to allow for precise optogenetic control of midbrain dopamine projections to different regions of the striatum. We also expressed dLight1.1 in the NAcc of wildtype (WT) mice as a control. Three different stimulation parameters were used in independent recordings: phasic 20 Hz stimulation, phasic 10-40 Hz variable stimulation, and tonic 2 Hz stimulation. Results First, we show that while dopamine release in response to rewarding behaviors is muted in OX KO animals, there is no difference in dopamine release across sleep state transitions between OX KO and WT animals. Second, we demonstrate that there is a striking increase in dopamine release in both the NAcc and NAcSh, but not in the DLS, at the onset of cataplexy. This increase occurs independently of the behaviors that the animals were engaged in immediately prior to cataplexy, suggesting that the increase is characteristic of all cataplexy episodes. Finally, we find that while high frequency phasic stimulation did not affect cataplexy frequency, low frequency, tonic stimulation reduced both cataplexy and REM occurrence. Conclusion We show that there are differences in dopamine release in response to rewarding events, but not changes in sleep states, in OX KO and WT animals. We also identify dopamine release in the ventral striatum as a key node in cataplexy regulation, which helps to further our understanding of the limbic circuitry that underlies narcolepsy symptoms. Support (if any) University of Michigan Gilmore Award and Rackham Graduate Research Award.

0609 Samelisant, Histamine 3 Receptor Agonist for the Potential Treatment of Excessive Daytime Sleepiness in Patients with Narcolepsy

Abstract Introduction Samelisant (SUVN-G3031) is a potent and selective histamine 3 receptor inverse agonist. In orexin knockout mice, samelisant produced wake-promoting and anticataplectic effects suggesting its potential therapeutic utility in the treatment of narcolepsy. Safety and tolerability studies in animals and healthy human volunteers suggested a favorable risk/benefit profile for samelisant. Methods Samelisant is currently being evaluated in a double blind, Phase-2 proof of concept study in USA and Canada for narcolepsy with or without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). The study is recruiting subjects diagnosed with narcolepsy as per ICSD-3 criteria, aged between 18 to 65 years with an Epworth Sleepiness Scale (ESS) score of ≥12 and mean Maintenance of Wakefulness Test (MWT) time of < 12 min. A total of 171 subjects are expected to be randomized into 3 treatment arms (placebo, samelisant 2 mg and samelisant 4 mg) in 1:1:1 ratio. Each subject will receive either placebo or study drug once daily for 2 weeks. The primary efficacy endpoint is change in MWT score from baseline to week 2. Key secondary endpoint is change in ESS from baseline to week 2. Safety will be monitored throughout the study by medical monitor and by data safety monitoring committee. As the study is still recruiting, demographics and baseline characteristic data for the currently enrolled subjects is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results At the data cutoff date of Nov 30, 2022, a total of 155 subjects were randomized in the study. The median age of subjects was 30 years (range: 18-57 years) with mean BMI of 28.5 kg/m2 (range: 18.3- 43.9 kg/m2). Overall, 57% subjects were of narcolepsy type-1, 71% were female and 71% were Caucasian. Mean (SD) baseline values of MWT and ESS scores were 6.1 (4.4) and 17.2 (2.8), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling subjects with narcolepsy and the data readout is expected in Q2 2023. Support (if any)

The role of orexin neuron activity in sleep/wakefulness regulation

Orexin (also known as hypocretin) is a neuropeptide exclusively synthesized in the neurons of the lateral hypothalamus (LH). Initially orexin was thought to be involved in the regulation of feeding behavior. However, it is now known to also be a critical regulator of sleep/wakefulness, especially the maintenance of wakefulness. Although the somas of orexin neurons are exclusively located in the LH, these neurons send axons throughout the brain and spinal cord. Orexin neurons integrate inputs from various brain regions and project to neurons that are involved in the regulation of sleep/wakefulness. Orexin knockout mice have a fragmentation of sleep/wakefulness and cataplexy-like behavior arrest, which is similar to the sleep disorder narcolepsy. Recent progress with manipulation of neural activity of targeted neurons, using experimental tools such as optogenetics and chemogenetics, has emphasized the role of orexin neuron activity on the regulation of sleep/wakefulness. Recording of orexin neuron activity in vivo using electrophysiological and gene-encoded calcium indicator proteins revealed that these cells have specific activity patterns across sleep/wakefulness state changes. Here, we also discuss not only the role of the orexin peptide, but also the role of other co-transmitters that are synthesized and released from orexin neurons and involved in sleep/wakefulness regulation.

OX2R-selective orexin agonism is sufficient to ameliorate cataplexy and sleep/wake fragmentation without inducing drug-seeking behavior in mouse model of narcolepsy.

Acquired loss of hypothalamic orexin (hypocretin)-producing neurons causes the chronic sleep disorder narcolepsy-cataplexy. Orexin replacement therapy using orexin receptor agonists is expected as a mechanistic treatment for narcolepsy. Orexins act on two receptor subtypes, OX1R and OX2R, the latter being more strongly implicated in sleep/wake regulation. However, it has been unclear whether the activation of only OX2R, or both OX1R and OX2R, is required to replace the endogenous orexin functions in the brain. In the present study, we examined whether the selective activation of OX2R is sufficient to rescue the phenotype of cataplexy and sleep/wake fragmentation in orexin knockout mice. Intracerebroventricular [Ala11, D-Leu15]-orexin-B, a peptidic OX2R-selective agonist, selectively activated OX2R-expressing histaminergic neurons in vivo, whereas intracerebroventricular orexin-A, an OX1R/OX2R non-selective agonist, additionally activated OX1R-positive noradrenergic neurons in vivo. Administration of [Ala11, D-Leu15]-orexin-B extended wake time, reduced state transition frequency between wake and NREM sleep, and reduced the number of cataplexy-like episodes, to the same degree as compared with orexin-A. Furthermore, intracerebroventricular orexin-A but not [Ala11, D-Leu15]-orexin-B induced drug-seeking behaviors in a dose-dependent manner in wild-type mice, suggesting that OX2R-selective agonism has a lower propensity for reinforcing/drug-seeking effects. Collectively, these findings provide a proof-of-concept for safer mechanistic treatment of narcolepsy-cataplexy through OX2R-selective agonism.

0301 Does Orexin Influence Sleep Fragmented Brain Inflammation? A Pilot Study

Abstract Introduction Fragmented sleep occurs when there are repetitive, short interruptions of sleep, resulting in less than six hours of sleep per day. Overall, however, in the United States, sleep fragmentation is reported by 30% of employed adults. Sleep fragmentation may be a risk factor for Alzheimer's disease, and orexin reduction appears effective in reducing amyloid plaque formation in mice with transgenic Alzheimer's disease. Orexin is a neuropeptide that regulates arousal, wakefulness, and appetite. Thus we hypothesized that loss of orexin might also be protective against sleep disruption injury in non-Alzheimer's mice. This study aims to combine sleep fragmentation with orexin loss to see if brain health is severely reduced. Methods The 24 mice in this study were split into four groups: B6 rested, B6 sleep fragmented, orexin knockout rested, orexin knockout sleep fragmented. The sleep fragmented mice were placed on a shaker table for 10½ weeks to initiate chronic sleep loss. The mice were all perfused within five to eight months of birth, and then the brains were cryopreserved and sliced. These sections were immunolabeled with different protein antibodies using immunohistochemistry techniques. The stained brains were either analyzed through microscope stereology counts or computer image analysis. Results Two-way ANOVA analysis for tyrosine hydroxylase, ionized calcium binding adaptor, and vesicular acetylcholine transporter had p<0.05 for the sleep fragmentation variable, showing differences in these antibodies for rested and sleep loss mice. ANOVA for cluster of differentiation 68, cofilin, postsynaptic density protein, and RanBP had p<0.05 for the genotype variable, showing differences in these antibodies for knockout and normal orexin mice. ANOVA for glial fibrillary acidic protein and amyloid-beta had p<0.05 for both variables, showing differences for sleep and orexin levels. There was no ANOVA significance for synapsin. Conclusion Our results show that knocking out orexinergic neurons causes hippocampal tissue damage, dampens the functioning of synapses, and diminishes the ability of the brain to adapt through plasticity and memory. Sleep fragmentation, however, increases phagocytic activity, and harms the acetylcholine and norepinepherine neurotransmitter pathways. When combined, cell communication worsens and the blood brain barrier loses function, resemblant of neurodegenerative diseases. Support (If Any) NIH AG054104; AG064231

0265 Samelisant (SUVN-G3031), a Histamine H3 Receptor Inverse Agonist in Animal Models of Narcolepsy

Abstract Introduction Samelisant (SUVN-G3031) is a potent and selective H3 receptor (H3R) inverse agonist with hKi of 8.7 nM. It lacks measurable affinity against 70 other targets which includes GPCRs, ion channels, transporters, enzymes, peptides, steroids, second messengers, growth factors and prostaglandins. Samelisant exhibited desired pharmacokinetic properties and favorable brain penetration in preclinical species. Samelisant blocked R-α-methylhistamine induced dipsogenia in rats and increased tele-methylhistamine levels in brain and cerebrospinal fluid as well. Samelisant is currently being evaluated in a Phase-2 study as monotherapy for the treatment of excessive daytime sleepiness (EDS) in patients with narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). Methods In brain microdialysis, samelisant was evaluated for its effects on modulation of neurotransmitters like histamine and norepinephrine in prefrontal cortex. In male orexin knockout mice, electroencephalography (EEG), electromyography and activity were monitored using telemetric device. Effects of samelisant on sleep/ wake profile and cataplexy episodes were evaluated during active period of animals. Animals were allowed three weeks of recovery from surgery prior to EEG recording. Results Samelisant significantly increased histamine, dopamine and norepinephrine levels in the prefrontal cortex. Samelisant did not change dopamine levels in the striatal and accumbal. These suggest that samelisant may not have propensity to induce abuse liability. Samelisant produced a significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleep in orexin knockout mice. It also significantly decreased number of cataplectic episodes in orexin knockout mice. Conclusion The results from non-clinical studies presented here provide a strong evidence for the potential utility of samelisant for the treatment of EDS and cataplexy in patients with narcolepsy. Support (If Any) None

0624 Samelisant (SUVN-G3031), Baseline Characteristics from a Phase-2 Study Evaluating Efficacy and Safety in Patients with Narcolepsy

Abstract Introduction Histamine H3 receptor (H3R) antagonists/inverse agonists increase histaminergic neurotransmission and offer a therapeutic option for the treatment of narcolepsy. Samelisant (SUVN-G3031) is a potent H3R inverse agonist and exhibits very high selectivity over other targets. In orexin knockout mice, samelisant produced wake-promoting and anticataplectic effects suggesting its potential therapeutic utility in the treatment of excessive daytime sleepiness and cataplexy associated with narcolepsy. It showed dose-dependent H3R occupancy at efficacy doses. Safety and tolerability studies in animals and healthy human volunteers suggested a favorable risk/benefit profile for samelisant.Samelisant is being evaluated in a Phase-2, multicenter, double-blind, placebo controlled, parallel-group study in patients with narcolepsy with or without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). Methods Eligibility criteria for the study include subjects diagnosed with narcolepsy as per ICSD-3, aged between 18 to 50 years with an Epworth Sleepiness Scale (ESS) score of ≥12 and mean maintenance of Wakefulness Test (MWT) time of <12 min. A total of 171 subjects will be randomized into 3 treatment arms (placebo, samelisant 2 mg and samelisant 4 mg) in a fixed 1:1:1. Further, the randomization will be stratified according to the type of narcolepsy (Type-1 or Type-2). Each subject will receive either placebo or study drug once daily for 2 weeks. The primary efficacy endpoint is change in MWT score from baseline to week 2. Secondary endpoints are change in ESS and Clinical Global Impressions of Severity (CGI-S) from baseline to week 2. Safety will be monitored throughout the study by medical monitor and by an independent data safety monitoring committee. Baseline clinical and demographic data for the currently enrolled study is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results At the data cutoff date of Nov30, 2021, a total of 108 subjects were randomized in the study. The median age of subjects was 30 years (range: 18-50 years) with mean BMI of 26.4 (range: 18.3- 43.1 kg/m2). Overall, 58% subjects were of narcolepsy type-1, 69% were female and 74% were Caucasian. Mean (SD) baseline values of MWT and ESS were 6.20 (4.53) and 17.17 (2.93), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling subjects with narcolepsy and the data readout is expected in Q3 2022. Support (If Any) None

005 Samelisant (SUVN-G3031), a histamine H3 receptor inverse agonist in animal models of sleep disorders

Abstract Introduction Narcolepsy is a chronic sleep disorder characterized by overwhelming daytime drowsiness, sudden attacks of sleep and sometimes accompanied by cataplexy. Although the orexin deficiency is considered to be the primary cause of this disorder, lot of attention has been diverted on targeting histaminergic neurotransmission by blockade of histamine H3 receptor (H3R). Samelisant (SUVN-G3031) is one of the potent and selective H3R inverse agonist currently being evaluated in a Phase 2 study as monotherapy for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). In the current research work, Samelisant was evaluated for neurotransmitter changes in rats and sleep EEG in orexin knockout mice, a reliable proof-of-concept study for treatment of excessive daytime sleepiness and cataplexy in narcolepsy. Methods Binding affinity of Samelisant towards human and rat histamine H3R was evaluated in in-vitro radioligand binding assay and functionality in GTP□S assay. Effect of Samelisant was studied in (R)-α-methyl histamine induced dipsogenia. In rat brain microdialysis, Samelisant was evaluated for its effects on modulation of neurotransmitters like histamine, dopamine and norepinephrine. Male orexin knockout mice were implanted with telemetric device for simultaneous monitoring of electroencephalography (EEG) and electromyography. Effects of Samelisant (3 and 10 mg/kg, p.o.) were evaluated during active period of animals. Results Samelisant is an inverse agonist at histamine H3 receptors with hKi of 8.7 nM and showed minimal binding against over 70 target sites. Samelisant produced significant increase in histamine, dopamine and norepinephrine levels in cortex. Samelisant produced no change in the striatal and accumbal dopamine levels in rats, suggesting no propensity to induce abuse liability. Samelisant blocked R-α-methyl histamine induced water intake and produced dose dependent increase in tele-methylhistamine levels in various brain regions and in cerebrospinal fluid of male Wistar rats. Samelisant produced significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleep in orexin knockout mice. Samelisant also significantly decreased number of cataplectic episodes in orexin knockout mice. Conclusion Samelisant is an inverse agonist at histamine H3 receptor and results from the preclinical studies presented here provide a strong evidence for the potential utility of Samelisant in the treatment of narcolepsy with and without cataplexy. Support (if any):

506 Samelisant: Baseline Characteristics from a Phase 2 Study Evaluating Efficacy and Safety in Patients with Narcolepsy

Abstract Introduction histamine H3 receptor (H3R) antagonists/ inverse agonists increase histaminergic neurotransmission and offer a therapeutic option for the treatment of narcolepsy. Samelisant (SUVN-G3031) is a potent and selective H3R inverse agonist exhibited selectivity over 70 other targets. Samelisant showed wake-promoting and anticataplectic effects in orexin knockout mice suggesting its potential therapeutic utility in the treatment of EDS and cataplexy associated with narcolepsy. Safety and tolerability studies in animals and healthy humans suggested a favorable risk/benefit profile. Methods The current study is a 2 week treatment, multicenter, double-blind, placebo controlled, parallel-group study in patients with Narcolepsy with or without Cataplexy. Eligibility criteria include age between 18 to 50 years old, an ESS score of ≥ 12; and mean MWT time of < 12 minutes and a confirm diagnosis of narcolepsy as per ICSD-3. Further, the randomization will be stratified according to type of narcolepsy (Type-1 or Type-2). Each subject will receive either placebo or study drug once daily for 2 weeks in a fixed ratio of 1:1:1. The primary efficacy endpoint is change in maintenance of wakefulness test (MWT) score from baseline to week 2. Key secondary endpoints include change from baseline to week 2 in ESS and an improvement in CGI-S scores. Safety will be monitored by medical monitor and by an independent data safety monitoring committee. Baseline clinical and demographic data for the currently enrolled study is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results As of data cutoff date of Dec 20, 2020, a total of 54 subjects were completed in the study. The median age of subjects was 30 years (range: 18 - 50 years) with mean BMI of 28.6 (range: 18.3 - 43.1 kg/m2). Overall, 74% of subjects were female and 83% were Caucasian. Mean (SD) baseline values of MWT and ESS are 5.65 (3.5) and 16.7 (2.5), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling the subjects with Narcolepsy with or without Cataplexy, and the Data readout is expected in the second half of 2021. Support (if any):

Orexin (hypocretin) participates in central autonomic regulation during fight-or-flight response

Our daily life does not only involve a calm resting state but is rather full of perturbations that induce active states such as moving, eating, and communicating. During such active conditions, cardiorespiratory regulation should be adjusted according to bodily demand, which differs from that during the resting state, by modulating or resetting the operating point. To explore neural mechanisms in the state-dependent adjustment of central autonomic regulation, my research group has recently focused on the fight-or-flight response because the stressor induces not only cognitive, emotional, and behavioral changes but also autonomic changes. In this brief review, I will summarize our discovery using orexin knockout mice and orexin neuron-ablated mice for the possible contribution of orexin, a hypothalamic neuropeptide, to the state-dependent adjustment of the central autonomic regulation. In addition, I will introduce some recent discovery using optogenetic manipulation of the orexin and related systems. The diversity of synaptic control of the cardiovascular and respiratory neurons appears necessary for animals to adapt themselves to ever-changing life circumstances and behavioral states. The orexin system is likely to function as one of the essential modulators for coordinating the circuits controlling autonomic functions and behaviors.

Induction of narcolepsy-like symptoms by orexin receptor antagonists in mice.

Orexins/hypocretins are hypothalamic neuropeptides that promote and stabilize wakefulness by binding to the orexin receptor type-1 (OX1R) and type-2 (OX2R). Disruption of orexinergic signaling results in the sleep disorder narcolepsy in mice, rats, dogs, and humans. The orexin receptor antagonist suvorexant promotes sleep by blocking both OX1R and OX2R. Whereas suvorexant has been clinically approved for the treatment of insomnia because it is well tolerated in experimental animals as well as in human patients, a logical question remains as to why orexin receptor antagonists do not induce overt narcolepsy-like symptoms. Here we show that acute and chronic suvorexant promotes both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep without inducing cataplexy in mice. Interestingly, chronic suvorexant increases OX2R mRNA and decreases orexin mRNA and peptide levels, which remain low long after termination of suvorexant administration. When mice are chronically treated with suvorexant and then re-challenged with the antagonist after a 1-week washout, however, cataplexy and sleep-onset REM (SOREM) are observed, which are exacerbated by chocolate administration. Heterozygous orexin knockout mice, with lower brain orexin levels, show cataplexy and SOREM after acute suvorexant administration. Furthermore, we find that acute suvorexant can induce cataplexy and SOREM in wild-type mice when co-administered with chocolate under stress-free (temporally anesthetized) conditions. Taken together, these results suggest that suvorexant can inhibit orexin synthesis resulting in susceptibility to narcolepsy-like symptoms in mice under certain conditions.

Samelisant (SUVN-G3031), a potent, selective and orally active histamine H3 receptor inverse agonist for the potential treatment of narcolepsy: pharmacological and neurochemical characterisation.

Samelisant (SUVN-G3031) is a potent and selective histamine H3 receptor (H3R) inverse agonist with good brain penetration and oral bioavailability. Pharmacological and neurochemical characterisation to support the utility of Samelisant (SUVN-G3031) in the treatment of sleep-related disorders like narcolepsy. Samelisant (SUVN-G3031) was tested in rat brain microdialysis studies for evaluation of modulation in histamine, dopamine and norepinephrine. Sleep EEG studies were carried out in orexin knockout mice to study the effects of Samelisant (SUVN-G3031) on the sleep-wake cycle and cataplexy. Samelisant (SUVN-G3031) has a similar binding affinity towards human (hH3R; Ki = 8.7 nM) and rat (rH3R; Ki = 9.8 nM) H3R indicating no inter-species differences. Samelisant (SUVN-G3031) displays inverse agonist activity and it exhibits very high selectivity towards H3R. Samelisant (SUVN-G3031) treatment in mice produced a dose-dependent increase in tele-methylhistamine levels indicating the activation of histaminergic neurotransmission. Apart from increasing the levels of histamine, Samelisant (SUVN-G3031) also modulates dopamine and norepinephrine levels in the cerebral cortex while it has no effects on dopamine levels in the striatum or nucleus accumbens. Treatment with Samelisant (SUVN-G3031; 10 and 30 mg/kg, p.o.) produced a significant increase in wakefulness with a concomitant decrease in NREM sleep in orexin knockout mice subjected to sleep EEG. Samelisant (SUVN-G3031) also produced a significant decrease in Direct REM sleep onset (DREM) episodes, demonstrating its anticataplectic effects in an animal model relevant to narcolepsy. Modulation in cortical levels of histamine, norepinephrine and dopamine provides the neurochemical basis for wake-promoting and anticataplectic effects observed in orexin knockout mice. Pre-clinical studies of Samelisant (SUVN-G3031) provide a strong support for utility in the treatment of sleep-related disorders related to EDS and is currently being evaluated in a phase 2 proof of concept study in the USA for the treatment of narcolepsy with and without cataplexy.

Autonomic mechanisms of blood pressure alterations during sleep in orexin/hypocretin-deficient narcoleptic mice.

Increase in arterial pressure (AP) during sleep and smaller differences in AP between sleep and wakefulness have been reported in orexin (hypocretin)-deficient mouse models of narcolepsy type 1 (NT1) and confirmed in NT1 patients. We tested whether these alterations are mediated by parasympathetic or sympathetic control of the heart and/or resistance vessels in an orexin-deficient mouse model of NT1. Thirteen orexin knock-out (ORX-KO) mice were compared with 12 congenic wild-type (WT) mice. The electroencephalogram, electromyogram, and AP of the mice were recorded in the light (rest) period during intraperitoneal infusion of atropine methyl nitrate, atenolol, or prazosin to block muscarinic cholinergic, β 1-adrenergic, or α 1-adrenergic receptors, respectively, while saline was infused as control. AP significantly depended on a three-way interaction among the mouse group (ORX-KO vs WT), the wake-sleep state, and the drug or vehicle infused. During the control vehicle infusion, ORX-KO had significantly higher AP values during REM sleep, smaller decreases in AP from wakefulness to either non-rapid-eye-movement (non-REM) sleep or REM sleep, and greater increases in AP from non-REM sleep to REM sleep compared to WT. These differences remained significant with atropine methyl nitrate, whereas they were abolished by prazosin and, except for the smaller AP decrease from wakefulness to REM sleep in ORX-KO, also by atenolol. Sleep-related alterations of AP due to orexin deficiency significantly depend on alterations in cardiovascular sympathetic control in a mouse model of NT1.

Enhancement of the rewarding effects of 3,4-methylenedioxymethamphetamine in orexin knockout mice

Orexinergic neurons, which are closely associated with narcolepsy, regulate arousal and reward circuits through the activation of monoaminergic neurons. Psychostimulants as well as 5-HT-related compounds have potential in the treatment of human narcolepsy. Previous studies have demonstrated that orexin receptor antagonists as well as orexin deficiencies affect the pharmacological effects of psychostimulants. However, little information is available on the consequences of psychostimulant use under orexin deficiency. Therefore, the present study was designed to investigate the abuse liability of psychostimulants in orexin knockout (KO) mice. In the present study, conditioned place preferences induced by methamphetamine and methylphenidate were not altered in orexin KO mice. Interestingly, we found that MDMA induced a conditioned place preference in orexin KO mice, but not in wild type (WT) mice. In addition, MDMA produced methylphenidate/methamphetamine-like discriminative stimulus effects in orexin KO mice, but not WT mice. Increases in 5-HT and dopamine release in the nucleus accumbens induced by MDMA were not altered by knockout of orexin; the steady-state level of G protein activation was higher in the limbic forebrain of orexin KO mice. In substitution tests using a drug discrimination procedure, substitution of 5-HT1A receptor agonist for the discriminative stimulus effects of methylphenidate was enhanced in orexin KO mice. These findings indicate that the orexinergic system is involved the rewarding effects of psychostimulants. However, there is a risk of establishing rewarding effects of psychostimulants even under orexin deficiency. On the other hand, deficiencies in orexin may enhance the abuse liability of MDMA by changing a postsynaptic signal transduction accompanied by changes in discriminative stimulus effects themselves.

0759 Phase 2 Proof Of Concept Study Of SUVN-G3031, A Histamine H3 Receptor Inverse Agonist For The Potential Treatment Of Narcolepsy

Abstract Introduction SUVN-G3031 is a potent inverse agonist at histamine H3 receptor (H3R) with selectivity over 70 other targets. SUVN-G3031 has excellent pharmacokinetics in rats and dogs. SUVN-G3031 demonstrated dose dependent receptor occupancy in rats with marked wake-promoting and anticataplectic effects in orexin knockout mice supporting its potential therapeutic utility in the treatment of narcolepsy. Long-term safety studies in animals and Phase 1 evaluation for safety, tolerability and pharmacokinetics demonstrated no concern for further development of SUVN-G3031. Methods SUVN-G3031 is currently being evaluated in a Phase 2 proof of concept study in USA for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). This is a double-blind, placebo-controlled, parallel-group, multicenter study to evaluate the safety, tolerability, pharmacokinetics and efficacy of SUVN-G3031 in comparison to placebo in patients with narcolepsy with and without cataplexy. Participants with an ESS score of ≥ 12; and mean MWT time of < 12 min are being randomized at a ratio of 1:1:1 to 2 mg SUVN-G3031, 4 mg SUVN-G3031 or placebo. Patients are to be stratified based on the type of narcolepsy. Each patient will receive study drug once daily for 14 days. The primary objective of the study is to evaluate the effectiveness of SUVN-G3031 compared to placebo as measured by an improvement in the maintenance of wakefulness test (MWT) score. Various secondary, exploratory, safety endpoints and steady state plasma concentrations will be evaluated. Results This study has been initiated in Q3 2019 and subject recruitment is expected to be completed by Q2 2020. Conclusion This study is a phase 2 clinical trial evaluating the efficacy and safety of SUVN-G3031 as a monotherapy in patients with narcolepsy with and without cataplexy. Safety and efficacy results from the study are expected in Q3 2020. Support None

0008 SUVN-G3031, A Histamine H3 Receptor Inverse Agonist Produces Robust Wake Promoting and Anticataplectic Activity in Orexin Knockout Mice

Abstract Introduction Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, sleep paralysis, hallucinations, and in some cases episodes of cataplexy. Results from animal studies indicate the involvement of deficient orexin transmission in narcolepsy which can be circumvented by the activation of histaminergic neurons. SUVN-G3031 is a potent and selective histamine H3 receptor inverse agonist with hKi of 8.7 nM and shows less than 50% inhibition at 1 µM against 70 other targets. SUVN-G3031 exhibited excellent pharmacokinetic properties and brain penetration in preclinical species. Oral administration of SUVN-G3031 produces significant increase in histamine, dopamine and norepinephrine levels in the rat cortex. Long-term safety studies in animals have been successfully completed without any concern for further development of SUVN-G3031. In the present study, the effects of SUVN-G3031 were evaluated in orexin knockout mice, a reliable animal model of narcolepsy as a proof-of-concept study for the treatment of narcolepsy with and without cataplexy. Methods Male orexin knockout mice (10 - 15 weeks old, 25 - 35 g at the time of surgery) were implanted with telemetric device for simultaneous monitoring of electroencephalography (EEG) and electromyography. Animals were allowed surgical recovery of 3 weeks prior to EEG recording. Effects of SUVN-G3031 (3 and 10 mg/kg, p.o.) were evaluated during active period of animals. Results SUVN-G3031 produced significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleep in orexin knockout mice. SUVN-G3031 also significantly decreased the number of cataplectic episodes in orexin knockout mice. Conclusion Results from the current preclinical study provide a strong basis for the utility of SUVN-G3031 for the treatment of narcolepsy with and without cataplexy. SUVN-G3031 is currently being evaluated in a Phase 2 study as monotherapy for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). Support None

The physiological signature of daily torpor is not orexin dependent.

Under conditions of scarce food availability and cool ambient temperature, the mouse (Mus Musculus) enters into torpor, a state of transient metabolic suppression mediated in part by the autonomic nervous system. Hypothalamic orexins are involved in the coordination of behaviors and autonomic function. We tested whether orexins are necessary for the coordinated changes in physiological variables, which underlie torpor and represent its physiological signature. We performed simultaneous measurements of brain temperature, electroencephalographic, and electromyographic activity allowing objective assessment of wake-sleep behavior, and cardiovascular, respiratory, and metabolic variables in orexin knockout mice (ORX-KO) and wild-type mice (WT) during torpor bouts elicited by caloric restriction and mild cold stress. We found that torpor bouts in WT are characterized by an exquisitely coordinated physiological signature. The characteristics of torpor bouts in terms of duration and rate of change of brain temperature and electromyographic activity at torpor entrance and exit did not differ significantly between ORX-KO and WT, and neither did the cardiovascular, respiratory, and metabolic characteristics of torpor. ORX-KO and WT also had similar wake-sleep state changes associated with torpor bouts, with the exception of a significantly higher rapid-eye movement sleep time in ORX-KO at torpor entrance. Our results demonstrate that orexins are not necessary either for the normal physiological adaptations occurring during torpor in mice or for their coordination, suggesting that mechanisms different from orexin peptide signaling may be involved in the regulation and the coordination of these physiological responses.

Role of hypothalamic orexin system in prevention of nonalcoholic steatohepatitis (NASH) in obese mice

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease induced by obesity. So far, no therapeutic drug is available against NASH, because the pathogenic mechanism remains unclear. Since hypothalamic orexin system is a main regulator of energy homeostasis, we investigated the role of orexin against NASH under obese conditions, using orexin knockout (ORX-KO) mice fed high fat diet (HFD). ORX-KO mice showed severer obesity and glucose intolerance on HFD, compared to wild-type controls. Also, remarkable NASH-like phenotypes were observed in the liver of ORX-KO mice, such as the accumulation of triglyceride and the increase in the levels of biomarkers for endoplasmic reticulum (ER) stress (phosphorylation of eIF2α, etc.), chronic inflammation (Tnfα mRNA, etc.), and hepatic fibrosis (Tgfβ mRNA, etc.). When the HFD-fed ORX-KO mice were treated with orexin A (i.c.v.), the hepatic ER stress and chronic inflammation were improved, whereas body weight was not altered. These results indicate that the central action of orexin is required to prevent the development of NASH by reducing ER stress and chronic inflammation in the liver under the obese condition. Hypothalamic orexin system may be a crucial therapeutic target to promote the brain-liver network functions for preventing the progression of NASH.

Effect of ambient temperature on sleep breathing phenotype in mice: the role of orexins.

The loss of orexinergic neurons, which release orexins, results in narcolepsy. Orexins participate in the regulation of many physiological functions, and their role as wake-promoting molecules has been widely described. Less is known about the involvement of orexins in body temperature and respiratory regulation. The aim of this study was to investigate if orexin peptides modulate respiratory regulation as a function of ambient temperature (Ta) during different sleep stages. Respiratory phenotype of male orexin knockout (KO-ORX, N=9) and wild-type (WT, N=8) mice was studied at thermoneutrality (Ta=30°C) or during mild cold exposure (Ta=20°C) inside a whole-body plethysmography chamber. The states of wakefulness (W), non-rapid eye movement sleep (NREMS) and rapid eye movement sleep (REMS) were scored non-invasively, using a previously validated technique. In both WT and KO-ORX mice, Ta strongly and significantly affected ventilatory period and minute ventilation values during NREMS and REMS; moreover, the occurrence rate of sleep apneas in NREMS was significantly reduced at Ta=20°C compared with Ta=30°C. Overall, there were no differences in respiratory regulation during sleep between WT and KO-ORX mice, except for sigh occurrence rate, which was significantly increased at Ta=20°C compared with Ta=30°C in WT mice, but not in KO-ORX mice. These results do not support a main role for orexin peptides in the temperature-dependent modulation of respiratory regulation during sleep. However, we showed that the occurrence rate of sleep apneas critically depends on Ta, without any significant effect of orexin peptides.

Involvement of orexin in lipid accumulation in the liver

ObjectivesOrexin, a hypothalamic neuropeptide, is involved in energy homeostasis and regulates motivated behaviors, including feeding and the awake-sleep cycle. Orexin-knockout (OX-KO) mice exhibit greater weight gain than wild-type (WT) mice, despite similar food intake, suggesting that OX-KO mice may have an altered metabolism for weight gain. However, the actual effects of orexin on the metabolism of mice remain unclear. MethodsWe compared the lipid metabolism of OX-KO and WT mice, with a focus on lipid metabolism in the liver. ResultsThe livers of OX-KO mice were significantly larger than those of WT mice and were yellowish in appearance. Oil red O staining revealed that lipid accumulation in the livers of OX-KO mice was higher than that in WT mice. Hepatic triglyceride content in OX-KO mice was higher than that in WT mice. The total cholesterol level in the peripheral blood was higher in OX-KO mice than in WT mice, although no significant differences in free fatty acid and serum triglyceride levels were observed. In comparison with WT mice, OX-KO mice showed higher transcript levels of hepatic lipogenic genes encoding sterol regulatory element binding protein 1 and Cd36 and lower transcript levels of genes encoding fatty acid transporter protein 2/5, peroxisomal proliferator-activated receptor α, STAT-induced suppressor of cytokine signaling 3, and hormone-sensitive lipase. Moreover, tumor necrosis factor (TNF) α mRNA levels in the liver were significantly higher in OX-KO mice than in WT mice. ConclusionsOrexin likely contributes to the regulation of lipid metabolism in the liver.