Discovery of Nivasorexant (ACT-539313): The First Selective Orexin-1 Receptor Antagonist (SO1RA) Investigated in Clinical Trials.

The orexin system consists of two neuropeptides (orexins A and B) and two receptors (OX1 and OX2). Selective OX1 receptor antagonists (SO1RA) are gaining interest for their potential use in the treatment of CNS disorders, including substance abuse, eating, obsessive compulsive, or anxiety disorders. While blocking OX2 reduces wakefulness, the expected advantage of selectively antagonizing OX1 is the ability to achieve clinical efficacy without the promotion of sleep. Herein we report our discovery efforts starting from a dual orexin receptor antagonist and describe a serendipitous finding that triggered a medicinal chemistry program that culminated in the identification of the potent SO1RA ACT-539313. Efficacy in a rat model of schedule-induced polydipsia supported the decision to select the compound as a preclinical candidate. Nivasorexant (20) represents the first SO1RA to enter clinical development and completed a first proof of concept phase II clinical trial in binge eating disorder in 2022.

Abuse potential assessment of the dual orexin receptor antagonist daridorexant in rats.

BACKGROUND: Drugs that act on the central nervous system (CNS) and have sedative effects can lead to abuse in humans. New CNS-active drugs often require evaluation of their abuse potential in dedicated animal models before marketing approval. Daridorexant is a new dual orexin receptor antagonist (DORA) with sleep-promoting properties in animals and humans. It was approved in 2022 in the United States and Europe for the treatment of insomnia disorder. AIMS: Nonclinical evaluation of abuse potential of daridorexant using three specific rat models assessing reinforcement, interoception, and withdrawal. METHODS: Reinforcing effects of daridorexant were assessed in an operant rat model of intravenous drug self-administration. Similarity of interoceptive effects to those of the commonly used sleep medication zolpidem was tested in an operant drug discrimination task. Withdrawal signs indicative of physical dependence were evaluated upon sudden termination of chronic daridorexant treatment. Rat experiments were conducted at a dose range resulting in daridorexant plasma concentrations equaling or exceeding those achieved at the clinically recommended dose of 50 mg in humans. RESULTS: Daridorexant had no reinforcing effects, was dissimilar to zolpidem in the drug discrimination task, and did not induce any withdrawal-related signs upon treatment discontinuation that would be indicative of physical dependence. OUTCOMES: Daridorexant showed no signs of abuse or dependence potential in rats. Our data indicate that daridorexant, like other DORAs, has a low potential for abuse in humans.

Failure of the dual orexin receptor antagonist suvorexant to engender drug discrimination in rats.

For abuse potential assessment, U.S. Food and Drug Administration (FDA) requests that new, brain-penetrating drugs are ideally compared with approved drugs that share the mechanism of action and are judged to have abuse liability by the Drug Enforcement Agency. For development of the dual orexin receptor antagonist (DORA) daridorexant, the FDA recommended conducting a rat drug discrimination paradigm against the approved, schedule IV, DORA suvorexant. Surprisingly, at suvorexant plasma levels up to three-fold the maximum concentration at the highest approved human dose, rats did not learn to discriminate the suvorexant stimulus from vehicle.

Nonclinical pharmacology of daridorexant: a new dual orexin receptor antagonist for the treatment of insomnia.

Dual orexin receptor antagonists (DORAs) represent a novel type of sleep medication that provide an alternative to the traditionally used positive allosteric gamma-aminobutyric acid (GABA)-A receptor modulators. Daridorexant is a new DORA that exhibited in phase 3 trials in insomnia not only a beneficial effect on sleep variables, measured objectively and assessed subjectively, but also an improvement in daytime functioning. Daridorexant was discovered through a tailored research program aimed at identifying an optimized sleep-promoting molecule with pharmacokinetic properties appropriate for covering the whole night while avoiding next-morning residual activity at efficacious doses. By specific binding to both orexin receptors, daridorexant inhibits the actions of the wake-promoting orexin (also called hypocretin) neuropeptides. This mechanism avoids a more widespread inhibition of neuronal pathways and associated side effects that are intrinsic to positive allosteric GABA-A receptor modulators. Here, we review the general pharmacology of daridorexant, based on nonclinical pharmacology studies of daridorexant, unpublished or already described, or based on work with other DORAs. Some unique features of daridorexant will be highlighted, such as the promotion of natural and surmountable sleep, the preservation of memory and cognition, the absence of tolerance development or risk of physical dependence, and how it can benefit daytime functioning.

Orexin neuropeptides contribute to the development and persistence of generalized avoidance behavior in the rat.

RATIONALE: Avoidance of contexts directly associated with fearful experiences represents an adaptive behavioral survival strategy. Over-interpretation of contextual cues leading to generalized avoidance of situations that are only remotely similar to the original fear context represents a pathologic process that contributes to anxiety disorders. Orexin neuropeptides modulate anxiety-like behavioral and physiological responses. OBJECTIVE: The objective of this paper was to investigate the impact of pharmacological orexin receptor blockade on generalized avoidance behavior. METHODS: Rats received a single electric foot-shock in the dark side of a two-compartment shuttle box followed by situational context reminders. After shock, rats were treated chronically (3 weeks) with the orexin receptor antagonist almorexant or with the selective serotonin reuptake inhibitor sertraline, used as positive anxiolytic control. In week 3, avoidance behavior was measured under conditions of high (dark-light (DL)-box) and low (elevated plus maze (EPM)) similarity to the original shock context. Avoidance behavior was re-assessed 5 and 17 weeks after treatment termination. RESULTS: Avoidance in the DL box (contextual fear memory) remained unaffected by any treatment and lasted 20 weeks post-shock exposure. Avoidance in the EPM (neophobic fear generalization) was partially attenuated during treatment with almorexant and sertraline at week 3. Following 5 and 17 weeks of drug washout, avoidance in the EPM was significantly reduced in almorexant- but not in sertraline-treated rats. Almorexant also reduced persistent avoidance in the EPM upon treatment initiation 3 weeks after shock exposure. CONCLUSION: Chronic orexin receptor blockade in rats reduces both the development and persistence of generalized avoidance in situations with low similarity to the initial shock context.

Structure-activity relationship, biological, and pharmacological characterization of the proline sulfonamide ACT-462206: a potent, brain-penetrant dual orexin 1/orexin 2 receptor antagonist.

The orexin system consists of two G-protein-coupled receptors, the orexin 1 and orexin 2 receptors, widely expressed in diverse regions of the brain, and two peptide agonists, orexin A and orexin B, which are produced in a small assembly of neurons in the lateral hypothalamus. The orexin system plays an important role in the maintenance of wakefulness. Several compounds (almorexant, SB-649868, suvorexant) have been in advanced clinical trials for treating primary insomnia. ACT-462206 is a new, potent, and selective dual orexin receptor antagonist (DORA) that inhibits the stimulating effects of the orexin peptides at both the orexin 1 and 2 receptors. It decreases wakefulness and increases non-rapid eye movement (non-REM) and REM sleep while maintaining natural sleep architectures in rat and dog electroencephalography/electromyography (EEG/EMG) experiments. ACT-462206 shows anxiolytic-like properties in rats without affecting cognition and motor function. It is therefore a potential candidate for the treatment of insomnia.

Discovery and characterization of ACT-335827, an orally available, brain penetrant orexin receptor type 1 selective antagonist.

Stress relief: Orexin neuropeptides regulate arousal and stress processing through orexin receptor type 1 (OXR-1) and 2 (OXR-2) signaling. A selective OXR-1 antagonist, represented by a phenylglycine-amide substituted tetrahydropapaverine derivative (ACT-335827), is described that is orally available, penetrates the brain, and decreases fear, compulsive behaviors and autonomic stress reactions in rats.

Examining the role of endogenous orexins in hypothalamus-pituitary-adrenal axis endocrine function using transient dual orexin receptor antagonism in the rat.

The orexin neuropeptide system regulates wakefulness and contributes to physiological and behavioral stress responses. Moreover, a role for orexins in modulating hypothalamus-pituitary-adrenal (HPA) axis activity has been proposed. Brain penetrating dual orexin receptor (OXR) antagonists such as almorexant decrease vigilance and have emerged as a novel therapeutic class for the treatment of insomnia. Almorexant was used here as a pharmacological tool to examine the role of endogenous orexin signaling in HPA axis endocrine function under natural conditions. After confirming the expression of prepro-orexin and OXR-1 and OXR-2 mRNA in hypothalamus, pituitary and adrenal glands, the effects of systemic almorexant were investigated on peripheral HPA axis hormone release in the rat under baseline, stress and pharmacological challenge conditions. Almorexant did not alter basal or stress-induced corticosterone release despite affecting wake and sleep stages (detected by radiotelemetric electroencephalography/electromyography) during the stress exposure. Moreover, almorexant did not affect the release of adrenocorticotropin (ACTH) and corticosterone at different time points along the diurnal rhythm, nor corticotrophin-releasing hormone (CRH)- and ACTH-stimulated neuroendocrine responses, measured in vivo under stress-free conditions. These results illustrate that dual OXR antagonists, despite modulating stress-induced wakefulness, do not interfere with endocrine HPA axis function in the rat. They converge to suggest that endogenous orexin signaling plays a minor role in stress hormone release under basal conditions and under challenge.

The selective orexin receptor 1 antagonist ACT-335827 in a rat model of diet-induced obesity associated with metabolic syndrome.

The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1) in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF) diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO) associated with metabolic syndrome (MetS). Rats were fed either standard chow (SC) or a cafeteria (CAF) diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S) diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks) and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure) were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% vs. controls) and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG) plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this model.

Sex comparison on long-lasting behavioral and physiological disturbances induced by single shock experience in rats.

Enhanced female vulnerability and symptom severity are described in post-traumatic stress disorder (PTSD). It remains largely unknown whether females present with more pronounced PTSD-like symptoms than males in rodent models of PTSD. A model of single electric foot-shock followed by situational reminders was used to investigate in rats the impact of sex on potential long-lasting changes in anxiety-like behavior, and in endocrine and physiological responses to stress and fearful situations. Three weeks after single shock exposure (in the dark side of a shuttle box) both male and female rats spent less time in the dark compartment of a dark-light box and in the closed arms of an elevated plus maze than non-shocked controls. Both behaviors were likely due to avoidance of places reminiscent of the initial shock context. The shock exposure had no long-term impact on social interaction behavior or on basal and restraint stress-induced increases in plasma corticosterone. Shock exposure increased sudden silence-induced freezing responses and hyperthermia during novelty stress and fear to a similar extent in both males and females and left heart rate responses unaffected. Non-shocked females generally showed a reduced response or faster recovery to baseline under stress- or fearful test conditions as compared to non-shocked males, which was likely due to differential sex-specific coping strategies. Taken together, our results suggest that, despite some baseline sex differences, both male and female rats are similarly affected in the long-term by the initial foot-shock exposure used in this particular simulation of PTSD.

The brain orexin system and almorexant in fear-conditioned startle reactions in the rat.

RATIONALE: The rat fear-potentiated startle (FPS) paradigm is a translational model of conditioned fear involving central amygdala pathways of the brain. Hypothalamic orexin neurons have input-output projections to the amygdala; they modulate vigilance and stress-related responses. OBJECTIVE: To investigate whether the transient pharmacological blockade of orexin receptors moderates the conditioned fear response. METHODS: F344 rats received acute oral treatment with the dual orexin receptor antagonist almorexant (30-300 mg/kg) or with one of the clinically effective anxiolytics diazepam (1-10 mg/kg), buspirone (10-100 mg/kg), fluoxetine (3-30 mg/kg), and sertraline (10-100 mg/kg). Drug effects on startle responses were assessed in both fear- and non-fear-conditioned rats; on forepaw grip and horizontal wire motor performance, and on elevated plus maze (EPM) behavior. RESULTS: Diazepam and almorexant both dose-dependently decreased FPS in the presence of the fear-conditioned stimulus (CS; light) more prominently than background startle in absence of the CS (dark). Diazepam induced myorelaxation and reduced startle responses in control non-fear-conditioned rats. Almorexant had no myorelaxant effects and left startle responses under light in non-fear-conditioned rats intact. On the EPM, diazepam showed anxiolytic-like effects, almorexant not. Buspirone demonstrated anxiolytic-like effects on FPS by simultaneously reducing CS-related startle and increasing no-CS-background startle. Fluoxetine did not affect FPS, whereas sertraline showed anxiogenic-like effects. CONCLUSIONS: Almorexant reduced FPS, but did not affect EPM behavior. Almorexant's overall pattern of effects on FPS was comparable to but less pronounced than that of the anxiolytic benzodiazepine diazepam. The endogenous orexin system actively contributes to fear-conditioned startle reactions in the rat.

Favoured genetic background for testing anxiolytics in the fear-potentiated and light-enhanced startle paradigms in the rat.

The fear-potentiated startle (FPS) and the light-enhanced startle (LES) paradigms are rodent tests of fear and anxiety, which combine face validity with predictive validity for clinically effective anxiolytic drugs. However, systematic strain comparisons aimed at identifying a rat strain that shows robust and reliable fear and anxiety responses in both models are missing. Here, we investigated four commonly used laboratory rat strains: Wistar, Sprague Dawley, Long-Evans and F344. Following strong cued fear conditioning training [60 conditioned stimulus-unconditioned stimulus (CS-US) pairings], all strains except Wistar exhibited significant FPS responses. F344 rats showed the strongest FPS response. Following milder cued fear conditioning protocols, designed to reduce the underlying component of contextual fear conditioning (by context pre-exposure or less CS-US pairings), also Wistar rats were able to show significant FPS, albeit still to a lesser extent than F344 rats tested under identical conditions. When tested in the LES protocol (light intensity ∼ 1500 lx), all strains except Long-Evans displayed significant light-enhanced startle responses. F344 and Wistar showed the strongest LES responses, which were of similar magnitude. The most sensitive strain in both paradigms, F344, was chosen for further pharmacological validation. The clinically active anxiolytic alprazolam (0.3, 1, 3mg/kg p.o.) dose-dependently reduced both fear-like responses in the FPS paradigm and anxiety-like responses in the LES paradigm at non-myorelaxant dosages. We propose that the F344 rat strain is particularly suited for the predictability assessment of novel anxiolytic drugs in both startle paradigms.

Differential effects of the dual orexin receptor antagonist almorexant and the GABA(A)-α1 receptor modulator zolpidem, alone or combined with ethanol, on motor performance in the rat.

Current insomnia treatments such as γ-aminobutyric acid (GABA) receptor modulators are associated with sedative and muscle-relaxant effects, which increase when drug intake is combined with alcohol. This study compared the novel sleep-enabling compound almorexant (ACT-078573-hydrochloride), a dual orexin receptor antagonist, with the positive GABA(A)-α1 receptor modulator zolpidem. Both compounds were administered alone or in combination with ethanol, and their effects on forced motor performance were determined in Wistar rats upon waking after treatment. To detect substance-induced sedation and myorelaxation, time spent on an accelerating rotating rod (rotarod) and forepaw grip strength were measured. Zolpidem (10, 30, and 100 mg/kg, p.o.) and ethanol (0.32, 1, and 1.5 g/kg, i.p.) dose-dependently decreased rotarod performance and grip strength, whereas almorexant (30, 100, and 300 mg/kg, p.o.) did not. Doses of ethanol (0.32 and 1 g/kg), which were ineffective when administered alone, showed interactions with zolpidem (10 and 30 mg/kg) leading to reduced rotarod performance and grip strength; in contrast, combination of ethanol (0.32 and 1 g/kg) with almorexant (100 and 300 mg/kg) did not reduce performance or grip strength below ethanol alone. We conclude that unlike zolpidem, almorexant does not interfere with forced motor performance or grip strength in the rat, nor does it further increase the sedative effects of ethanol. Our results suggest that the effect of almorexant can be immediately reversed to full alertness like under physiological sleep, and that almorexant is less likely to show strong sedation, excessive myorelaxation, or interaction with alcohol than commonly prescribed hypnotics such as zolpidem.

DeltaFosB in brain reward circuits mediates resilience to stress and antidepressant responses.

In contrast with the many studies of stress effects on the brain, relatively little is known about the molecular mechanisms of resilience, the ability of some individuals to escape the deleterious effects of stress. We found that the transcription factor DeltaFosB mediates an essential mechanism of resilience in mice. Induction of DeltaFosB in the nucleus accumbens, an important brain reward-associated region, in response to chronic social defeat stress was both necessary and sufficient for resilience. DeltaFosB induction was also required for the standard antidepressant fluoxetine to reverse behavioral pathology induced by social defeat. DeltaFosB produced these effects through induction of the GluR2 AMPA glutamate receptor subunit, which decreased the responsiveness of nucleus accumbens neurons to glutamate, and through other synaptic proteins. Together, these findings establish a previously unknown molecular pathway underlying both resilience and antidepressant action.

Role of the endocannabinoid system in regulation of the hypothalamic-pituitary-adrenocortical axis.

The endocannabinoid system has been recognized as a major neuromodulatory system, which functions to maintain brain homoeostasis. Endocannabinoids are synthesized and released from the postsynapse and act as retrograde neuronal messengers, which bind to cannabinoid type 1 receptors at the presynapse. Here, they inhibit the release of neurotransmitters, including glutamate and GABA. By these means, endocannabinoids control the activation of various neuronal circuits including those involved in neuroendocrine stress processing. Accordingly, exogenous cannabinoids such as the major active component of marijuana, Delta(9)-tetrahydrocannabinol, have long been known to activate the major neuroendocrine stress response system of mammals, the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the function of the endocannabinoid system in the regulation of stress hormone secretion has only recently begun to be understood. It is the focus of the present review to provide the reader with an overview of our current knowledge of the role of endocannabinoid signalling in HPA axis regulation under basal as well as under stressful conditions. This includes the specific sites of action, potential underlying neuronal pathways and interactions between behavioural and neuroendocrine stress coping. Furthermore, the potential role of HPA axis activity dysregulations, caused by deficits in the endocannabinoid system, for the pathophysiology of psychiatric diseases is discussed.

Conditional cannabinoid receptor type 1 mutants reveal neuron subpopulation-specific effects on behavioral and neuroendocrine stress responses.

The complete genetic loss or pharmacological blockade of cannabinoid receptor type 1 (CB1) in mice results in both altered behavioral performance and increased stress hormone secretion in response to stressful encounters such as forced swim test (FST) exposure. CB1 is expressed on nerve terminals belonging to different neurotransmitter systems, including the glutamatergic and GABAergic system, where it is able to suppress excitatory and inhibitory neurotransmission, respectively. In the current study, we used the conditional mutagenesis approach in mice to investigate the neurotransmitter systems involved in these behavioral and neuroendocrine phenotypes in regard to CB1 signaling. Mice lacking CB1 in cortical glutamatergic neurons (Glu-CB1(-/-)) showed decreased passive stress coping (decreased immobility) in the FST, whereas mice lacking CB1 in principal forebrain neurons (CaMK-CB1(-/-)) and GABAergic neurons (GABA-CB1(-/-)), respectively, behaved as littermate controls. However, we found increased FST-induced corticosterone secretion only in CaMK-CB1(-/-) mice, whereas Glu-CB1(-/-) and GABA-CB1(-/-) mice exhibited normal corticosterone release as compared to controls. Thus, behavioral and neuroendocrine acute stress coping in response to the FST is mainly influenced by CB1 signaling on different glutamatergic neuronal subpopulations, but not by CB1 on GABAergic neurons.

Increased water temperature renders single-housed C57BL/6J mice susceptible to antidepressant treatment in the forced swim test.

To investigate genotype x environment interactions in the forced swim test, we tested the influence of water temperature (20 degrees C, 25 degrees C, 30 degrees C) on floating behaviour in single-housed male C57BL/6J and BALB/c mice. We observed a contrasting relationship between floating and water temperature between the two strains, with C57BL/6J floating more and BALB/c floating less with increasing water temperature, independent of the lightening conditions and the time point of testing during the animals' circadian rhythm. Both strains showed an inverse relationship between plasma corticosterone concentration and water temperature, indicating that the differences in stress coping are unrelated to different perception of the aversive encounter. Treatment with desipramine (20mg/kg, i.p.) caused a reduction in immobility time in C57BL/6J mice if the animals were tested at 30 degrees C water temperature, with no effect at 25 degrees C and no effects on forced swim stress-induced corticosterone secretion. The same treatment failed to affect floating behaviour in BALB/c at any temperature, but caused a decrease in plasma corticosterone levels. Taken together we demonstrate that an increase in water temperature in the forced swim test exerts opposite effects on floating behaviour in C57BL/6J and BALB/c and renders single-housed C57BL/6J mice, but not BALB/c mice, susceptible to antidepressant-like behavioral effects of desipramine.

Antidepressant-like behavioral effects of impaired cannabinoid receptor type 1 signaling coincide with exaggerated corticosterone secretion in mice.

Hypothalamic-pituitary-adrenocortical (HPA) axis hyperactivity is associated with major depressive disorders, and treatment with classical antidepressants ameliorates not only psychopathological symptoms, but also the dysregulation of the HPA axis. Here, we further elucidated the role of impaired cannabinoid type 1 receptor (CB1) signaling for neuroendocrine and behavioral stress coping in the mouse forced swim test (FST). We demonstrate that the genetic inactivation of CB1 is accompanied by increased plasma corticosterone levels both under basal conditions and at different time points following exposure to the FST. The latter effect could be mimicked in C57BL/6N mice by acute, subchronic, and chronic administration of the selective CB1 antagonist SR141716. Further experiments confirmed the specificity of corticosterone-elevating SR141716 actions for CB1 in CB1-deficient mice. Subchronic and chronic pharmacological blockade of CB1, but not its genetic deletion, induced antidepressant-like behavioral responses in the FST that were characterized by decreased floating and/or increased struggling behavior. The antidepressant-like behavioral effects of acute desipramine treatment in the FST were absent in CB1-deficient mice, but the dampening effects of desipramine on FST stress-induced corticosterone secretion were not compromised by CB1 deficiency. However, antidepressant-like behavioral desipramine effects were intact in C57BL/6N mice pre-treated with SR141716, indicating potential developmental deficits in CB1-deficient mice. We conclude that pharmacological blockade of CB1 signaling shares antidepressant-like behavioral effects with desipramine, but reveals opposite effects on HPA axis activity.