Daridorexant is a dual orexin receptor antagonist (DORA) approved by FDA and EMEA in 2022 for the treatment of insomnia in adults. As requested by regulatory agencies for all CNS‑active drug candidates, abuse potential of daridorexant was assessed in a well-established specific set of behavioral animal studies, including drug-discrimination. Regulatory authorities' recommendations require that new drugs in development are ideally compared to approved drugs with the same mechanism of action with known abuse liability. Accordingly, FDA advised conducting a rat drug discrimination paradigm against the approved, schedule IV, DORA suvorexant, although at the time of testing there was no published evidence that suvorexant induced drug discrimination. Twelve female Sprague Dawley rats were submitted to an operant drug discrimination learning paradigm with daily training sessions, randomly alternating oral treatments of suvorexant (firstly at 30 mg/kg, subsequently increased to 100) with vehicle. Rats did not learn to discriminate the effects of suvorexant from those of vehicle at doses reaching total plasma levels up to 3‑fold Cmax at the highest therapeutic human dose. Therefore, testing the discriminative stimulus effects of daridorexant against suvorexant was not possible. Alternatively, we tested the interoceptive similarity of daridorexant to the positive allosteric GABA-A receptor modulator zolpidem that was used as training drug and as positive control. Daridorexant plasma exposures were measured at different timepoints to verify they were in line with the expected target exposure. In addition, we tested the suvorexant and the benzodiazepine lorazepam for their discriminative stimulus properties in relation to zolpidem. Drugs were administered orally in a cross-over design. Results showed that both daridorexant and suvorexant did not generalize to zolpidem as rats treated with different doses of both compounds almost exclusively selected the vehicle-associated lever. As expected, lorazepam caused stimulus generalization to zolpidem. In conclusion, suvorexant failed to serve as a robust reference drug of abuse as rats could not be fully trained to discriminate suvorexant from vehicle. Daridorexant and suvorexant, both acting as DORA, did not evoke any similarity to zolpidem. In contrast, lorazepam fully generalized to zolpidem as expected due to their specificity of action on GABA-A receptors.

Sinbaglustat is a brain-penetrant iminosugar under clinical investigation for glycosphingolipid (GSL) storage disorders, including GM2 gangliosidosis. It inhibits non-lysosomal glucosylceramidase (GBA2) with higher potency than glucosylceramide synthase (GCS). While efficacy of related GBA2/GCS inhibitors in mouse models of Sandhoff disease was previously demonstrated, the specific contribution of GBA2 inhibition to therapeutic outcome has remained unclear. We dissected the mechanism of Sinbaglustat in Sandhoff Hexb -/- mice using 30 or 300 mg/kg/day doses, designed to preferentially inhibit GBA2 alone or both GBA2 and GCS, respectively. Sinbaglustat's dose-dependent effects on GSLs in relation to both drug targets were consistent across enzymatic assays, patient-derived cells, and wild-type mouse brain. In Hexb -/- mice, GBA2 inhibition alone was sufficient to alter central GSL metabolism, attenuate neuroinflammatory gene expression, delay onset of motor symptoms by ≥ 2 weeks, and extend survival by 15%. High-dose treatment broadened substrate clearance via GCS inhibition and extended survival by 22%. These results reveal a therapeutic role of GBA2 inhibition in the brain and highlight sinbaglustat, an iminosugar without gastrointestinal side effects, as a promising candidate for GM2 gangliosidosis. By defining the mechanistic contribution of its dual targets, this study offers insight for dose optimization and therapeutic design in lysosomal storage disorders.

OBJECTIVE:Test the efficacy of the selective orexin 1 receptor (OX1R) antagonist (SO1RA) nivasorexant in an animal model of binge-eating disorder (BED) and study its dose-response relationship considering free brain concentrations and calculated OX1R occupancy. Compare nivasorexant's profile to that of other, structurally diverse SO1RAs. Gain understanding of potential changes in orexin-A (OXA) neuropeptide and deltaFosB (ΔFosB) protein expression possibly underlying the development of the binge-eating phenotype in the rat model used. METHOD:Binge-like eating of highly palatable food (HPF) in rats was induced through priming by intermittent, repeated periods of dieting and access to HPF, followed by an additional challenge with acute stress. Effects of nivasorexant were compared to the SO1RAs ACT-335827 and IDOR-1104-2408. OXA expression in neurons and neuronal fibers as well as ΔFosB and OXA-ΔFosB co-expression was studied in relevant brain regions using immuno- or immunofluorescent histochemistry. RESULTS:All SO1RAs dose-dependently reduced binge-like eating with effect sizes comparable to the positive control topiramate, at unbound drug concentrations selectively blocking brain OX1Rs. Nivasorexant's efficacy was maintained upon chronic dosing and under conditions involving more frequent stress exposure. Priming for binge-like eating or nivasorexant treatment resulted in only minor changes in OXA or ΔFosB expression in few brain areas. DISCUSSION:Selective OX1R blockade reduced binge-like eating in rats. Neither ΔFosB nor OXA expression proved to be a useful classifier for their binge-eating phenotype. The current results formed the basis for a clinical phase II trial in BED, in which nivasorexant was unfortunately not efficacious compared with placebo. PUBLIC SIGNIFICANCE:Nivasorexant is a new investigational drug for the treatment of binge-eating disorder (BED). It underwent clinical testing in a phase II proof of concept trial in humans but was not efficacious compared with placebo. The current manuscript investigated the drug's efficacy in reducing binge-like eating behavior of a highly palatable sweet and fat diet in a rat model of BED, which initially laid the foundation for the clinical trial.

In rodents, orexin neuropeptides regulate motivation and reward-seeking via orexin 1 receptor (OX1R) signaling in the mesolimbic dopaminergic system. This role is clearly established for rewards inherent to drugs of abuse but less so for natural rewards. Reported effects of the selective OX1R antagonist (SO1RA) SB-334867 on motivation for palatable food are ambiguous. In our experimental conditions neither SB-334867, nor two additional, structurally different SO1RAs, ACT-335827 and the clinical development candidate nivasorexant, affected effort-based responding for sucrose in rats. The positive control lisdexamfetamine, approved for psychiatric disorders associated with altered reward sensitivity such as binge eating disorder, increased effort-based responding.

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.

Dual orexin receptor antagonists (DORAs) belong to a novel class of sleep medications that function by blocking the actions of wakefulness-promoting orexin neuropeptides in sleep-wake centers of the brain. Orexins also transmit signals to brain nuclei that regulate emotions and stress responses. The effects of DORAs on anxiety-like reactions requires further exploration. The hyperarousal theory of insomnia suggests an underlying overactivation of the body’s stress response systems, and a considerable proportion of insomnia patients suffers from concurrent anxiety disorders. Hence, it is important for physicians to be certain that novel insomnia treatments do not exacerbate, but rather alleviate, patients’ anxiety and psychological stress responses. Our aim was to test the effect of the new DORA daridorexant on diverse anxiety- and fear-evoked behavioral and physiological reactions in rats to gain first insight into the drug’s potential in humans. Daridorexant, given orally at 10, 30 and 100 mg/kg 1 h before testing, resulted in a dose-dependent reduction of fear-potentiated acoustic startle (FPS) reactions, schedule-induced polydipsia (SIP) and social stress-induced hyperthermia and tachycardia (SIH/T). Furthermore, under non-stressful, basal conditions, daridorexant also reduced heart rate in spontaneously hypertensive rats, which are a model of endogenous sympathetic hyperactivation that can occur in insomnia or anxiety disorders. Daridorexant had no effect on ultrasound-induced, panic-like, defensive behavior (UIDB). We conclude that daridorexant rather attenuated and did not enhance fear/stress responses in rats. It was effective in models that simulate endophenotypes that are specific for post-traumatic stress, obsessive-compulsive, and social anxiety disorder.

Despite progress in understanding the pathological mechanisms underlying psychiatric disorders, translation from animal models into clinical use remains a significant bottleneck. Preclinical studies have implicated the orexin neuropeptide system as a potential target for psychiatric disorders through its role in regulating emotional, cognitive, and behavioral processes. Clinical studies are investigating orexin modulation in addiction and mood disorders. Here we review performance-outcome measures (POMs) arising from experimental medicine research methods which may show promise as markers of efficacy of orexin receptor modulators in humans. POMs provide objective measures of brain function, complementing patient-reported or clinician-observed symptom evaluation, and aid the translation from preclinical to clinical research. Significant challenges include the development, validation, and operationalization of these measures. We suggest that collaborative networks comprising clinical practitioners, academics, individuals working in the pharmaceutical industry, drug regulators, patients, patient advocacy groups, and other relevant stakeholders may provide infrastructure to facilitate validation of experimental medicine approaches in translational research and in the implementation of these approaches in real-world clinical practice.

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.

Background: Venglustat is a brain-penetrant, small molecule inhibitor of glucosylceramide synthase used in clinical testing for treatment of Parkinson's disease (PD). Despite beneficial effects in certain cellular and rodent models, patients with PD with mutations in GBA, the gene for lysosomal glucocerebrosidase, experienced worsening of their motor function under venglustat treatment (NCT02906020, MOVES-PD, phase 2 trial). Objective: The objective of this study was to evaluate venglustat in mouse models of PD with overexpression of wild-type alpha-synuclein. Methods: Mice overexpressing alpha-synuclein (Thy1-aSyn line 61) or Gba-mutated mice with viral vector-induced overexpression of alpha-synuclein in the substantia nigra were administered venglustat as food admixture. Motor and cognitive performance, alpha-synuclein-related pathology, and microgliosis were compared with untreated controls. Results: Venglustat worsened motor function in Thy1-aSyn transgenics on the challenging beam and the pole test. Although venglustat did not alter the cognitive deficit in the Y-maze test, it alleviated anxiety-related behavior in the novel object recognition test. Venglustat reduced soluble and membrane-bound alpha-synuclein in the striatum and phosphorylated alpha-synuclein in limbic brain regions. Although venglustat reversed the loss of parvalbumin immunoreactivity in the basolateral amygdala, it tended to increase microgliosis and phosphorylated alpha-synuclein in the substantia nigra. Furthermore, venglustat also partially worsened motor performance and tended to increase neurofilament light chain in the cerebrospinal fluid in the Gbadeficient model with nigral alpha-synuclein overexpression and neurodegeneration. Conclusions: Venglustat treatment in two mouse models of alpha-synuclein overexpression showed that glucosylceramide synthase inhibition had differential detrimental or beneficial effects on behavior and neuropathology possibly related to brain region-specific effects. (c) 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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.

IntroductionPeople with dementia (PwD) often present with neuropsychiatric symptoms (NPS). NPS are of substantial burden to the patients, and current treatment options are unsatisfactory. Investigators searching for novel medications need animal models that present disease-relevant phenotypes and can be used for drug screening. The Senescence Accelerated Mouse-Prone 8 (SAMP8) strain shows an accelerated aging phenotype associated with neurodegeneration and cognitive decline. Its behavioural phenotype in relation to NPS has not yet been thoroughly investigated. Physical and verbal aggression in reaction to the external environment (e.g., interaction with the caregiver) is one of the most prevalent and debilitating NPS occurring in PwD. Reactive aggression can be studied in male mice using the Resident-Intruder (R-I) test. SAMP8 mice are known to be more aggressive than the Senescence Accelerated Mouse-Resistant 1 (SAMR1) control strain at specific ages, but the development of the aggressive phenotype over time, is still unknown. MethodsIn our study, we performed a longitudinal, within-subject, assessment of aggressive behaviour of male SAMP8 and SAMR1 mice at 4, 5, 6 and 7 months of age. Aggressive behaviour from video recordings of the R-I sessions was analysed using an in-house developed behaviour recognition software. ResultsSAMP8 mice were more aggressive relative to SAMR1 mice starting at 5 months of age, and the phenotype was still present at 7 months of age. Treatment with risperidone (an antipsychotic frequently used to treat agitation in clinical practice) reduced aggression in both strains. In a three-chamber social interaction test, SAMP8 mice also interacted more fervently with male mice than SAMR1, possibly because of their aggression-seeking phenotype. They did not show any social withdrawal. DiscussionOur data support the notion that SAMP8 mice might be a useful preclinical tool to identify novel treatment options for CNS disorders associated with raised levels of reactive aggression such as dementia.

Agitation, which comprises verbal or physical aggression and hyperactivity, is one of the most frequent neuropsychiatric symptoms observed in patients with Alzheimer's disease (AD). It often co-occurs with dysregulated circadian rhythms. Current medications are associated with serious adverse effects, and novel therapeutics are therefore needed. Rodent models can be instrumental to provide a first signal for potential efficacy of novel drug candidates. Longitudinal data assessing the face validity of such models for AD-related agitation are largely missing. We employed telemeterized APPswe mice, a frequently used AD transgenic mouse line overexpressing the human beta-amyloid precursor protein (APP) with the Swedish KM670/671NL mutation, to study the occurrence and progression of changes in reactive aggressive behavior as well as the circadian profile of locomotor activity and body temperature. Analysis was conducted between 5 and 11 months of age, at regular 2-months intervals. The aggressivity of all mice was highest at 5 months and waned with increasing age. APPswe mice were more aggressive than WT at 5 and 7 months of age. The locomotor activity and body temperature of WT mice declined with increasing age, while that of APPswe mice remained rather constant. This genotype difference was solely evident during the active, dark phase. APPswe mice did not display a phase shift of their circadian rhythms. We conclude that the APPswe mouse line can recapitulate some of the behavioral disturbances observed in AD, including an agitation-relevant phenotype characterized by active phase hyperactivity and aggressivity. It does not recapitulate the nighttime disturbances (also characterized by hyperactivity) and the shift of circadian rhythms observed in AD patients. Therefore, the APPswe strain could be used at specific ages to model a subset of agitation-relevant behavioral problems and to test the modulatory effects of drugs.

Neuropsychiatric symptoms (NPS) affect people with dementia (PwD) almost universally across all stages of the disease, and regardless of its exact etiology. NPS lead to disability and reduced quality of life of PwD and their caregivers. NPS include hyperactivity (agitation and irritability), affective problems (anxiety and depression), psychosis (delusions and hallucinations), apathy, and sleep disturbances. Preclinical studies have shown that the orexin neuropeptide system modulates arousal and a wide range of behaviors via a network of axons projecting from the hypothalamus throughout almost the entire brain to multiple, even distant, regions. Orexin neurons integrate different types of incoming information (e.g., metabolic, circadian, sensory, emotional) and convert them into the required behavioral output coupled to the necessary arousal status. Here we present an overview of the behavioral domains influenced by the orexin system that may be relevant for the expression of some critical NPS in PwD. We also hypothesize on the potential effects of pharmacological interference with the orexin system in the context of NPS in PwD.

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

Abstract Introduction The ability to be fast alert and to interact with the environment without motor impairment upon waking up, is a critical feature of natural sleep. DORAs represent a new class of insomnia medications that specifically inhibit the wake-promoting effects of orexin neuropeptides. Daridorexant is a potent and selective DORA under late stage development for the treatment of insomnia. Here, we assessed the impact of sleep-promoting doses of daridorexant on rats’ and dogs’ behaviour upon forced awakening. Zolpidem (a positive GABAA receptor modulator) was used as active comparator in rats because of its known negative impact on motor functions. Methods Rats were woken up at different time points after oral administration of daridorexant (10, 30, 100 mg/kg) or zolpidem (30, 100 mg/kg) during their inactive phase, and repeatedly subjected to two motor tasks: 1) the rotating rod test (lasting 120 sec, at each time point) assessing gross motor skills and coordination, and 2) the forepaw grip strength test assessing fine motor skills and muscle strength. Dogs were presented with food as an external, salient stimulus, three hours after administration of daridorexant in gelatin capsules (10, 30 or 90 mg/dog) during their active phase. Behaviour and signs of muscle weakness, after having woken up, were assessed by manual inspection of video recordings and concomitant electroencephalogram/electromyogram recordings. Results In both the rotarod and grip tests, daridorexant treatment had no effect on motor behavior at any dose or time point tested, while zolpidem significantly reduced the time spent on the rotarod and the grip strength in a dose and time-dependent manner (N=12/group; p<0.001;) (e.g. at 30 min post-dose, time spent on the rotarod was 84, 79–89 and 10–19 sec for vehicle, daridorexant and zolpidem, respectively). Dogs treated with daridorexant were able to wake up easily upon food presentation. They behaved and ate normally and did not show any signs of muscle weakness. Conclusion The type of sleep promoted by daridorexant is surmountable in rats and dogs and similar to physiological sleep. It allows animals to easily wake up, to behave normally without motor impairment and to respond efficiently to the environmental conditions. Support (if any) Funded by Idorsia Pharmaceuticals Ltd

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.