Stimulation of the M4 muscarinic acetylcholine receptor reduces striatal hyperdopaminergia, suggesting its potential as a therapeutic target for schizophrenia. Emraclidine (CVL-231) is a novel, highly selective, positive allosteric modulator (PAM) of M4 muscarinic acetylcholine receptors i.e. acts as a modulator that increases the response of these receptors. First, we aimed to further characterize the positron emission tomography (PET) imaging and quantification performance of a recently developed M4 PAM radiotracer, [11C]MK-6884, in non-human primates (NHPs). Second, we applied these results to determine the receptor occupancy of CVL-231 as a function of dose. Using paired baseline-blocking PET scans, we quantified total volume of distribution, binding potential, and receptor occupancy. Both blood-based and reference region-based methods quantified M4 receptor levels across brain regions. The 2-tissue 4-parameter kinetic model best fitted regional [11C]MK-6884-time activity curves. Only the caudate nucleus and putamen displayed statistically significant [11C]MK-6884 uptake and dose-dependent blocking by CVL-231. For binding potential and receptor occupancy quantification, the simplified reference tissue model using the grey cerebellum as a reference region was employed. CVL-231 demonstrated dose-dependent M4 receptor occupancy in the striatum of the NHP brain and shows promise for further development in clinical trials.

Objective: To evaluate the panicolytic effect of 2 dose strengths of darigabat in reducing panic and fear symptoms following carbon dioxide (CO2) inhalation by healthy participants. Background: Panic disorder is associated with subjective and physiological symptoms which no single drug class adequately addresses. Darigabat, in development for neurological and psychiatric disorders, was designed to have nonsedative panicolytic potential by selectively enhancing the effect of GABA at α2/3/5, while sparing activity at α1 GABAA receptor subtypes. Design/Methods: This phase 1 randomized, double-blind, crossover, placebo- and active-controlled trial (NCT04592536) enrolled adults sensitive to anxiogenic effects of 35% CO2 double-breath inhalation. Participants were randomized to receive placebo and either 1 of 3 active treatments in 3 separate cohorts for 8 days: darigabat 7.5 or 25 mg twice daily (BID) or alprazolam 1 mg BID. Target darigabat doses were achieved following a 4-day titration. After each crossover period, CO2 challenge was performed 3 hours post-dose. Panic and fear symptoms were measured before and immediately after CO2 inhalation using the Panic Symptom List-IV total score (PSL-IV; primary endpoint) and fear visual analog scale (VAS Fear; secondary endpoint). Each participant's placebo treatment served as their own control. Results: Fifty-six participants were randomized. On Day 8, darigabat 7.5-mg and 25-mg BID groups demonstrated a 3.9-point (nominal P=0.036) and 4.5-point (nominal P=0.008) improvement on the PSL-IV versus placebo, respectively. Darigabat groups demonstrated a 12.8-point (nominal P=0.026) and 7.8-point (nominal P=0.282) improvement on the VAS Fear versus placebo, respectively. The positive control (alprazolam 1 mg BID) exhibited panicolytic effect compared with placebo, in line with expectations. Darigabat was generally well tolerated, with no serious adverse events and no discontinuations in darigabat cohorts. Conclusions: The panicolytic potential of darigabat was validated, warranting further evaluation in patients with panic disorder. Disclosure: Dr. Gurrell has received personal compensation for serving as an employee of Cerevel Therapeutics. Dr. Gurrell has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Cerevel Therapeutics. Dr. Gurrell has stock in Pfizer Ltd. Ms. Chang has received personal compensation for serving as an employee of Cerevel. Ms. Chang has stock in Cerevel Therapeutics. Dr. Dandurand has received personal compensation for serving as an employee of Cerevel. Dr. Dandurand has received personal compensation for serving as an employee of Otsuka. Dr. Dandurand has stock in Cerevel Therapeutics. An immediate family member of Dr. Dandurand has stock in Velocit . An immediate family member of Dr. Dandurand has stock in Cerebus. Dr. Duvvuri has received personal compensation for serving as an employee of Cerevel. Dr. Duvvuri has stock in Cerevel Therapeutics. Dr. Duvvuri has stock in Pfizer. Amy Guigliano has nothing to disclose. Dr. Pastino has received personal compensation for serving as an employee of Cerevel Therapeutics. Dr. Pastino has received personal compensation for serving as an employee of PRA Health Sciences. Dr. Pham has nothing to disclose. Dr. Versavel has received personal compensation for serving as an employee of Cerevel Therapeutics. Dr. Versavel has received stock or an ownership interest from Cerevel Therapeutics. Dr. Jacobs has nothing to disclose. Mr. Pour has nothing to disclose. Dr. Zuiker has nothing to disclose. Dr. Sanchez has received personal compensation for serving as an employee of Cerevel Therapeutics. Dr. Sanchez has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Coleman Research. Dr. Sanchez has received personal compensation in the range of $500,000-$999,999 for serving as an officer or member of the Board of Directors for Cerevel Therapeutics. Dr. Sanchez has received stock or an ownership interest from Cerevel Therapeutics. Dr. Sanchez has received personal compensation in the range of $500-$4,999 for serving as a Consultant with GLG. Dr. Renger has received personal compensation for serving as an employee of Cerevel Therapeutics.

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Background Emraclidine is a novel, brain-penetrant, highly selective M4 receptor positive allosteric modulator in development for the treatment of schizophrenia. We aimed to evaluate the safety and tolerability of multiple ascending doses of emraclidine in patients with schizophrenia.Methods We conducted a two-part, randomised, phase 1b trial in the USA. Eligible participants were aged 18-50 years (part A) or 18-55 years (part B) with a primary diagnosis of schizophrenia per the Diagnostic and Statistical Manual of Mental Disorders 5th edition, as confirmed by the Mini International Neuropsychiatric Interview, and extrapyramidal symptom assessments indicating normal to mild symptoms at screening. Part A evaluated the safety and tolerability of emraclidine in five cohorts of participants with stable schizophrenia who received ascending oral doses of emraclidine 5-40 mg (40 mg was administered as 20 mg twice daily) or placebo at a single US site. Part B was a double-blind, randomised, placebo-controlled study that enrolled adults with acute schizophrenia across five US sites; participants were randomly assigned (1:1:1) to receive emraclidine 30 mg once daily, emraclidine 20 mg twice daily, or placebo for 6 weeks (doses established in part A). The primary endpoint was safety and tolerability, assessed in the safety population (participants who received at least one dose of emraclidine or placebo). This trial is now complete and is registered with ClinicalTrials.gov, NCT04136873.Findings Between Sept 23, 2019, and Sept 17, 2020, 118 patients were assessed for eligibility and 49 were randomly assigned across five cohorts in part A. 44 participants completed the study, with 36 participants receiving emraclidine and eight receiving placebo. The two highest doses tested were selected for part B. Between Oct 12, 2020, and May 7, 2021, 148 patients were assessed for eligibility and 81 were randomly assigned to emraclidine 30 mg once daily (n=27), emraclidine 20 mg twice daily (n=27), or placebo (n=27) in part B. Incidence of adverse events (14 [52%] of 27 participants in the emraclidine 30 mg once daily group, 15 [56%] of 27 in the emraclidine 20 mg twice daily group, and 14 [52%] of 27 in the placebo group), clinical assessments, and weight changes were similar across groups. The most common adverse event was headache (15 [28%] of 54 participants in the emraclidine groups, seven [26%] of 27 in the placebo group). Modest, transient increases in blood pressure and heart rate in emraclidine groups observed at treatment initiation diminished over time and were not considered clinically meaningful by week 6.Interpretation These data support further investigation of emraclidine as a once-daily treatment for schizophrenia without need for titration and with a potentially favourable side-effect profile.

Tuesday, April 5May 3, 2022Free AccessPharmacokinetics, Pharmacodynamics, and Safety of the Highly Selective Dopamine D1/D5 Agonist Tavapadon: Summary of Phase 1 Clinical Studies (P10-11.001)Gina Pastino, Josh Yuan, Sridhar Duvvuri, Matthew Leoni, Kimberly Largay, Ih Chang, Amy Giugliano, Stacey Versavel, David Gray, Raymond Sanchez, and John RengerAuthors Info & AffiliationsMay 3, 2022 issue98 (18_supplement)https://doi.org/10.1212/WNL.98.18_supplement.2728 Letters to the Editor

BACKGROUND Recently the α1 adrenergic receptor antagonist terazosin was shown to activate PGK1, a possible target for the mitochondrial deficits in Parkinson disease related to its function as the initial enzyme in ATP synthesis during glycolysis. An epidemiologic study of terazosin users showed a lower incidence of Parkinson disease when compared to users of tamsulosin, an α1 adrenergic receptor antagonist of a different class that does not activate PGK1. However, prior research on tamsulosin has suggested that it may in fact potentiate neurodegeneration, raising the question of whether it is an appropriate control group. METHODS To address this question, we undertook an epidemiological study on Parkinson disease occurrence rate in 113,450 individuals from the U.S.A. with > 5 years of follow-up. Patients were classified as tamsulosin users (n = 45,380), terazosin/alfuzosin/doxazosin users (n = 22,690) or controls matched on age, gender and Charlson Comorbidity Index score (n = 45,380). RESULTS Incidence of Parkinson disease in tamsulosin users was 1.53%, which was significantly higher than that in both terazosin/alfuzosin/doxazosin users (1.10%; p<0.0001) and matched controls (1.01%; p < 0.0001). Terazosin/alfuzosin/doxazosin users did not differ in Parkinson disease risk from matched controls (p = 0.29). CONCLUSION These results suggest that zosins may not confer a protective effect against Parkinson disease, but rather that tamsulosin may in some way potentiate Parkinson disease progression. FUNDING This work was supported by Cerevel Therapeutics.

Orexins are neuropeptides synthesized in the lateral hypothalamus that influence arousal, feeding, reward pathways, and the response to stress. However, the role of orexins in repeated stress is not fully characterized. Here, we examined how orexins and their receptors contribute to the coping response during repeated social defeat and subsequent anxiety-like and memory-related behaviors. Specifically, we used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to stimulate orexins prior to each of five consecutive days of social defeat stress in adult male rats. Additionally, we determined the role of the orexin 2 receptor in these behaviors by using a selective orexin 2 receptor antagonist (MK-1064) administered prior to each social defeat. Following the 5 day social defeat conditioning period, rats were evaluated in social interaction and novel object recognition paradigms to assess anxiety-like behavior and recognition memory, respectively. Activation of orexin neurons by DREADDs prior to each social defeat decreased the average latency to become defeated across 5 days, indicative of a passive coping strategy that we have previously linked to a stress vulnerable phenotype. Moreover, stimulation of orexin signaling during defeat conditioning decreased subsequent social interaction and performance in the novel object recognition test indicating increased subsequent anxiety-like behavior and reduced recognition memory. Blocking the orexin 2 receptor during repeated defeat did not alter these effects. Together, our results suggest that orexin neuron activation produces a passive coping phenotype during social defeat leading to subsequent anxiety-like behaviors and memory deficits.

Safety pharmacology seeks to validate and refine methods for use in preclinical detection of new chemical entity adverse effect liability. It does so in accordance with the scientific method and seeks to organize the strategy of implementation of methods according to regulatory guidance documents. The discipline is in rapid evolution and its coverage is certain to expand to provide better guidance for the safety evaluation of more physiological systems. This effort is critical as the pursuit of drug development is costly and has high drug failure rates. Strategies and nonclinical methods and models are essential to prevent the development of adverse events during clinical trials. Technologies involved in the acquisition and analysis of safety pharmacology data are rapidly evolving and the safety pharmacologist often benefits from an in-depth understanding of such underlying technologies and associated methodologies. This gives a broad perspective to data interpretation and a better armamentarium with which to address adverse events related to the administration of the test article. Establishing the safety profile of a drug is a key responsibility in the conduct of safety pharmacology studies. To formulate a clear perspective, no effort should be spared to achieve high assay sensitivity and high predictivity of adverse event development in safety pharmacology studies. An emphasis must be placed on the development of translatable nonclinical safety pharmacology methods that identify and characterize not only cardiovascular but also related core battery (i.e., central nervous system and respiratory systems) study risks. If adverse events are observed in a safety pharmacology study, scientists need to develop a mitigation strategy that allows for either progression of the lead compound into further nonclinical development and eventual clinical evaluation or sufficient insight into why the compound (or program) should be recommended for termination. The future needs in issue resolution of problems that arise in drug development require highly skilled drug safety scientists with a broad understanding of essential biological principles, an awareness of novel methods and models and their application to discerning adverse event development and subsequent mitigation, and insight into clinical translational relevance.

To understand the transcriptomic organization underlying sleep and affective function, we studied a population of (C57BL/6J × 129S1/SvImJ) F2 mice by measuring 283 affective and sleep phenotypes and profiling gene expression across four brain regions. We identified converging molecular bases for sleep and affective phenotypes at both the single-gene and gene-network levels. Using publicly available transcriptomic datasets collected from sleep-deprived mice and patients with major depressive disorder (MDD), we identified three cortical gene networks altered by the sleep/wake state and depression. The network-level actions of sleep loss and depression were opposite to each other, providing a mechanistic basis for the sleep disruptions commonly observed in depression, as well as the reported acute antidepressant effects of sleep deprivation. We highlight one particular network composed of circadian rhythm regulators and neuronal activity-dependent immediate-early genes. The key upstream driver of this network, Arc, may act as a nexus linking sleep and depression. Our data provide mechanistic insights into the role of sleep in affective function and MDD.

While many preclinical models of Alzheimer's disease (AD) have been reported, none fully recapitulate the disease. In an effort to identify an appropriate preclinical disease model, we characterized age-related changes in 2 higher order species, the African green monkey (AGM) and the rhesus macaque. Gene expression profiles in the dorsolateral prefrontal cortex and the visual cortex showed age-related changes in AGMs that are strikingly reminiscent of AD, whereas aged rhesus were most similar to healthy elderly humans. Biochemically, age-related changes in AGM cerebrospinal fluid levels of tau, phospho-tau, and amyloid beta were consistent with AD. Histologically, aged AGMs displayed pathological hallmarks of the disease, plaques, and 2 AGMs showed evidence of neurofibrillary tangle-like structures. We hypothesized and confirmed that AGMs have age-related cognitive deficits via a prefrontal cortex-dependent cognition test, and that symptomatic treatments that improve cognition in AD patients show efficacy in AGMs. These data suggest that the AGM could represent a novel and improved translational model to assist in the development of therapeutics for AD.

Study objective: To assess differences in gene expression in cholinergic basal forebrain cells between sleeping and sleep-deprived mice sacrificed at the same time of day.Methods: Tg(ChAT-eGFP) 86Gsat mice expressing enhanced green fluorescent protein (eGFP) under control of the choline acetyltransferase (Chat) promoter were utilized to guide laser capture of cholinergic cells in basal forebrain. Messenger RNA expression levels in these cells were profiled using microarrays. Gene expression in eGFP(+) neurons was compared (1) to that in eGFP(-) neurons and to adjacent white matter, (2) between 7: 00 am (lights on) and 7: 00 pm (lights off), (3) between sleep-deprived and sleeping animals at 0, 3, 6, and 9 hours from lights on.Results: There was a marked enrichment of ChAT and other markers of cholinergic neurons in eGFP(+) cells. Comparison of gene expression in these eGFP(+) neurons between 7: 00 am and 7: 00 pm revealed expected differences in the expression of clock genes (Arntl2, Per1, Per2, Dbp, Nr1d1) as well as mGluR3. Comparison of expression between spontaneous sleep and sleep-deprived groups sacrificed at the same time of day revealed a number of transcripts (n = 55) that had higher expression in sleep deprivation compared to sleep. Genes upregulated in sleep deprivation predominantly were from the protein folding pathway (25 transcripts, including chaperones). Among 42 transcripts upregulated in sleep was the coldinducible RNA-binding protein.Conclusions: Cholinergic cell signatures were characterized. Whether the identified genes are changing as a consequence of differences in behavioral state or as part of the molecular regulatory mechanism remains to be determined.

Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA)A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABAA receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABAA modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABAA modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep.