M1 Positive Allosteric Modulators Research Articles

Potentiation of the muscarinic acetylcholine receptor 1 modulates neurophysiological features in a mouse model of Rett syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X chromosome-linked gene Methyl-CpG Binding Protein 2 (MECP2). Restoring MeCP2 expression after disease onset in a mouse model of RTT reverses phenotypes, providing hope for development of treatments for RTT. Translatable biomarkers of improvement and treatment responses have the potential to accelerate both preclinical and clinical evaluation of targeted therapies in RTT. Studies in people with and mouse models of RTT have identified neurophysiological features, such as auditory event-related potentials, that correlate with disease severity, suggesting that they could be useful as biomarkers of disease improvement or early treatment response. We recently demonstrated that treatment of RTT mice with a positive allosteric modulator (PAM) of muscarinic acetylcholine subtype 1 receptor (M1) improved phenotypes, suggesting that modulation of M1 activity is a potential therapy in RTT. To evaluate whether neurophysiological features could be useful biomarkers to assess the effects of M1 PAM treatment, we acutely administered the M1 PAM VU0486846 (VU846) at doses of 1, 3, 10 and 30 ​mg/kg in wildtype and RTT mice. This resulted in an inverted U-shaped dose response with maximal improvement of AEP features at 3 ​mg/kg but with no marked effect on basal EEG power or epileptiform discharges in RTT mice and no significant changes in wildtype mice. These findings suggest that M1 potentiation can improve neural circuit synchrony to auditory stimuli in RTT mice and that neurophysiological features have potential as pharmacodynamic or treatment-responsive biomarkers for preclinical and clinical evaluation of putative therapies in RTT.

Activation of M4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.

PET imaging of M4 muscarinic acetylcholine receptors in rhesus macaques using [11C]MK-6884: Quantification with kinetic modeling and receptor occupancy by CVL-231 (emraclidine), a novel positive allosteric modulator.

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.

SUVN‐I6107: A True Positive Allosteric Modulator (PAM) at Muscarinic M1 Receptors for the Treatment of Cognitive Disorders

AbstractBackgroundMuscarinic acetylcholine receptors belong to the family of G‐protein coupled receptors and are expressed widely throughout the body. Muscarinic receptors have been implicated in the pathophysiology of various central nervous system disorders. M1 subtype of muscarinic receptors play a crucial role in cognition, attention, and sensory processing. The discovery and development of selective ligands targeting M1 subtype have been unsuccessful largely due to the highly conservative orthosteric binding site. Thus, ligands that selectively bind to a less conserved allosteric site of M1 receptors and positively modulate the orthosteric site may circumvent the selectivity issues as posed by orthosteric ligands.MethodSUVN‐I6107 is a clinical candidate identified from the M1 positive allosteric modulator (M1‐PAM) program. The effects of SUVN‐I6107 on the allosteric site of the M1 receptor were evaluated using the reporter gene assay. Binding liability towards orthosteric sites across all the muscarinic receptor subtypes was also evaluated. The pharmacokinetic properties of SUVN‐I6107 were studied both in rodent and non‐rodent species. The pro‐cognitive potential of SUVN‐I6107 was evaluated in diverse animal models of learning and memory. The safety of SUVN‐I6107 was assessed using various in vitro and in vivo systems.ResultSUVN‐I6107 was found to be a selective M1‐PAM with no agonistic activity at Muscarinic M1 and selective across other muscarinic receptor subtypes. SUVN‐I6107 showed good oral bioavailability in rat, dog and monkey. It also has excellent brain penetration with adequate protein free fraction. SUVN‐I6107 reversed the delay‐induced memory deficit in object recognition task. It also attenuated scopolamine‐induced memory deficit in the object recognition as well as contextual fear conditioning task. SUVN‐I6107 enhanced cerebral blood flow, potentiated the effects of donepezil and promoted non‐amyloidogenic amyloid precursor protein processing in rats. SUVN‐I6107 is devoid of side effects associated with the stimulation of the cholinergic system. SUVN‐I6107 was found to be safe in the safety pharmacology and phase‐1 enabling animal toxicity studies.ConclusionSUVN‐I6107, a novel, potent and selective M1‐PAM demonstrated efficacy in animal models and devoid of cholinergic side effects

Characterization of a Novel M4 PAM PET Radioligand [11C]PF06885190 in Nonhuman Primates (NHP).

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer's disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around -10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.

M1-selective muscarinic allosteric modulation enhances cognitive flexibility and effective salience in nonhuman primates

Acetylcholine (ACh) in cortical neural circuits mediates how selective attention is sustained in the presence of distractors and how flexible cognition adjusts to changing task demands. The cognitive domains of attention and cognitive flexibility might be differentially supported by the M1 muscarinic acetylcholine receptor (mAChR) subtype. Understanding how M1 mAChR mechanisms support these cognitive subdomains is of highest importance for advancing novel drug treatments for conditions with altered attention and reduced cognitive control including Alzheimer's disease or schizophrenia. Here, we tested this question by assessing how the subtype-selective M1 mAChR positive allosteric modulator (PAM) VU0453595 affects visual search and flexible reward learning in nonhuman primates. We found that allosteric potentiation of M1 mAChRs enhanced flexible learning performance by improving extradimensional set shifting, reducing latent inhibition from previously experienced distractors and reducing response perseveration in the absence of adverse side effects. These procognitive effects occurred in the absence of apparent changes of attentional performance during visual search. In contrast, nonselective ACh modulation using the acetylcholinesterase inhibitor (AChEI) donepezil improved attention during visual search at doses that did not alter cognitive flexibility and that already triggered gastrointestinal cholinergic side effects. These findings illustrate that M1 mAChRpositive allosteric modulation enhances cognitive flexibility without affecting attentional filtering of distraction, consistent with M1 activity boosting the effective salience of relevant over irrelevant objects specifically during learning. These results suggest that M1 PAMs are versatile compounds for enhancing cognitive flexibility in disorders spanning schizophrenia and Alzheimer's diseases.

Opportunities and challenges for the development of M1 muscarinic receptor positive allosteric modulators in the treatment for neurocognitive deficits.

Targeting allosteric sites of M1 muscarinic acetylcholine receptors (M1 receptors) is a promising strategy to treat neurocognitive disorders, such as Alzheimer's disease and schizophrenia. Indeed, the last two decades have seen an impressive body of work focussing on the design and development of positive allosteric modulators (PAMs) for the M1 receptor. This has led to the identification of a structurally diverse range of highly selective M1 PAMs. In preclinical models, M1 PAMs have shown rescue of cognitive deficits and improvement of endpoints predictive of symptom domains of schizophrenia. Yet, to date only a few M1 PAMs have reached early-stage clinical trials, with many of them failing to progress further due to on-target mediated cholinergic adverse effects that have plagued the development of this class of ligand. This review covers the recent preclinical and clinical studies in the field of M1 receptor drug discovery for the treatment of Alzheimer's disease and schizophrenia, with a specific focus on M1 PAM, highlighting both the undoubted potential but also key challenges for the successful translation of M1 PAMs from bench-side to bedside. LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.

The M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM) VU0453595 and M4 PAM VU0467154 Normalize Sleep‐Wake Architecture Deficits in Aged Mice

AbstractBackgroundDeclining basal forebrain cholinergic integrity has been associated with disturbances in sleep‐wake architecture in Alzheimer’s Disease (AD). Acetylcholinesterase inhibitors (AChEIs) provide symptomatic treatment for the cognitive deficits in AD through nonselective enhancement of cholinergic signaling. Additionally, AChEIs promote wakefulness, but reduce NREM sleep quality and produce dose limiting side effects through nonselective activation of peripheral muscarinic acetylcholine receptors (mAChR). Selectively targeting different mAChRs with positive allosteric modulators (PAMs) provides an alternate approach. In the current study, we assessed the effects of the M1 PAM VU0453595 and the M4 PAM VU0467154 on sleep‐wake architecture and arousal following dosing across the circadian cycle in aged mice.MethodsYoung (3‐4 month‐old, n = 13‐14 per group) and aged (19‐21 month‐old, n = 14 per group) C57/BL6 mice were implanted with HD‐X02 telemetry devices (DSI) for electroencephalography (EEG) recording. The M1 PAM VU0453595 (3‐30mg/kg IP) or the M4 PAM VU0467154 (1‐30mg/kg IP) were dosed 2‐hours into the active or inactive phases of the circadian cycle. EEG was recorded for 24‐hours, with sleep stages scored in 5‐second epochs. Arousal was assessed through changes in gamma power during wake, and sleep quality was measured through changes in delta power during NREM sleep. Statistical comparisons involved repeated measures one‐ or two‐way ANOVAs as appropriate, with Dunnett’s or Sidak’s multiple comparisons applied in all cases.ResultsThe M1 PAM VU0453595 increased wakefulness and arousal with inactive phase dosing in young and aged mice. During the active phase, VU0453595 increased wakefulness and arousal only in aged mice. The M4 PAM VU0467154 increased NREM sleep in young and aged mice when dosed in both phases. In the inactive phase, suppression of REM sleep in young and aged mice is also observed. During NREM sleep, VU0467154 increased delta power in young and aged mice.ConclusionThese findings suggest that M1 and M4 PAMs may be valuable as treatments in AD at different phases of the circadian cycle. M1 PAMs could be beneficial with morning dosing in clinical populations as they enhanced wakefulness and arousal. M4 PAMs could be advantageous with evening dosing in clinical population as they enhanced NREM sleep quantity and quality.

The novel M4 PAM PET tracer [11C] MK‐6884: a novel biomarker for measuring target engagement of muscarinic M4 positive allosteric modulators (PAMs) as well as cholinergic tone in patients with Alzheimer’s disease

AbstractBackgroundThe muscarinic acetylcholine receptor 4 (M4) has attracted attention as a target for treating neuropsychiatric symptoms, such as agitation and aggression, associated with Alzheimer’s disease. We have developed novel selective, potent, and in vivo suitable M4 positive allosteric modulators (PAMs) and demonstrated robust pharmacology in preclinical models predictive of antipsychotic and pro‐cognitive effects. Importantly, we have also shown that these selective M4 PAMs produce greater therapeutic margins for cholinergic adverse effects compared to less selective muscarinic receptor agonists.MethodsTo enable translation in the clinic, we set out to discover an M4 PAM PET tracer suitable for measuring central receptor occupancy (RO).Result[11C]MK‐6884 binds with high affinity and selectivity to the allosteric site on the M4 receptor that our other M4 PAMs bind. The affinity of [11C]MK‐6884 was found to be influenced by the concentration of orthosteric agonist in vitro and in vivo, consistent with the high cooperativity of the molecule in vitro. PET studies successfully determined the target engagement of PAMs to enable modeling the relationship between drug plasma exposure, RO, and pharmacodynamic activity. Donepezil increased the tracer binding potential of [11C]MK‐6884 in rhesus and healthy human subjects, confirming in vivo the influence of cholinergic tone on [11C]MK‐6884 binding that was observed in vitro. Consistent with a presumed cortical deficit in synaptic acetylcholine, [11C]MK‐6884 PET studies in AD patients showed lower cortical binding potential compared to healthy elderly participants, as well as notable cortical heterogeneity and bilateral asymmetry of M4 receptor binding in AD patients. Striatal binding potential was unchanged in these different populations.ConclusionOur findings demonstrate that the M4 PAM PET tracer [11C]MK‐6884 is a useful tool for determining target engagement of M4 PAMs, as well serving as a unique probe to study changes in cholinergic neurotransmission in neurodegenerative disease. The Enigma Biomedical Group has an exclusive license agreement with Merck & Co., Inc., for the global development and commercialization of MK‐6884. This allows Enigma and its partner, Cerveau Technologies Inc. to provide biomarkers to industry and academia to accelerate research in the field of neurodegenerative and neuropsychiatric diseases.

Dissociating the pro‐cognitive effects of donepezil and an M1 PAM

AbstractBackgroundDonepezil, an FDA approved drug for dementia in Alzheimer’s disease, increases the extracellular concentration of acetylcholine non‐selectively stimulating cholinergic receptors indiscriminately leading to dose‐limiting side effects. Muscarinic M1 sub‐receptor specific positive allosteric modulators (PAMs) have shown promise in addressing symptoms in Alzheimer’s disease, Schizophrenia and more1. M1 PAMs have little to no agonistic properties, avoiding peripheral side effects, and are mostly centrally located allowing for the modulation of endogenous signaling of the brain2.MethodWe tested non‐human primates in two tasks measuring cognitive flexibility and attentional control3 at varying doses of donepezil and an M1 PAM (VU0453595). Attentional control was measured with a visual search task requiring animals to search for a learned target object among distractors of variable number and similarity to the target object. Cognitive flexibility was measured with a feature‐reward learning task that required animals to learn the value of objects of varying feature complexity by trial and error. Flexibility was indexed as the speed of learning, the degree of latent inhibition and the amount of target perseveration after switching the rewarded feature.ResultDonepezil enhanced attention control at a high dose (0.3 mg/kg) but enhance flexibility only moderately at a lower dose (0.06 mg/kg). In contrast, VU0453595 did not modulate attention, but at 0.1 mg/kg enhanced flexible learning, reduced target perseverations, and increased reaction time measures of decision confidence. At the dose with the most pronounced cognitive benefits donepezil resulted in gastrointestinal side effects, while VU0453595 did not cause side effects.ConclusionThese results suggest that donepezil engages the brain networks underlying cognitive flexibility and interference control at different doses4, while the M1 receptor plays a major role in facilitating flexible behavioral adaptation but with no role in controlling interference. Such receptor specific results can be used to better curate to patient‐specific needs in order to address the most debilitating symptoms with the highest efficacy.1. Melancon, B. J. et al. (2013) Drug Discovery Today https://doi.org/10.1016/j.drudis.2013.09.005.2. Ghoshal, A. et al. (2016) Neuropsychopharmacology doi:10.1038npp.2015.189 3. Womelsdorf, T. et al. (2021) Frontiers in Behavioral Neuroscience https://doi.org/10.3389/fnbeh.2021.721069 4. Hassani, S.A. et al. (2021) Biological Psychiatry Global Open Science https://doi.org/10.1016/j.bpsgos.2021.11.012.

M1 muscarinic acetylcholine receptor positive allosteric modulators do not disrupt sleep in aged cynomolgus macaques

AbstractBackgroundAge‐related changes in sleep may directly contribute to cognitive dysfunction and underlying pathological progression contributing to Alzheimer’s Disease or related dementias (AD/ADRDs). While acetylcholinesterase inhibitors (AChEIs) are FDA‐approved for the treatment of AD‐related cognitive impairments, these drugs also disrupt sleep. Positive allosteric modulators (PAMs) targeting the M1 muscarinic acetylcholine receptor (M1 mAChR) have produced cognitive and arousal enhancing as well as sleep‐modifying effects in aged and/or AD rodents models. The present study provided the first comparison of baseline sleep measures in young and old monkeys, then examined the effects of novel M1 mAChR PAMs VU0486846 and VU0453595, compared with the AChEI, Donepezil on sleep in aged monkeys.MethodEight young adult (4 male/4 female, 6‐8 years old) and 10 aged cynomolgus macaques (6 males/4 females, 19‐25 years old) were implanted with subcranial electrodes and a wireless transmitter (L04, DSI) for undisturbed telemetric recording of electroencephalography (EEG) from their home cage. After baseline sleep assessments, aged monkeys were tested once weekly with donepezil (3.0‐10 mg/kg, PO), VU0486846 (3.0‐30 mg/kg, PO), or VU0453595 (1.0‐10 mg/kg, PO), administered 2 hours prior to lights out (0600:1800 light:dark schedule). EEG was recorded and sleep stages including total sleep time (TST), % TST in rapid eye movement sleep (REM), N1, N2, and N3 non‐REM sleep were determined and summed across the 12 hour dark period.ResultAged monkeys showed significantly less % TST in N3 and REM sleep and increased N1 sleep compared to young monkeys. Donepezil dose‐dependently reduced TST, increased % REM and trended toward a decrease in N2 and N3 stage sleep. In contrast, M1 PAMs, within dose ranges known to be physiologically active in monkeys, did not alter sleep parameters.ConclusionAged‐related and donepezil‐mediated sleep disruptions in aged monkeys are consistent with the clinical literature. Lack of effects by M1 mAChR PAMs in aged monkeys when dosed prior to lights out may suggest fewer disruptive effects compared to AChEIs or that there is insufficient cholinergic tone for an M1 PAM to alter sleep. Future studies will assess the arousal enhancing effects of M1 PAMs when dosed during waking periods in aged monkeys.Funding: AG054622, DA017763

Clinical and Preclinical Evidence for M1 Muscarinic Acetylcholine Receptor Potentiation as a Therapeutic Approach for Rett Syndrome

SummaryRett syndrome (RTT) is a neurodevelopmental disorder that is characterized by developmental regression, loss of communicative ability, stereotyped hand wringing, cognitive impairment, and central apneas, among many other symptoms. RTT is caused by loss-of-function mutations in a methyl-reader known as methyl-CpG-binding protein 2 (MeCP2), a protein that links epigenetic changes on DNA to larger chromatin structure. Historically, target identification for RTT has relied heavily on Mecp2 knockout mice; however, we recently adopted the alternative approach of performing transcriptional profiling in autopsy samples from RTT patients. Through this mechanism, we identified muscarinic acetylcholine receptors (mAChRs) as potential therapeutic targets. Here, we characterized a cohort of 40 temporal cortex samples from individuals with RTT and quantified significantly decreased levels of the M1, M2, M3, and M5 mAChRs subtypes relative to neurotypical controls. Of these four subtypes, M1 expression demonstrated a linear relationship with MeCP2 expression, such that M1 levels were only diminished in contexts where MeCP2 was also significantly decreased. Further, we show that M1 potentiation with the positive allosteric modulator (PAM) VU0453595 (VU595) rescued social preference, spatial memory, and associative memory deficits, as well as decreased apneas in Mecp2+/- mice. VU595’s efficacy on apneas in Mecp2+/- mice was mediated by the facilitation of the transition from inspiration to expiration. Molecular analysis correlated rescue with normalized global gene expression patterns in the brainstem and hippocampus, as well as increased Gsk3β inhibition and NMDA receptor trafficking. Together, these data suggest that M1 PAMs could represent a new class of RTT therapeutics.

Optimized Administration of the M4 PAM VU0467154 Demonstrates Broad Efficacy, but Limited Effective Concentrations in Mecp2+/- Mice.

Hypofunction of cholinergic circuits and diminished cholinergic tone have been associated with the neurodevelopmental disorder Rett syndrome (RTT). Specifically, deletion of Mecp2 in cholinergic neurons evokes the same social and cognitive phenotypes in mice seen with global Mecp2 knockout, and decreased choline acetyltransferase activity and vesamicol binding have been reported in RTT autopsy samples. Further, we recently identified significant decreases in muscarinic acetylcholine receptor subtype 4 (M4) expression in both the motor cortex and cerebellum of RTT patient autopsies and established proof of concept that an acute dose of the positive allosteric modulator (PAM) VU0467154 (VU154) rescued phenotypes in Mecp2+/- mice. Here, we expand the assessment of M4 PAMs in RTT to address clinically relevant questions of tolerance, scope of benefit, dose response, chronic treatment, and mechanism. We show that VU154 has efficacy on anxiety, social preference, cognitive, and respiratory phenotypes in Mecp2+/- mice; however, the therapeutic range is narrow, with benefits seen at 3 mg/kg concentrations, but not 1 or 10 mg/kg. Further, sociability was diminished in VU154-treated Mecp2+/- mice, suggestive of a potential adverse effect. Compound efficacy on social, cognitive, and respiratory phenotypes was conserved with a 44-day treatment paradigm, with the caveat that breath rate was moderately decreased with chronic treatment in Mecp2+/+ and Mecp2+/- mice. VU154 effects on respiratory function correlated with an increase in Gsk3β inhibition in the brainstem. These results identify the core symptom domains where efficacy and adverse effects may present with M4 administration in RTT model mice and advocate for the continued evaluation as potential RTT therapeutics.

Effects of acute and repeated administration of the selective M4 PAM VU0152099 on cocaine versus food choice in male rats.

Ligands that stimulate muscarinic acetylcholine receptors 1 and 4 (M1 , M4 ) have shown promising effects as putative pharmacotherapy for cocaine use disorder in rodent assays. We have previously shown reductions in cocaine effects with acute M4 stimulation, as well as long-lasting, delayed reductions in cocaine taking and cocaine seeking with combined M1 /M4 receptor stimulation or with M1 stimulation alone. M4 stimulation opposes dopaminergic signalling acutely, but direct dopamine receptor antagonists have proved unhelpful in managing cocaine use disorder because they lose efficacy with long-term administration. It is therefore critical to determine whether M4 approaches themselves can remain effective with repeated or chronic dosing. We assessed the effects of repeated administration of the M4 positive allosteric modulator (PAM) VU0152099 in rats trained to choose between intravenous cocaine and a liquid food reinforcer to obtain quantitative measurement of whether M4 stimulation could produce delayed and lasting reduction in cocaine taking. VU0152099 produced progressively augmenting suppression of cocaine choice and cocaine intake, but produced neither rebound nor lasting effects after treatment ended. To compare and contrast effects of M1 versus M4 stimulation, we tested whether the M4 PAM VU0152100 suppressed cocaine self-administration in mice lacking CalDAG-GEFI signalling factor, required for M1 -mediated suppression of cocaine self-administration. CalDAG-GEFI ablation had no effect on M4 -mediated suppression of cocaine self-administration. These findings support the potential usefulness of M4 PAMs as pharmacotherapy to manage cocaine use disorder, alone or in combination with M1 -selective ligands, and show that M1 and M4 stimulation modulate cocaine-taking behaviour by distinct mechanisms.

Age and circadian rhythm‐dependent effects of M1 muscarinic acetylcholine receptor positive allosteric modulators and donepezil on sleep‐wake architecture and arousal

AbstractBackgroundDegeneration of basal forebrain cholinergic signaling in aging and Alzheimer's disease (AD) is linked with abnormalities in arousal, sleep/wake architecture, and cognition. While acetylcholinesterase inhibitors (AChEIs) are approved for the treatment of AD, they produce dose‐limiting adverse effects due to nonselective activation of peripheral muscarinic acetylcholine receptors (mAChRs). Selectively targeting the M1 mAChR using M1 positive allosteric modulators (PAMs) has shown promise for boosting cognitive performance and arousal across preclinical species. We evaluate the effects of the M1 PAM VU0453595 on arousal and sleep/wake architecture in aged and young mice across both the active and inactive phases of the circadian cycle. We hypothesize that dosing an M1 PAM during the active phase in aged mice will produce a greater enhancement of arousal and wakefulness, normalizing deficits observed in aged mice.MethodUtilizing electroencephalography, we examined the effects of the M1 PAM VU0453595 in comparison with the AChEI Donepezil on sleep‐wake architecture and state dependent spectral power in young and aged C57/BL6 mice following dosing in either the active or inactive phases of the circadian cycle.ResultWhen compared to young mice, aged mice displayed several alterations in normal sleep‐wake architecture; in the inactive phase, reductions in rapid eye movement (REM) sleep, while in the active phase increases in non‐REM (NREM) sleep and decreases in wakefulness. Aged mice also displayed reductions in slow delta power during NREM sleep and increased delta power during wake. In young mice, dosing with both compounds increased wakefulness and arousal during wake in the inactive phase, with reduced effects in the active phase. In the aged mice VU0453595 increased arousal during wake in the inactive phase, and both wakefulness and arousal during wake when dosed in the active phase. Donepezil in contrast produced increased wakefulness in the inactive period, but reduced wakefulness in the active period and limited effects on arousal during wake.ConclusionThe M1 PAM VU0453595 produced increases in wakefulness and arousal in aged mice during the active phase, suggesting that selective M1 PAMs may be suitable for dosing during the active phase in aging and AD populations to normalize disruptions in wakefulness and arousal.

Enantioselective Desymmetrization of 3-Substituted Oxetanes: An Efficient Access to Chiral 3,4-Dihydro-2H-1,4-benzoxazines.

Herein, we describe a versatile transition metal/oxidant free synthesis of the chiral 2H-1,4-benzoxazines through chiral phosphoric acid (CPA) catalyzed enantioselective desymmetrization of prochiral oxetanes (30 examples) in up to 99% yield and 99% enantioselectivity under mild reaction conditions. The reported strategy not only complements the conventional 2H-1,4-benzoxazine synthetic strategies but also provides access to key intermediates of therapeutic candidates, i.e., prostaglandin D2 receptor antagonist and M1 positive allosteric modulator (PAM) compound VU0486846.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes – Part 2

This Letter details our efforts to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a “tie-back” strategy to replace the 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core which lead to the discovery of two novel tricyclic cores: a 7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine core and 2,4-dimethylthieno[2,3-b:5,4-c′]dipyridine core. Both tricyclic cores displayed low nanomolar potency against the human M4 receptor.

Effects of a novel M4 muscarinic positive allosteric modulator on behavior and cognitive deficits relevant to Alzheimer's disease and schizophrenia in rhesus monkey

Alzheimer's disease (AD) is a profoundly debilitating neurodegenerative disorder characterized most notably by progressive cognitive decline, but also agitation and behavioral disturbances that are extremely disruptive to patient and caregiver. Current pharmacological treatments for these symptoms have limited efficacy and significant side effects. We have recently reported the discovery of Compound 24, an M4 positive allosteric modulator (PAM) that is potent, highly selective, and devoid of cholinergic-like side effects in rats. In order to further evaluate the translatability of the effects of compound 24 in primates, here we describe the effect of Compound 24 on three behavioral and cognition assays in rhesus monkeys, the stimulant induced motor activity (SIMA) assay, the object retrieval detour task (ORD), and the visuo-spatial paired-associates learning (vsPAL) task. As far as we know, this is the first such characterization of an M4 PAM in non-human primate. Compound 24 and the clinical standard olanzapine attenuated amphetamine induced hyperactivity to a similar degree. In addition, Compound 24 demonstrated procognitive effects in scopolamine-impaired ORD and vsPAL, and these effects were of similar magnitude to donepezil. These findings suggest that M4 PAMs may be beneficial to diseases such as Alzheimer's disease and schizophrenia, which are marked by behavioral disturbances as well as deficits in cognitive function.

Discovery of a novel class of heteroaryl-pyrrolidinones as positive allosteric modulators of the muscarinic acetylcholine receptor M1

This Letter describes the synthesis and optimization of a series of heteroaryl-pyrrolidinone positive allosteric modulators (PAMs) of the muscarinic acetylcholine receptor M1 (mAChR M1). Through the continued optimization of M1 PAM tool compound VU0453595, with a focus on replacement of the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one with a wide variety of alternative 4,5-dihydropyrrolo-fused heteroaromatics, the generation of M1 PAMs with structurally novel chemotypes is disclosed. Two compounds from these subseries, 8b (VU6005610) and 20a (VU6005852), show robust selectivity for the M1 mAChR, and no M1 agonism. Both compounds have favorable preliminary PK profiles in vitro;8b additionally demonstrates high brain exposure in a rodent IV cassette model.

Restoring Agonist Function at a Chemogenetically Modified M1 Muscarinic Acetylcholine Receptor.

Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M1 DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine-N-oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M1 mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M1 PAMs with distinct scaffolds is unknown. Here, we reveal that new M1 PAMs restore ACh function at the M1 DREADD while modulating ACh binding at the M1 wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M1 PAMs is translated in vivo using transgenic M1 DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of in vivo responses.