Selective M1 Research Articles

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan > enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

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.

An M1/sigma‐1 receptor agonist prevents cognitive deficits, reduces amyloid plaques and neuroinflammation in a transgenic rat model of Alzheimer’s amyloid pathology.

AbstractBackgroundAcetylcholinesterase inhibitors represent four out of six drugs for treating Alzheimer’s disease (AD), with time‐limited efficacy likely due to the progressive loss of cholinergic neurons. The selective allosteric M1 muscarinic and sigma‐1 receptor agonist Anavex 3‐71 (aka AF710B) [1] takes advantage of the fact that acetylcholine postsynaptic M1 muscarinic brain receptor levels remain unchanged in AD. Previously, Anavex 3‐71 treatment attenuated AD hallmarks in McGill‐R‐Thy1‐APP transgenic rats when administered at advanced stages of the AD‐like pathology [2]. However, preventive strategies instead of late treatment should be more effective in AD. Therefore, we tested whether Anavex 3‐71 administration during early amyloid pathology stages could prevent cognitive impairment, plaque deposition and neuroinflammation.MethodPre‐plaque, seven‐month‐old McGill‐R‐Thy1‐APP rats were subjected to daily oral administration of Anavex 3‐71 (10 µg/kg) for seven months. After one month of drug interruption (wash‐out), we performed Novel Object Recognition (NOR), Morris Water Maze (MWM) and Social Preference (SP) behavioural tests. Subsequently, brains were extracted, fixed and analyzed by histochemistry and immunohistochemistry.ResultMcGill‐APP rats had deficits in discriminating the novel object in the NOR and locating the hidden platform in the MWM compared to wild‐type littermates. Anavex 3‐71 restored the abilities of McGill‐APP rats in the NOR task to wild‐type levels and partially rescued the deficits in the MWM. In the SP task, Anavex 3‐71‐treated McGill‐APP rats exhibited diminished deficits in SP interaction time with a “stranger rat”. Treatment significantly reduced the number of mature Thioflavin‐S positive plaques in the subiculum compared to vehicle‐treated McGill‐APP rats and diminished the number of diffuse plaques in the cortex and hippocampus. Furthermore, Anavex 3‐71 treatment significantly reduced microglia and astrocyte recruitment towards CA1 hippocampal Aβ‐burdened neurons compared to untreated McGill‐APP rats.ConclusionThe long‐lasting effect of Anavex 3‐71 in preventing cognitive decline of McGill‐R‐Thy1‐APP rats, even after a wash‐out period would suggest some preventative, disease‐modifying, properties of the compound over the AD‐like amyloid pathology. This is supported by the attenuation of extracellular Aβ deposition and reduced glial cell recruitment towards hippocampal neurons. [1] Fisher et al., 2016; Neurodegenerative Diseases. https://doi.org/10.1159/000440864 [2] Hall et al., 2018; Alzheimer’s & Dementia. https://doi.org/10.1016/j.jalz.2017.11.009

Emraclidine, a novel positive allosteric modulator of cholinergic M4 receptors, for the treatment of schizophrenia: a two-part, randomised, double-blind, placebo-controlled, phase 1b trial

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. 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. 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. 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. Cerevel Therapeutics.

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.

Abstract 15072: Targeted Atrial Inhibition of Parasympathetic Signaling With GiGo Inhibitory Peptides Attenuates Electrical Remodeling and Development of Persistent Atrial Fibrillation in a Canine Model

Background: While vagal contribution to paroxysmal atrial fibrillation (AF) is well demonstrated, a role for parasympathetic signaling in persistent AF is less clear. We have shown marked atrial parasympathetic hyperinnervation in a canine rapid atrial pacing (RAP) model of AF. However, the role of these new nerves in AF development is unknown. Hypothesis: RAP-induced parasympathetic hyperinnervation contributes to the onset and maintenance of AF, and a gene therapy approach inhibiting muscarinic M2 signaling (Gα i and Gα o - GiGo) in the atria can prevent persistent AF. Methods: Plasmids expressing GiGo inhibitory peptides (GiGo_ct) were injected in the atria of 7 dogs followed by electroporation to facilitate gene delivery. RAP was performed for up to 12 weeks. At terminal EP study, residual AF was recorded in the posterior left atrium (PLA), left atrial free wall (LAFW) and left atrial appendage (LAA). Regional atrial ERP was measured. Results: Controls developed ≥30 minutes of AF after a median of 4 days, and persistent AF (≥8 hours) after a median of 14 days. GiGo_ct delayed onset of AF of ≥30 minutes, and attenuated development of persistent AF. ERP was markedly shortened in the PLA and LAA of controls, and this was attenuated by GiGo_ct (p<0.001). Residual AF was slower (lower dominant frequency; PLA 9.4±0.6Hz Vs 11.0±0.7Hz; LAFW 7.8±0.3Hz Vs 10.4±0.7Hz; LAA 8.9±0.6Hz Vs 9.9±0.5Hz p<0.01), less fractionated (longer fractionation interval; PLA 70.0.4±5.2ms Vs 68.1±5.2ms; LAFW 98.8±4.1ms Vs 70.9±3.1ms; LAA 88.9±7.8ms Vs 80.3±2.0ms p<0.01) and more organized (lower Shannon's entropy; p<0.05). Conclusion: Newly sprouted atrial parasympathetic nerves play a functional role in development of persistent AF. Gene-based, selective M2 signaling inhibition by GiGo_ct attenuates electrical remodeling and prevents initiation and maintenance AF. Future optimization of gene therapy targeting autonomic remodeling may lead to novel, mechanism-guided treatments for AF.

The interaction between hippocampal cholinergic and nitrergic neurotransmission coordinates NMDA-dependent behavior and autonomic changes induced by contextual fear retrieval.

Re-exposing an animal to an environment previously paired with an aversive stimulus evokes large alterations in behavioral and cardiovascular parameters. Dorsal hippocampus (dHC) receives important cholinergic inputs from the basal forebrain, and respective acetylcholine (ACh) levels are described to influence defensive behavior. Activation of muscarinic M1 and M3 receptors facilitates autonomic and behavioral responses along threats. Evidence show activation of cholinergic receptors promoting formation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) in dHC. Altogether, the action of ACh and NO on conditioned responses appears to converge within dHC. As answer about how ACh and NO interact to modulate defensive responses has so far been barely addressed, we aimed to shed additional light on this topic. Male Wistar rats had guide cannula implanted into the dHC before being submitted to the contextual fear conditioning (3footshocks/085mA/2s). A catheter was implanted in the femoral artery the next day for cardiovascular recordings. Drugs were delivered into dHC 10min before contextual re-exposure, which occurred 48h after the conditioning procedure. Neostigmine (Neo) amplified the retrieval of conditioned responses. Neo effects (1nmol) were prevented by the prior infusion of a M1-M3 antagonist (fumarate), a neuronal nitric oxide synthase inhibitor (NPLA), a NO scavenger (cPTIO), a guanylyl cyclase inhibitor (ODQ), and a NMDA antagonist (AP-7). Pretreatment with a selective M1 antagonist (pirenzepine) only prevented the increase in autonomic responses induced by Neo. The results show that modulation in the retrieval of contextual fear responses involves coordination of the dHC M1-M3/NO/cGMP/NMDA pathway.

Trihexyphenidyl increases delta activity in non-rapid eye movement sleep without impairing cognitive function in rodent models

Both human and rodent studies suggest the link between non-rapid eye movement (NREM) sleep and cognition. Recent study indicated that selective activation of cholinergic neurons in basal forebrain inhibits electroencephalogram (EEG) delta power and shortens NREM sleep. In the current study, we aimed to test the pharmacological effect of trihexyphenidyl (THP), a selective muscarinic M1 receptor antagonist, on EEG power spectra and sleep with or without the selective activation of basal forebrain cholinergic neurons. THP (1, 2, and 3 mg/kg) was administrated intraperitoneally in natural sleep phase. Basal forebrain cholinergic neurons expressing modified G protein-coupled muscarinic receptors (hM3Dq) were activated by intraperitoneal injection of clozapine-N-oxide in ChAT-IRES-Cre mice. EEG and electromyogram (EMG) signals were recorded in freely moving mice to analyze EEG power spectrum and sleep hypnogram. Y-maze and novel object recognition tests were used for testing cognition. THP 1 mg/kg significantly increased EEG delta power and facilitated NREM sleep in wildtype mice, while THP 3 mg/kg was required in ChAT-IRES-Cre mice treated with clozapine-N-oxide. THP with dosage up to 8 mg/kg did not induce cognitive impairments in wildtype mice. EEG delta power of NREM sleep is often used as an indicator of sleep depth or sleep quality, which tightly link with sleep-dependent cognition. Taken together, the data collected from rodents hinted that, THP could possibly be used as the NREM sleep facilitator in humans.

Effects of 4-DAMP on allergic rhinitis in guinea pigs and its potential mechanism.

BackgroundAllergic rhinitis (AR) is a non-infectious chronic inflammatory disease of the nasal mucosa mainly mediated by immunoglobulin E (IgE), which seriously affects the life quality of affected patients. This study aimed to observe the effects of 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a selective M3 receptor antagonist) on ovalbumin (OVA)-induced AR guinea pigs, and to explore its potential mechanism of involvement.MethodsAn AR model was established by inducing male guinea pigs (4–6 weeks of age) with OVA. AR guinea pigs were randomly divided into a model group, 0.6 mg/kg ipratropium bromide (IB) group, 0.12 and 0.6 mg/kg 4-DAMP group (n=18). The 0.6 mg/kg IB group, 0.12 and 0.6 mg/kg 4-DAMP group animals were treated with IB (0.6 mg/kg) and 4-DAMP (0.12 or 0.6 mg/kg) by intranasal instillation per nostril daily. Animals in the model group and normal group were treated with saline as control. The AR symptom scores were counted and nasal secretion weights were measured. Histopathological methods were used to observe nasal mucosa. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of histamine and cytokines. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expressions of mucin 5AC (MUC5AC), matrix metalloproteinase 9 (MMP9), and epidermal growth factor receptor (EGFR).ResultsCompared with model group animals, the AR symptom scores and nasal secretion weights of animals treated with 4-DAMP were reduced significantly, goblet cell metaplasia was reversed, and eosinophil infiltration was visibly alleviated. The levels of histamine and cytokines in nasal lavage fluid (NLF) were decreased, and the protein and messenger RNA (mRNA) expressions of MUC5AC, MMP9, and EGFR were inhibited.ConclusionsTreatment with 4-DAMP has a certain effect on AR, especially for mucus hypersecretion, which provides a new idea for clinical treatment of AR.

Glycopyrronium bromide regulates cigarette smoke-induced epithelial mesenchymal transition by mediating ACh production

Epithelial–mesenchymal transition (EMT) is one of the pathological features of chronic obstructive pulmonary disease (COPD) and lung cancer. Cigarette smoke (CS) stimulates the production of many factors including acetylcholine (ACh), leading to the progression of EMT in the lung. Our previous studies have shown the potential effects of glycopyrronium bromide (GB), a selective M3 receptor antagonist, on reducing acute lung inflammation induced by CS in mice. Here, we investigated the regulation of GB on CS-induced ACh production and EMT progression in a mouse model, where we found that inhalation of GB (600 µg/mL) significantly suppressed the ACh upregulation and reversed the changes of EMT-related indicators in the lung. In addition, GB prevented the EMT induced by ACh analogue methacholine (MCh) in vitro. We further explored the underlying mechanism, where we found that CS extract (CSE) and MCh exposure resulted in Akt phosphorylation, which were suppressed by GB. Moreover, the CSE or MCh exposure-induced EMT progression were restrained by Akt knockdown or inhibition. Thus, our results indicated that GB can be a potential, readily usable drug for the EMT-related diseases. Additionally, synergistic use of GB in clinics to strengthen the anti-tumor effects may provide novel therapeutic strategies for lung cancer patients with COPD.

Investigating Drivers for M1 Muscarinic Acetylcholine Receptor‐Mediated Adverse Events by M1 Positive Allosteric Modulators

IntroductionTargeting the allosteric site of the M1 muscarinic acetylcholine receptor (M1 mAChR) is a promising strategy to develop selective drugs to treat cognitive disorders. By binding to the allosteric site, positive allosteric modulators of the M1 mAChRs (M1 PAMs) can enhance the binding (α) and/or the efficacy (β) of the endogenous ligand acetylcholine (ACh) (Fig 1), therefore improving impaired cholinergic transmission that is evident in Alzheimer’s disease and Schizophrenia. In addition, M1 PAMs may also display direct allosteric agonism (tB) (Fig 1). To date, many highly selective M1 PAMs with diverse structures have been developed, unfortunately, the majority of them still caused M1‐mediated cholinergic adverse effects (AEs), including gastrointestinal (GI) disturbances, salivation, increased heart rate and convulsions. This results from the ability of the M1 mAChR to interact with multiple intracellular partners, leading to different functional outcomes, both the beneficial (i.e, improved cognition), and the detrimental (i.e, GI AEs) effects. Excitingly, M1 PAMs can bias the M1 mAChR towards specific signalling outcomes versus others, a phenomenon called biased modulation. However, to date, it is unknown which intracellular partner is the most predominant in M1 PAM activity, and how this may drive the preclinically observed AEs.AimTo determine the role(s) Gα protein(s) and β‐arrestins exert on the allosteric activity of 5 structurally distinct M1 PAMs and link these to their reported AEs.MethodsThe M1 PAMs including BQCA, MK7622, PF06767832, MIPS1780 which showed AEs in in vivo studies, and VU0486846 without observed AEs, were assessed against ACh, in parental or CRISPR HEK293A cells with deletion of specific G proteins or β‐arrestins (ΔGαq/11, ΔGα12/13or Δβ‐arrestin 1/2) expressing the human M1 mAChR. [3H]‐NMS binding assays were performed to determine the binding affinity of ACh (KA), and of the 5 M1 PAMs (KB) and their binding cooperativity (α) with ACh. IP1 accumulation and b‐arrestin 2 recruitment were performed to quantify the efficacy (τB) of the M1 PAMs and their functional cooperativity (αβ) with ACh.ResultsΔGαq/11, ΔGα12/13 or Δb‐arrestin 1/2 significantly reduced the binding affinity of ACh, and for 3 M1‐PAMs, MK7622, PF06767832 and MIPS1780. Interestingly, all these deletions significantly increased the binding cooperativity of these M1‐PAMs with ACh. Unsurprisingly, ΔGαq/11 completely abolished IP1 responses from both orthosteric and allosteric ligands. Notably, ΔGα12/13 or Δβ‐arrestin 1/2 both increased ACh efficacy (τA). While ΔG12/13 caused no change in efficacy of tested PAMs but increased functional cooperativity of PF06767832 and MIPS1780 with ACh, Δβ‐arrestin 1/2 affected the functional properties (τB and αβ) of MK7622 in IP1 pathway. In β‐arrestin 2 pathway, ΔGαq/11 increased MIPS1780 efficacy and ΔGα12/13 increased functional cooperativity of BQCA.ConclusionGαq/11, Gα12/13 and b‐arrestin 1/2 influence binding affinity and functional properties for both ACh and M1 PAMs. These transductors affect M1 PAMs with observed AEs more than the M1 PAM without AEs, VU0486846, and thus playing a potential role in driving AEs.

Development of Novel M4 Muscarinic Acetylcholine Receptor Antagonists for the Treatment of Movement Disorders

Non‐selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson’s disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti‐muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti‐parkinsonian and anti‐dystonic efficacy observed with the use of non‐selective anti‐muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non‐selective anti‐muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these non‐selective drugs. However, this hypothesis has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non‐selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor‐stimulating and anti‐parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of a series of first‐in‐class selective M4 antagonists. These novel compounds have ideal pharmacokinetic properties for in vivo evaluations, and we have confirmed that these optimized compounds have anti‐parkinsonian and anti‐dystonic efficacy in pharmacological and genetic models of movement disorders. These data provide critical pre‐clinical rationale for the development of M4 antagonists and represent a potential novel treatment mechanism to meet the unmet clinical need across several movement disorders.

Blockade of M4 muscarinic receptors on striatal cholinergic interneurons normalizes striatal dopamine release in a mouse model of TOR1A dystonia

Trihexyphenidyl (THP), a non-selective muscarinic receptor (mAChR) antagonist, is commonly used for the treatment of dystonia associated with TOR1A, otherwise known as DYT1 dystonia. A better understanding of the mechanism of action of THP is a critical step in the development of better therapeutics with fewer side effects. We previously found that THP normalizes the deficit in striatal dopamine (DA) release in a mouse model of TOR1A dystonia (Tor1a+/ΔE knockin (KI) mice), revealing a plausible mechanism of action for this compound, considering that abnormal DA neurotransmission is consistently associated with many forms of dystonia. However, the mAChR subtype(s) that mediate the rescue of striatal dopamine release remain unclear. In this study we used a combination of pharmacological challenges and cell-type specific mAChR conditional knockout mice of either sex to determine which mAChR subtypes mediate the DA release-enhancing effects of THP. We determined that THP acts in part at M4 mAChR on striatal cholinergic interneurons to enhance DA release in both Tor1a+/+ and Tor1a+/ΔE KI mice. Further, we found that the subtype selective M4 antagonist VU6021625 recapitulates the effects of THP. These data implicate a principal role for M4 mAChR located on striatal cholinergic interneurons in the mechanism of action of THP and suggest that subtype selective M4 mAChR antagonists may be effective therapeutics with fewer side effects than THP for the treatment of TOR1A dystonia.

Reimagining cholinergic therapy for Alzheimer's disease.

Currently, enhancement of cholinergic neurotransmission via cholinesterase inhibitors represents the main available approach to treat cognitive and behavioural symptoms of the early as well as late stages of Alzheimer's disease. Restoring the cholinergic system has been a primary means of improving cognition in Alzheimer's disease, as four of the six approved therapies are acetylcholinesterase inhibitors. Memantine is an N-methyl-d-aspartate antagonist with a well-documented clinical effect on behavioural symptoms, which is often added to cholinesterase inhibitors to potentiate their effect and aducanumab, targeting the amyloid pathology, has recently been approved. The early, progressive and selective degeneration of the cholinergic system together and its close relation to cognitive deficits supports the use of cholinergic therapy for Alzheimer's disease. This review provides an updated view of the basal forebrain cholinergic system, its relation to cognition and its relevance for therapy of Alzheimer's disease. It deals with the three main aspects that form the basis of the cholinergic-oriented therapy of Alzheimer's disease, its origin, its mechanism of action, its clinical effects, advantages and limits of a cholinergic therapeutic approach. It includes a new and updated overview of the involvement of muscarinic receptors in Alzheimer's disease as well as the recent development of new and highly selective M1 muscarinic receptor agonists with disease-modifying potential. It also addresses the discovery of a novel nerve growth factor metabolic pathway responsible for the trophic maintenance of the basal forebrain system and its deregulation in Alzheimer's disease. It discusses new clinical studies and provides evidence for the long-term efficacy of cholinesterase inhibitor therapy suggesting a disease-modifying effect of these drugs. The classical symptomatic cholinergic therapy based on cholinesterase inhibitors is judiciously discussed for its maximal efficacy and best clinical application. The review proposes new alternatives of cholinergic therapy that should be developed to amplify its clinical effect and supplement the disease-modifying effect of new treatments to slow down or arrest disease progression.

Design, Synthesis, and Biological Evaluation of 4,4'-Difluorobenzhydrol Carbamates as Selective M1 Antagonists.

Due to their important role in mediating a broad range of physiological functions, muscarinic acetylcholine receptors (mAChRs) have been a promising target for therapeutic and diagnostic applications alike; however, the list of truly subtype-selective ligands is scarce. Within this work, we have identified a series of twelve 4,4’-difluorobenzhydrol carbamates through a rigorous docking campaign leveraging commercially available amine databases. After synthesis, these compounds have been evaluated for their physico–chemical property profiles, including characteristics such as HPLC-logD, tPSA, logBB, and logPS. For all the synthesized carbamates, these characteristics indicate the potential for BBB permeation. In competitive radioligand binding experiments using Chinese hamster ovary cell membranes expressing the individual human mAChR subtype hM1-hM5, the most promising compound 2 displayed a high binding affinitiy towards hM1R (1.2 nM) while exhibiting modest-to-excellent selectivity versus the hM2-5R (4–189-fold). All 12 compounds were shown to act in an antagonistic fashion towards hM1R using a dose-dependent calcium mobilization assay. The structural eligibility for radiolabeling and their pharmacological and physico–chemical property profiles render compounds 2, 5, and 7 promising candidates for future position emission tomography (PET) tracer development.

A phase 1b/2a multicenter study of the safety and preliminary pharmacodynamic effects of selective muscarinic M1 receptor agonist HTL0018318 in patients with mild-to-moderate Alzheimer's disease.

IntroductionThis study examined the safety and pharmacodynamic effects of selective muscarinic M1 receptor orthosteric agonist HTL0018318 in 60 patients with mild‐to‐moderate Alzheimer's disease (AD) on background donepezil 10 mg/day.MethodsA randomized, double‐blind, placebo‐controlled 4‐week safety study of HTL0018318 with up‐titration and maintenance phases, observing exploratory effects on electrophysiological biomarkers and cognition.ResultsTreatment‐emergent adverse events (TEAEs) were mild and less frequently reported during maintenance versus titration. Headache was most commonly reported (7–21%); 0 to 13% reported cholinergic TEAEs (abdominal pain, diarrhea, fatigue, nausea) and two patients discontinued due to TEAEs. At 1 to 2 hours post‐dose, HTL0018318‐related mean maximum elevations in systolic and diastolic blood pressure of 5 to 10 mmHg above placebo were observed during up‐titration but not maintenance. Postive effects of HTL0018318 were found on specific attention and memory endpoints.DiscussionHTL0018318 was well tolerated in mild‐to‐moderate AD patients and showed positive effects on attention and episodic memory on top of therapeutic doses of donepezil.

The role of M3 receptors in regulation of electrical activity deteriorates in the rat heart during ageing.

Ageing is a complex process which affects all systems of the organism and therefore changes the environment where the heart is working. In this study we demonstrate the ageing-related changes in the mechanisms of parasympathetic regulation of mammalian heart. Electrophysiological effects produced by selective activation of M3-cholinoreceptors were compared in isolated cardiac preparations from young adult (4 months), adult (1 year) and ageing (2 years) rats using sharp glass microelectrode technique. M3-receptors were activated with muscarinic agonist pilocarpine (10-5M) in the presence of selective M2 antagonist AQ-RA741 (10-7M). In atrial and ventricular myocardium from young rats M3 stimulation induced shortening of action potentials(APs), while no significant effect was observed in both elder groups. The main mechanism of M3-induced AP shortening is inhibition of L-type Ca2+ current, estimated using whole-cell patch-clamp. It was negligible in atrial myocytes from ageing animals in comparison with young rats. The loss of sensitivity to stimulation of M3-receptors is due to decrease in M3 gene expression, shown by RT-PCR both in atrial and ventricular samples from ageing rats. Thus, in ageing rat heart M3-receptors are down-regulated and not involved in regulation of electrical activity.

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.

Neurocrine and Sosei partner for neuro drugs

Neurocrine Biosciences will pay Sosei Heptares $100 million to partner on developing small-molecule drugs for schizophrenia, dementia, and other neuropsychiatric disorders. Sosei’s compounds target specific muscarinic receptors and could have fewer side effects than drugs that target multiple receptors. Neurocrine plans a Phase 2 study next year of a selective M4 muscarinic receptor agonist for schizophrenia.

Discovery of the First Selective M4 Muscarinic Acetylcholine Receptor Antagonists with in Vivo Antiparkinsonian and Antidystonic Efficacy.

Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.