M1 Muscarinic Receptor Antagonist Research Articles

Involvement of Muscarinic M3 Receptor in the Development of M2 Macrophages in Allergic Inflammation.

The muscarinic M3 receptor antagonist, tiotropium, has a bronchodilatory effect on asthma patients. Additionally, tiotropium inhibits allergic airway inflammation and remodeling in a murine asthma model. However, the underlying mechanisms of this M3 receptor antagonist remain unclear. Therefore, we investigated the effect of muscarinic M3 receptor blockage on M2 macrophage development during allergic airway inflammation. BALB/c mice were sensitized and challenged with ovalbumin to develop a murine model of allergic airway inflammation mimicking human atopic asthma. During the challenge phase, mice were treated with or without tiotropium. Lung cells were isolated 24 h after the last treatment and gated using CD68-positive cells. Relm-α and Arginase-1 (Arg1) (M2 macrophage markers) expression was determined by flow cytometry. Mouse bone marrow mononuclear cell-derived macrophages (mBMMacs) and human peripheral blood mononuclear cells (PBMCs)-derived macrophages were stimulated with IL-4 and treated with a muscarinic M3 receptor antagonist in vitro. The total cells, eosinophils, and IL-5 and IL-13 levels in BAL fluids were markedly decreased in the asthma group treated with tiotropium compared to that in the untreated asthma group. The Relm-α and Arg1 expression in macrophages was reduced considerably in the asthma group treated with tiotropium compared to that in the untreated asthma group, suggesting that the development of M2 macrophages was inhibited by muscarinic M3 receptor blockage. Additionally, muscarinic M3 receptor blockage in vitro significantly inhibited M2 macrophage development in both mBMMacs- and PBMCs-derived macrophages. Muscarinic M3 receptor blockage inhibits M2 macrophage development and prevents allergic airway inflammation. Moreover, muscarinic M3 receptors might be involved in the differentiation of immature macrophages into M2 macrophages.

Biperiden reverses the increase in 50-kHz ultrasonic vocalizations but not the increase in locomotor activity induced by cocaine

Cocaine use disorder (CUD) is a worldwide public health problem, associated with severe psychosocial and economic impacts. Currently, no FDA-approved treatment is available for CUD. However, an emerging body of evidence from clinical and preclinical studies suggests that biperiden, an M1 muscarinic receptor antagonist, presents potential therapeutic use for CUD. These studies have suggested that biperiden may reduce the reinforcing effects of cocaine. It is well established that rodents emit 50-kHz ultrasonic vocalizations (USV) in response to natural rewards and stimulant drugs, including cocaine. Nonetheless, the effects of biperiden on the cocaine-induced increase of 50-kHz USV remains unknown. Here, we hypothesized that biperiden could antagonize the acute effects of cocaine administration on rat 50-kHz USV. To test this hypothesis, adult male Wistar rats were divided into four experimental groups: saline, 5 mg/kg biperiden, 10 mg/kg cocaine, and biperiden/cocaine (5 and 10 mg/kg, i.p., respectively). USV and locomotor activity were recorded in baseline and test sessions. As expected, cocaine administration significantly increased the number of 50-kHz USV. Biperiden administration effectively antagonized the increase in 50-kHz USV induced by cocaine. Cocaine administration also increased the emission of trill and mixed 50 kHz USV subtypes and this effect was antagonized by biperiden. Additionally, we showed that biperiden did not affect the cocaine-induced increase in locomotor activity, although biperiden administration per se increased locomotor activity. In conclusion, our findings indicate that administering biperiden acutely reduces the positive affective effects of cocaine, as demonstrated by its ability to inhibit the increase in 50-kHz USV.

The local GLP-1 system in the olfactory bulb is required for odor-evoked cephalic phase of insulin release in mice

ObjectiveThe olfactory bulb (OB) codes for sensory information and contributes to the control of energy metabolism by regulating foraging and cephalic phase responses. Mitral cells are the main output neurons of the OB. The glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) system in the OB (GLP-1OB) has been shown to be a major regulator of mitral cell activity but its function in vivo is unclear. Therefore, we investigated the role of GLP-1OB in foraging behavior and odor-evoked Cephalic Phase Insulin Release (CPIR). Methods and resultsBy fluorescent labeling, we confirmed the presence of GLP-1 producing neurons and the expression of GLP-1R in the mouse OB. In response to food odor presentation, we collected blood, quantified plasma insulin by ELISA and showed the existence of an odor-evoked CPIR in lean mice but its absence in obese animals. Expression of shRNA against preproglucagon mRNA in the OB resulted in blunted CPIR in lean mice. Injecting Exendin (9-39), a GLP-1R antagonist, into the OB of lean mice also resulted in decreased CPIR. Since parasympathetic cholinergic input to the pancreas is known to be partly responsible for CPIR, we systemically administered the muscarinic M3 receptor antagonist 4-DAMP which resulted in a reduced odor-evoked CPIR. Finally, local injection of Exendin (9-39) in the OB extinguished olfactory foraging in lean mice whereas the injection of the GLP-1R agonist Exendin-4 rescued the loss of foraging behavior in obese mice. ConclusionsOur results demonstrate that GLP-1OB controls olfactory foraging and is required for odor-evoked CPIR. We describe a new crucial brain function for GLP-1 and GLP-1R expressed within the brain.

Abstract #1401109: Quetiapine-induced Hypoglycemia in an Elderly Non-diabetic Patient with Dementia

Quetiapine is a second-generation antipsychotic agent (SGA) widely prescribed for the treatment of psychotic and mood disorders. In elderly patients, quetiapine is often used to treat psychotic symptoms associated with dementia. A metabolic syndrome phenotype of weight gain, hyperglycemia, and hyperlipidemia is a potential adverse effect of SGA’s, though hypoglycemia caused by SGA’s is documented. We report a case of quetiapine-induced hypoglycemia in a non-diabetic patient. A 79-year-old male with Lewy body dementia was admitted to hospital for management of a myocardial infarction. The patient had no history of diabetes mellitus, renal failure, or hepatic insufficiency, and nobody living with the patient was treated for diabetes. Plasma glucose on admission was 84 mg/dL. Quetiapine was started to control delirium, but within 24 h the patient had worsening confusion and a capillary blood glucose (CBG) measurement of 52 mg/dL. Symptoms improved with administration of dextrose, and the patient ultimately required a 10% dextrose infusion to maintain CBG’s >80 mg/dL. An ACTH-stimulation test (30 min stimulated cortisol 24.8 μg/dL, expected >18) and hypoglycemic medications panel (sulfonylureas and meglitinides) were unremarkable. Quetiapine was stopped, and hypoglycemia resolved. No hypoglycemia occurred during a subsequent 48-h diagnostic fast followed by a mixed meal tolerance test. Due to the patient’s advanced age and comorbidities, it was decided not re-challenge with quetiapine and observe for recurrence of hypoglycemia. Hypoglycemia is a rare complication of SGA’s, and diagnosis may be delayed or missed in patients with baseline confusion or agitation. In addition to quetiapine, there are case reports of hypoglycemia occurring during treatment with risperidone, olanzapine, aripiprazole, and paliperidone. Clozapine and olanzapine increase insulin release from isolated pancreatic islets in vitro, though quetiapine did not demonstrate this effect. However, endogenous hyperinsulinemia has been documented in case reports of quetiapine-induced hypoglycemia. The physiologic pathway by which SGA’s increase insulin release is unknown; some authors have focused on the antagonism of M1 muscarinic receptors, but the muscarinic M1 receptor antagonist pirenzepine has been demonstrated to reduce insulin secretion induced by the muscarinic receptor agonist carbachol from isolated rat pancreatic islets in vitro. Though the mechanism by which hypoglycemia occurs is unclear, this case demonstrates that hypoglycemia should be considered in patients with worsening mental status during treatment with SGA’s.

Unbinding Kinetics of Muscarinic M3 Receptor Antagonists Explained by Metadynamics Simulations.

The residence time (RT), the time for which a drug remains bound to its biological target, is a critical parameter for drug design. The prediction of this key kinetic property has been proven to be challenging and computationally demanding in the framework of atomistic simulations. In the present work, we setup and applied two distinct metadynamics protocols to estimate the RTs of muscarinic M3 receptor antagonists. In the first method, derived from the conformational flooding approach, the kinetics of unbinding is retrieved from a physics-based parameter known as the acceleration factor α (i.e., the running average over time of the potential deposited in the bound state). Such an approach is expected to recover the absolute RT value for a compound of interest. In the second method, known as the tMETA-D approach, a qualitative estimation of the RT is given by the time of simulation required to drive the ligand from the binding site to the solvent bulk. This approach has been developed to reproduce the change of experimental RTs for compounds targeting the same target. Our analysis shows that both computational protocols are able to rank compounds in agreement with their experimental RTs. Quantitative structure-kinetics relationship (SKR) models can be identified and employed to predict the impact of a chemical modification on the experimental RT once a calibration study has been performed.

ADME properties of CHF6366, a novel bi-functional M3 muscarinic receptor antagonist and ß2 adrenoceptor agonist (MABA) radiolabelled at both functional moieties

CHF6366, a dual action β2-receptor agonist and M3-muscarinic receptor antagonist developed for chronic obstructive pulmonary disease (COPD) was [14C]-radiolabelled on the two different functional moieties of the molecule (either aminobutanolic or carbamate) to characterise its ADME profile following intravenous (IV), intratracheal (IT) and oral (PO) administration. A very low oral bioavailability and a good balance between absorption and lung retention after IT administration were observed, together with a rapid distribution throughout the body and a complete metabolic transformation of the parent drug without relevant gender difference. CHF6366 was observed fully hydrolysed to alcohol (CHF6387) and carboxylic acid (CHF6361) in plasma and urine after IV and IT administration, and mainly unchanged in faeces only after oral administration. An important number of metabolites containing aminobutanolic moiety was excreted via urine, whereas carbamate-containing derivatives were excreted mainly by bile. The major metabolic routes of the alcoholic moiety (CHF6387) included isomerisation (Ma7), conjugation with glucuronic acid and dehydrogenation, while the carboxylic acid moiety (CHF6361) was mainly metabolised through oxidation, glucuronide conjugation and, in both pathways, combinations of those metabolic reactions. No major differences arose also from in vitro metabolism profiles investigated using liver microsomes and hepatocytes of different species.

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.

Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction.PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the β2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect.In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

Design, Synthesis, and Evaluation of Lung-Retentive Prodrugs for Extending the Lung Tissue Retention of Inhaled Drugs.

A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 μM. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.

Efficacy and Safety of DA-8010, a Novel M3 Antagonist, in Patients With Overactive Bladder: A Randomized, Double-Blind Phase 2 Study.

PurposeDA-8010 is a novel muscarinic M3 receptor antagonist with significant selectivity for bladder over salivary gland in preclinical studies. We evaluated the clinical efficacy and safety of DA-8010 in overactive bladder (OAB) patients.MethodsThis phase 2, randomized, double-blind, parallel-group, active reference- and placebo-controlled trial was conducted at 12 centers in South Korea (NCT03566134). Patients aged ≥19 years with OAB symptoms for ≥3 months were enrolled. Three hundred six patients (30.07% male) were randomized to 12 weeks of treatment among 4 groups; 2 experimental groups (DA-8010 2.5 or 5 mg), an active reference group (solifenacin 5 mg), and a placebo group. The change from the baseline of (=∆) 24-hour frequency at 12 weeks (primary endpoint), episodes of urgency, overall/urgency urinary incontinence, average/ maximum voided volume, nocturia, and patients’ subjective responses were analyzed.ResultsIn the full analysis set, the mean (standard deviation) [median] values for ∆ 24-hour frequency at 12 weeks were -1.01 (2.44) [-1.33] for placebo, -1.22 (2.05) [-1.33] for DA-8010 2.5 mg, and -1.67 (2.25) [-1.67] for DA-8010 5 mg; DA-8010 5 mg showed a significant decrease compared with placebo (P=0.0413). At 4 and 8 weeks, both DA-8010 2.5 mg (P=0.0391 at 4 weeks, P=0.0335 at 8 weeks) and DA-8010 5 mg (P=0.0001 at 4 weeks, P=0.0210 at 8 weeks) showed significant decrease in ∆ 24-hour frequency compared with placebo. DA-8010 5 mg achieved a significant decrease in ∆ number of urgency episodes, compared with placebo at 4 (P=0.0278) and 8 (P=0.0092) weeks. Adverse drug reactions (ADRs) were observed in 3.95% of placebo, 6.67% of DA-8010 2.5 mg, 18.42% of DA-8010 5 mg, and 17.33% of solifenacin 5 mg groups. No serious ADRs were observed in any patient.ConclusionsBoth DA-8010 2.5 mg and 5 mg showed therapeutic efficacy for OAB without serious ADRs. Therefore, both dosages of DA-8010 can advance to a subsequent large-scale phase 3 trial.

Antihypertensive Mechanism of Orally Administered Acetylcholine in Spontaneously Hypertensive Rats.

Acetylcholine (ACh) acts as a neurotransmitter and neuromodulator. A small dose of eggplant powder rich in ACh (equivalent to 22 g fresh eggplant/d) has been shown to reduce blood pressure (BP) in individuals with higher BP. Here, we investigated the mechanisms underlying the antihypertensive effects of low-dose orally administered ACh in spontaneously hypertensive rats (SHRs). The effects of ACh on BP and sympathetic nervous activity (SNA), including lumbar SNA (LSNA) and renal SNA (RSNA), were evaluated by subjecting conscious SHRs to a telemetry method. Single oral administration of ACh decreased LSNA and lowered BP. Repeated oral administration of ACh for 30 d decreased RSNA and suppressed the elevated BP. Noradrenaline levels in the urine also decreased. However, vagotomy and co-administration of M3 muscarinic ACh receptor antagonist reversed the BP-lowering effect; the dynamics of non-absorbable orally administered ACh was revealed using stable isotope-labeled ACh. In conclusion, ACh acts on the gastrointestinal M3 muscarinic ACh receptor to increase afferent vagal nerve activity, which decreases SNA by autonomic reflex, suppressing noradrenaline release and lowering BP. This study suggests the use of exogenous ACh as an antihypertensive food supplement for controlling the autonomic nervous system, without absorption into the blood.

The Acetylcholine-Muscarinic Receptor Pathway Promotes Type 2 Immune Responses to Inhaled Allergens

Non-neuronal cells, including airway epithelial cells and immune cells, are capable of producing acetylcholine (ACh), and they express muscarinic ACh receptors. M3 muscarinic receptor antagonists have been used clinically to treat asthma. However, our knowledge is limited regarding the roles of ACh and its receptors in immune responses.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Excitatory neurotransmission activates compartmentalized calcium transients in Müller glia without affecting lateral process motility.

Neural activity has been implicated in the motility and outgrowth of glial cell processes throughout the central nervous system. Here, we explore this phenomenon in Müller glia, which are specialized radial astroglia that are the predominant glial type of the vertebrate retina. Müller glia extend fine filopodia-like processes into retinal synaptic layers, in similar fashion to brain astrocytes and radial glia that exhibit perisynaptic processes. Using two-photon volumetric imaging, we found that during the second postnatal week, Müller glial processes were highly dynamic, with rapid extensions and retractions that were mediated by cytoskeletal rearrangements. During this same stage of development, retinal waves led to increases in cytosolic calcium within Müller glial lateral processes and stalks. These regions comprised distinct calcium compartments, distinguished by variable participation in waves, timing, and sensitivity to an M1 muscarinic acetylcholine receptor antagonist. However, we found that motility of lateral processes was unaffected by the presence of pharmacological agents that enhanced or blocked wave-associated calcium transients. Finally, we found that mice lacking normal cholinergic waves in the first postnatal week also exhibited normal Müller glial process morphology. Hence, outgrowth of Müller glial lateral processes into synaptic layers is determined by factors that are independent of neuronal activity.

Effects of the Partial M1 Muscarinic Cholinergic Receptor Agonist CDD-0102A on Stereotyped Motor Behaviors and Reversal Learning in the BTBR Mouse Model of Autism.

BackgroundAutism spectrum disorders (ASD) are a set of neurodevelopmental disorders marked by a lack of social interaction, restrictive interests, and repetitive behaviors. There is a paucity of pharmacological treatments to reduce core ASD symptoms. Various lines of evidence indicate that reduced brain muscarinic cholinergic receptor activity may contribute to an ASD phenotype.MethodsThe present experiments examined whether the partial M1 muscarinic receptor agonist, 5-(3-ethyl-1,2,4-oxadiazol-5-yl)-1,4,5,6-tetrahydropyrimidine hydrochloride (CDD-0102A), alleviates behavioral flexibility deficits and/or stereotyped motor behaviors in the BTBR mouse model of autism. Behavioral flexibility was tested using a reversal learning test. Stereotyped motor behaviors were measured by eliciting digging behavior after removal of nesting material in a home cage and by measuring repetitive grooming.ResultsCDD-0102A (0.2 and 0.6 mg/kg but not 1.2 mg/kg) injected prior to reversal learning attenuated a deficit in BTBR mice but did not affect performance in B6 mice. Acute CDD-0102A treatment (1.2 and 3 mg/kg) reduced self-grooming in BTBR mice and reduced digging behavior in B6 and BTBR mice. The M1 muscarinic receptor antagonist VU0255035 (3 mg/kg) blocked the effect of CDD-0102A on grooming behavior. Chronic treatment with CDD-0102A (1.2 mg/kg) attenuated self-grooming and digging behavior in BTBR mice. Direct CDD-0102A infusions (1 µg) into the dorsal striatum reduced elevated digging behavior in BTBR mice. In contrast, CDD-0102A injections in the frontal cortex were not effective.ConclusionsThe results suggest that treatment with a partial M1 muscarinic receptor agonist may reduce repetitive behaviors and restricted interests in autism in part by stimulating striatal M1 muscarinic receptors.

Effects of pirenzepine on vonoprazan-induced gastric acid inhibition and hypergastrinemia.

Compared to proton pump inhibitors, vonoprazan exerts a greater inhibitory effect on gastric acid secretion and is useful for treating acid-related diseases, such as gastro-esophageal reflux disease. However, there is a problem that vonoprazan causes hypergastrinemia, which confers a risk of carcinoid tumor. A previous report demonstrated that pirenzepine, an M1 muscarinic receptor antagonist, enhances the acid inhibitory effects while suppressing hypergastrinemia induced by omeprazole. Here, we examined whether pirenzepine enhances the gastric acid inhibitory effects of vonoprazan without further increasing serum gastrin levels. Eleven healthy volunteers were subjected to 24-h intragastric pH monitoring and serum gastrin measurements on day 7 of three different regimens: pirenzepine 75mg alone, vonoprazan 10mg alone, and vonoprazan 10mg plus pirenzepine 75mg administered in a randomized crossover fashion. Median pH 4 holding time ratios (range) achieved with pirenzepine 75mg, vonoprazan 10mg, and vonoprazan 10mg plus pirenzepine 75mg were 6.9% (2.4-32.8%), 88.4% (54.6-100%), and 84.2% (40.3-100%), respectively. Respective serum gastrin levels were 79 (75-210) pg/ml, 310 (110-870) pg/ml, and 170 (140-930) pg/ml. In cases with hypergastrinemia (gastrin ≥ 200pg/ml) induced by vonoprazan 10mg alone, concomitant treatment with pirenzepine significantly reduced serum gastrin levels from 370 to 180pg/ml (P = 0.028). Although pirenzepine does not enhance acid inhibition, it does improve hypergastrinemia induced by vonoprazan to some extent.

Selective or Specific M1 Muscarinic Receptor Antagonists Exhibit Biased Agonism at the M1 Receptor via β‐arrestin Signaling

Introduction Selective (pirenzepine; PZ) and specific (muscarinic toxin 7; MT7) antagonists of the muscarinic acetylcholine type 1 receptor (M1R) reverse nerve degeneration in rodent models of peripheral neuropathy. These drugs drive axonal outgrowth of adult sensory neurons and signaling is mediated via extracellular-regulated protein kinase (ERK) and β-arrestin. In order to understand the pharmacological mechanism of action of PZ and MT7, we hypothesized that these drugs exhibited biased agonism at the M1R mediated via b-arrestin signaling. Methods Nano bioluminescence resonance energy transfer (NanoBRET) assay: Human embryonic kidney (HEK293) cells co-expressing human M1R-Nluc and Halo-tagged β-arrestin 2 were treated with different doses of M1R agonists (muscarine or carbachol) or antagonists (PZ and MT7)at different time points. NanoBRET assay was employed to measure ligand-induced β-arrestin 2 recruitment to the M1R. Assays were performed in triplicate with 3 independent experiments completed. Data are presented as corrected milli-BRET (mBRET) units. Substrate was added 2 min before BRET detection, and drugs were applied at various time points. Data are reported as the mean ± SD (statistics by 1-way ANOVA with Tukey's post-hoc test). Inositol-phosphate one (IP1) measurement: Intracellular IP1 levels were measured using IP-One assay (Cisbio Bioassays, USA). HEK293 cells co-expressing hM1R-Nluc and Halo-tagged β-arrestin 2 were plated onto 384-well white microplates (20000 cells per well) and were treated with stimulation buffer with or without 1) different doses of M1R agonist (muscarine), 2) M1R antagonists (PZ and MT7) or 3) both M1R antagonist (different doses of PZ) and agonist (muscarine, 1mM) for different time points at 37 °C. Cells were lysed by addition of the supplied buffer containing d2-labeled IP1, followed by addition of terbium cryptate-labeled anti-IP1 antibody, according to the manufacturer's instructions. Plates were incubated for 1 h at room temperature and fluorescence signals were measured at 620 and 665 nm using the Biotek Synergy Neo2 plate reader. HTRF ratio 665 nm/620 nm of each well were extrapolated on the standard curve to obtain IP1 concentrations. Results Both carbachol (Fig. 1A) and muscarine (Fig. 1B) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at 5 min of treatment. pirenzepine (Fig. 1C) and MT7 (Fig. 1D) induced Halo-tagged β-arrestin 2 recruitment to hM1R-Nluc in a dose-dependent manner at longer time points (e.g. after 30 min). Interestingly, over short periods, e.g. 2-3 min, PZ and MT7 acted as classical antagonists with blockade of muscarine-induced b-arrestin 2 association with M1R. Unlike MT7 and PZ, muscarine increased IP1 levels in transfected cells (Fig. 2A). Also, PZ dose-dependently inhibited the formation of IP1 following muscarine (1mM) treatment (Fig. 2B). Conclusion Thisstudy for the first time provides molecular evidence to support a role for selective/specific muscarinic receptor antagonists acting as biased agonists at the M1R. This novel signaling pathway may contribute to driving axonal regeneration in adult sensory neurons and could be mobilized for nerve repair in neuropathic disease.

Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

The Firing of Theta State-Related Septal Cholinergic Neurons Disrupt Hippocampal Ripple Oscillations via Muscarinic Receptors.

The septo-hippocampal cholinergic system is critical for hippocampal learning and memory. However, a quantitative description of the in vivo firing patterns and physiological function of medial septal (MS) cholinergic neurons is still missing. In this study, we combined optogenetics with multichannel in vivo recording and recorded MS cholinergic neuron firings in freely behaving male mice for 5.5-72 h. We found that their firing activities were highly correlated with hippocampal theta states. MS cholinergic neurons were highly active during theta-dominant epochs, such as active exploration and rapid eye movement sleep, but almost silent during non-theta epochs, such as slow-wave sleep (SWS). Interestingly, optogenetic activation of these MS cholinergic neurons during SWS suppressed CA1 ripple oscillations. This suppression could be rescued by muscarinic M2 or M4 receptor antagonists. These results suggest the following important physiological function of MS cholinergic neurons: maintaining high hippocampal acetylcholine level by persistent firing during theta epochs, consequently suppressing ripples and allowing theta oscillations to dominate.SIGNIFICANCE STATEMENT The major source of acetylcholine in the hippocampus comes from the medial septum. Early experiments found that lesions to the MS result in the disappearance of hippocampal theta oscillation, which leads to speculation that the septo-hippocampal cholinergic projection contributing to theta oscillation. In this article, by long-term recording of MS cholinergic neurons, we found that they show a theta state-related firing pattern. However, optogenetically activating these neurons shows little effect on theta rhythm in the hippocampus. Instead, we found that activating MS cholinergic neurons during slow-wave sleep could suppress hippocampal ripple oscillations. This suppression is mediated by muscarinic M2 and M4 receptors.

Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists.

Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.