Cholinergic modulation of interhemispheric inhibition in the mouse motor cortex.

Interhemispheric inhibition of the homotopic motor cortex is believed to be effective for accurate unilateral motor function. However, the cellular mechanisms underlying interhemispheric inhibition during unilateral motor behavior remain unclear. Furthermore, the impact of the neuromodulator acetylcholine on interhemispheric inhibition and the associated cellular mechanisms are not well understood. To address this knowledge gap, we conducted recordings of neuronal activity from the bilateral motor cortex of mice during the paw-reaching task. Subsequently, we analyzed interhemispheric spike correlation at the cell-pair level, classifying putative cell types to explore the underlying cellular circuitry mechanisms of interhemispheric inhibition. We found a cell-type pair-specific enhancement of the interhemispheric spike correlation when the mice were engaged in the reaching task. We also found that the interhemispheric spike correlation was modulated by pharmacological acetylcholine manipulation. The local field responses to contralateral excitation differed along the cortical depths, and muscarinic receptor antagonism enhanced the inhibitory component of the field response in deep layers. The muscarinic subtype M2 receptor is predominantly expressed in deep cortical neurons, including GABAergic interneurons. These results suggest that GABAergic interneurons expressing muscarinic receptors in deep layers mediate the neuromodulation of interhemispheric inhibition in the homotopic motor cortex.

Identification of Psychoplastogenic Tropanes Lacking Muscarinic Activity.

Tropane-containing small molecules like scopolamine are a promising class of psychoplastogens. However, their potent antagonism of all muscarinic receptor subtypes presents the potential for undesirable anticholinergic side effects. In an effort to decouple their neuroplasticity-promoting effects from their muscarinic activity, we performed phenotypic structure-activity relationship studies across a variety of structurally distinct subclasses of tropanes. We discovered several novel tropanes capable of significantly increasing cortical neuronal growth while exhibiting drastically reduced activity at all muscarinic receptor subtypes compared to scopolamine.

Targeting the muscarinic M1 receptor with a selective, brain-penetrant antagonist to promote remyelination in multiple sclerosis.

Multiple sclerosis (MS) is a chronic and debilitating neurological disease that results in inflammatory demyelination. While endogenous remyelination helps to recover function, this restorative process tends to become less efficient over time. Currently, intense efforts aimed at the mechanisms that promote remyelination are being considered promising therapeutic approaches. The M1 muscarinic acetylcholine receptor (M1R) was previously identified as a negative regulator of oligodendrocyte differentiation and myelination. Here, we validate M1R as a target for remyelination by characterizing expression in human and rodent oligodendroglial cells (including those in human MS tissue) using a highly selective M1R probe. As a breakthrough to conventional methodology, we conjugated a fluorophore to a highly M1R selective peptide (MT7) which targets the M1R in the subnanomolar range. This allows for exceptional detection of M1R protein expression in the human CNS. More importantly, we introduce PIPE-307, a brain-penetrant, small-molecule antagonist with favorable drug-like properties that selectively targets M1R. We evaluate PIPE-307 in a series of in vitro and in vivo studies to characterize potency and selectivity for M1R over M2-5R and confirm the sufficiency of blocking this receptor to promote differentiation and remyelination. Further, PIPE-307 displays significant efficacy in the mouse experimental autoimmune encephalomyelitis model of MS as evaluated by quantifying disability, histology, electron microscopy, and visual evoked potentials. Together, these findings support targeting M1R for remyelination and support further development of PIPE-307 for clinical studies.

Discovery, Multiparametric Optimization, and Solid-State Driven Identification of CHF-6550, a Novel Soft Dual Pharmacology Muscarinic Antagonist and ß2 Agonist (MABA) for the Inhaled Treatment of Respiratory Diseases.

Clinical guidelines for COPD and asthma recommend inhaled ß-adrenergic agonists, muscarinic antagonists, and, for frequent exacerbators, inhaled corticosteroids, with the challenge of combining them into a single device. The MABA (muscarinic antagonist and ß2 agonist) concept has the potential to simplify this complexity while increasing the efficacy of both pharmacologies. In this article, we report the outcome of our solid-state driven back-up program that led to the discovery of the MABA compound CHF-6550. A soft drug approach was applied, aiming at high plasma protein binding and high hepatic clearance, concurrently with an early stage assessment of crystallinity through a dedicated experimental workflow. A new chemotype was identified, the diphenyl hydroxyacetic esters, able to generate crystalline material. Among this class, CHF-6550 demonstrated in vivo efficacy, suitability for dry powder inhaler development, favorable pharmacokinetics, and safety in preclinical settings and was selected as a back-up candidate, fulfilling the desired pharmacological and solid-state profile.

Muscarinic Modulation of Synaptic Transmission and Short-Term Plasticity in the Dorsal and Ventral Hippocampus.

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

LAMA improves tissue oxygenation more than LABA in patients with COPD.

The effect of bronchodilators is mainly assessed with forced expiratory volume in 1 s (FEV1) in chronic obstructive pulmonary disease (COPD). Their impact on oxygenation and lung periphery is less known. Our objective was to compare the action of long-acting ß2-agonists (LABA-olodaterol) and muscarinic antagonists (LAMA-tiotropium) on tissue oxygenation in COPD, considering their impact on proximal and peripheral ventilation as well as lung perfusion. FEV1, Helium slope (SHe) from a single-breath washout test (SHe decreases reflecting a peripheral ventilation improvement), frequency dependence of resistance (R5-R19), area under reactance (AX), lung capillary blood volume (Vc) from double diffusion (DLNO/DLCO), and transcutaneous oxygenation (TcO2) were measured before and 2 h post-LABA (day 1) and LAMA (day 3) in 30 patients with COPD (FEV1 54 ± 18% pred; GOLD A 31%/B 48%/E 21%) after 5-7 days of washout, respectively. We found that TcO2 increased more (P = 0.03) after LAMA (11 ± 12% from baseline, P < 001) compared with LABA (4 ± 11%, P = 0.06) despite a lower FEV1 increase (P = 0.03) and similar SHe (P = 0.98), AX (P = 0.63), and R5-R19 decreases (P = 0.37). TcO2 and SHe changes were negatively correlated (r = -0.47, P = 0.01) after LABA, not after LAMA (r = 0.10, P = 0.65). DLNO/DLCO decreased and Vc increased after LAMA (P = 0.04; P = 0.01, respectively) but not after LABA (P = 0.53; P = 0.24). In conclusion, LAMA significantly improved tissue oxygenation in patients with COPD, while only a trend was observed with LABA. The mechanisms involved may differ between both drugs: LABA increased peripheral ventilation, whereas LAMA increased lung capillary blood volume. Should oxygenation differences persist over time, LAMA could arguably become the first therapeutic choice in COPD.NEW & NOTEWORTHY Long-acting muscarinic antagonists (LAMAs) significantly improved tissue oxygenation in patients with COPD, while only a trend was observed with ß2-agonists (LABAs). The mechanisms involved may differ between drugs: increased peripheral ventilation for LABA and likely lung capillary blood volume for LAMA. This could argue for LAMA as the first therapeutic choice in COPD.

Cholinergic Neurotransmission Controls Orexigenic Endocannabinoid Signaling in the Gut in Diet-Induced Obesity.

The brain bidirectionally communicates with the gut to control food intake and energy balance, which becomes dysregulated in obesity. For example, endocannabinoid (eCB) signaling in the small-intestinal (SI) epithelium is upregulated in diet-induced obese (DIO) mice and promotes overeating by a mechanism that includes inhibiting gut-brain satiation signaling. Upstream neural and molecular mechanism(s) involved in overproduction of orexigenic gut eCBs in DIO, however, are unknown. We tested the hypothesis that overactive parasympathetic signaling at the muscarinic acetylcholine receptors (mAChRs) in the SI increases biosynthesis of the eCB, 2-arachidonoyl-sn-glycerol (2-AG), which drives hyperphagia via local CB1Rs in DIO. Male mice were maintained on a high-fat/high-sucrose Western-style diet for 60 d, then administered several mAChR antagonists 30 min prior to tissue harvest or a food intake test. Levels of 2-AG and the activity of its metabolic enzymes in the SI were quantitated. DIO mice, when compared to those fed a low-fat/no-sucrose diet, displayed increased expression of cFos protein in the dorsal motor nucleus of the vagus, which suggests an increased activity of efferent cholinergic neurotransmission. These mice exhibited elevated levels of 2-AG biosynthesis in the SI, that was reduced to control levels by mAChR antagonists. Moreover, the peripherally restricted mAChR antagonist, methylhomatropine bromide, and the peripherally restricted CB1R antagonist, AM6545, reduced food intake in DIO mice for up to 24 h but had no effect in mice conditionally deficient in SI CB1Rs. These results suggest that hyperactivity at mAChRs in the periphery increases formation of 2-AG in the SI and activates local CB1Rs, which drives hyperphagia in DIO.

A muscarinic receptor antagonist reverses multiple indices of diabetic peripheral neuropathy: preclinical and clinical studies using oxybutynin.

Preclinical studies indicate that diverse muscarinic receptor antagonists, acting via the M1 sub-type, promote neuritogenesis from sensory neurons in vitro and prevent and/or reverse both structural and functional indices of neuropathy in rodent models of diabetes. We sought to translate this as a potential therapeutic approach against structural and functional indices of diabetic neuropathy using oxybutynin, a muscarinic antagonist approved for clinical use against overactive bladder. Studies were performed using sensory neurons maintained in vitro, rodent models of type 1 or type 2 diabetes and human subjects with type 2 diabetes and confirmed neuropathy. Oxybutynin promoted significant neurite outgrowth in sensory neuron cultures derived from adult normal rats and STZ-diabetic mice, with maximal efficacy in the 1-100 nmol/l range. This was accompanied by a significantly enhanced mitochondrial energetic profile as reflected by increased basal and maximal respiration and spare respiratory capacity. Systemic (3-10 mg/kg/day s.c.) and topical (3% gel daily) oxybutynin reversed paw heat hypoalgesia in the STZ and db/db mouse models of diabetes and reversed paw tactile allodynia in STZ-diabetic rats. Loss of nerve profiles in the skin and cornea of db/db mice was also prevented by daily topical delivery of 3% oxybutynin for 8 weeks. A randomized, double-blind, placebo-controlled interventional trial was performed in subjects with type 2 diabetes and established peripheral neuropathy. Subjects received daily topical treatment with 3% oxybutynin gel or placebo for 6 months. The a priori designated primary endpoint, significant change in intra-epidermal nerve fibre density (IENFD) in skin biopsies taken before and after 20 weeks of treatments, was met by oxybutynin but not placebo. Secondary endpoints showing significant improvement with oxybutynin treatment included scores on clinical neuropathy, pain and quality of life scales. This proof-of-concept study indicates that muscarinic antagonists suitable for long-term use may offer a novel therapeutic opportunity for treatment of diabetic neuropathy. Trial registry number: NCT03050827.

Preliminary investigation of the administration of biperiden to reduce relapses in individuals with cocaine/crack user disorder: A randomized controlled clinical trial.

BACKGROUND: Several studies have demonstrated that ACh modulates the dopaminergic circuit in the nucleus accumbens, and its blockade appears to be associated with the inhibition of the reinforced effect or the increase in dopamine caused by cocaine use. The objective of this study was to evaluate the effect of biperiden (a muscarinic receptor antagonist with a relatively higher affinity for the M1 receptor) on crack/cocaine use relapse compared to a control group that received placebo. METHODS: This study is a double-blind, randomized, placebo-controlled clinical trial. The intervention group received 2 mg of biperiden, 3 times a day, for a period of 3 months. The control group received identical placebo capsules, at the same frequency and over the same period. All participants were followed for a period of six months. RESULTS: The sample comprised 128 people, with 61 in the control group and 67 in the biperiden group. Lower substance consumption was observed in the group that received biperiden treatment two (bT2 = -2.2 [-3.3; -1.0], p < 0.001) and six months (bT4 = -6, 2 [-8.6; -3.9], p < 0.001) after the beginning of the intervention. The biperiden group had a higher latency until a possible first day of consumption, in the same evaluation periods (bT2 = 0.26 [0.080; 0.44], p = 0.004; bT4 = 0.63 [0.32; 0.93], p < 0.001). CONCLUSIONS: Despite the major limitations of the present study, the group that received biperiden reduced the number of days of cocaine/crack use and showed an increase in the latency time for relapse. More studies are needed to confirm the utility of this approach.

The effect of combining an inhaled corticosteroid and a long-acting muscarinic antagonist on human airway epithelial cells in vitro.

BACKGROUND: Airway epithelial cells (AECs) are a major component of local airway immune responses. Direct effects of type 2 cytokines on AECs are implicated in type 2 asthma, which is driven by epithelial-derived cytokines and leads to airway obstruction. However, evidence suggests that restoring epithelial health may attenuate asthmatic features. METHODS: We investigated the effects of passive sensitisation on IL-5, NF-κB, HDAC-2, ACh, and ChAT in human bronchial epithelial cells (HBEpCs) and the effects of fluticasone furoate (FF) and umeclidinium (UME) alone and in combination on these responses. RESULTS: IL-5 and NF-κB levels were increased, and that of HDAC-2 reduced in sensitised HEBpCs. Pretreatment with FF reversed the effects of passive sensitisation by concentration-dependent reduction of IL-5, resulting in decreased NF-κB levels and restored HDAC-2 activity. Addition of UME enhanced these effects. Sensitized HEBpCs also exhibited higher ACh and ChAT levels. Pretreatment with UME significantly reduced ACh levels, and addition of FF caused a further small reduction. CONCLUSION: This study confirmed that passive sensitisation of AECs results in an inflammatory response with increased levels of IL-5 and NF-κB, reduced levels of HDAC-2, and higher levels of ACh and ChAT compared to normal cells. Combining FF and UME was found to be more effective in reducing IL-5, NF-κB, and ACh and restoring HDAC-2 compared to the individual components. This finding supports adding a LAMA to established ICS/LABA treatment in asthma and suggests the possibility of using an ICS/LAMA combination when needed.

A preliminary study of the effects of an antimuscarinic agent on anxious behaviors and white matter microarchitecture in nonhuman primates.

Myelination subserves efficient neuronal communication, and alterations in white matter (WM) microstructure have been implicated in numerous psychiatric disorders, including pathological anxiety. Recent work in rodents suggests that muscarinic antagonists may enhance myelination with behavioral benefits; however, the neural and behavioral effects of muscarinic antagonists have yet to be explored in non-human primates (NHP). Here, as a potentially translatable therapeutic strategy for human pathological anxiety, we present data from a first-in-primate study exploring the effects of the muscarinic receptor antagonist solifenacin on anxious behaviors and WM microstructure. 12 preadolescent rhesus macaques (6 vehicle control, 6 experimental; 8F, 4M) were included in a pre-test/post-test between-group study design. The experimental group received solifenacin succinate for ~60 days. Subjects underwent pre- and post-assessments of: 1) anxious temperament (AT)-related behaviors in the potentially threatening no-eye-contact (NEC) paradigm (30-min); and 2) WM and regional brain metabolism imaging metrics, including diffusion tensor imaging (DTI), quantitative relaxometry (QR), and FDG-PET. In relation to anxiety-related behaviors expressed during the NEC, significant Group (vehicle control vs. solifenacin) by Session (pre vs. post) interactions were found for freezing, cooing, and locomotion. Compared to vehicle controls, solifenacin-treated subjects exhibited effects consistent with reduced anxiety, specifically decreased freezing duration, increased locomotion duration, and increased cooing frequency. Furthermore, the Group-by-Session-by-Sex interaction indicated that these effects occurred predominantly in the males. Exploratory whole-brain voxelwise analyses of post-minus-pre differences in DTI, QR, and FDG-PET metrics revealed some solifenacin-related changes in WM microstructure and brain metabolism. These findings in NHPs support the further investigation of the utility of antimuscarinic agents in targeting WM microstructure as a means to treat pathological anxiety.

Design, synthesis and preclinical evaluation of muscarine receptor antagonists via a scaffold-hopping approach.

Our research group recently identified a rearrangement product of pirenzepine as starting point for a comprehensive rational drug design approach towards orthosteric muscarinic acetylcholine receptor ligands. Chemical reduction and bioscaffold hop lead to the development of sixteen promising compounds featuring either a benzimidazole or carbamate moiety, all exhibiting comparable pharmacophoric characteristics. The synthesized compounds were characterized by NMR, HR-MS, and RP-HPLC techniques. Subsequent evaluation encompassed binding affinity assessment on CHO-hM1-5 cells, mode of action determination, and analysis of physico-chemical parameters. The CNS MPO score indicated favorable drug-like attributes and potential CNS activity for the antagonistic ligands. The most promising compounds displayed Ki-values within a desirable low nanomolar range, and their structural features allow for potential carbon-11 radiolabeling. Our optimization efforts resulted in compounds with a remarkable 138-fold increase in binding affinity compared to the previously mentioned rearrangement product towards human M5, suggesting their prospective utility in positron emission tomography applications.

The role of the serotonergic system in atropine's anti-myopic effects.

The muscarinic cholinergic antagonist atropine is the most widely used pharmacological treatment for the visual disorder myopia (short-sightedness), the leading cause of low-vision worldwide. This study sought to better define the mechanism by which atropine inhibits myopic growth. Although classified as a muscarinic-cholinergic antagonist, atropine has been found to bind and modulate the activity of several non-cholinergic systems (e.g., serotonin). Thus, this study investigated whether the serotonergic system could underly atropine's anti-myopic effects. Using a chick model of myopia, we report that atropine's growth-inhibitory effects can be attenuated by pharmacological stimulation of the serotonin system. This may suggest that atropine can slow the development of myopia through inhibiting serotonergic receptor activity. We also observed that pharmacological antagonism of serotonergic receptors inhibits the development of experimental myopia in a dose-dependent manner, further demonstrating that modulation of serotonergic receptor activity can alter ocular growth rates. Finally, we found that neither experimental myopia, nor atropine treatment, induced a significant change in retinal serotonergic output (i.e., synthesis, transport, release and catabolism). This may suggest that, although myopic growth can be inhibited through modulation of serotonergic receptor activity (by atropine or serotonergic antagonists), this does not require a change in serotonin levels. These findings regarding a serotonergic mechanism for atropine may have significant ramifications for the treatment of human myopia. This includes assessing the use of atropine in patients who are also undergoing treatment to upregulate serotonergic signaling (e.g., serotonergic anti-depressants).

Activating M1 muscarinic cholinergic receptors induces destabilization of resistant contextual fear memories in rats.

Destabilization of previously consolidated memories places them in a labile state in which they are open to modification. However, strongly encoded fear memories tend to be destabilization-resistant and the conditions required to destabilize such memories remain poorly understood. Our lab has previously shown that exposure to salient novel contextual cues during memory reactivation can destabilize strongly encoded object location memories and that activity at muscarinic cholinergic receptors is critical for this effect. In the current study, we similarly targeted destabilization-resistant fear memories, hypothesizing that exposure to salient novelty at the time of reactivation would induce destabilization of strongly encoded fear memories in a muscarinic receptor-dependent manner. First, we show that contextual fear memories induced by 3 context-shock pairings readily destabilize upon memory reactivation, and that this destabilization is blocked by systemic (ip) administration of the muscarinic receptor antagonist scopolamine (0.3 mg/kg) in male rats. Following that, we confirm that this effect is dorsal hippocampus (dHPC)-dependent by targeting M1 receptors in the CA1 region with pirenzepine. Next, we show that more strongly encoded fear memories (induced with 5 context-shock pairings) resist destabilization. Consistent with our previous work, however, we report that salient novelty (a change in floor texture) presented during the reactivation session promotes destabilization of resistant contextual fear memories in a muscarinic receptor-dependent manner. Finally, the effect of salient novelty on memory destabilization was mimicked by stimulating muscarinic receptors with the selective M1 agonist CDD-0102A (ip, 0.3 mg/kg). These findings reveal further generalizability of our previous results implicating novel cues and M1 muscarinic signaling in promoting destabilization of resistant memories and suggest possible therapeutic options for disorders characterized by persistent, maladaptive fear memories such as PTSD and phobias.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor.

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights into this inhibitor remain unclear; specifically, the elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

The effect of adding Montelukast to oxybutynin on daily urination in children with pollakiuria: a randomized clinical trial.

PURPOSE: Pollakiuria is defined as a change in the pattern of daily urination. Students have mentioned wetting their pants at school as the third tragic event after the death of a parent or going blind. In this study, the effect of adding Montelukast to oxybutynin on the improvement of urinary symptoms of patients with pollakiuria was studied. MATERIALS AND METHODS: This study was a pilot clinical trial in which children with pollakiuria aged 3-18 years old were included. These children were randomly divided into two groups of intervention (Montelukast plus oxybutynin) and the control group (only oxybutynin). At the beginning and the end of the study (after 14 days), mothers were asked about the frequency of daily urination. Finally, the gathered data were compared between two groups. RESULTS: In the present study, 64 patients were examined in two intervention and control groups (32 in each group). The results revealed that although significant changes were observed in both groups before and after intervention, the average changes in the intervention group were significantly higher (p = 0.014). CONCLUSION: The results of this study showed that adding montelukast to oxybutynin has a significant decrease in frequency of daily urination in patients with pollakiuria, although further studies are recommended in this area.

Might It Be Appropriate to Anticipate the Use of Long-Acting Muscarinic Antagonists in Asthma?

A growing number of clinical trials are documenting that adding a long-acting muscarinic antagonist (LAMA) to established asthma treatment with an inhaled corticosteroid (ICS) and a long-acting ß2-agonist (LABA) is a treatment option that improves the health of patients with uncontrolled severe asthma even when therapy is optimized. These favorable results are the reason why the leading guidelines recommend triple therapy with ICS + LABA + LAMA in patients with asthma uncontrolled by medium- to high-dose ICS-LABA. However, we suggest adding LAMAs to ICS-LABAs at an earlier clinical stage. Such action could positively influence airflow limitation, exacerbations, and eosinophilic inflammation, conditions that are associated with acetylcholine (ACh) activity. It could also interrupt the vicious cycle related to a continuous release of ACh leading to the progressive expansion of neuronal plasticity resulting in small airway dysfunction. The utility of an earlier use of triple therapy in asthma should, in any case, be confirmed by statistically powered trials.

Experimental drugs in clinical trials for COPD: artificial intelligence via machine learning approach to predict the successful advance from early-stage development to approval.

INTRODUCTION: Therapeutic advances in drug therapy of chronic obstructive pulmonary disease (COPD) really effective in suppressing the pathological processes underlying the disease deterioration are still needed. Artificial Intelligence (AI) via Machine Learning (ML) may represent an effective tool to predict clinical development of investigational agents. AREAL COVERED: Experimental drugs in Phase I and II development for COPD from early 2014 to late 2022 were identified in the ClinicalTrials.gov database. Different ML models, trained from prior knowledge on clinical trial success, were used to predict the probability that experimental drugs will successfully advance toward approval in COPD, according to Bayesian inference as follows: ≤25% low probability, >25% and ≤50% moderate probability, >50% and ≤75% high probability, and >75% very high probability. EXPERT OPINION: The Artificial Neural Network and Random Forest ML models indicated that, among the current experimental drugs in clinical trials for COPD, only the bifunctional muscarinic antagonist - ß2-adrenoceptor agonists (MABA) navafenterol and batefenterol, the inhaled corticosteroid (ICS)/MABA fluticasone furoate/batefenterol, and the bifunctional phosphodiesterase (PDE) 3/4 inhibitor ensifentrine resulted to have a moderate to very high probability of being approved in the next future, however not before 2025.