M1 Receptor Antagonist Research Articles

Darifenacin: a selective M3muscarinic receptor antagonist for the treatment of overactive bladder

Overactive bladder is a common and distressing disorder that imposes significant financial and quality-of-life costs. The current therapeutic paradigm aims to decrease detrusor overactivity via blockade of bladder M3 muscarinic receptors, the primary cholinergic receptors responsible for detrusor contraction. However, systemic antimuscarinic adverse effects, such as dry mouth and constipation, limit the tolerability of antimuscarinic treatment. Therefore, a uroselective M3 receptor antagonist would be considered optimal therapy for overactive bladder. Darifenacin (Enablex®) is the most recent antimuscarinic drug approved for the treatment of overactive bladder. It demonstrates M3 receptor selectivity, but is not M3 specific. Despite animal data suggesting uroselectivity, it is not sufficiently uroselective in therapeutic use. While effective in reducing symptoms related to overactive bladder, dry mouth and constipation remain common. There are no comparative trials comparing darifenacin with existing agen...

UP-01.88: Darifenacin is also effective in neurogenic bladder dysfunction (multiple sclerosis)

60 percent of the patients suffering from multiple sclerosis (MS) show detrusor hyperactivity; 50 percent of the patients present with incomplete emptying of the bladder. Treatment must offer good efficacy without an increase in the formation of residual urine. Since more than 40 percent of the MS patients have an impaired hand function the objective of the study is to investigate whether treatment with darifenacin, a selective muscarinic M3 receptor antagonist, can prevent catheterization.

Dehydroepiandrosterone sulfate and estrone sulfate reduce GABA‐recurrent inhibition in the hippocampus via muscarinic acetylcholine receptors

Several recent studies have established a role for estrogens in ameliorating specific neurodegenerative disorders, mainly those associated with the cholinergic neurons of the basal forebrain and their targets in the cortex and hippocampus. We have previously demonstrated that endogenous and exogenous application of the neurosteroid dehydroepiandrosterone sulfate (DHEAS) markedly reduces GABA-mediated recurrent inhibition and synchronizes hippocampal unit activity to theta rhythm (Steffensen (1995) Hippocampus 5:320-328). In this study, we evaluated the role of muscarinic receptors in mediating the effects of DHEAS and estrone sulfate (ES), the principal circulating estrogen in humans, on short-latency-evoked potential responses, paired-pulse inhibition (PPI), paired-pulse facilitation, and GABA interneuron activity in the dentate gyrus and CA1 subfields of the rat hippocampus. In situ microelectrophoretic application of the muscarinic M2 subtype cholinergic receptor agonist cis-dioxolane, DHEAS, and ES markedly reduced PPI in the dentate and CA1 that was blocked by the M2 receptor antagonist gallamine. Similar to DHEAS, microelectrophoretic administration of ES increased population spike amplitudes, without increasing excitatory transmission, but this effect was not blocked by gallamine. Microelectrophoretic application of cis-dioxolane and ES markedly increased the firing rate of dentate hilar interneurons and CA1 oriens/alveus interneurons and enhanced their synchrony to hippocampal theta rhythm. These findings suggest that select GABA-modulating neurosteroids and neuroactive estrogen sulfates alter septohippocampal cholinergic modulation of hippocampal GABAergic interneurons mediating recurrent, but not feedforward, inhibition of hippocampal principal cell activity.

Selective blockade of central M1 muscarinic cholinergic receptors with pirenzepine impairs cardiovascular and respiratory function in rats with acute hemorrhage

Ultrasound studies showed that selective antagonist of central M1 muscarinic cholinergic receptors pirenzepine (50 mg/kg intravenously) causes transitory hypotension and respiratory depression in anesthetized intact rats. The M1 receptor antagonist had no effect on cardiac output and portal blood flow. Pretreatment with pirenzepine increased the sensitivity of rats with acute massive hemorrhage to circulatory hypoxia. After blockade of central M1 muscarinic cholinergic receptors, the posthemorrhagic period was characterized by primary decompensation of blood pressure, portal blood flow, and respiration and development of low cardiac output syndrome. The animals died over the first minutes after bleeding arrest. Our results indicate that central M1 muscarinic cholinergic receptors act as shock-limiting cholinergic structures under conditions of posthemorrhagic changes in systemic and portal blood flow, as well as during respiratory dysfunction.

Long-acting muscarinic M3 receptor antagonists

Two applications claim a novel class of muscarinic M3 receptor antagonists and their use as long-acting agents for the treatment of chronic obstructive pulmonary disease. The two applications claim closely related structures with one claiming quaternary amine salts of the tertiary amines claimed in the other. The claimed compounds comprise a biarylmethylamine core with the amino group modified by acylation or sulfonation and a tertiary or quaternary amine coupled to the distal ring of the biaryl system.

Long-acting muscarinic M3 receptor antagonists

A series of 10 closely related patent applications define Theravance’s approach to the development of long-acting M3 selective antagonists for the treatment of chronic obstructive pulmonary disease. The applications illustrate a modular approach to the design of such agents, comprising a muscarinic pharmacophore attached to a second, less functionalised moiety via a variable length spacer incorporating heteroatoms. Eight of the applications utilise a a 4-piperidinylester of biphenylcarbamic acid as the muscarinic pharmacophore and two utilise 2,2-diphenyl-2-(S)-pyrrolidin-3-ylacetamide. The first group of eight applications encompasses the use of two tail groups and four different sets of linking groups, whereas the second uses two very different tail groups and different spacers.

Fluorescent styryl dyes FM1‐43 and FM2‐10 are muscarinic receptor antagonists: intravital visualization of receptor occupancy

The fluorescent styryl dyes FM1-43 and FM2-10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular-independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1-43 and FM2-10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1-43 and FM2-10 bound with moderate affinity (23-220 nM) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1-M5). In functional studies in vitro, FM1-43 and FM2-10 inhibited electrical field stimulation (EFS) and acetylcholine-induced cholinergic contractions of guinea-pig tracheal strips (IC50: FM1-43, 0.4 +/- 0.1; FM2-10, 1.6 +/- 0.1 microM; concentration of antagonist producing a 2-fold leftward shift in the acetylcholine concentration-response curve (Kb): FM1-43, 0.3 +/- 0.1; FM2-10, 15.8 +/- 10.1 microM). Neither compound inhibited EFS-evoked, non-adrenergic non-cholinergic nerve-mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole-mount preparations with 5 microM FM1-43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.

Muscarinic regulation of neonatal rat bladder spontaneous contractions

In vitro preparations of whole urinary bladders of neonatal rats exhibit prominent myogenic spontaneous contractions, the amplitude and frequency of which can be increased by muscarinic agonists. The muscarinic receptor subtype responsible for this facilitation was examined in the present experiments. Basal spontaneous contractions in bladders from 1- to 2-wk-old Sprague-Dawley rats were not affected by M2 or M3 receptor antagonists. However, administration of 0.5 microM physostigmine, an anticholinesterase agent that increases the levels of endogenous acetylcholine, or 50-100 nM carbachol, a cholinergic agonist at low concentrations, which did not cause tonic contractions, significantly augmented the frequency and amplitude of spontaneous contractions. Blockade of M2 receptors with 0.1 microM AF-DX 116 or 1 microM methoctramine or blockade of M3 receptors with 50 nM 4-diphenylacetoxy-N-methylpiperidine methiodide or 0.1 microM 4-diphenylacetoxy-N-(2-chloroethyl)piperidine hydrochloride (4-DAMP mustard) reversed the physostigmine and carbachol responses. M2 and M3 receptor blockade did not alter the facilitation of spontaneous contractions induced by 10 nM BAY K 8644, an L-type Ca2+ channel opener, or 0.1 microM iberiotoxin, a large-conductance Ca2+-activated K+ channel blocker. NS-1619 (30 microM), a large-conductance Ca2+-activated K+ channel opener, decreased carbachol-augmented spontaneous contractions. These results suggest that spontaneous contractions in the neonatal rat bladder are enhanced by activation of M2 and M3 receptors by endogenous acetylcholine released in the presence of an anticholinesterase agent or a cholinergic receptor agonist.

Muscarinic receptors and salivary secretion

The release of acetylcholine from parasympathetic nerves and its interaction with muscarinic acetylcholine receptors (mAChRs) regulates many fundamental functions in the periphery (smooth muscle contraction, glandular secretion, modulation of cardiac output) and central nervous system (motor control

Central muscarinic cholinergic regulation of the systemic inflammatory response during endotoxemia.

TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this "cholinergic antiinflammatory pathway" inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents.

Increased Insulin Secretion by Muscarinic M1 and M3 Receptor Function from Rat Pancreatic Islets in vitro

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10(-8)-10(-5) M) with a maximum stimulation at 10(-7) M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.

μ- but not δ- and κ-opioid receptors in the ventrolateral orbital cortex mediate opioid-induced antiallodynia in a rat neuropathic pain model

Previous studies have indicated that the ventrolateral orbital cortex (VLO) is involved in opioid-mediated antinociception in the tail flick test and formalin test. The aim of the current study was to examine the effect of opioids microinjected into the VLO on allodynia in the rat L5/L6 spinal nerve ligation (SNL) model of neuropathic pain and determine the roles of different subtypes of opioid receptors in this effect. The allodynia was assessed by both mechanical (von Frey filaments) and cold plate (4 °C) stimuli. Morphine (1.0, 2.5, and 5.0 μg) microinjected into the VLO contralateral to the nerve ligation dose-dependently depressed the mechanical and cold allodynia and these effects were reversed by nonselective opioid receptor antagonist naloxone (1.0 μg) administrated into the same site. Microinjection of endomorphin-1 (5.0 μg), a highly selective μ-opioid receptor agonist, and [ d-Ala 2, d-Leu 5]-enkephalin (DADLE, 10 μg), a δ-/μ-opioid receptor agonist, also depressed the allodynia, and the effects of both drugs were blocked by selective μ-receptor antagonist β-funaltrexamine (β-FNA, 3.75 μg), but the effects of DADLE were not influenced by the selective δ-receptor antagonist naltrindole (5.0 μg). Microinjection of U-62066 (100 μg), a κ-opioid receptor agonist, into the VLO had no effect on the allodynia. These results suggest that the VLO is involved in opioid-induced antiallodynia and μ- but not δ- and κ-opioid receptor mediates these effects in the rat with neuropathic pain.

Bladder Volume Alters Cholinergic Responses of the Isolated Whole Mouse Bladder

The isolated bladder expresses autonomous activity, which may contribute to the generation of lower urinary tract sensation or pathophysiology. We evaluated how the effect of a cholinergic agonist on autonomous activity alters with increasing volume and in the presence of substances known to modulate functional bladder capacity. The bladder of 22 adult female C57 black mice were mounted in whole organ tissue baths. Recordings of intravesical pressure were performed under standardized conditions at different bladder volumes. At low volume the muscarinic agonist arecaidine elicited an initial peak response, which subsided to a sustained steady state pressure. At high volume phasic pressure fluctuations were also apparent. An M2-receptor antagonist caused a significantly greater decrease in peak and steady state responses than in pressure fluctuations. An M3-receptor antagonist decreased all 3 components. Alpha, beta-methylene adenosine triphosphate markedly decreased fluctuations, in contrast to norepinephrine, which eliminated the steady state response while preserving fluctuations. The response to cholinergic stimulation of the isolated bladder has 3 components. The initial tonic peak response increases with bladder distention and it is inhibited by M2 and M3 muscarinic receptor antagonists. The tonic steady state response does not vary with bladder volume and it is inhibited by M2 and M3-receptor antagonists, and by beta3-adrenergic receptor agonists. Phasic fluctuations are minimal at low bladder volume, and with alpha, beta-methylene adenosine triphosphate or an M3-receptor antagonist. Thus, the response to cholinergic stimulation varies with bladder volume. It can be differentially modulated by muscarinic antagonists and also by agents acting through nonmuscarinic receptors.

Chiesi Farmaceutici and Laboratorios SALVAT have signed a worldwide development and licensing agreement for new muscarinic M3 receptor antagonists.

Chiesi Farmaceutici and Laboratorios SALVAT have signed a worldwide development and licensing agreement for new muscarinic M3 receptor antagonists.

Tiotropium, a Novel Muscarinic M3 Receptor Antagonist, Improved Symptoms of Chronic Obstructive Pulmonary Disease Complicated by Chronic Heart Failure

A 77-year-old man was referred to hospital because of dyspnea on exertion. Although the patient had been fully medicated for chronic heart failure (CHF) caused by hypertensive heart disease, the echo-estimated left ventricular end-diastolic pressure (LVEDP) and brain natriuretic peptide (BNP) level had continued to be high for at least 2 years. Pulmonary functional examination revealed concomitant chronic obstructive pulmonary disease (COPD). Because beta-agonists were expected to exacerbate the CHF, inhalation of tiotropium, a non-beta-adrenergic bronchodilator and novel M3 muscarinic receptor antagonist, was used to treat the COPD. Not only did the pulmonary function improved but the treatment also safely ameliorated CHF signs including LVEDP and plasma BNP.

Galanin Receptor Antagonists

The pathophysiology of mood disorders involves several genetic and social predisposing factors, as well as a dysregulated response to chronic stress. Accumulated evidence during the last two decades has implicated disturbances in brain serotonin and/or noradrenaline (norepinephrine) neurotransmission in the aetiology of depression. In fact, current pharmacological treatment for mood disorders is based on the use of drugs that act mainly by enhancing brain serotonin and noradrenaline neurotransmission by blockade of the active reuptake mechanism for these neurotransmitters. However, current antidepressant drugs have a delayed onset of therapeutic action, and a substantial number of patients do not respond adequately to them. In addition, these drugs have a number of adverse effects that limit patient compliance. In view of this, there is an intense search to identify novel (receptor) targets for antidepressant therapy. Recent studies have indicated that several neuropeptides and their receptors are potential candidates for the development of novel antidepressant treatment. In this context, galanin is of particular interest, since it is co-localised with serotonin in the dorsal raphe nucleus and with noradrenaline in the locus coeruleus, nuclei known to play a major role in affective disorders and in the action of antidepressant drugs. The actions of galanin are mediated by three receptor subtypes (GAL1, GAL2 and GAL3), which are coupled to different intracellular effector systems. Studies in rats have shown that galanin administered intracerebroventricularly is a potent inhibitor of mesencephalic serotonergic neurotransmission, as indicated by a long-lasting reduction in the release of serotonin in the hippocampus. This inhibitory effect is related to activation of the galanin receptors located on the dorsal raphe neurons. Moreover, intracerebroventricular galanin alters the gene expression of serotonin 5-HT1A autoreceptors in the dorsal raphe and also changes their functional activity. In addition, galanin produces a functional blockade of postsynaptic 5-HT1A receptor-mediated responses. Both pharmacological and genetic studies suggest a role for galanin in depression-like behaviour in rodent models. Transgenic mice overexpressing galanin under the control of the platelet-derived growth factor-beta promoter display increased immobility in the forced swim test. Intracerebroventricular administration of galanin in the rat increases depression-like behaviour, and this is fully blocked by the nonselective peptide galanin receptor antagonist M35. Importantly, M35 alone administered intracerebroventricularly produces an antidepressant-like effect. Recently, newly developed receptor-specific nonpeptidergic galanin GAL3 receptor antagonists (SNAP-37889 and SNAP-398299), which cross the blood-brain barrier after systemic administration, have shown antidepressant-like activity in several animal models. On the other hand, stimulation of the GAL2 receptor at the raphe level by local application of the GAL2 receptor agonist galanin (2-11) has been shown to increase serotonin levels in the hippocampus and dorsal raphe. These results indicate an important (mainly inhibitory) role of galanin as a regulator of brain serotonin and 5-HT1A receptor-mediated transmission, which may be of potential importance for understanding mood disorders and for the development of antidepressant drugs. Taken together, the present evidence suggests that antidepressant efficacy may be associated with compounds acting as antagonists at the GAL3 and/or possibly GAL1 receptors, and/or agonists at the GAL2 receptor.

Excitatory muscarinic M1 receptors mediate in the regulation of guinea-pig terminal ileum motility

In vitro experiments were performed on smooth muscle strips cut out longitudinally or circularly from the terminal ileum about 5 cm from the ileo-coecal sphincter. The aim of this study was to determine the functional role of M1 muscarinic receptor subtype in the regulation of cholinergic contractions of the guinea-pig terminal ileum. Electrical field stimulation (EFS; 16 Hz) produced in vitro contractile responses of the guinea-pig terminal ileum muscle strips (longitudinal and circular muscle layer). Those contractions were inhibited by 1 μM tetrodotoxin (2±2% of the control response) and 1 μM atropine (1±1% of the control response), thus indicating the activation of intrinsic cholinergic nerves. Exogenous ACh (1 μM) induced contractions that were inhibited by 1 μM atropine, but not by 1 μM tetrodotoxin, indicating a direct effect on the smooth muscle. MCN-343 specific agonist of M1 receptor subtype (5 μM) induced contractions that were inhibited by 1 μM atropine and by 1 μM tetrodotoxin. M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh - induced contractions but reduced EFS - induced contractions by 11±3% and MCN-A-343 induced contractions by 17±4%. In conclusion, specific M1 receptors modulate terminal ileum contractions by regulating the release of ACh from cholinergic nerves. M1 receptors, present on cholinergic nerves, function as prejunctional facilitatory autoreceptors.

Central Muscarinic Receptor Subtypes Regulating Voiding in Rats

Muscarinic receptors are distributed widely in the brain. A recent study revealed that central muscarinic receptors are involved in voiding regulation. However, to our knowledge the role of each muscarinic receptor subtype has not been resolved. Therefore, we evaluated the effect of intracerebroventricular administration of selective muscarinic M1 to M4 receptor antagonists on voiding function in rats. Female Sprague-Dawley rats were cannulated for intracerebroventricular infusion under halothane anesthesia. In experiment 1 cystometry was performed in conscious rats, and BC and maximal voiding pressure were measured. In experiment 2 a catheter was inserted via the bladder dome to the bladder neck and UPP was measured by saline infusion. Repeat cystostomy was performed, and saline infusion and discharge saline, BC, maximal IVP and minimal UPP were measured in conscious rats. Pirenzepine, methoctramine, pFHHSiD and MT-3 were used as selective M1, M2, M3 and M4 muscarinic receptor antagonists, respectively, which were injected intracerebroventricularly. In experiment 1 pirenzepine and pFHHSiD increased BC and decreased maximal voiding pressure. Methoctramine and MT-3 decreased BC. In experiment 2 pirenzepine and pFHHSiD increased BC and minimal UPP, and decreased maximal IVP. Methoctramine and MT-3 decreased BC and maximal IVP. Minimal UPP remained unchanged. Intracerebroventricular administration of muscarinic M1 and M3 receptor antagonists inhibited urination in conscious rats, while M2 and M4 receptor antagonists induced excitatory changes.

Corticosteroids: Potential 2-Agonist and Anticholinergic Interactions in Chronic Obstructive Pulmonary Disease

Corticosteroids are often used in combination with beta2-agonist and anticholinergic bronchodilators in the treatment of chronic obstructive pulmonary disease (COPD). Corticosteroids activate the beta2-receptor gene, increasing receptor number and decreasing desensitization. Long-acting beta2-agonists prime the glucocorticoid receptor and enhance nuclear translocation via activation of CCAAT enhancer binding protein-alpha. Corticosteroids can also increase prejunctional auto-inhibitory M2-receptor gene expression in airway smooth muscle. There is evidence of a synergistic inhibition of cytokine and chemokine release from alveolar macrophages, epithelial cells, and mucosal glands and enhanced respiratory cytoprotection against viral and bacterial infection when a corticosteroid is combined with salmeterol. In airway smooth muscle, corticosteroids inhibit the contractile effects of acetylcholine, whereas M2-receptor antagonism increases the relaxant activity of isoproterenol. Complementary interactions between corticosteroids and long-acting beta2-agonists and between corticosteroids and anticholinergic bronchodilators may be important if these drugs are combined in the treatment of COPD.

Relationship of cocaine-induced c-Fos expression to behaviors and the role of serotonin 5-HT2A receptors in cocaine-induced c-Fos expression.

Serotonin 5-HT2A receptor antagonists have been shown to attenuate the locomotor stimulant effects of cocaine in rats. The present study used the expression of c-Fos protein as a marker to identify brain areas through which 5-HT2A receptors may modulate cocaine-induced behaviors. Significant correlations were observed between cocaine-induced hyperactivity and c-Fos expression in the nucleus accumbens core (NAcC), caudate-putamen (CPu), and subthalamic nucleus. In a separate experiment, a low, behaviorally relevant dose of cocaine was found to increase c-Fos immunoreactivity in the medial CPu, NAcC, and nucleus accumbens shell (NAcSh). The selective 5-HT2A receptor antagonist M100907 significantly attenuated cocaine-induced c-Fos expression in the medial CPu and in the NAcSh. These data suggest that 5-HT2A receptors in the NAcSh and CPu or in afferents to these regions may contribute to genomic responses to cocaine in the brain as well as to cocaine-induced locomotor activity.