Muscarinic receptor antagonists for the treatment of chronic obstructive pulmonary disease

Introduction: Long-acting muscarinic receptor antagonists (LAMAs) are central to the treatment of chronic obstructive pulmonary disease (COPD) because of the important role of the cholinergic system in the pathophysiology of this disorder.

Areas covered: LAMAs show clinically meaningful effects in lung function and other important supportive outcomes, such as exacerbations, health-related quality of life, dyspnea, rescue medication use and nighttime/early morning symptoms, and are safe. Muscarinic receptor antagonists could exert other useful actions such as the anti-inflammatory, anti-remodeling, mucus-modifying, and anti-cough effects. Concerns have been raised about possible associations of muscarinic receptor antagonists with cardiovascular morbidity and mortality. Muscarinic receptor antagonists can be combined with long-acting β₂-agonists (LABAs), inhaled corticosteroids (ICSs) and LABA + ICS. There are number of LAMAs that are being identified but only few have reached the clinical development. Fixed-dose combination formulations of both novel and established LAMAs with LABAs are being developed.

Expert opinion: There are important questions concerning the use of LAMAs in the treatment of patients suffering from stable COPD that need conclusive answers.     

Muscarinic acetylcholine receptors as therapeutic targets for obesity

Background: There are five types of muscarinic acetylcholine receptors (mAChRs), M₁ – M₅, which regulate several central and peripheral functions. Transgenic mice deficient in these receptors have been generated. Objective: To understand some processes in which these receptors are involved, mainly targeting obesity, which seems to be mediated by M₃ in the hypothalamus. Methods: The absence of M₃ has beneficial effects, which protect mice against some forms of obesity and ameliorate glucose and energy homeostasis. These findings suggest some relevance of muscarinic M₃ antagonists to the treatment of obesity, and also studies with new classes of M₃-receptor selective antagonists, to identify active/selective molecules, able to reach the CNS, which might have fewer side-effects compared with available muscarinic drugs. Results/conclusion: M₃ antagonists might have application to the design of antiobesity agents. However, this research is in its preliminary phases, and the lack of specific antagonists or agonists for M₃ receptors in CNS, make it impossible to validate this antiobesity target at present.     

Mapping the Ligand Binding Pocket of the Human Muscarinic Cholinergic Receptor Hml: Contribution of Tyrosine-82

The ligand binding pocket of many G protein-coupled receptors is thought to be located within the core formed by their seven transmembrane domains (TMDs). Previous results suggested that muscarinic antagonists bind to a pocket located toward the extracellular region of the TMDs, primarily at TMDs 2, 3, 6, and 7. Tyrosine-82 (Y82) is located in TMD2 only one helical turn from the presumed membrane surface of Hm1, whereas a phenylalanine (F124) is found in the equivalent position of the closely related Hm3. In order to determine the contribution of Y82 to Hm1 ligand binding and selectivity versus Hm3, we constructed the point mutation Y82 F of Hm1 and measured binding affinities of various ligands, with 3H-N-methylscopolamine (3H-NMS) as the tracer. The Hm1 wild-type receptor and the Y82F mutant were transfected into human embryonic kidney U293 cells. Whereas the affinities of NMS, carbachol, and atropine were either unchanged (carbachol) or enhanced by less than twofold (atropine and NMS), the affinity of the Hm1-selective piren-zepine was reduced threefold by the Y82 F mutation. These changes parallel affinity differences of Hm1 and Hm3, indicating that the Y82 F mutation affects the binding pocket and that Y82 contributes to the binding selectivity among closely related muscarinic receptors.     

Muscarinic receptor subtypes mediating positive and negative inotropy in the developing chick ventricle

The inotropic response to muscarinic receptor stimulation of isolated chick ventricular myocardium was examined at various developmental stages, and the receptor subtype involved was pharmacologically characterized. In embryonic chick ventricles, carbachol (CCh) produced positive inotropy at micromolar concentrations. In hatched chick ventricles, CCh produced negative inotropy at nanomolar concentrations. Neither positive nor negative inotropy was observed in the 19 - 21-day-old embryos. Both positive and negative inotropy were also observed with acetylcholine and oxotremoline-M. The CCh-induced positive inotropy in 7 - 9-day-old embryonic ventricles and the negative inotropy in 1 - 3-day-old hatched chick ventricles were antagonized by muscarinic receptor antagonists; pA(2) values for the positive and negative responses of pirenzepine were 7.5 and 7.2, those of AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepine-6-one) were 6.8 and 6.9, those of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were 9.0 and 8.5, and those of himbacine were 7.0 and 8.0, respectively. CCh had no effect on action potential configuration. In conclusion, the positive inotropy is most likely mediated by muscarinic M(1) receptors and the negative inotropy is mostly likely mediated by muscarinic M(4) receptors.     

Muscarinic receptor subtypes in neuronal and non-neuronal cholinergic function

1 Muscarinic M1-M5 receptors mediate the metabotropic actions of acetylcholine in the nervous system. A growing body of data indicate they also mediate autocrine functions of the molecule. The availability of novel and selective muscarinic agonists and antagonists, as well as in vivo gene disruption techniques, has clarified the roles of muscarinic receptors in mediating both functions of acetylcholine. 2 Selective M1 agonists or mixed M1 agonists/M2 antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonism, or mixed M2/M3 antagonists, are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease. Preclinical data suggest that selective agonism of the M4 receptor will provide novel anti-nociceptive agents, while therapeutics-based upon agonism or antagonism of the muscarinic M5 receptor have yet to be reported. 3 The autocrine functions of muscarinic receptors broadly fall into two areas - control of cell growth or proliferation and mediation of the release of chemical mediators from epithelial cells, ultimately causing muscle relaxation. The former particularly are involved in embryological development, oncogenesis, keratinocyte function and immune responsiveness. The latter regulate contractility of smooth muscle in the vasculature, airways and urinary bladder. 4 Most attention has focused on muscarinic M1 or M3 receptors which mediate lymphocyte immunoresponsiveness, cell migration and release of smooth muscle relaxant factors. Muscarinic M4 receptors are implicated in the regulation of keratinocyte adhesion and M2 receptors in stem cell proliferation and development. Little data are available concerning the M5 receptor, partly due to the difficulties in defining the subtype pharmacologically. 5 The autocrine functions of acetylcholine, like those in the nervous system, involve activation of several muscarinic receptor subtypes. Consequently, the role of these subtypes in autocrine, as well neuronal cholinergic systems, significantly expands their importance in physiology and pathophysiology.     

Muscarinic Receptor Activity of Some Malaysian Plant Species

Abstract

Muscarinic receptor binding activity was tested on 224 plant extracts obtained from more than 50 plant families found in Malaysia. The plant extracts were evaluated by a 96-well microplate filtration–based radioligand competitive assay, centered on the ability of the plant extracts to competitively displace the radioligand, [³H]N.-methylscopolamine, from binding to the muscarinic membrane receptors. The screening assay was initially carried out at 50 µg/assay point, and those showing inhibition at and above 61% were retested at 10 µg/assay point. The extracts of Ficus septica. Burm. f. (Moraceae) [65.85±3.75% inhibition; mean (n = 3)±SD], Polyalthia microtus. Miq. (Annonaceae) (32.63±1.38% inhibition), and Popowia odoardoi. Diels (Annonaceae) (35.79±7.11% inhibition) at 10 µg/assay point exhibited muscarinic properties, which are worthy of further investigation.     

Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors

Purpose This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1–M5), a region important for G protein coupling. Methods GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues. Results 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, Δ232–358. Conclusion Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.     

Muscarinic receptor antagonist-induced lenticular opacity in rats.

Investigations on compound A, an M2-sparing M3 muscarinic receptor antagonist, showed that focal polar anterior subcapsular lenticular opacities, characterized by focal epithelial proliferation, developed in Sprague-Dawley rats. The incidence and bilateral localization of this change increased generally with dose and time, though plateauing after 8 months of treatment; however the severity progressed very slightly. Over a 1-year period, no anterior cortical lens fiber changes or other histological ocular changes developed. A decreased severity of the change and apoptosis suggested some regression after a 26-week recovery period. Two nonselective muscarinic receptor antagonists, atropine and tolterodine, induced similar lenticular changes in rats. A hypothesis in relation to an indirect effect of the drug, such as increased illumination of the lens due to mydriasis observed with all these compounds, was investigated and disproven. Because these opacities are induced by structurally unrelated muscarinic receptor antagonists (atropine and tolterodine), it is likely that these lenticular changes are the result of muscarinic receptor inhibition. However, hypotheses regarding a direct effect of the drug on muscarinic receptors in the lens epithelium, possibly mediated by drug and/or metabolite(s) in the aqueous humor and/or lens epithelium, remain to be investigated. This lenticular opacity is similar to that observed spontaneously in Sprague-Dawley rats, although the latter occur at a lower incidence. No such lenticular opacities have been reported in other animal species, including man, after treatment with muscarinic receptor antagonists.     

Analogues of Carbacholine: Synthesis and Relationship between Structure and Affinity for Muscarinic and Nicotinic Acetylcholine Receptors

A series of acyclic and heterocyclic analogues of carbacholine ( 1 ) was synthesized using N-methylcarbacholine (MCC, 2 ), N,N-dimethylcarbacholine (DMCC, 3 ), and the corresponding tertiary amine ( 4 ) as leads. Whereas nicotinic acetylcholine receptor affinity was determined using [³H]nicotine as the radioactive ligand, [³H]oxotremorine-M ([³H]Oxo-M) and [³H]quinuclidinyl benzilate ([³H]QNB), in some cases supplemented with [³H]pirenzepine ([³H]PZ), were used as radioligands for muscarinic acetylcholine receptors on rat brain membranes. On the basis of receptor binding data, nicotinic/muscarinic (N/M) selectivity factors were determined, and muscarinic receptor efficacy (M agonist index) and M₁ selectivity (M₂/M₁ index) estimated. In most cases, quaternized analogues showed higher affinity than the corresponding tertiary amines for muscarinic and, in particular, nicotinic receptor sites. Among the new compounds, N,N-diethylcarbacholine ( 9e ) (IC₅₀ = 0.046 μM), (S)-1-methyl-2-(N,N-diethyl-aminocarbonyloxymethyl)pyrrolidine ( 17k ) (IC₅₀ = 0.068 μM), and the corresponding quaternized analogue, 18k (IC₅₀ = 0.018 μM) showed the highest nicotinic receptor affinity. The tertiary amine, 17k showed much higher nicotinic receptor affinity than the acyclic analogue, 4 (IC₅₀ = 5.7 μM), and the N/M selectivity factor determined for 17k (150) is an order of magnitude lower than that of nicotine (1400). The N/M selectivity factors for MCC ( 2 ) and DMCC ( 3 ), previously reported to be highly selective nicotinic receptor ligands, were shown to be 6.5 and 60, respectively, the latter value being comparable with that of 18k (89).     

毒蕈碱型M5受体亚型及生物学特性

M5是最后一个被克隆出的乙酰胆碱毒蕈碱型受体亚型,具有G蛋白偶联受体家族的基本结构特征, 其信号转导方式既可以与G蛋白偶联,激活多种效应酶 (腺苷酸环化酶、磷脂酶C、磷脂酶A2 )产生相应的生物学效应,又可作用于离子通道来调节细胞间的信号传递。M5生理作用主要与调节中枢NO的生成和DA的释放有关,可能为药物滥用成瘾、DA系统功能障碍性疾病、阿尔采末病、脑缺血等的药物治疗提供一个新靶标。     

Muscarinic cationic current in gastrointestinal smooth muscles: signal transduction and role in contraction

1 The muscarinic receptor plays a key role in the parasympathetic nervous control of various peripheral tissues including gastrointestinal tract. The neurotransmitter acetylcholine, via activating muscarinic receptors that exist in smooth muscle, produces its contraction. 2 There is the opening of cationic channels as an underlying mechanism. The opening of cationic channels results in influxes of Ca2+ via the channels into the cell and also via voltage-dependent Ca2+ channels which secondarily opened in response to the depolarization, providing an amount of Ca2+ for activation of the contractile proteins. 3 Electrophysiological and pharmacological studies have shown that the cationic channels as well as muscarinic receptors exist in many visceral smooth muscle cells. However, the activation mechanisms of the cationic channels are still unclear. 4 In this article, we summarize the current knowledge of the muscarinic receptor-operated cationic channels, focusing on the receptor subtype, G protein and other signalling molecules that are involved in activation of these channels and on the molecular characteristics of the channel. This will improve strategies aimed at developing new selective pharmacological agents and understanding the activation mechanism and functions of these channels in physiological systems.     

Muscarinic receptors: new potential for treating chronic pain

A new approach using small molecules that selectively activate a specific subtype of the muscarinic receptor is showing considerable promise at ACADIA Pharmaceuticals (San Diego, CA, USA) for the treatment of chronic pain.     

Muscarinic receptor-mediated activation of nitric oxide synthase

The potent vasodilator factor released from endothelial cells upon activation of muscarinic acetylcholine receptors has recently been identified as nitric oxide (NO). This discovery has sparked intensive research efforts aiming at understanding the functional role of this short-lived, highly reactive free radical. As a result, it has been shown that NO is a very important second messenger involved in a wide spectrum of physiological functions. One important aspect that differentiates NO from other second messengers is that NO is a ‘traveling’ messenger that diffuses out of the cells of its origin to produce marked effects in neighboring cells. This paper provides a synopsis of the diverse biological roles of NO and the mechanisms of its generation upon activation of muscarinic acetylcholine receptors. Drug Dev. Res. 40:205–214, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist

Herein, we report the SAR leading to the discovery of VU6028418, a potent M₄ mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.     

Muscarinic receptor agonists and antagonists

Although four different subtypes of the muscarinic acetylcholine (ACh) receptor with functional correlates are known to exist (function for M5 is still unclear), all muscarinic agonists and antagonists in clinical practice only show very weak selectivity. Therefore, intensive investigations are in progress to develop subtype selective ligands. This review describes the first M1 agonists and antagonists of the presynaptic M2 receptor, which can be used in the treatment of Alzheimer’s disease (AD), and M3 antagonists, which will be useful in the treatment of urinary urge incontinence. In addition, muscarinic agonists were found to exhibit analgesic effects and M4 antagonists may be useful in the treatment of movement disorders. However, the latter two pharmacological findings will need more research work to become established in clinical trials.     

M受体及相关选择性药物研究进展

毒草碱受体（M受体）是体内重要的G蛋白偶联受体之一,有M1～M5五种药理学亚型,各亚型在体内的分布和功能不同,受体蛋白结构和信号转导机制也有差异。对M受体、相关选择性药物及受体一配体作用位点的研究,将为设计以M受体各亚型为靶标的选择性药物提供帮助,对临床治疗多种M受体功能紊乱的疾病,如阿尔茨海默病等具有重要意义。     

Adenylate Cyclase Modulation by Ammonium Ion: GTP-like Effect on Muscarinic and α2-Adrenergic Receptors

The stimulatory influence of ammonium sulphate on adenylate cyclase activity has been investigated. By competition binding experiments on the β-adrenergic stimulatory receptor in rat myocardial membranes, no influence could be detected of ammonium sulphate neither in receptor coupling to the stimulatory guanine nucleotide binding protein nor in the GTP-induced uncoupling. In order to detect an impaired inhibition instead of an increased stimulation of adenylate cyclase activity by ammonium sulphate the investigation was extended to inhibitory receptors. The same type of effect by ammonium sulphate was detected on both the muscarinic cholinergic receptor in rat myocardial membranes as well as on the α₂-adrenergic receptor in human platelets. The influence of ammonium sulphate noted in competition binding studies and off-kinetics experiments was GTP-like, i.e. causing a decrease in agonist-receptor affinity leaving all the inhibitory receptors in the low affinity state. In conclusion, this paper indicates that the observed stimulatory effect of ammonium sulphate is exerted by the ammonium ion on the inhibitory guanine nucleotide binding protein, impairing the negative control of adenylate cyclase activity.