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It has been suggested that neuronal hyperexcitability contributes to Alzheimer’s disease (AD), so we asked how hyperexcitability develops in a common mouse model of β-amyloid neuropathology - Tg2576 mice. Using video-EEG recordings, we found synchronized, large amplitude potentials resembling interictal spikes (IIS) in epilepsy at just 5 weeks of age, long before memory impairments or β-amyloid deposition. Seizures were not detected, but they did occur later in life, suggesting that IIS are possibly the earliest stage of hyperexcitability. Interestingly, IIS primarily occurred during rapid-eye movement (REM) sleep, which is notable because REM is associated with increased cholinergic tone and cholinergic impairments are implicated in AD. Although previous studies suggest that cholinergic antagonists would worsen pathophysiology, the muscarinic antagonist atropine reduced IIS frequency. In addition, we found IIS occurred in APP51 mice which overexpress wild type (WT)-APP, although not as uniformly or as early in life as Tg2576 mice. Taken together with results from prior studies, the data suggest that surprising and multiple mechanisms contribute to hyperexcitability. The data also suggest that IIS may be a biomarker for early detection of AD.

Umeclidinium (UMEC), a long-acting muscarinic antagonist approved for chronic obstructive pulmonary disease (COPD), was investigated for primary hyperhidrosis as topical therapy. This study evaluated the pharmacokinetics, safety, and tolerability of a single dose of [(14) C]UMEC applied to either unoccluded axilla (UA), occluded axilla (OA), or occluded palm (OP) of healthy males. After 8 h the formulation was removed. [(14) C]UMEC plasma concentrations (Cp) were quantified by accelerator mass spectrometry. Occlusion increased systemic exposure by 3.8-fold. Due to UMEC absorption-limited pharmacokinetics, Cp data from the OA were combined with intravenous data from a phase I study. The data were described by a two-compartment population model with sequential zero and first-order absorption and linear elimination. Simulated systemic exposure following q.d. doses to axilla was similar to the exposure from the inhaled therapy, suggesting that systemic safety following dermal administration can be bridged to the inhaled program, and offering the potential for a reduced number of studies and/or subjects.

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) can be reset by the cholinergic agonist carbachol. In hamsters, intraSCN carbachol produces phase advances during the day. This phenomenon has previously been attributed to the muscarinic receptors, as carbachol-induced phase shifts are blocked by pretreatment with the muscarinic antagonist atropine. The SCN contains all five muscarinic receptors, leaving open the question as to which muscarinic receptors mediate these shifts. Here we test two selective muscarinic agonists, the M1/4 agonist McN-A-343 and the M2/3 agonist bethanechol, in addition to the non-selective cholinergic agonist carbachol. Consistent with previous reports, carbachol produced significant phase advances when injected to the SCN during the mid-subjective day. At the doses used here, McN-A-343, but not bethanechol, also produced significant phase shifts when injected to the SCN during the mid-subjective day. Phase shifts to McN-A-343 were as large as those produced by carbachol, suggesting that activation of the M1/4 receptors alone can fully account for the daytime phase advances produced by cholinergic agonists. Given acetylcholine's role in arousal, and the similarity between phase advances to carbachol/McN-A-343 and to exercise and arousal manipulations, it is possible that acetylcholine may contribute to non-photic resetting of the circadian clock.

BackgroundThe place of long-acting β agonist/long-acting muscarinic antagonist (LABA/LAMA) combinations in stable patients with COPD is not well defined. The purpose of this study was to systematically review the efficacy and safety of LABA/LAMA combinations.MethodsSeveral databases and manufacturers’ websites were searched for relevant clinical trials. Randomised control trials, at least 12 weeks duration, comparing a LABA/LAMA combination with placebo and/or monotherapy were included. The data were pooled using a network as well as a traditional direct comparison meta-analysis.ResultsTwenty-three trials with a total of 27 172 patients were included in the analysis. LABA/LAMA combinations were associated with a greater improvement in lung function, St. George's Respiratory Questionnaire (SGRQ) score, and Transitional Dyspnoea Index (TDI) than monotherapies. LABA/LAMA combinations were associated with a significantly greater proportion of SGRQ and TDI responders than monotherapies (OR 1.23 (95% credible interval (CrI) 1.06–1.39), OR 1.34 (95% CrI 1.19–1.50) versus LABAs and OR 1.24 (95% CrI 1.11–1.36), OR 1.31 (95% CrI 1.18–1.46) versus LAMAs, respectively) and fewer moderate-to-severe exacerbations compared with LABAs (HR 0.82 (95% CrI 0.73–0.93)), but not when compared with LAMAs (HR 0.92 (95% CrI 0.84–1.00)). There were no statistically significant differences associated with LABA/LAMA combinations compared with monotherapies in safety outcomes as well as in severe exacerbations.ConclusionsThe combination therapy was the most effective strategy in improving lung function, quality of life, symptom scores and moderate-to-severe exacerbation rates, and had similar effects on safety outcomes and severe exacerbations as compared with monotherapies.

Organophosphorus nerve agents exert their toxic action by inhibiting acetylcholinesterase, resulting in overstimulation of muscarinic and nicotinic receptors due to the accumulation of acetylcholine at cholinergic synapses. Historically, treatment of nerve agent poisoning has concentrated on the muscarinic effects, generally using the competitive muscarinic antagonist atropine. Due to the high toxicity of many nicotinic antagonists, nicotinic receptors have been largely neglected as targets for therapeutic intervention. Nicotinic effects are treated indirectly, using oximes to reactivate the inhibited enzyme, an approach that is only effective against certain nerve agents. The non-reactivating therapeutic action of certain oximes has been attributed to their anti-nicotinic properties, and novel noncompetitive nicotinic antagonists based on these oximes have recently shown efficacy against nerve agent poisoning in animal studies. The nicotinic antagonist mecamylamine has been reported to have beneficial effects against organophosphorus nerve agent poisoning. A recently synthesised compound, benthiactzine, has both anti-nicotinic and anti-muscarinic activity, and has been shown to afford better protection than atropine against VX and sarin poisoning. Novel nicotinic compounds are now being explored for the treatment of various diseases and it is possible that some of these may be beneficial for the treatment of poisoning by nerve agents.

Background Human gastric fundus relaxation is mediated by intrinsic inhibitory pathway. We investigated the roles of nitrergic and purinergic pathways, two known inhibitory factors in gastric motility, on spontaneous and nerve-evoked contractions in human gastric fundus muscles. Methods Gastric fundus muscle strips (12 circular and 13 longitudinal) were obtained from patients without previous gastrointestinal motility disorder who underwent gastrectomy for stomach cancer. Using these specimens, we examined basal tone, peak, amplitude, and frequency of spontaneous contractions, and peak and nadir values under electrical field stimulation (EFS, 150 V, 0.3 ms, 10 Hz, 20 s). To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine (muscarinic antagonist, 1 μM), MRS2500 (a purinergic P2Y1 receptor antagonist, 1 μM), and N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor, 100 μM) were added sequentially for spontaneous and electrically-stimulated contractions. Tetrodotoxin was used to confirm any neuronal involvement. Results In spontaneous contraction, L-NNA increased basal tone and peak in both muscle layers, while amplitude and frequency were unaffected. EFS (up to 10 Hz) uniformly induced initial contraction and subsequent relaxation in a frequency-dependent manner. Atropine abolished initial on-contraction and induced only relaxation during EFS. While MRS2500 showed no additional influence, L-NNA reversed relaxation (p = 0.012 in circular muscle, and p = 0.006 in longitudinal muscle). Tetrodotoxin abolished any EFS-induced motor response. Conclusions The relaxation of human gastric fundus muscle is reduced by nitrergic inhibition. Hence, nitrergic pathway appears to be the main mechanism for the human gastric fundus relaxation.

Background Long-acting bronchodilators are central in the pharmacological management of patients with chronic obstructive pulmonary disease (COPD). The aim of this systematic review is to provide an overview of the studies evaluating the safety and clinical efficacy of inhaled glycopyrronium bromide, a novel long-acting muscarinic antagonist, in patients with COPD. Methods This study was performed as a systematic literature review. Results Inhaled glycopyrronium bromide seems to be a safe and well tolerated long-acting muscarinic antagonist with a fast onset of action. In patients suffering from moderate to severe COPD, glycopyrronium bromide has clinically important effects on level of forced expiratory volume in one second, use of relief medication, percentage of days with no use of rescue medication, daytime dyspnea scores, and probably also on health status. Furthermore, in this group of patients, glycopyrronium bromide has beneficial effects on dynamic hyperinflation and exercise tolerance. Glycopyrronium bromide has been shown to reduce the rate of exacerbations in patients with moderate to severe COPD, but long-term controlled trials with exacerbation rate as the primary outcome variable have not been published yet. Conclusion Once-daily inhaled glycopyrronium bromide has characteristics important for use in COPD, including a fast onset of action, sustained 24-hour bronchodilatation, and improvement in exercise tolerance, and therefore appears to have the potential for a significant role in the future management of COPD.

These two patent applications each claim combination formulations comprising a single dual-acting muscarinic antagonist/beta 2 agonist (MABA), as a free base or salt, with a second class of drug and the use of these formulations for the treatment of asthma and COPD. The two specified compounds are close analogues of each other and were originally disclosed in the same patent application. They are, respectively, 3-(2-chloro-3-((4-(2-ethylthiazole-4-carbonyl)-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)methyl)phenethoxy)-N-cyclopentyl-N-(2-(2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[b]{1,4] oxazin-8-yl)ethylamino)ethyl)propanamide and N-butyl-N-(2-(2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[b][l,4]oxazin-8-yl)ethylamino)ethyl)-3-(3-(2-(4-(2-isopropylthiazole-4-carbonyl)-1-oxa-4,9-diazaspiro[5.5]undecan-9-yl)ethyl)phenethoxy)propanamide.

Aim To examine function of both cholinergic (muscarinic) and TRPV1 receptors in human bladder urothelial (HBUC) from non-neurogenic overactive bladder (OAB) patients as compared to control subjects. Methods Primary HBUC cultures were derived from cystoscopic biopsies from OAB and control subjects. Muscarinic and TRPV1 function was assessed by acetylcholine (5 μm) or capsaicin (0.5 μm) evoked ATP release, measured by luciferase assay. Overall, expression of TRPV1 and muscarinic M3 receptors in bladder urothelial cells was accomplished using western immunoblotting. Results Our findings revealed that the response to acetylcholine in OAB HBUC cultures (which was blocked by the nonselective muscarinic antagonist, atropine methyl nitrate or AMN) was not significantly different than from controls. The acetylcholine M3 receptor was slightly decreased as compared to control. In contrast, OAB HBUC cultures exhibited a capsaicin hypersensitivity and augmented release of ATP (3.2 fold higher), which was blocked by the antagonist capsazepine. The increase in capsaicin sensitivity correlated with increased urothelial TRPV1 expression. Conclusion Though characterized in a small number of subjects, augmented release of urothelial-derived transmitters such as ATP could ‘amplify’ signalling between and within urothelial cells and nearby afferent nerves.

NVA237 (glycopyrronium bromide) is a once-daily long-acting muscarinic antagonist (LAMA) in development for chronic obstructive pulmonary disease (COPD). The GLycopyrronium bromide in COPD airWays clinical Study 2 (GLOW2) evaluated the efficacy and safety of NVA237 in moderate-to-severe COPD over 52 weeks. Patients were randomised 2:1:1 to NVA237 50 μg, placebo or open-label tiotropium 18 μg for 52 weeks. Primary end-point was trough forced expiratory volume in 1 s (FEV1) at 12 weeks. 1,066 patients were randomised, 810 completed the study. At week 12, trough FEV1 increased significantly by 97 mL with NVA237 (95% CI 64.6–130.2; p

A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.

Summary Purpose: Our earlier findings of the modulation of cholinergic neurotransmission by an early life generalized seizure and the reported interaction between muscarinic and N-methyl-d-aspartate (NMDA) receptors prompted us to investigate the effects of endogenous acetylcholine (ACh) on the frequency (Hz) of the epileptiform discharges following NMDA-receptor activation in the hippocampal slice. Methods: A sustained (>20 min) generalized convulsion was induced in Sprague-Dawley juvenile rats by intraperitoneal injection with pentylenetetrazole (PTZ, 70–90 mg/kg) at postnatal day (P) 20. Temporal and septal hippocampal slices were prepared of normal (N) and PTZ-treated (PTZ) adult (≥P60) rats, and CA3 field potentials were recorded during perfusion with Mg2+-free artificial cerebrospinal fluid (ACSF) or with ACSF containing 50 μm 4-aminopyridine (4-AP). Key Findings: In Mg2+-free ACSF, spontaneous interictal-like epileptiform discharges (IEDs) were recorded in all slices, with significantly higher frequencies in temporal (0.46 ± 0.03 Hz, n = 85) versus septal slices (0.20 ± 0.02 Hz, n = 47, p

Jutta Beier1, Jan van Noord2, Amanda Deans3, Jean Brooks3, Claire Maden3, Suus Baggen4, Rashmi Mehta5, Anthony Cahn31INSAF Respiratory Research Institute, Germany; 2Atrium Medisch Centrum, The Netherlands; 3GlaxoSmithKline, Stockley Park, UK; 4GlaxoSmithKline, Zeist, The Netherlands; 5GlaxoSmithKline, RTP, NC, USABackground: GSK233705 is an inhaled, long-acting muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease (COPD). This study was performed to see if the addition of GSK233705 to salmeterol would provide greater bronchodilation than salmeterol or tiotropium alone in COPD.Methods: In an incomplete-block, three-period, crossover design, dually responsive patients received three of the following five treatments: GSK233705 20 µg plus salmeterol 50 µg twice-daily; GSK233705 50 µg plus salmeterol 50 µg twice-daily; salmeterol 50 µg or placebo, each twice-daily; and tiotropium 18 µg or placebo once-daily for 7 days. Each treatment period was separated by a 14-day washout. The primary efficacy endpoint was morning (trough) forced expiratory volume in 1 second (FEV1) on Day 8, following 7 days of treatment. Secondary endpoints included pulmonary function, plethysmography, pharmacokinetics of GSK233705 and salmeterol, adverse events (AEs), electrocardiograms (ECGs), vital signs, and laboratory parameters.Results: A total of 47 patients were randomized. The mean % predicted normal postbronchodilator FEV1 was 55% at screening. Compared with placebo (n = 24), the adjusted mean change from baseline in trough FEV1 on Day 8 was 215 mL higher with GSK233705 20 µg + salmeterol (n = 23) and 203 mL higher with GSK233705 50 µg + salmeterol (n = 27), whereas with salmeterol (n = 27) and tiotropium (n = 28) the changes were 101 mL and 118 mL higher, respectively. The primary efficacy results were supported by the results from the other secondary lung function assessments. AEs were reported by similar proportions of patients across the treatment groups, with headache the most frequently reported treatment-related AE reported by one subject receiving each of GSK233705 20 µg + salmeterol, tiotropium, and placebo. No significant differences were seen in vital signs, ECGs, or laboratory parameters between the groups.Conclusion: The addition of GSK233705 to salmeterol in partially reversible COPD patients resulted in greater bronchodilation than salmeterol or tiotropium alone and was well tolerated.Keywords: COPD, bronchodilation, dual therapy, LAMA, LABA

Background Resveratrol (3,4’,5-trihydroxystilbene) is a naturally occurring compound found in grapes, wine, peanuts and cranberries. Recently, in vitro and cell culture studies have reported beneficial effects of resveratrol in the neurodegenerative process in Alzheimer’s disease (AD). However, in vivo effect of resveratrol in models of learning and memory is not yet evaluated. The present study was performed to examine the effect of resveratrol on cognitive impairment induced by scopolamine, a muscarinic antagonist, in mice. Methods Scopolamine was administered in a dose of 1 mg/kg intraperitoneally ( ip ). Cognitive functions were assessed using transfer latency (TL) on elevated plus maze, step-down latency (SDL) on a passive avoidance apparatus and escape latency (EL) in Morris water maze test. Results Scopolamine produced significant prolongation of TL, reduction in SDL as well as EL showing cognitive impairment in mice. Pre-treatment with resveratrol (10 mg/kg and 20 mg/kg, ip ) for 21 days showed no difference in TL, SDL and EL. Conclusion Resveratrol treatment does not reverse scopolamine-induced deficit in cognitive functions in mice.

Objectives This study was conducted to evaluate the effects of exposure to pioglitazone, a peroxisome proliferator-activated receptor agonist, on cognitive impairment induced by scopolamine, a muscarinic antagonist, in mice. Methods Pioglitazone (9 mg/kg, 18 mg/kg) was orally administered for 9 days at 30 min before intraperitoneal injection with scopolamine (0.8 mg/kg, i.p.). Cognitive function was evaluated by the passive avoidance test and the Morris water maze test on the 10th day after treatment. Changes in cholinergic system reactivity were also examined by measuring the acetylcholine, acetylcholinesterase and choline acetyltransferase in the hippocampus and cortex. Key findings Scopolamine injection induced impaired performance in the passive avoidance test and the water maze test and severe decrease of cholinergic system reactivity, as indicated by reduced acetylcholine levels, decreased choline acetyltransferase activity and increased acetylcholinesterase activity. Daily administration of pioglitazone significantly increased step-through latency in passive avoidance test, and significantly decreased the escape latency, and increased the time spent in the platform quadrant in the Morris water maze test. Pioglitazone also protected against scopolamine-induced cholinergic system deficit, including reduced acetylcholine levels, decreased choline acetyltransferase activity and increased acetylcholinesterase activity in the hippocampus or cortex. Conclusions Pioglitazone demonstrates a significant neuroprotective effect against scopolamine-induced cholinergic system deficit and cognitive impairment.

We have previously reported that earlier blockade of protein kinase C (PKC) augments the suppressive effect of μ-opioid receptors (MORs) on the GABAergic inhibitory postsynaptic current (IPSC) in the MOR-rich striosomes of the striatum. Interestingly, striatal medium-spiny neurons have muscarinic acetylcholine receptor subtypes M1 and M4, among which M1 activates the phosphoinositide signaling pathway yielding PKC. In this study, we examined whether acetylcholine regulates the effects of MOR on presynaptic IPSC by binding to the M1 receptor, and found that IPSC suppression by the MOR agonist, [D-Ala-N-Me-Phe, Gly-ol]-enkephalin, was significantly augmented and prolonged by the PKC inhibitor chelerythrine and attenuated by the PKC activator, phorbol 12, 13-dibutyrate. This modulatory action by chelerythrine was mimicked by the muscarinic antagonist atropine and the M1-specific antagonist pirenzepine, whereas M2-M4 antagonists had no discernible effect. These results suggest that PKC activity modulates the effect of MOR by muscarinic receptors in the striosomes.

Introduction: GSK233705 is a recently developed inhaled anticholinergic being investigated for the potential treatment of chronic obstructive pulmonary disease (COPD). Objectives: This dose-ranging, parallel-group, double-blind study compared the bronchodilator efficacy, safety and pharmacokinetics of GSK233705 with placebo in patients with moderate-to-severe COPD. Methods: Patients were randomised to receive 12.5 µg, 25 µg, 50 µg, 100 µg or 200 µg of GSK233705 or placebo once daily for 28 days. The primary endpoint was change from baseline in trough forced expiratory volume in 1 s (FEV1) on day 29. Results: The intent-to-treat population consisted of 576 patients (mean predicted FEV1 51%; mean age 62 years). Treatment with GSK233705 produced statistically significant improvements in pulmonary function compared with placebo. Only the 200 µg dose exceeded the predefined target threshold of 130-mL difference compared with placebo for the primary endpoint of change from baseline in trough FEV1 on day 29. No clear pattern of dose response was observed for the other doses. Serial FEV1 (0–24 h) showed a peak effect around 2 h postdose and tended to decline to clinically insignificant levels compared with placebo at 23 and 24 h. Each dose of GSK233705 was well tolerated. The incidence of adverse events was low and similar across all treatment groups. There were no clinically significant effects on laboratory parameters, vital signs or electrocardiograms. Conclusion: All doses of GSK233705 demonstrated bronchodilatory activity and were well tolerated. Although the onset of bronchodilation was rapid, it was not sustained over 24 h making it unsuitable for once-daily dosing. Please cite this paper as: Bateman E, Feldman G, Kilbride S, Brooks J, Mehta R, Harris S, Maden C and Crater G. Efficacy and safety of the long-acting muscarinic antagonist GSK233705 delivered once daily in patients with COPD. Clin Respir J 2012; DOI:10.1111/j.1752-699X.2011.00278.x.

Aclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium.The effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10-1000 μg/kg, s.c.), anaesthetised Beagle dogs (1000 μg/kg, i.v.) and anaesthetised guinea pigs (3-100μg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only).Aclidinium 1000 μg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 μg/kg which significantly decreased this parameter (p0.05). Aclidinium 1000 μg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3-100 μg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 μg/kg decreased the peak micturition pressure (p0.05), increased the volume of urine retained in the bladder (p0.01) and showed a trend to decrease the volume of urine excreted.Aclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.

Bifunctional aromatic compounds are claimed to contain a β(2) agonist pharmacophore linked by a flexible lipophilic spacer incorporating a trans-4-aminocyclohexanol group to a muscarinic antagonist pharmacophore. The compounds display nanomolar affinity at both β(2) and M(3) receptors and a prolonged duration of action in animal models. This application is the first from Almirall claiming such compounds although it has recently disclosed that LAS-190792 is progressing toward clinical development.

Aclidinium bromide is a novel, inhaled long-acting muscarinic antagonist with low systemic activity developed for the treatment of COPD. It is an ester compound rapidly hydrolysed in plasma into inactive alcohol and acid metabolites. In this Phase I, open-label study, the rates and routes of elimination of radioactivity following intravenous administration of [14 C]-aclidinium bromide were determined. The metabolites of aclidinium were also characterized and identified in plasma and excreta. Twelve healthy males were randomized (1:1) to receive a single intravenous 400 µg dose of [phenyl-U-14 C]- or [glycolyl-U-14 C]-aclidinium bromide (via 5 min infusion) to label alcohol or acid metabolites of aclidinium, respectively. Safety and tolerability were assessed over a 9-day period. Following intravenous administration, the parent compound was rapidly hydrolysed into its acid and alcohol metabolites. Primary excretion routes for [phenyl-U-14 C]- and [glycolyl-U-14 C]-aclidinium were renal (urine: 65% and 54%, respectively; feces: 33% and 20%, respectively), with 1% excreted as unchanged aclidinium. A total of three treatment-emergent adverse events in two subjects were reported and were related to infusion site pain. Overall, aclidinium is rapidly hydrolysed into two main metabolites, which are predominantly excreted in urine. Aclidinium bromide 400 µg administered intravenously was safe and well tolerated in healthy subjects. Copyright © 2012 John Wiley & Sons, Ltd.

We searched for pre-junctional inhibitory muscarinic receptors in isolated bovine trachealis strips and bronchial rings. Electric stimulation (ES)-induced tritiated acetylcholine ([(3)H]-ACh)-release and isometric contractions were determined in muscles incubated with the non-selective muscarinic agonist pilocarpine, the non-selective muscarinic antagonist atropine, the selective M(2)-receptor antagonists methoctramine and gallamine, or the selective M(4)-receptor antagonist PD102807. Electric field stimulation (EFS)-induced isometric contractile responses were assessed in trachealis strips and bronchial rings treated with 10(-9)-10(-5)M methoctramine, gallamine or PD102807. Pilocarpine (10(-6) and 10(-5)M) and atropine (10(-7)M) significantly decreased and increased ES-evoked [(3)H]-ACh-release, respectively. The enhancing effect of atropine on [(3)H]-ACh-release prevailed over the inhibitory effect of pilocarpine. M(2)- and M(4)-receptor antagonists did not increase EFS-induced contraction or ES-induced [(3)H]-ACh-release. However, 10(-7)M methoctramine, gallamine or PD102807 significantly attenuated the inhibitory effects of pilocarpine 10(-5)M on ES-induced [(3)H]-ACh-release.Muscarinic autoregulation is present in bovine airways but is not fully accounted for by M(2)- and M(4)-receptor subtypes.

It has been suggested that neural mechanisms may contribute to effects of the incretin hormones, and, therefore, also to the effects of dipeptidyl peptidase (DPP-4) inhibition. We therefore examined whether muscarinic mechanisms are involved in the stimulation of insulin secretion by DPP-4 inhibition. Fasted, anesthetized mice were given intraperitoneal saline or the muscarinic antagonist atropine (5mg/kg) before duodenal glucose (75mg/mouse), with or without the DPP-4 inhibitor NVPDPP728 (0.095mg/mouse), or before intravenous glucose (0.35g/kg) with or without co-administration with GLP-1 or glucose-dependent insulinotropic polypeptide (GIP) (both 3nmol/kg). Furthermore, isolated islets were incubated (1h) in 2.8 and 11.1mM glucose, with or without GIP or GLP-1 (both 100nM), in the presence or absence of atropine (100µM). Duodenal glucose increased circulating insulin and this effect was potentiated by DPP-4 inhibition. The increase in insulin achieved by DPP-4 inhibition was reduced by atropine by approximately 35%. Duodenal glucose also elicited an increase in circulating intact GLP-1 and GIP and this was augmented by DPP-4 inhibition, but these effects were not affected by atropine. Atropine did also not affect the augmentation by GLP-1 and GIP on glucose-stimulated insulin secretion from isolated islets. Based on these findings, we suggest that muscarinic mechanisms contribute to the stimulation of insulin secretion by DPP-4 inhibition through neural effects induced by GLP-1 and GIP whereas neural effects do not affect the levels of GLP-1 or GIP or the islet effects of the two incretin hormones.

The atypical antipsychotic clozapine is widely used for treatment-resistant schizophrenic patients. Clozapine and its major active metabolite, N-desmethylclozapine (NDMC), have complex pharmacological properties, and interact with various neurotransmitter receptors. There are several biochemical studies reporting that NDMC exhibits a partial agonist profile at the human recombinant M1 muscarinic receptors. However, direct electrophysiological evidence showing the ability of NDMC to activate native M1 receptors in intact neurons is poor. Using rat hippocampal neurons, we previously demonstrated that activation of muscarinic receptors by a muscarinic agonist, oxotremorine M (oxo-M), induces a decrease in outward K(+)current at -40mV. In the present study, using this muscarinic current response we assessed agonist and antagonist activities of clozapine and NDMC at native muscarinic receptors in intact hippocampal excitatory and inhibitory neurons. Suppression of the oxo-M-induced current response by the M1 antagonist pirenzepine was evident only in excitatory neurons, while the M3 antagonist darifenacin was effective in both types of neurons. Muscarinic agonist activity of NDMC was higher than that of clozapine, higher in excitatory neurons than in inhibitory neurons, sensitive to pirenzepine, and partially masked when co-applied with clozapine. Muscarinic antagonist activity of clozapine as well as NDMC was not different between excitatory and inhibitory neurons, but clozapine was more effective than NDMC. These results demonstrate that NDMC has the ability to activate native M1 receptors expressed in hippocampal excitatory neurons, but its agonist activity might be limited in clozapine-treated patients because of the presence of excessive clozapine with muscarinic antagonist activity.

Afisi Segun Ismaila,1,2 Eline L Huisman,3 Yogesh Suresh Punekar,4 Andreas Karabis31Value Evidence and Outcomes, GlaxoSmithKline, Research Triangle Park, NC, USA; 2Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, ON, Canada; 3Real World Strategy and Analytics, Mapi Group, Houten, the Netherlands; 4Value Evidence and Outcomes, GlaxoSmithKline, Uxbridge, UKBackground: Randomized, controlled trials comparing long-acting muscarinic antagonist (LAMA) efficacy in COPD are limited. This network meta-analysis (NMA) assessed the relative efficacy of tiotropium 18 µg once-daily (OD) and newer agents (aclidinium 400 µg twice-daily, glycopyrronium 50 µg OD, and umeclidinium 62.5 µg OD).Methods: A systematic literature review identified randomized, controlled trials of adult COPD patients receiving LAMAs. A NMA within a Bayesian framework examined change from baseline in trough forced expiratory volume in 1 second (FEV1), transitional dyspnea index focal score, St George’s Respiratory Questionnaire score, and rescue medication use.Results: Twenty-four studies (n=21,311) compared LAMAs with placebo/each other. Aclidinium, glycopyrronium, tiotropium, and umeclidinium, respectively, demonstrated favorable results versus placebo, for change from baseline (95% credible interval) in 12-week trough FEV1 (primary endpoint: 101.40 mL [77.06–125.60]; 117.20 mL [104.50–129.90]; 114.10 mL [103.10–125.20]; 136.70 mL [104.20–169.20]); 24-week trough FEV1 (128.10 mL [84.10–172.00]; 135.80 mL [123.10–148.30]; 106.40 mL [95.45–117.30]; 115.00 mL [74.51–155.30]); 24-week St George’s Respiratory Questionnaire score (-4.60 [-6.76 to -2.54]; -3.14 [-3.83 to -2.45]; -2.43 [-2.92 to -1.93]; -4.69 [-7.05 to -2.31]); 24-week transitional dyspnea index score (1.00 [0.41–1.59]; 1.01 [0.79–1.22]; 0.82 [0.62–1.02]; 1.00 [0.49–1.51]); and 24-week rescue medication use (data not available; -0.41 puffs/day [-0.62 to -0.20]; -0.52 puffs/day [-0.74 to -0.30]; -0.30 puffs/day [-0.81 to 0.21]). For 12-week trough FEV1, differences in change from baseline (95% credible interval) were -12.8 mL (-39.39 to 13.93), aclidinium versus tiotropium; 3.08 mL (-7.58 to 13.69), glycopyrronium versus tiotropium; 22.58 mL (-11.58 to 56.97), umeclidinium versus tiotropium; 15.90 mL (-11.60 to 43.15), glycopyrronium versus aclidinium; 35.40 mL (-5.06 to 76.07), umeclidinium versus aclidinium; and 19.50 mL (-15.30 to 54.38), umeclidinium versus glycopyrronium. Limitations included inhaler-related factors and safety; longer-term outcomes were not considered.Conclusion: The new LAMAs studied had at least comparable efficacy to tiotropium, the established class standard. Choice should depend on physician’s and patient’s preference.Keywords: anticholinergics, muscarinic antagonist, bronchodilator, systematic review, meta-analysis

Acute inhalation of airborne pollutants alters cardiovascular function and evidence suggests that pollutant-induced activation of airway sensory nerves via the gating of ion channels is critical to these systemic responses. Here, we have investigated the effect of capsaicin [transient receptor potential (TRP) vanilloid 1 (TRPV1) agonist], AITC [TRP ankyrin 1 (TRPA1) agonist], and ATP (P2X2/3 agonist) on bronchopulmonary sensory activity and cardiovascular responses of conscious Sprague-Dawley (SD) rats. Single fiber recordings show that allyl isothiocyanate (AITC) and capsaicin selectively activate C fibers, whereas subpopulations of both A and C fibers are activated by stimulation of P2X2/3 receptors. Inhalation of the agonists by conscious rats caused significant bradycardia, atrioventricular (AV) block, and prolonged PR intervals, although ATP-induced responses were lesser than those evoked by AITC or capsaicin. Responses to AITC were inhibited by the TRP channel blocker ruthenium red and the muscarinic antagonist atropine. AITC inhalation also caused a biphasic blood pressure response: a brief hypertensive phase followed by a hypotensive phase. Atropine accentuated the hypertensive phase, while preventing the hypotension. AITC-evoked bradycardia was not abolished by terazosin, the α1-adrenoceptor inhibitor, which prevented the hypertensive response. Anesthetics had profound effects on AITC-evoked bradycardia and AV block, which was abolished by urethane, ketamine, and isoflurane. Nevertheless, AITC inhalation caused bradycardia and AV block in paralyzed and ventilated rats following precollicular decerebration. In conclusion, we provide evidence that activation of ion channels expressed on nociceptive airway sensory nerves causes significant cardiovascular effects in conscious SD rats via reflex modulation of the autonomic nervous system.

To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1 mg/kg) and DMXBA (0.3 mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment.

A novel muscarinic receptor agonist, SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), is a candidate therapeutic drug for xerostomia in Sjogren's syndrome. The general pharmacological properties of this drug on general behavior and the central nervous system were investigated in mice, rats and cats. 1. General behavior: When SNI-2011 was administered orally to mice at 100 mg/kg, mydriasis, a decrease of spontaneous motor activity, tremor, convulsions, salivation, abnormal posture, abnormal gait, reduced grip strength and reduced response against external stimulating were observed, and 2 out of 6 animals died. At 10 mg/kg or lower, no particular sign was observed except mydriasis, which appeared to be caused via the peripheral muscarinic acetylcholine receptors. 2. Central nervous system: SNI-2011 had no effect on the motor coordination in mice. Hypothermia was observed in rats and reduced spontaneous motor activity, analgesia and enhanced maximum electroshock-induced convulsions were observed in mice after oral administration of 30 mg/kg SNI-2011. Slight increase in the rate of theta-wave band in the hippocampal EEG of rats and spinal multisynaptic reflexes in cats were observed after intravenous injection of 10 mg/kg SNI-2011. At an oral dose of 10 mg/kg, prolongation of thiopental-induced sleeping time in mice was observed. The prolongation of sleeping time was inhibited by a peripheral muscarinic antagonist. These results suggest that SNI-2011 has muscarinic effects on general behavior and the central nervous system at the doses approximately 10-fold higher than the effective doses needed for saliva secretion.

Sexual behavior in male rats is thought to depend in part on central cholinergic activity. In particular, previous studies of responses to systemically administered cholinergic drugs suggest that male rat behavior can be facilitated by the muscarinic agonist oxotremorine but is disrupted by the muscarinic antagonist scopolamine. However, it is not clear how broadly these effects generalize across species. To address this issue, we observed the impact on sexual behavior in male hamsters of systemic treatment with oxotremorine or scopolamine. In each case, the peripheral muscarinic antagonist methylscopolamine was used as an auxiliary or control treatment to better isolate central cholinergic effects. Both oxotremorine and scopolamine disrupted male behavior in hamsters. For example, both increased the likelihood of failure to achieve intromission or ejaculation. Further, even on completed tests oxotremorine treatment led to changes including increases in mount latency and postejaculatory interval while scopolamine treatment caused changes including increases in ejaculation latency and intromission frequency. The many changes caused by these treatments suggest that acetylcholine helps to control many elements of male behavior, probably by acting at multiple brain sites. The generally similar responses to a cholinergic agonist and antagonist suggest the dependence of efficient mating behavior on optimal levels of central cholinergic activity.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the world today. Bronchodilators, particularly muscarinic antagonists and β2 agonists, are recommended for patients with moderate to severe COPD. Dual-pharmacology muscarinic antagonist– β2 agonist (MABA) molecules present an exciting new approach to the treatment of COPD by combining muscarinic antagonism and β2 agonism in a single entity. They have the potential to demonstrate additive or synergistic bronchodilation over either pharmacology alone. Due to this enticing prospect, several companies have now reported MABA discovery efforts through a conjugated/linked strategy with one candidate (GSK-961081) demonstrating clinical proof of concept. Several MABA crystal forms have been identified, satisfying the requirements for inhaled dosing devices. There are significant challenges in designing MABAs, but the potential to achieve enhanced bronchoprotection in patients and facilitate ‘triple therapy’ makes this an extremely important and exciting area of pharmaceutical research.

In 2002, the first long-acting muscarinic antagonist, tiotropium bromide (Spiriva®), was launched as a once-daily bronchodilating agent for the treatment of chronic obstructive pulmonary disease. Since then, there has been intense discovery research activity in this area and, currently, several alternative inhaled long-acting muscarinic antagonists are reported under clinical development by several pharmaceutical companies. This article will review the current inhaled development candidates, as well as literature reports of the most significant preclinical chemical series specifically designed as inhaled antimuscarinic agents.

Following interrogation of a wide-ligand profile database, a nonselective norepinephrin reuptake inhibitor was converted into a novel muscarinic antagonist using two medicinal chemistry transformations (M3/NRI selectivity of >1000). Conjugation to a β2 agonist motif furnished a molecule with balanced dual pharmacology, as demonstrated in a guinea pig trachea tissue model of bronchoconstriction. This approach provides new starting points for the treatment of chronic obstructive pulmonary disease and illustrates the potential for building selectivity into GPCR modulators that possess intrinsic promiscuity or reverse selectivity.

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.

The 5-hydroxytryptamine(6) (5-HT(6)) receptor has been suggested to play an important role in the regulation of memory and cognition. In the present study, our aim was to investigate whether the novel, selective 5-HT(6) antagonists compound (CMP) X and CMP Y and the reference 5-HT(6) antagonist GSK-742457 could ameliorate impairments in episodic memory in 3-months-old male Wistar rats. The acetylcholinesterase inhibitor (AChEI) donepezil (Aricept®, approved for symptomatic treatment of Alzheimer's disease, AD) was used as a positive reference compound. First, effects of the 5-HT(6) antagonists CMP X, CMP Y and GSK-742457 were investigated on object recognition task (ORT) performance in rats treated with the muscarinic antagonist scopolamine (0.1mg/kg, administered intraperitoneally, i.p., 30 min before trial 1). Second, effects of the combination of suboptimal doses of 5-HT(6) antagonists CMP X and CMP Y with the AChEI donepezil were studied, to determine whether the 5-HT(6) antagonists show additive synergism with donepezil in the ORT. Finally, effects of CMP Y, GSK-742457 and donepezil were investigated on object location task (OLT) performance in rats treated with scopolamine. Donepezil (1mg/kg, oral administration, p.o.), GSK-742457 (3mg/kg, i.p.), CMP X (3mg/kg, i.p.) and CMP Y (30 mg/kg, p.o.), all ameliorated the scopolamine-induced deficits in object recognition. In the ORT, we have found that combined administration of subthreshold doses of CMP X (1mg/kg, i.p.) and CMP Y (10mg/kg, p.o.) with the AChEI donepezil (0.1mg/kg, p.o.), enhanced memory performance in Wistar rats with deficits induced by scopolamine. Donepezil (0.1mg/kg, p.o.) alone had no discernable effects on performance. This suggests additive synergistic effects of the 5-HT(6) antagonists (CMP X and CMP Y) with donepezil on cognitive impairment. Finally, donepezil (1mg/kg, p.o.), GSK-742457 (10mg/kg, p.o.) and CMP Y (30 mg/kg, p.o.) also reduced scopolamine-induced deficits in the OLT. In conclusion, the 5-HT(6) antagonists were found to clearly improve episodic memory deficits induced by scopolamine. In addition, co-administration of the 5-HT(6) receptor antagonists CMP X and CMP Y with the AChEI donepezil to cognitively impaired rats also resulted in potentially additive enhancing effects on cognition. This suggests that these compounds could have potential as monotherapy, but also as adjunctive therapy in patients with AD treated with common treatments such as donepezil.

The atypical antipsychotics (AAPs) have been associated with increased risk of type-2 diabetes. Evidence suggests direct, drug-related effects independent of weight gain and although mechanisms underlying this phenomenon are unclear, it has been suggested that the heterogeneous receptor binding profile of the AAPs may influence receptors implicated in glucose metabolism. This study aimed to clarify weight gain-independent mechanisms of AAP-induced changes in insulin secretion by deconstructing their binding profile with representative antagonists. Healthy rats were pretreated with a single subcutaneous dose of darifenacin 6 mg/kg (n=10), a selective M(3) muscarinic antagonist; ketanserin 2mg/kg (n=10), a 5HT(2A) antagonist; raclopride 0.3mg/kg (n=11) a selective D(2)/D(3) antagonist; terfenadine 20mg/kg (n=9) a selective H(1) antagonist; or, vehicle (n=11). Hyperglycemic clamps were employed following injection, providing an index of secretory capacity of pancreatic β-cells. Acute treatment with darifenacin and ketanserin significantly decreased insulin response to glucose challenge as compared to controls, which was confirmed in the darifenacin group by reduced C-peptide levels. Treatment with raclopride resulted in an increased insulin response and a strong tendency to increased C-peptide levels. H(1) blockade did not result in effects on insulin or C-peptide. Results suggest that the effects of antipsychotics on glucose dysregulation may be related to direct inhibitory effects of muscarinic (M(3)) and serotonergic (5HT(2)) antagonism on insulin secretion. Based on the expression of D(2)-like receptors in β-cells, which mediate inhibition of insulin secretion, we propose that prolonged D(2) blockade with antipsychotics may predispose to depletion of insulin stores and an eventual defect in pancreatic compensation.

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T(9)-T(10) spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β-methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10 μM) prior to 4-DAMP (500 nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (P=0.041). This was accompanied by a substantial decrease in α,β-methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, P=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Vagus nerve stimulation (VNS) is an FDA approved treatment for drug-resistant epilepsy and depression. Recently, we demonstrated the capacity for repeatedly pairing sensory input with brief pulses of VNS to induce input specific reorganization in rat auditory cortex. This was subsequently used to reverse the pathological neural and perceptual correlates of hearing loss induced tinnitus. Despite its therapeutic potential, VNS mechanisms of action remain speculative. In this study, we report the acute effects of VNS on intra-cortical synchrony, excitability, and sensory processing in anesthetized rat auditory cortex. VNS significantly increased and decorrelated spontaneous multi-unit activity, and suppressed entrainment to repetitive noise burst stimulation at 6-8 Hz but not after application of the muscarinic antagonist scopolamine. Collectively, these experiments demonstrate the capacity for VNS to acutely influence cortical synchrony and excitability and strengthen the hypothesis that acetylcholine and muscarinic receptors are involved in VNS mechanisms of action. These results are discussed with respect to their possible implications for sensory processing, neural plasticity, and epilepsy.

M3 subtype muscarinic receptors (CHRM3) are over-expressed in colon cancer. In this study, we used Apc(min/+) mice to identify the role of Chrm3 expression in a genetic model of intestinal neoplasia, explored the role of Chrm3 in intestinal mucosal development and determined the translational potential of inhibiting muscarinic receptor activation. We generated Chrm3-deficient Apc(min/+) mice and compared intestinal morphology and tumor number in 12-week-old Apc(min/+)Chrm3(-/-) and Apc(min/+)Chrm3(+/+) control mice. Compared with Apc(min/+)Chrm3(+/+) mice, Apc(min/+)Chrm3(-/-) mice showed a 70 and 81% reduction in tumor number and volume, respectively (P < 0.01). In adenomas, β-catenin nuclear staining was reduced in Apc(min/+)Chrm3(-/-) compared with Apc(min/+)Chrm3(+/+) mice (P < 0.02). Whereas Apc gene mutation increased the number of crypt and Paneth cells and decreased villus goblet cells, these changes were absent in Apc(min/+)Chrm3(-/-) mice. To determine whether pharmacological inhibition of muscarinic receptor activation attenuates intestinal neoplasia, we treated 6-week-old Apc(min/+) mice with scopolamine butylbromide, a non-subtype-selective muscarinic receptor antagonist. After 8 weeks of continuous treatment, scopolamine butylbromide-treated mice showed a 22% reduction in tumor number (P = 0.027) and a 36% reduction in tumor volume (P = 0.004) as compared with control mice. Compared with Chrm3 gene ablation, the muscarinic antagonist was less efficacious, most probably due to shorter duration of treatment and incomplete blockade of muscarinic receptors. Overall, these findings indicate that interplay of Chrm3 and β-catenin signaling is important for intestinal mucosal differentiation and neoplasia and provide a proof-of-concept that pharmacological inhibition of muscarinic receptor activation can attenuate intestinal neoplasia in vivo.

Purpose: It is well established that the broad-band muscarinic antagonist, atropine is effective at inhibiting the progression of myopia and does so by preventing the elongation of the vitreous chamber of the eye. However, uncertainty remains as to whether this effect occurs through a receptoral mechanism and, if so, which muscarinic receptor subtype mediates this effect. Previous work, in avian and mammalian models of myopia, implicates the M1 and M4 receptors as potential targets. The current study used physiologically relevant concentrations of highly selective muscarinic antagonists (MT-3 and MT-7) to further characterise the role of the M4 receptor in the control of myopia in the chick model of refractive development. Methods: Nine groups of week-old chicks underwent 5 days of monocular deprivation, with a translucent occluder, to induce myopia. These animals had either no injection, scleral puncture with a needle, or daily intravitreal injections of MT-3 (M4-selective), MT-7 (M1-selective) or vehicle. Three concentrations of each antagonist were delivered (250 nm, 2.5 lm and 10 lm). After the treatment period, keratometry, retinoscopy and A-Scan ultrasound were used to assess ocular biometry. Results: MT-3 treatment produced a significant dose-dependent reduction in relative myopia (treated)control eye) compared to vehicle treatment (vehicle )10.1 ± 1.1 D vs 10 lm MT-3 )4.0 ± 1.5 D, p < 0.01). The majority of this effect was due to reduced relative vitreous chamber elongation in drug treated eyes (vehicle +0.26 ± 0.04 mm, 10 lm MT-3 +0.08 ± 0.07 mm, p < 0.05). In contrast, MT-7 had no significant effect on the development of myopia (MT-7 10 lm: myopia, )12.1 ± 0.8 D and vitreous chamber depth, +0.23 ± 0.07 mm). Calculations indicate that the experimentally achieved concentrations of MT-3 at intraocular receptors necessary to inhibit 50% of myopia development (between 5 and 50 nm) were consistent with published in vitro affinity constants for the M4 receptor and below those for the M1 receptor. Histology demonstrated that MT-3 at the doses used had no gross effects on the retina, indicating a non-toxic mode of action. Conclusions: In the chick, which lacks a homologue of the mammalian M1 receptor, the above findings represent compelling evidence that muscarinic antagonists prevent myopia progression through an M4-receptor mediated mechanism, most likely located in the retina.

NVA237 is a novel, once daily inhaled long-acting muscarinic antagonist administered via a dry powder inhaler. This study aimed to assess the 24-h bronchodilatory effect following 14 days of treatment with inhaled NVA237 in patients with mild, moderate or severe COPD. This was a randomized, double-blind, placebo-controlled, two-period, crossover, multicenter study. A total of 33 patients (≥ 40 years; smoking history of ≥ 10 pack-years) were randomized to receive NVA237 50 μg once daily followed by placebo or placebo followed by NVA237 50 μg for 14 days. Treatment periods were separated by a 7-14 day washout period. The primary variable was the mean forced expiratory volume in 1 s (FEV(1)) derived from the area under the curve (AUC) between 0 and 24 h post-dose on Day 14. The 24-h FEV(1) profiles showed a consistent bronchodilator effect for NVA237 versus placebo on Day 14. Least square (LS) mean difference in FEV(1) AUC(0-24 h) values between NVA237 and placebo was 163 mL (P < 0.001). There were significant increases in mean FEV(1) AUC(0-12 h) (LS mean difference 165 mL, P = 0.001) and FEV(1) AUC(12-24 h) (161 mL, P < 0.001) versus placebo. NVA237 significantly improved peak FEV(1) (by 208 mL, P < 0.001) and trough FEV(1) (by 154 mL, P = 0.003) versus placebo on Day 14. NVA237 was well tolerated; all adverse events were mild or moderate in intensity and not related to study drug. NVA237 50 μg once daily was well tolerated and showed significant and sustained 24-h bronchodilation in patients with COPD.

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na+ channel blocker) and ω-conotoxin GVIA (N-type Ca2+ channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent ω-conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl2, 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca2+-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.

BACKGROUND AND PURPOSE We have previously reported that galantamine, a weak acetylcholinesterase inhibitor, improves prepulse inhibition (PPI) deficits in mice reared in social isolation. ACh receptors are involved in the underlying mechanism of PPI, but whether rearing in social isolation causes dysfunction of the cholinergic system is unknown. In this study, we examined the involvement of muscarinic receptors in the improvement of PPI deficits induced by galantamine, and whether the cholinergic system is altered in mice reared in isolation. EXPERIMENTAL APPROACH Three-week-old male ddY mice were housed in isolated cages for 6 weeks before the initiation of experiments to create PPI deficits. Cholinergic functions were determined by measuring the behavioural and neurochemical responses to nicotinic and muscarinic receptor agonists. KEY RESULTS The improvement by galantamine of social isolation-induced PPI deficits was blocked by scopolamine, a non-selective muscarinic antagonist, and telenzepine, a preferential M1 receptor antagonist. Activation of M1 receptors improved social isolation-induced PPI deficits. Social isolation did not affect choline acetyltransferase and acetylcholinesterase activities in the prefrontal cortex and hippocampus, but it reduced the locomotor-suppressive response to muscarinic agonist oxotremorine, but not to nicotine. The isolation also attenuated the M1 receptor agonist N-desmethylclozapine-induced increase in prefrontal dopamine release. CONCLUSIONS AND IMPLICATIONS Galantamine improves PPI deficits of mice reared in social isolation via activation of M1 receptors. Social isolation reduces the muscarinic, especially M1, receptor function and this is involved in PPI deficits.

In small cell lung carcinoma (SCLC), acetylcholine (ACh) is synthesized and secreted, and it acts as an autocrine growth factor through activation of its receptors, muscarinic receptor (mAChR) and nicotinic receptor (nAChR). Alteration of tumor growth by blockade of M3 mAChR in a human SCLC cell line, NCI-H82, was investigated in the present study. We used a highly selective M3 muscarinic antagonist, N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propioamide (J-115311). Our results show that J-115311 inhibited the increased intracellular calcium elicited by carbachol, a muscarinic agonist, in SCLC cells. J-115311 also inhibited SCLC cell growth in vitro. In a mouse orthotopic xenograft model, J-115311 dose-dependently reduced tumor growth when NCI-H82 cells were inoculated into the upper left lobe of the lung. These findings indicate that blockade of M3 mAChR can suppress tumor growth in SCLC, suggesting the potential therapeutic utility of M3 muscarinic antagonists as anti-cancer agents. Keywords:: selective M3 muscarinic antagonist, M3 muscarinic receptor, small cell lung carcinoma, orthotopic xenograft model, non-neuronal cholinergic system

In the present study, we describe the cardiovascular effects of local acetylcholine (Ach) microinjection into both the ventrolateral (vlPAG) and dorsal (dPAG) periaqueductal gray areas of anesthetized rats and the possible local receptors involved with these responses. Microinjection of Ach (9, 27, 45 or 81nmol/50nL) into the vlPAG caused dose-related depressor responses. These hypotensive responses were blocked by local pretreatment with increasing doses of the nonselective muscarinic antagonist atropine (1, 3 or 9nmol/50nL). The microinjection of Ach into the dPAG caused no significant cardiovascular responses in anesthetized rats. In conclusion, the present findings suggest that a cholinergic system present in the vlPAG, but not in the dPAG, is involved with cardiovascular system control. Moreover, these cardiovascular responses evoked by Ach are mediated by muscarinic receptors.

Electroacupuncture (EA) possesses various therapeutic effects, including alleviation of pain, reduction of inflammation and improvement of sleep disturbance. The mechanisms of EA on sleep improvement, however, remain to be determined. It has been stated in ancient Chinese literature that the Anmian (EX17) acupoint is one of the trigger points that alleviates insomnia. We previously demonstrated that EA stimulation of Anmian acupoints in rats during the dark period enhances non-rapid eye movement (NREM) sleep, which involves the induction of cholinergic activity in the nucleus tractus solitarius (NTS). In addition to cholinergic activation of the NTS, activation of the endogenous opioidergic system may also be a mechanism by which acupuncture affects sleep. Therefore, this study was designed to investigate the involvement of the NTS opioidergic system in EA-induced alterations in sleep. Our present results indicate that EA of Anmian acupoints increased NREM sleep, but not rapid eye movement sleep, during the dark period in rats. This enhancement in NREM sleep was dose-dependently blocked by microinjection of opioid receptor antagonist, naloxone, and theμ-opioid receptor antagonist, naloxonazine, into the NTS; administrations ofδ-receptor antagonist, natrindole, and theκ-receptor antagonist,nor-binaltrophimine, however, did not affect EA-induced alterations in sleep. Furthermore,β-endorphin was significantly increased in both the brainstem and hippocampus after the EA stimuli, an effect blocked by administration of the muscarinic antagonist scopolamine into the NTS. Our findings suggest that mechanisms of EA-induced NREM sleep enhancement may be mediated, in part, by cholinergic activation, stimulation of the opiodergic neurons to increase the concentrations ofβ-endorphin and the involvement of theμ-opioid receptors.

Chlorogenic acid is a major polyphenolic component of many plants and beverages, and is particularly abundant in coffee. We evaluated the neuroprotective effects of chlorogenic acid on learning and memory impairment induced by scopolamine (0.5 mg/kg, i.p.), a muscarinic antagonist, using the Y-maze, passive avoidance, and Morris water maze tests. The chlorogenic acid significantly improved the impairment of short-term or working memory induced by scopolamine in the Y-maze test, and significantly reversed cognitive impairments in mice as measured by the passive avoidance test. In addition, chlorogenic acid decreased escape latencies in the Morris water maze test. In a probe trial session, chlorogenic acid increased the latency time in the target quadrant in a dose-dependent manner. Ex vivo, chlorogenic acid inhibited acetylcholinesterase activity in the hippocampus and frontal cortex. Chlorogenic acid also decreased malondialdehyde levels in the hippocampus and frontal cortex. In vitro, chlorogenic acid was found to inhibit acetylcholinesterase activity (IC₅₀=98.17 μg/ml) and free radical scavenging activity (IC₅₀=3.09 μg/ml) in a dose-dependent manner. These results indicate that chlorogenic acid may exert anti-amnesic activity via inhibition of acetylcholinesterase and malondialdehyde in the hippocampus and frontal cortex.

Alternative splicing is an important mechanism for expanding proteome diversity from a limited number of genes, especially in higher vertebrates. Brain-specific splicing factors play an important role in establishing specific patterns of alternative splicing in the brain and thereby contribute to its complex architecture and function. Nova proteins are splicing factors that are expressed specifically in the central nervous system, where they regulate a large number of pre-mRNAs encoding synaptic proteins that are important for the balance of neuronal excitation and inhibition. Since this balance is interrupted in epileptic seizures, we explored whether LiCl/pilocarpine- or kainate-induced epileptic seizures would induce changes in the levels of Nova mRNAs in the rat brain. We found that the muscarinic agonist, pilocarpine, but not the glutamatergic agonist, kainate, induced a significant downregulation of Nova2 mRNA and upregulation of all three Nova1 mRNA isoforms in the striatum. Treatment with the muscarinic antagonist, scopolamine, at the onset of pilocarpine-induced seizures inhibited the seizures and the changes in Nova mRNA levels. Therefore it seems likely that pilocarpine stimulation of muscarinic acetylcholine receptors was a prerequisite for the observed changes, while the contribution of other striatal neurotransmitter systems activated by seizures could not be excluded. We propose that the LiCl/pilocarpine seizure model could serve as a valuable tool for studying mechanisms of Nova-regulated alternative splicing in rat striatum.

Tiotropium bromide is a long acting muscarinic antagonist (LAMA), marketed under the brand name Spiriva, for the treatment of chronic obstructive pulmonary disease (COPD). Besides its proven direct bronchodilatory activity, recent clinical studies demonstrated that tiotropium is able to reduce the exacerbation rate and impact the clinical course of COPD. One significant pathological feature believed to be causative for the progressive nature of COPD is chronic pulmonary inflammation. The aim of the present study was to investigate the anti-inflammatory activity of tiotropium on cigarette smoke-induced pulmonary inflammation in mice. C57Bl/6 mice were exposed to cigarette smoke (CS) for four days with increasing exposure time for up to 6h per day to elicit pulmonary inflammation and mediator release. One hour before smoke exposure, animals were treated with tiotropium by inhalation (0.01-0.3mg/mL) for 5 min; 18h after the last CS exposure a bronchoalveolar lavage was performed. Tiotropium concentration-dependently inhibited pulmonary neutrophilic inflammation with an IC(50) of 0.058 mg/mL and a maximum inhibition of 60% at 0.3mg/mL. Furthermore, the CS-induced pulmonary release of leukotriene B(4), interleukin-6, keratinocyte-derived chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha and -2, and tumor necrosis factor alpha was dose-dependently reduced. The bronchodilatory activity of tiotropium against acetycholine-induced bronchoconstriction was found to be in the same dose range as the anti-inflammatory activity with an IC(50) of 0.045 mg/mL and a maximum bronchodilation of 90% at 0.3mg/mL. Our data suggest that the beneficial effects of tiotropium on the course of COPD shown in patients may be associated with an anti-inflammatory activity.

The acoustic startle reflex is strongly inhibited by a moderate-intensity acoustic stimulus that precedes the startling stimulus by roughly 10-1000 ms (prepulse inhibition, PPI). At long interstimulus intervals (ISIs) of 100-1000 ms, PPI in rats is reduced by the muscarinic receptor antagonist scopolamine. Here, we studied the role of GABA receptors in PPI at full ISI ranges in both mice and rats. In B6 mice, PPI begins and ends at shorter ISIs (4 and 1000 ms, respectively) than in Wistar rats (8 and 5000 ms). The GABA(A) antagonist bicuculline (1 mg/kg i.p.) reduced PPI at ISIs near the peak of PPI in both rats and mice. The GABA(B) antagonist phaclofen (10 or 30 mg/kg i.p. in rats or mice, respectively) reduced PPI only at long ISIs, similar to the effects of the muscarinic antagonist scopolamine (1 mg/kg i.p.). The effects of phaclofen and scopolamine were additive in rats, suggesting independent effects of GABA(B) and muscarinic receptors. Patch-clamp recordings of startle-mediating PnC (nucleus reticularis pontis caudalis) giant neurons in rat slices show that EPSCs evoked by either trigeminal or auditory fiber stimulation were inhibited by the GABA(A/C) agonist muscimol or the GABA(B) agonist baclofen via postsynaptic mechanisms. Hyperpolarization of PnC neurons by muscimol was reversed with bicuculline, indicating that postsynaptic GABA(A) receptors strongly inhibit PnC giant neurons needed for startle. Therefore, GABA receptors on PnC giant neurons mediate a substantial part of PPI, with GABA(A) receptors contributing at the peak of PPI, and GABA(B) receptors adding to muscarinic effects on PPI at long ISIs.