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LAS190792 is a novel muscarinic antagonist and β2-adrenoceptor agonist in development for chronic respiratory diseases. This study investigated the pharmacological profile of LAS190792 in comparison to batefenterol, tiotropium, indacaterol and olodaterol. LAS190792 is potent at the human M3 receptor (pIC50: 8.8 in binding assays). It is selective for the β2-adrenoceptor over the β1-and β3-adrenoceptor, and shows a functional potency in a similar range to batefenterol and LABA compounds (pEC50 in spontaneous tone isolated trachea: 9.6). The relaxant potency of LAS190792 in electrically stimulated tissue is similar to batefenterol, with an antimuscarinic activity in presence of propranolol slightly higher than batefenterol (pIC50 of 8.3 versus 7.9 in human tissue). LAS190792 exhibits a sustained duration of action in isolated tissue longer than that of batefenterol. Nebulized LAS190792 inhibits acetylcholine-induced bronchoconstriction in dog with minimal cardiac effects and sustained bronchodilation (t1/2: 13.3 h). In conclusion, these studies suggest that LAS190792 is a dual-acting muscarinic antagonist β2-adrenoceptor agonist that has the potential to be a next generation bronchodilator with long-lasting effects and wide safety margin in humans.

We previously demonstrated that 3-iodothyronamine (T1AM), a by-product of thyroid hormone metabolism, pharmacologically administered to mice acutely stimulated learning and memory acquisition and provided hyperalgesia with a mechanism which remains to be defined. We now aimed to investigate whether the T1AM effect on memory and pain was maintained in mice pre-treated with scopolamine, a non-selective muscarinic antagonist expected to induce amnesia and, possibly, hyperalgesia. Mice were pre-treated with scopolamine and, after 20 min, injected intracerebroventricularly (i.c.v.) with T1AM (0.13, 0.4, 1.32 μg/kg). 15 min after T1AM injection, the mice learning capacity or their pain threshold were evaluated by the light/dark box and by the hot plate test (51.5 °C) respectively. Experiments in the light/dark box were repeated in mice receiving clorgyline (2.5 mg/kg, i.p.), a monoamine oxidase (MAO) inhibitor administered 10 min before scopolamine (0.3 mg/kg). Our results demonstrated that 0.3 mg/kg scopolamine induced amnesia without modifying the murine pain threshold. T1AM fully reversed scopolamine-induced amnesia and produced hyperalgesia at a dose as low as 0.13 μg/kg. The T1AM anti-amnestic effect was lost in mice pre-treated with clorgyline. We report that the removal of muscarinic signalling increases T1AM pro learning and hyperalgesic effectiveness suggesting T1AM as a potential treatment as a “pro-drug” for memory dysfunction in neurodegenerative diseases.

Background Methacholine dose-response curves illustrate pharmacologic bronchoprotection against methacholine-induced airway hyperresponsiveness and can be used to quantitate changes in airway sensitivity (position), reactivity (slope), and maximal responsiveness following drug administration. Our objective was to determine the influence of single-dose glycopyrronium (long-acting muscarinic antagonist) and indacaterol (ultra-long acting β2 agonist), as monotherapy and in combination, on the methacholine dose-response curve of mild asthmatics and to compare these findings with a non-asthmatic control curve. Methods This was a randomized, double blind, double dummy, three-way crossover study. For asthmatic participants (n = 14), each treatment arm included a baseline methacholine challenge, drug administration, and repeat methacholine challenges at 1, 24, and 48 h. Non-asthmatic control participants (n = 15) underwent a single methacholine challenge and did not receive any study treatment. Methacholine dose-response curves were graphed as the percent fall in forced expiratory volume in 1 s (FEV1) for each methacholine concentration administered. Best-fit curves were then generated. Differences in airway reactivity were calculated through linear regression. Changes in airway sensitivity were assessed as the shift in the provocative concentration of methacholine causing a 20% fall in FEV1. Results Compared to baseline, all treatments significantly reduced airway sensitivity to methacholine at 1 h post-dose (indacaterol ~1.5 doubling concentrations; glycopyrronium ~5 doubling concentrations; combination ~5 doubling concentrations). Bronchoprotection at 24 and 48 h remained significant with glycopyrronium and combination therapy only. Airway reactivity was not influenced by indacaterol whereas glycopyrronium significantly reduced airway reactivity at all time-points (p = 0.003-0.027). The combination significantly decreased slope at 1 (p = 0.021) and 24 (p = 0.039) hours only. The non-asthmatic control and 1-h glycopyrronium curves are nearly identical. Only the non-asthmatic control and 1-h post-combination therapy curves appeared to generate a true response plateau (three data points within 5%), which occurred at a 14% fall in FEV1. Conclusions Methacholine dose-response curves differentiate the bronchoprotective mechanisms triggered by different classes of asthma medications. Assessment of bronchoprotection using methacholine dose-response curves may be useful during clinical development of respiratory medications when performing superiority, equivalence, or non-inferiority trials. Trial registration clinicaltrials.gov (NCT02953041). Retrospectively registered on October 24th 2016. Electronic supplementary material The online version of this article (doi:10.1186/s12931-017-0628-4) contains supplementary material, which is available to authorized users.

Background: The recent approval of several fixed-dose combination long-acting β2-agonist (LABA) and long-acting muscarinic antagonist (LAMA) products has increased the use of dual bronchodilators in the treatment of chronic obstructive pulmonary disease (COPD). Understanding the comparative safety of this combination is important for informing treatment decisions. Objective: To compare the risk of cardiovascular and cerebrovascular (CCV) events associated with LABA/LAMA compared with a combination of LABA and inhaled corticosteroid (ICS). Methods: This was a retrospective, observational cohort study using health insurance claims data to identify COPD patients initiating LABA/LAMA or LABA/ICS. CCV outcomes included hospitalizations with a primary diagnosis for acute coronary syndrome, heart failure, cardiac dysrhythmia, stroke, or transient ischemic attack. Patients were followed until they experienced an event, discontinued treatment, initiated medication from the opposite cohort, or lost enrollment. Patients were matched 1:4 on propensity scores, and time to event was compared using Cox proportional hazards models. Results: After matching, there were 3842 patients in the LABA/LAMA cohort and 15 225 in the LABA/ICS cohort. Cardiovascular events in the LABA/LAMA cohort were lower than in the LABA/ICS: hazard ratio (HR) = 0.794; 95% CI = 0.623-0.997. No significant difference in the risk of cerebrovascular events (HR = 1.166; 95% CI = 0.653-1.959) was observed. Conclusions: Despite concerns about the CCV effects of LAMA and LABA monotherapy, the LABA/LAMA combination had similar or lower risk of these events in comparison to LABA/ICS. Further studies are recommended to confirm these findings.

Several long-acting bronchodilators have been developed and are widely used as first-line treatment in patients with stable chronic obstructive pulmonary disease (COPD). However, the initial choice of therapy is still uncertain. The aim of this study was to examine the clinical efficacy and safety of long-acting muscarinic antagonist (LAMA) and long-acting beta2-agonist (LABA) in patients with stable COPD. We searched several databases and manufacturers’ websites to identify relevant randomized clinical trials for meta-analysis. Outcomes of interest were trough forced expiratory volume in 1 s (FEV1), acute exacerbations, transitional dyspnoea index (TDI) score, St George's Respiratory Questionnaire (SGRQ) score and adverse events. Sixteen trials with a total of 22 872 patients were included in this study. Compared with LABA, LAMA were associated with a greater reduction in acute exacerbations (OR: 0.84, 95% CI: 0.74–0.94, P = 0.003) and fewer adverse events (OR: 0.92, 95% CI: 0.86–0.97, P = 0.005). There were no significant differences in trough FEV1, TDI and SGRQ scores. In patients with stable COPD, LAMA were associated with a greater reduction in acute exacerbations and fewer adverse effects compared with LABA.

Objective: To compare the available literature regarding the use of long-acting muscarinic antagonist (LAMA)/long-acting β2 agonists (LABA) and inhaled corticosteroid (ICS)/LABA combination inhaler therapy in chronic obstructive pulmonary disease (COPD) maintenance therapy management. Data Sources: A MEDLINE literature search from database inception to February 2017 was conducted using the search terms chronic obstructive pulmonary disease, adrenergic beta-agonist, muscarinic antagonist, and inhaled corticosteroid. References from extracted sources were further searched for any relevant, missed data sources. Study Selection and Data Extraction: All English-language randomized-controlled trials comparing LAMA/LABA and ICS/LABA combination inhaler therapy were evaluated. Data Synthesis: A total of 10 randomized controlled trials have reviewed the use of LAMA/LABA compared with ICS/LABA therapy for COPD maintenance therapy. Results of clinical trials that evaluated LAMA/LABA and ICS/LABA maintenance therapy demonstrated superior improvements in pulmonary function tests via spirometry and improved clinical outcomes with LAMA/LABA therapy, specifically reduction in COPD exacerbation rates. The safety of LAMA/LABA combination therapy also is favorable compared with ICS/LABA combination therapy because of the increased infection risk with ICS therapy. Conclusions: COPD is a disease state with significant morbidity and mortality in the United States and is the third leading cause of death. Long-acting inhalers are recommended for the majority of COPD severities, and combination therapy is typically utilized. LAMA/LABA combination therapy has demonstrated superior improvements in pulmonary function and reduction in COPD exacerbation rates compared with ICS/LABA. LAMA/LABA combination therapy will have a larger future role in COPD maintenance management.

The glucocorticoid cortisol, the end product of hypothalamus-pituitary-interrenal axis in zebrafish (Danio rerio), is synthesized via steroidogenesis and promotes important physiological regulations in response to a stressor. The failure of this axis leads to inability to cope with environmental challenges preventing adaptive processes in order to restore homeostasis. Pesticides and agrichemicals are widely used, and may constitute an important class of environmental pollutants when reach aquatic ecosystems and nontarget species. These chemical compounds may disrupt hypothalamus-pituitary-interrenal axis by altering synthesis, structure or function of its constituents. We present evidence that organophosphorus exposure disrupts stress response by altering the expression of key genes of the neural steroidogenesis, causing downregulation of star, hsp70, and pomc genes. This appears to be mediated via muscarinic receptors, since the muscarinic antagonist scopolamine blocked these effects.

Background Chronic obstructive pulmonary disease (COPD) is a heterogeneous disorder characterized by usually progressive development of airflow obstruction that is not fully reversible. While most patients will experience symptoms throughout the day or in the morning upon awakening, many patients do not experience their symptoms as constant but report variability in symptoms during the course of the day or over time. Symptom variability adversely affects patients' health status and increases the risk of COPD exacerbations. Methods We examined data from the literature on symptom variability and control in patients with COPD, with focus on the use of inhaled bronchodilator therapy with long-acting muscarinic antagonist agents (LAMA) plus long-acting β 2 -agonists (LABA); in particular twice-daily fixed-dose combination LAMA/LABA therapy with aclidinium/formoterol. Results Correct diagnosis and assessment of COPD requires comprehensive clinical and functional evaluation and consideration of individual needs to support the clinical decisions necessary for effective long-term management. Combining bronchodilators from different and complementary pharmacological classes with distinct mechanisms of action can increase the magnitude of bronchodilation as opposed to increasing the dose of a single bronchodilator. Conclusions The use of inhaled bronchodilator therapy with LAMA/LABA fixed-dose combinations in patients with stable COPD is supported by current evidence. This treatment approach provides robust effects on lung function and symptom control and may improve patients' adherence to treatment. Administration of the long-acting bronchodilators aclidinium and formoterol as twice daily fixed-dose aclidinium/formoterol 400/12 μg has the potential to control symptoms throughout the 24 h in patients with stable moderate-to-severe COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity, mortality and health care expenditure throughout the world. COPD guidelines recommend the use of long-acting muscarinic antagonist (LAMA) either alone or in combination with a long-acting β2 agonist (LABA). For over 10 years, tiotropium was the only LAMA that was used in the management of COPD. Over the past few years, various new drugs have been identified that act on the muscarinic receptors and β2 receptors. Umeclidinium (Umec) is a new LAMA currently approved for use in patients with COPD either as monotherapy or in combination with vilanterol (Vil). Both Umec alone and in combination with Vil delivered through a multi-dose dry powder Ellipta™ device have shown improvement in lung function, health-related quality of life and exacerbation frequency in patients with COPD. This review provides an overview of the pharmacology, pharmacodynamics and pharmacokinetics of Umec, and evaluates the clinical efficacy and safety studies in patients with COPD.

Gustavo J Rodrigo,1 David Price,2,3 Antonio Anzueto,4,5 Dave Singh,6 Pablo Altman,7 Giovanni Bader,8 Francesco Patalano,8 Robert Fogel,7 Konstantinos Kostikas8 1Departamento de Emergencia, Hospital Central de las Fuerzas Armadas, Montevideo, Uruguay; 2Academic Primary Care, Division of Applied Health Sciences, University of Aberdeen, Aberdeen, Scotland, UK; 3Observational and Pragmatic Research Institute, Singapore; 4University of Texas Health Science Center, 5South Texas Veterans Health Care System, San Antonio, TX, USA; 6Medicines Evaluation Unit, National Institute for Health Research Respiratory and Allergy Clinical Research Facility, University Hospital of South Manchester NHS Foundation Trust, University of Manchester, Manchester, England, UK; 7Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA; 8Novartis Pharma AG, Basel, Switzerland Background: Randomized controlled trials (RCTs) indicate that long-acting bronchodilator combinations, such as β2-agonist (LABA)/muscarinic antagonist (LAMA), have favorable efficacy compared with commonly used COPD treatments. The objective of this analysis was to compare the efficacy and safety of LABA/LAMA with LAMA or LABA/inhaled corticosteroid (ICS) in adults with stable moderate-to-very-severe COPD. Methods: This systematic review and meta-analysis (PubMed/MEDLINE, Embase, Cochrane Library and clinical trial/manufacturer databases) included RCTs comparing ≥12 weeks’ LABA/LAMA treatment with LAMA and/or LABA/ICS (approved doses only). Eligible studies were independently selected by two authors using predefined data fields; the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed. Results: Eighteen studies (23 trials) were eligible (N=20,185). LABA/LAMA significantly improved trough forced expiratory volume in 1second (FEV1) from baseline to week 12 versus both LAMA and LABA/ICS (0.07L and 0.08L, P100mL (risk ratio [RR]: 1.33, 95% confidence interval [CI]: [1.20, 1.46] and RR: 1.44, 95% CI: [1.33, 1.56], respectively, the number needed to treat being eight and six, respectively). LABA/LAMA improved transitional dyspnea index and St George’s Respiratory Questionnaire scores at week 12 versus LAMA (both P

The effects of tiotropium, an inhaled long-acting muscarinic antagonist, on lung function were investigated in current smokers and nonsmokers with asthma treated with inhaled corticosteroids (ICSs) and other asthma controllers: inhaled long-acting β 2 agonists, leukotriene receptor antagonists, and/or theophylline. We conducted a double-blind, placebo-controlled study of an inhaled single dose of tiotropium in 9 asthmatics currently smoking and 9 asthmatics who have never smoked in a crossover manner. Lung function was measured before and 1, 3, and 24 h after inhalation of 18 μg of tiotropium or a placebo. The primary outcome was a change in forced expiratory volume in 1 s (FEV 1 ) from the baseline, and the secondary outcomes were changes in peak expiratory flow rate (PEFR), V ˙ 50 , and V ˙ 25 . At baseline, asthmatics with and without a smoking history had a mean FEV 1 of 2590 ml and 2220 ml and were taking a mean dose of ICSs of 1208 and 1000 μg/day, respectively. The increase from the baseline FEV 1 was 169 ml and 105 ml higher at 3 h after tiotropium than after the placebo in current smokers and nonsmokers, respectively. PEFR, V ˙ 50 , and V ˙ 25 were also significantly increased after tiotropium as compared with the placebo in both study groups. Changes in FEV 1 and PEFR tended to be greater in asthmatics currently smoking than in subjects who have never smoked, although there were no statistical differences at any time points. Tiotropium resulted in improved lung function and symptoms both in current smoker and nonsmoker asthmatics. These findings suggest that tiotropium will provide a new strategy for the treatment of bronchial asthma.

Quaternary ammonium analogues of atropine that are unable to cross the blood-brain barrier are used to alleviate peripheral muscarinic toxicity in animal models of epilepsy produced by systemic administration of pilocarpine or diisopropylfluorophosphate (DFP). Currently, methylatropine is the most popular and potent of these quaternary derivatives; however, it is expensive and produced in limited quantity. Here, we propose the use of ethylatropine bromide as an alternative to methylatropine. The synthesis of ethylatropine bromide is simple, inexpensive and has low environmental impact. We demonstrate the efficacy of ethylatropine bromide to antagonize the carbachol induced rise in intracellular calcium in a calcium mobilization assay, and its ability to prevent pilocarpine-induced total fluid secretions in mice without blocking pilocarpine-induced seizures. The ease of synthesis, cost effectiveness, and efficacy makes ethylatropine bromide a desirable alternative to methylatropine as a peripherally restricted acetylcholine receptor antagonist.

Active inspiration and expiration are opposing respiratory phases generated by two separate oscillators in the brainstem; inspiration driven by a neuronal network located in the preBotzinger Complex (preBotC) and expiration driven by a neuronal network located in the paraFacial Respiratory Group (pFRG). While continuous activity of the preBotC is necessary for maintaining ventilation, the pFRG behaves as a conditional expiratory oscillator, being silent in resting conditions and becoming rhythmically active in presence of increased respiratory drive (e.g., hypoxia, hypercapnia, exercise, or through release of inhibition). Recent evidence from our laboratory suggests that expiratory activity in the principal expiratory pump muscles, the abdominals, is modulated in a state-dependent fashion, frequently occurring during periods of REM sleep. We hypothesised that acetylcholine, a neurotransmitter released in wakefulness and REM sleep by mesopontine structures, contributes to the activation of pFRG neurons and thus acts to promote the recruitment of expiratory abdominal muscle activity. We investigated the stimulatory effect of cholinergic neurotransmission on pFRG activity and recruitment of active expiration in vivo under anaesthesia. We demonstrate that local application of the acetylcholinesterase inhibitor physostigmine into the pFRG potentiated expiratory activity. Furthermore, local application of the cholinomimetic carbachol into the pFRG activated late expiratory neurons and induced long lasting rhythmic active expiration. This effect was completely abolished by pre-application of the muscarinic antagonist scopolamine, and more selective M3 antagonists 4DAMP and J104129. We conclude that cholinergic muscarinic transmission contributes to excitation of pFRG neurons and promotes both active recruitment of abdominal muscles and active expiratory flow. This article is protected by copyright. All rights reserved

Marco Contoli,1 Paolo Solidoro,2 Fabiano Di Marco,3,4 Nicola Scichilone,5 Angelo Corsico,6 Fulvio Braido,7 Pierachille Santus4,8 1Research Centre on Asthma and COPD, Department of Medical Sciences, University of Ferrara, Ferrara, Italy; 2Cardiovascular and Thoracic Department, Città della Salute, Turin, Italy; 3Department of Health Sciences, University of Milan, Milan, Italy; 4Respiratory Unit, San Paolo Hospital, Milan, Italy; 5Department of Internal Medicine, Section of Pulmonology (DIBIMIS), University of Palermo, Palermo, Italy; 6Department of Molecular Medicine, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 7Allergy and Respiratory Diseases Clinic, DIMI, University of Genoa, IRCS AOU San Martino-IST, Genoa, Italy; 8Pulmonary Rehabilitation Unit, Fondazione Salvatore Maugeri, Scientific Institute of Milan-IRCCS, Milan, Italy Abstract: The pathophysiology of chronic obstructive pulmonary disease (COPD) includes persistent airflow limitation, altered gas exchange, and enhanced chronic inflammatory response. According to disease severity in individual patients, exacerbations and comorbidities frequently occur. The overall nocturnal and daily symptoms have a strong impact on patient quality of life and clinical outcomes. Bronchodilators, by targeting two important aspects of COPD pathophysiology, ie, bronchoconstriction and lung hyperinflation, are the mainstay of therapy for COPD. Aclidinium bromide in particular is an anticholinergic molecule, approved for maintenance bronchodilator treatment of stable COPD, that combines high antimuscarinic activity with strong kinetic selectivity for the M3 receptor subtype. Moreover, the elevated plasma clearance of aclidinium has been related to low systemic bioavailability and low incidence of anticholinergic adverse events, whereas the reduced residence time at M2 receptors provides good cardiovascular safety. Altogether, these characteristics result in a high safety and tolerability profile. This review aims to reappraise the contribution of symptoms and of the level of quality of life determinants on COPD severity and to evaluate how therapeutic strategies with aclidinium may positively impact on these specific determinants of disease severity. Keywords: COPD, quality of life, daily symptoms, LAMA, aclidinium

We have previously shown that muscarinic acetylcholine receptors (mAChRs) promote global protein biosynthesis in SNU-407 colon cancer cells. To gain insight into the molecular mechanisms underlying this event, we examined whether mAChRs regulate the phosphorylation of eIF4B (eukaryotic initiation factor 4B), an essential component of the translation machinery. When SNU-407 cells were treated with the cholinergic agonist carbachol, eIF4B was phosphorylated in a dose- and time-dependent manner. This carbachol effect was almost completely blocked by the muscarinic antagonist atropine, demonstrating that mAChRs specifically mediate the phosphorylation of eIF4B. Carbachol-stimulated eIF4B phosphorylation was significantly reduced by the MEK1/2 inhibitor U0126, indicating that the MEK1/2-ERK1/2 pathway plays an important role in mAChR-mediated eIF4B phosphorylation. However, treating the cells with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the mTORC1 (mammalian target of rapamycin complex 1) inhibitor rapamycin had little effect on carbachol-stimulated eIF4B phosphorylation, suggesting that PI3K and mTORC1 are not the key participants in this process. We also observed that the inhibition of protein kinase C (PKC) by GF109203X greatly diminished carbachol-stimulated eIF4B phosphorylation. Together, our data show that mAChRs modulate eIF4B phosphorylation via the ERK1/2 and PKC signaling pathways in SNU-407 colon cancer cells.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1–5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

This article contains information on the experimental design and methods on how the safety and tolerability data concerning patients with moderate to very severe chronic obstructive pulmonary disease (COPD) were obtained. This is in addition to our original research article. [1] We have also provided information on the clinical laboratory tests that were conducted.Further interpretation and discussion of the data are demonstrated in the article “Revefenacin, a Once-daily, Lung-selective, Long-acting Muscarinic Antagonist for Nebulized Therapy: Safety and Tolerability Results of a 52-week Phase 3 Trial in Moderate to Very Severe Chronic Obstructive Pulmonary Disease.” [1] Keywords: Chronic obstructive pulmonary disease, COPD, Long-acting muscarinic antagonist, Nebulization, Revefenacin

Muscarinic antagonist scopolamine has been extensively used to model amnesia in lab rodents, but most studies have focused on the effects of pre-training scopolamine administration. Here, we examined post-training scopolamine administration in C57BL/6JRj mice. Learning was assessed in three different procedures: odour discrimination in a digging paradigm, visual discrimination in a touchscreen-based setup, and spatial learning in the Morris water maze. Scopolamine administration affected performance in the odour discrimination task. More specifically, scopolamine decreased perseverance, which facilitated reversal learning. Similar results were obtained in the visual discrimination task, but scopolamine did not affect performance in the spatial learning task. It is unlikely that these results can be explained by non-memory-related cognitive effects (e.g., attention), non-cognitive behaviours (e.g., locomotor activity) or peripheral side-effects (e.g., mydriasis). They likely relate to the various neuropharmacological actions of scopolamine. ispartof: BRAIN RESEARCH BULLETIN vol:152 pages:52-62 ispartof: location:United States status: published

Triple inhaled therapy for maintenance of chronic obstructive pulmonary disease (COPD) consists of an inhaled corticosteroid, long-acting muscarinic antagonist, and long-acting beta2-agonist. Recent clinical studies evaluating triple therapy found a reduction in COPD exacerbations compared with other combination therapies. This article discusses the role of triple therapy in treating patients with COPD.

Introduction: Persistent airflow limitation (PAL) may predict a positive clinical response to add-on long-acting muscarinic antagonist (LAMA) in patients with asthma taking inhaled corticosteroids and long-acting β2-receptor agonists (ICS/LABA). Aims and Objectives: We evaluated the effect of extrafine MS beclometasone dipropionate/formoterol fumarate/glycopyrronium bromide (BDP/FF/GB) on asthma exacerbations in a subset of patients with PAL. Methods: TRIMARAN was a phase III multinational, randomized, parallel group trial comparing 52-week treatment with (BDP/FF/GB) 400/24/50 µg/d to BDP/FF 400/24 µg/d delivered via a pressurized metered dose inhaler (pMDI) in adult patients with uncontrolled asthma on medium dose ICS/LABA and a history of exacerbation in the past year. PAL was defined as an FEV1 ≤80% of predicted normal and FEV1/FVC ≤0.7 after 400 μg salbutamol pMDI. Results: 1155 subjects were randomized and 658 (57.0%) met the PAL criteria on BDP/FF/GB (n=331) and BDP/FF (n=327), respectively. Treatment of this subgroup with BDP/FF/GB compared to BDP/FF resulted in a 15.2% and 37.7% reduction in the annual rates of moderate-severe and severe asthma exacerbations respectively, (RR [95% CI] = 0.848 [0.694, 1.036], p=0.106; 0.623 [0.430; 0.902], p=0.012), vs. 15.4% (p=0.033) and 26.3% (p=0.040) reduction in the entire study population. Conclusions: In patients with asthma uncontrolled on medium dose ICS/LABA, PAL did not influence the efficacy of extrafine MS BDP/FF/GB in reducing moderate-severe exacerbations and was associated with a greater reduction in annual severe exacerbation rates.

Introduction: Persistent airflow limitation (PAL) may predict a positive clinical response to add-on long-acting muscarinic antagonist (LAMA) in asthmatics on inhaled corticosteroids and long-acting β2-receptor agonists (ICS/LABA) Objectives: We evaluated the effect of extrafine HS beclometasone dipropionate/formoterol fumarate/glycopyrronium bromide (BDP/FF/GB) on asthma exacerbations in a subset of patients with PAL Methods: TRIGGER was a phase III randomized, parallel group trial comparing 52-week treatment with BDP/FF/GB 800/24/50 µg/d to BDP/FF 800/24 µg/d via pressurized metered dose inhalers (pMDI) and open-label treatment consisting of BDP/FF plus tiotropium Respimat® 5 μg/d (TioR) in adult patients with uncontrolled asthma on high-dose ICS/LABA and a history of exacerbation in the past year. PAL criteria were FEV1 ≤80% of predicted normal and FEV1/FVC ≤0.7 after 400 μg salbutamol pMDI Results: 1437 subjects were randomized in 2:2:1 ratio; 880 (61.2%) met PAL criteria on BDP/FF/GB (n=357), BDP/FF (n=346), and BDP/FF+TioR (n=177), respectively. Treatment of this subgroup with BDP/FF/GB compared to BDP/FF resulted in a 25.9% and 31.8% reduction in annual moderate-severe and severe asthma exacerbations rates, respectively, (RR [95% CI] = 0.741 [0.611, 0.900], p=0.002; 0.682 [0.494; 0.940], p=0.019), vs. 12% and 20.5% reductions in the entire study population. There were no significant differences between BDP/FF/GB and BDP/FF+TioR. Conclusions: PAL is associated with a greater response to triple therapy with extrafine HS BDP/FF/GB in patients with asthma uncontrolled on high dose ICS/LABA

Background and Objectives: Atropine is a nonselective muscarinic antagonist which has been used to prevent worsening of myopia in children. Different concentrations of atropine were used for myopia, ranging from 0.01% to 1.0%. However, there are still potential toxicity of different doses of atropine to the cornea. Here, we present a study of investigating novel genes potentially involved in the effects of very low dose atropine treatment (0.003%) on corneal epithelial cells using next-generation sequencing (NGS) and bioinformatics approaches. Materials and Methods: Human corneal epithelial cells were treated with 0.003% atropine, cultured until confluence, and RNA extracted for differential expression profiling of mRNA and microRNA (miRNA) between control and atropine-treated corneal epithelial cells. The functional enrichment analysis for differentially expressed genes was performed using two bioinformatics databases, including Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity&reg, Pathway Analysis (IPA). In addition, potential miRNA-mRNA interactions involved in atropine-treated corneal epithelial cells were predicted and validated using different miRNA target prediction databases. Results: Our results showed 0.003% atropine might suppress the apoptosis of corneal epithelial cells, potentially through Ras and protein kinase A signaling pathways. We also validated the possible miRNA regulations by using TargetScan and miRDB databases. Hsa-miR-651-3p-EPHA7, hsa-miR-3148-TMEM108 and hsa-miR-874-5p-TBX6 were validated as possible miRNA regulations involved in corneal epithelial cells treated with 0.003% atropine. Conclusions: These findings may contribute novel insights into therapeutic strategies for treating cornea with 0.003% atropine.

Memory loss is one of the most tragic symptoms of Alzheimer’s disease. Our laboratory has recently demonstrated that ‘i-Extract’ of Ashwagandha (Withania somnifera) restores memory loss in scopolamine (SC)-induced mice. The prime target of i-Extract is obscure. We hypothesize that i-Extract may primarily target muscarinic subtype acetylcholine receptors that regulate memory processes. The present study elucidates key target(s) of i-Extract via cellular, biochemical, and molecular techniques in a relevant amnesia mouse model and primary hippocampal neuronal cultures. Wild type Swiss albino mice were fed i-Extract, and hippocampal cells from naïve mice were treated with i-Extract, followed by muscarinic antagonist (dicyclomine) and agonist (pilocarpine) treatments. We measured dendritic formation and growth by immunocytochemistry, kallikrein 8 (KLK8) mRNA by reverse transcription polymerase chain reaction (RT-PCR), and levels of KLK8 and microtubule-associated protein 2, c isoform (MAP2c) proteins by western blotting. We performed muscarinic receptor radioligand binding. i-Extract stimulated an increase in dendrite growth markers, KLK8 and MAP2. Scopolamine-mediated reduction was significantly reversed by i-Extract in mouse cerebral cortex and hippocampus. Our study identified muscarinic receptor as a key target of i-Extract, providing mechanistic evidence for its clinical application in neurodegenerative cognitive disorders.

Pilocarpine is an M3 muscarinic agonist that is widely used for the treatment of xerostomia caused by various diseases and medical conditions. Pilocarpine induced the secretion of salivary fluid in perfused submandibular glands of rats. The secretion of salivary fluid observed after removal of pilocarpine was referred to as residual fluid secretion. The volume of fluid and time of the residual secretion depended on the dose of pilocarpine. Such a residual effect of pilocarpine was observed on fluid secretion via the paracellular pathway and oxygen consumption. When a muscarinic antagonist was added to the perfusate immediately after cessation of pilocarpine, residual secretion of salivary fluid did not occur. These observations indicate that the residual secretion of salivary fluid is a characteristics of the interaction of pilocarpine with muscarinic receptors.

Mammals airways are extensively innervated by the vagus nerve, which controls the airway diameter and bronchial tone. However, very few studies described the respiratory function and lung morphology after vagal section. In the present study, we evaluated the respiratory mechanics after aerosolization of vehicle (to obtain control values), a muscarinic agonist (methacholine), a β2-adrenergic agonist (salbutamol) or a muscarinic antagonist (ipratropium bromide) in intact (Vi) and bilaterally vagotomized (Vx) Swiss male mice. Different group was established for morphometric analyze. The total lung resistance, airway resistance, elastance, compliance, lung tissue damping, lung tissue elastance, and morphological parameters (collagen and elastic fibers) were significantly different in the Vx group compared to the Vi group. Bronchoconstrictor and bronchodilators change the respiratory function of the Vx group. In conclusion, the vagus nerve modulates the lung function in response to bronchoconstriction and bronchodilation, as well as lung architecture of mice.

Clothianidin (CLO) is a neonicotinoid insecticide that produces toxic effects in experimental animals and humans. These effects are associated primarily to its action as a nicotinic agonist, acting on insect and vertebrate nicotinic acetylcholine receptors (nAChRs), but little is known about the mechanisms of action on the mammalian nervous system. In the rat striatum, CLO induces increases in the dopamine overflow in a concentration-dependent manner. In the present study, we evaluate, using in vivo brain microdialysis in adult Sprague-Dawley rats, the participation of specific nAChRs and muscarinic cholinergic receptors (mAChRs) on CLO-induced striatal dopamine release. We investigate the effects of selective antagonists of α4β2 heteromeric, β2 subunit, α7 nAChRs, and of broad-spectrum antagonist of mAChRs (atropine) on CLO-induced dopamine release. Intrastriatal administration of antagonists of α4β2 N-n-decilonicotinium iodide (NDNI), and of α7 methylcaconitine (MLA) significantly decreased the CLO-induced dopamine overflow in a concentration-dependent form, whereas pretreatment with the antagonist of β2 subunit DHβE not having effect. Pretreatment with the muscarinic antagonist atropine also blocked the increases in the extracellular dopamine levels. Taken together, these results suggest that the stimulatory effect of CLO on in vivo dopamine from rat striatum depends on the activation of α4β2 present in dopaminergic terminals and α7 nAChRs subtypes expressed in glutamatergic terminals in the striatum. On the other hand, the CLO-induced dopamine release also appears to involve the activation of mAChRs.

Changes in the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signaling are implicated in older people with dementia. Drugs that modulate the cAMP/cGMP levels in the brain might therefore provide new therapeutic options for the treatment of cognitive impairment in aging and elderly with dementia. Phosphodiesterase 2A (PDE2A), which is highly expressed in the forebrain, is one of the key phosphodiesterase enzymes that hydrolyze cAMP and cGMP. In this study, we investigated the effects of PDE2A inhibition on the cognitive functions associated with aging, such as spatial learning, episodic memory, and attention, in rats with a selective, brain penetrant PDE2A inhibitor, N-{(1S)-1-[3-fluoro-4-(trifluoromethoxy)phenyl]-2-methoxyethyl-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4(1H)-carboxamide (TAK-915). Repeated treatment with TAK-915 (3 mg/kg/day, p.o. for 4 days) significantly reduced escape latency in aged rats in the Morris water maze task compared to the vehicle treatment. In the novel object recognition task, TAK-915 (1, 3, and 10 mg/kg, p.o.) dose-dependently attenuated the non-selective muscarinic antagonist scopolamine-induced memory deficits in rats. In addition, oral administration of TAK-915 at 10 mg/kg significantly improved the attentional performance in middle-aged, poorly performing rats in the 5-choice serial reaction time task. These findings suggest that PDE2A inhibition in the brain has the potential to ameliorate the age-related cognitive decline.

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(-)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1 and 5, (R)-(-)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(-)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(-)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease characterized by progressive and persistent airflow limitation that is not fully reversible. Revefenacin is an investigational long-acting muscarinic antagonist (LAMA), in late-stage development as a nebulized inhalation solution, which has been designed to produce long-acting bronchodilation, consistent with once-daily dosing, and with a high degree of lung-selectivity. It is more selective for muscarinic type 3 (M3) than muscarinic type 2 (M2) or muscarinic type 1 (M1) receptors. Its dissociation half-life for M3 is 82 minutes and 6.9 minutes for M1, respectively. The bronchoprotective effect is seen as early as five-minute post-dose and is sustained for up to 24 hours. The estimated 24-hour potency (expressed as the concentration of dosing solution) is 45.0 mg/ml. Once-daily dose of revefenacin provided long-term improvement in trough forced expiratory volume in one second (FEV1). It improved day 28 trough FEV1 over placebo significantly (p < 0.001). M3: M2 receptor half-life is 12 compared to the other antagonists that have M3: M2 receptor half-life around 6.0. A 24-hour serial spirometry, on day 84, showed that revefenacin 88 or 175 µg was associated with significant (p

Asthma is the most common chronic disease in children. As suggested by international guidelines, the main goals of asthma treatment are symptoms control and lung function preservation, through a stepwise and control-based approach. The first line therapy based on inhaled corticosteroids may fail to reach control in more than one third of patients, especially adolescents, and in these lung function and quality of life may progressively worsen. Treatment with omalizumab, the first anti-immunoglobulin E recombinant humanized monoclonal antibody, has been definitely approved in pediatric uncontrolled asthma. In this review, we discuss the mechanisms and potential roles of emerging therapies for pediatric severe asthma. Novel biologic drugs (i.e., dupilumab, mepolizumab, reslizumab, and benralizumab) seem to be promising in reducing annual exacerbation rates and steroid-use in glucocorticoid-dependent cases, but available data are few and limited to adolescents and adults. Evidences on the use of the muscarinic antagonist tiotropium as controller medication in pediatric settings are progressively growing, sustaining an application as asthma maintenance treatment in children aged >6 years and in preschool children with persistent asthmatic symptoms, but well powered trials are needed to confirm its safety and efficacy. New inhaled corticosteroids (i.e., ciclesonide and mometasone) are effective as once-daily controller therapy, but long-term studies in the different pediatric ages are needed to compare effectiveness and safety to usual treatments. At present, the role of macrolides in pediatric severe asthma is controversial and their administration is not recommended routinely, but may be considered in children with neutrophilic asthma for reducing daily oral steroids administration and improving lung function. Despite the availability of several novel therapeutic strategies for uncontrolled asthma, future trials targeted at specific pediatric age subgroups are needed to support evidences of safety and efficacy also in children.

No mundo estima-se que existam cerca de 600 milhões de pessoas afectadas pela DPOC, na sua maioria com historial de fumadores, sendo que o número tende a aumentar todos os anos. Os sintomas provocados pela doença são limitativos e podem requerer hospitalização. A medicação utilizada para o controlo e alivio da sintomatologia pode diminuir a sua eficácia com a progressão da doença, havendo necessidade de novas respostas. O brometo de aclidínio é um antagonista muscarínico de longa duração de ação (LAMA), que atua nos recetores da acetilcolina envolvidos na expressão dos sintomas da doença. Esta revisão sistemática da literatura tem como objetivo comparar a eficácia do brometo de aclidínio em relação ao placebo. Efectuou-se umarevisão sistemática através de pesquisa nas bases de dados Pubmed, Conference Proceedings Citation Index - Science (CPCI-S) e Science Citation Index Expanded (SCI-Expanded), onde foram recolhidos ensaios clínicos randomizados e controlados. A pesquisa forneceu 6 artigos sobre ensaios clínicos para análise, comparação do brometo de aclidínio com o placebo. De acordo com os resultados, o brometo de aclidínio na dosagem de 400µg administrado duas vezes por dia, demonstrou ter eficácia, segurança e melhorar o estado de saúde. Esta revisão permitiu concluir que o brometo de aclidínio apresenta eficácia na redução dos sintomas da DPOC em comparação com o placebo, segurança no seu uso e produz uma melhoria do estado de saúde dos doentes.

Introduction Overactive bladder (OAB) is subtyped into OAB-wet and OAB-dry, based on the presence or absence, respectively, of urgency incontinence. Although women with OAB frequently have a higher risk for sexual dysfunction, a systematic review on the impact of OAB-wet on female sexuality is lacking. This may be evaluated by measuring the effect of the bladder condition on sexuality per se, or by the effect of OAB treatment on female sexual dysfunction. Aim To assess the role of OAB-wet on female sexual function. Methods A systematic review of the literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement recommendations. Research on PubMed, EMBASE, and SCOPUS was performed and concluded on October 15, 2018. A systematic computerized search was conducted on published literature from January 1, 2000–2018. Meta-analysis was performed with a meta-analysis program. Main Outcomes Measures The following search terms were used: (((“female sexual function”) OR (“female sexual dysfunction”) OR (“female sexuality”) OR (“dyspareunia”)) AND ((“overactive bladder”) OR (“coital incontinence”) OR (“detrusor instability”) OR (“detrusor overactivity”) OR (“urge urinary incontinence”) OR (onabotulinumtoxinA) OR (“botulinum”) OR (“sacral neuromodulation”) OR (SNM) OR (PTNS) OR (“stoller afferent neuro-stimulation”) OR (“SANS”) OR (“antimuscarinic drugs”) OR (“anticholinergic”) OR (“peripheral neuromodulation”) OR (beta-agonist))). Results 1,033 references were reviewed for inclusion and exclusion criteria. Final analysis identified 12 articles for systematic review. OAB-wet was reported as the most affecting factor on sexuality. OAB treatments showed improvement of both the OAB-wet and the sexual function. Results of the meta-analysis suggested that OAB therapies improving OAB-wet significantly reduced female sexual dysfunction (odds ratio 0.19; 95% CI 0.26–0.45). Conclusion OAB-wet represents a risk for sexual dysfunction; however, data available show low-quality evidence of the impact of OAB-wet on sexual dysfunction. Balzarro M, Rubilotta E, Mancini V, et al. Impact of Overactive Bladder-Wet Syndrome on Female Sexual Function: A Systematic Review and Meta-Analysis. Sex Med Rev 2019;7:565–574.

Fabiano Di Marco,1 Piero Balbo,2 Francesco de Blasio,3 Vittorio Cardaci,4 Nunzio Crimi,5 Giuseppe Girbino,6 Girolamo Pelaia,7 Pietro Pirina,8 Pietro Roversi,9 Pierachille Santus,10,11 Nicola Scichilone,12 Alessandro Vatrella,13 Patrizio Pasqualetti,14 Mauro Carone15 1Department of Health Sciences, University of Milan, Respiratory Unit, Papa Giovanni XXIII Hospital, Bergamo, Italy; 2SC Malattie dell’Apparato Respiratorio, AOU Maggiore della Carità, Novara, Italy; 3Respiratory Medicine and Pulmonary Rehabilitation Section, Clinic Center S.p.A. Private Hospital, Department of Medicine and Health Sciences “V Tiberio”, University of Molise, Campobasso, Italy; 4Unit of Pulmonary Rehabilitation, IRCCS “San Raffaele Pisana”, Rome, Italy; 5Unità Operativa Complessa di Pneumologia e Allergologia, Policlinico Rodolico Vittorio Emanuele, Università di Catania, Catania, Italy; 6Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali (BIOMORF), Università degli Studi di Messina, Messina, Italy; 7Department of Medical and Surgical Sciences, Unit of Respiratory Diseases, University Magna Graecia of Catanzaro, Catanzaro, Italy; 8Respiratory Unit, AOU Sassari, Sassari, Italy; 9Azienda Ospedaliera Universitaria, Policlinico di Modena, Modena, Italy; 10Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy; 11Division of Respiratory Diseases “L. Sacco” Hospital, ASST Fatebenefratelli Sacco, Milan, Italy; 12Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy; 13Department of Medicine, Surgery and Dentistry “Scuola Medica Salernitana”, University of Salerno, Salerno, Italy; 14Fondazione Fatebenefratelli per la Ricerca e la Formazione Sanitaria e Sociale, Rome, Italy; 15Istituti Clinici Scientifici Maugeri, IRCCS di Cassano delle Murge, Cassano delle Murge (BA), Italy Purpose: There is a lack of consensus on the most appropriate early diagnostic strategy, criteria for early access to treatment and follow-up approach for patients with COPD. Materials and methods: A Delphi consensus project investigated the early management of COPD. We formulated two questionnaires for completion by pneumologists in Italy. Results: A total of 207 specialists completed questionnaire 1 and 184 of them questionnaire 2, between November 2016 and October 2017. Early diagnosis of COPD was considered uncommon for 93.2% of the expert panel. Regardless of the definition of “early diagnosis” – a diagnosis made before the clinical manifestation of the disease for most responders (60.4%) – experts were confident of the positive effects of early disease management, which they consider is effective in modifying the natural history of the disease. Lack of awareness of the disease was considered the first limiting factor to early COPD management for 78% of respondents. The most effective steps to reduce functional decline were considered to be smoking cessation, followed by long-acting β2-agonist (LABA)/long-acting muscarinic antagonist (LAMA), LAMA, LABA, and finally inhaled corticosteroid/LABA(P

Introduction During the clinical development of a fixed-dose combination of drugs, it is best practice to conduct dose-finding studies to determine the optimal dose of each component. The aims of this phase II dose-finding study were to confirm the lung function benefit of adding olodaterol to tiotropium, describe the dose–response relationship of olodaterol in combination with tiotropium 5 μg, and compare it with the dose response of olodaterol monotherapy. Methods In this double-blind, parallel-group trial, patients were randomized to receive either tiotropium 5 μg or a fixed-dose combination of tiotropium 5 μg with olodaterol 2 μg, 5 μg, or 10 μg, delivered once daily via the Respimat® for 4 weeks (NCT00696020). Patients had a diagnosis of chronic obstructive pulmonary disease and post-bronchodilator forced expiratory volume in 1 s (FEV1) ≥ 30 and

Nerve agent exposure is generally treated by an antidote formulation composed of a muscarinic antagonist, atropine sulfate (ATR), and a reactivator of acetylcholinesterase (AChE) such as pralidoxime, obidoxime (OBI), methoxime, trimedoxime or HI-6 and an anticonvulsant. Organophosphates (OPs) irreversibly inhibit AChE, the enzyme responsible for termination of acetylcholine signal transduction. Inhibition of AChE leads to overstimulation of the central and peripheral nervous system with convulsive seizures, respiratory distress and death as result. The present study evaluated the efficacy and pharmacokinetics (PK) of ATR/OBI following exposure to two different VX dose levels. The PK of ATR and OBI administered either as a single drug, combined treatment but separately injected, or administered as the ATR/OBI co-formulation, was determined in plasma of naïve guinea pigs and found to be similar for all formulations. Following subcutaneous VX exposure, ATR/OBI-treated animals showed significant improvement in survival rate and progression of clinical signs compared to untreated animals. Moreover, AChE activity after VX exposure in both blood and brain tissue was significantly higher in ATR/OBI-treated animals compared to vehicle-treated control. In conclusion, ATR/OBI has been proven to be efficacious against exposure to VX and there were no PK interactions between ATR and OBI when administered as a co-formulation.

Ethnopharmacological relevance Pittosporum mannii (Pittosporaceae) is used in Africa traditional medicine to treat various ailments including pain and inflammation. Aim of the study The present work was undertaken to evaluate the antinociceptive effects of the aqueous (AEPM) and methanol (MEPM) extracts from the leaves of Pittosporum mannii. Methods High performance liquid chromatography coupled with mass spectrometry (LCMS) was used for the phytochemical analysis of AEPM prepared as decoction and MEPM prepared as cold maceration. The in vitro cytotoxicity of AEPM and MEPM were evaluated on Artemia salina larvae. AEPM and MEPM antinociceptive effects were evaluated at the doses of 35, 75, 150 and 300 mg/kg given orally, against pain induced by acetic acid, formalin, hot plate, capsaicin and glutamate. The rota rod test was also performed at the same doses. To determine the mechanism of action of these extracts, their antinociceptive effects were tested in animals pretreated with yohimbine (α2-adrenergic antagonist), atropine (muscarinic antagonist) or naloxone (an opioids antagonist). Result The LCMS analysis showed that both extracts contain pittovidoside and 1-O-rhamnopyranosyl-23-acetoxyimberbic acid 29-methyl ester, the aqueous extract being more concentrated. Oral administration of both extracts significantly reduced pain symptoms induced by acetic acid, formalin, capsaicin, glutamate and hot plate. The antinociceptive effect of AEPM was significantly inhibited by yohimbine, atropine and naloxone while these inhibitors tend to potentiate the activity of MEPM. Both extracts have no effect on Rota rod test. AEPM and MEPM showed respective LC50 of 2.44 and 0.70 mg/ml on Artemia larvae and were therefore, considered non-toxic. Conclusion These results indicate that AEPM and MEPM possesses analgesic effects with different mechanism of action. Although effects of both extracts may involve TRPV1 receptors and glutamatergic pathway, AEPM may in addition, interact with alpha-adrenergic, muscarinic and opioidergic pathways that are not involve in the effects of MEPM.

As disturbance in auditory steady-state response (ASSR) has been consistently found in many neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia, there is considerable interest in the development of translational rat models to elucidate the underlying neural and neurochemical mechanisms involved in ASSR. This is the first study to investigate the effects of the non-selective muscarinic antagonist scopolamine and the cholinesterase inhibitor donepezil (also in combination with scopolamine) on ASSR. We recorded the local field potentials through the chronic microelectrodes implanted in the auditory cortex of freely moving rat. ASSRs were recorded in response to auditory stimuli delivered over a range of frequencies (10-80 Hz) and averaged over 60 trials. We found that a single dose of scopolamine produced a temporal attenuation in response to auditory stimuli; the most attenuation occurred at 40 Hz. Time-frequency analysis revealed deficits in both power and phase-locking to 40 Hz. Donepezil augmented 40-Hz steady-state power and phase-locking. Scopolamine combined with donepezil had an enhanced effect on the phase-locking, but not power of ASSR. These changes induced by cholinergic drugs suggest an involvement of muscarinic neurotransmission in auditory processing and provide a rodent model investigating the neurochemical mechanism of neurophysiological deficits seen in patients.

In cases of organophosphate poisoning, patients are treated with a combination of antidotes. In addition to these poison-directed antidotes, patients may require extra oxygen and artificial ventilation; other modalities may also be needed due to the wide range of toxic effects. Anisodamine is a belladonna alkaloid, and like other drugs from this family is non subtype-selective muscarinic, and a nicotinic cholinoceptor antagonist, which has been employed in traditional Chinese medicine. As a muscarinic antagonist, it displays similar pharmacological effects to atropine and scopolamine. However, anisodamine is not only less potent than atropine and scopolamine but also less toxic. Current in vitro and animal model studies have demonstrated that anisodamine has protective effects in a variety of diseases. Organophosphate poisoning involves not only the central and peripheral nervous systems, but also the cardiac and respiratory systems, as well as activation of inflammatory processes and oxidative stress. Therefore, the anticholinergic and additional activities of anisodamine appear to be relevant and justify its consideration as an addition to the existing remedies. However, more research is needed, as at present data on the role of anisodamine in the management of organophosphate poisoning are limited. Here, we review the beneficial effects of anisodamine on processes relevant to organophosphate poisoning.

Coordination for the Improvement of Higher Education Personnel (CAPES) National Council of Scientific and Technological Development (CNPq) Research Incentive Fund Association (AFIP) Sao Paulo Research Foundation (FAPESP) Extensive research has shown the involvement of the central cholinergic system in the acquisition and consolidation of tasks involving conditioned fear responses, such as those observed in contextual fear conditioning (CFC), tone fear conditioning (TFC) and inhibitory avoidance (IA). However, there are few data concerning the role of this system in the memory retrieval process. Therefore, the present study aimed to compare the effects of the administration of an M1 antagonist on retrieval during these tasks. For each behavioral procedure, groups of male Wistar rats were trained. Twenty-four hr later, they were treated with different doses of dicyclomine (16, 32, or 64 mg/kg, i.p.) or with saline 30 min before the test session. The results showed that dicyclomine at doses of 16 and 32 mg/kg impaired CFC without interfering with IA performance. Moreover, only 64 mg/kg impaired TFC. These data suggest that M1 muscarinic receptors contribute to memory retrieval in CFC and TFC but are not essential for retrieval in IA. Univ Fed Sao Paulo, Dept Psychobiol, UNIFESP, Rua Botucatu 862,Edificio Ciencias Biomed 1 Andar, BR-04023062 Sao Paulo, Brazil Univ Fed Sao Paulo, Dept Psychobiol, UNIFESP, Rua Botucatu 862,Edificio Ciencias Biomed 1 Andar, BR-04023062 Sao Paulo, Brazil Web of Science

Chronic, high-frequency (>100 Hz) electrical stimulation, known as deep brain stimulation (DBS), of the internal segment of the globus pallidus (GPi) is a highly effective therapy for Parkinson's disease (PD) and dystonia. Despite some understanding of how it works acutely in PD models, there remain questions about its mechanisms of action. Several hypotheses have been proposed, such as depolarization blockade, activation of inhibitory synapses, depletion of neurotransmitters, and/or disruption/alteration of network oscillations. In this study we investigated the cellular mechanisms of high-frequency stimulation (HFS) in entopeduncular nucleus (EP; rat equivalent of GPi) neurons using whole cell patch-clamp recordings. We found that HFS applied inside the EP nucleus induced a prolonged afterdepolarization that was dependent on stimulation frequency, pulse duration, and current amplitude. The high frequencies (>100 Hz) and pulse widths (>0.15 ms) used clinically for dystonia DBS could reliably induce these afterdepolarizations, which persisted under blockade of ionotropic glutamate (kynurenic acid, 2 mM), GABAA (picrotoxin, 50 μM), GABAB (CGP 55845, 1 μM), and acetylcholine nicotinic receptors (DHβE, 2 μM). However, this effect was blocked by atropine (2 μM; nonselective muscarinic antagonist) or tetrodotoxin (0.5 μM). Finally, the muscarinic-dependent afterdepolarizations were sensitive to Ca2+-sensitive nonspecific cationic (CAN) channel blockade. Hence, these data suggest that muscarinic receptor activation during HFS can lead to feedforward excitation through the opening of CAN channels. This study for the first time describes a cholinergic mechanism of HFS in EP neurons and provides new insight into the underlying mechanisms of DBS.