Long-Acting Muscarinic Antagonists for the Treatment of Difficult-to-Treat and Severe Asthma: A Narrative Review Focusing on Inflammation from Bench to Bedside

Luigino Calzetta,¹ Gian Marco Manzetti,² Elena Pistocchini,² Renato Federico Lauro,² Shima Gholamalishahi,² Mario Cazzola,² Paola Rogliani²

¹Unit of Respiratory Clinical Pharmacology, Department of Clinical Science and Translational Medicine, University of Rome “Tor Vergata”, Rome, Italy; ²Unit of Respiratory Medicine, Department of Experimental Medicine, University of Rome “Tor Vergata”, Rome, Italy

Correspondence: Luigino Calzetta, Unit of Respiratory Clinical Pharmacology, Department of Clinical Science and Translational Medicine, University of Rome “Tor Vergata”, Rome, Italy, Email [email protected]

Abstract: Asthma is a heterogeneous disorder characterized by chronic airway inflammation and airway hyperresponsiveness (AHR). Inhaled corticosteroids (ICS) combined with long-acting β₂-agonists (LABA) remain the mainstay of therapy, yet a subset of patients with difficult-to-treat or severe disease remains uncontrolled and requires high-intensity treatment. Recommendations support adding a long-acting muscarinic antagonist (LAMA) (i.e., tiotropium, glycopyrronium, umeclidinium) as part of triple therapy for these patients. Robust clinical evidence from randomized controlled trials and meta-analyses indicates that open and fixed-dose triple combinations improve lung function and reduce exacerbation risk vs. ICS/LABA, although effects on moderate exacerbations are inconsistent. The established mechanism of action of LAMA is bronchodilation through antagonism of M₃ muscarinic acetylcholine (ACh) receptors (mAChR) on airway smooth muscle. However, evidence for anti-inflammatory effects derives mainly from preclinical investigations. These studies suggest that mAChR expressed on airway epithelial and immune cells may contribute to inflammation and remodeling via non-neuronal ACh signaling. In experimental models, LAMA reduce pro-inflammatory mediator release, limit neutrophil recruitment, and modulate tissue remodeling pathways, with potential synergistic effects when combined with ICS and LABA. Additional indirect anti-inflammatory mechanisms, including reduced airway stretch and potential antiviral activity, have been described. Overall, while clinical benefits of adding a LAMA in difficult-to-treat and severe asthma are well established, the evidence of anti-inflammatory effect of LAMA remains preliminary and requires confirmation in targeted ex vivo studies and further clinical trials.

Keywords: asthma, difficult-to-treat, inflammation, LAMA, severe, triple therapy

Background

Asthma is a heterogeneous and multifaceted respiratory disease, typically characterized by intermittent symptoms, chronic airway inflammation, and variable airflow obstruction.¹ Two pathophysiological hallmarks of asthma are airway inflammation and airway hyperresponsiveness (AHR). While airway inflammation involves the infiltration and activation of several immune cells, such as eosinophils, neutrophils, lymphocytes, and innate lymphoid cells (ILC) within the airways,^2–4 AHR refers to an exaggerated bronchoconstrictive response to external stimuli, leading to reversible airway narrowing.^5,6 Consequently, the core of asthma therapy is based on mimicking the endogenous adrenal function by eliciting the anti-inflammatory effects of cortisol produced by the adrenal cortex⁷ and the bronchodilator activity of adrenaline released by the adrenal medulla.⁸ Inhaled therapies, including inhaled corticosteroids (ICS), often combined with long-acting β₂-adrenoceptor agonists (LABA) in fixed-dose combinations (FDC), represent the cornerstone of pharmacological treatment for most individuals with asthma.⁹ The Global Initiative for Asthma (GINA, 2025) document¹ recommends a stepwise treatment approach for adolescents and adults (Steps 1–5), with therapy intensity adjusted according to symptom severity and disease control. Step-up therapy is considered when asthma remains uncontrolled despite good adherence and correct inhaler technique, as indicated by poor symptom control and/or frequent exacerbations which may necessitate oral corticosteroids (OCS).^1,10

Despite the widespread availability of ICS and LABA, a significant proportion of asthmatic patients remains uncontrolled.^10,11 According to GINA,¹ approximately 17% of patients are affected by difficult-to-treat asthma, defined as asthma that remains uncontrolled despite medium-dose (MD) or high-dose (HD) ICS in combination with a second controller (usually a LABA) or even maintenance OCS, or asthma that requires HD-ICS to achieve control. Importantly, an estimated 3.7% of patients have severe asthma, a subset of difficult-to-treat asthma, which remains uncontrolled despite optimal treatment with HD-ICS/LABA and management of contributory factors (i.e., optimization of inhaler technique, treatment adherence, and comorbidity control), or that worsens when HD treatment is reduced.^1,10,11 These two patient subgroups fall within GINA treatment Steps 4 and 5, which are included into the subgroup of patients at high-intensity treatment, representing approximately 24% of all asthmatic patients (Figure 1).¹

Figure 1 Proportions of patients with high-intensity treatment, difficult-to-treat asthma, and severe asthma. Percentages refer to total asthmatic patients. High-intensity treatment: high-dose ICS/LABA or medium-dose ICS/LABA plus OCS; difficult-to-treat asthma: GINA Step 4–5 treatment with poor symptom control; severe asthma: GINA Step 4–5 treatment with poor symptom control despite good adherence and correct inhaler technique.

Abbreviations: GINA, Global Initiative for Asthma; ICS, inhaled corticosteroids; LABA, long-acting β₂-adrenoceptor agonists; OCS, oral corticosteroids.

To address the complex treatment needs in asthma, GINA suggests adding a long-acting muscarinic antagonist (LAMA) to ICS/LABA, also as FDC, both as a controller option at GINA Step 4 and as preferred treatment at GINA Step 5 in adults and adolescents. Specifically, the LAMA compounds suggested by GINA for the management of asthma are tiotropium (TIO), glycopyrronium (GLY), and umeclidinium (UMEC).¹ TIO can be administered via soft mist inhaler as part of an open triple therapy, added to a separate ICS/LABA combination, and it is approved for patients aged ≥ 6 years. GLY and UMEC can be prescribed to subjects with at least 18 years and are available as FDC, both in pressurized metered-dose inhalers (beclomethasone dipropionate [BDP]/formoterol [FOR]/GLY) and dry powder inhalers (fluticasone furoate [FF]/vilanterol [VI]/UMEC and mometasone furoate [MF]/indacaterol [IND]/GLY).

Evidence from randomized controlled trials (RCTs) indicates that triple therapy improves bronchodilation and reduces exacerbation risk in patients with difficult-to-treat asthma patients.^12–21 However, the impact on exacerbations may not be explained exclusively by bronchorelaxant action of LAMA, as these agents may also exert anti-inflammatory effects, as suggested by preclinical studies in human bronchial tissue and cells, both in vitro and ex vivo.^22–24 The management of inhaled therapy in patients with difficult-to-treat and severe asthma remains challenging, particularly given the lack of clinical data directly comparing MD-triple FDC with HD-ICS/LABA in patients uncontrolled on MD-ICS/LABA.²⁵ A better understanding of the potential anti-inflammatory mechanisms of LAMA could therefore be crucial to optimize treatment strategies in this population. Therefore, the aim of this narrative review was to address the existing knowledge gap regarding the potential anti-inflammatory effect of LAMA, specifically in the difficult-to-treat and severe asthma population, by encompassing both preclinical and clinical evidence.

Pharmacological Characteristics of LAMA Approved for the Treatment of Asthma

The pharmacological rationale of using LAMA in the treatment of asthma lies in the pivotal role of the cholinergic pathway in regulating the bronchial tone, which is mainly mediated by the vagus nerve.²⁶ Acetylcholine (ACh) is released from both airway parasympathetic ganglia and postganglionic fibers innervating the bronchial tree, where it acts on airway smooth muscle (ASM) to induce bronchoconstriction, via the activation of muscarinic ACh receptors (mAChR). This cholinergic activity appears enhanced in patients with asthma, who exhibit increased vagally mediated baseline tone and heightened vagally mediated AHR.^27,28

The effect of ACh on airway function is not limited to its direct action on ASM, as supported by the broad expression of different mAChR subtypes throughout bronchial tree^29,30 (Table 1). More specifically, five different subtypes of mAChR, all G protein-coupled receptors, have been classified from M₁ to M₅. These mAChR subtypes are differentially coupled to intracellular signaling pathways. M₃ and M₅ mAChR subtypes are primarily linked to G_q proteins, resulting in increased intracellular calcium levels, whereas M₂ and M₄ mAChR subtypes are coupled to G_i/o proteins, exerting inhibitory effects on effector cells.^26,31,32 M₁ mAChR subtype is predominantly expressed in airway parasympathetic ganglia, where it facilitates ACh release, as well as in peripheral lung tissue and the alveolar wall. M₂ and M₃ mAChR subtypes constitute the major muscarinic receptor subtypes found in the larger airways.^31,32 Activation of M₂ mAChR subtype on postganglionic pre-synaptic cholinergic nerves inhibits further ACh release via an autoinhibitory feedback mechanism mediated by G_i/o signaling.^26,28 Accordingly, virus-induced M₂ mAChR subtype dysfunction appears to be associated with AHR in animal isolated airways.³³ M₃ receptor activation induces bronchoconstriction in ASM and stimulates secretion from submucosal glands.³⁴ The expression and function of M₄ and M₅ mAChR subtype in human lung remain poorly understood. Although their presence in human pulmonary tissue is still debated,³⁵ M₄ mAChR subtype has been identified in the alveolar walls, vasculature, and ASM of rabbits, as well as in airway postganglionic cholinergic nerves in other laboratory animals.^29,36,37

Table 1 Distribution and Functions of mAChR Subtypes.

Given the pivotal role of M₃ mAChR subtype on ASM contractility and mucus secretion, ideally a LAMA should selectively antagonize this mAChR subtype to induce bronchodilation and reduce mucus hypersecretion,^34,50 while minimizing adverse effects. However, since LAMA also block M₂ mAChR subtype expressed in the heart, they may lead to unwanted cardiovascular side effects such as tachycardia and QT interval prolongation.^50–52 The pharmacodynamic (PD) selectivity of LAMA for M₃ mAChR subtype over M₂ mAChR subtype can be evaluated using the receptor residence half-life ratio (t_1/2 M₃/M₂), with higher ratios indicating a more favorable efficacy/safety profile.⁵⁰

Overall, muscarinic signaling contributes directly to key features of asthma pathophysiology, including reversible airflow limitation, bronchial hyperresponsiveness and mucus hypersecretion, all of which drive symptoms and exacerbations. By blocking muscarinic receptors, particularly on ASM and submucosal glands, LAMA counteract these cholinergic mechanisms, thereby attenuating bronchoconstriction and mucus production that are amplified in asthmatic airways.⁵³ Much of this muscarinic pathway–driven biology is also relevant to COPD, underpinning the clinical utility of LAMA across both asthma and COPD, although the relative contribution of cholinergic tone and structural airway changes differs between the two diseases.^54,55

Detailed information on the PD and pharmacokinetic (PK) profile of LAMA approved for asthma treatment are summarized in Table 2.

Table 2 PD and PK Characteristics of LAMA Approved for Asthma Treatment.

Impact of LAMA for the Treatment of Asthma

The beneficial effect of LAMA in the management of asthma has been extensively demonstrated in several RCTs. A systematic review and meta-analysis by Sobieraj et al¹² indicated that adding a LAMA to ICS was associated with a significant reduction in the risk of asthma exacerbations requiring OCS and asthma worsening vs. placebo (PCB) in patients aged ≥12 years with uncontrolled asthma (risk ratio 0.67). This quantitative synthesis also demonstrated improvements in lung function, namely increase in peak, trough, and area under the curve (AUC) for forced expiratory volume in 1 second (FEV₁), in patients receiving LAMA as add-on therapy to ICS vs PCB.¹² These findings are consistent with a previous crossover trial, which showed that adding a LAMA to ICS in patients with uncontrolled asthma resulted in greater improvements in FEV₁ compared to doubling the ICS dose.¹³ However, no statistically significant difference was observed comparing ICS+LAMA to ICS+LABA in terms of asthma exacerbation requiring OCS, asthma worsening and lung function.¹² This evidence supports the use of LAMA as an add-on to ICS/LABA, rather than as an alternative to LABA in the management of uncontrolled asthma, as open and FDC triple therapy with an ICS and a LABA.

Several Phase III RCTs on triple FDC have been carried out enrolling patients with uncontrolled asthma despite MD or HD-ICS/LABA treatment,^14–16 consistent with the GINA difficult-to-treat asthma definition.¹

In the TRIMARAN trial,¹⁴ MD-BDP/FOR/GLY was associated with an increase in trough FEV₁ after 26 weeks (57 mL, P<0.01), along with a 15% reduction in the annualized moderate-to-severe exacerbation rate (rate ratio 0.85, P<0.05) vs. MD-BDP/FOR. On the other hand, in the TRIGGER trial,¹⁴ HD-BDP/FOR/GLY showed no statistically significant impact on the exacerbation rate (P>0.05), while confirming a 73 mL increase in trough FEV₁ (P<0.01) vs HD-BDP/FOR at week 26.

The IRIDIUM trial¹⁵ showed that MF/IND/GLY significantly (P<0.001) improved trough FEV₁ after 26 weeks regardless of ICS dose, when compared to MD and HD-MF/IND (76 mL and 65 mL, respectively). Consistently, in the ARGON trial¹⁷ both MD- and HD-MF/IND/GLY were non-inferior to the open triple therapy with HD-fluticasone propionate (FP)/salmeterol (SAL)+TIO in terms of lung function, asthma control and health status. Additionally, HD-MF/IND/GLY reduced in a clinically relevant manner the risk of exacerbations when compared to HD-MF/IND, HD-FP/SAL, and HD-FP/SAL+TIO.¹⁸

The CAPTAIN trial¹⁶ investigated the impact of different doses of FF/VI/UMEC (100/25/62.5 μg, 200/25/62.5 μg, 100/25/31.25 μg, 200/25/31.25 μg) vs FF/VI (100/25 μg or 200/25 μg) in uncontrolled asthmatic patients. While triple FDC was associated with a significant (P<0.001) increase in trough FEV₁ at week 24 when compared to ICS/LABA with the same dose of ICS (82–110 mL), it did not reduce the annualized moderate-to-severe exacerbation rate (P>0.05).

The impact of adding a LAMA to an ICS and a LABA, both as FDC and open triple therapy in uncontrolled asthma, was further assessed in a systematic review and meta-analysis by Kim et al.¹⁹ This quantitative synthesis, including 11,894 patients, indicated that triple therapy was associated with 80 mL improvement in trough FEV₁ and reduced risk of severe exacerbations, defined as those requiring OCS treatment for ≥3 days, hospitalization, or emergency department visits, compared to ICS/LABA (incidence rate ratio: 0.85). Similarly, a network meta-analysis by Rogliani et al²⁰ showed that triple FDC with HD-ICS was more effective than MD- and HD-ICS/LABA in increasing FEV₁ (91–114 mL) and reducing moderate and severe exacerbations (relative risk 0.57–0.88). It resulted also that HD-ICS/LABA/LAMA was particularly effective in reducing severe exacerbations, especially in patients with evidence of type 2 inflammation biomarkers.²¹ Overall, these findings highlight the impact of LAMA in enhancing bronchodilation and reducing the exacerbation risk in difficult-to-treat asthmatic patients.

The ongoing KALOS⁶² and LOGOS⁶³ Phase III RCTs are investigating the efficacy of the triple FDC with budesonide (BUD), FOR, and GLY compared to BUD/FOR in asthmatic patients who remain uncontrolled despite medium-to-high dose ICS/LABA. The outcomes include both lung function, namely the change from baseline in FEV₁ AUC from 0 to 3 hours (AUC_0-3h) at week 24, and the rate of severe asthma exacerbations up to 52 weeks.^62,63

The effectiveness of triple FDC appears to be supported in real-world studies enrolling difficult-to-treat^64,65 and severe⁶⁶ asthmatic patients. As a matter of fact, real-word evidence (RWE) suggests that ICS/LABA/LAMA can improve lung function, especially addressing small airways, which are characterized by an inner diameter <2 mm,⁶⁷ and reduce the risk of asthma exacerbations.²⁵ However, such results are mostly preliminary or reported at short time-points.²⁵

Anti-Inflammatory Effects of LAMA in Asthma Treatment

The beneficial effects of LAMA on lung function in asthmatic patients demonstrated in RCTs and RWE are supported by robust pharmacological evidence obtained from studies employing human isolated bronchial tissue.⁶⁷ The LAMA GLY induces a synergistic bronchorelaxant effect on hyperresponsive airways when co-administered with the ICS BDP.⁶⁸ This effect was observed in a validated ex vivo model asthma using human isolated bronchi and small airways passively sensitized with sera from patients during an atopic asthma exacerbation.^68,69 Similar synergistic interaction was detected combining BDP with the LABA FOR in the same experimental setting.⁷⁰ As expected, also BDP/FOR/GLY⁷¹ and MF/IND/GLY⁷² induced a synergistic bronchorelaxant effect in further experiments on passively sensitized human isolated airways, but the extent of synergy was greater compared to that induced by ICS/LABA combination. Preclinical evidence also indicates a sustained synergistic anti-inflammatory interaction of LAMA combined with ICS and LABA;⁷² however, synergistic anti-inflammatory effects have not yet been confirmed in clinical trials.

In any case, the protective effect of triple FDC against asthmatic exacerbations observed in RCTs and meta-analyses^19–21 may not be explained solely by the synergistic bronchorelaxant effect elicited by LAMA. In fact, muscarinic antagonist may have also anti-inflammatory effects. As a matter of fact, the role of LAMA in modulating airway inflammation was investigated in several preclinical studies in human bronchial tissue and cells, both in vitro and ex vivo.²²

In vitro studies showed that TIO^73–75 and GLY⁷⁶ reduced the levels of interleukin (IL)-8 in human bronchial epithelial cells stimulated with different irritants. TIO modulated the levels of metalloproteinases involved in airway remodelling⁷⁶ and of IL-8 secretion⁷⁷ from human asthmatic lung fibroblasts. TIO also reduced the alveolar macrophage mediated chemotaxis of neutrophils.⁷⁸ GLY synergistically reduced lipopolysaccharide-induced tumor necrosis factor (TNF)-α release from human monocytes when combined with BUD.⁷⁹

In ex vivo experimental setting, Rogliani et al⁷² investigated the effects of MF/IND/GLY and MF/IND combinations on the levels of inflammatory mediators in the supernatants of passively sensitized human isolated airways, namely IL-4, IL-5, IL-6, IL-9, IL-13, TNF-α, thymic stromal lymphopoietin, neurokinins, intracellular cyclic adenosine monophosphate, and neuronal e non-neuronal ACh. Notably, not only the addition of a LAMA was associated with a greater reduction in inflammatory mediators compared to ICS/LABA, but only MF/IND/GLY combination inhibited the release of non-neuronal ACh.

Non-neuronal ACh is synthesized via choline acetyltransferase (ChAT) by several cell types, including airway epithelial cells,⁸⁰ and it is implicated in airway remodelling, particularly at the level of small airways which express high levels of M₃ mAChR subtype even in the absence of direct vagal innervation.^81,82 Consistently, non-neuronal ACh signaling promotes the transition of lung fibroblast into myofibroblast.⁸³ Similarly to neuronal ACh, it appears to be involved in airway smooth cell contraction,³⁸ possibly by increasing Ca²⁺ sensitization in airway smooth muscle.^84,85 Beyond bronchoconstriction, non-neuronal ACh also contributes to airway immune regulation through both barrier and specific immune mechanisms.³⁸ At the barrier level, it promotes proliferation of human bronchial epithelial cells⁸⁶ and modulates epithelial ion transport in animal models,⁸⁷ while promoting airway inflammation via the activation of macrophages and lymphocytes.^88,89 Notably, epithelial-derived non-neuronal ACh can be released through non-vesicular mechanisms and secreted toward the luminal surface, thereby enabling signaling to airway macrophages that are not directly reached by neural fibers.⁹⁰

Several mechanisms are responsible for in the anti-inflammatory effect of LAMA in the airways of asthmatic patients (Figure 2). Resident immune cells express both ChAT and mAChR, suggesting that ACh is involved in a paracrine and/or autocrine pro-inflammatory signaling via non-neuronal pathways.^23,89 The activation of mAChR, especially M₃ mAChR subtype, is associated with increased cytotoxicity, cytokine production, and cell proliferation in T lymphocytes, cell proliferation in B lymphocytes, synthesis of leukotriene B₄ in neutrophils and macrophages, and enhanced neutrophilic chemotaxis (Table 1).^23,43,89 Such mechanism appears to be exaggerated in asthmatic patients, where the chronic presentation of allergens induces an upregulation of mAChR in T lymphocytes, hence enhancing their sensitivity to ACh.^91,92 Consistently, TIO was shown to attenuate type 2 airway inflammation, a hallmark of asthma, in mice, both by reducing eosinophils in bronchoalveolar lavage fluid and suppressing the activation of basophils and group 2 innate ILC in vitro.⁹³ Notably, group 2 innate ILC both express mAChR and ChAT, serving as a source of non-neuronal ACh and thereby amplifying inflammation.^94,95 Therefore, targeting mAChR with LAMA may play a crucial role in modulating the complex neuroimmune circuits involved in asthma pathogenesis.^96,97

Figure 2 Bronchorelaxant and anti-inflammatory effects of LAMA in asthma.

Abbreviations, ASM, airway smooth muscle; GLY, glycopyrronium; IL, interleukin; LAMA, long-acting muscarinic antagonists; mAChR, muscarinic acetylcholine receptor; SAD, small airway dysfunction; TGF-β, transforming growth factor-β; TIO, tiotropium; UMEC, umeclidinium.

Beside the direct inhibition of mAChR activation in immune cells, LAMA may exert their anti-inflammatory effect also via indirect mechanisms. ACh stimulates the release of neutrophil and eosinophil chemotactic activity through the activation of mAChR in airway epithelial cells, making airway epithelium a suitable target for LAMA activity.^45,46 Moreover, bronchorelaxation itself might reduce airway inflammation, since the cyclic stretching of human alveolar epithelial cells was demonstrated to induce the release of IL-8 and transforming growth factor (TGF)-β1 in vitro.⁹⁸ Finally, TIO was shown to reduce the replication of respiratory syncytial virus in human epithelial cells in vitro, which might reduce the inflammatory burden of viral infections in asthmatic patients.^99,100

The anti-inflammatory effects of LAMA could be particularly relevant, especially due to their impact on IL-4, IL-5, and IL-13,⁷² which are key mediators of type 2 inflammation.¹⁰¹ This inflammatory pathway has been extensively studied, particularly in severe asthma, and its modulation through biologic therapies has led to significant improvements in asthma management.¹⁰² Therefore, targeting type 2 inflammation with LAMA may represent an effective therapeutic approach, especially for patients with severe disease.

LAMA in Difficult-to-Treat and Severe Asthma

Airway remodelling in asthma is characterized by a complex interplay of structural changes, including epithelial dysfunction, goblet cell hyperplasia and metaplasia, subepithelial matrix thickening and fibrosis, increased ASM mass, and enhanced angiogenesis.¹⁰³ These pathological changes tend to progress with increasing disease severity,¹⁰⁴ and are associated with an increased risk of exacerbations and decline in lung function.¹⁰³ Importantly, the cholinergic pathway plays a key role airway remodelling through both direct and indirect mechanisms. ACh directly promotes airway remodelling in asthma by enhancing platelet-derived and epidermal growth factor-mediated ASM proliferation and by inducing a contractile phenotype in airway mesenchymal cells.⁸⁹ Indirectly, ACh-mediated airway inflammation might contribute to airway remodelling, especially through the release of TGF-β, which drives epithelial–mesenchymal transition and alters extracellular matrix structure.¹⁰⁵ Given that airway remodelling represents a hallmark of difficult-to-treat and severe asthma, targeting this process with the use of LAMA is associated with therapeutic benefits.¹⁰⁶

In asthmatic patients, small airways may exhibit increased infiltration of inflammatory cells, including activated eosinophils and lymphocytes.^25,107 Small airway dysfunction (SAD) is a key feature of difficult-to-treat and severe asthma^108–110 and it is associated with an enhanced risk of exacerbation.^111,112 Current evidence indicates that small airways represent the primary site of action for non-neuronal ACh.⁸¹ Epithelial cells synthetize ACh via ChAT in response to inflammatory stimuli, such as TNF-α, contributing to the inflammatory cascade and airway remodelling.¹¹³ Previous studies have demonstrated that triple FDC, particularly extrafine formulations with a mass median aerodynamic diameter <2 µm, achieve good penetration in the small airways.¹¹⁴ Moreover, both FDC and open triple therapy have been shown to improve functional outcomes related to SAD, such as ventilation heterogeneity and distal airway resistance.^25,115 Therefore, adding a LAMA to ICS/LABA delivered via extrafine formulations may offer a valuable therapeutic strategy for targeting SAD in patients with difficult-to-treat and severe asthma.

The therapeutic benefits of LAMA in uncontrolled asthmatic patients are not limited to inhibition on the non-neuronal cholinergic system. Some patients can remain uncontrolled with ICS/LABA due to vagal hypertone-related AHR.¹⁰⁶ Notably, this exaggerated vagal activity may be triggered by psychosocial stress and can be attenuated by the administration of the short-acting muscarinic antagonist ipratropium.^116,117 Furthermore, neuronal and non-neuronal cholinergic pathways appear to be interconnected. Inflammatory cytokines and eosinophil-derived cationic proteins induced by non-neuronal ACh may impair the function of inhibitory M₂ mAChR, thereby enhancing bronchoconstriction via the neuronal cholinergic pathway, as demonstrated in animal models of asthma.¹¹⁸

Overall, most of the available data on LAMA and triple extrafine formulations in difficult-to-treat and severe asthma derive from short-term studies focusing on functional outcomes (lung function and small airway indices), whereas evidence for sustained structural modification of airway remodelling or long-term anti-inflammatory effects remains limited. To date, such anti-inflammatory and remodelling effects have not been confirmed in randomized controlled trials.

Conclusion

LAMA represent a key therapeutic option within the pharmacological armamentarium for patients with difficult-to-treat and severe asthma. Evidence from RCTs indicates that open and triple FDC improve lung function and reduce exacerbation risk compared to ICS/LABA. These clinical benefits may reflect the anti-inflammatory effects of LAMA, particularly via inhibition of non-neuronal ACh-mediated airway inflammation. Non-neuronal ACh contributes to airway remodelling and SAD, which are pathological hallmarks of difficult-to-treat and severe asthma, further supporting LAMA use in asthmatic patients.

LAMA exert anti-inflammatory effects in asthma by blocking mAChR–mediated proinflammatory signaling, reducing release of cytokines and chemotactic factors, and limiting airway remodelling and small-airway inflammation, with the potential of complementing bronchodilation to improve disease control.

Overall, the findings of this narrative review endorse the anti-inflammatory activity of LAMA, especially in triple therapy with a LABA and an ICS. However, since evidence mainly derives from in vitro and ex vivo studies, additional studies enrolling difficult-to-treat and severe asthma are needed to confirm this preclinical evidence also in clinical setting.

If this anti-inflammatory effect is confirmed, triple therapy may reduce exacerbation rates while enabling ICS dose reduction, with a consequent decrease in dose-related adverse events.^119–121 According to the proven beneficial drug interaction across ICS, LABA, and LAMA,^71,72 adding a LAMA characterized by a fast onset of action such as GLY to ICS/FOR in a FDC may warrant a clinical trial to test a novel Triple MAintenance and Reliever Therapy (TriMART)^25,122 approach in asthma treatment.

Abbreviations

ACh, acetylcholine; AHR, airway hyperresponsiveness; AR, airway remodelling; ASM, airway smooth muscle; AUC, area under the curve; BDP, beclomethasone dipropionate; BUD, budesonide; ChAT, choline acetyltransferase; FDC, fixed-dose combination; FEV₁, forced expiratory volume in 1 second; FF, fluticasone furoate; FOR, formoterol; FP, fluticasone propionate; GINA, Global Initiative for Asthma; GLY, glycopyrronium; HD, high-dose; ICS, inhaled corticosteroids; IL, interleukin; ILC, innate lymphoid cells; IND, indacaterol; LABA, long-acting β₂-adrenoceptor agonists; LAMA, long-acting muscarinic antagonists; MD, medium-dose; MF, mometasone furoate; mAChR, muscarinic acetylcholine receptor; OCS, oral corticosteroids; PCB, placebo; PD, pharmacodynamic; PK, pharmacokinetic; RCTs, randomized controlled trials; RWE, real-world evidence; SAL, salmeterol; SA, small airways; SAD, small airway dysfunction; TGF-β, transforming growth factor β; TIO, tiotropium; TNF-α, tumor necrosis factor alpha; UMEC, umeclidinium; VI, vilanterol; t_1/2 M₃/M₂, receptor residence half-life ratio (M₃ over M₂).

Data Sharing Statement

Data availability is not applicable as no new dataset was generated/analyzed in this paper.

Author Contributions

L.C.: conceptualization; data curation, funding acquisition, resources, software, supervision, visualization, writing – original draft, writing – review and editing; G.M.M.: data curation, visualization, writing – original draft; E. P.: data curation, visualization, writing – review and editing; R. F. L.: data curation, visualization, writing – review and editing; S. G.: data curation, visualization, writing – review and editing; M. C.: data curation, visualization, writing – review and editing; P- R.: conceptualization; data curation, funding acquisition, resources, software, supervision, visualization, writing – original draft, writing – review and editing. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This research was not funded.

Disclosure

L.C. participated as an advisor in scientific meetings sponsored by Boehringer Ingelheim and Novartis, received non-financial support from AstraZeneca, a research grant partially funded by Chiesi Farmaceutici, Boehringer Ingelheim, Novartis, and Almirall, and is or was a consultant to ABC Farmaceutici, MSD, Recipharm, Zambon, Verona Pharma and Ockham Biotech. He also reports grants from Zambon, Verona Pharma, outside the submitted work. His department was funded by Almirall, Boehringer Ingelheim, Chiesi Farmaceutici, Novartis, and Zambon. M.C. has participated as a faculty member and advisor in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, Lallemand, Mundipharma, Novartis, Pfizer, Verona Pharma, and Zambon, and is or has been a consultant to ABC Farmaceutici, AstraZeneca, Chiesi Farmaceutici, Edmond Pharma, Lallemand, Novartis, Ockham Biotech, Verona Pharma, and Zambon. He also reports personal fees from Cipla, Neutec, Glenmark, and Abdi Ibrahim during the conduct of the study. His department was funded by Almirall. P.R. reported grants and personal fees from Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, MSD, Mundipharma, and Novartis, and participated as a lecturer and advisor in scientific meetings sponsored by Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, Edmond Pharma, GlaxoSmithKline, Menarini Group, Mundipharma, and Novartis. She also reports grants, personal fees, non-financial support from Arcede Pharma, Recipharm, Regeneron, Roche, Sanofi, Verona Pharma, and Zambon, outside the submitted work. Her department was funded by Almirall, Boehringer Ingelheim, Chiesi Farmaceutici, Novartis, and Zambon. The authors report no other conflicts of interest in this work. No sponsor had a role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

References

1. 2025 GINA strategy report - Global Initiative For Asthma - GINA n.d. Available from: https://ginasthma.org/2025-gina-strategy-report/. Accessed July 10, 2025.

2. Hammad H, Lambrecht BN. The basic immunology of asthma. Cell. 2021;184(9):2521–14. doi:10.1016/j.cell.2021.02.016

3. Maspero J, Adir Y, Al-Ahmad M, et al. Type 2 inflammation in asthma and other airway diseases. ERJ Open Res. 2022;8(3):00576–2021. doi:10.1183/23120541.00576-2021

4. Barnes PJ. The role of inflammation and anti-inflammatory medication in asthma. Respir Med. 2002;96:S9–S15. doi:10.1053/rmed.2001.1232

5. Cockcroft DW, Davis BE. Mechanisms of airway hyperresponsiveness. J Allergy Clin Immunol. 2006;118(3):551–559. doi:10.1016/j.jaci.2006.07.012

6. Nair P, Martin JG, Cockcroft DC, et al. Airway hyperresponsiveness in asthma: measurement and clinical relevance. J Allergy Clin Immunol. 2017;5. doi:10.1016/j.jaip.2016.11.030

7. Chiarella SE, Barnes PJ. Endogenous inhibitory mechanisms in asthma. J Allergy Clin Immunol. 2023. doi:10.1016/j.jacig.2023.100135

8. Warren J. The adrenal medulla and the airway. Br J Dis Chest. 1986;80:1–6. doi:10.1016/0007-0971(86)90002-1

9. Global Initiative for Asthma – GINA 2024 GINA main report Available from: https://ginasthma.org/2024-report/ Accessed 10 July 2025.

10. 2024 Severe Asthma Guide - Global Initiative for Asthma - GINA n.d. Available from: https://ginasthma.org/severe-asthma/. Accessed July 10, 2025.

11. Hekking PP, Wener R, Bouvy M, et al. The prevalence of adult severe refractory asthma in The Netherlands. Am J Respir Crit Care Med. 2014;189:e40159.

12. Sobieraj DM, Baker WL, Nguyen E, et al. Association of inhaled corticosteroids and long-acting muscarinic antagonists with asthma control in patients with uncontrolled, persistent asthma a systematic review and meta-analysis. JAMA. 2018;319. doi:10.1001/jama.2018.2757

13. Peters SP, Kunselman SJ, Icitovic N, et al. Tiotropium bromide step-up therapy for adults with uncontrolled asthma. N Engl J Med. 2010;363(18):1715–1726. doi:10.1056/nejmoa1008770

14. Virchow JC, Kuna P, Paggiaro P, et al. Single inhaler extrafine triple therapy in uncontrolled asthma (TRIMARAN and TRIGGER): two double-blind, parallel-group, randomised, controlled Phase 3 trials. Lancet. 2019;394(10210):1737–1749. doi:10.1016/S0140-6736(19)32215-9

15. Kerstjens HAM, Maspero J, Chapman KR, et al. Once-daily, single-inhaler mometasone–indacaterol–glycopyrronium versus mometasone–indacaterol or twice-daily fluticasone–salmeterol in patients with inadequately controlled asthma (IRIDIUM): a randomised, double-blind, controlled phase 3 study. Lancet Respir Med. 2020;8(10):1000–1012. doi:10.1016/S2213-2600(20)30190-9

16. Lee LA, Bailes Z, Barnes N, et al. Efficacy and safety of once-daily single-inhaler triple therapy (FF/UMEC/VI) versus FF/VI in patients with inadequately controlled asthma (CAPTAIN): a double-blind, randomised, phase 3A trial. Lancet Respir Med. 2021;9(1):69–84. doi:10.1016/S2213-2600(20)30389-1

17. Gessner C, Kornmann O, Maspero J, et al. Fixed-dose combination of indacaterol/glycopyrronium/mometasone furoate once-daily versus salmeterol/fluticasone twice-daily plus tiotropium once-daily in patients with uncontrolled asthma: a randomised, Phase IIIb, non-inferiority study (ARGON). Respir Med. 2020;170:106021. doi:10.1016/j.rmed.2020.106021

18. Rogliani P, Calzetta L. Clinical interpretation of efficacy outcomes in pharmacological studies on triple fixed-dose combination therapy for uncontrolled asthma: assessment of IRIDIUM and ARGON studies. J Exp Pharmacol. 2022;14:1–5. doi:10.2147/JEP.S336304

19. Kim LHY, Saleh C, Whalen-Browne A, et al. Triple vs dual inhaler therapy and asthma outcomes in moderate to severe asthma. JAMA. 2021;325(24):2466. doi:10.1001/jama.2021.7872

20. Rogliani P, Ritondo BL, Calzetta L. Triple therapy in uncontrolled asthma: a network meta-analysis of phase III studies. Eur Respir J. 2021;58(3):2004233. doi:10.1183/13993003.04233-2020

21. Laitano R, Calzetta L, Matino M, et al. Asthma management with triple ICS/LABA/LAMA combination to reduce the risk of exacerbation: an umbrella review compliant with the PRIOR statement. Expert Opin Pharmacother. 2024;25(8):1071–1081. doi:10.1080/14656566.2024.2366991

22. Calzetta L, Pistocchini E, Ritondo BL, et al. Muscarinic receptor antagonists and airway inflammation: a systematic review on pharmacological models. Heliyon. 2022;8(6):e09760. doi:10.1016/j.heliyon.2022.e09760

23. Agache I, Adcock IM, Akdis CA, et al. The bronchodilator and anti-inflammatory effect of long-acting muscarinic antagonists in asthma: an EAACI position paper. Allergy. 2025;80(2):380–394. doi:10.1111/ALL.16436

24. Muiser S, Gosens R, van den Berge M, et al. Understanding the role of long-acting muscarinic antagonists in asthma treatment. Ann Allergy Asthma Immunol. 2022;128. doi:10.1016/j.anai.2021.12.020

25. Rogliani P, Manzetti GM, De Guido I, et al. Triple extrafine fixed-dose combination in asthma: from randomized controlled trials to real-world evidence. Expert Opin Drug Deliv. 2025;22(10):1467–1485. doi:10.1080/17425247.2025.2527700

26. Cazzola M, Page CP, Calzetta L, et al. Pharmacology and therapeutics of bronchodilators. Pharmacol Rev. 2012;64(3):450–504. doi:10.1124/pr.111.004580

27. Coulson FR, Fryer AD. Muscarinic acetylcholine receptors and airway diseases. Pharmacol Ther. 2003;98(1):59–69. doi:10.1016/S0163-7258(03)00004-4

28. Costello RW, Jacoby DB, Fryer AD. Pulmonary neuronal M2 muscarinic receptor function in asthma and animal models of hyperreactivity. Thorax. 1998;53(7):613–618. doi:10.1136/thx.53.7.613

29. Barnes PJ. Muscarinic receptor subtypes in airways. Life Sci. 1993;52(5–6):521–527. doi:10.1016/0024-3205(93)90310-Y

30. Mak JCW, Barnes PJ. Autoradiographic visualization of muscarinic receptor subtypes in human and Guinea pig lung. Amer Rev Respir Dis. 1990;141(6):1559–1568. doi:10.1164/ajrccm/141.6.1559

31. Racké K, Matthiesen S. The airway cholinergic system: physiology and pharmacology. Pulm Pharmacol Ther. 2004;17. doi:10.1016/j.pupt.2004.03.001

32. Racké K, Juergens UR, Matthiesen S. Control by cholinergic mechanisms. Eur J Pharmacol. 2006;533(1–3):57–68. doi:10.1016/j.ejphar.2005.12.050

33. Matera MG, Calzetta L, Sanduzzi A, et al. Effects of neuraminidase on equine isolated bronchi. Pulm Pharmacol Ther. 2008;21(4):624–629. doi:10.1016/j.pupt.2008.02.003

34. Calzetta L, Ritondo BL, Zappa MC, et al. The impact of long-acting muscarinic antagonists on mucus hypersecretion and cough in chronic obstructive pulmonary disease: a systematic review. Eur Respir Rev. 2022;31(164):210196. doi:10.1183/16000617.0196-2021

35. Mak JC, Baraniuk JN, Barnes PJ. Localization of muscarinic receptor subtype mRNAs in human lung. Am J Respir Cell Mol Biol. 1992;7(3):344–348. doi:10.1165/ajrcmb/7.3.344

36. Lazareno S, Buckley NJ, Roberts FF. Characterization of muscarinic M4 binding sites in rabbit lung, chicken heart, and NG108-15 cells. Mol Pharmacol. 1990;38(6):805–815. doi:10.1016/s0026-895x(25)09592-6

37. D’Agostino G, Chiari MC, Grana E, et al. Muscarinic inhibition of acetylcholine release from a novel in vitro preparation of the Guinea-pig trachea. Naunyn Schmiedebergs Arch Pharmacol. 1990;342(2):141–145. doi:10.1007/BF00166956

38. Wessler IK, Kirkpatrick CJ. The non-neuronal cholinergic system: an emerging drug target in the airways. Pulm Pharmacol Ther. 2001;14(6):423–434. doi:10.1006/pupt.2001.0313

39. Fujii T, Kawashima K. An independent non-neuronal cholinergic system in lymphocytes. Jpn J Pharmacol. 2001;85(1):11–15. doi:10.1254/jjp.85.11

40. Strom TB, Deisseroth A, Morganroth J, et al. Alteration of the cytotoxic action of sensitized lymphocytes by cholinergic agents and activators of adenylate cyclase. Proc Natl Acad Sci U S A. 1972;69(10):2995–2999. doi:10.1073/PNAS.69.10.2995

41. Reinheimer T, Möhlig T, Zimmermann S, et al. Muscarinic control of histamine release from airways. Inhibitory M1-receptors in human bronchi but absence in rat trachea. Am J Respir Crit Care Med. 2000;162(2):534–538. doi:10.1164/AJRCCM.162.2.9911094

42. Reinheimer T, Baumgärtner D, Höhle KD, et al. Acetylcholine via muscarinic receptors inhibits histamine release from human isolated bronchi. Am J Respir Crit Care Med. 1997;156(2):389–395. doi:10.1164/AJRCCM.156.2.96-12079

43. Profita M, Di Giorgi R, Sala A, et al. Muscarinic receptors, leukotriene B 4 production and neutrophilic inflammation in COPD patients. Allergy. 2005;60(11):1361–1369. doi:10.1111/j.1398-9995.2005.00892.x

44. Hagforsen E, Einarsson A, Aronsson F, et al. The distribution of choline acetyltransferase- and acetylcholinesterase-like immunoreactivity in the palmar skin of patients with palmoplantar pustulosis. Br J Dermatol. 2000;142(2):234–242. doi:10.1046/J.1365-2133.2000.03290.X

45. Koyama S, Rennard SI, Robbins RA. Acetylcholine stimulates bronchial epithelial cells to release neutrophil and monocyte chemotactic activity. Am J Physiol Lung Cell Mol Physiol. 1992;262(4):L466–L471. doi:10.1152/ajplung.1992.262.4.l466

46. Koyama S, Sato E, Nomura H, et al. Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity. J Appl Physiol. 1998;84(5):1528–1534. doi:10.1152/jappl.1998.84.5.1528

47. Proskocil BJ, Sekhon HS, Jia Y, et al. Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells. Endocrinology. 2004;145(5):2498–2506. doi:10.1210/EN.2003-1728

48. Kanefsky J, Lenburg M, Hai CM. Cholinergic receptor and cyclic stretch-mediated inflammatory gene expression in intact ASM. Am J Respir Cell Mol Biol. 2006;34(4):417–425. doi:10.1165/RCMB.2005-0326OC

49. Roffel AF, Elzinga CRS, Van Amsterdam RGM, et al. Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function. Eur J Pharmacol. 1988;153(1):73–82. doi:10.1016/0014-2999(88)90589-4

50. Cazzola M, Page C, Matera MG. Long-acting muscarinic receptor antagonists for the treatment of respiratory disease. Pulm Pharmacol Ther. 2013;26(3):307–317. doi:10.1016/j.pupt.2012.12.006

51. Ora J, Coppola A, Cazzola M, et al. Long-acting muscarinic antagonists under investigational to treat chronic obstructive pulmonary disease. J Exp Pharmacol. 2020;12:559–574. doi:10.2147/JEP.S259330

52. Brodde OE, Bruck H, Leineweber K, et al. Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart. Basic Res Cardiol. 2001;96(6):528–538. doi:10.1007/s003950170003

53. Gosens R, Gross N. The mode of action of anticholinergics in asthma. Eur Respir J. 2018;52(4):1701247. doi:10.1183/13993003.01247-2017

54. Mahay G, Zysman M, Guibert N, et al. Long-acting muscarinic antagonists (LAMA) in asthma: what is the best strategy? Respir Med Res. 2025;87. doi:10.1016/j.resmer.2025.101157

55. Harrison EM, Kim V. Long-acting maintenance pharmacotherapy in chronic obstructive pulmonary disease. Respir Med X. 2019;1. doi:10.1016/j.yrmex.2019.100009

56. Disse B, Reichl R, Speck G, et al. Ba 679 BR, A novel long-acting anticholinergic bronchodilator. Life Sci. 1993;52(5–6):537–544. doi:10.1016/0024-3205(93)90312-Q

57. Hohlfeld JM, Sharma A, Van Noord JA, et al. Pharmacokinetics and pharmacodynamics of tiotropium solution and tiotropium powder in chronic obstructive pulmonary disease. J Clin Pharmacol. 2014;54(4):405–414. doi:10.1002/jcph.215

58. Casarosa P, Bouyssou T, Germeyer S, et al. Preclinical evaluation of long-acting muscarinic antagonists: comparison of tiotropium and investigational drugs. J Pharmacol Exp Ther. 2009;330(2):660–668. doi:10.1124/jpet.109.152470

59. Bartels C, Looby M, Sechaud R, et al. Determination of the pharmacokinetics of glycopyrronium in the lung using a population pharmacokinetic modelling approach. Br J Clin Pharmacol. 2013;76(6):868–879. doi:10.1111/bcp.12118

60. Salmon M, Luttmann MA, Foley JJ, et al. Pharmacological characterization of GSK573719 (Umeclidinium): a novel, long-acting, inhaled antagonist of the muscarinic cholinergic receptors for treatment of pulmonary diseases. J Pharmacol Exp Ther. 2013;345(2):260–270. doi:10.1124/jpet.112.202051

61. Cahn A, Tal-Singer R, Pouliquen IJ, et al. Safety, tolerability, pharmacokinetics and pharmacodynamics of single and repeat inhaled doses of Umeclidinium in healthy subjects: two randomized studies. Clin Drug Investig. 2013;33. doi:10.1007/s40261-013-0088-7

62. NCT04609878 study to assess PT010 in adult and adolescent participants with inadequately controlled asthma (KALOS). Available from: https://clinicaltrials.gov/study/NCT046098782025. Accessed February 27, 2026.

63. Wise R. NCT04609904 study to assess pt010 in adult and adolescent participants with inadequately controlled asthma (LOGOS). Available from: https://clinicaltrials.gov/study/NCT046099042025. Accessed February 27, 2026.

64. Trinkmann F, Bogoevska V, Nachtigall D, et al. Exacerbation reduction and improved quality of life in asthma with extrafine formulation single-inhaler triple therapy (efSITT): six-month results of the trimaximize study (abstract). Eur Respir J. 2024;64(3). doi:10.1183/13993003.00347-2024

65. Gessner C, Bogoevska V, Nachtigall D, et al. Improvement in Lung Function after Six Months of Treatment with Extrafine Formulation Single-Inhaler Triple Therapy (efSITT) in Patients with Asthma - TriMaximize Study (abstract). Eur Respir J. 2024.

66. Carpagnano GE, Portacci A, Dragonieri S, et al. Managing small airway disease in patients with severe asthma: transitioning from the “Silent Zone” to Achieving “Quiet asthma. J Clin Med. 2024;13(8):2320. doi:10.3390/jcm13082320

67. Calzetta L, Page C, Matera MG, et al. Use of human airway smooth muscle in vitro and ex vivo to investigate drugs for the treatment of chronic obstructive respiratory disorders. Br J Pharmacol. 2024;181(5):610–639. doi:10.1111/bph.16272

68. Cazzola M, Calzetta L, Rogliani P, et al. Interaction between corticosteroids and muscarinic antagonists in human airways. Pulm Pharmacol Ther. 2016;36:1–9. doi:10.1016/j.pupt.2015.11.004

69. Rabe KF. Mechanisms of immune sensitization of human bronchus. Am J Respir Crit Care Med. 1998;158(supplement_2):S161–S170. doi:10.1164/AJRCCM.158.SUPPLEMENT_2.13TAC130

70. Calzetta L, Matera MG, Facciolo F, et al. Beclomethasone dipropionate and formoterol fumarate synergistically interact in hyperresponsive medium bronchi and small airways. Respir Res. 2018;19(1). doi:10.1186/s12931-018-0770-7

71. Rogliani P, Matera MG, Facciolo F, et al. Beclomethasone dipropionate, formoterol fumarate and glycopyrronium bromide: synergy of triple combination therapy on human airway smooth muscle ex vivo. Br J Pharmacol. 2020;177(5):1150–1163. doi:10.1111/bph.14909

72. Rogliani P, Ritondo BL, Facciolo F, et al. Indacaterol, glycopyrronium, and mometasone: pharmacological interaction and anti-inflammatory profile in hyperresponsive airways. Pharmacol Res. 2021;172:105801. doi:10.1016/j.phrs.2021.105801

73. Asano K, Suzaki I, Shikama Y, et al. Suppression of IL-8 production from airway cells by tiotropium bromide in vitro. Int J COPD. 2011;6:439–448. doi:10.2147/COPD.S23695

74. Anzalone G, Gagliardo R, Bucchieri F, et al. IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: effect of Tiotropium. Life Sci. 2016;152:107–116. doi:10.1016/j.lfs.2016.03.031

75. Albano GD, Bonanno A, Moscato M, et al. Crosstalk between mAChRM3 and β2AR, via acetylcholine PI3/PKC/PBEP1/Raf-1 MEK1/2/ERK1/2 pathway activation, in human bronchial epithelial cells after long-term cigarette smoke exposure. Life Sci. 2018;192:99–109. doi:10.1016/j.lfs.2017.11.034

76. Asano K, Shikama Y, Shoji N, et al. Tiotropium bromide inhibits TGF-β-induced MMP production from lung fibroblasts by interfering with Smad and MAPK pathways in vitro. Int J Chron Obstruct Pulmon Dis. 2010;5:277–286. doi:10.2147/copd.s11737

77. Costa L, Roth M, Miglino N, et al. Tiotropium sustains the anti-inflammatory action of olodaterol via the cyclic AMP pathway. Pulm Pharmacol Ther. 2014;27(1):29–37. doi:10.1016/j.pupt.2013.11.001

78. Vacca G, Randerath WJ, Gillissen A. Inhibition of granulocyte migration by tiotropium bromide. Respir Res. 2011;12(1). doi:10.1186/1465-9921-12-24

79. Pahl A, Bauhofer A, Petzold U, et al. Synergistic effects of the anti-cholinergic R,R-glycopyrrolate with anti-inflammatory drugs. Biochem Pharmacol. 2006;72(12):1690–1696. doi:10.1016/j.bcp.2006.07.025

80. Kummer W, Krasteva-Christ G. Non-neuronal cholinergic airway epithelium biology. Curr Opin Pharmacol. 2014;16:43–49. doi:10.1016/j.coph.2014.03.001

81. Cazzola M, Calzetta L, Matera MG. Long-acting muscarinic antagonists and small airways in asthma: which link? Allergy. 2021;76(7):1990–2001. doi:10.1111/all.14766

82. Kistemaker LEM, Gosens R. Acetylcholine beyond bronchoconstriction: roles in inflammation and remodeling. Trends Pharmacol Sci. 2015;36(3):164–171. doi:10.1016/j.tips.2014.11.005

83. Milara J, Serrano A, Peiró T, et al. Aclidinium inhibits human lung fibroblast to myofibroblast transition. Thorax. 2012;67(3):229–237. doi:10.1136/thoraxjnl-2011-200376

84. Chiba Y, Takada Y, Miyamoto S, et al. Augmented acetylcholine-induced, Rho-mediated Ca 2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol. 1999;127(3):597–600. doi:10.1038/sj.bjp.0702585

85. Ozaki H, Kwon SC, Tajimi M, et al. Changes in cytosolic Ca2+ and contraction induced by various stimulants and relaxants in canine tracheal smooth muscle. Pflugers Arch. 1990;416(4):351–359. doi:10.1007/BF00370740

86. Klapproth H, Reinheimer T, Metzen J, et al. Non-neuronal acetylcholine, a signalling molecule synthezised by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol. 1997;355(4):515–523. doi:10.1007/PL00004977

87. Acevedo M. Effect of acetyl choline on ion transport in sheep tracheal epithelium. Pflugers Arch. 1994;427(5–6):543–546. doi:10.1007/BF00374272

88. Koarai A, Traves SL, Fenwick PS, et al. Expression of muscarinic receptors by human macrophages. Eur Respir J. 2012;39(3):698–704. doi:10.1183/09031936.00136710

89. Gosens R, Zaagsma J, Meurs H, et al. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res. 2006;7(1). doi:10.1186/1465-9921-7-73

90. Saracino L, Zorzetto M, Inghilleri S, et al. Non-neuronal cholinergic system in airways and lung cancer susceptibility. Transl Lung Cancer Res. 2013;2(4):284–294. doi:10.3978/j.issn.2218-6751.2013.06.01

91. Gwilt CR, Donnelly LE, Rogers DF. The non-neuronal cholinergic system in the airways: an unappreciated regulatory role in pulmonary inflammation? Pharmacol Ther. 2007;115(2):208–222. doi:10.1016/j.pharmthera.2007.05.007

92. Ricci A, Amenta F, Bronzetti E, et al. Expression of peripheral blood lymphocyte muscarinic cholinergic receptor subtypes in airway hyperresponsiveness. J Neuroimmunol. 2002;129(1–2):178–185. doi:10.1016/S0165-5728(02)00177-7

93. Matsuyama T, Machida K, Motomura Y, et al. Long-acting muscarinic antagonist regulates group 2 innate lymphoid cell-dependent airway eosinophilic inflammation. Allergy. 2021;76(9):2785–2796. doi:10.1111/all.14836

94. Chu C, Parkhurst CN, Zhang W, et al. The ChAT-acetylcholine pathway promotes group 2 innate lymphoid cell responses and anti-helminth immunity. Sci Immunol. 2021;6(57). doi:10.1126/sciimmunol.abe3218

95. Roberts LB, Schnoeller C, Berkachy R, et al. Acetylcholine production by group 2 innate lymphoid cells promotes mucosal immunity to helminths. Sci Immunol. 2021;6(57). doi:10.1126/sciimmunol.abd0359

96. Chen Q, Jia N, Liu J, et al. Neuroimmune circuits in respiratory pathophysiology: decoding molecular crosstalk for precision therapeutic targeting. Ann Med. 2026;58(1). doi:10.1080/07853890.2026.2620337

97. Zhang N, Xu J, Jiang C, et al. Neuro-immune regulation in inflammation and airway remodeling of allergic asthma. Front Immunol. 2022;13. doi:10.3389/fimmu.2022.894047

98. Yamamoto H, Teramoto H, Uetani K, et al. Cyclic stretch upregulates interleukin-8 and transforming growth factor- β 1 production through a protein kinase C-dependent pathway in alveolar epithelial cells. Respirology. 2002;7(2):103–109. doi:10.1046/j.1440-1843.2002.00377.x

99. Iesato K, Tatsumi K, Saito K, et al. Tiotropium bromide attenuates respiratory syncytial virus replication in epithelial cells. Respiration. 2008;76(4):434–441. doi:10.1159/000151729

100. Ma R, Zhang C, Zhang Y, et al. The impact of respiratory syncytial virus on asthma development and exacerbation. Ann Allergy Asthma Immunol. 2025:1–8. doi:10.1016/j.anai.2025.05.011

101. Pavord ID, Afzalnia S, Menzies-Gow A, et al. The current and future role of biomarkers in type 2 cytokine-mediated asthma management. Clin Exp Immunol. 2017;47(2):148–160. doi:10.1111/cea.12881

102. Rogliani P, Calzetta L, Matera MG, et al. Severe asthma and biological therapy: when, which, and for whom. Pulm Ther. 2020;6(1):47–66. doi:10.1007/s41030-019-00109-1

103. Varricchi G, Brightling CE, Grainge C, et al. Airway remodelling in asthma and the epithelium: on the edge of a new era. Eur Respir J. 2024;63(4):2301619. doi:10.1183/13993003.01619-2023

104. Bonsignore MR, Profita M, Gagliardo R, et al. Advances in asthma pathophysiology: stepping forward from the maurizio vignola experience. Eur Respir Rev. 2015;24(135):30–39. doi:10.1183/09059180.10011114

105. Tiotiu A, Steiropoulos P, Novakova S, et al. Airway remodeling in asthma: mechanisms, diagnosis, treatment, and future directions. Arch Bronconeumol. 2025;61(1):31–40. doi:10.1016/J.ARBRES.2024.09.007

106. Cazzola M, Braido F, Calzetta L, et al. The 5T approach in asthma: triple therapy targeting treatable traits. Respir Med. 2022;200:106915. doi:10.1016/j.rmed.2022.106915

107. Roche WR. Inflammatory and structural changes in the small airways in bronchial asthma. Am J Respir Crit Care Med. 1998;157(5):S191–S194. doi:10.1164/ajrccm.157.5.rsaa-5

108. Usmani OS. Small airways dysfunction in asthma: evaluation and management to improve asthma control. Allergy Asthma Immunol Res. 2014;6(5):376. doi:10.4168/aair.2014.6.5.376

109. Berry M, Hargadon B, Morgan A, et al. Alveolar nitric oxide in adults with asthma: evidence of distal lung inflammation in refractory asthma. Eur Respir J. 2005;25(6):986–991. doi:10.1183/09031936.05.00132404

110. Bittar HET, Doberer D, Mehrad M, et al. Histologic findings of severe/therapy-resistant asthma from video-assisted thoracoscopic surgery biopsies. Am J Surg Pathol. 2017;41. doi:10.1097/pas.0000000000000777

111. Sagmen SB, Eraslan BZ, Demirer E, et al. Small airway disease and asthma control. J Asthma. 2023;60(9):1761–1766. doi:10.1080/02770903.2023.2185894

112. Van Der Wiel E, Ten Hacken NHT, Postma DS, et al. Small-airways dysfunction associates with respiratory symptoms and clinical features of asthma: a systematic review. J Allergy Clin Immunol. 2013;131(3):646–657. doi:10.1016/j.jaci.2012.12.1567

113. Barnes PJ. The role of anticholinergics in chronic obstructive pulmonary disease. Am J Med. 2004;117(Suppl 12A):24S–32S. doi:10.1016/j.amjmed.2004.10.018

114. Usmani OS, Baldi S, Warren S, et al. Lung deposition of inhaled extrafine beclomethasone dipropionate/formoterol fumarate/glycopyrronium bromide in healthy volunteers and asthma: the STORM study. J Aerosol Med Pulm Drug Deliv. 2022;35(4):179–185. doi:10.1089/jamp.2021.0046

115. Rogliani P, Ritondo BL, Puxeddu E, et al. Impact of long-acting muscarinic antagonists on small airways in asthma and COPD: a systematic review. Respir Med. 2021;189:106639. doi:10.1016/j.rmed.2021.106639

116. Liccardi G, Salzillo A, Calzetta L, et al. Can bronchial asthma with an highly prevalent airway (and systemic) vagal tone be considered an independent asthma phenotype? Possible role of anticholinergics. Respir Med. 2016;117:150–153. doi:10.1016/j.rmed.2016.05.026

117. Ritz T, Kullowatz A, Goldman MD, et al. Airway response to emotional stimuli in asthma: the role of the cholinergic pathway. J Appl Physiol. 2010;108(6):1542–1549. doi:10.1152/japplphysiol.00818.2009

118. Haddad EB, Rousell J. Regulation of the expression and function of the M2 muscarinic receptor. Trends Pharmacol Sci. 1998;19(8):322–327. doi:10.1016/S0165-6147(98)01231-0

119. Patel R, Naqvi SA, Griffiths C, et al. Systemic adverse effects from inhaled corticosteroid use in asthma: a systematic review. BMJ Open Respir Res. 2020;7(1):e000756. doi:10.1136/bmjresp-2020-000756

120. Bloom CI, Yang F, Hubbard R, et al. Association of dose of inhaled corticosteroids and frequency of adverse events. Am J Respir Crit Care Med. 2025;211(1):54–63. doi:10.1164/rccm.202402-0368OC

121. Rogliani P, Calzetta L. Dose of inhaled corticosteroids and cardiovascular disease in asthma: an unexpected misstep? Am J Respir Crit Care Med. 2025;211(2):292–293. doi:10.1164/rccm.202409-1788LE

122. Calzetta L, Chetta A, Aiello M, et al. The impact of corticosteroids on human airway smooth muscle contractility and airway hyperresponsiveness: a systematic review. Int J Mol Sci. 2022;23(23):15285. doi:10.3390/ijms232315285

Creative Commons License © 2026 The Author(s). This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms and incorporate the Creative Commons Attribution - Non Commercial (unported, 4.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.