Preclinical investigations using isolated human bronchial tissues have demonstrated a synergistic inhibitory effect on ASM tone when ensifentrine is coadministered with either atropine or glycopyrronium [13]. In contrast, its interaction with salbutamol appears to be additive rather than synergistic [13], possibly owing to overlapping mechanisms. Both β2-agonists and dual PDE3/4 inhibition converge on increasing intracellular cyclic adenosine monophosphate (cAMP) levels, thereby promoting ASM relaxation [11, 14]. Specifically, PDE3 and PDE4 are responsible for cAMP degradation; their inhibition results in preserved cAMP signaling and increased bronchial smooth muscle relaxation. Β2-Agonists exert their effects via G-protein coupled receptor-mediated activation of adenylate cyclase, directly stimulating cAMP synthesis [14]. In contrast, muscarinic antagonists act through distinct pathways involving the attenuation of intracellular calcium release and suppression of protein kinase C activity, culminating in ASM relaxation [14]. Thus, the observed synergism between ensifentrine and muscarinic antagonists is underpinned by the engagement of distinct, but complementary, signaling mechanisms [15].

The added value of combining ensifentrine with other COPD medications, compared with using other COPD drugs alone, is improvement in lung function and symptoms, with a possible reduction in exacerbation rates (Table 3). Indeed, data from a small study suggested that ensifentrine, when added to a LABA/LAMA regimen, amplifies bronchodilation and reduces both residual volume and functional residual capacity [16]. In a more extensive study, Ferguson et al. showed that adding ensifentrine to tiotropium resulted in a placebo-corrected increase in peak FEV1 at week 4, ranging from 77.5 mL (0.375 mg) to 124.2 mL (3 mg). The highest dose was also associated with a significant increase in average FEV1 over 12 h [17]. Clinically meaningful improvements in quality of life, as measured by the St. George’s Respiratory Questionnaire (SGRQ), were observed with the 1.5 mg and 3 mg doses

A pooled post-hoc analysis of the phase 3 ENHANCE-1 and ENHANCE-2 trials confirms the bronchodilator efficacy of ensifentrine, demonstrating statistically significant improvements in lung function (forced expiratory volume in 1 second—area under the curve from 0 h to 12 h [FEV₁ AUC0–12 h]) compared with placebo after 12 weeks of treatment. This effect was observed in patients concomitantly receiving LAMA as well as in patients on LABA ± ICS regimens. The placebo-adjusted least-squares mean increase in FEV₁ was slightly greater in the LAMA subgroup (92 mL versus 74 mL), but the difference is unlikely to be clinically significant [18]. These findings, together with mechanistic insights, support the rationale for combining ensifentrine with a bronchodilator in a single inhaler to potentiate bronchodilator effects [11].

The effects on exacerbation rates are less clear. While some studies and reviews suggest a reduction in exacerbation frequency, these findings are limited by the fact that the trials were not designed or powered to assess exacerbation outcomes, and the enrolled populations were not enriched for exacerbation risk. According to the GOLD 2025 report, ensifentrine demonstrated a reduction in exacerbation rate; however, the patient populations studied were not enriched for exacerbation risk, and the studies did not assess the impact of ensifentrine on top of LABA+LAMA or LABA+LAMA+ICS regimens [1].

Nevertheless, in the pooled post-hoc analysis of the phase 3 ENHANCE-1 and ENHANCE-2 trials, ensifentrine was associated with substantial reductions in both the rate and risk of exacerbations in all treatment subgroups, by 48% and 50% in the LAMA cohort and by 51% and 56% in the LABA+ICS group, respectively [18]. These results are likely owing to the anti-inflammatory activity of ensifentrine. In addition, consistent with hypotheses previously proposed for dual bronchodilation [19], the observed reduction in exacerbation frequency may also reflect improvements in lung mechanics, such as reduced dynamic hyperinflation and decreased mechanical stress, thereby raising the threshold for symptom onset. Furthermore, it cannot be excluded that the potential improvement in mucociliary clearance induced by ensifentrine's ability to reduce mucus hypersecretion and facilitate sputum clearance may have played a role [20].

It is noteworthy that the incidence of pneumonia was highest in the LABA+ICS alone group (2.7%), followed by the LABA+ICS+ensifentrine group (1.3%), with both LAMA-containing groups (LAMA+ensifentrine and LAMA alone) showing the lowest rates (0.6% each) [18]. This pattern is consistent with broader evidence that ICS-containing regimens are associated with a higher risk of pneumonia compared with LAMA-based regimens [21].

The cumulative evidence suggests that the therapeutic efficacy of ensifentrine, particularly in reducing the risk of exacerbations, may be optimized by the coadministration with a LAMA in a single inhaler. A solid pharmacological rationale supports this possibility. The cholinergic neurotransmitter acetylcholine (ACh), while traditionally regarded as a neuronal mediator, plays a central role in airway inflammation in COPD [22]. In addition to its neuronal sources, ACh is synthesized by non-neuronal cells, including airway epithelial cells, in response to inflammatory stimuli [23]. Structural cells such as fibroblasts and mast cells, as well as immune cells such as macrophages, lymphocytes, and granulocytes, have been shown to express choline acetyltransferase and produce ACh [23]. These inflammatory cells also express functional muscarinic receptors [24], which mediate proinflammatory signaling cascades upon ACh binding, enhance chemokine release, and promote neutrophil chemotaxis and activation [25]. Thus, inhibition of cholinergic signaling via muscarinic receptor blockade may confer anti-inflammatory benefits, further reducing the risk of exacerbation [2].