D108-05 Modulating Inflammation in the Target Tissue: Anti-Inflammatory Effects of Ensifentrine in Medium Bronchi and Small Airways of COPD Donors

Abstract Rationale Ensifentrine is an inhaled dual PDE3/4 inhibitor approved after Phase III trials that showed reduced COPD exacerbations. Although its high affinity for PDE3 explains bronchodilation, PDE4 is predominantly expressed in inflammatory cells and epithelium; therefore ensifentrine may exert clinically relevant anti-inflammatory effects in target airway tissues that contribute to exacerbation reduction. We evaluated the anti-inflammatory activity of ensifentrine in human medium bronchi, small airways, and primary bronchial epithelial cells from COPD donors, and its interactions with commonly used inhaled therapies. Methods Human medium bronchial rings, small airways, and primary bronchial epithelial cells from COPD lobectomy donors were treated with concentration-response curves (10nM-1mM) of ensifentrine or roflumilast (a PDE4 inhibitor used as reference), with epithelium-intact and epithelium-denuded preparations where specified. Tissues were challenged with LPS (300ng/ml, 2h). The EC30 and EC50 concentrations for ensifentrine refer to its effect on airway smooth muscle relaxation, determined in preliminary studies, and were used to assess anti-inflammatory effects in combination experiments. Ensifentrine was combined at these isoeffective concentrations with formoterol, glycopyrronium, formoterol+glycopyrronium, formoterol+beclometasone, and formoterol+glycopyrronium+beclometasone. Supernatants were assayed by Luminex for cytokines, alarmins, and growth factors. Anti-inflammatory effects were expressed as percent reductions vs. LPS (control). Interactions were analyzed using the BLUR (Bliss-Loewe Uncertainty Range) model, which assesses if observed effects exceed expected additive responses, indicating synergy. N = 5; P 0.05. Results Ensifentrine produced a concentration-dependent anti-inflammatory response across medium bronchi (Emax ∼104%, pEC50 7.36), small airways (Emax ∼102%, pEC50 6.26), and primary epithelial cells (Emax ∼99%, pEC50 6.29), with effects comparable to roflumilast. At EC30 (0.63µM), ensifentrine reduced LPSinduced inflammation by 84.3±8.7% in medium bronchi and 64.8±9.0% in small airways; at EC50 (14.13µM), reductions reached 94.4±5.3% and 90.9±7.5%, respectively. BLUR analysis revealed synergistic anti-inflammatory interactions in approximately 31.4% of tested combinations, with the highest synergy observed for IL-10 (mean delta effect: 15.3%) and in small airways for IL-25, GM-CSF, and TNF-α, with deltas ranging from 10% to 18%. Combinations with glycopyrronium containing regimens yielded larger and more frequent synergistic effects (mean IL-10 delta = 15.2%), significantly exceeding the expected additivity (P 0.05). Conclusions In COPD target tissues, inhaled ensifentrine exerts potent, concentration-dependent anti-inflammatory effects, with glycopyrronium including treatments showing greater synergy on key inflammatory mediators. These findings support further clinical evaluation of optimized combinations that leverage synergistic interactions to enhance therapeutic efficacy in COPD management. This study was funded by Verona Pharma plc. This abstract is funded by: Verona Pharma plc