Abstract
Asthma, a chronic airway inflammatory disease, manifests as excessive obstruction of the airways and airway hyperresponsiveness (AHR) due to airway smooth muscle (ASM) contraction. Conventional bronchodilator treatments, such as β2-adrenergic receptor (β2-AR) agonists, often lose efficacy due to receptor desensitization, while corticosteroid-resistant neutrophilic airway inflammation remains a major challenge in severe asthma. To identify novel bronchodilators, we screened the Sigma LOPAC1280 compound library using β2-agonist-desensitized precision-cut lung slices (PCLS) as an ex vivo model and identified pirfenidone (PFD), an FDA-approved drug for idiopathic pulmonary fibrosis, as a novel bronchodilator. We then developed CXN-8, a more potent PFD derivative. Both compounds produced rapid, concentration-dependent airway relaxation in human, mouse, and rat PCLS preconstricted with multiple bronchoconstrictors, and maintained efficacy under β2-AR-insensitive conditions. CXN-8 was ∼50-fold more potent than PFD while achieving comparable maximal relaxation, with EC50 values ranging from 0.8-4.7 μM across the different bronchoconstrictors. Mechanistically, PFD and CXN-8 suppressed RhoA-mediated Ca2 + sensitization by restoring myosin light chain phosphatase activity, which reduced phosphorylation of myosin regulatory light chain (MLC20) and promoted ASM relaxation. In vivo, acute oral administration of PFD (300 mg/kg) or CXN-8 (15 mg/kg) reduced airway resistance and AHR in house dust mite-sensitized mice. Chronic oral treatment (daily, Days 14-24) further attenuated airway inflammation and remodeling, decreasing eosinophil and neutrophil infiltration, Th2 and Th17 cytokines, ASM thickening, and peribronchial collagen deposition. Collectively, these findings identify PFD and CXN-8 as dual-acting bronchodilator and anti-inflammatory agents that also alleviate airway remodeling, offering a promising therapeutic strategy for β2-agonist-insensitive and corticosteroid-resistant asthma.