Breathing-induced stretch enhances the efficacy of an inhaled and orally delivered anti-fibrosis drug in vitro

Mechanical forces, which are crucial for most downstream signaling pathways in lung (patho-)physiology, may also regulate the efficacy of drugs. We investigated the role of mechanical forces on the effectiveness of inhaled and systemic (oral) administration of an anti-fibrosis drug. We established an induced triple coculture fibrosis model of a tight alveolar endothelial-epithelial barrier combined with pro-fibrotically stimulated primary fi-broblasts derived from healthy donors and compared it to an analogous triple coculture model with fibroblasts from idiopathic pulmonary fibrosis (IPF) patients (innate IPF model). The 3D in vitro fibrosis models were established on a biomimetic, stretchable basement (BETA) membrane and cultured at an air-liquid interface (ALI). These fibrosis models were treated with an FDA-approved anti-fibrosis drug (oral and aerosolized appli-cation of Nintedanib) under static and dynamic culture conditions - including cyclic mechanical stretch and medium-flow induced shear stress - leveraging our advanced millifluidic CIVIC mini-lung technology. Fibrosis markers were characterized by protein and immunofluorescence analysis supplemented with real-time mea-surement of pulmonary compliance as a functional assay. Nintedanib shows more potent anti-inflammatory (IL1 beta, IL-6, and IL8) and anti-fibrotic (alpha SMA, soluble and deposited (type I) collagen, and compliance) effects on our IPF models under dynamic culture conditions for both delivery methods. Mechanotransduction enhanced the restoration of alveolar phenotypes after drug delivery, as indicated by surfactant protein C and Yes-associated protein levels. Our findings suggest that cyclic mechanical stretch plays a crucial role in the drug efficacy of Nintedanib. Albeit Nintedanib's anti-fibrotic and anti-inflammatory potency in both delivery routes is similar, the inhaled administration has a lower off-target dose fraction than oral application. Thus, inhaled Nintedanib showed a superior therapeutic index to systemic (oral) application.