Hoffman E1, Kelsall J1, Mahendran R1, Zeqiri E1, Silva M2, Schaffer K3, Jötten A4, Paulitschke P3,4 Victoria Hutter1.
1ImmuONE, Stevenage, UK.
2Technical University of Munich, Klinikum der Technischen Universität München, Munich, Germany
3PHIO scientific GmbH, Munich, Germany
4Ludwig-Maximilians-University, Faculty of Physics, Munich, Germany
Summary
While in vivo studies currently remain the regulatory standard for assessing the safety and efficacy of inhaled drug candidates, the role of in vitro methods is rapidly evolving for inhaled candidate screening. Traditional in vitro testing for inhaled drug candidates typically focuses on aerosol characterization, cytotoxicity assays, epithelial barrier integrity, and permeability studies using airway epithelial cell lines or primary human cells. While these models provide valuable early insights, they lack the physiological complexity needed to predict long-term toxicity, immune responses and persistence in the lung. Key areas of unmet need are particularly models for the pulmonary region and include models that better represent interactions with immune cells, immune cell and early inflammatory endpoints as well as surrogate endpoints for lung clearance. The aim of this study was to evaluate if novel endpoints including macrophage motility (surrogate marker for clearance) and detailed individual subcellular morphological feature characterisation could provide better mechanistic insights to the fate and safety of medicines in the lung. Human alveolar macrophages in monoculture and co-culture were exposed to a panel of 12 compounds including inhaled medicines, therapeutics with established off-target lung toxicities and mechanistic control substances. A motility assay was developed tracking the velocity of macrophages alongside high content image analysis of intracellular features and established techniques for assessing early inflammation (cytokine production, polarisation), cell stress and phagocytic activity. Concentration and time dependent effects could be discriminated using macrophage motility and morphology techniques and added to a battery of in vitro tests that was able to comprehensively evaluate the safety and fate of inhaled medicines.