Microliter-scale in vitro aerosol-cell delivery of inhalable antisense oligonucleotide (aso) induces “Burst-like” Pharmacokinetics

Unlike traditional submerged cell cultures, nebulized drug delivery to air-liquid interface (ALI)-cultured lung cells provides a clinically relevant route for in vitro testing of inhalable drugs. Current nebulizer systems deliver non-physiologically large drug volumes. Here, we introduce the Cloud MAX technology, characterize its accuracy of dosimetry, and apply it to toxicity, efficacy, and biokinetics or permeability testing of a locked nucleic acid-modified antisense oligonucleotide (ASO). On average the Cloud MAX delivers 44% of 3–20 μL aerosolized liquid to ALI-cultured cells, the coefficient of variation (CV) of repeated drug dosing is <11%-rel. and as little as 1 μL of drug/insert (=0.25 μL/cm 2 ) can be delivered to ALI-cultured cells. As proof of concept for dose-controlled in vitro drug testing, we demonstrated target engagement of a LIM and Calponin Homology domain 1 (LIMCH1)-specific ASO in primary lung fibroblasts from Idiopathic Pulmonary Fibrosis (IPF) patients “shielded” by an A549 alveolar epithelial layer, mimicking inhalation therapy. LIMCH1-ASO was effective in LIMCH1-silencing even at the lowest dose (0.64 nmol/cm 2 ). Nebulization did neither impair LIMCH1-ASO efficacy nor induce cytotoxicity for the highest tested dose (3.21 nmol/cm 2 .) The permeability (biokinetics) of nebulized ASO through the A549 cell barrier was 14-fold faster (“burst-like”) than for larger volume (pipetted) ASO application. Computational modelling revealed that this well-known “burst-like” pharmaco−/biokinetics profile is due to small-volume drug application, not due to nebulizer-induced changes on barrier tightness (apparent permeability P app ). Thus, the Cloud MAX is a substance-efficient platform for in vitro pharmacokinetics/−dynamics testing of inhalable drugs for targeted pulmonary therapy.