Inhaled dry powder formulations achieved higher drug permeability than their solution formulations in ALI. A high local concentration of drugs on the cell layer, caused by direct attachment of the inhaled dry powder, contributed to increased drug permeability via both trans- and paracellular routes.

Most safety evaluations of dry powder inhalers (DPIs) using cultured cells have been performed with dry powder formulations dissolved in a medium. However, this method is not considered to be suitable to evaluate the safety of inhaled dry powder formulations correctly since it cannot reflect the actual phenomenon on the respiratory epithelial surface. In this study, we established a novel in-vitro safety evaluation system suitable for DPIs by combining an air-liquid interface cultured cell layer and a device for dispersing dry powders, and evaluated the safety of candidate excipients of dry powders for inhalation. The safety of excipients (sugars, amino acids, cyclodextrins, and positive controls) in solutions was compared using submerged cell culture systems with a conventional 96-well plate and Transwell ® . The sensitivity of the cells grown in Transwell ® was lower than that of those grown in the 96-well plate. Dry powders were prepared by spray-drying and we evaluated their safety with a novel in-vitro safety evaluation system using an air-liquid interface cultured cell layer. Dry powders decreased the cell viability with doses more than solutions. On the other hand, dissolving the dry powders attenuated their cytotoxicity. This suggested that the novel in-vitro safety evaluation system would be suitable to evaluate the safety of DPIs with high sensitivity.Key words dry powder; safety evaluation; air-liquid interfaced culture; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; A549 cell Pulmonary drug delivery has been investigated as not only a local treatment for respiratory diseases such as asthma or chronic obstructive pulmonary disease, but also as an alternative route for the systemic delivery of macromolecule drugs such as insulin, calcitonin, and heparin. 1-4)Among the three major systems for pulmonary deliverynebulizers, pressurized metered-dose inhalers (pMDIs), and dry powder inhalers (DPIs)-DPIs have several advantages, including favorable portability, a low cost, and the lack of a need for propellants. Furthermore, the handling of DPIs is easier than that of pMDIs because of breath-actuated passive aerosolization. 5)For effective pulmonary deposition after inhalation, in general, the optimal aerodynamic diameter of drug particles is less than 6 µm.6,7) However, micronized drug particles tend to be highly cohesive and poorly flowable, leading to low-level performance. To solve these problems, it is usual for an excipient, such as coarse lactose monohydrate, to be formulated to physically attach to fine active ingredients. 8,9) It has been recently reported that the addition of amino acids or cyclodextrins (CyDs) as excipients improves pulmonary delivery. 10,11) As for fundamental research on DPIs, evaluation of the physical properties such as particle size and pulmonary deposition is mainstream, whereas biological evaluation such as of the efficacy and safety is essential for clinical application. 12)These biological evaluations of dry powder formulations are often conduct...