**1. Introduction**

Inhaled corticosteroids were first discovered 50 years ago and are used as anti-inflammatory drugs. They are very effective controllers of asthma and largely used in chronic obstructive pulmonary disease (COPD) to prevent exacerbations and improve quality of life in COPD patients [1,2] despite the appearance of corticosteroid insensitivity [3]. Several alternatives to glucocorticoids have been developed in the past few years [4–6] but efforts are still essential to address the lack of treatment

options in COPD smoking patients for whom a loss of sensitivity to glucocorticoids is observed [7]. The cellular and molecular mechanisms underlying steroid insensitivity in severe asthma and COPD are still not fully understood [7]. Oxidative stress, an increase in phosphoinositide-3-kinase/Akt (PI3K/Akt) signaling leading to the phosphorylation of HDAC2, associated with a loss of HDAC2 activity, could be critical [3,8].

Budesonide (BUD) is one of the most extensively used inhaled glucocorticoids including in the prophylactic management of asthma [9] and smoking-induced COPD [10,11]. However, frequent dosing remains a major concern in the use of budesonide. Moreover, the therapeutic potential of budesonide might be limited by its low solubility at a physiological pH. The development of budesonide formulations that can enhance drug solubility and the dissolution rate in biological fluids will likely achieve higher tissue concentrations and effectiveness.

With the aim to improve the use of inhaled corticoids with sustained release, Dufour et al. [12] evaluated in a mouse model of asthma a new formulation where budesonide was complexed with cyclodextrin (2-hydroxypropyl-β-cycodextrin; HPβCD). In a model of smoking-induced COPD in mammals, Cataldo et al. (Cataldo et al., Patent, 2014) suggested a potential interest in a pharmaceutical preparation resulting from the complexation of budesonide with HPβCD.

Cyclodextrins are typically cone-shaped cyclic oligosaccharides of six (α-CD), seven (β-CD) or eight (γ-CD) glucose units. They possess a hydrophobic cavity allowing them to host hydrophobic molecules. They are widely used as complexing agents for low water-soluble drugs to improve their physicochemical properties including solubility, bioavailability and stability, but they also have many other applications in food, cosmetics, or textiles, for example [13–16]. β-CD and its derivatives can form a soluble inclusion complex with cholesterol and are often used to extract it from biological material [15]. Among β-CDs, methyl-β-CD (MβCD) is the most effective and the most used method to extract cholesterol but has limited clinical application, unlike HPβCD whose clinical application is broader [17–20].

The main anti-inflammatory mechanism of glucocorticoids involves the activation of glucocorticoid receptors in the cytosol after glucocorticoid binding, leading to their translocation to the nucleus, where they recruit histone deacetylase 2 (HDAC2) to the activated inflammatory gene complex. HDAC2 then reduces the acetylation of histones and glucocorticoid receptors, allowing chromatin condensation and the trans-repression of inflammatory transcription factors, respectively [21,22]. Through a decrease in activity and expression of HDAC2 in lung airways and alveolar macrophages, corticosteroid treatment is poorly effective for patients suffering from COPD [21,22]. Based on the characterized interaction between cyclodextrins and cholesterol, we hypothesized that this interaction could be involved in the effects of the BUD:HPβCD complex. Cholesterol is largely known for its effect on biophysical membrane properties and cholesterol-enriched domains are linked to membrane signaling [23–26] including pathways involved in PI3K/Akt signaling and inflammation processes. On giant unilamellar vesicles (GUVs) and lipid monolayers, BUD:HPβCD induced the disruption of cholesterol-enriched raft-like liquid ordered domains—an increase in membrane permeability and fluidity [27]. Except for membrane fluidity, all these effects were enhanced when HPβCD was complexed with budesonide as compared with HPβCD [27]. On cellular models, this could involve signal transduction pathways such as ROS generation, inflammatory cytokines expression and cell death.

The current study aimed to characterize the effect of the BUD:HPβCD complex in comparison with BUD and HPβCD on the response of human alveolar epithelial cells (A549) or human monocytes (A-THP1) to a mix of hydrogen peroxide and lipopolysaccharide (H2O<sup>2</sup> + LPS) mimicking stressful effects including those from cigarette smoke [28–30] or from environmental toxicants. In detail, we pursued four objectives: first, to establish the potential interest of BUD:HPβCD on the cytotoxicity induced by oxidative and inflammatory stressors; second, to investigate the cellular effect of BUD:HPβCD on the signaling pathway involved in corticosteroid effects including ROS generation, PI3K/Akt activation, HDAC2 activity and the release of pro-inflammatory cytokines such as IL-8; third, to question the role of cholesterol in the effect induced by BUD:HPBCD on ROS generation and PI3K/Akt activation, with the two first membranous events leading to inflammation, and fourth, to determine the effect of BUD:HPβCD as compared to BUD in glucocortioid resistance and the role of HDAC2 in mediating the loss of the glucocorticoid anti-inflammatory effect. BUD:HPβCD on the signaling pathway involved in corticosteroid effects including ROS generation, PI3K/Akt activation, HDAC2 activity and the release of pro-inflammatory cytokines such as IL-8; third, to question the role of cholesterol in the effect induced by BUD:HPBCD on ROS generation and PI3K/Akt activation, with the two first membranous events leading to inflammation, and fourth, to determine the effect of BUD:HPβCD as compared to BUD in glucocortioid resistance and the role

induced by oxidative and inflammatory stressors; second, to investigate the cellular effect of

This study is a part of the continuing efforts to develop novel drug delivery systems, as the complex between budesonide and cyclodextrins, with the aim to improve the treatment of patients suffering from smoking-induced COPD. of HDAC2 in mediating the loss of the glucocorticoid anti-inflammatory effect. This study is a part of the continuing efforts to develop novel drug delivery systems, as the complex between budesonide and cyclodextrins, with the aim to improve the treatment of patients
