Co-spray dried resveratrol and budesonide inhalation formulation for reducing inflammation and oxidative stress in rat alveolar macrophages

https://doi.org/10.1016/j.ejps.2016.02.018Get rights and content

Abstract

Oxidative stress is instrumental in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Novel therapeutic strategies that target macrophages, based on the use of antioxidant compounds, could be explored to improve corticosteroid responses in COPD patients. In this study, inhalable microparticles containing budesonide (BD) and resveratrol (RES) were prepared and characterized. This approach was undertaken to develop a multi-drug inhalable formulation with anti-oxidant and anti-inflammatory activities for treatment of chronic lung diseases. The inhalable microparticles containing different ratios of BD and RES were prepared by spray drying. The physico-chemical properties of the formulations were characterized in terms of surface morphology, particle size, physical and thermal stability. Additionally, in vitro aerosol performances of these formulations were evaluated with the multi-stage liquid impinger (MSLI) at 60 and 90 l/min, respectively. The cytotoxicity effect of the formulations was evaluated using rat alveolar macrophages. The biological responses of alveolar macrophages in terms of cytokine expressions, nitric oxide (NO) production and free radical scavenging activities were also tested. The co-spray dried (Co-SD) microparticles of all formulations exhibited morphologies appropriate for inhalation administration. Analysis of the deposition profiles showed an increase in aerosol performance proportional to BD concentration. Cell viability assay demonstrated that alveolar macrophages could tolerate a wide range of RES and BD concentrations. In addition, RES and BD were able to decrease the levels of tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in lipopolysaccharide (LPS) induced alveolar macrophages.

This study has successfully established the manufacture of Co-SD formulations of RES and BD with morphology and aerosol properties suitable for inhalation drug delivery, negligible in vitro toxicity and enhanced efficacy to control inflammation and oxidative stress in LPS-induced alveolar macrophages.

Introduction

Chronic obstructive pulmonary disease (COPD) represents one of the leading causes of morbidity and mortality worldwide (Viegi, Scognamiglio et al., 2001). COPD is a lung disease characterized by chronic inflammation, airflow limitation, hyper mucous production, emphysema, bronchoconstriction, a decline of respiratory activity and eventual death (Barnes, 2007). The pathogenesis of COPD is multi-factorial which includes genetic predisposition, age, inhaled pollution and cigarette smoke. Previous studies have shown that cigarette smoke (CS) is the main risk factor for the development and progression of COPD (Rabe, Hurd et al., 2007). This is because CS causes a production of reactive oxygen species (ROS) that increase oxidative stress and for this reason it is implicated in the pathogenesis and in irreversible airway inflammation. Oxidative stress causes airway inflammation by stimulating the release of inflammatory mediators such as IL-6, IL-8 and TNF-α. These inflammatory mediators result in an increase of ROS and hence an increase in oxidative stress in the lungs (Rahman and Adcock, 2006). Furthermore, COPD exacerbations caused by chronic bacterial infection can result in additional airway inflammation owing to the further release of pro-inflammatory mediators (Khair, Davies et al., 1996).

Alveolar macrophages are one of the first lines of defence of the respiratory tract against inhaled noxious agents. Although airway epithelial cells as a whole are involved in COPD, several studies have demonstrated that alveolar macrophages play an important role in the pathogenesis of the disease (Tetley, 2002; Hodge, Hodge et al., 2007), mainly in smokers, by regulating the release of inflammatory mediators that attract neutrophils into the airway (Kent, Smyth et al., 2008). Corticosteroid molecules are able to suppress the release of these inflammatory mediators in alveolar macrophages but these drugs are relatively ineffective in COPD patients (Barnes et al., 2004, Bhavsar et al., 2008). For this reason a novel therapeutic strategy is needed.

The current first-line therapy for COPD involves the use of bronchodilators such as long acting beta agonists (LABA) in combination with inhaled corticosteroids. However, unlike other inflammation-based diseases, such as asthma, corticosteroids are less effective in improving the lung function of COPD patients, and a have limited effect in reversing the progression of tissue damage (Pauwels et al., 1999, Pauwels et al., 2001, Dahl et al., 2010, Vogelmeier et al., 2011). Furthermore, previous studies have shown that ROS have been implicated in initiating inflammatory responses in the lungs through the activation of transcription factors such as nuclear factor-kappa B (NF-κB) (Rahman and MacNee, 1998). This results in a vicious cycle of oxidative stress by ROS and airway inflammation. Histone deacetylase activities are required for NF-κB blockade by corticosteroid receptors (Barnes et al., 2004, Barnes, 2009). In several cases COPD patients became non-responsive to corticosteroid treatment as histone deacetylase (HDAC2) activities can become inhibited in the presence of oxidative stress (Barnes, Ito et al., 2004).

For these reasons, the use of anti-oxidant compounds in association with one of the corticosteroid drugs could provide a new therapeutic approach for the treatment and management of COPD.

Polyphenolic compounds are potential candidate molecules since these compounds naturally exhibit potent anti-oxidant and anti-inflammatory activities (Biswas, Hwang et al., 2013). Resveratrol (3,5,4-trihydroxystilbene) (RES) is a naturally occurring polyphenolic compound found in a large number of plant species (e.g. grapes, berries and legumes) and in red wine. Resveratrol is a light sensitive molecule with two isoforms, cis-resveratrol and trans-resveratrol, the trans form being more stable and also the more biologically active form (Neves, Lucio et al., 2012). The anti-oxidant activity of this polyphenol is due to its ability to scavenge free radicals (Arts and Hollman, 2005). Furthermore, different studies have shown resveratrol as anti-inflammatory, anti-allergic, anti-viral, anti-carcinogenic and anti-asthmatic (Cheong et al., 1999, Docherty et al., 1999, Manna et al., 2000, Alarcón de la Lastra and Villegas, 2005, Faith et al., 2006, Athar et al., 2009, Lee et al., 2009). Specifically in the lungs, in vitro and in vivo experiments have shown that RES can reduce inflammation in lung cells, scavenging oxygen-derived free radicals; subsequently, RES maybe a potential adjunct therapy in the treatment of COPD (Trotta, Lee et al., 2015). In addition, RES has been shown to inhibit the release of inflammatory cytokines from alveolar macrophages in COPD and therefore can be considered a suitable candidate for pharmacotherapy of macrophages (Culpitt, Rogers et al., 2003).

The aim of this study was to develop inhalable microparticles containing RES and budesonide (BD), a common anti-inflammatory corticosteroid. To the authors' knowledge, this is the first attempt to deliver a combination formulation containing anti-oxidant and anti-inflammatory compounds for the improvement of COPD. Different series of co-spray dried (Co-SD) formulations were prepared and the physico-chemical characteristics and in vitro aerosol performance were investigated. Importantly, the biological responses of alveolar macrophage cell lines in terms of cell viability, anti-inflammatory and anti-oxidant activities were evaluated with the prepared spray dried formulations.

Section snippets

Materials

Resveratrol, trans-3,4′,5-trihydroxystilbene, (RES) was purchased from Fagron Italia (Bologna, Italy). Budesonide (BD) used in this study was supplied by Yicheng Chemical Corp, Jiangsu, China. Nitro-l-arginine methyl ester (l-NAME), α-lipoic acid, l-ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), lipopolysaccharide (LPS) from Escherichia coli and 2,3-diaminonaphthalene (DAN) were purchased from Sigma-Aldrich (Sydney, Australia). Other cell culture reagents including phosphate buffer saline

Assessment of particle size and morphology

In this study, inhalable microparticles containing anti-oxidant RES and anti-inflammatory BD compounds were formulated using spray-drier. Particle sizes with uniform distributions within inhalable range were obtained via manipulation of the spray-drying parameters, which included inlet temperature, outlet temperature, atomisation, and feed rate of solution. Additional factors such as ratios of co-solvent and concentration feed solutions led to significant changes in particle distribution as

Conclusions

In this study novel co-spray dried formulations of an anti-oxidant (RES) and anti-inflammatory (BD) compounds were produced. The spray dried powders showed appropriate morphologies and suitable aerosol properties for inhalation drug delivery. In vitro studies showed that alveolar macrophages could tolerate RES and BD in the range of concentrations investigated (0.612 to 50 μM). Moreover, RES and BD showed to have anti-inflammatory activities due to their ability to reduce the levels of TNF-α

Declaration of interest

Professor Traini is the recipient of an Australian Research Council Future Fellowship (project number FT12010063). Professor Young is the recipient of an Australian Research Council Future Fellowship (project number FT110100996). WH Lee is the recipient of Early Career Fellowship Cancer Institute NSW (Project reference 14/ECF/1-12).

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