Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model

Abstract Glucocorticoids are among the most effective drugs to treat asthma. However, the severe adverse effects associated generate the need for its therapeutic optimization. Conversely, though histamine is undoubtedly related to asthma development, there is a lack of efficacy of antihistamines in controlling its symptoms, which prevents their clinical application. We have reported that antihistamines potentiate glucocorticoids’ responses in vitro and recent observations have indicated that the coadministration of an antihistamine and a synthetic glucocorticoid has synergistic effects on a murine model of allergic rhinitis. Here, the aim of this work is to establish if this therapeutic combination could be beneficial in a murine model of asthma. We used an allergen‐induced model of asthma (employing ovalbumin) to evaluate the effects of the synthetic glucocorticoid dexamethasone combined with the antihistamine azelastine. Our results indicate that the cotreatment with azelastine and a suboptimal dose of dexamethasone can improve allergic lung inflammation as shown by a decrease in eosinophils in bronchoalveolar lavage, fewer peribronchial and perivascular infiltrates, and mucin‐producing cells. In addition, serum levels of allergen‐specific IgE and IgG1 were also reduced, as well as the expression of lung inflammatory‐related genes IL‐4, IL‐5, Muc5AC, and Arginase I. The potentiation of dexamethasone effects by azelastine could allow to reduce the effective glucocorticoid dose needed to achieve a therapeutic effect. These findings provide first new insights into the potential benefits of glucocorticoids and antihistamines combination for the treatment of asthma and grants further research to evaluate this approach in other related inflammatory conditions.


| INTRODUC TI ON
Over 300 million people worldwide suffer from asthma and it is expected that 100 million more will do it in the year 2025. 1 Asthma prevalence is variable and growing in the last decades, ranking between 2% and 10%. 2 It is also an important global issue due to its morbidity and mortality. Asthma is the cause of 1 in every 250 global deaths and it is associated with an estimated loss of 15 million years of productive life per year (measured as disability-adjusted life years or DALYs). 3 We have previously reported that antihistamines, acting on the histamine H 1 receptor, are capable of enhancing the transcriptional activity of the glucocorticoid receptor (GR), 4 an effect that could have clinical relevance, particularly for inflammation-associated conditions. Importantly, the GR and the H 1 receptor are the targets of the largest number of drugs currently approved for clinical treatment. 5 The combination of a synthetic glucocorticoid and an antihistamine is commonly administered in allergic rhinitis and atopic dermatitis. 6,7 Furthermore, coadministration of the antihistamine azelastine and the synthetic glucocorticoid mometasone synergistically ameliorated allergic inflammation in a murine model of allergic rhinitis. 8 Therefore, coadministration therapies could lead to the design of new strategies to treat inflammation-associated diseases. In this sense, asthma is an inflammatory disease that represents an interesting experimental scenario to extend this concept.
Glucocorticoids (GCs) are among the most effective current therapy for asthma. 9 However, the existence of important adverse effects as well as asthmatics patients unable to control symptoms generates the need of new therapeutic strategies. 10 Histamine has been consistently related to the development of the disease, since its identification as a potent constrictor of the smooth-muscle airway and its increased presence in diseased tissue. 11 Histamine levels are augmented in the airways of asthmatic patients, increasing vascular permeability, acting as chemoattractant of eosinophils and neutrophils, modulating the immune response, overall representing a key mediator between allergen, immunoglobulin E, mast cells, and asthma. 12 However, the use of antihistamines for the treatment of asthma is an enigma. Despite the abundance of preclinical information endorsing histamine's role in asthma, there is a lack of efficacy of antihistamines in controlling its symptoms. Several clinical studies have shown that antihistamines were unable to control some of asthma symptoms in adults at regular doses, 13 while an improvement of the symptoms was observed at higher doses. 14 Based on previous observations in vitro 4 and in vivo, 8 we hypothesized that the enhancement of GR transcriptional activity by antihistamines could be used to reduce GC's effective doses used to counteract asthma symptoms, possibly resulting in new (and safer) therapeutic strategies to treat asthma. To this aim, we used an allergen-induced murine model of asthma, as it represents the most prevalent form of asthma (allergic asthma), ideal to study the combination of a corticoid and an antiallergic drug. In this model, we specifically selected the antihistamine azelastine (AZE) to study its coadministration with the synthetic GR agonist dexamethasone (DEX) because AZE reduced the frequency of administration of inhaled GCs in chronic bronchial asthma patients. 15 Importantly, three formulations containing AZE and GCs have been patented to treat allergic rhinitis, highlighting the potential therapeutic application of this drug combination in some inflammation-associated respiratory conditions that could be linked to asthma. [16][17][18][19] Our results show that AZE potentiates DEX-induced GR transcriptional activity in vivo, and that the combination of both drugs results in a reduction of the effective GC concentration needed to achieve a therapeutic effect in an allergen-induced murine model of asthma. These findings provide insight into the potential benefits of GCs and antihistamines combination for the treatment of asthma symptoms and grant further research to evaluate this approach for the treatment of other inflammatory conditions.

| Materials
Dulbecco's modified Eagle's medium (DMEM) medium, antibiotics, phosphate-buffered saline (PBS), DEX, and AZE were obtained from Sigma Chemical Company. Fetal calf serum (FCS) was purchased from Natocor (Córdoba, Argentina). All other chemicals were of analytical grade and obtained from standard sources.

| Plasmid construct
pRsSV-GR was a gift from Dr Keith Yamamoto. 20 pCEFL-H 1 receptor and TAT3-Luc were previously generated in our laboratory. 21

| Cell culture
HEK293T (human embryonic kidney stably transfected with SV40 T-antigen) cells were obtained from the American Type Culture Collection (ATCC: Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal calf serum and 5-μg/mL gentamicin. Cells were incubated at 37°C in humidified atmosphere containing 5% CO 2 . For cell passaging or plating, cells were first washed out with phosphate-buffered saline (Invitrogen, Thermo Fisher Scientific) and then trypsinized using 1X trypsin-EDTA.

| Transfection and reporter gene assays
HEK293T cells seeded on 24-well plates were cotransfected with the pRSV-GR, pCEFL-H 1 receptor, and the luciferase reporter plasmids TAT3-Luc or IL6-Luc using the K2 Transfection System (Biontex, Munich, Germany) according to the manufacturer's instructions. After 4 hours, cells were seeded in 96-well plates, and 24 hours later were starved overnight and then stimulated with ligands. After a kinetic assessment, luciferase activity was measured at the optimal time of 24 hours using the Steady-Glo Luciferase Assay System according to the manufacturer's instructions (Promega Biosciences Inc San Luis Obispo) using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices).
As shown before, no differences were observed in results normalized to Renilla-Luc or to protein expression levels. 4  Mice were used at the age of 6 to 8 weeks. Randomization was used to assign animals to different experimental groups and to collect and process data, with analysis performed by investigators blinded to the treatment groups. One week after the last injection, animals were exposed to aerosols of 5 mL of allergen OVA in PBS (3% (w/v)) during 20 min for 3 consecutive days. Aerosol exposure was performed within individual compartments of a mouse pie chamber using a nebulizer (SAN-UP, Argentina, OVA solution flux 0.33 mL/min in air flux of 6 to 8 L/min).

| Development of the experimental protocol
One hour after the last exposure, mice were intranasally treated with 7 μL of a water solution containing the different drugs. The experimental protocol is summarized as follows: To perform the experiment, 30 animals were randomized and divided into six different experimental groups as follows:   It has been described that the allergen and the adjuvant in this particular strain promotes an antigen-specific Th2 immune response, involving the induction of allergic parameters that reflect some pathological changes observed in bronchial asthma, such as high levels of allergenspecific IgE, eosinophilic infiltrate in the airways and bronchial hyperreactivity. 32 According to this, animals were euthanized and evaluated 48 hours after initiation of the treatment.

| Pathologic analysis
Animals were euthanized with isoflurane. The chest wall was opened, and the animals were exsanguinated by cardiac puncture.
Serum was prepared and stored at −20°C. The trachea was cannulated after blood collection. Bronchoalveolar lavage (BAL) was performed four times with 1 mL of sterile PBS. Lavage fluid was collected, centrifuged at 300g for 10 min, and the pellet was resuspended in 0.5-mL PBS. BAL differential cell counts were performed sample was similarly generated (reverse transcriptase was replaced with water). cDNA as well as -RT samples were kept at −20°C.

| Quantitative polymerase chain reaction (QPCR)
Forward and reverse primer pairs were generated using the primer3Input online software (https ://prime r3plus.com/prime r3web/ prime r3web_input.htm) and designs were based on publicly available mouse mRNA sequences. Primers were designed to have approximately 50% G/C content and to generate 75-150-bp amplicons. Primer pair specificity against target sequence was checked in the NCBI Genbank database using Primer-BLAST  To estimate the efficiency of the amplification reaction, serial half logarithm unit dilutions of cDNA were used and standard curves were generated. The linear slope of the standard curve for each primer pair was estimated using GraphPad Prism 6 software and the efficiency was calculated based on this following formula (1).
Additionally, the -RT samples and a water template were included in the analysis to confirm the absence of any residual DNA or contamination. All cDNA samples were analyzed in triplicates.
Finally, the following formula (2) was used to calculate the fold induction of gene expression.

| Compliance with design and statistical analysis requirements
All animal groups have n = 5. Samples obtained from each animal were measured three times to test precision. Randomization was used to assign animals to different experimental groups and to (1) Efficiency = 10 −(1∕slope) collect and process data, with analysis performed by investigators blinded to the treatment groups.
The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology. 36 Graphs and statistical analysis were performed using GraphPad

| AZE enhances DEX response in vitro
We in combination with plasmids encoding for GR and H 1 receptor.
In this system we found that, while 10-μmol/L AZE alone had no

| AZE and DEX cotreatment reduces IgG and IgE
As it was mentioned, atopic or allergic asthma is the most prevalent form of the disease and it is characterized by the presence of hypersensitivity reactions mediated by allergen-specific IgE antibodies. We therefore analyzed the effects of the cotreatment on the serum levels of OVAspecific IgG1, IgG2a, and IgE of the different experimental groups. Mice sensitized with OVA showed an increase in the specific immunoglobulin levels, which were significantly reduced by the cotreatment with 0.1mg/kg DEX and 0.5-mg/kg AZE for IgE and IgG1. Remarkably, none of the treatments had an effect by themselves, suggesting a higher effectivity of the cotreatment (Figure 2A, 2, and 2). In this regard, it is worth mention that differently from what was previously reported, the optimal dose of DEX did not significantly reduced IgE levels.

| AZE and DEX cotreatment reduces eosinophilia in BAL
Accumulation of eosinophils in alveoli is a hallmark of allergic asthma and inflammation of the airways mediated by these cells is charac-

| AZE and DEX cotreatment improves lung histopathology
Aerosol exposure of the animals to the allergen OVA produces an allergic-inflammatory reaction of the airways that is reflected in of the allergic infiltration (from 100.0 ± 5.3% to 30.7 ± 6.4%), while the cotreatment with a 0.1-mg/kg DEX and 0.5-mg/kg AZE reduced it by 30% (from 100.0 ± 5.3% to 71.2 ± 6.3%). Treatments with the individual drugs did not significantly reduce this parameter ( Figure 4G). Similar observations were made for PAS-stained lungs sections in which the histological goblet cell score was reduced by 64% in animals who received a 1-mg/kg DEX (from 100.0 ± 12.2% to 35.9 ± 11.8%); by 40% in those who received the cotreatment of 0.1-mg/kg DEX and 0.5-mg/kg AZE (from 100.0 ± 5.3% to 59.4 ± 8.6%); while no significant differences were observed in animals who received the treatments alone with respect to vehicle ( Figure 4H), supporting the qualitative changes described above.

| AZE and DEX cotreatment reduces the expression of asthma-inflammatory genes
One key feature of this model is the development of an antigen-specific Th2 immune response. Th2 cells are characterized

| D ISCUSS I ON
We The efficacy of antihistamines for the treatment of asthma has been intensively studied over the last 50 years. Since mast cells were identified as key players in allergic asthma development, first and second generation of antihistamines were evaluated. Due to its proinflammatory effects histamine has been related to asthma development and abundant preclinical observations support this relationship. 12 However, antihistamines have failed to be clinically effective for the management of asthma due to limited efficacy observed in several studies. 13 We have previously reported a crosstalk mechanism between H 1 receptor and GR-mediated signaling pathways. This mechanism involves a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory its use as part of a treatment is limited by the existence of important adverse effects, such as osteoporosis, dyslipidaemias, body fat redistribution, muscular weakness and atrophy, insulin resistance, glucose intolerance and even diabetes. 46 Given that anti-inflammatory and adverse effects share the same molecular mechanisms and, as we described, antihistamines could enhance DEX-induced GR activity both for gene transactivation and transrepression, it should be critical to address the potential modulation of GR adverse effects by antihistamines in order to assure their complete safety. Induction of adverse effects by long-term use of corticoids at high doses might be reduced by diminishing corticoid dosage. However, corticoids' adverse effects could be also enhanced by antihistamines.
There are some limitations to this study that warrant discussion. in the decision to submit the article for publication.

D I SCLOS U R E
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.