Nasal immune gene expression in response to azelastine and fluticasone propionate combination or monotherapy

1 INTRODUCTION

Allergic rhinitis (AR) is associated with significant global medical and economic burden.1-3 Intranasal antihistamines and corticosteroids are first-line treatments for AR management. A combination spray is available for moderate-to-severe AR sufferers who require multiple therapies to achieve symptomatic relief.4 Clinical trials comparing each active agent (antihistamine or corticosteroid) versus the combination therapy, have demonstrated that the combination spray was more effective than either monotherapy at reducing AR symptoms.5-9 As antihistamines and corticosteroids have distinct mechanisms of action, potential additive, or synergistic effects may contribute to the enhanced symptomatic relief observed. Experimental studies examining the mechanism of action behind these enhanced effects are limited. The purpose of this study was to investigate the potential mechanisms through which antihistamine and corticosteroid nasal sprays provide relief from AR symptoms and to determine if combining an antihistamine and corticosteroid provides any synergistic effects on gene expression profiles in the nasal mucosa and blood samples.

2 METHODS 2.1 Study design

This study was a randomized, double-blind, three-armed parallel-group study where combination therapy was compared against single active ingredients as control groups. Clinical assessments were conducted at the Queensland Allergy Services Clinic (Gold Coast, Australia) and the Clinical Trial Unit at Griffith University (Gold Coast, Australia) from November 2016 to May 2018. Participants attended a screening visit (Day 14) for evaluation of allergen sensitivities and provision of blood samples. Following screening, eligible participants were instructed to complete a 14-day washout period and cease use of all intranasal and immune modulating medications. Participants were advised to use the following as rescue medication in the event of a considerable symptomatic episode: (1) nasal irrigation with saline solution, (2) oral decongestants, and (3) oral antihistamines. Participants were requested not to take any allergy medications in 48 h before the screening (Day 14) and baseline (Day 0).

At baseline, participants provided nasal lavage/brush and blood samples for gene expression analysis. Participants were randomized using a block randomization method stratified by allergen sensitivity (dust mite only or dust mite and grass allergy) and sex to one of three treatment groups (1) Azep® nasal spray [Mylan Health Pty Ltd], azelastine (AZE) 125 µg/spray, (2) Flixonase® nasal spray [Mylan Health Pty Ltd], fluticasone propionate (FP) 50 µg/spray, or (3) Dymista® nasal spray [Mylan Health Pty Ltd], 125 µg of azelastine, and 50 µg fluticasone propionate/spray (AZE/FP). Participants were instructed to administer the allocated nasal spray (provided in a sealed envelope to maintain blinding) 1 spray per nostril, twice daily, for 7 days. Participants were discouraged from using any allergy medications other than the study medication during the treatment period. Following the treatment period, participants returned for the final visit (Day 7) for the provision of nasal lavage/brushing and blood samples. Participant compliance was assessed based on a self-report of the number of doses missed and was also estimated by measuring the amount of study medication remaining (by weight) relative to the amount (weight) before dispensing.

This study was approved by the Griffith University Human Research Ethics Committee (Ref:2016/279) and was registered with the Australian and New Zealand Clinical Trial Registry (ACTRN12616001439437) before commencement. All participants provided written and informed consent before participation.

2.2 Participant selection: Inclusion and exclusion criteria

Men and women, 18–65 years of age, with a more than 2-year history of moderate-severe AR (as defined by the Allergic Rhinitis and Its Impact on Asthma (ARIA) guidelines)10 were recruited to the study. Participants were also required to have a Total Nasal Symptom Score (TNSS)11 of at least six, a score of at least 50 mm on a Visual Analog Scale (VAS) for overall symptom severity12 in the previous 24 h, and a positive allergic response to dust mites determined with a skin prick test and/or serum specific IgE radioallergosorbent test (RAST) (QML Pathology, Murarrie, Queensland, Australia) to Dermatophagoides pteronyssinus or D. farinae.

Individuals suffering from nonallergic rhinitis; who consumed probiotics in the previous 12 weeks, used oral corticosteroids within the previous 6 months or antibiotics within the previous 30 days; used anti-inflammatory/immune-modulating medications; had the existing respiratory disease (including asthma, nasal polyposis, or chronic obstructive pulmonary disorder); had existing immune dysfunction (other than allergies); had recent nasal surgery/trauma that could affect sampling; were ill at time of enrollment; reported hepatic impairment or excessive alcohol consumption13; had known hypersensitivity to steroids or antihistamines; or were pregnant at the time of enrolment were excluded from participating.

2.3 Symptom assessment

Participants completed the mini Rhinoconjunctivitis Quality of Life Questionnaire (mRQLQ)14 at the beginning and end of the intervention and maintained a symptom and medication diary (SMD) daily for the duration of the study. The SMD consisted of three symptom questionnaires: TNSS, total ocular symptom score (TOSS)15 and Other Allergic Rhinitis Symptom Score (OARSS), in addition to a VAS for overall symptom severity. The use of allergy and non-allergy-related medications were also recorded in the diary.

2.4 Sample collection and laboratory analysis

A screening blood sample was collected and the following tests conducted: full blood count, white cell differential, erythrocyte sedimentation rate (ESR), and specific IgE to dust mites (D. pteronyssinus and D. farinae.) and grass pollen mix (Bermuda, Timothy, Meadow, Johnson, Rye and Paspalum) (QML Pathology). For those individuals who met the inclusion criteria nasal washing and brushing, and blood samples were collected at Day 0 and Day 7 visits as described previously.16 Briefly nasal samples included both washing with 100 ml of phosphate-buffered saline and brushing of the mucosa between the nasal septum and inferior turbinate of each nostril. Recovered washing and brushing material was combined and cellular material concentrated by centrifugation. The cell pellet was directly lyzed using a commercially available lysis buffer (RLT; Qiagen) and the lysate was stored frozen until gene expression analysis. For whole blood samples, RNA was extracted from PAXgene tubes using a Maxwell® RSC automated RNA extraction instrument using the commercially available Maxwell® RSC miRNA Tissue Kit (Promega Corporation).

2.5 Gene expression analysis

Immune gene expression analysis of nasal cell lysate and extracted RNA from blood was performed using a commercially available NanoString nCounter PanCancer Immune Profiling panel (NanoString Technologies). This panel contained 40 references (housekeeping) genes and 730 immune genes and was used in combination with the nCounter panel plus probe set which contained an additional 30 immune genes relating to the allergic response and mechanism of action of steroids and antihistamines (760 immune genes in total). Gene expression data underwent imaging quality control and normalization checks before analysis and interpretation of data. Genes that were expressed at counts below 20 in 80% or more samples were excluded from further analysis. Reference (housekeeping) normalization was performed using the GeNorm Algorithm where 20 out of 40 housekeeping genes were used for the nasal lysate samples and 33/40 housekeeping genes were used for the peripheral blood samples.

2.6 Statistical analysis

Based on a standard deviation of gene expression intensity of 0.6, an α of 0.001, and at least two-fold difference in gene expression, a sample size of 16 patients per group was estimated to achieve 95% power. Differences in demographic and clinical measures between groups were assessed with a one-way analysis of variance and a χ2 test for categorical variables. Variables were log-transformed where appropriate to approximate a normal distribution. Change (pre-post) in symptom severity questionnaires was measured with a paired t-test. Differences in absolute change in symptom severity questionnaires between groups were measured with an analysis of covariance with baseline score as the covariate. Differentially expressed genes were identified using R package Limma,17 where moderated t-tests were performed to compare the gene expression levels between groups. The significantly differentially expressed genes (p < .05) in each treatment group were assessed for enrichment into Reactome pathways. Statistical significance of all clinical measures differentially expressed genes and pathway enrichment was accepted at p < .05.

3 RESULTS 3.1 Study cohort

Forty-eight participants were randomized to the study. Two participants did not complete the treatment period and were withdrawn (Figure 1). Reasons for withdrawal included the development of the ear infection in one participant and an unexpected adverse event considered unrelated to the intervention in a second participant that prevented compliance with the study protocol. The demographic and baseline characteristics of the study groups based on a per-protocol analysis are given in Table 1. The groups were matched with the exception of blood eosinophil counts which were significantly different between the FP and AZE groups.

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Consort diagram depicting flow and retention of study participants FP, fluticasone propionate “Flixonase®” group; AZE/FP, azelastine/fluticasone propionate “Dymista®” group; and AZE, azelastine “Azep®” group

Table 1. Baseline demographic and clinical measures for the per-protocol population FP AZE/FP AZE p value n 14 16 16 – Age (years) 37.63 ± 14.60 39.42 ± 10.03 37.26 ± 15.08 .889 Sex F/M (% female) 10/4 (71%) 10/6 (63%) 11/5 (69%) .864 Height (cm) 169.11 ± 9.35 171.03 ± 10.26 171.94 ± 8.63 .710 Weight (kg) 76.69 ± 15.72 72.16 ± 15.33 73.46 ± 13.20 .694 BMI (kg/m2) 26.65 ± 3.95 24.45 ± 3.37 24.78 ± 3.64 .226 Ethnicity (% Caucasian) 78.60% 68.80% 87.50% .437 Immune measures (Day-14) White cell count (×109/L) 7.18 ± 1.70 6.98 ± 2.09 5.90 ± 1.50 .112 Lymphocytes (×109/L) 2.32 ± 0.80 2.26 ± 0.73 1.98 ± 0.63 .366 Eosinophils (×109/L) 0.53 ± 0.37 0.44 ± 0.30 0.26 ± 0.16 .038 Neutrophils (x109/L) 3.74 ± 1.22 3.68 ± 1.33 3.18 ± 1.00 .366 Basophils (×109/L) 0.06 ± 0.05 0.07 ± 0.03 0.04 ± 0.03 .085 ESR (mm/h) 14.50 ± 13.78 7.94 ± 6.17 7.94 ± 8.27 .267 Allergen sensitivity (Day-14) Co-allergy to dust mites and pollen (%) 50% 62.50% 62.50% .731 IgE Dermatophagoides pteronyssinus (kU/L) 39.28 ± 40.39 17.12 ± 21.60 11.37 ± 20.34 .088 IgE D. farinae (kU/L) 35.11 ± 39.85 13.41 ± 18.31 8.56 ± 17.31 .093 IgE grass pollen mix (kU/L) 2.13 ± 4.29 10.44 ± 25.84 6.20 ± 21.29 .526 IgG4 D. pteronyssinus (kU/L) 0.50 + 0.54 0.47 ± 0.42 0.38 ± 0.43 .772 IgG4 D. farinae (kU/L) 0.42 ± 0.42 0.39 ± 0.26 0.30 ± 0.38 .617 IgG4 grass pollen mix (kU/L) 0.67 ± 0.35 1.07 ± 0.97 0.80 ± 0.66 .469 Symptom severity (Day 0) Total Nasal Symptom Score (0–12 U) 5.93 ± 3.95 4.00 ± 1.86 7.06 ± 3.64 .299 Total Ocular Symptom Score (0–9 U) 3.57 ± 2.44 2.00 ± 2.00 3.19 ± 2.74 .182 mRQLQ Score (0–6 U) 2.90 ± 1.25 2.66 ± 0.79 3.00 ± 1.12 .653 Other allergic rhinitis symptoms (0–12 U) 4.51 ± 3.89 3.07 ± 2.16 5.21 ± 3.47 .865 Visual Analog Scale (0–100 mm) 54.18 ± 33.51 48.16 ± 22.31 60.50 ± 30.10 .485 Medication Usage (Day 14–Day 0) Allergy medication use; % of total diary responses (washout period) 0.37 ± 0.29 0.25 ± 0.20 0.40 ± 0.31 .237 Abbreviations: AZE, azelastine “Azep®” group; AZE/FP, azelastine/fluticasone propionate “Dymista ®” group; FP, fluticasone propionate “Flixonase ®” Group; mRQLQ, miniRhinoconjunctivitis quality of life.

Treatments were well tolerated by participants. Adverse events were mild, with minimal impact on daily activities, were consistent with previously reported findings7, 18 and are presented in Table S1. Self-reported compliance was 96% ± 6% of total scheduled doses for the FP group, 96% ± 6% for the AZE/FP group, and 99% ± 3% for the AZE group. All participants administered ≥86%. Compliance was verified from weights of returned medications and ranged from 81% (AZE group) to 93% (AZE/FP group) missing less than two doses.

Eight of the participants reported needing to follow the rescue medication strategy during the 7-day intervention period. For five of these participants (two from the FP group, one from the AZE/FP group, and two from the AZE group) this included use of nasal irrigation only. One participant (FP group) reported use of a nasal decongestant, one participant (FP group) reported use of oral antihistamines, and one participant (AZE group) reported use of eye drops.

3.2 Symptom assessment

All symptom severity measures were significantly improved following treatment in all groups (Table S2). mRQLQ improvement was significantly greater in AZE/FP compared with AZE (p = .014) (Figure 2). Both FP (p = .013) and AZE/FP (p = .016) treatments had a significantly greater effect on TNSS improvement when compared to AZE. Improvement in OARSS was significantly greater in FP (p = .029) and AZE/FP (p = .044) when compared with the AZE. Reduction in overall symptom severity (based on VAS) was greater in the AZE/FP (p = .022) and FP (p = .040) when compared with AZE.

image

Clinical response to treatment. Dots and lines represent change in symptom scores for each participant from pre-nasal spray application (Day 0) to post-nasal spray application (Day 7). The data are shown in the unadjusted values. Asterisk (*) indicates significance at p < .05 following nasal spray application. Hash (#) indicates raw change in symptoms scores (pre-post) was significant between groups (ANCOVA with baseline score as the covariate). ANCOVA, analysis of covariance; AZE, azelastine “Azep®” group; AZE/FP, azelastine/fluticasone propionate “Dymista®” group; FP, fluticasone propionate “Flixonase®” group

3.3 Differentially expressed genes

Demographic and clinical characteristics of the subjects who had samples that met the quality control criteria for gene expression studies were not different from the per-protocol population.

3.3.1 Nasal mucosa

Nasal lysate samples from 13 FP, 11 AZE/FP, and 11 AZE participants were available for analysis (Table S3). A total of 588 genes included in the NanoString nCounter panel were expressed above background. FP had a strong downregulatory effect on gene expression, while AZE/FP and AZE had a mostly upregulatory effect on immune gene expression (Figure 3). The top 10 differentially expressed genes for all treatment groups are shown in Table 2.

image

Heat map of Log2 expression all genes in the nasal mucosa samples included in the analysis (n = 588) by treatment group. Each row represents a gene and each column represents a sample. The Log2 gene expression counts are represented on a Z scale whereby blue indicates low expression (downregulation) and yellow indicates high expression (upregulation). AZE, azelastine “Azep®” group; AZE/FP, azelastine/fluticasone propionate “Dymista®'”group; FP, fluticasone propionate “Flixonase®” group

Table 2. Top 10 differentially expressed genes in nasal lysate and blood samples for the three treatment groups Gene Log2 fold change Linear fold change Lower confidence limit (log2) Upper confidence limit (log2) p value p adjust FP Group Nasal mucosa samples AMICA1 −2.46 0.18 −3.10 −1.83 3.16 × 10−7 1.86 × 10−4 GZMB −2.54 0.17 −3.30 −1.78 2.37 × 10−6 6.96 × 10−4 LTB −2.41 0.19 −3.20 −1.62 6.83 × 10−6 1.03 × 10−3 FCER1A −2.55 0.17 −3.38 −1.71 7.03 × 10−6 1.03 × 10−3 SOCS1 −2.20 0.22 −3.01 −1.40 2.49 × 10−5 2.92 × 10−3 PTGDR2 −2.29 0.20 −3.20 −1.38 6.19 × 10−5 5.43 × 10−3 IL1RL1 −2.36 0.19 −3.33 −1.40 8.39 × 10−5 5.43 × 10−3 HLA-DRA −1.75 0.30 −2.47 −1.03 9.22 × 10−5 5.43 × 10−3 CXCR3 −2.04 0.24 −2.89 −1.19 1.02 × 10−4 5.43 × 10−3 CD1C −1.62 0.33 −2.30 −0.94 1.07 × 10−4 5.43 × 10−3 Peripheral blood samples IL3RA −0.47 0.72 −0.65 −0.29 4.95 × 10−5 2.40 × 10−2 IL1B 0.33 1.26 0.15 0.51 1.20 × 10−3 2.91 × 10−1 MS4A2 −0.38 0.77 −0.62 −0.14 4.45 × 10−3 5.14 × 10−1 IL5RA −0.33 0.79 −0.55 −0.12 5.08 × 10−3 5.14 × 10−1 NFKB1 0.22 1.16 0.07 0.37 6.29 × 10−3 5.14 × 10−1 RNASE3 −0.47 0.72 −0.79 −0.15 7.12 × 10−3 5.14 × 10−1 HRH4 −0.29 0.82 −0.49 −0.09 7.43 × 10−3 5.14 × 10−1 CD24 −0.45 0.73 −0.77 −0.13 9.39 × 10−3 5.61 × 10−1 SLPI 0.30 1.23 0.08 0.52 1.04 × 10−2 5.61 × 10−1 NFKB2 0.20 1.15 0.05 0.34 1.22 × 10−2 5.91 × 10−1 AZE/FP group Nasal mucosa samples TNFSF10 −0.92 0.53 −1.48 −0.36 3.58 × 10−3 9.85 × 10−1 NOS2A −1.53 0.35 −2.57 −0.49 7.43 × 10−3 9.85 × 10−1 PPBP 1.52 2.87 0.48 2.57 7.66 × 10−3 9.85 × 10−1 ABCB1 1.23 2.35 0.32 2.14 1.17 × 10−2 9.85 × 10−1 KIT −0.98 0.51 −1.76 −0.21 1.66 × 10−2 9.85 × 10−1 IFNA8 1.40 2.64 0.25 2.55 2.10 × 10−2 9.85 × 10−1 IL33 −1.07 0.47 −1.97 −0.18 2.25 × 10−2 9.85 × 10−1 CD274 −0.98 0.51 −1.82 −0.14 2.62 × 10−2 9.85 × 10−1 TNFSF13 −0.83 0.56 −1.55 −0.11 2.65 × 10−2 9.85 × 10−1 TPSAB1 −1.12 0.46 −2.17 −0.07 3.81 × 10−2 9.85 × 10−1 Peripheral blood samples CXCR6 −0.26 0.84 −0.43 −0.08 7.35 × 10−3 9.43 × 10−1 CLEC4C −0.25 0.84 −0.44 −0.06 1.12 × 10−2 9.43 × 10−1 LY9 0.17 1.12 0.04 0.30

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