Pilot Randomized Controlled Trial Evaluating the Effect of Hypertonic Saline With and Without Hyaluronic Acid in Reducing Inflammation in Cystic Fibrosis

Introduction

Lung disease is the most important cause of morbidity and mortality in cystic fibrosis (CF) patients and is the end result of defective activity of the CF Transmembrane Conductance Regulator (CFTR) protein, serving as a chloride channel in the epithelium of airways, and consequent hyperactivation of the epithelial sodium channel. Chloride hyposecretion and sodium hyperabsorption eventually bring liquid depletion, periciliary liquid collapse, and mucus dehydration to the surface of airways, causing formation of mucus plugs and reducing mucociliary clearance.⁽¹⁾ This leads to progressive bronchial obstruction that undermines inhaled pathogen elimination and triggers a vicious cycle of chronic infection and inflammation, the last characterized by neutrophil influx in the airways and high levels of cytokines/chemokines.⁽²⁾

The role of epithelial cells, macrophages, and other immune cells in the pathogenesis of CF lung disease has been revealed in recent years. However, these cell-mediated immune responses may not remove pathogens or are diverted and/or disabled. For example, neutrophils are activated and give off high amounts of elastase when they enter the airways attracted by high levels of chemotactic factors. Increased neutrophil elastase levels are considered a biomarker of disease progression, predicting the decline of lung function and bronchiectasis.⁽²⁾ The dysfunction in the inflammatory and immune responses leads to secretion of growth factors, including vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), which are believed to contribute to the remodelling of CF airways.⁽³⁾

Current therapy for CF targets the restoration of the proper intraluminal hydration of the airways.⁽⁴⁾ Thus, regular inhalation of hypertonic saline (HS) improves lung function within 2 weeks,^(5,6) and its beneficial effects are maintained as long as inhalation is continued, at least for 48 weeks.⁽⁷⁾ For patients with CF older than 6 years of age, the Cystic Fibrosis Foundation recommends the chronic use of inhaled HS to improve lung function and to reduce exacerbations.⁽⁸⁾ However, a European consensus document concluded that, based on large individual differences in response to dornase alfa and hypertonic saline found in a comparative study,⁽⁹⁾ long-term prescription patients should firstly be tested on an individual basis.⁽¹⁰⁾ Moreover, tolerability of HS treatment is still an issue, since a meta-analysis of studies examining regular HS inhalation for patients with CF indicated intolerance due to cough or other respiratory symptoms only in 3% and 8% of patients, respectively.⁽¹¹⁾

The potential usefulness of hyaluronic acid (HA), a glycosaminoglycan responsible for water content and homeostasis in the extracellular matrix, in improving airway hydration and reducing the effect of neutrophil elastase has been described in vitro and in vivo.^(12–14) In addition, treatment of CF mice with nebulized high molecular weight HA for 3 days was sufficient to reduce lung inflammation.⁽¹⁵⁾ Clinical experience of HS with or without HA in CF so far indicates higher tolerability and pleasantness of the former,^(16,17) documented as perceived acceptability of inhalation, as well as reduction in the need for other medications.^(18,19) So far, no study has been carried out on the effect of HS+HA on the inflammatory response in CF patients' airways.

The aim of the present study is to estimate the treatment effects of HS with or without HA in reducing airway inflammation, as measured by cytokine levels in sputum. Secondary aims are safety outcomes (adverse effects with the treatment), assessment of the prevalence of commonly reported symptoms like cough, throat irritation, and saltiness, and changes in pulmonary function.

Materials and Methods

Results

Demographic characteristics, pulmonary function tests, presence of Pseudomonas aeruginosa respiratory infection, and inflammatory marker levels at baseline are shown in Table 1. Groups were similar with the exception of age, pancreatic sufficiency, and disease severity, as expressed as FEV₁ pred. and FEF_25-75. Patients in HS+HA group were younger, with normal pulmonary function (mean FEV₁ pred. >90%), while 25% of patients in the HS group showed moderate lung disease. Because this was a randomized trial, this difference occurred purely by chance. The flow diagram⁽²³⁾ for this clinical trial is displayed in Figure 1.

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FIG. 1.

Flow chart for randomization procedure.

One subject in each treatment arm did not receive allocated treatment due to scanty hemoptysis after first inhalation. One episode of bronchospasm occurred after first inhalation in the HS arm while one pulmonary exacerbation led to treatment discontinuation in the HS+HA arm (Fig. 1).

ITT analysis for 39 subjects showed no influence of HA+HS on inflammatory markers (Fig. 2) versus HS alone, and this was confirmed by analysis of confidence intervals (Table 2). A within-group analysis showed that, in the HS+HA group, the highest decrement from baseline was observed for IL-1β (−58.8%) followed by VEGF (−49.9%) and IL-8 (−12.9%). Within-group analysis of the HS revealed that only IL-10 median level increased significantly from baseline (+83%; p = 0.0111) (Table 3). In the HS group IL-1β levels did not show any variation and VEGF levels increased.

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FIG. 2.

Box and Whiskers Plot of inflammatory markers (pg/mL) at baseline (before) and at the end of the study (after). The length of the box represents the interquartile range, horizontal line in the box represents the median, end of the whiskers are adjacent values. Outliers, defined as observations more than 1.5 (IQR) beyond the first or third quartile, are plotted individually (circles).

With regard to secondary outcomes, prevalence of unfavorable scores (VAS <6) was 36.8% in HA+HS versus 55% in HS group; no between-group differences were detected in the prevalence of moderate/severe symptoms of cough, saltiness, and throat irritation after 28 days (Table 4).

Pulmonary function showed the same behavior in both groups with the only exception of total airway resistance (SR tot%) (Table 5): no clear influence of HA+HS on pulmonary functions tests over confidence intervals was detected.

Subjects were considered fully adherent to test products inhalation if the number of empty vials returned after 28 days was zero. 50% of patients in the HS arm were fully adherent versus 57.9% in the HS+HA group (p = 0.397) with 2.5 ± 5.6 and 3.1 ± 5.5 vials not administered, respectively.

Discussion

Hypertonic saline has been established as a therapy aimed at restoring airway surface liquid, improving lung function, and reducing the rate of pulmonary exacerbations in patients with CF.^{(5–9,24,25)} Besides its positive effect on mucus rheology,^(26,27) HS has mucolytic, antimicrobial, and anti-inflammatory properties, and has been shown to inhibit growth, motility, and biofilm formation of Pseudomonas aeruginosa, partially explaining its favorable clinical effects.^(28,29) Furthermore, HS was shown to stimulate mucin secretion via a direct effect on secretory cells or via the output of inflammatory mediators by glandular serous cells.⁽³⁰⁾ Nevertheless, two drawbacks of HS use remain to be resolved, namely its effectiveness in the long term and its unpleasantness. A formula containing HS +0.1% hyaluronic acid is also available and used in CF patients, basing use on its greater pleasantness.^(16,17)

HA can prevent elastolysis in airways, decrease chemotaxis by reducing elastin fragmentation, and block elastase secretion by neutrophils and macrophages in humans.⁽¹²⁾ Treatment of CF mice with high molecular weight HA for 3 days was sufficient to reduce lung inflammation, and this effect did not decrease during the 4 subsequent days of treatment, as well as after 24 hours following drug withdrawal.⁽¹⁵⁾ Levels of TNF-α and MIP-2, analogous to human IL-8, mainly responsible for neutrophils recruitment into the mice lungs, were significantly reduced as well as leukocytes lung infiltration.

In this study, we sought to explore the effects of HS+HA on lung inflammation in CF patients. After 28 days of treatment, no clear influence of HS+HA on cytokine levels in sputum as compared to HS alone was observed. In addition, there was no significant change in cytokine levels within the two groups, with the only exception of IL-10, which increased in the HS group. Even removing the 25% of patients with moderate lung disease in the HS group (n = 5), the overall cytokine behavior was not modified (data not shown).

The airway inflammation in CF is directed by many cell types belonging to the innate immune system, including airway epithelial cells and macrophages. Some studies have pointed out that the CF lungs are characterized by an imbalance between pro-inflammatory and anti-inflammatory mediators, such as IL-10, which is produced by alveolar macrophages.⁽³¹⁾ In this study, the most important finding was that sputum IL-10 levels were significantly increased only in the HS arm. Whether VEGF, a cytokine involved in monocyte activation and therefore in chronic inflammation,⁽³²⁾ is determining the increase in IL-10 sputum levels, should be the focus of further research in the HS field.

In a single-dose unblind randomized trial, HS+HA was preferred by CF patients with respect to HS alone, since HS+HA intervention reduced severity of cough, throat irritation, and salty taste, and also increased overall pleasantness.⁽¹⁶⁾ In our study, we did not find a clear preference in favor of HS+HA treatment concerning tolerability, since reduction of unfavorable symptoms in HS+HA group vs. HS alone could not be documented. Our study is methodologically different from the study of Ros and colleagues, in which they reported that the addition of HA was beneficial in CF patients⁽¹⁷⁾ who had already been exposed to HS and had shown poor tolerance. Furnari and colleagues also reported in a prospective, randomized, double-blind study a reduction in the incidence of throat irritation and in the use of bronchodilator after inhalation of HS+HA.⁽¹⁹⁾ Although they did not enroll patients using HS in the 15 days preceding enrolment, these subjects cannot be considered blinded to HS taste.

Although an imbalance in the randomization flow occurred for age and spirometric values, we did not observe any influence of HS+HA over confidence intervals of pulmonary function tests, both static and dynamic, as already reported elsewhere.^(17,19) However, the same conclusions can be drawn after removing the 25% of patients with moderate disease from HS group.

This is the first study to explore the influence of HS with or without HA on inflammatory markers. Concentration of inflammatory cytokines in different body fluids have been used for the early detection of pulmonary exacerbation and to predict the response to antibiotic therapy in CF.⁽³³⁾ Indeed, if we based our calculation on the 24.5% decrement of IL-8 associated to a significant improvement in lung function⁽³⁴⁾ and considered the IL-1β and VEGF sputum level detected in the group of CF patients enrolled in the present study (IL-1β mean 1613.573 with standard deviation 2983.893 pg/mL, VEGF mean 1544.113 with standard deviation 1538.335 pg/mL), we would need more than 300 subjects in each group to be able to detect that 24.5% meaningful reduction of IL-1β and VEGF under HS+HA treatment, with a power (1-β) of 90% and a significance level (α) of 5%.

In conclusion, according to our results HS+HA treatment is safe for CF patients, but we were unable to show any effects that influence lung inflammation and function as compared to HS alone.

Although previous studies showed that HA significantly improves the tolerability and efficacy of HS, we could not confirm this finding. It is worth noting that a one-month treatment with HS did not cause an increase in sputum inflammatory markers from baseline levels.