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. Author manuscript; available in PMC: 2012 May 1.
Published in final edited form as: Respir Med. 2010 Dec 18;105(5):667–673. doi: 10.1016/j.rmed.2010.11.025

Cysteinyl Leukotriene Antagonism Inhibits Bronchoconstriction in Respose to Hypertonic Saline Inhalation in Asthma

Shamsah Kazani 1, Jonathan Sadeh 1, Sreedhar Bunga 1, Michael E Wechsler 1, Elliot Israel 1
PMCID: PMC3080101  NIHMSID: NIHMS256652  PMID: 21169002

Abstract

Background

In asthma, cysteinyl leukotrienes (CysLTs) play varying roles in the bronchomotor response to multiple provocative stimuli. The contribution of CysLTs on the airway's response to hypertonic saline (HS) inhalation in asthma is unknown. Whether polymorphisms in the leukotriene biosynthetic pathway affect the contribution of CysLTs to this response is also unknown.

Methods

In a prospective, randomized, double blind, placebo-controlled cross-over study, mild and moderate asymptomatic asthmatics underwent inhaled 3% HS challenge by doubling the duration of nebulization (0.5, 1, 2, 4, and 8 min) two hours after one dose of montelukast (a CysLT receptor 1 [CysLTR1] antagonist) or placebo, and after three week courses. We examined the effect of the leukotriene C4 synthase (LTC4S) polymorphism (A-444C) on the efficacy of montelukast against HS inhalation in an exploratory manner.

Results

In 37 subjects, two hours after administration of montelukast, the mean provocative dose of HS required to cause a 20% drop in FEV1 (HS-PD20) increased by 59% (9.17 after placebo vs. 14.55 ml after montelukast, p = 0.0154). Three weeks of cysLTR1 antagonism increased the HS-PD20 by 84% (10.97 vs. 20.21 ml, p = 0.0002). Three weeks of CysLTR1 antagonism appeared to produce greater effects on blocking bronchial hyper responsiveness (two hour vs. three week HS-PD20 values 14.55 vs. 20.21 ml respectively, p = 0.0898). We did not observe an effect of the LTC4S polymorphism on the response to CysLTR1 antagonism in this cohort.

Conclusions

A significant proportion of HS-induced bronchoconstriction is mediated by release of leukotrienes as evidenced by substantial acute inhibition with a CysLTR1 antagonist. There was a trend toward greater inhibition of bronchial responsiveness with three weeks of therapy as opposed to acute CysLTR1 antagonism.

Keywords: Asthma; Leukotriene-C4 synthase; Montelukast; Polymorphism, Single Nucleotide; Saline Solution, Hypertonic

Introduction

Asthma is a chronic inflammatory disorder characterized by reversible obstruction of the airways. Bronchial hyper-responsiveness to inhaled stimuli is a characteristic feature of asthma. Direct stimuli such as methacholine lead to bronchoconstriction predominantly by their action on effector cells such as airway smooth muscle. In contrast, indirect stimuli such as exercise, and inhalational challenges with adenosine, cold air, and hyperosmolar aerosols including mannitol and hypertonic saline provoke bronchospasm via the release of mediators from inflammatory cells.1,2 Cysteinyl leukotrienes, prostaglandins, histamine and acetylcholine are some of the mediators implicated in the pathophysiology of bronchoconstriction caused by indirect challenges in asthma.1 Cysteinyl leukotrienes (CysLTs, which include LTC4, LTD4 and LTE4) are arachidonic acid-derived biologically-active mediators of inflammation.3 Upon release by resident and inflammatory airway cells, they activate CysLT receptor 1 (CysLTR1) which leads to initiation and amplification of a variety of patho-physiologic changes observed in asthmatic airways, including eosinophil recruitment, stimulation of mucus secretion, bronchial smooth muscle contraction and airway edema.4 CysLTR1 blockers such as montelukast antagonize the effects of CysLTs at this receptor and have been shown to be efficacious in asthma.5,6 Montelukast is specifically helpful against exercise and allergen induced bronchial hyper-responsiveness.7,8,9 The role of CysLTs and leukotriene modifying drugs including LT receptor antagonists and 5-lipoxygenase inhibitors have been studied extensively in most indirect bronchoprovocative challenges in asthma.2,10-13 Their role in hypertonic saline (HS) induced bronchospasm is suggested by elevated urinary LTE4 after HS inhalation.14 However their quantitative contribution and thus their significance in an airway's bronchomotor response to inhalation of HS is unknown.15

To determine whether CysLTs play a significant role in HS-induced airway narrowing in asthma, we examined the effect of a single dose of montelukast, a CysLTR1 antagonist, in inhibiting the response to HS. Since CysLTs can also mediate a variety of patho-physiologic inflammatory events that occur in asthma including eosinophil recruitment, stimulation of mucus secretion, and airway edema,4 we also examined whether prolonged therapy would amplify the efficacy seen after a single dose.

In addition to potential stimulus-response heterogeneity, we also examined whether genetic heterogeneity might affect the role CysLTs play in mediating airway responses by examining the difference in protection conferred by a CysLTR1 antagonist in a genotype-stratified manner. A single nucleotide polymorphism in the promoter region of a gene which is involved in CysLT synthesis, LTC4 synthase (LTC4S A-444C), has been associated with an increase in CysLT production.16 The dominant variant C polymorphism has also been associated with increased effectiveness of CysLTR1 antagonism against bronchoconstriction in asthma17 and a reduced risk for asthma exacerbations.18 In contrast to polymorphisms in other genes involved in leukotriene synthesis/receptor pathways, this one has been most extensively examined for its effect on clinical outcomes after therapy with leukotriene antagonists. In an exploratory manner, we examined whether this particular polymorphism modulated the efficacy of montelukast against HS-induced bronchospasm. We included this pharmacogenetic component into the clinical trial in a prospective, double-blind placebo-controlled crossover study design, which has not been done in the past.

Methods and Materials

Subjects

The protocol received IRB approval from our institutional review board and written informed consent was obtained from all participants. Subjects had to be between 18 and 65 years old, and diagnosed with mild or moderate asthma by their physicians prior to enrollment in the study. They had to be off all controller medications such as inhaled corticosteroids, long acting beta agonists and leukotriene receptor antagonists for at least four weeks, with FEV1 ≥ 60 % of the predicted value at the baseline visit. Subjects were required to demonstrate hyper-responsiveness to HS with a baseline PD20 ≤ 18 ml to be eligible for analysis. A cut off of 18 ml was set pre hoc to enable us to detect a 100% increase (doubling dose) in the amount of HS tolerated after randomization, as 36.82 ml of HS were inhaled during the last stage of the challenge. Exclusion criteria included smoking history ≥ 10 pack-years or any cigarette use within the last 12 months, respiratory tract infection within the last six weeks, and asthma exacerbation within the last four weeks of study enrollment. We consented 136 subjects who were found to be eligible by these screening criteria.

Study design

We conducted a prospective, randomized, double-blind, placebo-controlled, cross-over study. After obtaining baseline PD20 to HS at the screening visit, eligible subjects were randomized as depicted in figure 1. At visit two, subjects were challenged with HS two hours after receiving one oral dose of blinded drug (montelukast [Singulair®, Merck & Co., Inc, USA] 10 mg dose or matching placebo). Subsequent to a washout period of at least one week, subjects were re-challenged two hours after receiving the alternate study drug at visit three. Subjects returned for a repeat challenge at visit four after a minimum three week course of the same study medication which they received at visit three (montelukast or placebo). They then entered a minimum four week washout phase following which they returned at visit five to repeat a baseline spirometry and receive the alternate study medication. Subjects returned thereafter at visit six after taking this medication for at least three weeks. The order of study medications was determined by random allocation in blocks of two by the investigational pharmacy.

Figure 1. Study design.

Figure 1

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M = montelukast, P = placebo, HS = hypertonic saline challenge

Hypertonic saline challenge

3% HS was administered for inhalation through a DeVilbiss Ultraneb 99 nebulizer via a protocol of doubling duration of exposure from 30 seconds to eight minutes. The result of the inhalation challenge was measured as HS-PD20, i.e. provocative dose of HS in ml required to cause a 20% drop in FEV1. PD20 was calculated by logarithmic interpolation of the amount of saline delivered. When the drop in FEV1 was < 20% at the end of the eight minute inhalation, the PD20 was assigned a value of twice the amount of saline inhaled during the last step, i.e.73.64 ml.

Sample collection and genotype analysis

Genomic DNA was extracted from whole blood by the PureGene DNA Purification system (Qiagen, Valencia, CA). The LTC4S gene promoter polymorphism rs730012 was genotyped using either Sequenom MassARRAY system (Sequenom, San Diego, CA) or Taqman analysis on the Applied Biosystems 7900HT system (Applied Biosystems, Forster City, California, USA) based on assay availability in the laboratory. Quality control was assured by running internal and external controls on all genotyping plates.

Statistical analyses

Sample size was determined a priori to be 14 in each genotype group (A homozygotes and C allele carriers) to have 80% power to detect a doubling dose difference in PD20 after receiving montelukast compared to that after receiving placebo at a 95% significance level. In the manuscript, PD20 data are presented as geometric means with the 95% confidence intervals in brackets. We used Student's t-tests to compare normally distributed data, Wilcoxon rank sum tests for unpaired non-parametric data and Wilcoxon signed rank tests for paired non-parametric data. Categorical data were analyzed using two-sided Fisher's exact tests. Data were analyzed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA) where the significance level is set at 95%.

Results

Subject enrollment

136 mild to moderate asthmatics using only rescue inhaler on an as-needed basis signed informed consent between April 2003 and September 2006 (Figure 2). Ninety one (67%) of these subjects did not qualify to continue the study as they were unresponsive to the 3% HS challenge. Further tests to confirm their diagnosis of asthma were not conducted prior to their exit from the study. Five subjects were excluded prior to analysis because they were unresponsive to HS inhalation at all visits subsequent to screening. Three subjects were unable to complete sufficient study visits to be included in comparative analysis. Of the 37 subjects thus included in data analysis, two dropped out after visit three (one in each genotype group); one due to an asthma exacerbation unrelated to study medications or procedures, and the other due to unknown reasons. Their available data have been included in the results below. Table 1 presents their baseline demographics and genotype-specific data.

Figure 2. Subject enrollment.

Figure 2

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Table 1. Characteristics of study subjects.

All subjects A homozygotes C allele carriers p value*
N 37 17` 19`
Age (yr) 30 ± 10
(18 to 57)		 32 ± 12
(18 – 57)		 29 ± 7
(21 – 44)		 1.00
Sex (% female) 51 65 37 0.18
Race (% Caucasians) 73 53 90 0.03
Ethnicity (% Hispanic) 11 6 16 0.61
FEV1 (L) 3.26 ± 0.83
(1.63 to 4.86)		 3.00 ± 0.78
(1.66 – 4.18)		 3.51 ± 0.84
(1.63 – 4.86)		 0.07
FEV1 (% pred) 87 ± 13
(61 to 115)		 86 ± 15
(61 – 115)		 88 ± 13
(62 – 114)		 0.64
FVC (L) 4.37 ± 1.08
(2.44 to 6.75)		 4.05 ± 1.11
(2.44 – 6.75)		 4.69 ± 1.01
(2.45 – 6.05)		 0.08
FVC (% pred) 98 ± 14
(69 to 134)		 97 ± 16
(69 – 134)		 98 ± 13
(79 – 125)		 0.87
Baseline PD20 to HS inhalation (ml)ˆ 7.45
(5.88 to 9.44)		 7.81
(5.32 to 11.47)		 7.53
(5.44 to 10.42)		 0.87
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Data presented as mean ± SD (range) unless otherwise noted

*

p value is for difference between A homozygotes and C allele carriers

`

Sum not equal to 37 because one subject could not be successfully genotyped. 19 C allele carriers include two homozygotes for the C allele

ˆ

Data presented as geometric mean (95% CI)

Change in lung function after CysLTR1 antagonism

The mean post-drug baseline FEV1 was similar during placebo and montelukast visits, both two hours after one dose (both 86%), and after three week courses (84 vs. 86%, p = 0.48) The post-drug FVC was similar as well (90 vs. 91%, p = 0.61 two hours after one dose; (89 vs. 87%, p = 0.61 after three week courses). There was no change in post-drug FEV1 nor FVC (after both placebo and montelukast and after both one dose and three week courses) when compared with baseline established at visit 1, either.

Change in HS-PD20 after CysLTR1 antagonism

Acute CysLTR1 antagonism (two hours after administration) increased the PD20 by 59% (9.17 [95% CI 6.65 to 12.65] after placebo vs. 14.55 [9.87 to 21.46] ml after montelukast, p = 0.0154) (Figure 3a).

Figure 3.

Figure 3

Figure 3

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Figure 3a Difference in HS-PD20 two hours after a single dose of study medications. Horizontal bars represent geometric means, 9.17 ml (95% CI 6.65 to 12.65 ml) and 14.55 ml (9.87 to 21.46 ml) respectively. N = 37

Figure 3b. Difference in HS-PD20 after three week courses of study medications. Horizontal bars represent geometric means, 10.97 ml (95% CI 7.36 to 16.35 ml) and 20.01 ml (13.15 to 31.06 ml) respectively. N = 35

Three weeks of CysLTR1 antagonism over three weeks increased the PD20 by 89% (10.97 [7.36 to 16.35] after placebo vs. 20.21 [13.15 to 31.06] ml after montelukast, p = 0.0002) (Figure 3b).

There was a trend towards statistical significance in the difference between the mean PD20 after a single dose of montelukast versus that after a three week course (14.55 [9.87 to 21.46] ml vs. 20.21 [13.15 to 31.06], p = 0.0898). There was no period effect of the order of study medications on outcomes.

Genotype-specific response

Seventeen subjects were homozygous for the A allele while 19 carried at least one copy of the C allele. Since only two of the 19 C allele carriers were homozygous for the C allele, we combined the AC and CC genotypes into one group labeled as the “variant C allele carriers”, similar to how this rare genotype has been handled in previous studies.18,19 A blood sample from a 28 year old Caucasian female subject failed DNA extraction and thus her polymorphism status could not be determined. We excluded her from the genotype stratified data. There was no difference in baseline PD20 values between the two genotype groups (Table 1).

Genotype status did not affect lack of response of baseline FEV1 to CysLTR1 antagonism either at two hours or at three weeks. Two hours after montelukast, HS-PD20 increased by 64% (p = 0.1928 compared with placebo) in the A homozygotes, and by 57% in the C allele carriers (p = 0.0494 compared with placebo, p = 0.6094 for the difference between the genotypes). After three weeks of montelukast, HS-PD20 increased by 84% in the A homozygotes (p = 0.0134 compared with placebo), and by 72% in the C allele carriers (p = 0.0250 compared with placebo, p = 0.5580 for the difference between the genotypes).

Discussion

In this prospective, randomized, double-blind placebo-controlled cross-over study, we found indirect evidence that CysLTs mediate a significant proportion of the bronchoconstrictor response in asthmatics after inhalation of hypertonic saline. Further we found that three weeks CysLTR1 antagonist therapy provides additional benefit in terms of protection against this provocative stimulus.

Similar to exercise and cold air challenges, the presence of a hyperosmolar environment stimulates inflammatory cells in the airways such as epithelial cells, eosinophils, basophils, mast cells and sensory nerve cells.1,20,21 Upon activation, these cells release a variety of mediators including histamine and acetyl-choline.21-24 CysLTs are unique arachidonic acid derived pro-inflammatory mediators which are purported to be released from resident airway and inflammatory cells upon exposure of the bronchial epithelium to hyperosmolar stimuli. Mai et al. demonstrated this by measuring elevated levels of urinary LTE4 levels after HS inhalation challenges.14 However, the relative contribution of CysLTs to the patho-physiology of the resultant bronchospasm was unknown. The 59% change that we found in HS-PD20 after one dose of a CysLTR1 antagonist indicates both the qualitative and the relative quantitative role that CysLTs play in HS-induced bronchospasm. This degree of bronchoprotection offered by montelukast against HS-induced bronchospasm is similar to that which it offers against exercise-induced bronchospasm. Leff et al. demonstrated that twelve weeks of therapy with montelukast led to a 47.4 % improvement in the AUC (area under the curve) of change in FEV1 as compared with placebo (p=0.002).10 Similar degree of protection from montelukast was observed in early response to allergen challenges (53% improvement in post-challenge FEV1).25 In contrast, montelukast does not effect the airway sensitivity to mannitol, but does improve recovery from the challenge.26

The increased effect of CysLTR1 antagonism that we noted after three weeks of administration is of interest. Since montelukast has very high affinity for the CysLTR1,27 it is unlikely that further receptor blockade occurs after multiple doses. It is possible that the increased response after three weeks is related to anti-inflammatory effects of long term CysLTR1 antagonism.28 Prior reports have supported this as well. Seven day treatment with zafirlukast (another CysLTR1 antagonist) was found to be associated with reduced number of lymphocytes and basophils and indices of inflammation in BAL fluid after an antigen challenge.29 Montelukast therapy for four weeks has been shown to reduce sputum and blood eosinophils in addition to improving asthma control,30 while daily therapy with pranlukast (another CysLTR1 antagonist) therapy was associated with significant decreases in mast cells, eosinophils and T lymphocytes in endobronchial biopsy specimens.31

Variability in the prevalence of hyper-responsiveness to hypertonic saline is attributed to the differences in severity of asthma in the subjects studied, in the concentration of HS used (3 or 4.5%) and the cut-off for the drop in FEV1 required to define a positive challenge (15 or 20). We suspect that our relatively high rate of screen failures may have been due to the relatively dilute hypertonic saline (3%) which we used. However, Leuppi et al. found that only 234 out of 604 asthmatics (99% with mild disease) i.e. 39% achieved a PD15 to 4.5% hypertonic saline challenge.32 54.8% of children with a diagnosis of asthma did not demonstrate the prevalence of hyper-responsiveness to 4.5% saline when studied by Riedler et al.33 Thus, our data are comparable to prior reports.

There are several reasons to believe that the LTC4S promoter polymorphism might identify a subgroup of asthmatics who would preferentially respond to CysLTR1 antagonists. The variant C allele is associated with enhanced gene transcription and increased cellular capacity for CysLT synthesis in a dominant manner.16,34 Sampson et al. demonstrated significantly increased LTC4 synthesis in stimulated eosinophils from C allele carriers.17 Further, in a study in 23 patients with severe asthma, there was a trend toward greater response to 2 weeks of therapy with zafirlukast in C allele carriers as compared to A homozygotes. The C allele has also been associated with decreased risk of asthma exacerbations18 - the authors observed an 80% reduction in risk of asthma exacerbation in C allele variants of the LTC4S polymorphism when compared with the A homozygotes (p < 0.001) after six months of montelukast therapy. We were unable to demonstrate a difference in the acute or three week response to montelukast between the genotypes. It is likely that our study was underpowered to detect a difference. Another explanation for this observation is that the secondary effects of prolonged CysLTR1 antagonism of decrease in the airway inflammatory milieu referred to earlier28-31 overwhelm the difference in genotype-specific CysLT release between the two groups.

We did not find an effect of a single dose or a three week course of montelukast on baseline lung function in the cohort as a whole or in the genotype-stratified analysis. This contrasts with an open label trial of 4 week therapy with the CysLTR1 antagonist pranlukast in moderate asthmatics with a baseline FEV1 of 65% where a 14.3% increase in FEV1 in C allele carriers occurred compared to a 3.1% increase in A homozygotes (p<0.01).19 The lack of improvement in baseline lung function in our subjects may have been due to the fact that we studied asymptomatic asthmatics with an average baseline FEV1 of 87% (range 61 - 115%) of predicted and thus little improvement in FEV1 would have been expected.

To summarize, we found indirect evidence that cysteinyl leukotrienes mediate a significant portion of the acute airway narrowing that occurs in response to hyperosmolar stimuli. The degree of protection offered by a CysLTR1 antagonist in this setting is similar to that seen in response to exercise and allergen-induced bronchoconstriction. The bronchoprotection increases with three weeks of CysLTR1 antagonist therapy, possibly secondary to additional anti-inflammatory or disease-modifying effects that occur with prolonged CysLTR1 antagonism.

Acknowledgments

We thank the Channing laboratory at the Brigham and Women's Hospital, Boston MA for genotyping services.

Abbreviations

CysLT

Cysteinyl leukotriene

CysLTR1

Cysteinyl leukotriene receptor 1

FEV1

Forced expiratory volume in one second

HS

Hypertonic saline

LTC4S

Leukotriene C4 synthase

PD20

Provocative dose of hypertonic saline required to decrease FEV1 by 20%

Footnotes

Clinicaltrials.gov registration number NCT00116324

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