INTRODUCTION

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Histamine and leukotrienes are potent mediators of bronchoconstriction and exert their effects through histamine and CysLT₁ receptors located on bronchial smooth muscle. Although the mast cell is likely to be the primary source of these mediators, other inflammatory cells such as eosinophils contain leukotrienes. Both histamine and the leukotrienes are involved in the airway response to exercise, as their specific receptor antagonists are effective in reducing, but not abolishing, exercise-induced asthma (EIA) (1). Histamine is a preformed mediator and is rapidly metabolized. It is likely the major contributor to the maximum fall observed after exercise (2). Leukotrienes probably act to sustain the airway response to exercise because rapid recovery to baseline lung function occurs after exercise in the presence of a leukotriene antagonist (3, 5, 6). EIA is thought to occur as a result of evaporative water loss increasing the osmolarity of the airway surface liquid and causing inflammatory cells to release their mediators (8, 9).

Mannitol, given as a dry powder for inhalation, is a new bronchial provocation test (10) and is also thought to increase the osmolarity of the airway surface (11). In vitro, mannitol causes a rapid release of histamine from human lung mast cells, with the maximum release occurring at two to three times physiological osmolarity (12). Subjects with asthma responsive to exercise and hypertonic saline are also responsive to inhaled mannitol (10, 11). As with exercise (13), the response to mannitol is markedly inhibited or abolished by premedication with nedocromil sodium (14). These findings support the suggestion that mannitol and EIA share a common mechanism of airway narrowing and, although there is evidence implicating histamine and leukotrienes in the airway response to exercise, their role in response to mannitol is unknown.

To investigate whether histamine and leukotrienes are involved in the airway response to inhaled mannitol, we assessed the effect of the specific receptor antagonists fexofenadine hydrochloride and montelukast sodium on the airway sensitivity to mannitol in subjects with clinically diagnosed asthma. We also wanted to know whether the rapid airway recovery after exercise, in the presence of a leukotriene antagonist, also occurs after administration of mannitol. To do this we measured recovery to baseline lung function after mannitol challenge. Two separate studies were performed. In one, fexofenadine hydrochloride (180 mg) or its matched placebo was administered and in the other, montelukast (10 mg) or its matched placebo was administered. Both the active agent and placebo were administered randomly, double blind before challenge with inhaled mannitol.

	METHODS

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Advertisements circulated in the local community and the Pulmonary Function Laboratory (Royal Prince Alfred Hospital, Camperdown, Australia) were used to recruit subjects with asthma. After an interview by telephone or in person, subjects were asked to attend the laboratory if they were nonsmokers and had no chest infection in the 4 wk preceding the study. Subjects were asked to refrain from taking short-acting ₂-adrenoceptor agonists for 6 h and nedocromil sodium or sodium cromoglycate for 36 h before the study. Subjects were not permitted to take either long-acting ₂-adrenoceptor agonists or antihistamines throughout the study period. No inhaled corticosteroids were taken on the day of the study and no vigorous exercise was permitted before testing on the study day. The dose of inhaled corticosteroids remained unchanged for at least 4 wk before the study. Subjects had a brief medical history taken and were examined by a physician to confirm a clinical diagnosis of asthma.

The Central Sydney Area Health Service Ethics Committee approved the studies (Protocol Nos. X99-0090 and X99-0091) and all subjects signed a consent form before commencement of the study. The studies were performed under the Clinical Trials Notification Scheme (No.1999/149) of the Therapeutic Goods Administration of Australia.

Study Design

Subjects attended the laboratory on three separate occasions at approximately the same time of day. The first day served as a control day for familiarization and a mannitol challenge was performed to assess airway responsiveness. To enter the study, the provoking dose of mannitol required to cause a 15% reduction in FEV₁ (PD₁₅) was required to be less than 290 mg. The second and third days consisted of pretreatment with the administration of the active drug or its placebo followed by a mannitol challenge.

Fexofenadine Hydrochloride

Fexofenadine HCl or a matched placebo (Hoechst Marion Roussel, Lane Cove, NSW, Australia) was administered before challenge testing. If the studies were conducted in the morning, subjects were instructed to take the standard dose of one capsule (180 mg) in the evening before bed and one 1.5-2 h before the scheduled mannitol challenge. If the studies were conducted in the afternoon subjects were required to take one capsule on waking and the other 1.5-2 h before the scheduled mannitol challenge, to ensure adequate blood concentrations of the drug were reached. A minimum of 48 h separated the control and initial fexofenadine/placebo day and a minimum of 5 d separated the initial and final fexofenadine/placebo day to permit adequate washout of the fexofenadine or placebo. All three challenges were completed by each subject within a 4-wk period (7-28 d).

Montelukast Sodium

Montelukast sodium or its placebo (Merck and Co., Whitehouse Station, NJ) was administered before challenge testing according to a dosing schedule different from that recommended for clinical use. Subjects were instructed to take the standard dose of one tablet (10 mg) according to the standard schedule (i.e., before bed) on the two evenings before the study, but they took an additional dose on the morning of the study. There was a minimum of 48 h between the control and first challenge day (montelukast/placebo) and a minimum of 5 d separated the following challenge to ensure adequate washout of montelukast or placebo. All challenges were completed by each subject within a 4-wk period (7-22 d).

Lung Function Measurements

Spirometry was performed with a Microlab 3300 spirometer (Micro Medical, Kent, UK). Forced expiratory volume in 1 s (FEV₁) was used as an index of change in airway caliber. On each day the baseline FEV₁ had to be greater than 65% of predicted for the challenge to proceed (15, 16).

Subjects

For the fexofenadine trial 22 subjects with asthma were recruited. Twenty subjects (14 females and 6 males) aged between 17 and 68 yr met all the inclusion criteria and were entered into and completed the study (Table 1). Eighteen subjects were currently taking asthma medication. Nine were taking inhaled corticosteroids on a daily basis and 18 were taking ₂-adrenoceptor agonists (Table 1). All except one subject (no. 17) had at least one positive skin prick test (3-mm wheal or more) in response to a common aeroallergen.

For the montelukast trial 22 subjects with asthma were recruited. Nineteen subjects (11 females and 8 males) aged between 13 and 68 yr met all the inclusion criteria and were entered into and completed the study (Table 2). Seventeen subjects were currently taking asthma medication, with 11 taking inhaled corticosteroids on a daily basis and 17 taking ₂-adrenoceptor agonists (Table 2). All except one subject (no. 12) had at least one positive skin prick test (3-mm wheal or more) in response to a common aeroallergen.

Mannitol Capsule Challenge

The preparation of the dry powder mannitol has been described in detail previously (10). In brief, mannitol (Mannitol BP; Rhône Poulenc Chemicals, Brookvale, NSW, Australia) was prepared by spray drying (190 mini spray dryer; Buchi, Flawil, Switzerland) a solution of 15 mg/ml. The particle size of the mannitol was measured with a multistage liquid impinger (Astra Pharmaceuticals, Lund, Sweden). The mannitol powder used had 66% of the particles by mass under 7 µm in diameter. A Halermatic (Rhône-Polenc Rorer, Collegeville, PA) was used to deliver the mannitol.

On arrival at the laboratory each day, subjects had their FEV₁ measured in triplicate and this was repeated 10 min later to confirm stability. On the treatment days patients were asked to confirm the times the tablets were taken. After spirometry, a nose clip was applied and subjects then performed the challenge with doses consisting of 0 (empty capsule acting as a placebo), 5, 10, 20, 40, 80, 160, 160, and 160 mg of mannitol via the Halermatic. The 80- and 160-mg doses were given in multiple doses of 40-mg capsules. After inhalation of each capsule, patients were instructed to hold their breath for 5 s. At least two repeatable FEV₁ maneuvers were performed 60 s after each dose and the highest FEV₁ was used in the calculation. The FEV₁ value measured after the 0-mg capsule was taken as the prechallenge FEV₁ and used to calculate the percentage decrease in FEV₁ in response to the mannitol challenge. If the subject had a greater than 10% decrease in FEV₁ in response to a single dose, the same dose was repeated. The challenge ceased when a positive response, that is, a 15% decrease in FEV₁, was documented or a cumulative dose of 635 mg had been administered. The PD₁₅ was calculated by linear interpolation of the relationship between the percent decrease in FEV₁ and the cumulative dose of mannitol required to provoke this decrease. The time of each challenge was recorded with a stopwatch. Timing commenced on administration of the first dose (0-mg capsule) and stopped when the desired airway response was obtained.

For the subjects who enrolled in the montelukast protocol, immediately after the challenge on the control day 200 µg of salbutamol was administered via a metered dose inhaler (Airomir; Hoechst Marion Roussel) actuated into a spacer device (Breath-A-Tech; Scott-Dibben, Newcastle, NSW, Australia).

After the active and placebo challenges patients recovered spontaneously. Recovery from all challenges was monitored by obtaining repeatable measurements of FEV₁ at 5-min intervals for 30 min after completion of the challenge. If the subjects had not achieved their baseline FEV₁ by 30 min after completing the challenge then 200 µg of salbutamol was administered.

Statistical Analysis

The geometric mean (Gmean) and 95% confidence interval (CI) of the PD₁₅ were calculated, using the log-transformed values that were normally distributed. The Student paired t test was used to compare the values between active drug and its placebo (17). The repeatability of the PD₁₅ to mannitol was calculated in 10 subjects who took part in both studies and who thus performed two control day challenges. The repeatability was calculated to be within ±1.0 doubling dose and a median (range) of 15 (13) d separating the two challenge tests (18). Thus, in an individual subject, the drug was considered to have a significant effect if the PD₁₅ was beyond one doubling dose of the PD₁₅ obtained in the presence of placebo.

For the montelukast protocol the area under the percent reduction in FEV₁-versus-time curve over the 30-min recovery period (AUC_{0- 30}) after the mannitol challenges was calculated, using trapezoidal integration. For this analysis values that exceeded the baseline FEV₁ were considered as if the FEV₁ had returned to the baseline value. Tests for significance in recovery of lung function were performed, using the AUC_0-30 values and using an analysis of variance (ANOVA) with repeated measures. Percent protection, using the values for AUC_0-30, was calculated for those subjects who were administered the same total dose of mannitol. FEV₁ values are expressed as the percent predicted normal FEV₁. Unless otherwise stated values are expressed as means ± SD.

	RESULTS

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In the presence of fexofenadine, all subjects except one had decreased airway sensitivity to inhaled mannitol compared with placebo (Figure 1 and Table 1). After fexofenadine administration, the PD₁₅ to mannitol was 138 [95, 201] mg compared with placebo (51 [25, 106] mg) (p < 0.001). There was a significant increase in the PD₁₅ in 13 subjects, with the PD₁₅ in the presence of fexofenadine being greater than one doubling dose compared with the PD₁₅ with placebo. The two doses of fexofenadine were taken and the mean ± SD times before the challenges were 13.3 ± 3.4 and 2.2 ± 0.5 h.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Individual values for the provoking dose of mannitol to cause a 15% fall in FEV1 (PD15) after administration of fexofenadine and placebo to 20 subjects with asthma. Geometric mean PD15 (95% CI) values were 51 (25, 106) mg and 138 (95, 201) mg for placebo and fexofenadine, respectively (p < 0.001).

The total dose of mannitol administered was significantly greater in the presence of fexofenadine (237 ± 161 mg) compared with placebo (126 ± 103 mg) (p < 0.001). There was no difference in the final percent reduction in FEV₁ at the end of the challenge period (fexofenadine, 20.8 ± 5.4%; placebo, 20.1 ± 5.3%) (p = 0.7) with the higher dose of mannitol. However, in the presence of fexofenadine the FEV₁ continued to fall for a further 5 min to 27.8 ± 13.8%.

Because the dose of mannitol given on the fexofenadine and placebo days was different it was not considered valid to analyze the AUC_0-30. However, the time to recover to baseline lung function spontaneously was significantly slower after the larger dose of mannitol given in the presence of fexofenadine compared with placebo (p = 0.0001) (Figure 2). After 30 min the percentage decrease in FEV₁ after fexofenadine was 16 ± 12% compared with placebo, 9 ± 9% (p < 0.01) (n = 19). One subject was not included in the analysis of recovery, as she required a ₂-agonist to assist recovery on the day fexofenadine was administered. The value for baseline FEV₁ was significantly higher in the presence of fexofenadine (92.9 ± 11.0%) compared with placebo (87.7 ± 12.0%) (p = 0.002). There was, however, no relationship between improvement, that is, change in FEV₁ %pred and improvement, that is, change in PD₁₅ (r_p = 0.25) (p = NS).

View larger version (19K): [in this window] [in a new window]   Figure 2.   Percentage reduction from baseline FEV1 (mean ± SEM) and spontaneous recovery in 20 subjects for 30 min after challenge with inhaled mannitol following the administration of 180 mg of fexofenadine (solid circles) or placebo (open circles). Note: There was an increase in the mean (SD) cumulative dose of mannitol administered after fexofenadine pretreatment (237 ± 161 mg) compared with placebo (126 ± 103 mg) (p < 0.001).

Montelukast sodium had no effect on the airway sensitivity to inhaled mannitol. There was no difference in the dose of mannitol required to cause a 15% reduction in FEV₁ in the presence of montelukast or placebo and no subject had a significant improvement in PD₁₅ in the presence of montelukast (p = 0.35) (Figure 3 and Table 3). The three doses of montelukast were taken and the mean ± SD times before the challenges were 4.9 ± 3.1 h and 10.6 ± 2.0 and 23.9 ± 0.7 h.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Individual values for the provoking dose of mannitol to cause a 15% fall in FEV1 (PD15) after administration of montelukast and placebo to 19 subjects with asthma. Geometric mean PD15 (95% CI) were 87 (51, 148) mg and 71 (36, 144) mg for placebo and montelukast, respectively (p = 0.35).

There was no difference in the final percent reduction in FEV₁, the total dose of mannitol administered, or the time of delivery of mannitol (Table 3). The spontaneous recovery of lung function to the baseline value was more rapid in the presence of montelukast, with a significant reduction in AUC_0-30 compared with placebo (p < 0.001) (Figure 4 and Table 3).

View larger version (22K): [in this window] [in a new window]   Figure 4.   Percentage reduction from baseline FEV1 (mean ± SEM) and spontaneous recovery in 19 subjects for 30 min following challenge with inhaled mannitol after the administration of montelukast (solid circles) or placebo (open circles). After the mannitol challenge on the control day, subjects recovered with 200 µg of salbutamol (shaded squares).

Fourteen subjects were administered the same cumulative dose of mannitol on the montelukast and placebo study days. In these subjects the percent protection of the AUC_0-30 with montelukast was (mean [range]) 53% [78 to 91%] and this improvement in AUC_0-30 was observed in all except one subject (no. 1) (Table 2). In the presence of montelukast after 15 min of spontaneous recovery the FEV₁ had returned to baseline values in 10 of the 19 subjects, whereas at that time no subject had returned to baseline in the presence of placebo. By 30 min all subjects had returned to baseline FEV₁ in the presence of montelukast compared with 2 of 19 subjects after placebo.

The recovery to baseline lung function in the presence of montelukast over 30 min was comparable to the recovery on the control day when 200 µg of salbutamol was given to hasten recovery (p = 0.14) (Figure 4). Even after 15 min of recovery, there was no significant difference in percent reduction in FEV₁ after the mannitol challenge in the presence of montelukast (4.6 ± 4.6%) and after recovery with the ₂ adrenoceptor agonist (2.4 ± 4.4%) (p = 0.17).

There was no significant difference between the mean baseline FEV₁ values on the placebo and montelukast study days (Table 3). However, montelukast did appear to have a beneficial effect on resting lung function in subjects 1 and 9, who showed an 18 and 10% improvement in FEV₁, respectively, in the presence of montelukast compared with placebo.

	DISCUSSION

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This study clearly demonstrates that, in subjects with asthma, the histamine (H₁) receptor antagonist is effective in reducing the airway sensitivity, as measured by PD₁₅, to inhaled mannitol. By contrast, montelukast had no effect on airway sensitivity but recovery from challenge with mannitol was significantly faster compared with placebo.

The beneficial effect of fexofenadine appeared to be independent of the small improvement it had on baseline FEV₁. However, the extent of reduction in sensitivity to mannitol, afforded by fexofenadine, could be considered similar to that reported for the inhibition by terfenadine against similar exercise and hypertonic saline (1, 2, 19). This finding suggests that mast cells, which are the major source of histamine in the lung, contribute to airway narrowing after challenge with inhaled mannitol.

The finding that montelukast sodium had no effect on the airway sensitivity to inhaled mannitol, but that spontaneous recovery to baseline lung function was significantly faster, suggests that leukotrienes are important mediators for sustaining the airway response to this osmotic stimulus. This is similar to the findings with montelukast when exercise is the stimulus to airway narrowing (5, 6).

The finding of a reduced sensitivity to mannitol with fexofenadine and not montelukast suggests that histamine, a preformed mediator, is likely to be important in the initial airway response to mannitol and that leukotrienes, which need to be synthesized, are released later and sustain the bronchoconstriction to mannitol after challenge. In a previous study we reported that a single dose of 8 mg of nedocromil sodium, given by inhalation 15 min before challenge, reduces the airway sensitivity to mannitol and enhances recovery from mannitol challenge (14). In light of the findings in this study the concept that nedocromil inhibits the release of mediators is supported.

Leukotriene antagonists are effective at shortening the airway response to exercise (5). Montelukast has been shown to reduce, but not abolish, the maximum percent reduction in FEV₁ after exercise; however, airway recovery to baseline lung function is significantly faster when compared with placebo (5, 6). This decreased duration of bronchoconstriction after exercise in the presence of a leukotriene antagonist also suggests that leukotrienes are released later than histamine. We have demonstrated that subjects with asthma responsive to exercise and eucapnic hyperventilation are also responsive to mannitol, and we have suggested that mannitol causes airway narrowing via a similar mechanism (11). The findings in this study suggest that the profile of mediator release to mannitol is similar to that which occurs with exercise.

Many studies investigating the effects of leukotriene antagonists on exercise have demonstrated a large variation in individual responses, with only some patients showing complete inhibition (3, 4, 20). This has led to the suggestion that some subjects with asthma may produce a different array of bronchoconstricting mediators compared with others (20). By contrast, in all but one subject who performed the montelukast protocol, the recovery to baseline lung function after mannitol was significantly faster in the presence of montelukast compared with placebo. The importance of this finding is highlighted by the fact that there was a wide range of airway sensitivity to mannitol in this group of subjects (PD₁₅ from 3 to 392 mg). This suggests that leukotrienes have the potential to play a role in the airway response to an osmotic stimulus in the majority of subjects with asthma.

To ensure that there was an adequate drug level we studied the effect of the drug when two doses had been taken in the last 24 h, with the last one taken 5 h before the challenge. This is a shorter interval of dosing than is clinically recommended. Thus beneficial effects may have been greater than might be expected if the mannitol challenge had been performed in the middle or at the end of a 24-h dosing schedule, as has been done with exercise (5, 6). One study, however, has demonstrated that there was no difference in the extent of protection against EIA at either 1, or 4, or 8 or 12 h after a single dose (10 mg) of montelukast (7).

The source of the leukotrienes is not known but there are several possibilities, and these include mast cells and the eosinophils (21). In vitro experiments in which mannitol was used as an osmotic stimulus demonstrated release of histamine from human airway mast cells and basophils (12), but not leukotrienes (22). This suggests that other cellular sources may be important for the osmotic release of leukotrienes. Mannitol, acting as an osmotic stimulus, has the potential to affect all cells in the airways. It is thought that regulatory volume increase following cell shrinkage is the important event that leads to release of mediators (23), and this release can be from a wide variety of cells (24). What appears to make the airways sensitive to osmotic stimuli is the presence of cells containing inflammatory mediators. Thus responsiveness to mannitol may reflect the presence of inflammatory cells and be an indirect index of severity of airway inflammation.

We also found that the airway recovery to baseline lung function after mannitol in the presence of montelukast was similar to the responses after rescue with a standard dose of inhaled ₂-adrenoceptor agonist. The rapid reversal of the bronchoconstriction in response to a ₂-agonist demonstrates the safety of using mannitol as a provoking agent, as the airway narrowing appears to be primarily the result of bronchial smooth muscle contraction.

We found no significant improvements in baseline FEV₁ in the presence of montelukast in the majority of subjects studied. All subjects had good resting lung function and the majority were taking inhaled corticosteroids on a daily basis. As a result, it is likely that no additional benefit on the resting lung function of these subjects could be obtained with a leukotriene antagonist.

It is likely that the initial bronchoconstriction after mannitol administration is due to the release of preformed mediators such as histamine, and occurs before the release of newly synthesized mediators such as leukotrienes that sustain the airway response to mannitol. This concept is in keeping with our observation of a slower spontaneous recovery of FEV₁ after mannitol administration in the presence of fexofenadine compared with placebo. By blocking the initial bronchoconstricting effect of inhaled mannitol with fexofenadine and allowing the administration of a greater dose of mannitol it is possible that more leukotrienes were released with a consequent further significant reduction in FEV₁ at 5 min after challenge.

The greater reduction in lung function after mannitol, in the presence of fexofenadine, serves to question the therapeutic benefit of taking a drug that simply reduces sensitivity to a stimulus, but does not affect the outcome if the same stimulus also causes the release of other mediators that sustain the response. Our findings here may explain why some investigators have not found H₁ antagonists protective against exercise challenge (25, 26) and others have found only partial protection (1, 2).

In the presence of both antagonists, the airway response to mannitol was similar to the response to exercise, where the maximal response usually occurs about 5 min after the cessation of exercise (27). Previous studies investigating airway recovery after mannitol administration have shown that the maximum airway response is similarly transient, and that the FEV₁ starts to improve 5 min after administration of the last dose (10, 28). The reason for the transient nadir in FEV₁ normally observed with mannitol and exercise may be due to histamine being rapidly metabolized.

In conclusion, this study demonstrates that histamine and leukotrienes both contribute to the airway response to inhaled mannitol in subjects with asthma. Histamine would appear to be more important for the immediate response than it is for sustaining the response. In addition to contributing to the severity of the airway response leukotrienes appear to sustain and prolong the airway narrowing to mannitol. The findings of this study also supports the suggestion that the mechanism of airway narrowing to inhaled mannitol is similar to exercise (11). The findings also suggest that a positive response to mannitol challenge reflects the presence of inflammatory cells in the airways and mast cells and eosinophils in particular.