INTRODUCTION

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Corticosteroids are the most effective drugs for reducing asthma severity, as judged by exacerbations and airway inflammation (1). Consequently, the numbers of inhaled corticosteroids and delivery devices available for the management of asthma are increasing. To date, there has been no approved method for comparing the relative potency of these different compounds or delivery devices. Such a method would require an outcome measurement that is relevant and displays a measurable dose- response relationship with treatment (4).

There have been few demonstrations of relevant outcome measurements that display dose-response relationships with inhaled corticosteroids in the dose range used in the management of asthma. Recently, dose-response effects have been observed for budesonide (200 to 1,600 µg) (5) and beclomethasone (100 to 800 µg) (6) in improving lung function after 12 wk of treatment, and differences were observed between the effects of 1 yr of treatment with 200 or 800 µg budesonide daily in the prevention of asthma exacerbations (1). However, the sample size and duration of treatment required to observe such relationships make these methods unattractive for comparing different treatments. Pedersen and Hansen have observed a dose-response effect for inhaled budesonide over a 4-wk period to protect against exercise-induced bronchoconstriction in 19 asthmatic children (7). Methods such as this, where fewer subjects and shorter treatment periods are required, offer a major advantage in potency studies. Other attempts with small sample sizes were unable to detect dose-dependent differences in lung function or markers of inflammation in blood or induced sputum (8, 9), although significant effects on exhaled nitric oxide have been reported (10).

In one study, a dose-response relationship for three inhaled corticosteroids against the early bronchoconstrictor response after allergen inhalation challenge was established, allowing relative potency comparisons to be made (11). However, other studies attempting to demonstrate dose response relationships for inhaled corticosteroids against allergen-induced responses have not been successful (12, 13). In all of these studies, the investigators chose to observe a single outcome as the basis for establishing a dose-response relationship. It is known, however, that there are several measurable responses after allergen challenge, including early and late asthmatic responses, increased airway hyperresponsiveness, and increased airway eosinophilia (14). To date, there have been no studies attempting to establish dose-response relationships for inhaled corticosteroids on each of these outcomes.

Mometasone furoate (MF) is a corticosteroid originally developed as a topical anti-inflammatory agent to treat dermatologic disorders (Elocon, Elocom, and Elomet) (15) and more recently as a nasal spray for allergic rhinitis (Nasonex) (16). MF has also been reformulated as a MF/lactose agglomerate to be administered via a novel breath-actuated dry powder inhaler (MF-DPI) for use in the management of asthma (17). MF-DPI has been shown to be efficacious and well tolerated by patients with mild to moderate persistent asthma; improving lung function while reducing adenosine monophosphate (AMP) hyperresponsiveness and asthma symptoms (17).

In this study, we investigated the effects of three doses of MF-DPI on allergen-induced early and late responses, airway hyperresponsiveness, and airway inflammation, and whether a dose-response relationship could be detected for any of these outcomes.

	METHODS

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Subjects

Twelve nonsmoking subjects (six male, six female) (Table 1) with stable mild atopic asthma were included in the study. Inclusion was based on having a provocative concentration producing a 20% reduction in FEV₁ (PC₂₀) of less than 16 mg/ml methacholine and allergen-induced early and late bronchoconstrictor responses of at least 15% reduction in FEV₁ during screening challenges. Subjects who had been treated with any asthma medication other than inhaled ₂-agonists, or who used inhaled ₂-agonists more frequently than once daily during the 4-wk period before screening were not permitted to enter the study. Two subjects failed to complete all treatment arms of the study, one after completing one treatment arm (unstable asthma) and one after completing two treatment arms (protocol violation). Subjects who had completed at least two arms of the study were included in the analysis. Thus, the analysis was based on 11 subjects.

Study Design and Protocol

The study was carried out using a double-blind, placebo-controlled, randomized, four-period crossover design. After successful screening, subjects were randomized to receive MF-DPI at three different dosages (50 µg twice daily, 100 µg twice daily, and 400 µg twice daily) and placebo during four treatment phases of 6 d each, separated by washout periods of at least 21 d (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   The protocol used within each treatment phase of the study. EAR = early asthmatic response; LAR = late asthmatic response; Sput = sputum; trt = treatment; Ag = allergen.

During each treatment phase, subjects inhaled their first dose of study medication after a methacholine inhalation challenge to determine baseline airway responsiveness and sputum induction to determine pretreatment baseline airway inflammatory status. Treatment was continued twice daily thereafter, and on the fourth day, the methacholine challenge and sputum induction were repeated to establish preallergen baseline levels. On the fifth treatment day, after the morning dose of study medication, subjects received an allergen inhalation challenge and early and late asthmatic responses were measured. Seven hours after allergen inhalation, sputum induction was repeated to assess airway inflammation. Study medication was taken on the evening of the fifth day. On the sixth day, 24 h after allergen inhalation, subjects again received a methacholine challenge and sputum induction 30 min after the morning dose of study medication.

Laboratory Procedures

Methacholine inhalation challenge. Methacholine inhalation challenge was performed as described by Cockcroft and coworkers (20). Subjects inhaled through a mouthpiece, attached to a Wright nebulizer. Normal saline, then doubling concentration increases in methacholine were nebulized for 2 min each. FEV₁ was measured at 30, 90, 180, and 300 s after each inhalation using a Collins water-sealed spirometer and kymograph. The test was terminated when FEV₁ had fallen to a level at least 20% below the postsaline measurement. The concentration of methacholine required to achieve a decrease in FEV₁ of 20% (MCh PC₂₀) was calculated through linear interpolation of %fall in FEV₁ against the log-transformed MCh concentration (20).

Allergen inhalation challenge. Allergen challenge was performed as described by O'Byrne and coworkers (21). The allergen producing the largest skin wheal diameter after skin prick testing was diluted in sterile normal saline. Allergens used were house dust mite (7), grass (3), tree (1), and cat (1). The concentration of allergen extract for inhalation was determined using a formula derived by Cockcroft and coworkers (22) using the results from skin test titrations and the MCh PC₂₀. During the screening allergen challenge, the starting concentration of allergen extract for inhalation was two doubling concentrations below that predicted to cause a 20% decrease in FEV₁. Doubling increases in allergen concentration were inhaled every 10 min until a 15% reduction in FEV₁ was achieved. FEV₁ was then measured at 10, 20, 30, 45, 60, 90, and 120 min after allergen inhalation, then each hour until 7 h after allergen inhalation. The early bronchoconstrictor response was taken to be the largest %fall in FEV₁ within 2 h after allergen inhalation, and the late response was taken to be the largest %fall in FEV₁ in the period beginning 3 h and ending 7 h after allergen inhalation. Maximal decreases in FEV₁ were chosen to quantify the early and late response magnitudes based on our earlier studies indicating that these measurements have utility in detecting treatment effects (23). Only subjects who achieved a 15% or greater decline in FEV₁ during both of these periods at screening progressed to randomization. All allergen challenges performed after randomization were performed with subjects inhaling the same allergen concentrations used during the screening challenge.

Sputum analysis. Sputum was induced using the method of Pin and coworkers (24). Subjects inhaled 3%, 4%, then 5% saline for 7 min each and attempted to expectorate sputum after each inhalation period. Sputum was manually separated from saliva and weighed as described by Pizzichini and coworkers (25). Samples were aspirated and rocked in four times their weight of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA) and then in four times their weight of Dulbecco's phosphate-buffered saline (DPBS) (GIBCO, Mississauga, ON, Canada). The cell suspension was filtered through a 52-µm nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, then centrifuged at 1,500 rpm for 10 min. The total cell count was determined using a hemocytometer (Neubauer Chamber) and expressed as the number of cells per ml sputum. Cells were resuspended in DPBS at 0.75 to 1.0 × 10⁶/ml. Cytospins were prepared on glass slides using 50 µl of cell suspension and a Shandon III cytocentrifuge (Shandon Southern Instruments, Sewickly, PA), at 300 rpm for 5 min. Slides were stained using Diff-Quik and differential cell counts were performed on all slides by a single observer. The mean count obtained from two slides with 400 cells counted per slide was used for analysis. Cell types were enumerated by multiplying the differential, expressed as a fraction, by the total cell count per ml sputum.

Analysis

MCh PC₂₀ measurements were log₂-transformed to normalize the data (26). The choice of base 2 for the transformation allows differences between PC₂₀ values to be expressed as doubling concentrations.

Changes in log-transformed MCh PC₂₀ and sputum eosinophilia in response to treatment or allergen challenge were evaluated using analysis of variance (ANOVA) allowing for effects due to subject, period, and treatment. Pairwise treatment and mean differences were estimated using linear contrasts of the least means squares from the ANOVA. Comparisons between treatment effects of placebo and the three doses of MF-DPI on preallergen sputum eosinophilia and MCh PC₂₀ were made using separate ANOVAs with the pretreatment to preallergen deltas. Treatment effects on allergen-induced changes in sputum eosinophilia and MCh PC₂₀ were made using separate ANOVAs of the pre- to postallergen deltas. In the case of methacholine PC₂₀, these deltas were calculated using the log₂-transformed data and are therefore expressed in the text as doubling concentration changes. Comparisons of treatment effects of placebo and the three doses of MF-DPI on allergen-induced airway responses were made using separate ANOVAs to compare the largest %fall in FEV₁ during each time period. Measurement variability was expressed using standard deviation (SD) for baseline subject characteristics and standard error of the mean (SEM) for outcome variables.

In addition to the protocol specified analysis, further analyses were performed to determine whether dose-response relationships existed for the effects of MF-DPI on allergen-induced responses. Separate ANOVAs were performed, as previously described, but using only those data from the active treatment arms. A dose-response relationship was then formally tested by comparing the 50-µg twice-daily dose with the 400-µg twice-daily dose using the linear contrast of the least square means from the ANOVAs. Additionally, slopes were calculated for each subject for the relationship between each outcome variable (early asthmatic response, late asthmatic response, PC₂₀, and  sputum eosinophils) and the log of the MF-DPI dose. All comparisons were made with  set at 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subject Demographics

Subject demographics, measured at the initial screening visit are presented in Table 1.

Early and Late Responses

There were no significant differences between the prechallenge FEV₁ values in each treatment arm, being 3.30 L ± 0.18, 3.40 L ± 0.19, 3.54 L ± 0.20, and 3.41 L ± 0.20 after placebo, 50-, 100-, and 400-µg twice-daily treatment respectively. The maximal early %fall in FEV₁ after placebo treatment was 36.8% ± 3.0 (mean ± SEM). This was significantly reduced by all three doses of inhaled MF, to 29.6% ± 3.3, 24.2% ± 3.2, and 26.4% ± 3.4 after the 50-, 100-, and 400-µg twice-daily treatments respectively (p < 0.05) (Figure 2). The maximal late %fall in FEV₁ after placebo treatment was 23.5% ± 4.4. This was significantly reduced by all three doses of inhaled MF, to 12.3% ± 2.2, 11.0% ± 1.8, and 5.9% ± 1.9 after the 50-, 100-, and 400-µg twice-daily treatments respectively (p < 0.005 versus placebo) (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 2.   Time course of the mean decline in FEV1 (expressed as percent below the prechallenge value) after allergen challenges in each treatment phase. There was significant attenuation of the maximal early response and late response by all three doses when compared with placebo (p < 0.05).

Airway Responsiveness

On Day 4 (preallergen), the MCh PC₂₀ increased relative to preallergen values after treatment with 50, 100, and 400 µg twice-daily MF-DPI by 0.49 ± 0.34, 0.62 ± 0.34, and 0.8 ± 0.33 doubling concentrations respectively, but these increases were not significantly different than the 0.22 ± 0.23 doubling concentration increase after placebo treatment (p > 0.05) (Figure 3). The allergen-induced decreases in MCh PC₂₀ (postallergen  preallergen) after treatment with 50, 100, and 400 µg twice-daily MF-DPI were 0.62 ± 0.30, 0.64 ± 0.30, and 0.41 ± 0.32 doubling concentrations respectively, which were all significantly less than the 1.71 ± 0.32 doubling concentration decrease following allergen in the placebo treatment phase (p < 0.05) (Figure 3).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Airway responsiveness, as measured by MCh PC20 before and after treatment and allergen challenge in each phase of the study. *Different than preallergen value in same arm (p < 0.05). Allergen-induced decrease in PC20 less than in placebo arm (p < 0.05).

Sputum Eosinophilia

Sputum eosinophils did not change significantly during the pretreatment to preallergen period in any treatment phase, nor were there differences in this response between treatment phases (p > 0.05) (Figure 4). The increase in sputum eosinophilia between preallergen and 7 h postallergen in the placebo treatment arm was 42.6 ± 11.0 × 10⁴ cells/ml sputum, which was significantly greater than the increases of 11.7 ± 5.1, 16.4 ± 6.9, and 3.7 ± 2.0 × 10⁴ cells/ml sputum in the 50, 100, and 400 µg twice-daily MF-DPI treatment arms respectively (all p < 0.005) (Figure 4). The increase in sputum eosinophilia between preallergen and 24 h postallergen in the placebo treatment arm was 60.2 ± 19.7 × 10⁴ cells/ml sputum, which was significantly greater than the increases of 24.0 ± 11.0, 15.3 ± 3.1, and 6.8 ± 2.6 × 10⁴ cells/ml sputum in the 50, 100, and 400 µg twice-daily MF-DPI treatment arms respectively (p < 0.05) (Figure 4). Similar findings were observed when sputum eosinophils were expressed as a percentage of the total sputum cell count (see Table 2 for eosinophil differentials).

View larger version (18K): [in this window] [in a new window]   Figure 4.   Sputum eosinophil numbers before and after treatment and allergen challenge in each phase of the study. *Allergen-induced increase in eosinophils less than the corresponding increase during the placebo phase (p < 0.05).

Dose-Response Relationships

The effects of MF-DPI on the magnitude of the early response and allergen-induced decreases in PC₂₀ were not significantly different between the three dose phases (p > 0.05), nor were the mean dose-outcome slopes for these outcomes different than zero (p > 0.05). There was, however, a significant dose- response relationship (p = 0.007, ANOVA) for the late response measurements; the mean slope of the regression performed on the late response data was 8.0 (Figure 5). The allergen-induced increases in sputum eosinophilia at 7 h during the active treatment arms did not exhibit a dose-response relationship (p > 0.05). There was, however, a marginally significant dose- response relationship for the allergen-induced increased in sputum eosinophils at 24 h (p = 0.063); the regression performed on the 24-h increase in sputum eosinophils yielded a slope of 18.6 (Figure 5). Similar results were observed when eosinophils were expressed as a percentage of the sputum total cell count (not shown).

View larger version (23K): [in this window] [in a new window]   Figure 5.   Individual dose-response curves for the late response and the change in eosinophils 24 h after allergen (i.e., 24-h value minus the prechallenge value). The squares represent the mean value at each dose. The slopes of the late response curves were significantly different than zero (p < 0.05), whereas the slopes of the eosinophil responses were marginally significantly different than zero (p = 0.08).

Adverse Events

There were no severe adverse events in any subject in any treatment arm. Adverse events were reported by six, four, four, and five subjects during the placebo, 50-µg, 100-µg, and 400-µg twice-daily treatment phases respectively. Adverse events rated as mild or moderate that were reported by more than one subject included headache, abdominal pain, nausea, dysmenorrhea, and pharyngitis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we have observed that three different doses of MF reduced the magnitude of allergen-induced early and late asthmatic responses, allergen-induced airway hyperresponsiveness, and allergen-induced increases in sputum eosinophilia. These findings offer support for the usefulness of MF-DPI in the management of asthma. The finding that a dose-response effect was observed for the attenuation of allergen-induced late asthmatic responses supports the use of allergen inhalation challenges as a tool for inhaled corticosteroid relative potency studies, comparing different compounds, formulations, or delivery devices.

Treatment with the highest dose of MF-DPI (400 µg twice a day) for 4 d before allergen inhalation challenge resulted in attenuation of the early asthmatic response by 28.2%; the late asthmatic response by 75%; allergen-induced hyperresponsiveness by 76%; and allergen-induced increases in sputum eosinophilia by 91.3% and 88.7% at times 7 h and 24 h after challenge respectively. Even with the lowest dose of MF-DPI (50 µg twice a day), the attenuation of the 7-h and 24-h postallergen increases in sputum eosinophils was 72.5% and 60.1% respectively. This degree of attenuation compares favorably with results we have obtained using inhaled budesonide, where 200 µg inhaled twice daily attenuated the 7-h and 24-h increases in eosinophils by 66% and 59% respectively (14). However, it should be stated that any conclusions about the relative potency of these or other compounds in reducing or preventing airway inflammation would require a direct comparison.

The primary purpose of this study was to observe whether dose-response relationships exist for the effects of inhaled MF on outcomes after allergen inhalation challenge. We observed a significant dose-response effect on the allergen-induced late response and a trend toward significance for the 24-h sputum eosinophilia. These findings indicate that allergen inhalation challenge may be a useful tool for performing relative potency studies on antiasthma compounds or delivery devices. These findings are to some extent in agreement with the observations of McCubbin and coworkers (11) who observed similar dose- response curves for flunisolide, triamcinolone, and beclomethasone on the magnitude of allergen-induced early responses. In that study, there were no measurements of other outcomes after allergen, nor is it clear whether the full time course of the early response was followed in each treatment arm.

Previous attempts to detect dose-response relationships for the efficacy of inhaled steroids to improve lung function or reduce markers of inflammation in blood or sputum have not been successful (8, 9). Our findings suggest that this is made possible through the addition of an allergen challenge, likely through increasing the signal-to-noise ratio of the outcome measurements.

Although we have observed a significant dose response curve, for the late response, it is clear that large sample sizes would be required to determine relative potency between compounds. This is likely to a large extent due to the fact that even the lowest dose of MF-DPI that we used was close to maximally effective. The attenuating effect of the 50-µg twice-daily dose, as a percentage of the effect of the 400-µg twice-daily dose was 69% for the early response, 64% for the late response, 84% for allergen-induced hyperresponsiveness, and 79% and 68% respectively for the 7-h and 24-h increase in eosinophils. Thus, it is clear that the doses studied cover a small range of attenuating effects, near the top of the dose- response curve. If smaller doses were available, then a greater range of attenuation could be achieved, making relative potency studies more feasible.

Holgate and coworkers failed to observe a difference between treatment with MF-DPI 50 µg twice daily or 100 µg twice daily for 2 wk with respect to reversal of airway responsiveness to the indirectly acting mediator AMP (18). Possibly the lack of an observed dose-response effect in that study was a result of treating the subject with two doses different by only a doubling, as opposed to the current study where the difference between the 50-µg twice-daily and 400-µg twice-daily doses was three doublings. It is also possible that protection against allergen-induced airway responses is a more sensitive means for assessing steroid treatment effects than is measuring AMP airway responsiveness.

In the current study, we were able to detect a difference in the attenuating effects of 50 µg and 400 µg MF-DPI twice daily on the allergen-induced late response. The effectiveness of 200 µg and 800 µg daily of budesonide have been shown to be different in the prevention of mild and severe asthma exacerbations over the course of 1 yr (1). In other recent studies, dose-dependent effects in improving lung function were observed when patients were treated with inhaled budesonide over the range 200 to 1,600 µg for 12 wk or beclomethasone dipropionate over the range 100 to 800 µg for 6 wk (6). Although it is tempting based on these findings to infer that efficacy in an allergen challenge translates to effectiveness in practice, it is not clear that the mechanisms responsible for these two outcomes are the same, or that efficacy will always translate to effectiveness. However, the number of subjects required to demonstrate effectiveness in the previously cited studies was in the hundreds, compared with 11 used in the current study (1). Clearly, relative potency studies would be impossible if researchers were to rely on effectiveness as the primary outcome. Although efficacy in allergen challenge may prove a useful method for determining relative potency, demonstration of efficacy in preventing responses to allergen challenge does not always predict effectiveness in asthma management (e.g., frusemide [27]).

In summary, we have demonstrated that inhaled MF attenuates early and late asthmatic responses and allergen-induced airway hyperresponsiveness and sputum eosinophilia. The detection of a dose-response effect for the attenuation of the late asthmatic response, as well as the borderline dose-response for the effect on sputum eosinophilia indicate that allergen challenge may prove useful in relative potency studies of inhaled corticosteroids for the management of asthma.