Dose-response protective effect of salbutamol on methacholine airway responsiveness using pressurized metered dose inhalers and Turbuhalers.

The purpose of this study was to estimate the relative dose potency of salbutamol Turbuhaler compared with salbutamol pressurized metered dose inhaler (pMDI) with respect to the protective effect against methacholine bronchoconstriction. Twenty-three asthmatic subjects with stable asthma participated in the study. Baseline forced expiratory volume in 1 s (FEV1) was 70% or more of predicted, and baseline methacholine provocative concentration causing a 20% fall in FEV1 (PC20) was 4 mg/mL or less. The design was randomized, double-blind, double-dummy, crossover and placebo controlled and was conducted over seven study days. On each study day, the subjects inhaled 50 microg or 100 microg of salbutamol via Turbuhaler, 100 microg, 200 microg, 400 microg or 800 microg of salbutamol via pMDI, or placebo in randomized order. PC20 was determined 30 mins after inhalation. Increasing doses of salbutamol pMDI increased the PC20 in a dose-dependent fashion from 3.9 mg/mL after placebo to 13.3 mg/mL after pMDI 100 microg, 19.0 mg/mL after 200 microg, 32.6 mg/mL after 400 microg, and 35.1 mg/mL after 800 microg. The half-maximum response dose for pMDI (ED50) was 104 microg. Salbutamol Turbuhaler 50 microg increased the PC20 to 10.0 mg/mL and 100 microg to 12.6 mg/mL. Salbutamol pMDI 200 microg provided significantly greater protection to methacholine than pMDI 100 microg or Turbuhaler 100 microg and significantly less protection than pMDI 400 microg (P<0. 05). This study demonstrates that the relative protective dose potency of inhaled beta-agonists can be determined by comparing their effects on methacholine airway responsiveness. The estimated relative protective dose potency for salbutamol Turbuhaler in comparison with pMDI was 1.38 (95% CI 0.67 to 2.87) at 50 microg and was 0.96 (95% CI 0.56 to 1.64) at 100 microg.

The purpose of this study was to estimate the relative dose potency of salbutamol Turbuhaler compared with salbutamol pressurized metered dose inhaler (pMDI) with respect to the protective effect against methacholine bronchoconstriction. Twenty-three asthmatic subjects with stable asthma participated in the study. Baseline forced expiratory volume in 1 s (FEV 1 ) was 70% or more of predicted, and baseline methacholine provocative concentration causing a 20% fall in FEV 1 (PC 20 ) was 4 mg/mL or less. The design was randomized, double-blind, double-dummy, crossover and placebo controlled and was conducted over seven study days. On each study day, the subjects inhaled 50 µg or 100 µg of salbutamol via Turbuhaler, 100 µg, 200 µg, 400 µg or 800 µg of salbutamol via pMDI, or placebo in randomized order. PC 20 was determined 30 mins after inhalation. Increasing doses of salbutamol pMDI increased the PC 20 in a dose-dependent fashion from 3.9 mg/mL after placebo to 13.3 mg/mL after pMDI 100 µg, 19.0 mg/mL after 200 µg, 32.6 mg/mL after 400 µg, and 35.1 mg/mL after 800 µg. The half-maximum response dose for pMDI (ED 50 ) was 104 µg. Salbutamol Turbuhaler 50 µg increased the PC 20 to 10.0 mg/mL and 100 µg to 12.6 mg/mL. Salbutamol pMDI 200 µg provided significantly greater protection to methacholine than pMDI 100 µg or Turbuhaler 100 µg and significantly less protection than pMDI 400 µg (P<0.05). This study demonstrates that the relative protective dose potency of inhaled beta-agonists can be determined by comparing their effects on methacholine airway responsiveness. The estimated relative protective dose potency for salbutamol Turbuhaler in comparison with pMDI was 1.38 (95% CI 0.67 to 2.87) at 50 µg and was 0.96 (95% CI 0.56 to 1.64) at 100 µg. S albutamol is a selective beta-agonist that provides 4 to 6 h of bronchodilation upon inhalation. Until recently, this drug was administered mainly as aerosols from pressurized metered dose inhalers (pMDI). Many available pMDIs contain chlorofluorocarbons as propellants for aerosol generation (1). Problems exist with respect to coordination of actuation and inhalation, and, in some patients, the propellants can provoke acute transient bronchoconstriction (2). Turbuhaler (Astra Pharma Inc) is an inspiratory flow-driven, multidose, dry powder inhaler that provides the medication without the need for propellants. A randomized crossover comparison of five inhaler systems determined an overall preference for Turbuhaler with respect to ease of use (3).
Salbutamol inhaled via Turbuhaler and pMDI have been compared in recent studies. The same nominal dose of salbutamol given via Turbuhaler has been shown to be more potent than salbutamol via pMDI with respect to bronchodilator response and systemic effects (4,5). In addition, Turbuhaler gives a higher pulmonary deposition of terbutaline compared with pMDI (6) and significantly greater bronchodilation compared with terbutaline pMDI plus Nebuhaler (7).
The potency of beta-agonists can be expressed not only by their bronchodilator responses but also by their protective effect to bronchoconstrictor stimuli such as inhaled methacholine. Large doses of inhaled salbutamol pMDI have been demonstrated to shift the methacholine dose-response curve by four-to eightfold (8). However, by using different drug doses, it may be possible to construct a dose-response curve of airway responsiveness versus dose of salbutamol. In this way, the relative protective potency may be determined for salbutamol when delivered from different devices, such as salbutamol pMDI and salbutamol Turbuhaler. The relative protective potency concept assumes that the curves are parallel. The differences of one curve relative to the other may be represented by a constant, which is a measure of the relative protective potency. Developing a method of estimating relative protective potency is important for comparing single dose therapy of salbutamol Turbuhaler with salbutamol pMDI.
The purpose of this study was to construct a doseresponse curve of salbutamol pMDI against methacholine airway responsiveness and estimate the relative protective potency of salbutamol Turbuhaler compared with salbutamol pMDI with respect to the protective effect against methacholine bronchoconstriction.

Subjects:
Twenty-three adults with asthma volunteered to participate in the study (Table 1). Each subject's asthma was stable, forced expiratory volume in 1 s (FEV 1 ) was 70% predicted or greater and methacholine provocation concentration to cause a fall in FEV 1 of 20% (PC 20 ) was 4 mg/mL or less. In addition, after salbutamol pMDI 200 µg was inhaled, PC 20 had to increase at least fourfold. Four subjects were on regular treatment with inhaled steroid (mean 837 µg daily dose) and the dose had been stable for at least three months, and was kept constant throughout the study; no study subjects were on treatment with beta-receptor antagonists, prednisone, antihistamine or immunotherapy. Before each study day the following washout periods were applied: long-acting inhaled beta-agonists for 72 h; oral beta-agonists for 12 h; long-acting oral beta-agonists and methylxanthines for 48 h; anticholinergics for 12 h. No patient was taking regular short-acting beta-agonists at the time of the study. Oral and parenteral corticosteroids were not permitted for one month before visit 1. None of the subjects had symptoms of a respiratory tract infection or exposure to allergens to which they were sensitized for six weeks before or during the study, and none had exercised vigorously before any study visit. The study was approved by the Hospital Research Ethics Committee, written informed consent was obtained for each subject, and the performance of the study was in accord with the principles of the Declaration of Helsinki. Study design: The study was of randomized, double-blind, double-dummy, crossover and placebo controlled design. There were two enrolment days (visits 1 and 2) and seven study days (visits 3 to 9). At visit 1, informed consent was obtained. At visit 2, criteria for entry into the study were 120 Can Respir J Vol 5 No 2 March/April 1998
checked. The baseline methacholine PC 20 was measured and, if it was 4 mg/mL or less and if no salbutamol was required to treat the methacholine bronchoconstriction, the protective effect of salbutamol pMDI 200 µg was examined. The latter was determined 115 mins after the last inhalation of methacholine, if the FEV 1 was greater than 90% of baseline. If the FEV 1 was not greater than 90% of baseline, FEV 1 was measured every 15 mins until this value was reached. Then salbutamol pMDI 200 µg was inhaled, and 10 mins later the methacholine inhalation test was repeated, beginning with a twofold dose below the baseline methacholine PC 20 . If the postsalbutamol methacholine PC 20 increased by more than fourfold, the subject was entered into the study. At visits 3 to 9, the protective effect of the study drugs on the methacholine PC 20 was examined, and between these visits, salbutamol 100 µg was allowed only when needed. At each visit, the subject had to be stable as indicated by symptoms, need for salbutamol and an FEV 1 that did not vary by more than 10% from visit 2. If FEV 1 was lower than this, the subject was re-examined on another day; if it was still abnormal, the subject was withdrawn. The washout period between treatments was two to seven days.
At each visit, the subjects inhaled, in randomized order, salbutamol 50 or 100 µg by Turbuhaler, or 100, 200, 400 or 800 µg by pMDI, or placebo. The active medication and placebo were administered in a double-dummy fashion to maintain blinding. The methacholine inhalation test was repeated 10 mins later, beginning with a twofold dilution below baseline on visit 2 so that the methacholine PC 20 value would be determined about 30 mins after inhalation of the test medication. On each day, pulse rate, blood pressure and history of symptoms were recorded before and after the methacholine test.

Spirometric measurements and methacholine tests:
The FEV 1 and slow vital capacity (SVC) were measured with a Vitalograph Compact Spirometer (Vitagraph Ltd, Buckingham, United Kingdom) according to American Thoracic Society guidelines (9). Methacholine inhalation tests were performed as described by Juniper et al (10) using a Wright Nebulizer (English Wright, Aerosol Medical Ltd, Colchester, United Kingdom) attached to a three-way Hans Rudolph Valve (Hans Rudolph Inc, Missouri), doubling concentrations of methacholine between 0.6 and 256 mg/mL and tidal breathing for 2 mins. The response was measured by change in FEV 1 recorded at 30 and 90 s, and then at 3 mins and every 2 mins thereafter until it stopped falling. The fall was recorded between the highest post-test drug, premethacholine value and the lowest postmethacholine value. The results were expressed as the methacholine PC 20 obtained from linear interpolation of the last two methacholine doses below and above a 20% fall in FEV 1 . A methacholine PC 20 value was obtained from each subject at each visit, so no data censoring was necessary. Statistical analysis: A dose-response curve for the pMDI doses was established by estimating a nonlinear regression of log methacholine PC 20 versus dose of salbutamol pMDI. The standard dose-response curve was represented by the following equation: log PC20 = log PC20 (placebo) + Max D k /(K k + D k ) where D is the dose of salbutamol, Max is the maximal effect, K is the dose giving 50% of the maximal effect (ED 50 ) and k was the slope parameter. The same model was applied for Turbuhaler, and a different K value was obtained which was called rK. This model assumes that each Turbuhaler dose lies on an individual curve that is parallel to the pMDI dose-   response curve. The relative dose potency was represented by the r value. Analysis of variance was used to obtain period-adjusted mean values and a correct variability. These data were then analyzed using nonlinear regression in order to estimate the r value and give confidence limits. For all analyses, a probability value of P<0.05 was considered significant. The adverse events were analyzed by means of descriptive statistics and qualitative analysis.

RESULTS
The mean baseline FEV 1 was 3.13 L, and the SVC was 3.93 L. The mean difference in baseline FEV 1 was 7.0% (range 3.0% to 13.2%) with a coefficient of variation (CV) of 2.7% (range 1.4% to 5.7%). The baseline PC 20 was 1.8 mg/mL (range 0.4 to 3.8) and increased by 11.3-fold after two times 100 µg salbutamol pMDI to 17.0 mg/mL (range 4.6 to 56).
Salbutamol was well tolerated at all doses. There were no significant adverse effects nor were there any significant changes in pulse rate or blood pressure.

DISCUSSION
This study has demonstrated the use of bronchoprotection against methacholine challenge as a method to determine relative protective potencies of different salbutamol formulations. The study suggests that the relative dose potency for salbutamol Turbuhaler was 1.36 at the 50 µg dose compared with pMDI and 0.96 at the 100 µg dose compared with pMDI.
A dose-response curve of methacholine PC 20 versus doses of salbutamol was obtained for salbutamol pMDI. The ED 50 response occurred at 104 µg which was close to the lowest dose of 100 µg. Methacholine PC 20 increased significantly at doses of 100 µg, 200 and 400 µg, but did not change further with 800 µg. The demonstration of ED 50 close to the lowest dose delivered by pMDI was unexpected and indicates that a major portion of the dose-response curve is located at 100 µg and below. Therefore, future studies could incorporate the use of lower doses of salbutamol pMDI (eg, 50 µg) to measure this part of the curve more accurately.
Studies have shown that Turbuhaler is effective in delivering a higher proportion of the nominal dose to the lungs compared with pMDI (11,12). In the present study, the relative dose protective potency of 0.96 for Turbuhaler 100 µg was not significantly different from that of salbutamol pMDI. Thus, Turbuhaler 100 µg would produce the same amount of bronchoprotection against inhaled methacholine as the pMDI. One explanation for this similarity could be a 'ceiling effect' where both devices produce the maximal possible bronchoprotection and, therefore, do not allow differences to be demonstrated between these devices. This is clearly not the case because a dose of 100 µg is close to the ED 50 . This result does demonstrate the advantage of constructing dose-response curves, as was done in this study, because comparison of single doses from the two devices could not have eliminated this possibility.
Mildly asthmatic subjects (baseline methacholine PC 20 of 1.8 mg/mL) with normal baseline pulmonary function were selected for this study. Dose-response curves may differ for asthmatics with moderate or severe asthma, or heightened Figure 1) Geometric mean methacholine provocative concentration causing a 20% fall in forced expiratory volume in 1 s (PC 20 ) ratio (adjusted relative to placebo) for salbutamol Turbuhaler (TBH) and pressurized metered dose inhaler (pMDI) following each salbutamol dose is shown on a logarithmic scale. Dose-response curve for salbutamol pressurized metered dose inhaler is shown by the solid line. The half-maximal response is represented by the dotted lines airway responsiveness. In these patients, pretreatment with salbutamol may produce additional bronchodilation that may interfere with the bronchoconstricting effects of methacholine. It is also possible that methacholine exerts its effect at locations different from the site of action of salbutamol when given by either Turbuhaler or pMDI or that there are differences in lung distribution pattern between Turbuhaler and pMDI. Several factors, such as inhalation pattern, airway calibre and degree of obstruction, have been shown to influence the site of deposition in the lung (13,14).
Because methacholine exerts its effects on the central airways, which are the main sites of the cholinergic receptors (15), the bronchodilating effects of a medication may not be representative of the protection it provides against a cholinergic bronchoconstrictor mediator, such as methacholine. This caveat also applies when comparing the results of the present study with those achieved using other provocation agents acting through other mechanisms.

CONCLUSIONS
This study has demonstrated that the relative dose protective potency of salbutamol given via Turbuhaler and via pMDI may be determined by comparing its effects on methacholine airway responsiveness. The relation between dose-protective potency obtained using other provocation agents, as well as the relation between protective and brochodilating potency, needs to be further investigated.