INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Formoterol and salmeterol are two long-acting ₂-agonists given by inhalation, with bronchodilating effects lasting for at least 12 h after a single administration (1). Both of these drugs have become valuable complements in the regular treatment of asthmatic patients who are not satisfactorily controlled with inhaled corticosteroids (4). Formoterol and salmeterol have similar pharmacological properties in the sense that they are highly selective and potent ₂-adrenoceptor agonists, with relaxant effects on bronchial smooth muscle in vitro (5). However, some important pharmacological differences between these drugs have been documented in vitro and in patients. First, formoterol has a faster onset of action compared with salmeterol, which has been documented both in airway smooth muscle preparations (6, 7) as well as in asthmatic patients (3, 8, 9). Second, studies using isolated smooth muscle preparations from both experimental animals and humans show that salmeterol is less efficacious than formoterol, and thus a partial agonist. For example, a strongly contracted smooth muscle will relax to a larger extent if formoterol is added to the preparation, compared with addition of salmeterol (7, 10). Importantly, a partial agonist has the capacity to act as a relative blocker of the receptor, in the presence of an agent with similar or greater efficacy and intrinsic activity, which has been shown in vitro for salmeterol in relation to both salbutamol and formoterol (11). Formoterol, on the other hand, does not show this characteristic, confirming that salmeterol but not formoterol acts a partial ₂-adrenoceptor agonist under these basic experimental conditions. However, this relatively weaker efficacy of salmeterol in comparison with formoterol, has not been objectively evaluated in asthmatic patients.

The aim of the present study was therefore to investigate whether the partial agonistic characteristics of salmeterol that have been documented in different systems in vitro, also apply to human asthmatic airways in vivo. It would not be possible to test this question by evaluating the bronchodilating effects of the drugs because quite low doses result in near-maximal bronchodilation. However, all ₂-adrenoceptor agonists are protective against bronchoconstrictor stimuli, which may be used to further evaluate the efficacy of salmeterol and formoterol. We therefore hypothesized that salmeterol is less efficacious than formoterol in protecting against methacholine- induced bronchoconstriction. The study was thus designed to evaluate the shift of methacholine responsiveness induced by increasing doses of formoterol or salmeterol, in an attempt to evaluate the maximal protective effect of each drug. When performing comparisons of efficacy of drugs, it is important to choose drugs with similar duration of action, which has been documented for formoterol and salmeterol (8, 9, 12).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The local Ethics Committee of Göteborg University approved the study. The study was performed according to Good Clinical Trial Practice.

Inclusion criteria for the participating patients were age between 18 and 70 yr with a confirmed diagnosis of asthma and a stable inhaled dose of corticosteroids 200 to 1,600 µg/d (budesonide or equivalent), as well as stable medication for at least 1 mo. The patients were not allowed to be current smokers. Patients on oral bronchodilators, long-acting inhaled ₂-agonists, or oral glucocorticoids were not allowed to participate in the study. The baseline FEV₁ had to be at least 70% predicted and the provocative dose of methacholine producing a 20% fall in FEV₁ (PD₂₀) less than 200 µg on a screening visit. No asthma exacerbation, oral steroid course, or respiratory infection was allowed within 1 mo before the study start. No history of myocardial infarction or other clinically significant condition was allowed and corrected Q-T interval (Q-T_c) had to be less than 0.46 s. Patient characteristics are presented in Table 1.

Design

On a screening day the patients were interviewed, blood samples were taken, and electrocardiogram (ECG) (Q-T_c), spirometry (FEV₁), and bronchial methacholine challenge test were performed. If all inclusion criteria were fulfilled, including lung function FEV₁ 70% of predicted value, and threshold dose of methacholine responsiveness 200 µg, the patient was included in the study. In order to standardize for any tolerance to ₂-agonists, the patients were instructed to use Ventoline Diskhaler (Glaxo Wellcome, Ware, UK) 0.2 mg twice daily plus extra when needed, and no other ₂-agonist therapy was allowed.

On study days, the patients were given cumulative doses of formoterol (Foradil; Novartis, Basel, Switzerland) via the Aerolizer (12 + 48 + 60 µg, total dose 120 µg), or salmeterol (Serevent; Glaxo Wellcome) via the Diskhaler (50 + 200 + 250 µg, total dose 500 µg) or placebo, in a randomized, double-blind, double-dummy and crossover design. The visits were separated with a 3- to 12-d wash-out period. Before the first dose, and 50 min after each individual dose, ECG was recorded, samples for S-potassium taken, and scoring of tremor performed. These tests were done also 110 min after the last of the three cumulative doses. Sixty minutes after each individual dose, a methacholine provocation procedure was initiated.

Bronchial Challenges

The bronchial challenges were performed with methacholinechloride, purchased from the hospital pharmacy in the concentration of 256 mg/ml. This high concentration solution was diluted with physiological saline in half strengths to 128 mg/ml, 64 mg/ml, etc., to a lowest concentration of 0.03 mg/ml. The diluted solutions were instantly put in a freezer (70° C) and 30 min before each challenge, the solutions were taken out to achieve room temperature. The dosimeter ME.FAR MB3 (Mefar s.p.a., Bovezzo, Italy) was used for the methacholine challenges. With an inspiratory capacity breath, the patient inhaled the aerosol dose slowly, followed by 5 s of breath holding. The output of aerosol was 10 µl per breath, with a nebulization time set to 1 s. With an air pressure of 1.65 kg/cm² and an airflow rate of 70 to 75 L/min, the particle size of the aerosol is 0.5 to 5 µm. Five inhalations of each concentration of methacholine was given (total volume given is 50 µl).

On the screening day, FEV₁ was measured 90 and 180 s (one maneuver at each time-point) after inhalation of the vehicle (physiological saline), and the lowest value was used as baseline value. The challenge started with inhalation of methacholine chloride 0.03 mg/ml, and FEV₁ was measured after 90 and 180 s. The lower of the two recorded FEV₁ values was regarded to be the reaction to the given dose. Every fifth minute, the methacholine concentration was increased twofold, until a fall in FEV₁ of at least 20% was reached, and the challenge was then stopped. Methacholine up to a concentration of 256 mg/ml (delivered dose 12,800 µg), could be given as a highest dose. On the study days, the methacholine challenges started at a methacholine dose of three doubling doses (DD) below the threshold dose recorded at the screening visit, but were otherwise performed in the same way. The PD₂₀ value at each provocation was calculated using a log-linear scale, as recommended by the European Respiratory Society guidelines (15). If the methacholine-induced drop in FEV₁ was not reversed to at least 90% of the study day baseline value, after the last of the three methacholine challenges, 80 µg of ipratropium bromide was given by inhalation to block prolonged cholinergic bronchial smooth muscle contraction.

Baseline FEV₁ for subsequent study days was required to be within 15% of the baseline FEV₁ value measured on the first study day. In presence of a greater fluctuation, the patient was asked to return on another day.

Spirometry

The spirometries (FEV₁) were performed either with the Jaeger spirometer (Jaeger Pneumoscreen II/1; Epich Jaeger GmbH, Hoechberg, Germany) or with the Vitalograph VICA-test 3 (Vitalograph Ltd., Buckingham, UK), but the same individual equipment was used for each patient during the whole study.

Tremor Score

The patients were asked to state the degree of tremor, by giving it a number from 0 to 4 (0, no tremor; 4, very strong tremor).

Statistics

The primary efficacy variable was methacholine responsiveness, measured as PD₂₀. The patient sample size was determined to give 80% power to observe at least 1 DD difference in efficacy (shift in PD₂₀) between the two active treatments. Differences in PD₂₀ between active treatment and placebo were calculated based on the ²log of the PD₂₀ for each measurement (the difference in ²log PD₂₀ between active treatment and placebo is a measurement of protective effect, as DD of methacholine). The statistics was performed initially by comparing these values for corresponding time-points for the different doses of formoterol, salmeterol, and placebo, using analysis of variance (ANOVA) to test for variance among groups, and subsequent paired two-way t test to test for testing differences between individual groups. If a protective effect of formoterol and salmeterol was observed, versus placebo, we proceed to step two, comparing the efficacy of each active treatment with each other. The primary end-point of this study is thus a two-value comparison, testing the difference in maximal PD₂₀ methacholine between salmeterol and formoterol, subtracted by the corresponding placebo day value, as doubling doses. If the PD₂₀ was not reached with the maximal dose of methacholine (256 mg/ml; delivered dose 12,800 µg), analysis is performed as if the PD₂₀ was this dose. Secondary variables, including FEV₁, S-potassium, heart rate, and Q-T_c time (1 h after dosing) are tested using ANOVA, and subsequent paired two way t tests. The tremor score is evaluated by a nonparametric test, the Wilcoxon signed rank test. For all statistics, the level of significance was determined to be p < 0.05. Values of protective effect of methacholine responsiveness by formoterol and salmeterol are presented as mean and standard deviations (SD), as suggested previously (16). Other values are presented as mean ± standard error of the mean (SEM).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Eighteen patients fulfilled all inclusion criteria and were randomized into the study. Fifteen patients participated on all study days, and were therefore used in the paired statistical analysis. One patient dropped out because of a sudden and severe airflow obstruction induced during a methacholine provocation on the first study day, one because of repeated and probable ₂-adrenoceptor-mediated adverse events including anxiety, tremor, tachycardia, and dizziness, and one because of a deterioration of asthma between study days, documented as a decrease in baseline FEV₁.

FEV₁

The baseline percent predicted FEV₁ on the methacholine challenge days measured prior to study medication were not significantly different (86.9 ± 3.8, 88.5 ± 3.3, and 88.0 ± 3.3% before formoterol, salmeterol, and placebo, respectively). The cumulative doses of formoterol (12, 60, and 120 µg) and salmeterol (50, 250, and 500 µg) both significantly increased FEV₁ compared with placebo, although no dose-dependent effect was observed on this measurement by either drug (Figure 1). Furthermore, no significant differences in FEV₁ were observed between each formoterol and salmeterol dose (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Baseline FEV1 before each methacholine challenge, and after each dose of formoterol, salmeterol, and placebo. Data are shown as mean ± SEM.

Shift in PD₂₀ Methacholine

The geometric mean PD₂₀ after each dose of placebo during the placebo day was 67.0, 71.2, and 83.9 µg (ANOVA p = not significant [NS]). The protective effects of formoterol and salmeterol, minus the PD₂₀ after respective placebo dose, are shown as DD protection in Figure 2. The protective effect of formoterol (12, 60, and 120 µg) shows a clear dose-response relationship, with maximally measured protective effect at the highest dose (4.60 DD, SD 1.97). The maximal protective effect of salmeterol was found after 250 µg (2.84 DD, SD 1.17), with no further additive effect of 500 µg salmeterol (2.68 DD, SD 1.57). Thus, the maximal protective effect on methacholine PD₂₀ was close to two doubling doses higher for formoterol compared with salmeterol (Figure 2; p < 0.003). A 20% decrease in FEV₁ was not reached with the maximal dose of methacholine (256 mg/ml; delivered dose 12,800 µg) on five occasions with formoterol, on three occasions with salmeterol, and on one occasion with placebo, using 12,800 µg as the PD₂₀ value in these instances.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Shift in PD20 methacholine after increasing doses of formoterol (Fo) and salmeterol (Sm), minus the corresponding placebo day values. There was no significant difference between the shift in PD20 at the lowest doses of each drug (p = 0.70). However, after both of the two higher doses, formoterol caused a significantly higher shift in PD20 compared with salmeterol (p = 0.0021 and 0.0001, respectively). Data are shown as mean; for SD, see text.

Systemic Effects

S-potassium. There were no significant differences in baseline S-potassium on each study day (4.2 ± 0.1, 4.1 ± 0.1, and 4.1 ± 0.1 mmol/L before formoterol, salmeterol, and placebo, respectively, p = NS). The lowest S-potassium value after formoterol 120 µg was 3.4 ± 0.1 mmol/L (range 3.1 to 3.9 mmol/ L) and after salmeterol 500 µg 3.7 ± 0.1 mmol/L (range 3.3 to 3.9 mmol/L), which is statistically different (p = 0.001; Figure 3). After the highest dose of formoterol, one patient had S-potassium 3.1 mmol/L), and after the highest dose of salmeterol three patients had 3.3 mmol/L.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Changes from baseline in S-potassium after increasing doses of formoterol, salmeterol, and placebo. There was a significantly more pronounced drop in S-potassium 50 min after the medium dose of formoterol versus salmeterol (p = 0.008), and 110 min after the highest doses of formoterol versus salmeterol (p = 0.002). Data are shown as mean ± SEM.

Heart rate. There were no significant differences in baseline heart rate on each study day (66 ± 2, 66 ± 2, and 68 ± 3 beats/ min before formoterol, salmeterol, and placebo, respectively, p = NS). The highest heart rate values were observed 50 min after 120 µg of formoterol, 79 ± 3 beats/min, and 110 min after 500 µg salmeterol, 80 ± 3 beats/min, but these values are not statistically different (p = NS).

Q-T_c . Mean Q-T_c times were similar before treatment with formoterol, salmeterol, and placebo (0.393 ± 0.005, 0.402 ± 0.005, and 0.393 ± 0.005 s respectively, p = NS). The longest mean Q-T_c was found 50 min after 120 µg of cumulative doses of formoterol (0.419 ± 0.007 ms, range 0.370 to 0.480) and 110 min after the third dose-step of salmeterol (0.423 ± 0.006 s, range 0.380 to 0.450), the difference between salmeterol and formoterol not being statistically different.

Tremor. Significant subjective tremor was recorded after the second dose-step for both formoterol and salmeterol versus placebo (p < 0.05 versus placebo), but no significant difference was found between the two drugs. However, after the third dose-step, 13 patients reported tremor after both formoterol and salmeterol, but the severity was significantly greater after formoterol (p < 0.03; Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Individual subjective tremor scores after the highest dose of formoterol (120 µg) and salmeterol (500 µg), versus placebo. A significant difference in tremor score was found between formoterol and salmeterol, p < 0.03.

Adverse events. Headache was reported by four patients during the formoterol day, by three patients during the salmeterol day, and by no patient during the placebo day.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study shows that formoterol in a dose-dependent way protects against bronchial hyperresponsiveness, measured as methacholine responsiveness, and that salmeterol has a smaller dose-dependency and a weaker maximal protective effect. The higher efficacy of formoterol, compared with salmeterol, shows that salmeterol is a partial agonist versus formoterol in human airways in vivo. This higher airway efficacy of formoterol tended to be associated with a slightly larger drop in serum potassium, and more pronounced finger tremor.

Both formoterol and salmeterol slightly, but significantly, improved FEV₁ compared with placebo. However, the higher doses of both drugs did not improve FEV₁ further, thus not showing dose-dependent effects of the drugs on this measurement. These data confirm that this end-point, at least in these patients, is inappropriate to evaluate efficacy differences between these two drugs. This may however be an advantage in the present study, because it has been shown that baseline FEV₁ is one important factor affecting the degree of bronchial hyperresponsiveness (17, 18). Because we did not observe any difference in baseline FEV₁ between the two drugs, we can conclude that the detected differences in shift in PD₂₀ between the two treatments is a result of their different pharmacological characteristics, and not due to different baseline lung function.

Clinically, formoterol and salmeterol are recommended as regular treatment in doses up to 48 and 200 µg daily. Previously, no differences in efficacy were found between formoterol and salmeterol on methacholine responsiveness, as reported by Rabe and colleagues (14), comparing the effects of formoterol 24 µg and salmeterol 100 µg. However, we did detect differences in efficacy between the drugs at doses 25 to 150% higher than those used clinically. We also suggest that the doses used with our present study over the study day may correspond to concentrations reached during regular treatment, because both drugs have a long residence time in the airway wall, evident as a prolonged bronchodilation (5). Thus, to reach a local concentration similar to that reached during regular treatment, higher single doses would be needed. It has in fact been shown that lipophilic drugs such as fluticasone propionate have been shown to remain in the airways for many hours after inhalation (19), a phenomenon that also is likely to be valid for the lipophilic drugs formoterol and salmeterol. Therefore, we suggest that regular treatment with formoterol and salmeterol may cause higher local concentrations of the drugs in the airway wall than a single dose, which further implies that the effects observed in the present study with slightly higher doses may be relevant in some clinical situations.

The degree of efficacy difference on the shift of methacholine responsiveness amounted to almost 2 DD. This degree of shift would be regarded to be quite large, because only a 1-DD shift in responsiveness is sufficient to affect asthma symptoms (20). Therefore, the difference in efficacy of the two drugs is likely to be important physiologically.

The mechanism behind the protection against inhaled methacholine-induced bronchoconstriction by a ₂-agonist is generally regarded to be functional antagonism (23). In principal, increasing doses of any ₂-agonist increase the intracellular levels of cyclic adenosine monophosphate (cAMP), resulting in bronchial smooth muscle relaxation, or in a relaxed state, inhibition of any induced bronchial smooth muscle contraction. Therefore, in these experiments, it is likely that high doses of formoterol resulted in higher levels of intracellular cAMP in the bronchial smooth muscle, compared with salmeterol.

During regular treatment with a short-acting or long-acting ₂-agonist, tolerance to the protective effects of these drugs on bronchial responsiveness has been documented (24). In the present study, we decided to treat the patients with regular doses of salbutamol during the whole study period, to avoid pronounced fluctuations in the tolerance induced by pulsatile salmeterol and formoterol treatments on the experimental days. The dose of salbutamol chosen has been proven to be sufficient for the induction of tolerance in asthmatic patients (28).

The higher efficacy of formoterol versus salmeterol observed in the airways was also reflected in a tendency to more pronounced systemic side effects, such as more pronounced decreased S-potassium and induced finger tremor. However, the differences observed in S-potassium did not result in excessively low values, despite the high doses of formoterol and salmeterol used. This does not exclude, however, that some patients, perhaps with additional diseases, may get more pronounced changes in S-potassium when treated with formoterol compared with salmeterol. Importantly, we did not observe any significant differences in heart rate and Q-T_c intervals between the two drugs, implying that formoterol is not more arrhythmogenic than salmeterol in these patients (29, 30).

The clinical implications of formoterol being a full agonist in relation to salmeterol may be several. First, it has been suggested in a case report that a subpopulation of patients find formoterol, but not salmeterol, effective (31), which may be due to salmeterol being less efficacious than formoterol in these individuals. Second, a partial agonist has the capacity to attenuate the effect of an agonist with similar or greater intrinsic activity (11), but no report of this being the case in the clinical situation has been published. In fact, one study has failed to document that salmeterol in doses up to 200 mg attenuates the bronchodilating effects of additional salbutamol (32), in stable asthmatic patients with moderate reversibility. The more important implication, however, is that pretreatment with a partial ₂-agonist in patients experiencing severe acute bronchoconstriction, may partly block the effect of a short-acting ₂-agonist used as rescue medication (i.e., salbutamol or terbutaline), which may be detrimental in this situation. Studies evaluating any such hypothetical consequence of regular salmeterol treatment should therefore be of high priority.

Despite these data showing that salmeterol is less efficacious in human asthmatic airways in vivo, it is possible that there may be some clinical advantages with the use of a partial agonist. For example, it is possible that a partial agonist causes less side effects, as demonstrated in this study (Figures 3 and 4). It has also been suggested that a partial agonist may cause less tolerance on the ₂-receptors, but this has not been confirmed (26, 27).

This study, for the first time, shows strong evidence of salmeterol being a partial agonist on the ₂-receptor in comparison with formoterol in human airways in vivo. This difference in efficacy has several important clinical implications, especially in relation to acute asthma, which warrants further investigation.