It is now generally accepted that desensitization of β-adrenoceptors develops after long-term exposure to β-adrenoceptor agonists. Desensitization occurs both with long-term exposure to exogenously applied agonists (as is the case in the therapy of bronchial asthma, see 1 for review) and with chronically increased endogenous catecholamines [e.g., in chronic heart failure (see 2 for review)]. Thus desensitization limits the therapeutic efficacy of exogenously applied β-adrenoceptor agonists (asthma) and may even contribute to progression of the disease (chronic heart failure). Drugs that can prevent or at least attenuate desensitization of β-adrenoceptor mediated functions may therefore be of particular therapeutic value. Ketotifen, an antianaphylactic drug used in the therapy of bronchial asthma, may be such a drug. We and others previously showed that in human lymphocytes, ketotifen is able to prevent or at least attenuate the development of β2-adrenoceptor desensitization and to restore blunted β2-adrenoceptor responsiveness induced by prolonged exposure to β2-adrenoceptor agonists (3-5). Studies in healthy volunteers (6) and asthmatic patients (4) suggested that the therapeutic efficacy of ketotifen when given in combination with β2-adrenoceptor stimulants in the therapy of asthma is-at least in part-due to this effect. Moreover, we previously demonstrated that in healthy volunteers, prolonged administration of the selective β2-adrenoceptor agonist terbutaline also selectively desensitizes β2-adrenoceptor-mediated cardiovascular functions and that ketotifen is able to attenuate this desensitization markedly (7,8). The mechanisms underlying this modulating effect of ketotifen on β-adrenoceptor function are not fully elucidated at present.
Disodium cromoglycate (cromolyn sodium) is another antiallergic drug established in the therapy of mild to moderate bronchial asthma (9) with a mode of action different from that of ketotifen. As for ketotifen, however, a number of observations provide indirect evidence for a possible interaction between disodium cromoglycate and the function of the β-adrenergic system. In animals it potentiates the relaxation of tracheal strips induced by β2-adrenoceptor agonists (10). In trials in patients, disodium cromoglycate has no bronchodilator activity when given as monotherapy (11,12), but it potentiates the bronchodilatory effect of the β2-adrenoceptor agonist reproterol (12,13). The combination of disodium cromoglycate with the β2-adrenoceptor agonist terbutaline is more effective in antagonizing cold-induced bronchoconstriction than is monotherapy alone (11); furthermore, disodium cromoglycate protects against the bronchoconstrictor effects of the nonselective β-adrenoceptor blocker propranolol but not against histamine-induced bronchoconstriction (14). Finally, in healthy volunteers, it prevents downregulation of lymphocyte β2-adrenoceptors induced by 1-week administration of inhaled fenoterol (15). Therefore we hypothesized that disodium cromoglycate might also interact with β2-adrenoceptor-mediated cardiovascular functions in humans. The primary aim of this study was therefore to investigate whether or not disodium cromoglycate is able to influence the desensitization of β2-adrenoceptor-mediated cardiovascular functions during long-term administration of the β2-adrenoceptor-selective agonist terbutaline. Isoprenaline-induced changes in heart rate and diastolic blood pressure were assessed as mixed β1/β2-adrenoceptor-mediated cardiovascular responses (16-19), and exercise-induced tachycardia was analyzed as a predominantly β1-adrenoceptor-mediated cardiovascular function (16,17,20). As a secondary end point, the effect on the β2-adrenoceptor-mediated noncardiovascular function of tremulousness (21) also was studied.
SUBJECTS AND METHODS
Ten young, healthy, male volunteers (mean age ± SEM, 25 ± 2 years) who were drug free and had no clinical, electrocardiographic, hematologic, or biochemical evidence of disease at prestudy evaluation were studied. The study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the ethical committee of the University Hospital, Essen. All subjects had given written, informed consent.
The study followed a double-blind, randomized, placebo-controlled crossover design. We compared the effects of either placebo or disodium cromoglycate on β-adrenoceptor-mediated cardiovascular and noncardiovascular functions after a 2-week treatment with the selective β2-adrenoceptor agonist terbutaline. The two treatment phases were separated by a 4-week washout period. The study protocol is summarized in Fig. 1. At days 1 and 2 (i.e., before the commencement of any drug therapy), subjects underwent a baseline exercise and isoprenaline infusion test. Thereafter they were treated with disodium cromoglycate 4 × 200 mg orally/day (Colimune capsules; Fisons Arzneimittel GmbH, Köln, Germany; at 6 a.m., 12 noon, 6 p.m., and 12 midnight) or matching placebo, respectively, for 3 weeks (i.e., until the end of the study period). At days 8 and 9 (i.e., after 1-week therapy with disodium cromoglycate or placebo), the exercise and isoprenaline-infusion test were repeated before terbutaline administration was started. From day 9 to 23, subjects received terbutaline, 3 × 5 mg orally/day (Bricanyl forte tablets; Astra Chemicals, Wedel, Germany; at 7 a.m., 2 p.m., and 9 p.m.). At days 22 and 23, with the volunteers still receiving treatment, the exercise and isoprenaline-infusion test were repeated for a last time 2 h after intake of the morning dose of terbutaline. The order in which the exercise and isoprenaline-infusion tests were performed (exercise on days 1, 8, 22; isoprenaline infusion on days 2, 9, 23, or vice versa) was randomly allocated between subjects but kept constant for each subject during the entire study. All tests were performed after an overnight fast between 8 and 11 a.m., with the subjects in the supine position in a quiet room at a constant ambient temperature after subjects had been resting for ≥30 min.
The exercise test was conducted on a bicycle (Bosch, Berlin, Germany). Subjects started with an initial workload of 50 W, which was increased every 3 min until a maximal workload of 150 W was attained.
The nonselective β-adrenoceptor agonist isoprenaline (Aleudrina; Boehringer Ingelheim, Germany) was administered as a continuous intravenous (i.v.) infusion with five incremental doses of 3.5, 7.0, 17.5, 35, and 70 ng/kg/min by means of a perfusion pump (Braun, Melsungen, Germany). Each dose was infused for 8 min; during the last 5 min of each dose step, blood pressure and heart rate were measured at 1-min intervals. The mean of these five recordings was chosen for statistical analysis. Systolic and diastolic (phase V) blood pressures were measured with a standard mercury sphygmomanometer, and heart rate was calculated from the RR interval of an ECG hard copy.
Throughout the entire study period, volunteers were asked to complete a self-administered questionnaire daily and to rate their subjective awareness of tremulousness on a score graded from 0 to 3 with 0, tremulousness not present; 1, mildly present; 2, moderately present; and 3, severely present.
One volunteer developed frequent ventricular ectopic beats during the isoprenaline infusion on days 1 and 8 of the study, necessitating termination of the infusion. For safety reasons, he was therefore excluded from further participation in the study, and data evaluation was performed on the remaining nine subjects.
Supine resting heart rate and blood pressure were measured immediately before start of the isoprenaline infusion and exercise test, respectively. These recordings served as baseline for the isoprenaline infusion and exercise test. The effect of i.v. isoprenaline and exercise on heart rate and blood pressure were described as change from this baseline. The effect of placebo and disodium cromoglycate on resting heart rate and blood pressure and on the isoprenaline and exercise-induced changes were statistically analyzed by two-way analysis of variance (ANOVA) analyzing the effects of treatment (placebo vs. cromoglycate), time (day 1 vs. day 8 vs. day 22) and treatment-time interaction. If ANOVA indicated a significant effect, this was further investigated by post hoc paired t tests with the Bonferroni method to correct for multiple comparisons.
For the highest dose of isoprenaline of 70 ng/kg/min, complete measurements of blood pressure and heart rate at all six study days could be obtained in only four of the nine subjects. In the remaining five subjects, this dose was either not administered at all when the increase in heart rate exceeded 40-45 beats/min at the dose of 35 ng/kg/min or because the subject had experienced side effects (nausea, palpitations) when the dose of 70 ng/kg/min had been delivered during a preceding study day. Therefore ANOVA for the isoprenaline-induced effects was generally restricted to the first four doses (3.5, 7.0, 17.5, and 35 ng/kg/min).
The effects of placebo or disodium cromoglycate on terbutaline-induced tremulousness were analyzed by comparison of the tremulousness score by the Wilcoxon signed-rank test. This analysis was restricted to the first 8 days of terbutaline administration (study days 9-16; i.e., to the period for which the median score during placebo treatment differed from zero; compare Fig. 2).
All hemodynamic data are provided as mean ± standard error of the mean (SEM) unless otherwise indicated. Throughout a p value of <0.05 (two-tailed) was considered statistically significant. In figures displaying isoprenaline dose-response curves, doses of isoprenaline are presented on a logarithmic scale.
The most prominent side effect of terbutaline was tremulousness, which occurred in all subjects after initiation of terbutaline administration. There was a significant (p < 0.001 by Wilcoxon signed-rank test) difference in tremulousness score between placebo and disodium cromoglycate during the first 8 days of terbutaline administration (study days 9-16; Fig. 2). When terbutaline was administered with placebo, tremulousness disappeared only after 8 days of terbutaline administration. When terbutaline was given with disodium cromoglycate, the tremulousness score returned to baseline (score 0) already after 4 days of terbutaline treatment (Fig. 2).
On day 22, after 2 weeks of treatment with terbutaline, resting heart rate and systolic blood pressure were significantly higher compared with days 1 and 8 (Table 1). There was, however, no difference between placebo and disodium cromoglycate on any of the 3 study days. Mean resting diastolic blood pressure pooled across all 3 study days was slightly lower with disodium cromoglycate (66.8 ± 1.6 mm Hg) compared with placebo (68.7 ± 1.3 mm Hg; ANOVA main treatment effect, p = 0.046). There was, however, no significant difference between placebo and disodium cromoglycate on any of the 3 individual study days. Diastolic blood pressure on day 22 was slightly lower compared with days 1 and 8, but this trend was not statistically significant.
Isoprenaline-mediated cardiovascular effects
Isoprenaline dose-dependently increased heart rate and systolic blood pressure and decreased diastolic blood pressure (Figs. 3 and 4; Table 2). For the increase in heart rate (Fig. 3) and systolic blood pressure and the decrease in diastolic blood pressure (Fig. 4), ANOVA indicated a significant time effect (p < 0.001) with a significantly reduced increase in heart rate and systolic blood pressure and a significantly smaller decrease in diastolic blood pressure on day 22 (i.e., after 2 weeks of terbutaline administration) compared with day 8 (i.e., the last day before administration of terbutaline). Averaging the isoprenaline-induced changes from baseline for the first four dose levels (3.5, 7.0, 17.5, and 35 ng/kg/min), the mean percentage inhibition of the response on day 22 relative to day 8 was 53.3% for heart rate, 34.3% for systolic, and 55.6% for diastolic blood pressure (Table 2). There were, however, no significant treatment-time interactions by ANOVA (i.e., this time-dependent change in cardiovascular responsiveness to isoprenaline was not different between placebo and cromoglycate for both heart rate and systolic and diastolic blood pressures). The overall decrease in diastolic blood pressure pooled across all 3 study days and all doses (3.5-35 ng/kg/min) was less with cromoglycate (−8.3 ± 0.9 mm Hg) than with placebo (−11.3 ± 1.2 mm Hg; p = 0.03, ANOVA main treatment effect).
Exercise-mediated cardiovascular effects
For the exercise-induced tachycardia, there was likewise a significant time effect (p < 0.001 by ANOVA) with a reduced increase in heart rate on day 22 compared with day 8 (Fig. 5). The magnitude of this reduction in exercise-induced tachycardia was very small (mean percentage inhibition, day 22 vs. day 8, of 5.6%) but statistically significant. ANOVA also indicated a significant treatment-time interaction (p < 0.01) with a significant difference between day 1 of the placebo phase and day 1 of the cromoglycate phase, with a slightly larger increase in heart rate during cromoglycate (mean difference between placebo and cromoglycate in exercise-induced increase in heart rate across all workload levels of 2.3 ± 0.7 beats/min). Responses on days 8 and 22 did, however, not differ between placebo and disodium cromoglycate.
After 2 weeks' administration of the β2-adrenoceptor-selective agonist terbutaline, we observed a significant rightward shift of the dose-response curve for the isoprenaline-induced decrease in diastolic blood pressure, for the isoprenaline-induced increase in heart rate and systolic blood pressure, and for the bicycle exercise-induced increase in heart rate. These results demonstrate that β-adrenoceptor-mediated cardiovascular responsiveness was blunted after 2 weeks' administration of terbutaline. The magnitude of this rightward shift was greatest for the isoprenaline-induced reduction in diastolic blood pressure and isoprenaline-induced tachycardia (compare Figs. 3, 4, and Table 2). It was very small-although statistically significant-for exercise-induced tachycardia (compare Fig. 5 and Table 2). Isoprenaline-induced reduction in diastolic blood pressure and increase in heart rate are "mixed" β1/β2 responses (16-19), whereas exercise-induced tachycardia is a predominantly β1-adrenoceptor-mediated response (16,17,20). Therefore these results confirm our previous findings in healthy volunteers (7,8) that prolonged stimulation of cardiovascular β2-adrenoceptors by terbutaline results in a desensitization of cardiovascular β-adrenoceptor-mediated functions and that this desensitization predominantly affects β2-adrenoceptors. We also observed development of tolerance to the noncardiovascular side effect of tremulousness (Fig. 2). This is in keeping with previous findings of development of tolerance to the subjective awareness of tremulousness during prolonged bronchodilator therapy in asthmatic patients (22). Because tremulousness has been shown to be another very specific parameter of β2-adrenoceptor agonism (16,18,21), this result provides direct evidence that terbutaline-induced desensitization is not limited to β-adrenoceptor-mediated cardiovascular effects but also involves noncardiovascular functions mediated by β2-adrenoceptors.
In two previous studies in healthy volunteers, we were able to demonstrate that ketotifen significantly attenuated terbutaline-induced desensitization of β2-adrenoceptor-mediated cardiovascular responses (7,8). Evidence from in vitro studies investigating guinea pig tracheal strips (10) and lymphocyte β2-adrenoceptors in healthy volunteers (15) and therapeutic trials in asthmatic patients (11-14) suggest that disodium cromoglycate might interfere with β-adrenoceptor function in a way that increases β2-adrenoceptor responsiveness. We therefore hypothesized that disodium cromoglycate might also be able to attenuate desensitization of β2-adrenoceptor-mediated cardiovascular function in humans. However, in this study there was no evidence for such an effect. If anything, inspection of Figs. 3-5 would rather suggest a promoting influence of disodium cromoglycate on the development of tolerance; the rightward shifts of the dose-response curves for the cardiovascular effects of isoprenaline and exercise were slightly more pronounced after 2 weeks' treatment with terbutaline when disodium cromoglycate was administered concomitantly, although this effect was not statistically significant. The significant difference between placebo and disodium cromoglycate on tremulousness score with a significant shorter period of tremulousness during concomitant administration of cromoglycate also suggest a promoting rather than an inhibitory effect of disodium cromoglycate on the development of tolerance (Fig. 2). The failure of disodium cromoglycate to influence β2-adrenoceptor-mediated cardiovascular effects of terbutaline could be due to insufficient plasma levels of disodium cromoglycate, a drug that is only poorly absorbed after oral administration (23,24). However, the finding of a significant treatment difference between placebo and disodium cromoglycate with respect to their effect on tremulousness score (see earlier) clearly argues against this pharmacokinetic explanation.
The pharmacologic mechanism by which ketotifen is able to attenuate desensitization of β2-adrenoceptor-mediated effects is not well understood. The therapeutic efficacy of both disodium cromoglycate and ketotifen in the treatment of bronchial asthma and allergic disorders is attributed to their ability to prevent the release of inflammatory mediators from mast cells (23-25). The negative findings of our study with disodium cromoglycate would therefore suggest that pharmacologic properties other than this "mast cell stabilizing effect" are responsible for the effectiveness of ketotifen to blunt β2-adrenoceptor desensitization.
In summary, this study first confirms that long-term selective β2-adrenoceptor stimulation desensitizes β2-adrenoceptor-mediated cardiovascular in vivo functions in humans. It demonstrates also that this desensitization affects noncardiovascular β2-adrenoceptor-mediated responses, and third, shows that disodium cromoglycate is not able to prevent or attenuate desensitization of β2-adrenoceptor-mediated cardiovascular and noncardiovascular functions in humans.
Acknowledgment: This study was supported by a grant of the Deutsche Forschungsgemeinschaft (O-E.B.: DFG: Br 526/3-2). The assistance of the hospital pharmacy staff for the preparation of placebo capsules is gratefully acknowledged.
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