Eur J Clin Pharmacol (1996) 50 : 269–273

© Springer-Verlag 1996

P H A R M AC O DY NAMICS

D. Schmidt · R. A. Jörres · K. F. Rabe · H. Magnussen

Reproducibility of airway response to inhaled bradykinin and effect of the neurokinin receptor antagonist FK-224 in asthmatic subjects

Received: 18 August 1995 /Accepted in revised form: 3 January 1996

Abstract Objective: Inhaled neurokinins have been shown to induce bronchoconstriction in asthmatic subjects. We have investigated the effect of a neurokinin receptor antagonist, FK-224, on bradykinin (BK)induced bronchoconstriction, and have compared its effect with the spontaneous variability of BK responsiveness. Methods: Thirteen subjects with mild extrinsic bronchial asthma participated in the study. Four BK inhalation challenge tests (Study Days 2 to 5) were performed over a period of several weeks. On Study Days 4 and 5 subjects inhaled either 2 mg FK-224 or placebo 30 min before the BK challenge. Results: The geometric mean PC20FEV1 of BK was 0.04, 0.06, and 0.10 mg ·ml[1 on the first and second BK challenge and after placebo. Mean PC20FEV1 after FK-224 was 0.20 mg ·ml[1 and was not different from placebo, whereas there was a significant effect in PC15FEV1. The mean shift in PC20FEV1 after FK-224 vs placebo was 1.0 doubling concentrations. The mean changes in BK responsiveness on the second BK challenge and placebo days compared to the first BK challenge were 0.6 and 1.3 doubling concentrations. We observed a significant fall in FEV1 after inhalation of saline plus ethanol, which was the diluent for BK (mean decrease 4.2%). Conclusion: The data demonstrate that inhalation of 2 mg FK-224 is only marginally effective against BKinduced bronchoconstriction in mild asthmatic subjects and that its effect is similar to the variability in BK responsiveness assessed over several weeks. Key words Airway hyperresponsiveness, Bradykinin; bronchial challenge, neurokinins, ethanol, bronchoconstriction, lung function, FK-224.

D. Schmidt · R. A. Jörres · K. F. Rabe (*) · H. Magnussen Krankenhaus Grosshansdorf, Zentrum für Pneumologie und Thoraxchirurgie, LVA - Freie und Hansestadt Hamburg, Wöhrendamm 80, D-22927 Grosshansdorf, Germany

Introduction Bronchial asthma is characterised by respiratory symptoms associated with variable airway calibre and airway hyperresponsiveness, which can be demonstrated in response to different stimuli, such as histamine, methacholine, prostaglandin D2 and bradykinin (BK). Patients with bronchial asthma show increased levels of kinins in plasma and bronchoalveolar lavage fluid [1–3]. These findings, and the observation that inhalation of BK causes asthma-related symptoms, such as cough, sore throat and retrosternal discomfort, as well as bronchoconstriction, arteriolar vasodilatation, increased mucus secretion, and stimulation of sensory non-myelinated nerves, suggest that kinin release is involved in asthma [4]. It has been suggested that BK stimulates sensory nerve endings and releases tachykinins. Reproducibility of the response to inhaled BK has been reported to be within one doubling concentration over a period of about 3–7 days [5]. It is not known whether BK responsiveness shows increased variation over a longer period of time; histamine and methacholine responsiveness exhibit similar short-term and longterm variation [6]. Several selective and non-selective neurokinin receptor antagonists have been developed to investigate the role of tachykinins in bronchial asthma. Inhalation of 4 mg FK-224, a neurokinin antagonist with NK-1 and NK-2 receptor selectivity [7], reduced BK-induced bronchoconstriction in asthmatic patients [8]. In order to evaluate the potential utility of a drug, it appears reasonable to compare the magnitude of its effects with the spontaneous variability, independently of statistical considerations. Following this approach, the aim of our study was to assess the effect of 2 mg FK-224 on BK-induced bronchoconstriction and to compare these data with the variabilty of BK responsiveness over a period of several weeks.

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Materials and methods Subjects We studied 13 subjects (Table 1) with mild, stable bronchial asthma [10] and a positive skin prick test to at least one common allergen. Inhaled b2-adrenoceptor agonists were withheld for at least 6 h and disodium cromoglycate for at least 48 h before each test, whereas inhaled corticosteroids were continued. None of the subjects was a smoker or had had an upper respiratory tract infection within the last month prior to the study. Subjects with pollen allergy were studied out of season. The study was approved by the Ethical Committee of the Chamber of Physicians of Schleswig-Holstein, Germany. Subjects were instructed about the aim of the study and gave their written informed consent to it. Lung function measurements FEV1 (forced expiratory volume in one second) was measured with a wedge spirometer (Vitalograph, Hamburg, Germany), accepting the best of three measurements as the true value. Upon entry into the study (Table 1), FEV1 and inspired vital capacity (VC) were determined by a pneumotachygraph (Bodytest, Jaeger, Würzburg, Germany). Bronchial challenges Bronchial challenges with BK or methacholine were performed in a standardised manner [11, 12], using a DeVilbiss nebuliser (No. 646). The aerosol was generated for 0.6 s at the beginning of 5 slow inspirations from functional residual to total lung capacity (total volume of aerosol 60 µl). Solutions of BK acetate salt (Sigma Chemie, Deisenhofen, Germany) in saline plus 10 % (v /v) ethanol, or methacholine chloride in 0.9 % saline were prepared immediately before use. After inhaling saline (BK- and methacholine challenge) followed by saline plus ethanol (BK challenge only), subjects inhaled doubling concentrations of BK or methacholine. BK concentrations ranged from 0.075 µg·ml[1 to 10 mg·ml[1, yielding a total of 18 doubling concentrations. In some subjects, starting concentrations were higher according to their known individual responsiveness. Lung function was measured 1, 3, and 5 min after inhalation. Data evaluation was based on the maximal responses.

The challenge was stopped once at least a 20 % fall in FEV1 had occurred as compared to the values after inhalation of the corresponding solvent. Dose-response curves were constructed by plotting FEV1 against log concentration and linear interpolation was used to compute provocative concentrations which produced a 20 or 15 % fall in FEV1 (PC20FEV1 and PC15FEV1).

Test medication FK-224 2 mg or placebo (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) were administered as two puffs from metered-dose inhalers. Subjects were instructed to inhale the test medication deeply with synchronised actuation and then to hold the breath for 10 s.

Study design Each subject was studied at the same time of the day, on five different occasions within a 2 month period. On Day 1, lung function measurements, methacholine challenge, and skin prick test were performed. On Days 2 and 3, BK challenges without test medication were performed. On Days 4 and 5, either placebo or FK-224 were administered in a randomised, double-blind cross-over manner. FEV1 was measured immediately before and 15 and 30 min after medication. BK challenges were started 30 min after drug inhalation. The FEV1 at 30 min served as the baseline value for the challenge. Study days 1, 2, and 3 were separated by a time period of at least 3 (average 5) days, and Days 3, 4, and 5 by at least 7 days. On average, Days 1 and 2 were separated by 5, Days 2 and 3 by 11, Days 3 and 4 by 24, and Days 4 and 5 by 16 days. Between Day 2 and the placebo day, there were on average 39 days, and between Day 3 and the placebo day 28 days.

Data analysis Arithmetic means and standard deviations of lung function parameters were calculated. Provocative concentrations were logarithmically transformed for analysis and geometric mean values were computed; standard deviations (SD) and changes of BK responsiveness were expressed as numbers of doubling concentrations. Reproducibility was quantified as the mean of the absolute values of these differences determined on Days 2 and 3 and the placebo day.

Table 1 Characteristics of the subjects. Arithmetic mean and standard deviation Patient No.

Sex

Age (y)

Height (cm)

VC (l)

FEV1 (l)

FEV1 (%)c

PC20FEV1 Methacholine (mg ·ml[1)

Therapyb

1 2 3 4 5 6 7 8 9 10 11 12 13

H.K. O.B. J.G. K.R. S.K. O.H. O.G. S.F. K.T. S.B. A.W. F.H. C.R.

F M M M F M M M F F F M F

24 24 24 34 33 23 27 21 25 26 25 24 24

173 183 183 187 176 182 188 188 167 165 170 181 160

4.16 6.26 6.97 5.80 4.42 6.44 5.40 7.45 4.19 3.89 3.69 4.91 3.73

3.15 5.00 4.65 4.91 3.19 4.60 3.66 4.75 2.99 2.68 2.70 2.89 2.90

87.3 107.4 99.9 107.6 90.4 99.8 76.1 97.6 88.7 82.0 77.4 63.3 93.7

0.63 0.97 0.07 1.11 0.37 0.17 0.21 0.06 1.30 0.02 2.00 1.02 0.10

Ø B B,D Ø B,Ci B Ø B Ci Ø B B B

Mean (SD)

–

26 (4)

177 (9)

5.17 (1.29)

3.70 (0.93)

a

90 (13)

Geometric mean value, standard deviation as number of doubling concentrations B = b-2-adrenoceptor agonists, Ci = inhaled corticosteroids, D = disodium cromoglycate c percentage of predicted value according to Reference 9 b

0.31 (2.1)a

271 Lung function parameters and provocative concentrations were analysed by the paired t-test. To evaluate the effect of inhalation of saline and saline plus ethanol, we also applied two-way and threeway ANOVA and post-hoc Newman-Keuls tests. To study the relationship between lung function and BK provocative concentrations, linear correlation coefficients were computed. Tests were performed two-tailed and statistical significance was assumed at P ≤ 0.05. P values are given explicitely, so correction for multiple comparisons has not been applied.

Results Subjects 19 subjects entered the study. Four subjects with bronchial asthma were excluded as they did not demonstrate a sufficient response to BK. These subjects, who showed PC20FEV1 of methacholine of 0.6–2.0 mg · ml[1, had a maximum fall in FEV1 of 1–6 % after inhalation of BK. Therefore, subjects with high methacholine responsiveness were more likely to respond to BK than subjects with low responsiveness. One subject had to be excluded from the final analysis as he demonstrated a more than a 20 % fall in FEV1 at the lowest BK concentration both on Days 2 and 3, and another subject was excluded due to his response to ethanol (see below). Baseline measurements Baseline FEV1 did not show significant differences between study days, the overall mean value (SD) being 3.79 (0.81) l (Table 2). FEV1 decreased after inhalation of saline on Study Day 1 (P = 0.011) but not on the other study days. Summarising over all BK challenges, there was no significant effect of saline inhalation on FEV1 (overall (SD) change, [1.5 (3.3) %). Effect of FK-224 on lung function FEV1 measured 15 or 30 min after the administration of placebo was not significantly different from baseline,

mean changes (SD) being [0.9 (3.4)% at 15 min and [0.8 (3.0)% at 30 min (Table 2). After inhalation of FK-224, there was a statistically significant fall in FEV1 of 2.3 (2.9)% at 15 min (P = 0.013) but not at 30 min (fall of 1.3 (4.4)%). Effect of ethanol inhalation FEV1 was evaluated after inhalation of saline or saline plus ethanol on Days 2, 3, and after placebo inhalation. There was a statistically significant fall in FEV1after ethanol compared to baseline (mean (SD), 4.2 (6.0)%, P = 0.0043). After FK-224, the mean response to ethanol was 3.7 (4.3)% (P = 0.017), (Table 2). Reproducibility of bradykinin responsiveness and effect of FK-224 PC20FEV1 of BK did not differ significantly between Days 2, 3 or after placebo (Table 2, Fig. 1). After FK224, PC20FEV1 was not significantly changed compared to placebo (P = 0.075), whereas PC15FEV1 was significantly increased by FK-224 (P = 0.016). After FK-224 compared to placebo, the mean increase in PC20FEV1 was 0.97 (1.79) and in PC15FEV1 1.35 (1.75) doubling concentrations. Reproducibilty of PC20FEV1 on Day 3 vs Day 2 was 1.29, on Day 3 vs the placebo day 2.52, and on Day 2 vs the placebo day 2.64-doubling concentrations. For PC15FEV1, the corresponding values were 1.07, 2.08, and 1.74-doubling concentrations. Correlation analysis There was no statistically significant correlation between baseline lung function parameters and methacholine or BK responsiveness, neither after placebo nor after FK-224. Furthermore, PC20FEV1 of BK and methacholine were not significantly correlated (analysis of covariance, P = 0.08; Fig. 2). The airway

Table 2 Lung function parameters (FEV1) and provocative concentrations of bradykinin (PC20FEV1). BK(1) on Day 2 and BK(2) on Day 3. Mean and standard deviations. % = percent change with respect to baseline

FEV1 Baseline 15 min after medication 30 min after medication After saline Change after saline After ethanol + saline Change after ethanol + saline

(L) (L) (L) (L) (%) (L) (%)

PC20FEV1 (BK)a PC15FEV1 (BK)a

(mg·ml[1) (mg·ml[1)

a

BK (1)

BK (2)

Placebo

FK-224

3.82 (0.85) – – 3.75 (0.84) [ 1.9 (2.3) 3.56 (0.86) [ 6.5 (9.1)

3.78 (0.75) – – 3.75 (0.74) [ 0.7 (2.5) 3.68 (0.78) [ 3.0 (3.4)

3.76 (0.85) 3.74 (0.92) 3.74 (0.90) 3.75 (0.87) [ 0.5 (3.0) 3.66 (0.90) [ 3.0 (3.0)

3.78 (0.88) 3.70 (0.90) 3.75 (0.96) 3.67 (0.87) [ 2.9 (4.7) 3.65 (0.90) [ 3.7 (4.3)

0.040 (5.7) 0.026 (5.6)

0.060 (5.5) 0.037 (5.5)

0.100 (5.6) 0.032 (5.5)

0.196 (4.8) 0.081 (5.2)

Geometric mean and standard deviation as number of doubling concentrations

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Fig. 1 Provocative concentration of bradykinin, PC20FEV1, in the 1st (open circle) and 2nd (open square) provocation testing (Days 2 and 3) and after inhalation of FK-224 (closed circle) vs PC20FEV1 after inhalation of placebo

Fig. 2 Provocative concentrations of bradykinin, PC20FEV1, on Day 2 (open circle) and Day 3 (open square) and after inhalation of placebo (open triangle) vs PC20FEV1 of methacholine

response to ethanol showed a significant correlation with that to saline (analysis of covariance, P < 0.01; Fig. 3). Adverse effects of bradykinin inhalation Inhalation of BK caused retrosternal discomfort and cough in all subjects, even those who did not show a response to BK. FK-224 had no effect on these symptoms.

Discussion Our study demonstrated that a single inhaled dose of 2 mg of the neurokinin receptor antagonist FK-224 was

Fig. 3 Percent changes of FEV1 from baseline after inhalation of ethanol vs corresponding percentage change after inhalation of saline on Study Days 2 (open circle) and 3 (open square) after placebo ( filled circle) and FK-224 (cross). Note the different scales of the abscissa and ordinate

only marginally effective in inhibiting BK-induced bronchoconstriction. The effect of FK-224 and the variability of the BK response assessed over a period of several weeks, were in the same range. Ichinose et al. [8] reported that 4 mg FK-224 administered 30 min before a BK-challenge exerted a significant protective effect. We found that 2 mg FK224 was only weakly effective, as PC20FEV1 showed only a non-significant trend in favour of FK-224 (0.05 < P < 0.10). The small effect of FK-224 could be attributed to fact that the dose administered was half of that given in the previous study [8], and because we used a different measure of airway hyperresponsiveness. The effect of 4 mg FK-224 [8] was to shift PC35sGaw of BK (provocative concentration causing a 35 % fall in specific airway conductance) by about 3 doubling concentrations. In contrast, in our study 2 mg FK-224 caused a shift of about 1–1.5-doubling concentrations. On average, a fall in sGaw of 35 % can be considered as equivalent to a fall in FEV1 of 10–15 % [13]. It is interesting that the PC15FEV1 indicated a slightly larger effect of FK-224 than the conventional PC20FEV1. In order to evaluate the protective effects of antiasthma drugs, it is reasonable to compare drug-induced changes with spontaneous variability. According to the results of Ichinose et al. [8], changes PC35sGaw after FK-224 ranged from 0.0 to 4.7-doubling concentrations, and in our data from [1.4 to 4.2 and [0.5 to 5.6-doubling concentrations, for PC20FEV1 and PC15FEV1, respectively. Unfortunately, the previous study did not address the variability of BK responses. We found that the differences in BK responsiveness ranged between [2.5 and 3.1-doubling concentrations over a period of 1–2 weeks and between [3.8 and 11doubling concentrations over a period of 5–6 weeks. All subjects were clinically stable throughout the course of the study. The fact that the protective effect of 2 mg FK-224 did not exceed 1.5-doubling concentrations,

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which is similar to the spontaneous variability of BK responsiveness, underlines our conclusion that 2 mg FK-224 is of minimal effectiveness in protection against BK-induced bronchoconstriction. In previous studies [4, 5, 14], a correlation between the airway response to BK and methacholine or histamine has been reported. Although there was an association between the degree of methacholine and BK responsiveness, there was no statistically significant correlation between the provocative concentrations of BK and methacholine within the range of airway hyperresponsiveness encountered in our group of subjects. Our data are in accordance with those of Rajakulasingam et al. [15], who was also unable to find a correlation in terms of the provocative concentration. Given the multiplicity of actions of BK, it is reasonable to assume that BK responsiveness is not closely related to histamine or methacholine responsiveness. Two subjects were on inhaled corticosteroids and one took disodium cromoglycate. When data were analysed with and without these three subjects, the results were virtually identical. Inhaled b2-adrenoceptor agonists were withheld at least 6 h before each testing. Therefore, it is unlikely that our results have been biased by the current medication of our subjects. Whereas FK-224 did not show significant protection against BK-induced bronchoconstriction, there was a significant effect on bronchial tone. These results are in contrast to the previous findings [8] that inhalation of FK-224 did not change lung function, using the same inhalation procedure as in our study. As the containers used here were identical, except for the presence or absence of the drug, it is reasonable to assume that observed a drug-related effect, which cannot be explained at present. We wish to emphasise, however, that the mean change in FEV1 amounted to [2.0 ([6.3 to 2.9)%, which seems not to be of clinical significance. We demonstrated a small but consistent bronchoconstrictor effect of low doses of ethanol. It may be noteworthy that one subject, who was excluded from the study, demonstrated a 48 % fall in FEV1 after inhalation of 10% ethanol plus saline, but without any response to saline alone.The same subject showed a 25 % fall in FEV1 after inhalation of an ethanol concentration as low as 0.1 %. The effect of ethanol was not due to an irritant effect of the inhaled aerosol. In normal subjects we did not observe any effect of ethanol on lung function (data not shown). This is consistent with the study of Zuskin et al. [16] who showed that, after inhalation of ethanol in normal subjects, there was no significant change in FEV1 , although there appeared to be an effect on flow rate. We do not know the mechanism by which ethanol may cause bronchoconstriction in asthmatics. A physical mechanism such as cooling of the airways by evaporation appear to be irrelevant. It is possible that the response is mediated by acetaldehyde. Our results emphasise the need to consider ethanol-induced bronchoconstriction.

When ethanol is used as a diluent for inhaled substances. In summary, we conclude that in subjects with mild asthma, inhalation of 2 mg FK-224 did not inhibit bronchoconstriction induced by inhaled bradykinin to a clinically relevant degree. Airway responsiveness to bradykinin showed considerable variability over a period of several weeks. In addition, we observed mild bronchoconstriction after inhalation of low doses of ethanol. Acknowledgement The study was supported by the Fujisawa Pharmaceutical Company, Japan. This paper forms part of the MD thesis of Dunja Sormidt

References 1. Abe K, Watanabe N, Kumagai N, Mouri T, Seki T, Yoshinaga K (1967) Circulating plasma kinin in patients with bronchial asthma. Experientia 23 :626–627 2. Christiansen SC, Proud D, Cochrane CG (1987) Detection of tissue kallikrein in the bronchoalveolar lavage fluid of asthmatic subjects. J Clin Invest 79 : 188–197 3. Christiansen SC, Zuraq BL, Proud D, Cochrane CG (1989) Inhibition of human bronchial kallikrein in asthma. Am Rev Respir Dis 139 :1125–1131 4. Fuller RW, Dixon CM, Barnes PJ (1987) Bradykinin-induced bronchoconstriction in human. Am Rev Respir Dis 135 :176–180 5. Polosa R, Phillips GD, Lai CKW, Holgate ST (1990) Contribution of histamine and prostanoids to bronchoconstriction provoked by inhaled bradykinin in atopic asthma. Allergy 45 :174–182 6. Wiebicke W, Jörres R, Magnussen H (1990) Comparison of the effects of inhaled corticosteroids on the airway response to histamine, methacholine, hyperventilation, and sulfur dioxide in subjects with asthma. J Allergy Clin Immunol 86 :915–923 7. Murai M, Morimoto H, Maeda Y, Kiyotoh S, Nishikawa M, Jujii T (1992) Effect of FK224, a novel compound NK1 and NK2 receptor antagonist, on airway constriction and airway edema induced by neurokinins and sensory nerve stimulation in guinea-pigs. J Pharmacol Exp Ther 262 :403–408 8. Ichinose M, Nakajima M, Takahashi T, Yamauchi H, Inoue H, Takishima T (1992) Protection against bradykinin-induced bronchoconstriction in asthmatic patients by neurokinin receptor antagonist. Lancet 340 :1248–1251 9. Quanjer PH (1993) Standardized lung function. Eur Respir J 6 [Suppl 16]: 5–40 10. American Thoracic Society (1987) Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 136 : 225–244 11. Bonnet R, Jörres R, Heitmann U, Magnussen H (1991) Circadian rhythm in airway tone in patients with mild asthma. J Appl Physiol 71 : 1598–1605 12. Jörres R, Nowak D, Rabe K, Magnussen H (1992) Variability in aerosol output of the DeVilbiss 646 jet nebulizer. Chest 102 :1636 13. Fish JE, Kelly JF (1979) Measurement of responsiveness in bronchoprovocation testing. J Allergy Clin Immunol 64 :592–596 14. Polosa R, Holgate ST (1990) Comparative airway response to inhaled bradykinin, kallidin and [des-arg9]-bradykinin in normal and asthmatic subjects. Am Rev Respir Dis 142 : 1367–1371 15. Rajakulasingam K, Church MK, Howarth PH, Holgate ST (1993) Factors determining bradykinin bronchial responsiveness in asthma. J Allergy Clin Immunol 92 : 140–142 16. Zuskin E, Bouhuys A, Saric M (1981) Lung function changes by ethanol inhalation. J Clin Allergy 11 : 243–248