Effect of KW-4679, an Orally Active Anti-allergic Drug, on Antigen-Induced Airway Hyperresponsiveness in Actively Sensitized Guinea Pigs

Effect

of KW-4679,

Induced

Airway

an Orally

Active

Hyperresponsiveness

in Actively

Hirokazu Kawasaki, Haruhiko Pharmaceutical

Research

Laboratories,

Kyowa

Hakko

Received

Kogyo

November

Anti-allergic

Co.,

Ltd.,

24, 1995

Drug,

on Antigen

Sensitized

Guinea

Pigs

Manabe and Kenji Ohmori 1188,

Shimotogari,

Accepted

January

Nagaizumi-cho,

Sunto-gun,

Shizuoka

411, Japan

19, 1996

ABSTRACT-We investigated the effect of KW-4679 ((Z)-11-[(3-dimethylamino)propylidene]-6,11-dihydro dibenz[b,e]oxepin-2-acetic acid monohydrochloride), an orally active anti-allergic drug, on antigen-in duced airway hyperresponsiveness using two different indicators, pulmonary resistance (RL) and dynamic lung compliance (Cdyn), in actively sensitized guinea pigs. Oral administration of KW-4679 (0.1 and 1 mg/kg) 1 hr before aerosolized antigen exposure significantly inhibited the development of airway hyper responsiveness to inhaled acetylcholine in RL and Cdynin a dose-dependent manner. Terfenadine (10 mg/kg) also inhibited the development of airway hyperresponsiveness. These results indicate that KW-4679 could be useful in the treatment of bronchial asthma. Keywords:

KW-4679,

Airway

hyperresponsiveness

Airway hyperresponsiveness (AHR) is one of the most important pathophysiological features of asthma. Ac cordingly, it is very essential to find a way to alleviate the AHR. To date, virus (1)-, ozone (2)-, platelet activating factor (PAF) (3)-, lipopolysaccharide (4) and antigen (5) induced AHR models in various animals are widely used in many laboratories. The model of antigen-induced AHR in particular is an essential model for evaluating the efficacy of drugs. KW-4679 ((Z)-11-[(3-dimethylamino)propylidene]-6,11 dihydrodibenz[b,e]oxepin-2-acetic acid monohydrochlo ride) is an orally active anti-allergic drug that inhibits both the release and the action of histamine (6, 7). KW-4679 also inhibits the production and/or release of PAF and leukotriene B4 (LTB4) in human neutrophils (8). KW-4679 reduces the electrical field stimulation induced tachykininergic contraction through reduction in the release of transmitters from sensory nerve endings in the isolated guinea pig main bronchus (9). Receptor binding studies have shown this compound to be a hista mine H1-receptor antagonist with a K; value of 16 nM (10). KW-4679 had neither anti-cholinergic, ganglion blocking, a-adrenergic blocking nor ~-adrenergic recep tor stimulating properties. In the present

study,

we investigated

the effect of KW

4679 on antigen-induced AHR to inhaled acetylcholine by using RL and Cdyn as indicators of functional pulmonary alterations

in

actively

sensitized

guinea

pigs

under

anesthetization.

Active sensitization and antigen exposure were per formed as previously described by Boichot et al. (5), with a slight modification. For active sensitization, male Hart ley guinea pigs (Charles River, Yokohama), weighing 350 to 500 g, were exposed twice for 30 min to an aerosol of 0.2% ovalbumin (OA, chicken egg, grade III; Sigma, St. Louis, MO, USA) at a 48-hr interval in a plastic chamber (30 x 50 x 30 cm). The aerosol was generated by an ultra sonic nebulizer (Ultra-NEB 99; DeVilbiss, Sommerset, PA, USA). Under these conditions, the aerodynamic diameter of the nebulized particles was between 0.5 and 3 pm. At 15 20 days after the initial sensitization pro cedure, the guinea pigs were challenged in the plastic chamber as described above by exposure to aerosols of five successive increasing concentrations of OA (0.01, 0.1, 0.5, 1 and 5 mg/ml) for 15 min each. One hour after the last challenge with the highest concentration of OA, no apparent sign of respiratory failure was observed in the guinea pigs. KW-4679, which was synthesized in our laboratories, was dissolved in distilled water. Terfenadine (Sigma), which is a non-sedative histamine H1 antagonist, was sus pended in 0.3% carboxymethylcellulose (CMC) solution. Drugs were administered orally 1 hr before the first anti gen challenge. As a vehicle-treated group, sensitized guinea pigs were administered distilled water or 0.3% CMC solution instead of drugs.

Twenty-four

hours

after the antigen

challenge,

the

guinea pigs were anesthetized with urethane (1.2 g/kg, i.p.; Tokyo Kasei Kogyo, Tokyo) and artificially venti lated by a respiratory pump (Ugo Basile, Varese, Italy) with a constant volume of 1 ml laboratory air/ 100 g body weight at 60 breaths/min. Spontaneous breathing was abolished by injection of gallamine triethiodide (Sigma, 3 mg/animals) into the jugular vein. After a sufficient stabilization period, five successive 1-min aerosol ad ministrations of acetylcholine chloride (ACh; Daiichi Pharmaceutical Co., Ltd., Tokyo; 20, 50, 100, 150 and 200 pg/ml) were performed. The aerosol was generated by an ultrasonic nebulizer connected to the inspiratory line of the respiratory pump using three-way breathing valves. The airflow signal and the pressure signal meas ured by pressure transducers were electronically inte grated toring USA). system

using a computerized pulmonary function moni system (Model 6; Buxco Electronics, Sharon, CT, RL and Cdyn were simultaneously calculated by this according to the method of Aoki et al. (11).

Results indicated

are each expressed as the mean±S.E.M. number of experiments. The statistical

of the differ

ences were analyzed by Student's t-test or the Aspin Welch's test. In the case of the experiment of KW-4679 pretreatment, test followed

Dunnett's or Steel's multiple comparison by the Bartlett test was used. A value of P

less than 5076 was accepted

as statistically

significant.

First, the following four groups were examined: unsen sitized and saline-challenged animals (saline-saline), un sensitized and OA-challenged animals (saline-OA), sensi tized and saline-challenged animals (OA-saline) and sen sitized and OA-challenged animals (OA-OA). The groups of unsensitized animals and saline-challenged animals were exposed to saline instead of OA under the same conditions in the sensitization procedure and in the challenge procedure, respectively. Inhalation of ACh produced a concentration-dependent increase in RL and reduction in Cdyn (Fig. 1). In the group of OA-OA, the AHR to ACh was clearly observed; that is, the alterations in both RL and Cdyn were significantly enhanced at 20, 50 and 100 pg/ml of ACh compared to the other groups. In contrast, no significant alterations were observed in both RL and Cd,,n at each concentration of ACh between the groups of saline-saline, saline-OA and OA-saline. In ad dition, there were slight but significant reductions in the basal Cdyn values measured just before ACh exposure in the group of OA-OA. The oral treatment of 0.1 and 1 mg/kg of KW-4679 1 hr before antigen challenge significantly prevented the de velopment of AHR (Fig. 2). A significant inhibition was observed in RL in a dose-dependent manner. In the ex periment on the OA-saline group, treatment of 1 mg/kg of KW-4679 1 hr before the saline challenge almost never

Fig. 1. Airway responsiveness to inhaled acetylcholine (ACh) 24 hr after saline or antigen exposure in unsensitized or actively sensitized guinea pigs. The concentrations of ACh used were 20, 50, 100, 150 and 200 pg/ml. Airway responsiveness was assessed by pulmonary resistance (RL, upper) and dynamic lung compliance (Cdyn„lower). Groups are as follows: unsensitized and saline-challenged animals (saline-saline) (0), unsensitized and OA-challenged animals (saline OA) (A), sensitized and saline-challenged animals (OA-saline) (O), sensitized and OA-challenged animals (OA-OA) (0). Distilled water was orally administered 1 hr before the antigen challenge in each guinea pig. Each value represents the mean±S.E.M. of 7 to 26 guinea pigs.

influenced the airway responsiveness to ACh in both RL and Cdyn (data not shown). It is therefore considered that the alterations of airway responsiveness caused by pretreatment of KW-4679 were due to the prevention of the development of AHR, not a bronchodilative action or an anti-cholinergic action. The oral pretreatment of 10 mg/kg of terfenadine also significantly inhibited the de velopment of AHR (Fig. 3). In the present

study,

we demonstrated

inhibited the development cited by the five successive

that

KW-4679

of AHR to inhaled ACh eli antigen challenges in actively

Fig. 2. Effect of KW-4679 on antigen-induced airway hyperrespon siveness in actively sensitized guinea pigs. Airway responsiveness was assessed by pulmonary resistance (RL, upper) and dynamic lung compliance (Cdyn,lower) 24 hr after antigen exposure. Vehicle (•) or KW-4679 at doses of 0.1 (0) and 1 mg/kg (A) was orally ad ministered 1 hr before antigen challenge. Each value represents the mean±S.E.M. of 6 to 8 guinea pigs. * and **: Statistically sig nificant difference from the vehicle group at P<0.05 and P<0.01, respectively.

Fig. 3. Effect of terfenadine on antigen-induced airway hyper responsiveness in actively sensitized guinea pigs. Airway responsive ness was assessed by pulmonary resistance (RL, upper) and dynamic lung compliance (Cdyn,lower) 24 hr after antigen exposure. Vehicle (0) or terfenadine at a dose of 10 mg/kg (O) was orally ad ministered 1 hr before antigen challenge. Each value represents the mean ±S.E.M. of 7 to 9 guinea pigs. *, ** and ***: Statistically sig nificant difference from the vehicle group at P < 0. 05, P < 0.01 and P<0.001, respectively.

sensitized guinea pigs. The present experimental model was developed without any pharmacological modulation, which involved various mediators including histamine. In

bronchoscopy at 24 hr after antigen challenge showed clear signs of inflammation in peripheral airways such as tissue edema and hyperemia. Moreno et al. (13) demon strated by morphological and physiological studies that airway wall thickness is one of the mechanisms of the de velopment of AHR. We also observed a significant base line reduction in Cdyn. Accordingly, it is likely that suc cessive antigen exposures induce the development of AHR through the manifestation of edema in peripheral airways, although it has not been fully elucidated. It has been demonstrated that a variety of mediators such as thromboxanes (TXs), leukotrienes, PAF and tachykinins participate in the development of AHR.

addition, we measured RL and Cdyn simultaneously assess airway responsiveness separately in the more

to cen

tral airways and in the more peripheral airways. Accord ingly, we considered that it is a more favorable ex perimental

model of asthma

as a pulmonary

disease and a

good model for evaluation of drugs that possess anti histaminic properties such as KW-4679 and terfenadine. From our observations, Cdyn was more sensitive than RL as an indicator of airway responsiveness to ACh.

In a recent

study,

Mauser et al. (12) reported

that

Recently, Santing et al. (14) reported that histamine may contribute to the antigen-induced AHR in guinea pigs. The development of AHR, not surprisingly, was marked ly reduced by drugs that inhibit both the release and the action of histamine. It is therefore likely that the inhibi tory effect of drugs such as KW-4679 and terfenadine on the development of AHR is based on their anti-histaminic

5

6

properties. We also found that KW-4679 (1 mg/kg, p.o.) inhibited the production and/or release of TXB2 from leukocytes in bronchial alveolar lavage fluid of guinea pigs (Y. Sasaki, unpublished data). On the other hand, we recently demonstrated that KW-4679 inhibits the an tigen-induced development of AHR through the preven tion of eosinophil infiltration into guinea pig airways using a mild-symptoms experimental model that utilizes a single-antigen challenge with metyrapone (15). Conse quently, although the anti-asthmatic mechanism of KW 4679 has not been fully elucidated, it is considered that not only the inhibition of leukocyte infiltration but also the prevention of the action of chemical mediators (e.g., edema formation) such as TXA2, LTB4, PAF, tachykinins and especially histamine could contribute to the inhibi tory effect of KW-4679 on the development of AHR. In conclusion,

we demonstrated

that

pretreatment

by

the oral administration of KW-4679 markedly inhibited the development of AHR elicited by the successive anti gen challenge in actively sensitized guinea pigs. These findings suggest that KW-4679 will be beneficial for the treatment

of bronchial

asthma.

7

8

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Mauser antibody

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