Life Sciences, vol. Printed in the U S A
51, pp.
1577-1583
Pergamon
Press
EFFECT OF A PEPTIDE LEUKOTRIENE A N T A G O N I S T , ONO-1078 ON A N T I G E N - I N D U C E D A I R W A Y M I C R O V A S C U L A R L E A K A G E IN A C T I V E L Y SENSITIZED GUINEA PIGS
Takaaki Obata, Tadamasa Kobayashi, Yutaka Okada, Naoki Nakagawa, Tamiya Terawaki and Hideki Aishita Preclinical Research Department, Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618, Japan. (Received
in final
form September
8, 1992)
Summarv
We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma. Airway microvascular leakage is a primary feature of inflammation and leads to the plasma exudation into airways (1). The formation of mucosal edema is the result of increased microvascular leakage in the bronchial circulation (2). Furthermore, the exudation of plasma proteins into the bronchial interstitium results in the activation of several biochemical pathways followed by bronchial narrowing and epithelial damage (3). Therefore, it is important to reduce microvascular leakage with plasma exudation into airway tissues for the treatment of bronchial asthma. Peptide leukotrienes are 5-1ipoxygenase
products of
the arachidonic acid
Corresponding Author : Takaaki Obata, Preclinical Research Department, Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618, Japan.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1578
ONO-I078:
A P e p t i d e LT A n t a g o n i s t
Vol.
51, No. 20,
1992
metabolism and major constituents of slow reacting substance of anaphylaxis (SRS-A) (4). Peptide leukotrienes can increase vascular permeability (5) and cause airway smooth muscle contraction (6,7). These properties have led to the hypothesis that peptide leukotrienes are important mediators of bronchial asthma. The pharmacological manipulation of these activities of peptide leukotrienes by receptor antagonists or biosynthesis inhibitors may provide a novel therapeutic approach for the treatment of bronchial asthma. ONO-1078 (4-oxo-8-[4-(4-phenylbutoxy)benzoylamino]2-(tetrazol-5-yl)-4H-l-benzopyran hemihydrate) is a potent and orally active peptide leukotriene antagonist that inhibits peptide leukotriene-induced bronchoconstriction and LTD4-induced increase in cutaneous vascular permeability (8). In the present study, we examined the effect of ONO-1078 on antigen-induced airway microvascular leakage assessed by the extravasation of Evans blue dye into airway tissues in sensitized guinea pigs.
Materials and Methods Materials : Male Hartley guinea pigs (300-520g, Keari Co. and Nihon Rabbit Co.) were used throughout the experiments. Ovalbumin, indomethacin (Sigma Chemical Co.), mepyramine maleate (May & Baker Co.), azelastine (Eisai Co.), killed organisms Bordetella pertussis (The Chemo-Sero-Therapeutic Research Institute), Evans blue (Tokyo Kasei Co.) and formamide (Katayama Chemical Co.) were purchased from commercial sources. Indomethacin was dissolved in 7% sodium bicarbonate solution (Meylon, Ohtsuka Pharmaceutical Co.) and other chemicals were dissolved in 0.9% saline. ONO-1078 was synthesized by Ono Pharmaceutical Co., Ltd. and suspended in 0.5% sodium carboxymethylcellulose (CMC). Azelastine was dissolved in distilled water or suspended in 0.5% CMC. They were given at a volume of 10 ml/kg. Measurement of airway microvascular leakage: Male guinea pigs were sensitized with intraperitoneal injection of 0.5 ml saline containing 1 mg of ovalbumin and 5x109 killed organisms Bordetella pertussis. Two to three weeks later, guinea pigs were anesthetized with sodium pentobarbital 75 mg/kg, intraperitoneally. A tracheal cannula was inserted and attached to a constant volume respirator (Shinano Apparatus, Model SN-480-7). Animals were ventilated with constant volume of 5 ml at a rate of 70 strokes/min. A catheter was inserted into jugular vein for the administration of ovalbumin solution containing Evans blue dye (20 mg/kg, 1 ml/kg). The animal was challenged with 1 mg/kg ovalbumin and 10 min later exsanguinated, and the chest cavity was opened. Evans blue dye was washed out by perfusing with saline from the pulmonary artery into the left atrium. The airways and lung were then removed and cleared of extraneous connective tissues. The extrapulmonary airway was separated into trachea and main bronchi, and the intrapulmonary airways stripped of parenchyma. Wet weights of all tissues were taken. Evans blue dye was extracted by 2 ml of formamide for more than 24 hrs. The amount of Evans blue dye was determined by measuring the optical density at 620 nm with a spectrophotometer (Shimadzu, UV240) and expressed as ng/mg tissue. The baseline was determined by the administration of saline containing Evans blue dye (20 mg/kg, 1 ml/kg). ONO-1078 and azelastine were given orally 1 hr prior to challenge with ovalbumin. To avoid fatal anaphylactic shock, guinea pigs were pretreated with intravenous injection of 1 mg/kg mepyramine 1 min prior to antigen challenge. In some experiments, the side arm of the cannula was connected to a pressure transducer (Nihon Kohden, MFP-1T) to record lung overflow as an increase in pressure for the measurement of bronchoconstriction and guinea pigs were pretreated with intravenous injection of 5 mg/kg indomethacin in combination with mepyramine. The antigen-induced bronchoconstriction was measured for 10 min and was expressed as a percentage of maximum
Vol.
51, No. 20, 1992
ONO-I078:
A Peptide LT Antagonist
1579
bronchoconstriction obtained by clamping off the trachea. This bronchoconstriction is reported to be mainly mediated by SRS-A (9,10). S t a t i s t i c a l a n a l y s i s : Results were expressed as the means + SEM. Statistical significance (P
51, pp.
1577-1583
Pergamon
Press
EFFECT OF A PEPTIDE LEUKOTRIENE A N T A G O N I S T , ONO-1078 ON A N T I G E N - I N D U C E D A I R W A Y M I C R O V A S C U L A R L E A K A G E IN A C T I V E L Y SENSITIZED GUINEA PIGS
Takaaki Obata, Tadamasa Kobayashi, Yutaka Okada, Naoki Nakagawa, Tamiya Terawaki and Hideki Aishita Preclinical Research Department, Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618, Japan. (Received
in final
form September
8, 1992)
Summarv
We examined the effect of ONO-1078, a peptide leukotriene antagonist, on antigen-induced airway microvascular leakage in ovalbumin-sensitized guinea pigs. When guinea pigs were pretreated with mepyramine, ovalbumin challenge increased vascular permeability to Evans blue dye in trachea, main bronchi and intrapulmonary airways. Oral administration of ONO-1078 significantly reduced microvascular leakage in intrapulmonary airways at doses more than 3 mg/kg, but not in trachea. Moreover, oral administration of ONO-1078 significantly reduced SRS-A mediated microvascular leakage into all airway tissues and was more effective in intrapulmonary airways at 3 mg/kg. Simultaneously, ONO-1078 also inhibited SRS-A mediated bronchoconstriction. On the other hand, azelastine (10 mg/kg, p.o.), an anti-asthma agent, failed to inhibit microvascular leakage into the airways. These results suggest that peptide leukotrienes may be important mediators of airway microvascular leakage, and that the inhibitory effect of ONO-1078 on antigen-induced airway microvascular leakage in addition to the blockade of bronchoconstriction may have therapeutic implications for bronchial asthma. Airway microvascular leakage is a primary feature of inflammation and leads to the plasma exudation into airways (1). The formation of mucosal edema is the result of increased microvascular leakage in the bronchial circulation (2). Furthermore, the exudation of plasma proteins into the bronchial interstitium results in the activation of several biochemical pathways followed by bronchial narrowing and epithelial damage (3). Therefore, it is important to reduce microvascular leakage with plasma exudation into airway tissues for the treatment of bronchial asthma. Peptide leukotrienes are 5-1ipoxygenase
products of
the arachidonic acid
Corresponding Author : Takaaki Obata, Preclinical Research Department, Minase Research Institute, Ono Pharmaceutical Co., Ltd., 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618, Japan.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1578
ONO-I078:
A P e p t i d e LT A n t a g o n i s t
Vol.
51, No. 20,
1992
metabolism and major constituents of slow reacting substance of anaphylaxis (SRS-A) (4). Peptide leukotrienes can increase vascular permeability (5) and cause airway smooth muscle contraction (6,7). These properties have led to the hypothesis that peptide leukotrienes are important mediators of bronchial asthma. The pharmacological manipulation of these activities of peptide leukotrienes by receptor antagonists or biosynthesis inhibitors may provide a novel therapeutic approach for the treatment of bronchial asthma. ONO-1078 (4-oxo-8-[4-(4-phenylbutoxy)benzoylamino]2-(tetrazol-5-yl)-4H-l-benzopyran hemihydrate) is a potent and orally active peptide leukotriene antagonist that inhibits peptide leukotriene-induced bronchoconstriction and LTD4-induced increase in cutaneous vascular permeability (8). In the present study, we examined the effect of ONO-1078 on antigen-induced airway microvascular leakage assessed by the extravasation of Evans blue dye into airway tissues in sensitized guinea pigs.
Materials and Methods Materials : Male Hartley guinea pigs (300-520g, Keari Co. and Nihon Rabbit Co.) were used throughout the experiments. Ovalbumin, indomethacin (Sigma Chemical Co.), mepyramine maleate (May & Baker Co.), azelastine (Eisai Co.), killed organisms Bordetella pertussis (The Chemo-Sero-Therapeutic Research Institute), Evans blue (Tokyo Kasei Co.) and formamide (Katayama Chemical Co.) were purchased from commercial sources. Indomethacin was dissolved in 7% sodium bicarbonate solution (Meylon, Ohtsuka Pharmaceutical Co.) and other chemicals were dissolved in 0.9% saline. ONO-1078 was synthesized by Ono Pharmaceutical Co., Ltd. and suspended in 0.5% sodium carboxymethylcellulose (CMC). Azelastine was dissolved in distilled water or suspended in 0.5% CMC. They were given at a volume of 10 ml/kg. Measurement of airway microvascular leakage: Male guinea pigs were sensitized with intraperitoneal injection of 0.5 ml saline containing 1 mg of ovalbumin and 5x109 killed organisms Bordetella pertussis. Two to three weeks later, guinea pigs were anesthetized with sodium pentobarbital 75 mg/kg, intraperitoneally. A tracheal cannula was inserted and attached to a constant volume respirator (Shinano Apparatus, Model SN-480-7). Animals were ventilated with constant volume of 5 ml at a rate of 70 strokes/min. A catheter was inserted into jugular vein for the administration of ovalbumin solution containing Evans blue dye (20 mg/kg, 1 ml/kg). The animal was challenged with 1 mg/kg ovalbumin and 10 min later exsanguinated, and the chest cavity was opened. Evans blue dye was washed out by perfusing with saline from the pulmonary artery into the left atrium. The airways and lung were then removed and cleared of extraneous connective tissues. The extrapulmonary airway was separated into trachea and main bronchi, and the intrapulmonary airways stripped of parenchyma. Wet weights of all tissues were taken. Evans blue dye was extracted by 2 ml of formamide for more than 24 hrs. The amount of Evans blue dye was determined by measuring the optical density at 620 nm with a spectrophotometer (Shimadzu, UV240) and expressed as ng/mg tissue. The baseline was determined by the administration of saline containing Evans blue dye (20 mg/kg, 1 ml/kg). ONO-1078 and azelastine were given orally 1 hr prior to challenge with ovalbumin. To avoid fatal anaphylactic shock, guinea pigs were pretreated with intravenous injection of 1 mg/kg mepyramine 1 min prior to antigen challenge. In some experiments, the side arm of the cannula was connected to a pressure transducer (Nihon Kohden, MFP-1T) to record lung overflow as an increase in pressure for the measurement of bronchoconstriction and guinea pigs were pretreated with intravenous injection of 5 mg/kg indomethacin in combination with mepyramine. The antigen-induced bronchoconstriction was measured for 10 min and was expressed as a percentage of maximum
Vol.
51, No. 20, 1992
ONO-I078:
A Peptide LT Antagonist
1579
bronchoconstriction obtained by clamping off the trachea. This bronchoconstriction is reported to be mainly mediated by SRS-A (9,10). S t a t i s t i c a l a n a l y s i s : Results were expressed as the means + SEM. Statistical significance (P
