Yoshinori Sakuma

Effect of Azelastine on the Release and Action of Leukotriene C4 and D4

The effect of azelastine on the release of leukotriene C4 and D4 (LTC4 and LTD4), and the antagonistic action of the drug against the leukotrienes were determined by using in vitro tests and compared with those of ketotifen and chlorpheniramine. Azelastine inhibited LTC4 and LTD4 release from guinea pig lung fragments passively sensitized with homologous anti-ovalbumin IgGl-b antibody. The 50% inhibitory concentration (IC50) of azelastine was 6.4 X 10(-5) M for a 15-min preincubation or 4.7 X 10(-5) M for a 30-min preincubation. Ketotifen and chlorpheniramine were inhibitory only at the highest concentration tested (3 X 10(-4) M), giving inhibitions of 35.6 and 21.3%, respectively. Azelastine also inhibited calcium ionophore A23187-induced release of leukotrienes from human polymorphonuclear leukocytes; the IC50 values were 3.6 X 10(-5) M for 15 min and 2.3 X 10(-6) M for 30 min of preincubation. Ketotifen and chlorpheniramine were inhibitory only after a 30-min preincubation, with IC50 values of 2.1 X 10(-5) and 5.9 X 10(-5) M, respectively. The potent inhibition by azelastine might be partly a result of the inhibition of 5-lipoxygenase, since 5-hydroxyeicosatetraenoic acid formation in rat basophilic leukemia cell homogenate was inhibited by azelastine. Pretreatment of guinea pig ileum with azelastine antagonized LTC4- and LTD4-induced contraction of the ileum with IC50 values of 7.0 X 10(-6) and 1.1 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of a novel PAF antagonist E6123 on anaphylactic responses in passively and actively sensitized guinea pigs and passively sensitized mice

The effects of the platelet-activating factor (PAF) antagonist, E6123, on anaphylactic responses in guinea pigs and mice were investigated. E6123 inhibited i.v. antigen (Ag)- or inhaled Ag-induced bronchoconstriction in passively and actively sensitized guinea pigs after oral administration at 3 and 10 micrograms/kg, respectively. E6123 inhibited Ag inhalation-induced airway hyperreactivity in guinea pigs after oral administration at 30 micrograms/kg. E6123 protected mice from anaphylactic death with an ED50 value (p.o.) of 7 micrograms/kg. The inhibitory effects of E6123 described above were very potent compared to those of the PAF-antagonists WEB2347 and Y-24180. The present results suggest that E6123 may be beneficial for the treatment of asthma, a condition in which PAF is assumed to be involved.

Involvement of leukotriene C4 in PAF-induced death in mice

The role of leukotrienes (LTs) in the pathogenesis of platelet-activating factor (PAF)-induced death in mice was reinvestigated, since previously reported results are in conflict. A novel 5-lipoxygenase inhibitor, E6080, and a leukotriene antagonist, LY17883, protected mice from PAF-induced death in a dose-dependent manner, while the well-known 5-lipoxygenase inhibitor, AA861, was less effective than E6080. After the intravenous injection of PAF in mice, immunoreactive leukotriene C4 (i-LTC4), which was co-eluted with authentic LTC4 in HPLC, was significantly increased in bronchoalveolar lavage fluid (BALF). Oral administration of E6080 suppressed the increase in i-LTCM4. The results suggest that LTs may play an important role in PAF-induced lethality in mice.

Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.

Effect of a novel 5-lipoxygenase inhibitor, E6080 on bronchospasm, airway cellular infiltration and leukotriene production in guinea pigs

6-Hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride (E6080), an orally active and selective 5-lipoxygenase inhibitor, dose-dependently inhibited the bronchospasm induced by antigen (ovalbumin) inhalation in sensitized conscious guinea pigs. The inhibitory effect of E6080 was more potent than that of a typical 5-lipoxygenase inhibitor, AA861, but less than that of a leukotriene (LT) antagonist, LY171883. When airway infiltration of neutrophils and eosinophils was measured in bronchoalveolar lavage fluid (BALF) at 6 h after antigen inhalation by passively sensitized guinea pigs, the inhibitory effect of E6080 on neutrophil infiltration was more marked than that on eosinophil infiltration. The inhibitory effect of E6080 on bronchoalveolar cellular infiltration and bronchoepithelial damage was confirmed by examination of photomicrographs of the lung. In addition to the above pharmacological effects, E6080 inhibited the increase in BALF levels of both i-LTC4 and i-LTB4. These results suggest that E6080 may prove to be effective for the treatment of asthma, in which large amounts of leukotrienes (LTs) are elaborated.

Inhibition of IgE-mediated leukotriene generation and bronchoconstriction in primates with a new 5-lipoxygenase inhibitor, E6080.

The inhibitory effects of a novel compound, 6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride (E6080) on IgE-mediated reactions in vitro and in vivo were determined in rhesus monkeys. E6080 inhibited both antigen- and anti-human IgE-induced leukotriene C4 generations from lung fragments at similar concentrations: the IC50s of E6080 were 1.11 and 0.96 microM, respectively. AA861 also inhibited both leukotriene C4 generations with IC50s of 0.79 and 0.76 microM, respectively. All 9 monkeys demonstrating positive cutaneous reactivity against Ascaris antigen at < 10(-7) g protein/0.1 ml showed reproducible bronchoconstriction upon aerosolized antigen challenge, but 5 monkeys demonstrating weak or no reactivity to the antigen at levels > 10(-5) g protein/0.1 ml showed no significant bronchoconstriction. The high responder monkeys showed a marked increase in lung resistance (RL 335.3 +/- 85.3%, n = 11) and a decrease in dynamic lung compliance (67.7 +/- 4.2%, n = 11) after antigen challenge following pretreatment with diphenhydramine. These pulmonary changes lasted for more than 30 min. E6080 at oral doses of 3 and 10 mg/kg showed dose-dependent inhibition of both pulmonary changes. Ten milligrams per kilogram of E6080, administered 1.5 h prior to antigen challenge, significantly inhibited the changes in both parameters, while 3 mg/kg showed significant inhibition of only the RL change. These results demonstrate that E6080 inhibited immunologically stimulated leukotriene production in the lung, resulting in inhibition of the antigen-induced airway response in primates.