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  • Tetracaine HCl br Materials and methods br Results


    Materials and methods
    Discussion In this study, we developed a new CysLT1 and CysLT2 receptors-mediated Tetracaine HCl guinea pig model for screening both CysLT2 receptor and CysLT1/2 receptor antagonists. Unlike human CysLT2 receptors, which are equally stimulated by both LTC4 and LTD4[9], [24], guinea pig CysLT2 receptors are activated mainly by LTC4[15]. However, because LTC4 is rapidly metabolized to LTD4 by γ-GTP, we used S-hexyl Tetracaine HCl GSH [19], a synthetic substrate of γ-GTP to mediate guinea pigs anaphylactic response via CysLT2 receptors. In S-hexyl GSH non-treated guinea pigs, antigen-induced anaphylactic response was suppressed by montelukast, but not by BayCysLT2RA, indicating that this asthmatic response was basically CysLT1-receptor-dependent. On the other hand, the suppressive effect of montelukast was weakened by treatment with S-hexyl GSH. In fact, treatment with S-hexyl GSH at 30mg/kg completely shifted antigen-induced anaphylactic response to be CysLT1 receptor-independent. However, this shift does not reflect clinical settings, and is therefore not suitable for screening CysLT1/2 receptor dual antagonists. In contrast, BayCysLT2RA improved survival rate in the presence of S-hexyl GSH. Furthermore, treatment with S-hexyl GSH potentiated the effect of combination treatment with montelukast and BayCysLT2RA as compared to treatment with montelukast alone. Taken together, these findings indicate that CysLT2 receptors contribute to antigen-induced asthmatic response in S-hexyl GSH-treated animals. LTC4 to LTD4 ratio in the blood and lung tissue in S-hexyl GSH-treated guinea pigs was higher than that in S-hexyl GSH-non-treated guinea pigs. Considering the fact that LTC4 has high binding affinity for CysLT2 receptors in guinea pigs, it is further suggested that contribution of CysLT2 receptors to asthmatic response was strengthened by treatment with S-hexyl GSH. In the animals that received S-hexyl GSH at 5mg/kg, the dual CysLT1/2 antagonist ONO-6950 dose-dependently suppressed systemic anaphylaxis, further indicating that the response was both CysLT1 and CysLT2 receptors-dependent. As indicated above, human CysLT2 receptors are activated by not only LTC4 but also LTD4. Thus, simultaneous antagonism of both CysLT1 and CysLT2 receptors may have a beneficial effect on asthma. Zileuton, a selective inhibitor of 5-lipoxygenase, has been shown to be clinically superior to a CysLT1 receptor antagonist in a 12-week study aimed at treating mild to moderate chronic stable asthma [25]. Because the pharmacological effect of zileuton is due to inhibition of endogenous CysLTs production, treatment with zileuton does not activate CysLT1 and CysLT2 receptors. Accordingly, the efficacy of dual antagonism for CysLT1 and CysLT2 receptors is expected to be comparable to that of zileuton. In a post hoc analysis of combined phase III pivotal trials, zileuton showed greater improvement in morning trough FEV1, asthma symtoms, daily β-agonist use and oral corticosteroid rescue in patients whose baseline FEV1 values were less than 50% compared to all patients [26]. However, zileuton was used at a high dose, i.e. 2400mg/person, causing liver function abnormalities, including increase in liver enzyme [27], [28], [29]. It is therefore expected that the dual CysLT1/2 receptor antagonist ONO-6950 would have a better safety profile than zileuton. Recent clinical studies in mild asthma subjects have revealed no significant difference in the efficacy of ONO-6950 and montelukast against allergen-induced asthmatic response [30]. Thus, CysLT2 receptors may contribute to the pathogenesis of relatively severe asthma rather than mild asthma. Corticosteroids and adrenergic β2 receptor agonists have been clinically used as effective pharmacotherapy for asthma. In this study, asthmatic response in both S-hexyl GSH-non-treated and treated animals was attenuated by single treatment with dexamethasone or salmeterol. Combination treatment with these two agents completely suppressed asthmatic response. Therefore, the effectiveness of dexamethasone and salmeterol against asthmatic response suggests that the anaphylactic model used could reproduce clinical settings. In addition, asthmatic response under treatment with S-hexyl GSH generally be to screen for anti-asthma drugs.