The synergistic effects of clopidogrel with montelukast may be beneficial for asthma treatment

Abstract

Platelets modulate asthma pathogenesis by forming the platelet-eosinophil aggregation (PEA), which facilitates the activation of eosinophils. Platelets exhibit the purinergic receptor (P2Y12R), which responds to cysteinyl leukotriene E4 (LTE₄ ). We have suggested that the combination of an antiplatelet drug (clopidogrel, [Clo]) and montelukast (Mon) would synergistically suppress asthma. BALB/c mice were intraperitoneally sensitized with ovalbumin (OVA) on days 0 and 14 and subsequently challenged on days 28-30 and 42-44. Mice were administered with Clo (10 mg/kg), Mon (10 mg/kg) or both drugs (Clo/Mon) orally 30 minutes before the OVA (1%) challenge on days 42-44. Mice were assayed for airway hyper-responsiveness (AHR) to methacholine and airway inflammation. Clopidogrel and montelukast attenuated the increased AHR; the combined treatment was more effective than a single treatment for total and eosinophil counts (all P < 0.05). Levels of interleukin (IL)-4, IL-5, IL-13, platelet factor 4, eosinophil peroxidase and LTE₄ increased in the bronchoalveolar lavage fluid of asthmatic mice, but these levels decreased in mice treated with Clo/Mon (all P < 0.05). Goblet cell hyperplasia decreased in response to Clo/Mon. Mouse platelets and eosinophils were isolated and co-cultured for an in vitro assay with 10 µmol/L adenosine diphosphate (ADP), LTE₄ (200 nmol/L), Mon (1 µmol/L), Clo (1 µmol/L) and Clo/Mon (1 µmol/L). Flow cytometry revealed that the increased formation of the PEA (%) was fully mediated by ADP and partly mediated by LTE₄ . Clo/Mon reduced ADP-induced PEA formation and P-selectin expression (P < 0.05). In conclusion, Clo/Mon synergistically relieved asthma by inhibiting ADP-mediated PEA formation.

Keywords: P2Y12R; asthma; clopidogrel; cysteinyl leukotriene receptors; eosinophil; montelukast; rodent.

Figure 1

Additional effects of clopidogrel and montelukast (Clo/Mon) in reducing airway hyper‐responsiveness and inflammation. A, Airway hyper‐responsiveness was recorded by the FlexiVent 48 h after the last challenge. B, Total and differential cell counts were calculated using a haemocytometer. P values were analysed by one‐way ANOVA with Tukey's post‐hoc test. *, **, and *** indicate P < 0.05, 0.01 and 0.001, respectively, in the comparisons between the indicated groups; ^# P < 0.05 in comparisons involving the drug‐treated groups; NS, not significant

Figure 2

Effects of clopidogrel and montelukast (Clo/Mon) on Th2 cytokines and the activation of platelets and eosinophils. Bronchoalveolar lavage (BAL) fluid was harvested and stored at −70°C until further analysis. The BAL fluid levels of (A) interleukin (IL)‐4, IL‐5, IL‐13; (B) platelet factor 4 (PF4); and (C) EPX were measured by enzyme‐linked immunosorbent assay. Data are means ± SD. N = 10 mice per group. P values were analysed by one‐way ANOVA with Tukey's post hoc test, except the IL‐4 and PF4 levels were analysed by the Mann‐Whitney U‐test. *, **, and *** indicate P < 0.05, 0.01 and 0.001, respectively, in the comparisons between the indicated groups

Figure 3

Effects of clopidogrel and montelukast (Clo/Mon) in lung tissue. A, Lung tissues were stained with haematoxylin and eosin (H&E) for inflammation and (B) with Periodic acid‐Schiff (PAS) for mucus‐containing cells. P values were analysed by one‐way ANOVA with Tukey's post‐hoc test. *, **, and *** indicate P < 0.05, 0.01 and 0.001, respectively, in the comparisons between the indicated groups; NS, not significant

Figure 4

Up‐regulation of platelet‐eosinophil aggregation (PEA) in asthma. A, The percentage of PEA in mouse whole blood. Platelet‐eosinophil aggregation was defined as Siglec‐F⁺/CD41⁺ cells based on flow cytometry. Data are means ± SD P values were analysed by one‐way ANOVA with Tukey's post hoc test. N = 5 for each group. B, Representative image of the flow cytometric data. From the leucocytes gate (P1), eosinophils were selected based on Siglec‐F+ cells (P3). Gate P4 contains CD41+ eosinophils (Siglec‐F+/CD41+ cells). C, In whole blood and (D) bronchoalveolar lavage fluid, the activated platelets (P‐selectin) bound to the eosinophil surfaces were identified by P‐selectin and EPX. Representative images from at least three independent experiments are shown

Figure 5

Effects of clopidogrel and montelukast (Clo/Mon) on the ligands of platelet‐eosinophil aggregations (PEA) in vitro. A, The percentages of Siglec‐F+/CD41+ cells were calculated. Data are means ± SD. B, Clopidogrel and montelukast significantly reduced PEA formation and (C) P‐selectin expression on platelets. Data are presented as folds of induction (means ± SD) compared to untreated group, from three independent experiments with duplicates. P values were analysed with the Wilcoxon signed‐rank test. * and ** indicate P < 0.05 and 0.01, respectively, in the comparison between the indicated groups; NS, not significant

Figure 6

Additional effects of clopidogrel and montelukast (Clo/Mon) on reducing leukotriene E4 (LTE₄)_. The levels of LTE₄ in (A) bronchoalveolar lavage fluid (BALF) and (B) serum were measured by ELISA. P values were analysed by one‐way ANOVA with Tukey's post hoc test. Data are means ± SEM; n = 10 per group for BALF and n = 5 per group for serum. * and ** indicate P < 0.05 and 0.01, respectively, in comparisons between the two groups; NS, not significant

Figure 7

Correlations between platelet‐eosinophil aggregations (PEA) and eosinophil counts with inflammatory mediators and leukotriene E4 (LTE₄). Platelet‐eosinophil aggregations correlated with (A) bronchoalveolar lavage fluid (BALF) level of platelet factor 4 (PF4) and (B) eosinophil count in BALF. The eosinophil count also correlated with BALF levels of (C) PF4, (D) eosinophil peroxidase (EPX), and (E) LTE₄. Analyses were performed by Spearman's rank correlation coefficient