Combination of Clopidogrel and Montelukast may be Beneficial in the Management of Asthma

Abstract

Background: Cysteinyl leukotrienes (LT), C4, D4, and E4 which are lipid mediators released from the immune cells, regulate the airway inflammation, hyperreactivity and permeability in allergic inflammation by interacting with LT-related receptors, including CysLTR1, CysLTR2 and purinergic receptor P2Y12 (P2Y12R). The latter, P2Y12R expressed on surface of platelets and eosinophils, is involved in cell activation. Platelets can modulate asthma pathogenesis by releasing intracellular granules, priming leukocytes for activation and facilitating eosinophil migration by forming the platelet-eosinophil aggregation (PEA) via surface ligands. The adherent platelets in PEA are the plentiful source for LT overproduction, thereby possibly enhancing the inflammatory response. To date the antiplatelet drugs, such as prasugrel, and clopidogrel, were found to inhibit the bronchial-hyperreactivity, platelet activation and eosinophil degranulation in animal studies and clinical trials. Therefore, we hypothesized that the combination of the antagonists to CysLTR1 (montelukast, [Mon]) and to P2Y12R on platelets (clopidogrel, [Clo]) could elicit the synergistic effect on suppressing the airway inflammation. Objective: Firstly, we investigated the distribution ratio and characteristics of the CysLT-related receptors in our asthma mouse model. Secondly, we evaluated the synergistic effects of the combination therapy (Clo/Mon) in a mouse model of asthma and elucidated the mechanism by which the combination therapy exerts the effects. Lastly, we attempted to find the clinical effectiveness of Clo on asthma patients who used clopidogrel. Materials & Methods: To investigate the localization and interactions of CysLTRs, BALB/c mice were subjected to ovalbumin (OVA) to induce allergic asthma. Some mice were administered the antagonists of CysLTR1, CysLTR2, and P2Y12R, which is montelukast sodium hydrate, HAMI 3379 and clopidorgrel hydrogen sulfate, respectively. The expression levels of CysLTR1, CysLTR2, and P2Y12R on lung tissues and inflammatory cells were evaluated by Western blot, flow cytometry, and double fluorescence staining. To assess the therapeutic effect of Clo/Mon, a mouse model of eosinophilic asthma was established. In addition to two times of sensitization on days 0, 14, BALB/c mice were challenged with OVA 0.2% on days 28-30 and with OVA 1% on days 42-44. Mice were either administered Clo (10mg/kg), Mon (10mg/kg), both drugs or dexamethasone (1 mg/kg) (Dex) for 30 minutes before OVA (1%) challenges on days 42-44. Mice were assayed for airway hyperresponsiveness (AHR) to methacholine (MCh) by FlexiVent® and sampling. The levels of interleukin (IL)-4, IL-5, IL-13, platelet factor 4 (PF4) and eosinophil peroxidase (EPX) in bronchoalveolar lavage (BAL) fluid were measured by ELISA kits. Histological characteristics were analyzed in mouse lung tissues. As in in vitro assay, platelets and eosinophils were isolated separately and co-cultured, in addition to adenosine diphosphate (ADP) 10µM, LTC4 (200 nM), LTE4 (200 nM), Mon (1 µM), Clo (1 µM), Clo/Mon (1 µM), anti-CD40L antibody (100 ng/mL), tirofiban hydrogen sulfate (1 µM). The percentages of PEA and P-selectin expression in whole blood, BAL fluid and in in vitro assay were assessed by flow cytometry and immunohistochemistry. To assess the effects of Clo in patients with asthma, we conducted a retrospective, cross-sectional study on the basis of electronic medical records from 1998 to 2015 in Ajou Medical Center, Suwon, South Korea. The inclusion criteria were patients who had been diagnosed as having asthma and/or allergic rhinitis (AR) and had been administered Clo more than 7 days after the asthma diagnosis. Patients without the complete blood count (CBC) during the exposure period to Clo were excluded from the study. A total of 596 subjects were recruited and changes of the eosinophil count, including the percentage (%) and absolute counts (x104 cells) were analyzed. Results: Expression of CysLTR1 and P2Y12R were significantly up-regulated in lung tissues (P<0.05 for each) of mice after being sensitized and challenged (OVA/OVA). The ratio of CysLTR1: CysLTR2: P2Y12R in lungs of negative control (NC) mice was shifted from 1:0.43:0.35 to 1:0.65:1.34 in OVA/OVA mice. The administration of Mon significantly diminished the up-regulation of CysLTR1, CysLTR2, and P2Y12R (P<0.05 for each), while the effects of HAMI 3379 and Clo suppressed predominantly the expression of CysLTR2 and P2Y12R, respectively. On the basis of CysLTR1: P2Y12R expression ratio (1:1.34) in the asthma mouse, we introduced Mon:Clo at the ratio 1:1. Clo/Mon attenuated the increased AHR at higher concentration of MCh; the total and eosinophil counts were more effectively diminished than single treatment (P<0.05 for each). The levels of IL-4, IL-13, PF4 and EPX were reduced in BAL fluid from the mice treated with Clo/Mon (P<0.05 for each). The inflammatory cell count, mucus containing cells in the bronchi were attenuated significantly by Clo/Mon (P<0.05 for each). Clo/Mon reduced ADP-induced PEA formation in whole blood (P<0.05) and BAL fluid. In patients with asthma and/or AR, we observed a decrease in the percentage of blood eosinophil (%) and absolute eosinophil counts (x104 cells) after using Clo for more than 60 days, however, no significant difference was observed. Conclusions: The upregulation of CysLTR1 and P2Y12R may involve in the allergic inflammation of asthmatic airway. Combination of Mon and Clo attenuated synergistically the airway inflammation and hyperresponsiveness by inhibiting the ADP-mediated PEA formation. Further studies are required to elucidate the effectiveness of antiplatelet drugs in asthma treatment.