Methods for Treating Asthma

Abstract

The present invention relates to a method for treating asthma, comprising administering to a subject in need thereof an effective amount of 3-methanesulfonylpropionitrile (dapanstril) or a pharmaceutically acceptable solvate thereof. Preferred routes of administration are oral or topical.

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to the use of 3-methanesulfonylpropionitrile (dapanstril) or a pharma- ceutically acceptable solvate thereof for the treatment of asthma. [Background technology]

[0002] background Asthma is a common chronic disease of the airways, characterized by variable and recurrent symptoms, reversible airway obstruction, bronchial hyperresponsiveness, and underlying inflammation.Acute symptoms of asthma include coughing, wheezing, shortness of breath, and nighttime awakenings.Asthma is considered a chronic disease based on a state of chronic airway inflammation together with airway hyperresponsiveness, at least partially accompanied by reversible airway obstruction.

[0003] Central to the pathophysiology of asthma is the presence of causative airway inflammation mediated by the recruitment and activation of multiple cell types, including mast cells, eosinophils, T lymphocytes, macrophages, dendritic cells, and neutrophils. Type 2 T helper (Th2) cells appear to play a central role in the activation of the immune cascade leading to inflammation. Th2-inducing cytokines include IL-5 (required for eosinophil differentiation and survival) and IL-4 (important for Th2 cell differentiation and, together with IL-13, important for IgE formation and leading to IgE overproduction and eosinophilia). IgE-driven activation of mucosal mast cells releases bronchoconstrictor mediators such as histamine and cysteinyl leukotrienes, as well as inflammatory cytokines. Eosinophils contain inflammatory enzymes, produce leukotrienes, and express a variety of proinflammatory cytokines. Airway epithelial cells also play a role in the inflammatory process through the release of cytokines such as eotaxin that direct and modify the inflammatory response. Acute and chronic inflammation can not only affect airway caliber and airflow, but can also enhance pre-existing bronchial hyperresponsiveness to various stimuli (increasing susceptibility to bronchospasm).

[0004] As a result of airway inflammation and production of growth factors, airway smooth muscle cells can undergo proliferation, activation, contraction, and hypertrophy; events that can affect airway airflow limitation. In asthma, the primary physiological event that leads to clinical symptoms is airway narrowing and subsequent interference with airflow. In acute asthma exacerbations, bronchial smooth muscle contraction (bronchoconstriction) occurs rapidly, narrowing the airways in response to exposure to various stimuli, including allergens or irritants. Allergen-induced acute bronchoconstriction results from IgE-dependent release of mediators from mast cells, including histamine, tryptase, leukotrienes, and prostaglandins, which directly contract airway smooth muscle. The mechanisms that affect airway hyperresponsiveness are complex and include inflammation, neuroregulatory dysfunction, and airway remodeling. Airway remodeling involves structural changes such as sub-basement membrane thickening, subepithelial fibrosis, airway smooth muscle hypertrophy and hyperplasia, vascular hyperplasia and dilation, with subsequent permanent airway changes that increase airflow obstruction.

[0005] Airway epithelial and endothelial cell function is also highly involved in asthma. With disease progression, the epithelial basement membrane thickens due to mucus hypersecretion and formation of mucus plugs. Alterations in endothelial cell integrity lead to edema, another major pathophysiology that defines asthmatic changes in the airways. These factors act to further restrict airflow.

[0006] Asthma is characterized by a predominant T helper type 2 (Th2) immune response, including enhanced IL-4, IL-5, and IL-13 responses, allergen-specific immunoglobulin production, eosinophilia, airway inflammation, bronchoconstriction, and airway hyperresponsiveness. The current standard of care for asthma is a combination of corticosteroids and β2 agonists (anti-inflammatory drugs and bronchodilators). These drugs provide acceptable disease control for many asthma patients. However, despite treatment with this combination of corticosteroids and β2 agonists, it is estimated that 5-10% of asthma patients have symptomatic disease.

[0007] There is a need to develop new methods to effectively treat asthma, which must be effective with minimal side effects. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows the total numbers (mean ± SEM) of leukocyte specific cell type populations (from left to right: macrophages, lymphocytes, neutrophils, and eosinophils) in bronchoalveolar lavage fluid 24 hours after the last ovalbumin (OVA) aerosol challenge and intraperitoneal dapanstril dosing on day 29. Eosinophils, ****p<0.0001 between asthmatic and treated mice (n=8 per group). Mice were treated with 60 mg / kg dapanstril intraperitoneally. [Diagram 2] Figure 2 shows the volume of inflammatory cell infiltration per basement membrane in lung tissue of mice treated with dapanstril. **p<0.01 between asthmatic and treated mice (n=8 per group). Mice were treated with 60 mg / kg dapanstril intraperitoneally. [Diagram 3] Figure 3 shows the area of ​​epithelial basement membrane covered by goblet cells (mean ± SEM) in healthy, asthmatic, and treated mice (n = 8 per group). **p < 0.01 between asthmatic and treated mice. Mice were treated with dapanstril at 60 mg / kg intraperitoneally. [Figure 4] Figure 4 shows the mean ± SEM airway resistance to methylcholine in healthy, asthmatic, and dapanstril-treated mice. ****p<0.0001 between asthmatic and treated mice. MCh (acetyl-β-methylcholine chloride) challenge was initiated with PBS followed by increasing concentrations of MCh aerosol from 0 to 100 mg / mL (n=8 per group). Mice were treated with 60 mg / kg dapanstril intraperitoneally. [Diagram 5]Figure 5 shows the total number of leukocyte specific cell type populations (left to right: macrophages, lymphocytes, neutrophils, and eosinophils, mean ± SEM) in bronchoalveolar lavage fluid 24 hours after the last ovalbumin (OVA) aerosol challenge and oral dapanstril dose on day 29. Eosinophils, ****p<0.0001 between asthmatic and treated mice (n=8 per group). [Figure 6] Figure 6 shows the volume of inflammatory cell infiltration per basement membrane in lung tissue of mice treated with dapanstril. *p<0.05 between asthmatic and treated mice (n=8 per group). [Figure 7] Figure 7 shows the mean ± SEM airway resistance to methylcholine in healthy, asthmatic, and dapanstril-treated mice. **p<0.01 between asthmatic and treated mice. MCh (acetyl-β-methylcholine chloride) challenge was initiated with PBS followed by increasing concentrations of MCh aerosol from 0 to 100 mg / mL (n=8 per group). Mice were orally treated with intravenous dapanstril at 7.5 g / kg food. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description of the Invention The inventors have discovered that 3-methanesulfonylpropionitrile, which reduces IL-1β and IL-6 levels in several whole animal models of local and systemic inflammation, is effective in treating asthma, reducing airway inflammation, reducing airway resistance, improving lung function, improving asthma symptoms, and improving patient quality of life. The present invention relates to a method of treating asthma, comprising administering to a subject in need thereof an effective amount of the compound 3-methanesulfonylpropionitrile (dapansutrile), or a pharma- ceutically acceptable solvate thereof, to treat asthma.

[0010] [ka]

[0011] As used herein, a "solvate" is an addition complex in which a compound is combined in a fixed ratio with an acceptable solvent, including but not limited to water, acetic acid, ethanol, and other suitable organic solvents.

[0012] In one embodiment, the method is effective in prophylactic treatment, which is a protective process against the onset of asthma by treatment with dapanstril to affect the pathogenesis prior to the onset of asthma. By prophylactic treatment, dapanstril is administered to a patient in need thereof prior to the onset of asthma. In another embodiment, the method is effective in therapeutic treatment after the onset of asthma, when the patient begins to show clinical signs and / or symptoms.

[0013] The main functional changes in the lungs associated with asthma include immune system dysfunction, cellular infiltration composed mainly of eosinophils and neutrophils, acute and chronic inflammation, fluid accumulation (edema), excessive mucus secretion, and changes in the airway wall that may lead to bronchial epithelial injury, fibrosis, and increased sensitivity to agents that cause bronchoconstriction. These features need to be considered to develop treatments for the underlying disease process. Small animal models can be designed to mimic changes in airway inflammation, increased responsiveness to the bronchial sphincter, changes in the airway wall, and migration of eosinophils and neutrophils into the lungs. A mouse model of asthma with ovalbumin sensitization (Lunding, 2015b) can be used to evaluate the bronchodilator efficacy of, for example, dapanstril.

[0014] The present methods for treating asthma are based on the property of dapanstril to reduce at least one of the following processes that contribute to the pathophysiology associated with this disorder: inflammation, excessive cell proliferation, airway and / or pulmonary tissue edema, airway hyperresponsiveness, and bronchoconstriction.

[0015] Evidence of efficacy of the present method for treating asthma includes measurable improvements in signs, symptoms, and other variables that are clinically relevant to asthma, such as increases in blood oxygen saturation, reductions in hypoxia and hypercapnia, decreased need for supplemental oxygen, decreased frequency of wheezing and / or coughing, and reduced forced expiratory volume in 1 second (FEV 1 improved respiratory function, reduced need for mechanical ventilation, reduction in the amount of inflammatory cells infiltrating the lungs, reduction in proinflammatory cytokine and chemokine levels, improved rate of alveolar flow clearance, reduction in pulmonary edema, e.g., amount of epithelial lining fluid, dry and wet lung weights, alveolar fluid clearance and / or radiographic visualization, improved general quality of life, symptoms reported by the patient or observed by the physician, e.g., ease of breathing, and reduced severity of wheezing and / or coughing.

[0016] The method treats asthma by (i) improving symptoms (daytime and nighttime symptoms, activity limitations, use of rescue medications), (ii) improving lung function, e.g., peak expiratory flow (PEF) and / or forced expiratory volume in one second (FEV1), and / or (iii) reducing exacerbations (rate and severity). The method improves Asthma Quality of Life Questionnaire (AQLQ) scores (including scores for symptoms, activity limitations, emotional functioning, and environmental exposures).

[0017] The present invention demonstrated that dapanstril reduces airway resistance, reduces inflammatory cells (eosinophils and neutrophils) and mucus hyperproduction in bronchoalveolar lavage fluid, and reduces airway inflammation in ovalbumin-induced allergic airway inflammation in mice.

[0018] The present invention provides pharmaceutical compositions comprising one or more pharma- ceutically acceptable carriers and an active compound of 3-methanesulfonylpropionitrile, or a pharma- ceutically acceptable solvate thereof. The active compound or a pharma- ceutically acceptable solvate thereof in the pharmaceutical composition is generally in an amount of about 1-90% for tablet formulations, 1-100% for capsule formulations, about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, or 0.5-10%, or 1-5% (w / w) for topical formulations, about 0.1-5% for injectable formulations, and 0.1-5% for patch formulations. Generally, the active compound used in the pharmaceutical compositions is at least 90%, preferably 95%, or 98%, or 99% (w / w) pure.

[0019] In one embodiment, the pharmaceutical composition is in the form of tablets, capsules, granules, powders, syrups, suppositories, injections, patches, and the like. In one embodiment, the pharmaceutical composition is in the form of an aerosol suspension of inhalable particles comprising the active compound (to be inhaled by the subject). The inhalable particles can be liquid or solid with a particle size small enough to pass through the mouth and larynx during inhalation. In general, particles having a size of about 1-10 microns (1-10 μm), preferably 1-5 microns (1-5 μm), are considered to be respirable. Inhalable particles comprising dapanstril can be prepared into a dry powder using well-known techniques of supercritical fluid technology. In such cases, the compound is mixed with suitable excipients and milled into a homogenous mass using suitable solvents or adjuvants. This mass is then subjected to mixing using supercritical fluid technology to achieve a suitable particle size distribution. The particles in the formulation should be within the desired particle size range such that they can be inhaled directly into the lungs using a suitable inhalation method or introduced into the lungs via a ventilator.

[0020] Pharmaceutically acceptable carriers for the inactive ingredients can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers include, but are not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, dextrose; pH adjusters and buffers such as hydroxides, phosphates, citrates, acetates, borates, and the like; and trolamine; antioxidants such as salts of bisulfite, acids and / or bases, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and ascorbyl palmitate; surfactants such as lecithin, phospholipids including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; polyethers such as poloxamers and poloxamines, polysorbates such as polysorbate 80, polysorbate 60, polysorbate 20, polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and povidone; It may also contain ingredients including cellulose derivatives and their salts, such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose; petroleum derivatives, such as mineral oil and white petrolatum; fats, such as lanolin, peanut oil, soybean oil; mono-, di-, and triglycerides; acrylic acid polymers, such as carboxypolymethylene gel and hydrophobically modified crosslinked acrylate polymers, polysaccharides, such as dextran, and glycosaminoglycans, such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be protected from bacterial contamination using well-known preservatives, including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as non-preserved preparations for either single or multiple use.

[0021] For example, tablet or capsule formulations of active compounds may contain other excipients that are not biologically active and do not react with the active compounds. Tablet excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Binders promote adhesion of the particles of the formulation and are important for tablet formulations. Examples of binders include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, starch, and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone.

[0022] The patch formulation of the active compound may contain several inactive ingredients, such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone (polyvinylpyrrolidone), propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. The patch formulation may also contain a skin permeation enhancer, such as a lactate ester (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

[0023] The topical formulation containing active compound can be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution and suspension.The inactive ingredients in topical formulation include, but are not limited to, for example, lauryl lactate (emollient / penetration enhancer), diethylene glycol monoethyl ether (emollient / penetration enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), caprylic / capric triglyceride (emollient), octisalate (emollient / UV filter), silicone oil (emollient / diluent), squalene (emollient), sunflower oil (emollient), and silicon dioxide (thickener).

[0024] Method of administration The present invention relates to a method of treating asthma, the method comprising the steps of first identifying a subject suffering from asthma or prone to developing asthma, and administering to the subject an active compound, dapanstril, in an amount effective to treat asthma. As used herein, an "effective amount" is an amount effective to treat asthma by alleviating pathology, reducing airway inflammation, reducing airway hyperresponsiveness, improving lung function, and / or alleviating asthma symptoms.

[0025] Any method for delivering compounds to pulmonary tissue is suitable for the present invention, including local and systemic administration. The pharmaceutical composition of the present invention can be applied by systemic or local administration. Systemic administration includes oral, parenteral (intravenous, intramuscular, subcutaneous or rectal, etc.), inhalation, and other systemic administration routes. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissue.

[0026] In one embodiment, the active compound is delivered by local administration to the lungs.Local administration includes inhalation and targeted drug delivery.Inhalation methods include liquid instillation, instillation as a pressurized fluid preparation by metered dose inhaler or equivalent, inhalation of an atomized solution by a nebulizer, inhalation of dry powder, and induction of soluble or dry substances into the air stream during artificial respiration.

[0027] In one embodiment, the pharmaceutical composition is administered to a subject by inhalation of an aerosol suspension of inhalable particles comprising the active compound. The inhalable particles can be liquid or solid (e.g., dry powder) and have a particle size small enough to pass through the mouth and pharynx when inhaled. Generally, particles ranging in size from about 1 to 10 microns (1-10 μm), more preferably 1 to 5 microns (1-5 μm), are considered respirable. Surface concentrations of the active compound delivered by inhalation can vary by compound; however, generally, they are greater than 1×10 -10 ~1×10 -4 moles / liter, preferably 1 x 10 -8 ~1×10 -5It is in moles per liter.

[0028] In one embodiment, the pharmaceutical composition is orally administered to a subject. The dosage for oral administration is generally at least 0.1 mg / kg / day and less than 100 mg / kg / day or 200 mg / kg / day. For example, the dosage for oral administration is 1-100, 5-50, or 10-50 mg / kg / day for a human subject. For example, the dosage for oral administration is 100-10,000 mg / day, preferably 500-2000, 500-4000, 500-4000, 1000-5000, 2000-5000, 2000-6000, or 2000-8000 mg / day for a human subject. The drug may be taken orally once, twice, three times, or four times a day.

[0029] In one embodiment, the pharmaceutical composition is administered to a subject intravenously. The dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 20, preferably 0.03 to 10 mg / kg / day. In one embodiment, the pharmaceutical composition is administered subcutaneously to a subject. The dosage for subcutaneous administration is usually 0.3 to 20, preferably 0.3 to 3 mg / kg / day.

[0030] Those of skill in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention. The invention is useful for treating mammalian subjects, such as humans, horses, and dogs. The invention is particularly useful for treating humans. The following examples further illustrate the invention. These examples are intended merely to illustrate the invention and should not be construed as limiting. EXAMPLES

[0031] Example 1. Dapanstril treatment (intraperitoneal) reduced allergic airway inflammation and mucus production in mice Using a well-established experimental mouse model of allergic asthma (Sel 2008, Wegmann 2005, Wegmann 2007, Lunding 2015a), we evaluated dapanstril as a therapeutic option in allergic bronchial asthma and determined whether it influenced the development of airway inflammation and airway hyperresponsiveness (AHR). Inflammation in this model is characterized by infiltration of eosinophils and Th2 cells followed by AHR and mucus hyperproduction, such that this model resembles the main pathophysiological hallmark of human bronchial asthma.

[0032] Methods and Materials C57BL / 6j mice were sensitized to OVA (ovalbumin) by three intraperitoneal (ip) injections of 10 μg OVA adsorbed to 150 mg aluminum hydroxide on days 1, 14, and 21. This sensitization resulted in an adoptive immune response to OVA involving OVA-specific TH2 cells and the production of OVA-specific antibodies of subclasses IgE and IgG4.

[0033] Mice were exposed to OVA aerosol (1% w / v in PBS) three times on days 26, 27, and 28 to induce acute allergic airway inflammation. Healthy control animals (healthy group) were mock sensitized to PBS and subsequently challenged with PBS aerosol. Non-drug treated animals (asthma group) and drug treated animals (treatment group) were sensitized with OVA aerosol and subsequently challenged with OVA aerosol. The treatment group was treated with 60 mg / kg dapanstril by intraperitoneal (ip) injection on days 25, 26, 27, and 28, while the healthy and asthma groups were administered saline by intraperitoneal injection on days 25, 26, 27, and 28.

[0034] Eight animals were used per group. All animals were sacrificed on day 29. The following measurements were taken according to Lunding, 2015b: Global infiltration of inflammatory cells into the bronchoalveolar lumen Histological differentiation of bronchoalveolar lavage (BAL) cells to determine the specific infiltration of eosinophils, neutrophils, and lymphocytes Goblet cell hyperplasia using the CAST system (Computer Assisted Stereological Toolbox) including airway microscopy Airway resistance in response to methacholine to measure airway hyperresponsiveness

[0035] In particular, inflammatory cell subpopulations (eosinophils, macrophages, neutrophils, lymphocytes) infiltrating the bronchoalveolar lumen were quantified using Cytospind and Quick-Diff stained cells from bronchoalveolar lavage fluid (BALF). In addition, inflammatory cell infiltration was recorded from hematoxylin and eosin (HE) stained lung / airway cross sections. AHR was assessed by recording airway resistance during methacholine (MCh) provocation in mechanically ventilated mice with a Buxco FinePoint RC unit. Mucus hyperproduction was quantified in PAS (periodic acid-Schiff) stained airway cross sections that underwent consistent, fixed random sampling and subsequent stereological analysis of mucus volume in the airways and mucus-producing goblet cells in the airway mucosa.

[0036] result Allergic airway inflammation and mucus hyperproduction were assessed 24 hours after the final OVA aerosol challenge and dapanstril dosing on day 29. Figure 1 shows the total numbers of leukocyte specific cell type populations (from left to right: macrophages, lymphocytes, neutrophils, and eosinophils) on day 29.

[0037] Dapanstril (60 mg / kg) administered as an ip injection four times daily prior to and concomitantly with three daily OVA aerosol challenges led to a significant reduction in eosinophils in the BALF (Figure 1). Compared to asthmatic mice, treatment with dapanstril resulted in an approximately 60% reduction in eosinophils in the BALF (21.96 × 10 4 cells / ml to 7.30 x 10 4 cells / ml; ****p<0.0001), a 70% reduction in neutrophils (2.41 x 10 4cells / ml to 0.70 x 10 4 cells / ml; p < 0.01), and a 32% reduction in lymphocytes (0.80 × 10 4 cells / ml to 0.55 x 10 4 cells / ml). Macrophage numbers did not show a significant decrease (Figure 1).

[0038] Figure 2 shows that the number of inflammatory cells in the lung tissue was significantly lower in asthmatic mice versus dapanstril-treated mice (**p<0.01). The labels on the Y-axis indicate "volume of inflammatory cell infiltration per basement membrane area (μm2)." 3 / μm 2 ) is shown. Inflammatory cells were counted within a specific distance around the airways using a microscope equipped with a Computer-Aided Stereological Analysis Toolbox (CAST) system. These counts were ratioed to the basement membrane for normalization within each microscope slide to avoid slide-dependent differences.

[0039] Figure 3 shows the area of ​​epithelial basement membrane covered by goblet cells in healthy, asthmatic, and treated mice. Compared to asthmatic mice, dapanstril-treated animals showed a significant reduction in airway mucosa covered by goblet cells (22.74% reduced to 17.67%, p<0.01) as quantified by stereological analysis of PAS-stained airway cross sections.

[0040] The results in Figures 1 to 3 show that dapanstril treatment in the OVA-induced allergic airway inflammation model resulted in a significant reduction in allergic airway inflammation and mucus production. Airway hyperresponsiveness was determined by measuring airway resistance in response to 100 mg / mL methacholine on day 29. The results are shown in FIG.

[0041] Airway resistance in response to methacholine was 5.61 cm H in asthmatic mice. 2 O sec ml -1 and 3.93 cm H in dapanstril-treated mice. 2 O sec ml -1Dapanstril treatment reduced airway resistance by approximately 60% when compared to asthmatic mice. ****p<0.0001 between asthmatic and treated mice. Baseline airway resistance in healthy animals was 2.83 cm H 2 O sec ml -1 It was.

[0042] Example 2. Cytokines in BALF Cytometric bead arrays were used as a method to assess the concentrations of cytokines (IFN-γ, TNFα, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, and IL17A) in the BALF of Example 1. The beads in the array were coated with specific antibodies against various cytokines, some of which are related and known to be affected by NLRP3 signaling. The concentrations of all cytokines measured showed some reduction between asthmatic and dapanstril-treated mice. Both IL-1β and IL-6 concentrations showed a statistically significant reduction between asthmatic and dapanstril-treated mice (p<0.001 and p<0.05, respectively).

[0043] Example 3. Dapanstril treatment (oral) reduced allergic airway inflammation and airway resistance in mice The experimental protocol for the mouse model was the same as that described in Example 1, except that dapanstril was administered orally via food.

[0044] Already sensitized mice were fed ad libitum with food pellets containing 7.5 g / kg dapanstril for one week, starting on day 22; the first aerosol challenge was on day 26. Mice typically ate about 4 g of food per day, resulting in an approximate daily dose of 0 mg / kg / day for the control group and 1,000 mg / kg / day for the treatment group. This food pellet concentration in the mouse diet (7.5 g / kg dapanstril in food) resulted in blood concentrations almost identical to those in humans treated orally with dapanstril at a dose of 1000 mg / day (blood concentrations of 40 μg / mL; Marchetti 2018b).

[0045] Sham-sensitized, OVA-challenged mice served as healthy controls (healthy). OVA-sensitized, OVA-challenged asthmatic controls (asthmatic) were fed control food pellets that did not contain dapanstril.

[0046] Compared to asthmatic mice, treatment with dapanstril (food) resulted in approximately a 75% reduction in eosinophils in BALF (15.93 × 10 4 cells / ml to 3.77 x 10 4 cells / ml; p < 0.0001), with an approximately 75% reduction in neutrophils (1.74 × 10 4 cells / ml to 0.43 x 10 4 cells / ml; p < 0.05), and a reduction of approximately 75% in lymphocytes (1.00 × 10 4 cells / ml to 0.26 x 10 4 cells / ml; p<0.05). Macrophage numbers did not show a significant decrease (Figure 5).

[0047] Figure 6 shows that the number of inflammatory cells in the lung tissue was significantly lower in asthmatic mice versus dapanstril-treated mice (p<0.05). The labels on the Y-axis indicate "volume of inflammatory cell infiltration per basement membrane area (μm2)." 3 / μm 2) is shown. Inflammatory cells were counted within a specific distance around the airways using a microscope equipped with a Computer-Aided Stereological Analysis Toolbox (CAST) system. These counts were ratioed to the basement membrane for normalization within each microscope slide to avoid slide-dependent differences.

[0048] Compared with asthmatic mice, dapanstril-treated mice also showed a significant reduction in airway mucosa lined with goblet cells (-29%; 15.75% reduced to 11.16%) as quantified by stereological analysis of PAS-stained airway cross sections (data not shown).

[0049] Airway resistance in dapanstril-treated animals in response to MCh was significantly reduced, showing an approximate 60% reduction (4.57 cm H) at 100 mg / mL MCh when compared to sham-treated asthmatic controls. 2 Os / mL is 3.38 cm H 2 Os / mL) (Figure 7 ). **p<0.01 between asthmatic and treated mice. Baseline airway resistance in healthy animals was 2.58 cm H at 100 mg / mL MCh. 2 Os / mL.

[0050] The results show that dapanstril treatment via dietary intake resulted in a significant reduction in allergic airway inflammation.

[0051] References: Lunding L, Webering S, Vock C, et al. IL-37 requires IL-18Ra and SIGIRR / IL-1R8 to diminish allergic airway inflammation in mice. Allergy 2015a Apr;70(4):366-73. Lunding LP, Webering S, Vock C, et al. Poly(inosinic-cytidylic) acid-triggered exacerbation of experimental asthma depends on IL-17A produced by NK cells. J Immunol 2015b;194:5615-5625 . Marchetti C, Swartzwelter B, Koenders MI, et al. The NLRP3 Inflammasome Inhibitor OLT1177TM Suppresses Joint Inflammation in Murine Models of Acute Arthritis. Arthritis Research and Therapy 2018b;20:169. Sel S, Wegmann M, Dicke T, et al. Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme. J Allergy Clin Immunol 2008;121:910-916.e5. Wegmann M, Fehrenbach H, Held T, et al. Involvement of distal airways in a chronic model of experimental asthma. Clin Exp Allergy 2005 Oct;35(10):1263-71. Wegmann M, Goggel R, Sel S, et al. Effects of a low-molecular-weight CCR-3 antagonist on chronic experimental asthma. Am J Respir Cell Molec Bio 2007;36(1):61-7.

[0052] The invention, and the manner and process of making and using the same, are described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the invention, and that changes can be made without departing from the scope of the invention as set forth in the appended claims. To clearly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

1. A pharmaceutical composition for treating asthma, comprising an amount of dapansutrile or a pharma- ceutical acceptable solvate thereof effective to treat asthma, and administered by oral administration.

2. 10. The pharmaceutical composition of claim 1, which reduces airway inflammation, reduces airway hyperresponsiveness, improves lung function, and / or reduces the symptoms of asthma.

3. 2. The pharmaceutical composition of claim 1, wherein the treatment is a therapeutic treatment and dapanstril is administered to the subject when the subject exhibits clinical signs and / or symptoms of asthma.

4. 2. The pharmaceutical composition of claim 1, wherein the treatment is a prophylactic treatment and dapanstril is administered to the subject prior to the onset of asthma.

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