Inhaled statins as bronchodilators to improve lung function in respiratory diseases

Abstract

To provide: methods for relaxing airway smooth muscle tissue, alleviating or preventing bronchospasm and treating lung diseases by administering an HMG-CoA reductase inhibitor (statin) directly to lung tissue by inhalation; and formulations and compositions useful for the practice of methods of the disclosure.SOLUTION: A method for reducing airway smooth muscle contraction in a subject comprises: administering a formulation by inhalation to a subject having a lung disease; and administering one, two or three additional therapeutic agents. The formulation comprises: a therapeutically effective amount of a statin, or an isomer, enantiomer or diastereomer thereof; and a pharmaceutically acceptable carrier.SELECTED DRAWING: Figure 18

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This international application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 826,620, filed March 29, 2019, and U.S. Provisional Patent Application No. 62 / 906,427, filed September 26, 2019, the contents of each of which are incorporated herein by reference in their entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant No. NIH / NHLBI K08 HL114882-01A1 and California Regional Primate Center Pilot Grant (P51 OD011107). The government has certain rights in this invention. [Background technology]

[0003] (background) Asthma affects approximately 20 million people in the United States and over 339 million people worldwide, and is characterized by symptoms of wheezing and shortness of breath due to excessive narrowing of the airways. See, e.g., S.S. An et al., Eur Respir J (2007) 29(5):834-60; Y. Amrani et al., Int J Biochem Cell Biol (2003) 35:272-76 (http: / / www.globalasthmareport.org / , accessed March 29, 2019). Despite widespread use of long-acting beta-2 agonists and high-dose inhaled corticosteroids, 55% of asthma patients experience inadequate symptom control, leading to increased hospitalizations, lost work days, disability, and death, at an estimated annual cost of approximately $82 billion in 2013 (KR Chapman et al., Eur Respir J (2008) 31(2):320-25; SP Peters et al., J Allergy Clin Imunol (2007) 119:1454-61; S. Webb, Nat Publ Gr (2011) 29(10):860-63; T. Nurmagambetov et al., Ann Am Thorac Soc (2018) 15(3):348-56). This stark reality highlights the persistent unmet therapeutic need in asthma.

[0004] Steroids act by suppressing proinflammatory cytokines and chemokines, blocking immune cell recruitment to the airways and local inflammation that can sensitize airways to proconstrictor agonists, resulting in airway hyperresponsiveness. However, many patients with COPD and severe asthma exhibit steroid insensitivity, even at high doses, eliminating the direct bronchodilatory effect on airway smooth muscle, an important mechanism for maintaining lung function and controlling disease. Indeed, in COPD, standard treatment often omits steroids and relies solely on antimuscarinic or beta-agonist bronchodilators to maintain disease control. The importance of bronchodilatory airway smooth muscle for maintaining disease control has been recognized in a variety of other respiratory disorders, including cystic fibrosis (DP Cook et al., Am J Respir Crit Care Med (2016) 193(4):417-26; CD Pascoe et al., Am J Respir Cell Mol Biol (2018) doi: 10.1165 / rcmb.2018-0378ED). However, many patients remain poorly controlled and experience frequent exacerbations despite regular use of these existing bronchodilators, indicating the need for new medications.

[0005] Airway smooth muscle cells exhibit phenotypic plasticity, exhibiting either a proliferative or contractile state. Bronchodilators target the contractile state to relax the airways, providing patients with acute relief of breathlessness, improved lung function, and disease control. Increased airway smooth muscle mass has also been observed in respiratory diseases, and potential therapies to reduce airway smooth muscle proliferation and mass are also under preclinical study. However, no drugs have progressed to human clinical studies, and it remains unclear whether reducing airway smooth muscle proliferation alone is sufficient to improve patients' lung function and disease control.

[0006] During asthma exacerbations, airway smooth muscle (ASM) contraction is a major cause of acute bronchoconstriction (SS An et al., supra; RK Lambert et al., J Appl Physiol (1997) 83(11):140-47; PT Macklem, Am J Respir Crit Care Med (1996) 153:83-89). Furthermore, ASM mass increases significantly in severe and life-threatening asthma (L. Benayoun et al., Am J Respir Crit Care Med (2003) 167(10):1360-68; N. Carroll et al., Am Rev Respir Dis (1993) 147(2):405-10). Therefore, the therapeutic potential of ASM-targeting agents is, in principle, even greater in these subpopulations. However, current treatments aimed at overcoming ASM contraction, such as β2 agonists, muscarinic antagonists, and cysteinyl leukotriene receptor antagonists, do not fully control symptoms because they target complex, indirect, and receptor-mediated pathways that are susceptible to desensitization (EJ Whalen et al., Cell (2007) 129(3):511-22).Targeting the ASM cytoskeleton is an alternative approach to achieve ASM relaxation, and strong preclinical data are accumulating to support targeting specific pathways, such as actin, myosin, zyxin, cofilin, and Rho kinase (ROCK)-mediated signaling (S. Chen et al., Am J Respir Cell Mol Biol (2014) 50:1076-83; W. T. Gerthoffer et al., Curr Opin Pharmacol (2013) 13:324-30; T. L. Lavoie et al., Proc Am Thor Soc (2009) 6:295-300; S. R. Rosner et al., PLoS One (2017) 12:e0171728; B. Lan et al., Am J Physiol Lung Cell Mol Physiol (2018) 314(5):L799-807; W. Zhang et al., J Physiol (2018) 596:3617-35). However, further development of these cytoskeletal targets has been hindered by concerns regarding their safety, specificity, and efficacy in ASM in asthmatic patients.

[0007] Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors that block the biosynthesis of mevalonate (MA) and the downstream isoprenoid lipids farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP). Currently, in the United States, they are only approved for oral administration as lipid-lowering agents. In asthma models, statins have pleiotropic effects, including anti-inflammatory, anti-fibrotic, anti-proliferative, and immunomodulatory effects. Despite clear laboratory and epidemiological data, clinical trials using oral statins to improve asthma symptoms have yielded conflicting and / or negative results. Summary of the Invention [Problem to be solved by the invention]

[0008] While statins are recognized to be able to reduce inflammation, it is unclear whether these anti-inflammatory effects can benefit patients by improving lung function beyond other already widely used anti-inflammatory treatments, including steroids. However, excessive smooth muscle bronchoconstriction is a daily problem for patients suffering from respiratory diseases, directly leading to airway narrowing and reduced lung function. There remains a need for new bronchodilators for patients whose disease is not well controlled with existing therapies. [Means for solving the problem]

[0009] (Brief summary of the invention) In one embodiment, the present disclosure provides a method for reducing airway smooth muscle contraction in a subject, the method comprising administering a formulation by inhalation to a subject with non-inflammatory pulmonary airway disease, wherein the formulation comprises a therapeutically effective amount of a statin, or its isomer, enantiomer, or diastereoisomer, and a pharmaceutically acceptable carrier.In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and their isomers, enantiomers, and diastereoisomers.In some embodiments, the statin is a hydrophobic statin.In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of pitavastatin and its isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of pitavastatin and simvastatin. In some embodiments, the statin is pitavastatin. In some embodiments, the statin is simvastatin.

[0010] In some embodiments, the therapeutically effective amount is about 0.005 μg to about 40 mg. In some embodiments, the therapeutically effective amount is about 0.5 μg to about 15 mg. In some embodiments, the therapeutically effective amount is about 1.0 μg to about 10 mg. In some embodiments, the therapeutically effective amount is about 1.0 μg to about 5 mg.

[0011] In some embodiments, the subject has been diagnosed with a pulmonary airway disease. In some embodiments, the pulmonary airway disease is selected from the group consisting of exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligogranulocytic asthma, obesity-related airway hyperresponsiveness, and post-viral airway hyperresponsiveness.

[0012] In some embodiments, the pulmonary airway disease is characterized by bronchospasm. In some embodiments, the pulmonary disease is selected from the group consisting of bronchospasm following infection with a viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing wheezing due to bronchospasm); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

[0013] In some embodiments, administration is performed using a mechanical inhaler. In some embodiments, the mechanical inhaler is a metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol metered dose inhaler. In some embodiments, the metered dose inhaler is a dry powder inhaler. In some embodiments, the mechanical inhaler is a nebulizer. In some embodiments, the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, and an Ellipta® inhaler.

[0014] In some embodiments, the method further comprises administering one, two, or three additional therapeutic agents. In some embodiments, the one, two, or three additional therapeutic agents are administered in the same formulation as the statin. In some embodiments, the one, two, or three additional therapeutic agents are not administered in the same formulation as the statin. In some embodiments, at least one of the additional therapeutic agents is administered in a separate formulation from the statin. In some embodiments, the statin and the one, two, or three additional therapeutic agents are administered simultaneously. In some embodiments, the statin and the one, two, or three additional therapeutic agents are administered at different times.

[0015] In some embodiments, the additional therapeutic agent is a beta agonist; a corticosteroid; a muscarinic antagonist; a RhoA inhibitor; a GGTase-I or -II inhibitor; a ROCK1 and / or ROCK2 inhibitor; a soluble epoxide hydrolase inhibitor; a fatty acid amide hydrolase inhibitor; a leukotriene receptor antagonist; a phosphodiesterase-4 inhibitor, such as roflumilast; a 5-lipoxygenase inhibitor, such as zileuton; a mast cell stabilizer, such as nedocromil; theophylline; an anti-IL5 antibody; an anti-IgE antibody; an anti-IL5 receptor antibody; an anti-IL13 / 4 receptor and combinations of beta agonists and corticosteroids, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; and combinations of beta agonists, corticosteroids, and muscarinic antagonists, including both long-acting and short-acting formulations. In some embodiments, the additional therapeutic agent is selected from the group consisting of albuterol, aformoterol, formoterol, salmeterol, indacaterol, levalbuterol, salbutamol, terbutaline, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, bambuterol, clenbuterol, formoterol, salmeterol, abesiterol, and carmoterol, buphenine, bopexamine, epinephrine, fenoterol, isoetharine, isoproterenol, and benzocaine. The beta-agonist is selected from the group consisting of proterenol, orciprenaline, levoalbutamol, pirbuterol, procaterol, ritodrine, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and gintero.

[0016] In some embodiments, the additional therapeutic agent is beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, flucortolone, fluprednidene, fluticasone, fluticasone furoate In some embodiments, the additional therapeutic agent is a corticosteroid selected from the group consisting of ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, rebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin. In some embodiments, the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A. In some embodiments, the additional therapeutic agent is the RhoA inhibitor rosin.

[0017] In some embodiments, the one, two, or three additional therapeutic agents are augmented with a statin. In some embodiments, the one, two, or three additional therapeutic agents are administered at sub-therapeutic doses.

[0018] In some embodiments, the pharmaceutically acceptable carrier comprises a component selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, polyalcohols, cyclodextrins, DexSol, amino acids, salts, and mixtures thereof. In some embodiments, the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose. In some embodiments, the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose. In some embodiments, the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose. In some embodiments, the component comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol. In some embodiments, the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, Captisol, and sulfobutyl-β-cyclodextrin. In some embodiments, the component comprises arginine or arginine hydrochloride. In some embodiments, the component comprises a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

[0019] In another embodiment, the present disclosure provides a method for treating bronchospasm in a subject in need thereof by administering a formulation by inhalation to the subject, wherein the formulation contains an effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof, and a pharmaceutically acceptable carrier. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of simvastatin and pitavastatin. In some embodiments, the statin is selected from the group consisting of pitavastatin and its isomers, enantiomers, and diastereoisomers. In some embodiments, the statin comprises pitavastatin.

[0020] In some embodiments, the subject has been diagnosed with a pulmonary airway disease. In some embodiments, the pulmonary airway disease is selected from the group consisting of asthma; exercise-induced bronchoconstriction (or exercise-induced asthma); COPD, which may include emphysema, chronic bronchitis, and / or alpha-1 antitrypsin deficiency (AATD); ACOS; cystic fibrosis; and bronchiectasis. In some embodiments, the pulmonary airway disease is a non-inflammatory pulmonary airway disease. In some embodiments, the pulmonary airway disease is selected from the group consisting of exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligogranulocytic asthma, obesity-related airway hyperresponsiveness, and post-viral airway hyperresponsiveness. In some embodiments, the pulmonary airway disease is characterized by airway smooth muscle contraction. In some embodiments, the pulmonary disease is selected from the group consisting of bronchospasm following infection with a viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing wheezing due to bronchospasm); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

[0021] In some embodiments, the pulmonary airway disease is characterized by bronchospasm. In some embodiments, administration is performed using a mechanical inhaler. In some embodiments, the mechanical inhaler is a metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol metered dose inhaler. In some embodiments, the metered dose inhaler is a dry powder inhaler. In some embodiments, the mechanical inhaler is a nebulizer. In some embodiments, the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, a PulmoSphere carrier inhaler, and an Ellipta® inhaler.

[0022] In some embodiments, the formulation further comprises one, two, or three additional therapeutic agents. In some embodiments, at least one of the additional therapeutic agents is augmented with a statin. In some embodiments, the additional therapeutic agents are administered at subtherapeutic doses. In some embodiments, one, two, or three additional therapeutic agents are administered at subtherapeutic doses. In some embodiments, the additional therapeutic agents are selected from the group consisting of beta-agonists; corticosteroids; muscarinic antagonists; RhoA inhibitors; GGTase-I or -II inhibitors; ROCK1 and / or ROCK2 inhibitors; soluble epoxide hydrolase inhibitors; fatty acid amide hydrolase inhibitors; leukotriene receptor antagonists; phosphodiesterase-4 inhibitors such as roflumilast; 5-lipoxygenase inhibitors such as zileuton; mast cell stabilizers such as nedocromil; theophylline; anti-IL5 antibodies; anti-IgE antibodies; anti-IL5 receptor antibodies; anti-IL13 / 4 receptor and combinations of beta agonists and corticosteroids, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; and combinations of beta agonists, corticosteroids, and muscarinic antagonists, including both long-acting and short-acting formulations.

[0023] In some embodiments, the additional therapeutic agent is a beta agonist selected from the group consisting of arformoterol, buphenine, clenbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, levalbuterol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and zinterol. In some embodiments, the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A. In some embodiments, the second therapeutic agent is the RhoA inhibitor rosin.

[0024] In some embodiments, the pharmaceutically acceptable carrier comprises a component selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, polyalcohols, cyclodextrins, amino acids, salts, and mixtures thereof. In some embodiments, the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose. In some embodiments, the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose. In some embodiments, the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose. In some embodiments, the component comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol. In some embodiments, the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin. In some embodiments, the component comprises arginine or arginine hydrochloride, hi some embodiments, the component comprises a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

[0025] In another embodiment, the present disclosure provides a pharmaceutical formulation for treating pulmonary airway diseases, the composition comprising a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof, and a pharmaceutically acceptable carrier suitable for administration by inhalation. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of pitavastatin and simvastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is pitavastatin. In some embodiments, the statin is simvastatin.

[0026] In some embodiments, the effective amount is about 0.005 mg to about 80 mg. In some embodiments, the effective amount is about 0.5 mg to about 15 mg. In some embodiments, the effective amount is about 1.0 mg to about 10 mg. In some embodiments, the effective amount is about 1.0 mg to about 5 mg.

[0027] In some embodiments, the formulation further comprises one, two, or three additional therapeutic agents. In some embodiments, at least one of the additional therapeutic agents is augmented with a statin. In some embodiments, the additional therapeutic agents are administered at subtherapeutic doses. In some embodiments, the one, two, or three additional therapeutic agents are administered at subtherapeutic doses. In some embodiments, the formulation further comprises one, two, or three additional therapeutic agents ... and combinations of beta agonists and corticosteroids, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; and combinations of beta agonists, corticosteroids, and muscarinic antagonists, including both long-acting and short-acting formulations.

[0028] In some embodiments, the additional therapeutic agent is a beta agonist selected from the group consisting of arformuterol, buphenine, clenbuterol, levalbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, pirbuterol, procaterol, ritododrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and zinterol. In some embodiments, the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A. In some embodiments, the additional therapeutic agent is the RhoA inhibitor rosin.

[0029] In some embodiments, the pharmaceutically acceptable carrier comprises a component selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, polyalcohols, cyclodextrins, amino acids, salts, and mixtures thereof. In some embodiments, the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose. In some embodiments, the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose. In some embodiments, the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose. In some embodiments, the component comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol. In some embodiments, the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin. In some embodiments, the component comprises arginine or arginine hydrochloride, hi some embodiments, the component comprises a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

[0030] In another embodiment, the present disclosure provides a pre-filled inhalation device for treating a pulmonary airway disease in a subject, the device comprising: a delivery device for delivering a therapeutic dose of a formulation to the pulmonary airways of a subject in need thereof; and a pharmaceutically acceptable formulation described herein. In some embodiments, the device comprises a pressurized inhaler, a metered dose inhaler, a dry powder inhaler, or a nebulizer.

[0031] In some embodiments, the device comprises a plurality of therapeutic doses. In some embodiments, the delivery device is a metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol inhaler. In some embodiments, the metered dose inhaler is a dry powder inhaler. In some embodiments, the delivery device is a nebulizer. In some embodiments, the delivery device is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, a PulmoSphere carrier inhaler, and an Ellipta® inhaler.

[0032] In another embodiment, the present disclosure provides a pre-filled cartridge for use with an inhaler, the cartridge comprising: a container including a coupling means for attaching the container to an inhalation device; and a pharmaceutically acceptable formulation as described herein. In some embodiments, the inhalation device further comprises a pharmaceutically acceptable propellant.

[0033] In another embodiment, the present disclosure provides any of the above methods, wherein the therapeutically effective amount is effective for maintaining lung function; reducing asthma exacerbations; reducing the subject's need for corticosteroids; reducing bronchoconstriction and mucus accumulation in the subject; or enhancing breathing-induced bronchodilation. In some embodiments, the therapeutically effective amount is effective for maintaining lung function; reducing asthma exacerbations; reducing bronchoconstriction and mucus accumulation in the subject; or enhancing breathing-induced bronchodilation.

[0034] In another embodiment, the present disclosure provides a method for reducing airway hyperresponsiveness (AHR) or ASM hypercontractility in a subject, the method comprising administering by inhalation to a subject in need thereof a formulation of the present disclosure, wherein the therapeutically effective amount is effective to reduce AHR or ASM hypercontractility in the subject.

[0035] In another embodiment, the present disclosure provides a method for increasing stretch-induced airway smooth muscle (ASM) relaxation in a subject, the method comprising administering by inhalation to a subject in need thereof a formulation of the present disclosure, wherein the therapeutically effective amount is effective to increase stretch-induced ASM relaxation in the subject.

[0036] In another embodiment, the present disclosure provides a method for enhancing the bronchodilatory effect of a β2 agonist on ASM, the method comprising contacting ASM with an enhancing amount of a statin and contacting ASM with an enhancing amount of a β2 agonist, wherein the resulting enhanced effect comprises ASM relaxation. In some embodiments, the ASM is contacted with the β2 agonist for about 2 hours to about 24 hours after contact with the statin. In some embodiments, the ASM is in a human subject in need of ASM relaxation. In some embodiments, the statin and β2 agonist are administered by inhalation. In some embodiments, the enhanced effect reduces ASM contraction by at least about 10% more than the sum of the reduction in ASM contraction caused by the β2 agonist alone and the reduction in ASM contraction caused by the statin alone. In some embodiments, the enhanced effect reduces ASM contraction by about 10% to about 30% more than the reduction in ASM contraction in the absence of the statin.

[0037] In some embodiments, administration is performed using a mechanical inhaler. In some embodiments, the mechanical inhaler is a metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized nebulized aerosol inhaler. In some embodiments, the metered dose inhaler is a dry powder inhaler. In some embodiments, the mechanical inhaler is a nebulizer. In some embodiments, the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, and an Ellipta® inhaler.

[0038] In another embodiment, the disclosure provides a method for treating a symptom of interstitial lung disease, the method comprising administering by inhalation a formulation of the disclosure to a subject in need thereof, wherein the interstitial lung disease causes an airway symptom selected from the group consisting of ASM contraction, ASM overgrowth or thickening, bronchospasm, bronchoconstriction, airway mucus accumulation, or ASM release of inflammatory mediators, and wherein the therapeutically effective amount is effective to reduce the severity of the symptom by at least 10%.

[0039] In another embodiment, the present disclosure provides a method for treating a pulmonary airway disease in a subject by administering a formulation by inhalation to the subject having the pulmonary disease and administering one, two, or three additional therapeutic agents, wherein the formulation comprises a pharmaceutically acceptable carrier and a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof. In some embodiments, the one, two, or three additional therapeutic agents are selected from the group consisting of a beta-agonist, a corticosteroid, a muscarinic antagonist, a RhoA inhibitor, a GGTase-I or -II inhibitor, a ROCK1 and / or ROCK2 inhibitor, a soluble epoxide hydrolase inhibitor, a fatty acid amide hydrolase inhibitor, a leukotriene receptor antagonist, a phosphodiesterase-4 inhibitor such as roflumilast, a 5-lipoxygenase inhibitor such as zileuton, a mast cell stabilizer such as nedocromil, theophylline, an anti-IL5 antibody or antibody derivative, an anti-IgE antibody or antibody derivative, an anti-IL5 receptor antibody, or anti-IL13 / 4 receptor antibodies or antibody derivatives; biologics such as mepolizumab, reslizumab, benralizumab, omalizumab, and dupilumab; beta-agonist and muscarinic antagonist combinations, including both long-acting and short-acting formulations; beta-agonist and corticosteroid combinations, including both long-acting and short-acting formulations; corticosteroid and muscarinic antagonist combinations, including both long-acting and short-acting formulations; and beta-agonist, corticosteroid, and muscarinic antagonist combinations, including both long-acting and short-acting formulations.

[0040] In some embodiments, the additional therapeutic agent is selected from the group consisting of albuterol, aformoterol, formoterol, salmeterol, indacaterol, levalbuterol, salbutamol, terbutaline, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, bambuterol, clenbuterol, formoterol, salmeterol, abesiterol, and carmoterol, buphenine, bopexamine, epinephrine, fenoterol, isoetharine, isoproterenol, and benzocaine. The beta-agonist is selected from the group consisting of proterenol, orciprenaline, levoalbutamol, pirbuterol, procaterol, ritodrine, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and gintero.

[0041] In some embodiments, the additional therapeutic agent is beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, flucortolone, fluprednidene, fluticasone, fluticasone furoate In some embodiments, the additional therapeutic agent is a corticosteroid selected from the group consisting of ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, rebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin. In some embodiments, the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A. In some embodiments, the additional therapeutic agent is the RhoA inhibitor rosin.

[0042] In some embodiments, the additional therapeutic agents are beta-agonists, corticosteroids, muscarinic antagonists, or any combination thereof. In some embodiments, one, two, or three additional therapeutic agents are augmented with a statin. In some embodiments, one, two, or three additional therapeutic agents are administered at subtherapeutic doses.

[0043] In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is a hydrophobic statin. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of pitavastatin and their isomers, enantiomers, and diastereoisomers. In some embodiments, the statin is selected from the group consisting of pitavastatin and simvastatin. In some embodiments, the statin is pitavastatin. In some embodiments, the statin is simvastatin.

[0044] In some embodiments, the therapeutically effective amount is about 0.005 μg to about 40 mg. In some embodiments, the therapeutically effective amount is about 0.5 μg to about 15 mg. In some embodiments, the therapeutically effective amount is about 1.0 μg to about 10 mg. In some embodiments, the therapeutically effective amount is about 1.0 μg to about 5 mg.

[0045] In some embodiments, the subject has been diagnosed with a pulmonary airway disease. In some embodiments, the pulmonary airway disease is selected from the group consisting of asthma, exercise-induced bronchoconstriction, COPD, emphysema, chronic bronchitis, alpha 1 antitrypsin deficiency (AATD), ACOS, cystic fibrosis, bronchiectasis, exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, post-viral airway hyperresponsiveness, post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection, airway edema due to congestive heart failure, and airway edema due to pulmonary edema. In some embodiments, the pulmonary disease is selected from the group consisting of exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, and post-viral airway hyperresponsiveness.

[0046] In some embodiments, the pulmonary airway disease is characterized by bronchospasm. In some embodiments, the pulmonary disease is selected from the group consisting of bronchospasm following infection with a viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing wheezing due to bronchospasm); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

[0047] In some embodiments, administration is performed using a mechanical inhaler. In some embodiments, the mechanical inhaler is a metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol metered dose inhaler. In some embodiments, the metered dose inhaler is a pressurized aerosol metered dose inhaler. In some embodiments, the metered dose inhaler is a dry powder inhaler. In some embodiments, the mechanical inhaler is a nebulizer. In some embodiments, the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, and an Ellipta® inhaler.

[0048] In one embodiment, the present disclosure provides a method for reducing future symptoms caused by an event that has already occurred or is expected to be experienced in the future, the method comprising administering a formulation of the present disclosure to a subject at risk of experiencing the future symptoms. In some embodiments, the method is for the treatment of a future condition characterized by bronchospasm caused by post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing localized edema and bronchospasm; pulmonary embolism; airway trauma; surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; or bronchospasm associated with a paraneoplastic syndrome. [Brief explanation of the drawings]

[0049] [Figures 1A-1D]Figures 1A-1D show that the differential effects of statins on inhibiting basal ASM cell contractility occur through a mevalonate (MA)-dependent mechanism. ASM were treated with 1 μM statin for 24 hours. P values: **p<0.01, ***p<0.001. NT: untreated. Pra: pravastatin; Ros: rosuvastatin; Sim: simvastatin (biologically active form, β-hydroxy acid, "SA"); Pit: pitavastatin. Pitavastatin and simvastatin were more potent at the concentrations used. Figure 1A shows the effect in the absence of mevalonate. Figure 1B shows the effect in the presence of 100 μM mevalonate, which abolishes the beneficial effects of statins on ASM relaxation. This indicates that the statin effect occurs via inhibition of the mevalonate pathway. Of note, simvastatin is a prodrug (lactone form). Once absorbed, it is biotransformed to the active metabolite, β-hydroxysimvastatin acid. In the blood circulation, there is a constant equilibrium between the lactone and the hydroxy acid.

[0050] Figure 1C shows the dose response of statins across a range of drug lipophilicity. Simvastatin and pitavastatin are highly lipophilic, atorvastatin is moderately lipophilic, and pravastatin is the least lipophilic (most hydrophilic) statin. After 24 h of treatment at a dose of 0.4 μM, simvastatin and pitavastatin showed a significant decrease in strain energy (energy imparted to the substrate by a contracting cell = contraction), corresponding to an increase in ASM relaxation. In comparison, pravastatin showed no effect at these concentrations. At 2 and 10 μM, simvastatin, pitavastatin, and atorvastatin significantly (even more) reduced strain energy compared to pravastatin. Compared to untreated (0 μM), statistically significant decreases in strain energy occurred with: 0.4 and 10 μM simvastatin, 2 and 10 μM pitavastatin, and 2 and 10 μM atorvastatin. P values: ***p<0.001, ****p<0.0001.

[0051] Figure 1D shows that pitavastatin is a more potent inhibitor of ASM cell contraction than other statins. Pretreatment with pitavastatin (1 μM, 24 h) strongly and significantly inhibited basal ASM contraction (also known as strain energy). Pitavastatin was also more potent than the same dose of simvastatin, further confirming the enhanced efficacy of pitavastatin compared to the more lipophilic simvastatin. P values: **p<0.01, ***p<0.001 compared to NT.

[0052] [Figures 2A-2E] Figures 2A-2E show the results of apoptosis and necrosis studies using statins compared to a positive control known to induce apoptosis (Figure 2A). Individual statins are shown in Figures 2B-E: (2B) simvastatin (SA), (2C) pitavastatin, (2D) rosuvastatin, and (2E) pravastatin. There was no evidence of apoptosis or cell death at any of the doses tested, including those at which the statins demonstrated a relaxing effect on ASM cells. Abbreviations: Sim (simvastatin), Pra (pravastatin), Pit (pitavastatin), Ros (rosuvastatin).

[0053] [Figure 3] FIG. 3 shows the dose-dependent effects of simvastatin acid (SA) and pitavastatin on primary ASM cells obtained from three different human donors.

[0054] [Figures 4A-4C]Figures 4A-4C show that statins inhibit histamine-induced ASM contraction. Figure 4A: Pretreatment with pitavastatin (24 h) potently and significantly inhibited both basal and histamine-induced (10 μM histamine for 30 min) ASM contraction, indicating that pitavastatin can prevent agonist-induced contraction and airway narrowing. A 0.4 μM dose of pitavastatin abolished histamine-induced ASM contraction (basal vs. post-histamine, p = NS), and higher doses (2, 100, and 50 μM) produced similar effects. A similar pattern was observed with simvastatin (Figure 4B) and atorvastatin, with pitavastatin being the most potent of the three drugs. Abbreviations: NT - untreated, NS - not significant. P values: *p < 0.05, **p < 0.01, ****p < 0.0001, ####p < 0.0001. Figure 4C shows ASM relaxation over time. Pitavastatin caused greater ASM relaxation than 1 μM simvastatin at all time points, including 24 h, with or without medium deprivation. H = histamine applied. I = isoproterenol (a β2 agonist) applied. Histamine causes ASM contraction, while isoproterenol causes ASM relaxation. SE = strain energy (ASM contraction).

[0055] [Figure 5A-5B]Figures 5A-5B show that statins inhibit contractile function in ASM cells. Figure 5A shows that pitavastatin inhibits Rho-kinase (ROCK-1) phosphorylation in a mevalonate-dependent manner in human ASM cells. Histamine (10 μM, 5 min) induces ROCK-1 phosphorylation, which is inhibited by pretreatment with pitavastatin (Pit, 1 μM) for 24 h. Cotreatment with mevalonate (MA, 200 μM, 24 h) eliminates the inhibitory effect of Pit on ROCK-1 phosphorylation, confirming that the MA pathway mediates ROCK-1 activation. Figure 5B shows that pitavastatin inhibits myosin light chain-2 (MLC-2) phosphorylation in human ASM cells. Thrombin (2 units, 30 min) induces MCL-2 phosphorylation, which is inhibited by pretreatment with pitavastatin (1 or 10 μM for 24 h). P values: **p<0.01, ***p<0.001. The data indicate that statins block the ASM contractile machinery. MLC proteins directly control smooth muscle contraction.

[0056] [Figure 6] Figure 6 shows that the bronchodilatory effect of intratracheally instilled pitavastatin is independent of any anti-inflammatory activity. Using a non-inflammatory mouse model of methacholine (MCh)-induced hyperconstriction, 1-hour pretreatment with intratracheal pitavastatin (5 mg / kg for 5 days) before each MCh infusion significantly reduced airway contraction (control 22.3% vs. statin 7.3%, *p=0.0361). Airway contraction in response to 500 nM (0.5 μM) MCh was measured using precision-cut lung sections (n=2 mice per group, 13 airways per group).

[0057] [Figure 7] FIG. 7 shows the experimental design for the non-human primate inhaled statin study.

[0058] [Figure 8]FIG. 8 shows that inhaled (nebulized) simvastatin inhibits basal levels of eicosanoid lipids (LTB4 and TXB2) that cause bronchoconstriction.

[0059] [Figure 9] Figure 9 shows the tissue distribution of inhaled simvastatin. Mass spectrometry and metabolomics were used to examine the tissue distribution of inhaled (nebulized) simvastatin (1 mg / kg) and the effects of lung lipids, simvastatin, and its active metabolite, simvastatin acid (SA), on concentrations in the mainstem bronchi and lower lung lobes (approximately 0.8–1 μg / 100,000 epithelial cells). Up to 405 ng / g of SA was detected in the liver, while 25 ng / g and 7 ng / g of SA were detected in the large intestine and muscle, respectively.

[0060] [Figures 10A-10F]Figures 10A-10F show that pitavastatin inhibits basal, histamine-, and MCh-induced ASM contraction. Figure 10A shows that, compared to untreated (0 μM), a statistically significant decrease in contraction occurred with 0.4, 2, and 10 μM pitavastatin (Pit) and 0.4 and 10 μM simvastatin (Sim). Pravastatin had no significant effect on ASM cell relaxation. Figure 10B shows that both 1 μM Sim and 1 μM Pit reduced ASM contraction in a time-dependent manner compared to the control, but Pit was significantly more effective than Sim at 24 h (indicated by #). Experiments were performed under serum-deprived medium conditions. Figure 10C shows that 0.4 μM Pit inhibited histamine-induced ASM contraction, whereas 0.4 μM Sim did not. Statistical comparisons were performed using Student's t-test. Figure 10D shows that resuspending Pit-free medium in the wells reversed the force-inhibitory effect of 1 μM Pit. Figure 10E shows that statin treatment did not induce cell apoptosis. Digitonin (50 μg / ml) was used as a positive control. Figure 10F shows that no reduction in viability was observed in mouse PCLS compared to untreated (0 μM). 2 hours of 0.01% Triton® treatment was included as a positive control. All cell experiments were performed in serum-containing (10% FBS) media conditions unless otherwise noted. One non-asthmatic primary human ASM donor line was used in Figures 10A–D; Figure 10E used one non-asthmatic hTERT ASM cell line. In all graphs, ASM contraction is plotted as a fold change relative to the baseline value before treatment at time 0. p-values: *, #p<0.05; **, ##p<0.01; ###p<0.001. n=4-8 individual wells per condition for each group. All data are reported as mean and standard error of the mean (SEM).

[0061] [Figures 11A-11C]Figures 11A-11C show that pitavastatin inhibits ASM contraction in human cells, human PCLS, and mice. Figure 11A shows that ASM cells from asthmatic patients had greater basal contraction (in the absence of additional agonist) than ASM cells from nonasthmatic patients. Despite these differences in basal capacity, pitavastatin dose-dependently inhibited ASM contraction across both asthmatic and nonasthmatic donor cells (p<0.0001 compared to 0 μM treatment). For each donor, n=4-8 separate wells per condition. Figure 11B shows that in a non-inflammatory mouse model of MCh-induced ASM hyperconstriction, pretreatment with intratracheal pitavastatin (5 mg / kg for 5 days) 1 h before each MCh nebulization significantly reduced % airway contraction (control = 24.9%, vehicle = 27.2%, pitavastatin = 14.2%, *p<0.05). Figure 11C shows that human PCLS airways pretreated with 5 μM Pit (n = 5) or vehicle (n = 3) for 24 hours and posttreated with 0, 0.1, and 1 μM histamine (hist) for 15 minutes each reduced stenosis. Compared with vehicle, Pit significantly reduced 1 μM hist-induced bronchoconstriction. Changes in luminal narrowing are reported as the % change in luminal area compared to 0 μM hist. Absolute values ​​of luminal area were not statistically different between the Pit and vehicle groups at 0 μM histamine. All data are reported as the mean and standard error of the mean (SEM).

[0062] [Figures 12A-12B]Figures 12A-12B show that pitavastatin potentiates the ASM relaxation effect of simulated deep breathing, a beneficial effect of pitavastatin notably absent from isoproterenol. Figure 12A shows that pretreatment with 1 μM Pit (24 h) (n = 7) or 10 μM isoproterenol (30 min) (n = 6) significantly inhibited basal ASM contractions compared with untreated controls (n = 7). Contraction values ​​normalized to the untreated control group are shown. Figure 12B shows that in response to a subsequent single stretch-unstretch maneuver (10% amplitude, 4 s duration) mimicking deep breathing, ASM cells rapidly and dramatically eliminated their contractions. Force then recovered over 180 s. Force removal was similar in all three groups, but subsequent force recovery was significantly inhibited by pitavastatin treatment (*p < 0.05; ****p < 0.0001). n indicates the number of individual wells of the ASM monolayer. All data are reported as the mean and standard error of the mean (SEM).

[0063] [Figure 13] Figure 13 shows that pitavastatin (Pit), isoproterenol (Iso), and the combination of Pit and Iso reduced histamine (Hist)-induced bronchoconstriction (*p=0.0298). This also indicates that the combination is at least additive, and that pitavastatin does not interfere with the action of isoproterenol, and vice versa. Precision-cut human lung slices from a single human donor were pretreated with 5 μM pitavastatin (Pita) or vehicle (control) for 24 h and posttreated with histamine (Hist, 10 μM for 15 min) followed by isoproterenol (Iso, 30 μM for an additional 30 min). Changes in luminal narrowing are reported as % change (±SEM) from the pretreatment condition. Absolute values ​​of luminal airway area were not statistically different between the Pita and control groups at pretreatment. Experiments were performed under serum-deprived medium conditions. n = 3–7 airways per group.

[0064] [Figures 14A-14B]Figures 14A-14B show that pitavastatin inhibits ASM cell secretion of pro-inflammatory cytokines in a GGPP-dependent manner. Non-asthmatic primary human ASM cells were grown to confluence and either untreated (Con. (NT)) or pretreated with 2 mM pitavastatin (Pit or PIT) and GGPP (10 mM) for a total of 72 hours. Cells were treated with either IL17 and TNFα or IL13 and TNFα at 10 ng / mL for 18 hours. Figure 14A: PIT inhibited IL13 / TNFα-induced eotaxin-3 peptide secretion through a GGPP-dependent mechanism. Figure 14B: Pit inhibited IL17 / TNFα-induced IL6 peptide secretion through a GGPP-dependent mechanism. The IL13 / IL17 / TNFα cocktail is labeled "CytoMix (CM)" in the figure. All experiments were performed in serum-containing media (10% FBS). P value: *p<0.05, **p<0.01, ****p<0.0001.

[0065] [Figures 15A-15C] Figures 15A-15C show that pitavastatin inhibits the ASM cytoskeleton through MA- and GGPP-dependent mechanisms. Non-asthmatic primary human ASM cells were treated with 1 μM pitavastatin (Pit) in vehicle or vehicle alone for 24 hours. Wells were then immunostained for F-actin expression. Pit significantly reduced basal F-actin expression (Figure 15A). Non-asthmatic primary human ASM cells were co-treated with 1 μM pitavastatin (Pit), Pit containing 10 μM GGPP, or Pit containing 10 μM GGPP and 100 μM MA for 24 hours. Pit reduced F-actin expression and ASM contraction; these reductions were abrogated by GGPP and MA (Figure 15B). Cell lysates were analyzed by Western blot for total ROCK-1 and total ROCK-2. Pit reduced the basal expression of both ROCK-1 and ROCK-2 (Fig. 15C).

[0066] [Figure 16]Figure 16 shows that simvastatin and dexamethasone synergistically inhibit eotaxin-3 secretion from HBE1 cells. Pretreatment of HBE1 cells with simvastatin (Sim, 5 μM) and / or dexamethasone (DEX, 10 M) for 72 hours independently inhibited IL-13-induced extracellular secretion of eotaxin-3, and simvastatin and dexamethasone together exhibited a synergistic inhibitory effect on eotaxin-3 secretion.

[0067] [Figures 17A-17B] Figure 17A shows that pretreatment with appropriate concentrations of statins enhances the relaxant effect of relevant concentrations of isoproterenol. Figure 17B details the data shown in the boxed portion of Figure 17A.

[0068] [Figure 18] Figure 18 shows that pretreatment with appropriate concentrations of statins enhances the relaxant effect of dexamethasone. Primary human airway smooth muscle cells were cultured in serum-containing medium (10% FBS) until confluent and then pretreated with 0.1, 1, or 5 μM dexamethasone ("Dex") for 60 hours, with or without 0.1, 0.5, or 1 μM pitavastatin ("Pit"). Next, ASM cells were exposed to a cytokine mixture (10 ng / mL IL-13, IL-17, and TNFα, "CM") for 15 hours, and eotaxin-3 expression was measured. "NT" means untreated. When pitavastatin was added to any concentration of dexamethasone, a significant decrease in eotaxin-3 expression was observed. DETAILED DESCRIPTION OF THE INVENTION

[0069] (Detailed explanation) Overview : The need for novel bronchodilators is met by new methods of using statins, which provide a novel mechanism for bronchodilation of the airways, improving lung function and alleviating symptoms when delivered directly to the airways by inhalation. The present disclosure demonstrates that inhaled statins have therapeutic effects separate and distinct from their anti-inflammatory activity. Direct administration of statins to the airways delivers effective amounts of statins to airway smooth muscle (ASM) that are not achieved by oral administration. However, when inhaled, statins induce ASM relaxation, reduce bronchoconstriction and airway mucus accumulation, reduce bronchospasm, reduce hyperreactivity and hyperconstriction, enhance deep breathing-induced ASM relaxation and bronchodilation, enhance the bronchodilatory effects of drugs such as β2 agonists and inhaled corticosteroids, reduce the need for inhaled corticosteroids, maintain lung function, and improve lung function. Thus, the present disclosure includes novel methods and formulations for treating diseases and disorders of the pulmonary airways. Inhaled statins directly reduce the contractile force exerted by ASM, disrupt the ASM cytoskeleton, and inhibit the release of inflammatory cytokines and mediators by ASM. Inhaled statins can be combined with additional therapeutic agents to enhance the therapeutic effect of the additional therapeutic agent, thereby allowing for lower doses and / or increased efficacy.

[0070] definition : The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all the alternatives of "A," "B," "A or B," and "A and B."

[0071] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value is defined to the tenth of the unit of the lower limit between the upper and lower limits of that range, and any other stated or intervening value within that stated range is included within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included within the disclosure, subject to the excluding any particular limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0072] All ranges disclosed herein also encompass any and all possible subranges and combinations of these subranges. Any listed range can be recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be easily broken down into a lower third, middle third, upper third, etc. As will be understood by those of skill in the art, all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can be subsequently divided into subranges as described above. Finally, as will be understood by those of skill in the art, ranges include individual members. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0073] It is understood that certain features of the present disclosure that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if all combinations were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0074] "Statins" are small molecule HMG-CoA reductase inhibitors. Statins are designed to block the mevalonate metabolic pathway, thereby reducing cholesterol production in the body. Suitable statins of the present disclosure include, but are not limited to, simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, as well as their isomers, enantiomers, and diastereoisomers. Hydrophobic statins include simvastatin, pitavastatin, and other statins with similar hydrophobicity. Hydrophilic statins include pravastatin and other statins with similar hydrophilicity.

[0075] The term "airway smooth muscle" ("ASM") refers to the smooth, involuntary muscle tissue that lines the bronchi and bronchioles. Contraction of ASM reduces the diameter of the airways, while relaxation of ASM dilates the airways.

[0076] The term "therapeutically effective amount" refers to an amount of a statin (or isomer, enantiomer, diastereoisomer) or mixture thereof sufficient to achieve a measurable beneficial effect when administered by inhalation. The beneficial effect can be reduced airway smooth muscle contraction, reduced bronchospasm or bronchoconstriction, prophylactic maintenance of lung function, reduced mucus accumulation, reduced corticosteroids needed to control symptoms, reduced severity and / or frequency of asthma exacerbations, improved respiratory-induced bronchodilation, etc.

[0077] A "sub-therapeutic dose" refers to a dose of one or more agents in a synergistic or enhanced combination formulation, method, or system, where the dose of an agent is insufficient or sub-therapeutic when administered alone or as part of a non-synergistic combination formulation, method, or system, but is reduced to a level sufficient for therapeutic use when administered as part of a synergistic combination formulation, method, or system. A sub-therapeutic dose of an agent can be about 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the effective dose of the agent when the agent is administered by inhalation as part of a non-synergistic formulation, method, or system according to the present disclosure.

[0078] The term "pharmaceutically acceptable carrier" refers to an excipient that is non-toxic to a subject in the amounts and concentrations at which it is administered and in which a statin can be dissolved and / or suspended. In the practice of the present disclosure, a pharmaceutically acceptable carrier is suitable for administration by inhalation.

[0079] The term "pulmonary airway disease" refers to a disease or disorder in which pulmonary obstruction or restricted or obstructed airflow into or out of the lungs is a substantial symptom. This obstruction may be due to constriction of ASM (bronchoconstriction) and / or excessive mucus secretion. Pulmonary airway diseases may include, but are not limited to, asthma; exercise-induced bronchoconstriction (or exercise-induced asthma); chronic obstructive pulmonary disease (COPD), which may include emphysema, chronic bronchitis, and / or alpha-1 antitrypsin deficiency (AATD); asthma-COPD overlap syndrome (ACOS) (also known as asthma-COPD overlap or ACO); cystic fibrosis; acute bronchitis; eosinophilic bronchitis; constrictive bronchiolitis; and bronchiectasis. The use of inhaled statins can reduce compressive forces, thereby reducing airway remodeling, mucus hypersecretion, and mucus plug formation. Pulmonary airway diseases considered "non-inflammatory" can include exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, post-viral airway hyperresponsiveness, and other pulmonary airway diseases that are not initiated or maintained by inflammation.

[0080] "Bronchoprotection" is pulmonary "protective" activity or administration using the disclosed methods, formulations, or systems to reduce harm from future symptoms or effects already occurring or expected to be experienced in the future, where the subject receiving the method, formulation, or system is at risk of experiencing future symptoms. These future manifestations may include post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infections; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing bronchospasm-induced wheezing); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes; and other events that may cause bronchospasm. A subject is "expected" to experience a future symptom if there is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about a 100% chance that the subject will experience one or more symptoms due to the subject's health, physical condition, genetics, occupation, age, or other factors that are causally related to the expected future symptom.

[0081] A pulmonary airway disease is "characterized" by a given factor if that factor is a hallmark of the disease, i.e., if that factor is commonly associated with that factor, regardless of whether it is also found in other diseases. For example, asthma can be characterized by bronchoconstriction because bronchoconstriction is present in the majority of asthma cases, despite the fact that bronchoconstriction is also a hallmark of emphysema.

[0082] "Interstitial lung diseases" are those that occur in the lung tissue and spaces between the lung airways, such as the basement membrane, perivascular, and perilymphatic tissue. Despite the fact that these diseases do not directly affect the lung airways, they can have indirect effects and symptoms that affect the airways and lung function.

[0083] formulation: The disclosed compositions are formulated for inhalation, in which the composition is inhaled or sprayed into the lungs. Ideally, the composition is administered so as to distribute evenly throughout the respiratory tract and provide an effective dose of the statin directly to the ASM. This is generally achieved by administering the formulation as a population of small particles suspended in air or gas, where the particle size distribution affects the distance the particles penetrate distally into the trachea. The composition may be in the form of a solution, suspension, powder, or other form suitable for pulmonary administration. See, for example, HM Mansour et al., Int J Nanomed (2009) 4:299-319. These compositions are administered to the lungs via a suitable device known in the art, for example, in aerosol, pulverized, nebulized, or vaporized form. The amount of composition administered can be controlled by providing a valve to deliver a metered amount, such as in a metered-dose inhaler (MDI), which delivers a fixed dose in a spray with each actuation of the device. In this way, an appropriate dose (e.g., a therapeutically effective amount) of the composition can be reliably delivered from a multi-dose device.

[0084] The formulation used for delivery will typically be designed to work with a particular mode of administration, such as an aerosol, nebulizer, or dry powder formulation.

[0085] The formulations of the present disclosure comprise a therapeutically effective amount of a statin. In some embodiments, the therapeutically effective amount is at least about 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg. In some embodiments, the therapeutically effective amount is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 12, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg. In some embodiments, the therapeutically effective amount will be about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or 0.005 mg or less.

[0086] The formulation may comprise any pharmaceutically active statin or a mixture thereof. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, atorvastatin, lovastatin, and pravastatin. In some embodiments, the statin is selected from the group consisting of simvastatin and pitavastatin.

[0087] The formulations of the present disclosure may further comprise additional therapeutic agents, such as beta-agonists; corticosteroids; muscarinic antagonists; RhoA inhibitors; GGTase-I or -II inhibitors; ROCK1 and / or ROCK2 inhibitors; soluble epoxide hydrolase inhibitors; fatty acid amide hydrolase inhibitors; leukotriene receptor antagonists; phosphodiesterase-4 inhibitors, such as roflumilast; 5-lipoxygenase inhibitors, such as zileuton; mast cell stabilizers, such as nedocromil; theophylline; anti-IL5 antibodies or antibody derivatives; anti-IgE antibodies or antibody derivatives; anti-IL5 receptor antibodies or antibodies. anti-IL13 / 4 receptor antibodies or antibody derivatives; biologics such as mepolizumab, reslizumab, benralizumab, omalizumab, and dupilumab; beta-agonist and muscarinic antagonist combinations, including both long-acting and short-acting formulations; beta-agonist and corticosteroid combinations, including both long-acting and short-acting formulations; corticosteroid and muscarinic antagonist combinations, including both long-acting and short-acting formulations; and beta-agonist, corticosteroid, and muscarinic antagonist combinations, including both long-acting and short-acting formulations.

[0088] An antibody derivative is a protein capable of binding to an antigen that resembles or is based on an antibody. Examples of antibody derivatives include nanobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, F(ab)v fragments, single-chain variable fragments (scFv), single-domain antibodies (sdAb), and functional fragments thereof.

[0089] Because the additional therapeutic agent also does not undergo hepatic first-pass metabolism, it too may be administered at a lower dose than is generally effective for oral or parenteral administration. In some embodiments, the effective dose when administered by inhalation is less than about 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the dose typically recommended for oral administration.

[0090] Corticosteroids suitable for use as additional therapeutic agents include, but are not limited to, beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, fluocortolone, fluoxetine ... These may include prednidene, fluticasone, fluticasone furoate, halometasone, meprednisone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol, amcinonide, ciclesonide, deflazacort, desonide, formocortal, fluchlorone acetonide, fludroxycortide, fluocinolone acetonide, fluocinonide, halcinonide, and triamcinolone acetonide.

[0091] Muscarinic antagonists are anticholinergic agents that block muscarinic acetylcholine receptors and can therefore block bronchoconstriction. Muscarinic antagonists suitable for use as additional therapeutic agents include, but are not limited to, ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, rebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin.

[0092] Beta-agonists are compounds that activate β2 adrenergic receptors and are used to relax ASM. Beta-agonists (β-agonists) suitable for use as additional therapeutic agents include, but are not limited to, albuterol, arformoterol, buphenine, clenbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, levalbuterol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and zintero.

[0093] ROCK inhibitors inhibit the enzyme Rho kinase (ROCK1 and / or ROCK2). Suitable ROCK inhibitors include, for example, 1-methyl-5-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-indazole ("TS-f22", M. Shen et al., Sci Rep (2015) 5:16749), (1S)-2-amino-1-(4-chlorophenyl)-1-[4-(1H-pyrazol-4-yl)phenyl]ethanol ("AT13148", T. A. Yap et al., Clin Cancer Res (2012) 18(14):3912-23), N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide ("GSK429286A", E. Ahler et al., Mol Cell (2019) 74(2):393-408e20), 1-[(3-hydroxyphenyl)methyl]-3-(4-pyridin-4-yl-1,3-thiazol-2-yl)urea ("RKI-1447", H. Wang et al., Cancer Res (2017) 77(8):2148-60), and 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide ("Y-27632", YC. Liao et al., Cell (2019) 179(1):147-64.e20). Suitable RhoA inhibitors include compounds such as N-[1-(4-chloroanilino)-1-oxopropan-2-yl]oxy-3,5-bis-(trifluoromethyl)benzamide ("CCG-1423", DA Lionarons et al., Cancer Cell (2019) 36(1):68-83.e9).Suitable GGTI inhibitors include compounds such as N-(1-amino-1-oxo-3-phenylpropan-2-yl)-4-[2-(3,4-dichlorophenyl)-4-(2-methylsulfanylethyl)-5-pyridin-3-yl-pyrazol-3-yl]oxybutanamide ("GGTI-DU40", YK Peterson et al., J Biol Chem (2006) 281:12445-50), and (2S)-2-[[4-[[(2R)-2-amino-3-sulfanylpropyl]amino]-2-naphthalen-1-ylbenzoyl]amino]-4-methylpentanoic acid 2,2,2-trifluoroacetic acid ("GGTI-297", PA Subramani et al., Bioinformation (2015) 11(5):248-53). Suitable soluble epoxide hydrolase inhibitors include compounds such as 1-(1-acetylpiperidin-4-yl)-3-(1-adamantyl)urea ("AR9281", R.H. Ingraham et al., Curr Med Chem (2011) 18(4):587-603), 1-(1-propanoyl-piperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea ("TPPU", Y.M. Kuo et al., Mol Neurobiol (2019) 56:8451-74).

[0094] Suitable fatty acid amide hydrolase inhibitors include, but are not limited to, 4-hydroxy-N-[(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenyl]benzamide ("AM-1172", C. J. Hillard et al., J. Mol. Neurosci. (2007) 33:18-24), N-phenyl-4-(3-phenyl-1,2,4-thiadiazol-5-yl)-1-piperazinecarboxamide ("JNJ1661010", T. Lowin et al., Arth. Res. Ther. (2015) 17:321), and N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide ("PF-3845", S. Ghosh et al., J. al., J Pharmacol Exp (2015) 354(2):111-20). Suitable leukotriene receptor antagonists include, but are not limited to, compounds such as zafirlukast, montelukast, and zileuton.

[0095] (a) Aerosol formulation Aerosol is a suspension of small solid particles or droplets, typically with an average diameter of less than 10 μm, suspended in air or another gas. Aerosol formulations for delivering drugs to the respiratory tract are known in the art. For example, see A. Adjei et al., J Pharm Res (1990) 1:565-69; P. Zanen et al., J Int J Pharm (1995) 114:111-15; I. Gonda, Crit Rev Ther Drug Carrier Syst (1990) 6:273-313; Anderson et al., Am Rev Respir Dis, (1989) 140:1317-24 (the contents of all of which are incorporated herein by reference in their entirety).

[0096] Compositions for aerosol administration via pressurized metered dose inhalers (pMDIs) can be formulated as solutions or suspensions. Solution compositions can be more convenient to manufacture because the active agent is completely dissolved in the propellant vehicle, avoiding physical stability issues (such as particle aggregation) sometimes associated with suspension compositions. If the drug is not sufficiently soluble in the propellant, a cosolvent such as ethanol can be used to increase its solubility in pharmaceutical compositions for administration via pMDI. In some embodiments, the formulation comprises a statin dissolved in a propellant and a cosolvent.

[0097] Suspension formulations can contain small solid particles of the drug, typically having an average diameter of less than about 10 μm. Such formulations can be prepared by grinding or milling the drug in crystalline form or by spray-drying a solution containing the drug. In some embodiments, the formulation contains a powdered statin, a propellant, and a suspension vehicle. In some embodiments, the suspension vehicle is selected from PEG400, PEG1000, and propylene glycol (1,2-propanediol). In some embodiments, the statin comprises pitavastatin.

[0098] The pharmaceutical composition may be formulated with one or more suitable propellants, such as hydrofluoroalkanes, CO2, or other suitable gases. In some embodiments, a surfactant may be added to reduce the surface and interfacial tension between the composition, the propellant, and, if present, the cosolvent. The surfactant may be any suitable non-toxic compound that is non-reactive with other components of the pharmaceutical composition and reduces the surface and / or interfacial tension between the composition, the propellant, and the cosolvent to the desired extent. In some embodiments, the formulation does not require or may be surfactant-free to produce and / or maintain a stable pharmaceutical composition solution under normal operating conditions.

[0099] (b) Nebulizer formulation "Atomization" refers to the reduction of a liquid into a fine spray or mist. Small, uniformly sized droplets are produced in a controlled manner from a larger body of liquid formulation, typically with an average particle size of about 0.5 μm to about 10 μm. Atomization can be achieved by any suitable means, including mechanical nebulizers, including Respimat® Soft Mist nebulizers, in which the formulation is squeezed through a nozzle under spring pressure; jet nebulizers, in which a compressor compresses air or oxygen to flow at high velocity through a liquid, producing a mist; ultrasonic nebulizers, in which a piezoelectric transducer vibrating at ultrasonic frequencies is placed in contact with the liquid formulation, and the vibrations produce a mist or aerosol; or vibrating mesh nebulizers, in which a mesh or membrane with small holes vibrates on the surface of a liquid reservoir, producing a fine mist. Nebulizers using any of these technologies are commercially available. When the active ingredients are adapted to be administered together or separately via a nebulizer, they may be in the form of a nebulized aqueous suspension or solution, with or without appropriate pH or tonicity adjustment, as a single-dose or multi-dose device.

[0100] Formulations used for nebulizer administration are usually, but not necessarily, primarily aqueous. If the administered drug is only slightly soluble in water, a pharmaceutically acceptable cosolvent such as ethanol can be added to dissolve or aid in dissolving the drug. Alternatively, the formulation can be a suspension of appropriately sized particles suspended in a primarily aqueous carrier. The drug can also be formulated as solid lipid microparticles (SLMs), solid lipid nanoparticles (SLNs), or liposomes, and can be suspended in a liquid carrier for nebulization or aerosolization. See, for example, M. Paranjpe et al., Int J Mol Sci (2014) 15:5852-73; MJ de Jesus Valle et al., J Antibiot (Tokyo) (2013) 66(8):447-51 (both of which are incorporated herein by reference). The particle size of the nebulized droplets can be adjusted by several parameters, including, for example, the viscosity and surface tension of the formulation and the characteristics of the nebulizer, as taught in the art.

[0101] (c) Dry powder formulation: Dry powder formulations, as the name suggests, have no liquid carrier. Instead, the active substance and excipients are crushed or ground into a fine powder with a particle size suitable for inhalation. The formulation is designed to be delivered to the lungs by sharp inhalation and / or a puff of compressed air or gas. Dry powder formulations are particularly convenient for administering drugs that are difficult to dissolve or suspend in conventional liquid carriers.

[0102] Dry powder formulations often contain excipients in addition to one or more active substances. These excipients are often included to improve the flow properties of the product, such as dispersion and absorption, and for chemical stability during storage. The formulations can be prepared, for example, by spray drying (AA Ambike et al., Pharm Res (2005) 22(6):990-98), milling or grinding, extrusion, precipitation, and / or screening using methods known in the art to obtain inhalable powders. The excipients used can also be a mixture of milled excipients obtained by mixing excipient fractions of different average particle sizes.

[0103] Examples of physiologically acceptable excipients that can be used to prepare inhalable powders for use in an inhaler (or cartridge thereof) include monosaccharides (e.g., glucose, fructose, or arabinose), disaccharides (e.g., lactose, saccharose, maltose, trehalose), oligosaccharides, and polysaccharides (e.g., dextran, dextrin, maltodextrin, starch, cellulose), polyalcohols (e.g., sorbitol, mannitol, xylitol), cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, Excipients include statins, χ-cyclodextrin, methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin (Captisol®, Dexolve®), amino acids (e.g., arginine hydrochloride), salts (e.g., sodium chloride, calcium carbonate), or mixtures thereof. Lactose, glucose, and other compounds can be used in the form of their hydrates. Excipients can be combined with the statin before, during, or after the powdering process.

[0104] Within the inhalable powder range, excipients can have a maximum average particle size of up to about 250 μm, 10-150 μm, or 15-80 μm. Finer excipient fractions with an average particle size of 1-9 μm can also be added to the above excipients. The average particle size can be determined using methods known in the art (e.g., WO 02 / 30389). Finally, to prepare the inhalable powder, micronized crystalline statin, which can be characterized by an average particle size of about 0.5 to about 10 μm, or about 1 to about 5 μm, is added to the excipient mixture (see, e.g., WO 02 / 30389). Processes for grinding and micronizing active substances are known in the art. In the absence of a specially prepared excipient mixture to be used as an excipient, an excipient with an average particle size of 10-50 μm and a 10% fine content of 0.5-6 μm can be used. In some embodiments, the maximum average particle size is less than about 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μm. In some embodiments, the average particle size is at least about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 19, 20, 25, 30, 35, 40, 45, or 50 μm. In some embodiments, the average particle size is less than about 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μm.

[0105] In one method for preparing a dry powder formulation, excipients and active substances are placed in a suitable mixing vessel. In some embodiments, the active substance has an average particle size of 0.5-10 μm, 1-6 μm, or 2-5 μm. The excipients and active substance are added using a sieve or granulating sieve with a mesh size of 0.1-2 mm, 0.3-1 mm, or 0.3-0.6 mm. The excipients can be added first, followed by the active substance. During this mixing process, the two components can be added in batches, and the two components can be sieved in alternating layers. Mixing of the excipients and active substance can occur while the two components are still being added.

[0106] Inhalable powders can also be formulated as PulmoSpheres (see, e.g., J.G. Weers et al., Ther Deliv (2014) 5(3):277-95; J.G. Weers et al., AAPS PharSciTech (2019) 20(3):103; and U.S. Pat. No. 9,452,139, all of which are incorporated herein by reference), in which a suspension of micronized drug particles is spray-dried to form a powder. Alternatively, powders and suspensions can be formulated from self-assembled nanoparticles (see, e.g., N.J. Kenyon et al., PLOS One (2013) https: / / doi.org / 10.1371 / journal.pone.0077730).

[0107] inhaler The three main types of inhalers are nebulizers, pressurized metered-dose inhalers (pMDIs), and dry powder inhalers (DPIs). Nebulizers convert a liquid solution or suspension of drug into a fine mist of droplets, which are then inhaled into the lungs. Nebulizers typically take longer to administer medication than pMDIs or DPIs, and are less accurate regarding the exact dose of drug absorbed due to drug loss within the device and to the surrounding air. However, they are typically the easiest to use and can be used on subjects too young to operate a pMDI or DPI, or on unconscious subjects. Nebulizers typically include a reservoir containing the drug formulation, a nebulizing chamber, a face mask, and a mechanism for nebulizing the formulation. In jet nebulizers, this mechanism includes a nozzle through which air passes at high velocity, drawing the liquid formulation up through a capillary tube. Droplets of the formulation are entrained in the air jet and impinge on a baffle to reduce droplet size, or excessively large droplets are sieved and removed. The baffle also reduces air velocity, so the resulting mist leaves the nebulizer at a slower rate, making it more likely to reach the lower respiratory tract. The nebulization process in these devices also typically reduces the temperature of the formulation due to evaporation of the droplets. Jet nebulizers typically require a compressor to generate the airflow, making them noisier and less portable than other inhalers.

[0108] Ultrasonic nebulizers use an element that vibrates at ultrasonic frequencies to break up a liquid formulation into droplets. The vibrating element is often a rigid mesh or perforated membrane. These nebulizers are generally quieter than jet nebulizers and do not require a compressor, although they do require a power source. Ultrasonic vibrations often increase the temperature of the formulation.

[0109] pMDIs contain a solution or suspension of a drug in a propellant under pressure and include a valve that delivers a precisely measured amount of the formulation when actuated. The propellant is often a gas, such as a hydrofluoroalkane propellant, combined with the drug and, optionally, a cosolvent such as ethanol and / or a surfactant. The formulation is compressed to a liquid state and loaded into a pMDI or pMDI cartridge. A typical pMDI releases the liquid formulation into a metering chamber, which determines the dose. Upon actuation of the device, the measured formulation is released into an expansion chamber, where the propellant volatilizes. To efficiently and consistently deliver medication, subjects using a pMDI must synchronize their breathing with the actuation of the device to ensure the maximum possible amount of aerosol reaches the lower airways. Modern pMDIs may also include a valve or sensing mechanism that releases the aerosol only when the subject is inhaling. Most pMDIs also employ a spacer, which is essentially a tube between the pMDI and the subject, to improve the efficiency of aerosol delivery and increase the time it takes for the propellant to evaporate (leading to smaller droplets).

[0110] DPIs generally contain a measured amount of medication as a dry powder, optionally with a dry powder carrier such as powdered lactose. DPIs rely on a sharp inhalation by the subject to dispense the powder formulation, rather than the generation of a mist or aerosol. They are generally easier to use than pMDIs, but the efficiency of delivery depends in part on the air velocity the subject can generate. Newer breath-activated, but power-assisted, DPIs are under development.

[0111] The formulations of the present disclosure can be administered using commercially available inhalation devices such as, for example, nebulizers, including, but not limited to, the Respimat® Soft Mist™ inhaler, RespiClick® inhaler, Breezhaler® inhaler, Genuair® inhaler, and Ellipta® inhaler. The inhaler can be provided pre-filled with one or more therapeutic doses of the formulations of the present disclosure or can be configured to accept cartridges pre-filled with one or more therapeutic doses of the formulations of the present disclosure.

[0112] Inhalable powders and aerosols can be administered using an inhaler that meters a single dose from a reservoir, for example, by means of a metering chamber (see, e.g., U.S. Pat. No. 4,570,630) or other means (see, e.g., DE 3625685). In some embodiments, the inhalable powders are packaged in capsules or cartridges for use in inhalers such as those described in WO 94 / 28958.

[0113] Capsules and cartridges for use in an inhaler may be formulated into a powder mix comprising a disclosed compound or pharmaceutical composition and a suitable powder base such as lactose or starch.

[0114] system The methods of the present disclosure can also be carried out using the systems of the present disclosure, which include a statin or statin formulation and one or more additional therapeutic agents or a formulation containing one or more additional therapeutic agents. In the systems of the present disclosure, the statin and the additional therapeutic agent do not need to be in the same formulation and can be administered at different times. In some embodiments, the system includes a statin selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof. In some embodiments, the statin is selected from the group consisting of simvastatin, pitavastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, and tenivastatin. In some embodiments, the statin is a hydrophobic statin. In some embodiments, the statin is simvastatin or pitavastatin. In some embodiments, the statin is pitavastatin. In some embodiments, the statin is simvastatin.

[0115] In some embodiments, the formulation is a dry powder formulation. In some embodiments, the formulation is an aerosol formulation. In some embodiments, the formulation is a sprayable formulation. In some embodiments, the sprayable formulation comprises an aqueous solution of the statin. In some embodiments, the sprayable formulation further comprises a pharmaceutically acceptable alcohol. In some embodiments, the pharmaceutically acceptable alcohol comprises ethanol.

[0116] The additional therapeutic agent can be any of the additional therapeutic agents described in this disclosure. In some embodiments, the additional therapeutic agent is beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, fluocortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, meprednisone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol, amcinonide, ciclesonide, deflazacort, desonide, formocortal, fluchlorolone acetonide, fludroxycortide, fluocinolone acetonide, fluocinonide, halcinonide, or triamcinolone acetonide, or a combination thereof. In some embodiments, the additional therapeutic agent is albuterol, arformoterol, buphenine, clenbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, levalbuterol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, or zintero, or a combination thereof. In some embodiments, the additional therapeutic agent is albuterol.

[0117] In some embodiments, the additional therapeutic agent is ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, rebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, darifenacin, or a combination thereof. In some embodiments, the additional therapeutic agent is roflumilast, zileuton, nedocromil, theophylline, an anti-IL5 antibody or antibody derivative, an anti-IgE antibody or antibody derivative, an anti-IL5 receptor antibody or antibody derivative, an anti-IL13 / 4 receptor antibody or antibody derivative, mepolizumab, reslizumab, benralizumab, omalizumab, dupilumab, or a combination thereof. In some embodiments, the additional therapeutic agent is TS-f22, AT13148, GSK429286A, RKI-1447, Y-27632, CCG-1423, GGTI-DU40, GGTI-297, AR9281, TPPU, AM-1172, JNJ1661010, PF-3845, zafirlukast, montelukast, zileuton, or a combination thereof.

[0118] In some embodiments, the additional therapeutic agent is provided in a formulation comprising the additional therapeutic agent and a pharmaceutically acceptable carrier or vehicle. In some embodiments, the formulation is suitable for administration by inhalation. In some embodiments, the formulation is suitable for administration orally or by injection.

[0119] The additional therapeutic agent may treat the same disease, disorder, or symptom as the statin, or may treat a different symptom of the same disease or disorder. Combinations of one or more statins with one or more additional therapeutic agents can, in some cases, exhibit an additive effect, where the degree of response from the combined formulation is substantially the same as the sum of the degree of response from each agent when administered alone. The combination can also produce sub-additive effects, where the combination produces a degree of response that is less than the sum of the degree of response from each agent when administered alone (but greater than the response produced by either agent alone), or a synergistic effect, where the combination produces a degree of response that is greater than the sum of the degree of response from each agent when administered alone. Thus, combinations of one or more statins with one or more additional therapeutic agents can be used to achieve a greater response while administering a given dose, while achieving the same response while administering a lower dose, or any combination thereof.

[0120] If the degree of effect produced by the combination is greater than desired or necessary, the dose of one or both agents can be reduced until the desired degree of effect is achieved. The amount of dose reduction does not necessarily have to be the same amount or percentage for each agent. This can be used to reduce side effects or minimize the likelihood of side effects occurring. Thus, the dose of one or more agents in a synergistic combination can be reduced to a level that is insufficient or subtherapeutic when administered alone or as part of a non-synergistic combination, but is sufficient for therapeutic use when administered as part of the synergistic combination. A sub-therapeutic dose of an agent in a synergistic combination formulation can be about 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the effective amount of the agent when administered by inhalation as part of a non-synergistic formulation according to the present disclosure.

[0121] In some systems, administration of an inhaled statin enhances the effect of an additional therapeutic agent administered over a subsequent predetermined period, providing a greater therapeutic effect than either the statin or the additional therapeutic agent alone. In some systems, administration of an inhaled statin enhances the effect of the additional therapeutic agent other than ASM relaxation. In some systems, the additional therapeutic agent is administered after the statin. In some embodiments, the period is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or about 1, 2, or 3 days. In some embodiments, the period is about 72, 48, 36, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 hours or less.

[0122] In some systems, the inhaled statin enhances or increases the anti-inflammatory effect of the additional therapeutic agent, hi some embodiments, the degree of enhancement is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45, 50, 75, or 100 times the effect of the additional therapeutic agent at the administered dose. In some embodiments, the synergistic therapeutic effect is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, or 1,000% greater than the effect produced by the sum of the agents of the combination when those agents are administered alone at the same dosages as present in the combination.

[0123] In some embodiments, the effect is an anti-inflammatory effect. In some embodiments, the additional therapeutic agent is a beta-2 agonist or an anti-inflammatory corticosteroid. In some embodiments, the additional therapeutic agent is a beta-2 agonist. In some embodiments, the beta 2 agonist is albuterol, arformoterol, buphenine, clenbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, levalbuterol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, or zintero, or a combination thereof. In some embodiments, the beta 2 agonist is albuterol or isoproterenol.

[0124] In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the corticosteroid is beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, fluocortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, meprednisone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol, amcinonide, ciclesonide, deflazacort, desonide, formocortal, fluchlorolone acetonide, fludroxycortide, fluocinolone acetonide, fluocinonide, halcinonide, or triamcinolone acetonide, or a combination thereof.

[0125] Treatment method (a) Inhalation administration The disclosed therapeutic method is based on the administration of an appropriate statin by inhalation. The disclosed methods, formulations, and systems treat lung diseases not directly caused by inflammation, thus providing a treatment for diseases not effectively or completely treated with existing therapeutics. Furthermore, the disclosed methods, formulations, and systems enhance the activity of other therapeutic agents, such as β2 agonists and corticosteroids, and increase the activity of other therapeutic agents, which may include anti-inflammatory activity. Administration by inhalation has the advantages of (a) direct contact with the respiratory tract, (b) avoidance of first-pass hepatic metabolism, and (c) avoidance of injection (JL Rau, Resp Care (2005) 50(3):367-82; M. Ibrahim et al., Med Dev Evidence Res (2015) 8:131-39). Because the drug does not undergo first-pass metabolism and is administered locally to the lungs rather than systemically, the dose of an inhaled drug is often lower than that administered orally. Additionally, the disclosed methods, formulations, and systems that enhance the activity of other therapeutic agents can provide treatments with greater activity or can match existing treatments using lower doses of other therapeutic agents, or combinations thereof.

[0126] As described herein, the formulations of the present disclosure are administered with the aid of an inhalation device ("inhaler"), which may be a nebulizer, pMDI, DPI, or other device capable of delivering the formulation to the lower respiratory tract. The frequency of administration depends on the clearance rate of the statin and / or additional therapeutic agent from the subject's lungs. In some embodiments, the statin formulation is administered no more than 8, 7, 6, 5, 4, 3, 2, or 1 time per day, or no more than once every 2, 3, 4, 5, 6, or 7 days. In some embodiments, the statin formulation is administered at least once every 4, 3, or 2 days, or at least 1, 2, 3, 4, 5, or 6 times per day.

[0127] In the disclosed method, the therapeutic composition is administered directly to the lungs and therefore does not undergo first-pass metabolism in the liver. As a result, the active agent in the formulation is not diluted in the subject's system and is not metabolized by the liver, so that therapeutic concentrations are reached in the subject's respiratory tract in smaller amounts than would be required for conventional oral administration. The therapeutically effective amount will depend on the condition being treated, the severity of the condition, the subject's general health and condition, and the particular statin (and / or isomer, enantiomer, and / or diastereoisomer) selected. Thus, a therapeutically effective amount of a statin in the practice of the present disclosure may be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin in the practice of the present disclosure may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0128] In some embodiments, the therapeutically effective amount of the statin is at least about 0.005 μg / kg, about 0.008 μg / kg, about 0.01 μg / kg, about 0.05 μg / kg, about 0.08 μg / kg, about 0.1 μg / kg, about 0.5 μg / kg, about 0.8 μg / kg, about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 18 μg / kg, or about 20 μg / kg. In some embodiments, the therapeutically effective amount of the statin is less than or equal to about 40 mg / kg, 20 mg / kg, 18 mg / kg, 15 mg / kg, 12 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.

[0129] (b) Decreased airway smooth muscle contraction The airways of mammalian lungs are lined with airway smooth muscle (ASM) covered by a thin layer of airway epithelial cells (AECs). ASM is an involuntary muscle tissue that contributes to regulating lung function by contracting and relaxing, controlling the diameter of the airways (bronchi and bronchioles). In some disorders, ASM inappropriately contracts, narrowing the airways and increasing the effort required for breathing. Conventional treatments attempt to inhibit contraction by blocking or reducing inflammatory stimuli. In the methods of the present disclosure, ASM is relaxed by directly administering a formulation of the present disclosure to the airways. Without intending to be bound by a particular theory, the methods of the present disclosure reduce the contractile response, decrease the force exerted by ASM, and inhibit ASM overgrowth (which also causes airway narrowing). In some embodiments, the present disclosure provides a method of reducing ASM contraction by administering an effective amount of a formulation of the present disclosure by inhalation.

[0130] In some embodiments, the present disclosure provides a method for reducing airway smooth muscle (ASM) contraction in a subject by inhaling a therapeutically effective amount of a statin (or its isomer, enantiomer, or diastereoisomer) together with a pharmaceutically acceptable carrier. Relaxation of ASM can reduce obstruction to breathing and increase lung capacity at rest. Thus, the methods of the present disclosure are useful for treating pulmonary diseases characterized by or otherwise involving obstruction of the subject's airways, such as bronchoconstriction or bronchospasm inherent in asthma, COPD, ACOS, cystic fibrosis, bronchiectasis, idiopathic pulmonary fibrosis, alpha-1 antitrypsin deficiency (AATD), etc. Additional pulmonary diseases and disorders that may directly or indirectly affect the pulmonary airways (and thus may be ameliorated by the methods of the present disclosure) include, but are not limited to, interstitial lung diseases (ILDs), such as pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), desquamative interstitial pneumonia (DIP), acute interstitial pneumonia (AIP), nonspecific interstitial pneumonia (NSIP), respiratory bronchitis-associated interstitial lung disease (RB-ILD), latent organizing pneumonia (COP), and lymphocytic interstitial pneumonia (LIP). Additional diseases and disorders that may involve the lungs and may benefit from the methods of the present disclosure include sarcoidosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), systemic sclerosis, polymyositis, dermatomyositis, antisynthetase syndrome, pulmonary infections, hypersensitivity pneumonitis, and responses to acute or chronic exposure to foreign bodies such as asbestos, beryllium, silica, industrial chemicals, and irritant particles. In these additional lung diseases and disorders, symptoms such as increased airway wall thickening (see, e.g., JM Oldham, Ann Am Thorac Soc (2019) 16(4):432-33; ER Miller et al., Ann Am Thorac Soc (2019) 16(4):447-54) may be improved by administration of inhaled statins.

[0131] ASM relaxation can be determined in vitro, for example, but not limited to, by methods for measuring cellular force and by measuring airway lumen changes in lung tissue samples. For example, ASM contractile force can be measured using the method described in R. Rokhzan et al., Lab Invest (2019) 99(1):138-45, which measures the displacement of fluorescent beads on a substrate of known stiffness. Measurement of airway lumen changes in lung tissue samples can be performed using the method described in KR Patel et al., FASEB J (2017) 31(10):4335-46, which measures airway diameter in precision-cut lung sections.

[0132] ASM relaxation can be measured in vivo by standard pulmonary function tests, including, but not limited to, spirometry and lung volume measurements. Spirometry is the measurement of respiration, including the volume and / or rate of air inhaled or exhaled. Typical measurements include forced vital capacity (FCV), in which a subject takes as deep a breath as possible and exhales it as forcefully and as long as possible into a spirometer; forced expiratory volume in 1 second (FEV1), which is a measurement of the amount of air a subject can exhale in 1 second; maximum ventilation (MVV); and forced expiratory flow (FEF). Other parameters include lung volume, lung volume, and vital capacity, which are also commonly measured by spirometry. An increase in any of the aforementioned values ​​indicates ASM relaxation, in that ASM relaxation may decrease obstruction and increase vital capacity. An increase in any of these values ​​can be measured relative to the subject's measurements before treatment and / or relative to standard expected values ​​for subjects of similar height, weight, and sex. The disclosed methods induce ASM relaxation of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of ASM relaxation is about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50%.

[0133] A therapeutically effective amount of a statin for inducing ASM relaxation can be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for inducing ASM relaxation can be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0134] (c) Decreased bronchospasm Bronchospasm is a sudden contraction or constriction of the bronchi, usually in response to inflammatory stimuli, such as exposure to allergens, mast cell degranulation, and administration of certain drugs. It occurs in asthma, chronic bronchitis, and anaphylaxis and can be life-threatening. Bronchospasm is a hallmark of asthma exacerbations ("asthma attacks"). Inhalation administration of a formulation of the present disclosure treats bronchospasm (and asthma exacerbations) by reducing airway hyperactivity and hyperresponsiveness, making bronchospasm less likely to occur, and reducing ASM contractile force, thereby reducing the severity of bronchospasm if it does occur. In some embodiments, the present disclosure provides a method for reducing bronchospasm by administering an effective amount of a formulation of the present disclosure by inhalation.

[0135] Bronchospasm may include, for example, post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing wheezing due to bronchospasm); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; bronchospasm associated with paraneoplastic syndromes.

[0136] A therapeutically effective amount of a statin for reducing bronchospasm may be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0137] A reduction in bronchospasm can be measured by counting the number of bronchospasm events (e.g., asthma exacerbations) over a period of time and comparing this frequency to the frequency observed before treatment. A reduction in bronchospasm can also be measured by measuring a subject's pulmonary function (e.g., FEV1) before challenge, then administering a dose (or series of increasing doses) of nebulized methacholine or histamine, and then measuring the subject's pulmonary function again. After obtaining a baseline value, the subject is treated with inhalation of a formulation of the present disclosure and challenged again after an appropriate period of time. The challenge can occur about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours or more after inhalation of a formulation of the present disclosure. A reduction in bronchoconstriction is determined by an improvement in pulmonary function after challenge, for example, by comparing the FEV1 after administration and challenge to the FEV1 before administration but after challenge. The disclosed methods reduce bronchospasm by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of reduction in bronchospasm is about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50%.

[0138] (d) Reduced bronchoconstriction and mucus accumulation Some airway diseases cause chronically constricted or blocked bronchi and bronchioles, which further leads to the accumulation of mucus.This is characteristic of diseases such as emphysema, asthma, COPD, cystic fibrosis, allergen-induced bronchoconstriction, and exercise-induced bronchoconstriction (also known as exercise-induced asthma).Administering the formulation of the present disclosure by inhalation relaxes ASM and expands airways, thereby treating bronchoconstriction and mucus accumulation.In some embodiments, the present disclosure provides a method for reducing bronchoconstriction and / or mucus accumulation by administering an effective amount of the formulation of the present disclosure by inhalation.

[0139] Bronchoconstriction, bronchospasm, and ASM relaxation are clear signs, and improvements in each can be measured by pulmonary function tests such as spirometry, as described above. The disclosed methods reduce bronchoconstriction by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of reduction in bronchoconstriction is about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50%. A therapeutically effective amount of a statin for reducing bronchoconstriction may be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for reducing bronchoconstriction may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0140] (e) Decreased bronchial hyperresponsiveness Bronchial hyperresponsiveness (BH, also known as airway hyperresponsiveness, AHR, or airway hyperactivity) is a condition in which bronchospasm is easily triggered or induced. The disclosed method of administering the disclosed formulation by inhalation reduces BH by relaxing ASM and reducing ASM sensitivity. In some embodiments, the disclosure provides a method of reducing bronchial hyperresponsiveness by administering an effective amount of the disclosed formulation by inhalation.

[0141] Because BH is an airway disorder, it is typically measured by spirometry and other measures of lung function. For example, a subject's FEF can be measured after administration of a formulation and after challenge with a provocative substance, such as nebulized methacholine or histamine, and compared to the FEF after stimulation with the same amount of provocative substance without administration of a formulation of the present disclosure. The challenge can be administered about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours or more after inhalation of a formulation of the present disclosure. The methods of the present disclosure reduce BH by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of BH reduction is about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50%. A therapeutically effective amount of a statin for reducing BH can be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for lowering BH may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0142] (f) Stretch-induced increase in ASM relaxation ASM is induced to relax by deep breathing ("deep inspiration"), which stretches the ASM. The methods of the present disclosure enhance, increase, and / or prolong this breathing-induced ASM relaxation (also called breathing-induced bronchodilation, or deep inspiratory bronchodilation, DIB). In some embodiments, the present disclosure provides a method for enhancing deep breathing-induced ASM relaxation by administering an effective amount of a formulation of the present disclosure by inhalation.

[0143] Because DIB is a function of the airways, it is also commonly measured by spirometry and other measures of lung function. For example, a subject's vital capacity (VC) can be measured after administration of a formulation and compared to VC without administration of the formulation of the present disclosure, and an increase in VC correlates with enhanced respiratory-induced ASM relaxation. VC can be measured about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 24 hours or more after inhalation of the formulation of the present disclosure. The method of the present disclosure increases DIB by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of DIB increase is about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50%. A therapeutically effective amount of a statin for enhancing DIB can be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for enhancing DIB may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0144] (g) Decreased corticosteroid use Inhaled corticosteroids (ICS) are commonly used to treat severe asthma. However, chronic use of ICS can also cause serious side effects, such as dysphonia, decreased bone density, thinning and bruising of the skin, and cataracts. The methods of the present disclosure reduce and treat asthma and other airway diseases, reducing the need for a subject to take ICS. In some embodiments, the present disclosure provides methods of reducing the need for ICS by administering an effective amount of a formulation of the present disclosure by inhalation.

[0145] A reduction in the need for ICS can be determined by placing a subject on a treatment regimen of regular administration of a formulation of the present disclosure by inhalation while using ICS, and gradually reducing the dosage or frequency of ICS use until the subject's pulmonary function (if this point can be reached) is equal to the pulmonary function of the subject before initiating inhaled statin therapy, and measuring the subject's pulmonary function, e.g., by spirometry. The methods of the present disclosure reduce the need for ICS by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (i.e., at which point the statin completely replaces the ICS). The upper limit of the reduction in the need for ICS is about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, or 25%. A therapeutically effective amount of a statin to reduce the need for ICS can be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin to reduce the need for ICS can be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0146] (h) Methods for maintaining or improving lung function Pulmonary function varies from subject to subject and is generally higher (higher than average) in athletes, mountain climbers, and subjects living at high altitudes. Improved pulmonary function can be measured by spirometry and is often manifested as an increase in VC or lung capacity (LC). The methods of the present disclosure are also useful for improving pulmonary function in healthy subjects as well as subjects with airway disorders. For example, it is advantageous to improve the pulmonary function of athletes, mountain climbers, soldiers, wind instrument players, public speakers, etc. Furthermore, the methods of the present disclosure allow subjects to maintain a certain degree of pulmonary function during periods when exercise is impossible, for example, due to injury. Improved (or maintained) pulmonary function can be measured by spirometry, for example, but not limited to, as an increase in VC or lung capacity (LC). In some embodiments, the present disclosure provides a method for maintaining or improving pulmonary function by administering an effective amount of a formulation of the present disclosure by inhalation.

[0147] The disclosed methods increase lung function by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, or 150%. The upper limit of increase in lung function is about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, or 20%. A therapeutically effective amount of a statin for increasing lung function may be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for increasing lung function may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0148] In some cases, maintaining lung function requires less statin than increasing lung function, for example, doses may be as low as about 0.001, 0.005, 0.008, 0.01, 0.05, 0.08, 0.1, 0.5, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, or 20 μg. A therapeutically effective amount of a statin for increasing lung function may be as high as about 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0149] (i) Methods for reducing ASM growth COPD, ACOS, cystic fibrosis, and chronic asthma can all exhibit narrowing of the airways due to excessive proliferation and thickening of ASM associated with pathological airway remodeling, excluding bronchoconstriction. The methods of the present disclosure also reduce ASM proliferation, thereby treating such disorders. Inhibition of ASM proliferation can be measured by spirometry as maintained VC or lung capacity (LC), or by imaging methods such as X-ray or MRI. In some embodiments, the present disclosure provides a method of reducing ASM proliferation by administering an effective amount of a formulation of the present disclosure by inhalation.

[0150] The disclosed methods reduce ASM growth by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% (i.e., no ASM growth is observed at that time). The upper limit of reduction in ASM growth is about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, or 25%. A therapeutically effective amount of a statin for reducing ASM proliferation can be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for reducing ASM proliferation can be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0151] (j) Method for treating interstitial lung disease Interstitial lung disease (ILD) directly affects lung tissue outside the airways. However, ILD can affect the airways and ASM, causing one or more symptoms that can be treated with the methods of the present disclosure. These symptoms include, for example, ASM contraction, ASM hyperproliferation, and lung volume loss. In some embodiments, the present disclosure provides a method for helping treat ILD affecting the airways of a subject by administering by inhalation a formulation of the present disclosure in an amount sufficient to alleviate one or more symptoms. Examples of specific ILDs are described above.

[0152] The reduction in one or more ILD symptoms can be measured using spirometry, such as FEV1 and LC, and imaging techniques, such as X-ray and MRI. The disclosed methods reduce at least one ILD symptom by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% (i.e., the symptom is no longer observed at that time). The upper limit of reduction in an ILD symptom is about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, or 25%. A therapeutically effective amount of a statin for reducing one or more ILD symptoms may be as low as about 0.005 μg, about 0.008 μg, about 0.01 μg, about 0.05 μg, about 0.08 μg, about 0.1 μg, about 0.5 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 14 μg, about 15 μg, about 16 μg, about 18 μg, or about 20 μg. A therapeutically effective amount of a statin for reducing one or more ILD symptoms may be as high as about 40 mg, 20 mg, 18 mg, 15 mg, 12 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, or 1 mg.

[0153] In some embodiments, the present disclosure provides a method for treating COPD in a subject. COPD can be characterized as the destruction of both small airways and parenchyma, resulting in progressive impairment of lung function. This disease is divided into two subgroups: chronic bronchitis and emphysema. Chronic bronchitis is characterized by mucus hypersecretion from the conducting airways, inflammation, and eventual scarring of the bronchi (airways). Many people with COPD have components of both of these conditions.

[0154] The interplay between parenchymal disease and the vasculature is often evident clinically by the observation that patients with severe COPD have mild or moderate pulmonary hypertension at rest. Histopathologically and microscopically, the pulmonary vasculature in COPD is typically characterized by early thickening accompanied by smooth muscle deposition and loss of both alveolar septal architecture and microvasculature. Apoptosis of both alveolar septal cells and endothelial cells has also been observed in COPD.

[0155] The presenting symptom of COPD is typically shortness of breath accompanied by a decrease in FEV1 (i.e., forced expiratory volume in 1 second) and / or forced vital capacity (FVC). COPD patients develop smaller, inflamed airways and partial destruction of alveoli, making breathing difficult. Chronic bronchitis can also be diagnosed by asking patients whether they have a "productive cough," i.e., a cough that produces phlegm. Patients' symptoms include coughing and phlegm production. Chronic bronchitis can lead to more frequent and severe respiratory infections, bronchial narrowing and obstruction, difficulty breathing, and obstruction.

[0156] In some embodiments, the present disclosure provides a method for treating emphysema in a subject. Emphysema is a chronic lung disease that affects the alveoli and / or the ends of the smallest bronchi. This condition is characterized by destructive changes and enlargement of the alveoli (air sacs) in the lungs. The lungs lose elasticity, and these areas of the lungs therefore enlarge. These enlarged areas trap stale air, which is not effectively exchanged with fresh air. This can lead to breathing difficulties and insufficient oxygen delivery to the blood. The primary symptom of emphysema patients is shortness of breath.

[0157] In some embodiments, the present disclosure provides methods of treating a subject who is not adequately controlled by current drug therapy by administering a formulation of the present disclosure in place of or in combination with the subject's current drug therapy.

[0158] In practicing the methods of the present disclosure, a pulmonary disease is treated by reducing airway smooth muscle contraction in a subject in need thereof by administering to the subject by inhalation a formulation, wherein the formulation comprises a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof, and a pharmaceutically acceptable carrier. [Example]

[0159] The following examples are provided as a guide and are not intended to limit the scope of the claims herein.

[0160] Primary human airway smooth muscle (ASM) cells from both non-asthmatic and asthmatic donors were obtained from the Gift of Hope Organ and Tissue Donor Network. These cells have been extensively characterized (see, e.g., H. Yoshie et al., Biophys J (2018) 114(9):2194-99). All measurements were performed using passages 5–8 cells from three non-asthmatic donors. Cells were grown in either complete F12 medium containing 10% serum or serum-deficient medium supplemented with insulin, transferrin, and selenium (Corning, Tewksbury, MA).

[0161] To enhance cellular uptake and predictable cell relaxation properties, simvastatin was activated by alkaline hydrolysis, chemically converting simvastatin lactone to simvastatin acid (SA). In vivo, hydrolysis can also occur spontaneously within cells via lactonase, paraoxonase, alkaline hydrolase, and carboxylesterase. Simvastatin was activated by opening the lactone ring using a protocol provided by Merck. Briefly, 8 mg of simvastatin (0.019 mM) was dissolved in 0.2 mL of 100% ethanol, followed by the addition of 0.3 mL of 0.1 N NaOH. The solution was then heated in a sand bath at 50 °C for 2 hours and then neutralized with HCl to pH 7.2 (CC Ghosh et al., Crit Care Med (2015) 43(7):e230-40).

[0162] Example 1: Relaxation of ASM contraction force The following experiments demonstrate that statins can cause relaxation of ASM in resting, unstimulated cells.

[0163] Contractile Force ScreeningHuman ASM cells were grown to confluence on custom NuSil® 8100 elastic substrates (Avantor, Inc., Radnor, PA) in 96-well plates (R. Rokhzan et al., Lab Invest (2019) 99(1):138-45). Fluorescent beads (approximately 400 nm in diameter) were embedded on the substrate surface to allow for calculation of traction forces based on their displacement. To measure traction forces, an inverted epifluorescence microscope (DMI 6000B, Leica Inc., Germany) equipped with a heated chamber (37°C), a monochrome camera (Leica DFC365 FX), and a motorized stage was used. Spatial images of the fluorescent beads embedded in the substrate were recorded at 10x magnification. Based on the bead displacement (resolution, approximately 15 μm) compared to the acellular model, along with knowledge of the substrate stiffness and thickness, traction forces were calculated using the approach of Fourier transform traction cytometry (B. Yeganeh et al., Pharmacol Ther (2014) 143(1):87-110), modified for the case of cell monolayers (EJ Whalen et al., Cell (2007) 129(3):511-22). From each traction force map, root mean square traction force (RMST) values ​​and strain energy were calculated and reported as a measure of normal contraction in the monolayer. For each well, the ratio of post-treatment to pre-treatment strain energy was calculated, and all values ​​for a particular treatment group were normalized to the mean for each treatment group.

[0164] Comparison of statinsTo determine the effects of statins on the contractile mechanism independent of inflammation, primary ASM cells were grown on bead-embedded NuSil™ (3 kPa hardness) and treated with simvastatin acid (SA), rosuvastatin, pravastatin, and pitavastatin (1 μM each) with or without mevalonate (MA, 100 μM). Because MA is a direct product of the action of HMGCR on HMB-CoA, the addition of MA would reverse the effects of statin inhibition of HMGCR. Contractile force screening (CFS) demonstrated that statins directly relax human ASM cells under basal conditions (Figure 1A). Lipophilic statins were the most potent (simvastatin < pitavastatin). Conversely, other hydrophilic statins, such as rosuvastatin and pravastatin, had little or no effect on ASM cell relaxation, confirming the differential effects of statin drugs, likely due to differences in lipophilicity. Coadministration of MA and statins eliminated the relaxant effect of statins on ASM, suggesting that ASM tone depends on MA or the MA pathway and confirming the MA-dependent mechanism of statin-induced relaxation in ASM cells (Fig. 1B).

[0165] Simvastatin acid (SA), atorvastatin, pravastatin, and pitavastatin were examined in CFS to determine the dose-response of their ASM relaxant effects. Simvastatin acid (SA), atorvastatin, pravastatin, and pitavastatin were added to primary ASM cells at 0, 0.08, 0.4, 2, and 10 μM, respectively, as described above. In a second experiment, simvastatin acid (SA), rosuvastatin, pravastatin, and pitavastatin were added to primary ASM cells at 0, 1, and 10 μM, respectively, as described above. These dose-response experiments further demonstrated that the inhibitory potency of statins on ASM contraction varied according to lipophilicity (simvastatin ≈ pitavastatin > atorvastatin >> pravastatin), where the most lipophilic statins, atorvastatin and pitavastatin, had the most pronounced effects compared with the less lipophilic atorvastatin and the hydrophilic pravastatin (Figures 1C and 1D).

[0166] We next compared the dose-dependent effects of SA and pitavastatin on primary ASM cells obtained from three different human donors and showed that both SA and pitavastatin relaxed ASM in a dose-dependent manner (Figure 3).

[0167] Cell force measurements were performed in prepared custom 96-well plates (substrate stiffness = 3 kPa) using a contractile force screening method performed with an inverted fluorescence microscope (10x microscope objective, Leica DMI6000V, Leica Microsystems, Buffalo Grove, IL). From each ASM driving force map, strain energy (i.e., the energy imparted to the substrate by the contractile cell, in pJ) was calculated and taken as the average cell contraction.

[0168] Using cell mapping rheometry, well-defined biaxial stretches (4 s duration, 10% magnitude) were imposed. Strain energy from each ASM driving force map was calculated as a metric of average cell contraction and reported as a fold change from the baseline value before stretch.

[0169] Using a contractile force screening method, we determined that the lipophilic statins pitavastatin and simvastatin inhibited ASM contraction, but the hydrophilic statin pravastatin did not (Figure 10A). Furthermore, the relaxant effect of pitavastatin was more pronounced at 24 h compared with simvastatin (Figure 10B). The driving force-inhibitory effect of pitavastatin was reversible after cessation of treatment (Figure 10D).

[0170] Pitavastatin showed no cytotoxicity (Figure 10E) or lung tissue toxicity (Figure 10F). To establish clinical relevance, we evaluated the effects of pitavastatin in multiple ASM cell lines derived from the lungs of both non-asthmatic and asthmatic human donors. Furthermore, asthmatic ASM were more contractile than non-asthmatic ASM (Figure 11A). Regardless of donor (asthmatic donors: D1–D3; non-asthmatic donors: D4–D6) or disease state (non-asthmatic vs. asthmatic), pitavastatin dose-dependently inhibited ASM contraction (Figure 11A).

[0171] Lack of apoptosis Several studies have shown that sufficiently high doses of statins can reduce the viability of vascular smooth muscle cells, epithelial cells, and endothelial cells (S. Ghavami et al., Biochim Biophys Acta (2014) 1843(7):1259-71; T. P. Miettinen et al., Cell Rep (2015) 13(11):2610-20). To confirm that statin-mediated cell relaxation is independent of apoptosis or loss of cell viability, we performed a cell-based apoptosis / necrosis assay after dose-dependent treatment of ASM with simvastatin (SA), pitavastatin, rosuvastatin, and pravastatin according to the manufacturer's protocol for 24 hours (Figure 2).

[0172] The RealTime-Glo® Annexin V apoptosis and necrosis assay was performed by growing ASM cells in 96-well white cell culture plates according to the manufacturer's instructions (Promega Inc.). Cells were treated with simvastatin, rosuvastatin, pitavastatin, and pravastatin at doses ranging from 0.08 μM to 100 μM. In this real-time Annexin V binding assay, apoptosis was detected using a luminescent signal and necrosis was detected using a fluorescent signal using a SpectraMax® plate reader. Digoxigenin (20 μg / mL) was used as a positive control (Figure 2).

[0173] No apoptosis was observed in cells treated with up to 30 μM simvastatin (treated as SA), pitavastatin, or pravastatin, or up to 100 μM rosuvastatin (Figure 2). This suggests that lipophilic statins are well tolerated by ASM. This further indicates that the observed positive effect of statin-induced ASM relaxation was not due to cytotoxicity or cell death.

[0174] Example 2: Histamine-induced contractions These experiments were performed to determine the ability of statins to reduce ASM contraction induced by exposure to histamine.

[0175] Cells were pretreated with 0, 0.08, 0.4, 2, 10, or 50 μM pitavastatin and simvastatin acid for 24 hours and then challenged with histamine (10 μM). Both pitavastatin (Figure 4A) and simvastatin (Figure 4B) significantly reduced histamine-induced ASM contraction in both complete and serum-starved media (Figure 4C). In complete media, 0.08 μM pitavastatin was sufficient to inhibit histamine-mediated ASM contraction (Figure 4A). However, a 5-fold molar excess of simvastatin (SA) was required to achieve similar protection (Figure 4B). In serum-starved media, 0.4 μM pitavastatin and 10 μM simvastatin provided comparable protection from histamine-induced contraction.

[0176] The time-dependent effects of both SA and pitavastatin indicate that the ASM-relaxing effect begins as early as 4 h (Figure 4C). Thus, this experiment demonstrates that a) pitavastatin is at least 5-fold more potent than SA in preventing histamine-induced ASM contraction and b) nanomolar availability of statins in the lung may be sufficient to inhibit histamine-mediated bronchoconstriction.

[0177] Lungs from non-transplantable, non-asthmatic human donors were obtained through the Gift of Hope / Regional Organ Bank in Illinois and sliced ​​according to published protocols. Briefly, lung lobes were filled with 1.5% low-melting-point agarose (Type IX; Sigma, St. Louis, MO) in Hank's balanced salt solution (pH = 7.4; Invitrogen, Carlsbad, CA) and sliced ​​using a VT1200S vibrating blade microtome (Leica Microsystems, Bannockburn, IL) to generate 250 μm-thick sections. Sections were stored frozen until the day of the experiment.

[0178] Slices were treated with pitavastatin (2 μM) for 24 hours and then challenged with histamine (1 μM). Airway contraction was measured as the change in luminal area in response to increasing doses of histamine. For both sets of PCLS samples, airway luminal area was quantified from bright-field images using Fiji image analysis software.

[0179] In the bronchial airways of human PCLS, pitavastatin significantly inhibited 1 μM histamine-induced airway contraction (Figure 11C). Pitavastatin enhanced stretch-induced force relaxation of ASM (Figure 12B). Surprisingly, this bronchodilatory effect was not conferred to ASM by isoproterenol. Thus, pitavastatin offers novel and additive therapeutic effects beyond those of existing β2-agonist bronchodilators.

[0180] Example 3: Deep breathing relief This experiment demonstrates that pitavastatin, in contrast to the β2 agonist isoproterenol, enhances the ASM relaxant effect of simulated deep breathing.

[0181] Normal ASM cells were treated with pitavastatin (1 μM, 24 h, n = 7), isoproterenol (10 μM, 30 min, n = 6), or vehicle (n = 7) and examined by CFS as described above. Figure 12A shows that pretreatment with pitavastatin significantly inhibited basal ASM contractions compared with untreated controls. Shown are contraction values ​​normalized to the untreated control group. Figure 12B shows that ASM cells rapidly and dramatically eliminated their contractions in response to a subsequent single stretch-unstretch maneuver mimicking a deep breath (10% amplitude, 4 s duration). Contractile force gradually recovered over 180 s. Force elimination was similar in all three groups, but subsequent force recovery was significantly inhibited by pitavastatin treatment (*p < 0.05; ****p < 0.0001). All data are reported as the mean and standard error of the mean (SEM).

[0182] Example 4: RHO kinase inhibition Statins inhibit the activation of Rho-kinase (ROCK) in animals (A. Nohria et al., Atherosclerosis (2009) 205(2):517-21). One of the major substrates of ROCK in regulating actin-myosin contraction is myosin light chain 2 (MLC2) (Y. Kureishi et al., J Biol Chem (1997) 272(19):12257-60). These experiments were conducted to demonstrate that statins significantly reduced the activation of ROCK by histamine or thrombin, which in turn reduced ASM contractile force.

[0183] Antibodies for Western blot analysis against total and phospho-MLC2 were obtained from Santa Cruz Biotechnology and Cell Signaling Technology, respectively. Antibodies against pROCK1, total ROCK1, and GAPDH were obtained from Abcam. Pitavastatin was obtained from Santa Cruz Biotechnology.

[0184] Human ASM cells were treated with pitavastatin (1 μM) with or without mevalonate (MA, 200 μM) for 24 hours, followed by histamine (10 μM) for 5 minutes. As shown in Figure 5, pitavastatin-treated ASM significantly reduced histamine-induced ROCK1 phosphorylation (Figure 5A), an effect that was eliminated by MA.

[0185] Human ASM cells were treated with pitavastatin (1 or 10 μM) for 24 hours, followed by thrombin treatment (2 U, 30 min). As shown in Figure 5B, pitavastatin (1 or 10 μM) inhibited thrombin-induced MLC2 phosphorylation.

[0186] Example 5: Cytoskeletal Inhibition Contractile forces in ASM are mediated by the cytoskeleton, and these experiments were performed to demonstrate that statins inhibit the expression of F-actin, a cytoskeletal component.

[0187] Non-asthmatic primary human ASM cells were treated with either vehicle or pitavastatin (1 μM) for 24 hours. Cells were then immunostained for F-actin expression. As shown in Figure 15A, pitavastatin significantly reduced basal F-actin expression. Cell lysates were analyzed by Western blot for total ROCK-1, total ROCK-2, total MLC-2, and phosphorylated MLC-2. As shown in Figures 15C and 15D, pitavastatin reduced the total expression of ROCK-1, ROCK-2, and MLC-2 (total and phosphorylated).

[0188] Non-asthmatic primary human ASM cells were co-treated with 1 μM pitavastatin (Pit) and 10 μM GGPP, or Pit and 10 μM GGPP plus 100 μM MA for 24 h. Pit reduced F-actin expression and ASM contraction; these reductions were abrogated by GGPP and MA (Fig. 15B).

[0189] Example 6: Decrease / prevent hypercontraction of ASM This experiment was performed to demonstrate that statins reduce or prevent the development of airway hyperconstriction, independent of their anti-inflammatory effects. In this model, administration of nebulized methacholine (MCh) to mice during postnatal ASM maturation induces a hyperconstriction phenotype without eliciting an inflammatory response.

[0190] All mouse experiments were approved by the Institutional Animal Care and Use Committee at Brigham & Women's Hospital, Harvard Medical School. pastThe constriction mouse model was described in KR Patel et al., FASEB J (2017) 31(10):4335-46. Briefly, mice (C57BL / 6) were exposed to nebulized MCh (30 mg / mL) for 10 minutes daily between P15 and P20 (5 days). Control mice received nebulized normal saline. This established an MCh hyperconstrictive asthma phenotype, a non-inflammatory model of asthmatic airway hyperresponsiveness (AHR). Before each MCh nebulization, mice were administered intratracheal (it) pitavastatin (5 mg / kg for 5 days) or vehicle control for 1 hour. For the airway constriction assay, mouse precision-cut lung slices (PCLS) were stimulated to constrict using increasing concentrations of MCh (0.1–100 μM). For each measurement, at least n = 4 mice from two independent experiments were used, with a total of 20–30 airways per lung.

[0191] Pretreatment with intratracheal (it) pitavastatin before each MCh nebulization caused a statistically significant reduction in airway contraction (%) (vehicle control 22.3% vs. pitavastatin 7.3%, p = 0.0361, Figure 6). This suggests that pitavastatin reduces airway contraction independently of its anti-inflammatory effects. This further confirms its direct effect on an intact ASM contractile apparatus. At the molecular level, pitavastatin reduced MLC-2 phosphorylation, a key contractile branch point in regulating airway hyperconstriction (Figure 5B).

[0192] A non-inflammatory mouse model of ASM hypercontraction demonstrated that inhaled statins could target ASM and prevent the MCh-induced hypercontractile phenotype (Figure 11B). These effects were achieved without evidence of airway damage or toxicity (Figure 10F). Thus, direct delivery of pitavastatin to the airways via inhalation simultaneously attenuated both airway inflammation and ASM contraction, hallmarks of asthma.

[0193] Example 7: Combination therapy This experiment was performed to demonstrate that direct administration of statins to ASM does not interfere with the activity of β2 agonists.

[0194] Precision-cut human lung slices from a single human donor lung were pretreated with 5 μM pitavastatin or vehicle (control) for 24 h and posttreated with histamine (10 μM for 15 min) followed by isoproterenol (30 μM for an additional 30 min). Experiments were performed under serum-deprived media conditions, with n = 3–7 airways per group. Changes in luminal narrowing were reported as percentage changes (± SEM) from the pretreatment state (see Figure 13). Absolute values ​​of luminal airway area were not statistically different between the pitavastatin and control groups at the pretreatment state. This indicated that pitavastatin did not interfere with the β2-agonist effect of isoproterenol.

[0195] This experiment was performed to demonstrate that contacting ASM with statins inhibits the release of inflammatory cytokines such as eotaxin and IL6 in response to IL13, IL17, and TNFα.

[0196] Non-asthmatic primary human ASM cells were grown to confluence and either untreated or pretreated with 2 mM pitavastatin and GGPP (10 mM) for a total of 72 hours. Total cytokine stimulation was at 10 ng / mL for 18 hours. As shown in Figure 14A, pitavastatin inhibited IL13 / TNFα-induced eotaxin-3 peptide secretion through a GGPP-dependent mechanism. As shown in Figure 14B, pitavastatin also inhibited IL17 / TNFα-induced IL6 peptide secretion through a GGPP-dependent mechanism. All experiments were performed in serum-containing media (10% FBS).

[0197] Normal human bronchial epithelial cells (cell line HBE1) were grown to confluence and treated with simvastatin (5 μM) and / or dexamethasone (10 μM). -7 M) for 72 hours. The cells were then treated with IL-13 (10 ng / mL). As shown in Figure 16, each treatment independently inhibited IL-13-induced extracellular secretion of eotaxin-3, and the combination of simvastatin and dexamethasone exhibited a synergistic inhibitory effect on eotaxin-3 secretion.

[0198] Figure 18 shows that pretreatment with appropriate concentrations of statins enhances the relaxant effect of dexamethasone. Primary human airway smooth muscle cells were cultured in serum-containing medium (10% FBS) until confluent and then pretreated with 0.1, 1, or 5 μM dexamethasone ("Dex") for 60 hours, with or without 0.1, 0.5, or 1 μM pitavastatin ("Pit"). Next, ASM cells were exposed to a cytokine mixture (10 nM IL-13, IL-17, and TNFα, "CM") for 15 hours, and eotaxin-3 expression was measured. "NT" means untreated. When pitavastatin was added to each concentration of dexamethasone, a significant decrease in eotaxin-3 expression was observed. This indicates that statins such as pitavastatin enhance the therapeutic effect of dexamethasone, and that inhaled statins can enhance the therapeutic effect of inhaled corticosteroids.

[0199] This experiment was performed to demonstrate the enhancing effect of statins on β2-agonist relaxation of histamine-induced ASM contraction.

[0200] Primary ASM cells from non-asthmatic donors were serum-starved for 2 days in tissue culture flasks and then cultured in serum-free medium in 96-well traction force measurement plates (3 kPa hardness) for an additional 24 hours until confluent. Pretreatment contractile force was measured as described above. Cells were then cultured in either vehicle (PEG400), control (serum-free medium), or 10 -5 M, 10 -6 M, 10 -7 M, and 10 -8 ASM cells were treated with either 10 μM or 10 μM pitavastatin for 24 hours, and baseline contractile force was measured. Next, ASM cells were acutely treated with histamine (10 μM) for 30 minutes, and histamine-induced contractile force was measured. Next, cells were treated with isoproterenol (10 μM). -6 M, 10 -7 M, 10 -8 M, 10 -9 , and 10 -11M) for 30 minutes, and the isoproterenol-relaxed ASM contractile force was measured. The % "histamine contraction" was calculated as the ratio of the isoproterenol-relaxed ASM contractile force to the histamine-induced contractile force. The results are shown in Figure 17 and demonstrate that pretreatment with appropriate concentrations of statins enhances the relaxant effect of relevant concentrations of isoproterenol. Figure 17A shows the results of pretreatment with 10 -11 At 10 M isoproterenol, all concentrations of statin provided essentially the same degree of relaxation that was not substantially different from vehicle. -7 M, 10 -6 M, and 10 -5 Pitavastatin in M ​​is 10 -9 M and 10 -8 It significantly enhanced the effect of isoproterenol in M ​​more than vehicle. Figure 17B shows details of the boxed data in Figure 17A.

[0201] Example 8: Inhibition of eicosanoid mediators Six female rhesus macaques from the California National Primate Research Center (CNPRC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), were used in this study. All protocols were approved by the University of California-Davis Institutional Animal Care and Use Committee and complied with the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Animals were treated humanely, and care was taken to minimize and / or alleviate pain and discomfort. The experimental design is shown in Figure 7. The rhesus macaques (n = 6) used in this study were age-matched at the start of the study, aged 9 years and 1 month, and divided into two cohorts: control and drug-treated. Animals were administered 1 mg / kg simvastatin or simvastatin vehicle, 10% ethanol (PBS), via aerosol mask nebulization for 40–45 min for 7 consecutive days. Plasma, airway epithelial cells, and bronchoalveolar lavage fluid (BALF) were sampled 1 day after the final exposure treatment of each phase (day 8). Tracheal and bilateral lung tissues were obtained after sacrifice 5 days after the end of statin exposure (day 12) (Figure 8).

[0202] Anesthetized (10 mg / kg ketamine + 0.1 mg / kg / min propofol) adult female rhesus monkeys were administered 10% ethanol by inhalation while ventilation was measured using a simultaneous flow spirometry aerosol inhalation system modified from that described by H.C. Yeh et al., Environ Health Perspect (1976) 15:147-56.

[0203] Aerosols were generated using a jet nebulizer (MiniHEART®, Westmed, Inc., Tucson, AZ) with particle size MMAD = 2.5 μm and σg = 2. Aerosols were delivered through a conical, clear plastic face mask that effectively sealed over each animal's nose and mouth with a flexible rubber diaphragm and a secondary seal of a latex dental dam. Ventilation fluctuations were measured using a pressure transducer (model MP45-14, Validyne Engineering Corp., Northridge, CA) and a heated air pressure graph (model 8300A, Hans Rudolph, Inc., Kansas City, MO) connected to a computer-based pulmonary physiology platform (Ponemah, DSI, Inc., St. Paul, MN), providing real-time measurements of respiratory flow, mean minute ventilation, and total ventilation during the inhalation exposure period. Dose was estimated using aerosol concentration, deposition fraction estimated from aerodynamic size, and total volume inhaled.

[0204] Rhesus monkeys were anesthetized with ketamine (10 mg / kg) and maintained with propofol (0.1 mg / kg / min). As previously described (E.S. Schelegle et al., Am J Pathol (2001) 158(1):333-41), 10 mL of endotoxin-free PBS (Sigma, St. Louis, MO) was infused through the bronchoscope. The bronchoscope was flushed with PBS three times before each sample to clear the channels and avoid cross-contamination. The bronchoscope and cytobrush were inserted and removed from the airway as a single unit. After the cytobrush was collected, bronchoalveolar lavage fluid (BALF) was collected. A 24 mL or 32 mL aliquot of PBS was instilled and then withdrawn, recovering approximately 30–50% of the BALF for most samples.

[0205] BALF samples were stored on ice immediately after collection, centrifuged at 6,000 rpm for 5 min to obtain lavage supernatants, and then stored at −80° C. BALF was collected first from the right middle lobe and then from the left upper lobe.

[0206] Treatment of rhesus monkey NHPs with nebulized simvastatin (1 mg / kg) inhibits leukotriene B4 (LTB4) and thromboxane B2 (TXB2), two eicosanoids known to be potent airway bronchoconstrictors. Simvastatin significantly reduced LTB4 in BAL fluid (*p=0.0143) on day 12 (5 days after the last statin administration), but there were no significant changes in lung tissue (Figure 9A-B). Simvastatin significantly inhibited TXB2 in lung tissue (*p=0.0358), with a positive trend (p=0.051) toward reduced TXB2 levels in BAL fluid on day 12 (Figure 9C-D). These results demonstrate that even basal levels of pro-bronchoconstrictor lipid agonists are reduced in the airways of non-inflamed rhesus monkey lungs, suggesting a bronchoprotective effect of inhaled statins in vivo.

[0207] On day 12, the rhesus monkeys were euthanized, and organs, including the lungs, were subsequently collected. A mass spectrometry-based method was used to determine the distribution of simvastatin (lactone) and its active metabolite, SA. Both forms were primarily present in the mainstem bronchi and lower lobes, with relatively low levels in the intestine, liver, or muscle tissue (Figure 9). Thus, administration of statins by inhalation appears to be a safe and feasible method for achieving high airway distribution (Figure 10).

[0208] All publications, patents, and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. No admission is made that any reference cited herein constitutes prior art. The discussion of a reference states what its author asserts, and the inventor reserves the right to challenge the accuracy and pertinence of the cited documents. It is expressly understood that many sources of information, such as scientific journal articles, patent documents, textbooks, and the like, are referenced herein. This reference is not an admission that any of these documents form part of the general knowledge in the art.

[0209] While specific alternatives of the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims, and therefore, it is not intended to be limited to the precise summary and disclosure presented herein.

Claims

1. 1. A method of reducing airway smooth muscle contraction in a subject, comprising: administering the formulation by inhalation to a subject having a pulmonary disease; and administering one, two, or three additional therapeutic agents, the formulation comprising: a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof; and a pharmaceutically acceptable carrier.

2. The one, two, or three additional therapeutic agents are selected from the group consisting of beta-agonists; corticosteroids; muscarinic antagonists; RhoA inhibitors; GGTase-I or -II inhibitors; ROCK1 and / or ROCK2 inhibitors; soluble epoxide hydrolase inhibitors; fatty acid amide hydrolase inhibitors; leukotriene receptor antagonists; phosphodiesterase-4 inhibitors, such as roflumilast; 5-lipoxygenase inhibitors, such as zileuton; mast cell stabilizers, such as nedocromil; theophylline; anti-IL5 antibodies; anti-IgE antibodies; anti-IL5 receptor antibodies; anti-IL13 / 4 receptor antibodies; 10. The method of claim 1, wherein the therapeutic agent is selected from the group consisting of therapeutic agents such as mepolizumab, reslizumab, benralizumab, omalizumab, and dupilumab; combinations of beta-agonists and muscarinic antagonists, including both long-acting and short-acting formulations; combinations of beta-agonists and corticosteroids, including both long-acting and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long-acting and short-acting formulations; and combinations of beta-agonists, corticosteroids, and muscarinic antagonists, including both long-acting and short-acting formulations.

3. 3. The method of claim 1 or 2, wherein the additional therapeutic agent is a beta agonist, a corticosteroid, a muscarinic antagonist, or any combination thereof.

4. The additional therapeutic agent may be albuterol, aformoterol, formoterol, salmeterol, indacaterol, levalbuterol, salbutamol, terbutaline, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, bambuterol, clenbuterol, formoterol, salmeterol, abesiterol, and carmoterol, buphenine, bopexamine, epinephrine, fenoterol, isoetharine, isoproterenol, orciprenaline.

4. The method of any one of claims 1 to 3, wherein the beta agonist is selected from the group consisting of levoalbutamol, pirbuterol, procaterol, ritodrine, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and gintero.

5. The additional therapeutic agent may be beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, flucortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, meprednisolone, 5. The method of any one of claims 1 to 4, wherein the corticosteroid is selected from the group consisting of flucloxone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol, amcinonide, ciclesonide, deflazacort, desonide, formocortal, fluchlorolone acetonide, fludroxycortide, fluocinolone acetonide, fluocinonide, halcinonide, and triamcinolone acetonide.

6. 6. The method of any one of claims 1 to 5, wherein the additional therapeutic agent is a muscarinic antagonist selected from the group consisting of ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, lebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin.

7. 7. The method of any one of claims 1 to 6, wherein the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A.

8. The method of any one of claims 1 to 7, wherein the additional therapeutic agent is the RhoA inhibitor rosin.

9. 9. The method of any one of claims 1 to 8, wherein the one, two, or three additional therapeutic agents are augmented with a statin.

10. 10. The method of any one of claims 1 to 9, wherein one, two, or three additional therapeutic agents are administered in sub-therapeutic doses.

11. 11. The method of any one of claims 1 to 10, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof.

12. 12. The method of any one of claims 1 to 11, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and isomers, enantiomers, and diastereoisomers thereof.

13. The method of any one of claims 1 to 12, wherein the statin is selected from the group consisting of pitavastatin and simvastatin.

14. The method of any one of claims 1 to 13, wherein the therapeutically effective amount is from about 0.005 μg to about 40 mg.

15. 15. The method of any one of claims 1 to 14, wherein the therapeutically effective amount is from about 0.5 μg to about 15 mg.

16. 16. The method of any one of claims 1 to 15, wherein the therapeutically effective amount is from about 1.0 μg to about 10 mg.

17. 17. The method of any one of claims 1 to 16, wherein the therapeutically effective amount is from about 1.0 μg to about 5 mg.

18. 18. The method of any one of claims 1 to 17, wherein the pulmonary airway disease is characterized by airway smooth muscle contraction.

19. 19. The method of any one of claims 1 to 18, wherein the pulmonary airway disease is characterized by bronchospasm.

20. The pulmonary disease is selected from the group consisting of asthma, exercise-induced bronchoconstriction, COPD, emphysema, chronic bronchitis, alpha 1 antitrypsin deficiency (AATD), ACOS, cystic fibrosis, bronchiectasis, exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, post-viral airway hyperresponsiveness, post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection, airway edema due to congestive heart failure, airway edema due to pulmonary edema, airway edema due to cardiogenic pulmonary edema, and airway edema due to non-cardiogenic pulmonary edema.

20. The method of any one of claims 1 to 19, wherein the pulmonary airway disease is selected from the group consisting of: airway edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; aspiration of foreign body; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing localized edema and bronchospasm; pulmonary embolism; airway trauma; surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

21. The method of any one of claims 1 to 20, wherein the administration is by mechanical inhaler.

22. The method of any one of claims 1 to 21, wherein the mechanical inhaler is a metered dose powder inhaler.

23. The method of any one of claims 1 to 21, wherein the metered dose powder inhaler is a pressurized aerosol inhaler.

24. The method of any one of claims 1 to 21, wherein the metered dose powder inhaler is a dry powder inhaler.

25. The method of any one of claims 1 to 21, wherein the mechanical inhaler is a nebulizer.

26. 22. The method of any one of claims 1 to 21, wherein the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, and an Ellipta® inhaler.

27. 1. A method of reducing airway smooth muscle contraction in a subject, comprising: administering by inhalation to a subject having a non-inflammatory lung disease a formulation comprising: a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof; and a pharmaceutically acceptable carrier.

28. 28. The method of claim 27, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof.

29. 29. The method of claim 27 or 28, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and isomers, enantiomers, and diastereoisomers thereof.

30. 30. The method of any one of claims 27 to 29, wherein the statin is selected from the group consisting of pitavastatin and simvastatin.

31. 31. The method of any one of claims 27 to 30, wherein the therapeutically effective amount is from about 0.005 μg to about 40 mg.

32. 32. The method of any one of claims 27 to 31, wherein the therapeutically effective amount is from about 0.5 μg to about 15 mg.

33. 33. The method of any one of claims 27 to 32, wherein the therapeutically effective amount is from about 1.0 μg to about 10 mg.

34. 34. The method of any one of claims 27 to 33, wherein the therapeutically effective amount is from about 1.0 μg to about 5 mg.

35. The method of any of claims 27 to 34, wherein the subject has been diagnosed with a pulmonary airway disease or an interstitial lung disease.

36. 36. The method of any one of claims 27 to 35, wherein the pulmonary disease is selected from the group consisting of exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, and post-viral airway hyperresponsiveness.

37. 37. The method of any one of claims 27 to 36, wherein the pulmonary airway disease is characterized by airway smooth muscle contraction.

38. 38. The method of any one of claims 27 to 37, wherein the pulmonary airway disease is characterized by bronchospasm.

39. 39. The method of any one of claims 27 to 38, wherein the pulmonary disease is selected from the group consisting of post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing bronchospasm-induced wheezing); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

40. 39. The method of any one of claims 27 to 38, wherein the administration is by mechanical inhaler.

41. 41. The method of any one of claims 27 to 40, further comprising administering one, two, or three additional therapeutic agents.

42. 42. The method of any one of claims 27 to 41, wherein the one, two, or three additional therapeutic agents are administered in the same formulation as the statin.

43. 42. The method of any one of claims 27-41, wherein the one, two, or three additional therapeutic agents are not administered in the same formulation as the statin.

44. The additional therapeutic agent may be a beta-agonist; a corticosteroid; a muscarinic antagonist; a RhoA inhibitor; a GGTase-I or -II inhibitor; a ROCK1 and / or ROCK2 inhibitor; a soluble epoxide hydrolase inhibitor; a fatty acid amide hydrolase inhibitor; a leukotriene receptor antagonist; a phosphodiesterase-4 inhibitor, such as roflumilast; a 5-lipoxygenase inhibitor, such as zileuton; a mast cell stabilizer, such as nedocromil; theophylline; an anti-IL5 antibody; an anti-IgE antibody; an anti-IL5 receptor antibody; an anti-IL13 / 4 receptor antibody; a biologic, such as mepolipoprotein B12; 44. The method of any one of claims 27 to 43, wherein the therapeutic agent is selected from the group consisting of ibuprofen, reslizumab, benralizumab, omalizumab, and dupilumab; combinations of beta-agonists and muscarinic antagonists, including both long- and short-acting formulations; combinations of beta-agonists and corticosteroids, including both long- and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long- and short-acting formulations; and combinations of beta-agonists, corticosteroids, and muscarinic antagonists, including both long- and short-acting formulations.

45. The additional therapeutic agent may be albuterol, aformoterol, formoterol, salmeterol, indacaterol, levalbuterol, salbutamol, terbutaline, olodaterol, vilanterol, isoxsuprine, mabuterol, zilpaterol, bambuterol, clenbuterol, formoterol, salmeterol, abesiterol, and carmoterol, buphenine, bopexamine, epinephrine, fenoterol, isoetharine, isoproterenol, orciprenaline, The method of any one of claims 27 to 44, wherein the beta agonist is selected from the group consisting of levoalbutamol, pirbuterol, procaterol, ritodrine, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and gintero.

46. The additional therapeutic agent is selected from the group consisting of beclomethasone, fluticasone, budesonide, mometasone, flunisolide, alclometasone, beclomethasone, betamethasone, clobetasol, clobetasone, clocortolone, desoximetasone, dexamethasone, diflorasone, difluocortolone, flurchlororone, flumethasone, fluocortin, fluocortolone, fluprednidene, fluticasone, fluticasone furoate, halometasone, and meprednisolone.

46. ​​The method of any one of claims 27 to 45, wherein the corticosteroid is selected from the group consisting of flucloxone, mometasone, mometasone furoate, paramethasone, prednylidene, rimexolone, urobetasol, amcinonide, ciclesonide, deflazacort, desonide, formocortal, fluchlorolone acetonide, fludroxycortide, fluocinolone acetonide, fluocinonide, halcinonide, and triamcinolone acetonide.

47. 47. The method of any one of claims 27-46, wherein the additional therapeutic agent is a muscarinic antagonist selected from the group consisting of ipratropium bromide, tiotropium, glycopyrrolate, glycopyrronium bromide, lebefenacin, umeclidinium bromide, aclidinium, trospium chloride, oxitropium bromide, oxybutynin, tolterodine, solifenacin, fesoterodine, and darifenacin.

48. 48. The method of any one of claims 27 to 47, wherein the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A.

49. 49. The method of any one of claims 27 to 48, wherein the additional therapeutic agent is the RhoA inhibitor rosin.

50. 50. The method of any one of claims 27 to 49, wherein the one, two, or three additional therapeutic agents are augmented with a statin.

51. 51. The method of claim 50, wherein one, two, or three additional therapeutic agents are administered in sub-therapeutic doses.

52. 52. The method of any one of claims 27 to 51, wherein the pharmaceutically acceptable carrier comprises a component selected from the group consisting of mono-, di-, oligo-, and polysaccharides, polyhydric alcohols, cyclodextrins, amino acids, salts, and mixtures thereof.

53. 53. The method of any one of claims 27 to 52, wherein the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose.

54. 54. The method of any one of claims 27 to 53, wherein the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose.

55. 55. The method of any one of claims 27 to 54, wherein the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose.

56. 56. The method of any one of claims 27 to 55, wherein the ingredient comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol.

57. 57. The method of any one of claims 27 to 56, wherein the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, and hydroxypropyl-β-cyclodextrin, Captisol, and sulfobutyl-β-cyclodextrin, or DexSol.

58. 58. The method of any one of claims 27 to 57, wherein the component comprises arginine or arginine hydrochloride.

59. 59. The method of any one of claims 27 to 58, wherein the component comprises a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

60. 1. A method of treating bronchospasm in a subject, comprising: administering by inhalation to a subject having a non-inflammatory pulmonary airway disease characterized by bronchospasm a formulation comprising: a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof; and a pharmaceutically acceptable carrier.

61. 61. The method of claim 60, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof.

62. 62. The method of claim 60 or 61, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and isomers, enantiomers, and diastereoisomers thereof.

63. 63. The method of any one of claims 60 to 62, wherein the statin is selected from the group consisting of pitavastatin and simvastatin.

64. 64. The method of any one of claims 60 to 63, wherein the subject has been diagnosed with a pulmonary airway disease or an interstitial lung disease.

65. The pulmonary airway disease is selected from the group consisting of exercise-induced bronchospasm, exercise-induced asthma, aspirin-exacerbated respiratory disease, NSAID-exacerbated respiratory disease, oligocytic asthma, obesity-related airway hyperresponsiveness, post-viral airway hyperresponsiveness, post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection, airway edema due to congestive heart failure, airway edema due to pulmonary edema, airway edema due to cardiogenic pulmonary edema, airway edema due to non-cardiogenic pulmonary edema, bronchiolitis due to airway edema, bronchiectasis due to anatomical distortion rather than inflammation, foreign body aspiration, food, liquid, and / or aspiration of gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism (which may release local factors causing wheezing due to bronchospasm); airway trauma, including surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; and bronchospasm associated with paraneoplastic syndromes.

66. 66. The method of any one of claims 60 to 65, wherein the pulmonary airway disease is characterized by airway smooth muscle contraction.

67. 67. The method of any one of claims 60 to 66, wherein the pulmonary airway disease is characterized by bronchospasm.

68. 68. The method of any one of claims 60 to 67, wherein the administration is performed using a mechanical inhaler.

69. 69. The method of any one of claims 60 to 68, wherein the mechanical inhaler is a metered dose inhaler.

70. 70. The method of any one of claims 60 to 69, wherein the metered dose inhaler is a pressurized aerosol inhaler.

71. 70. The method of any one of claims 60 to 69, wherein the metered dose inhaler is a dry powder inhaler.

72. 69. The method of any one of claims 60 to 68, wherein the mechanical inhaler is a nebulizer.

73. 73. The method of any one of claims 60 to 72, wherein the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, a PulmoSphere carrier inhaler, and an Ellipta® inhaler.

74. 74. The method of any one of claims 60-73, wherein the formulation further comprises one, two, or three additional therapeutic agents.

75. The additional therapeutic agent may be a beta-agonist; a corticosteroid; a muscarinic antagonist; a RhoA inhibitor; a GGTase-I or -II inhibitor; a ROCK1 and / or ROCK2 inhibitor; a soluble epoxide hydrolase inhibitor; a fatty acid amide hydrolase inhibitor; a leukotriene receptor antagonist; a phosphodiesterase-4 inhibitor, such as roflumilast; a 5-lipoxygenase inhibitor, such as zileuton; a mast cell stabilizer, such as nedocromil; theophylline; an anti-IL5 antibody; an anti-IgE antibody; an anti-IL5 receptor antibody; an anti-IL13 / 4 receptor antibody; a biologic, such as mepolipoprotein B12; 75. The method of any one of claims 60 to 74, wherein the therapeutic agent is selected from the group consisting of ibuprofen, reslizumab, benralizumab, omalizumab, and dupilumab; combinations of beta-agonists and muscarinic antagonists, including both long- and short-acting formulations; combinations of beta-agonists and corticosteroids, including both long- and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long- and short-acting formulations; and combinations of beta-agonists, corticosteroids, and muscarinic antagonists, including both long- and short-acting formulations.

76. 76. The method of any one of claims 60-75, wherein the additional therapeutic agent is a beta-agonist selected from the group consisting of arformoterol, buphenine, clenbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levosalbutamol, levalbuterol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and zinterol.

77. 77. The method of any one of claims 60 to 76, wherein the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A.

78. 78. The method of any one of claims 60 to 77, wherein the additional therapeutic agent is the RhoA inhibitor rosin.

79. 79. The method of any one of claims 60-78, wherein the pharmaceutically acceptable carrier comprises a component selected from the group consisting of mono-, di-, oligo-, and polysaccharides, DexSol, polyalcohols, cyclodextrins, amino acids, salts, and mixtures thereof.

80. 80. The method of any one of claims 60 to 79, wherein the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose.

81. 81. The method of any one of claims 60 to 80, wherein the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose.

82. 82. The method of any one of claims 60 to 81, wherein the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose.

83. 83. The method of any one of claims 60 to 82, wherein the ingredient comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol.

84. 84. The method of any one of claims 60 to 83, wherein the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, Captisol, and hydroxypropyl-β-cyclodextrin.

85. 85. The method of any one of claims 60 to 84, wherein the component comprises arginine or arginine hydrochloride.

86. 86. The method of any one of claims 60 to 85, wherein the component comprises a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

87. a therapeutically effective amount of a statin, or an isomer, enantiomer, or diastereoisomer thereof; and a pharmaceutically acceptable carrier suitable for administration by inhalation.

88. 88. The formulation of claim 87, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, atorvastatin, lovastatin, fluvastatin, mevastatin, cerivastatin, tenivastatin, and pravastatin, and isomers, enantiomers, and diastereoisomers thereof.

89. 89. The formulation of claim 87 or 88, wherein the statin is selected from the group consisting of simvastatin, pitavastatin, rosuvastatin, and atorvastatin, and isomers, enantiomers, and diastereoisomers thereof.

90. 90. The formulation of any one of claims 87 to 89, wherein the statin is selected from the group consisting of pitavastatin and simvastatin.

91. 91. The formulation of any one of claims 87-90, wherein the effective amount is from about 0.005 mg to about 80 mg.

92. 92. The formulation of any one of claims 87-91, wherein the effective amount is from about 0.5 mg to about 15 mg.

93. 93. The formulation of any one of claims 87-92, wherein the effective amount is from about 1.0 mg to about 10 mg.

94. 94. The formulation of any one of claims 87-93, wherein the effective amount is from about 1.0 mg to about 5 mg.

95. 95. The formulation of any one of claims 87-94, wherein the formulation further comprises one, two, or three additional therapeutic agents.

96. 96. The formulation of any one of claims 87-95, wherein the formulation comprises sub-therapeutic amounts of one, two, or three additional therapeutic agents.

97. The additional therapeutic agent may be a beta-agonist; a corticosteroid; a muscarinic antagonist; a RhoA inhibitor; a GGTase-I or -II inhibitor; a ROCK1 and / or ROCK2 inhibitor; a soluble epoxide hydrolase inhibitor; a fatty acid amide hydrolase inhibitor; a leukotriene receptor antagonist; a phosphodiesterase-4 inhibitor, such as roflumilast; a 5-lipoxygenase inhibitor, such as zileuton; a mast cell stabilizer, such as nedocromil; theophylline; an anti-IL5 antibody; an anti-IgE antibody; an anti-IL5 receptor antibody; an anti-IL13 / 4 receptor antibody; a biologic, such as mepolipoprotein B12; 97. The formulation of any one of claims 87 to 96, wherein the active ingredient is selected from the group consisting of ibuprofen, reslizumab, benralizumab, omalizumab, and dupilumab; combinations of beta-agonists and muscarinic antagonists, including both long- and short-acting formulations; combinations of beta-agonists and corticosteroids, including both long- and short-acting formulations; combinations of corticosteroids and muscarinic antagonists, including both long- and short-acting formulations; and combinations of beta-agonists, corticosteroids, and muscarinic antagonists, including both long- and short-acting formulations.

98. 98. The formulation of any one of claims 87-97, wherein the additional therapeutic agent is a beta-agonist selected from the group consisting of arformoterol, buphenine, clenbuterol, levalbuterol, bopexamine, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, orciprenaline, levoalbutamol, pirbuterol, procaterol, ritodrine, albuterol, salmeterol, terbutaline, albutamine, befunolol, bromoacetylalprenolol menthone, broxaterol, cimaterol, cirazoline, etilefrine, hexoprenaline, higenamine, isoxsuprine, mabuterol, methoxyphenamine, oxyfedrine, ractopamine, reproterol, rimiterol, tretoquinol, tulobuterol, zilpaterol, and zinterol.

99. 99. The formulation of any one of claims 87 to 98, wherein the additional therapeutic agent is a ROCK inhibitor selected from the group consisting of fasudil, ripasudil, netarsudil, RKI-1447, Y-27632, Y-30141, and GSK429286A.

100. 100. The formulation of any one of claims 87 to 99, wherein the additional therapeutic agent is the RhoA inhibitor rocin.

101. 101. The formulation of any one of claims 87-100, wherein the pharmaceutically acceptable carrier comprises a component selected from the group consisting of mono-, di-, oligo-, and polysaccharides, polyhydric alcohols, DexSol, cyclodextrins, amino acids, salts, and mixtures thereof.

102. 102. The formulation of any one of claims 87 to 101, wherein the component comprises a monosaccharide selected from the group consisting of glucose, fructose, and arabinose.

103. 103. The formulation of any one of claims 87 to 102, wherein the component comprises a disaccharide selected from the group consisting of lactose, sucrose, maltose, and trehalose.

104. 104. The formulation of any one of claims 87 to 103, wherein the component comprises an oligosaccharide or polysaccharide selected from the group consisting of dextran, dextrin, maltodextrin, starch, and cellulose.

105. 105. The formulation of any one of claims 87 to 104, wherein the ingredient comprises a polyalcohol selected from the group consisting of sorbitol, mannitol, and xylitol.

106. 106. The formulation of any one of claims 87-105, wherein the component comprises a cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, χ-cyclodextrin, methyl-β-cyclodextrin, Captisol, and hydroxypropyl-β-cyclodextrin.

107. 107. The formulation of any one of claims 87 to 106, wherein the component comprises arginine or arginine hydrochloride.

108. 108. The formulation of any one of claims 87 to 107, wherein the ingredients comprise a salt selected from the group consisting of sodium chloride, potassium chloride, sodium bromide, and calcium carbonate.

109. a delivery device for delivering a therapeutic dose of the formulation to the pulmonary airways of a subject in need thereof; A pre-filled inhalation device for treating a pulmonary airway disease in a subject, comprising a pharmaceutically acceptable formulation according to any one of claims 87 to 108.

110. 110. The inhalation device of claim 109, wherein the device comprises a pressurized inhaler, a metered dose inhaler, a dry powder inhaler, or a nebulizer.

111. 111. An inhalation device according to claim 109 or 110, wherein the device comprises a plurality of therapeutic doses.

112. 112. An inhalation device according to any one of claims 109 to 111, wherein the delivery device is a metered dose inhaler.

113. 113. The inhalation device of any one of claims 109 to 112, wherein the metered dose inhaler is a pressurized aerosol inhaler.

114. 113. An inhalation device according to any one of claims 109 to 112, wherein the metered dose inhaler is a dry powder inhaler.

115. 113. An inhalation device according to any one of claims 109 to 112, wherein the delivery device is a nebulizer.

116. 116. The inhalation device of any one of claims 109 to 115, wherein the delivery device is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, a PulmoSphere carrier inhaler, and an Ellipta® inhaler.

117. a container including a connecting means for attaching the container to an inhaler device; A pre-filled cartridge for use in an inhaler comprising a pharmaceutically acceptable formulation according to any one of claims 87 to 108.

118. 118. A pre-filled cartridge according to claim 117, further comprising a pharmaceutically acceptable propellant.

119. 87. The method of any of claims 27-86, wherein the therapeutically effective amount is effective to maintain lung function; reduce asthma exacerbations; reduce bronchoconstriction and mucus accumulation in a subject; or enhance respiratory-induced bronchodilation.

120. 109. A method of reducing airway hyperresponsiveness (AHR) in a subject in need thereof, comprising administering by inhalation to a subject the formulation of any one of claims 87-108, wherein the therapeutically effective amount is effective to reduce AHR in the subject.

121. 109. A method of reducing airway smooth muscle (ASM) hypercontractility in a subject in need thereof, comprising administering by inhalation to a subject the formulation of any one of claims 87-108, wherein the therapeutically effective amount is effective to reduce ASM hypercontractility in the subject.

122. 109. A method of increasing stretch-induced airway smooth muscle (ASM) relaxation in a subject in need thereof, comprising administering by inhalation to a subject a formulation of any one of claims 87-108, wherein the therapeutically effective amount is effective to increase stretch-induced airway smooth muscle relaxation in the subject.

123. 123. The method of any one of claims 119 to 122, wherein the administering is performed using a mechanical inhaler.

124. 124. The method of any one of claims 119 to 123, wherein the mechanical inhaler is a metered dose inhaler.

125. 125. The method of any one of claims 119 to 124, wherein the metered dose inhaler is a pressurized aerosol inhaler.

126. 126. The method of any one of claims 119 to 125, wherein the metered dose inhaler is a dry powder inhaler.

127. 125. The method of any one of claims 119 to 124, wherein the mechanical inhaler is a nebulizer.

128. 128. The method of any one of claims 119 to 127, wherein the mechanical inhaler is selected from the group consisting of a Respimat® Soft Mist™ inhaler, a RespiClick® inhaler, a Breezhaler® inhaler, a Genuair® inhaler, and an Ellipta® inhaler.

129. 109. A method of treating a symptom of interstitial lung disease comprising administering by inhalation to a subject in need thereof a formulation according to any one of claims 87 to 108, the interstitial lung disease causes airway symptoms selected from the group consisting of ASM contraction, ASM overgrowth or thickening, bronchospasm, bronchoconstriction, airway mucus accumulation, or ASM release of inflammatory mediators; The above method, wherein the therapeutically effective amount is effective to reduce the severity of said symptoms by at least 10%.

130. A method for reducing future symptoms caused by an event that has already occurred or is expected to be experienced in the future, comprising administering to a subject at risk of experiencing the future symptoms a formulation according to any one of claims 87 to 108.

131. The method of claim 130, wherein the future condition is bronchospasm caused by post-infectious bronchospasm due to viral, bacterial, fungal, and / or mycobacterial infection; airway edema due to congestive heart failure; airway edema due to pulmonary edema; airway edema due to cardiogenic pulmonary edema; airway edema due to non-cardiogenic pulmonary edema; bronchiolitis due to airway edema; bronchiectasis due to anatomical distortion rather than inflammation; foreign body aspiration; aspiration of food, liquid, and / or gastric contents; gastroesophageal reflux disease; lung cancer or metastatic cancer to the lung causing local edema and bronchospasm; pulmonary embolism; airway trauma; surgery; anaphylaxis and anaphylactoid reactions; nerve-mediated cough and / or bronchospasm; bronchospasm associated with inhalation injury; bronchospasm associated with endocrine dysfunction; or bronchospasm associated with a paraneoplastic syndrome.

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