Methods of reducing lung inflammation

Abstract

A method of treating or preventing lung inflammation by administering high concentrations of inhaled chelators, in particular CaEDTA.

Description

[0001] This application is a divisional application of Chinese invention patent application (application date: June 20, 2018; application number: 201880041015.9 (international application number: PCT / AU2018 / 050609); invention title: method for relieving lung inflammation). Technical Field

[0002] This invention relates to a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, and to formulations for use in this method. In one embodiment of the invention, the lung inflammation is related to or caused by cystic fibrosis. Background Technology

[0003] Cystic fibrosis is characterized by its susceptibility to infection, which leads to inflammation and lung damage. However, inflammation and lung damage can also occur in the absence of bacterial infection (Sly et al., Am J Respir Crit Care Med. 2009 180(2):146-52).

[0004] Inflammation is the body's response to harm, including infection, trauma, and allergies. The inflammatory response is complex and involves multiple mechanisms for defending against pathogens and repairing tissues. In the lungs, inflammation is typically caused by pathogens or exposure to toxins, pollutants, irritants, and allergens.

[0005] During inflammation, multiple types of inflammatory cells are activated. Each cell releases cytokines and mediators to alter the activity of other inflammatory cells. The arrangement of these cells and molecules leads to the progression of inflammation. Clinically, acute inflammation is seen in conditions such as pneumonia and acute respiratory distress syndrome (ARDS), while chronic inflammation manifests in conditions such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Because the lungs are vital organs for gas exchange, excessive inflammation can be life-threatening. The delicate balance between inflammation and anti-inflammation is crucial for lung homeostasis.

[0006] Immunity involves both innate and adaptive systems. Innate immunity is nonspecific and elicits a rapid response, including inflammation in the face of pathogen invasion. Adaptive immunity is antigen-specific. It first detects a specific antigen and then mobilizes inflammatory cells to target that specific antigen. Innate and adaptive systems share components and work together to fight off pathogens.

[0007] Airway epithelium secretes various substances, such as mucin, defensins, lysozyme, lactoferrin, and nitric oxide, which nonspecifically protect the respiratory tract from microbial invasion. Epithelial cells also produce various mediators, such as reactive oxygen species, cytokines (TNF-α, IL-1β, granulocyte / macrophage colony-stimulating factor [GM-CSF]), and platelet-activating factor, to recruit inflammatory cells to sites of inflammation. Cytokines stimulate the release of arachidonic acid from membrane lipids, leading to the production of arachidic acid, which further stimulates goblet cells and mucus secretion induced by tissue inflammation.

[0008] Surfactants are located on the alveolar surface and comprise four surfactant proteins (SP-A and SP-D). These proteins play a crucial role in surfactant uptake onto the alveolar surface, which is important for reducing lung surface tension. SP-A and SP-D are also involved in host defense. They bind to bacterial surface molecules, regulate leukocyte activity, and contribute to pathogen opsonization.

[0009] IgA secreted by plasma cells forms an additional protective barrier for the epithelium, preventing microorganisms from adhering to the epithelial surface. It also binds to pathogens, inducing phagocytosis and antibody-dependent cell-mediated cytotoxicity. Immunoglobulin E (IgE) induces immediate hypersensitivity reactions in the respiratory tract. It produces severe responses by binding to IgE receptors on the surfaces of mast cells, basophils, eosinophils, and B lymphocytes. Repeated exposure to the same antigen leads to degranulation and the release of pro-inflammatory mediators, including histamine, prostaglandins, leukotrienes, and trypsin inhibitors. These effects increase vascular permeability, bronchoconstriction, and inflammatory cell infiltration.

[0010] US20160263151 teaches the use of inhaled antibiotics in combination with acidified nitrites and iron chelators for the treatment of bacterial infections. The iron chelators present in the formulations of US20160263151, when combined with acidified nitrites, provide a synergistic effect, thereby enhancing the efficacy of the antibiotics.

[0011] Current treatments for lung inflammation include oral or inhaled steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) that target the host's inflammatory response. However, these effects are temporary, require continuous treatment, and have significant side effects.

[0012] A method is needed to treat or prevent lung inflammation; or at least one method that complements or provides an alternative to a previously known treatment.

[0013] The present invention aims to provide an improved or alternative method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent.

[0014] The preceding discussion of the background art is intended only to facilitate understanding of the present invention. This discussion is not an admission or endorsement that any material mentioned was or was part of common general knowledge prior to the priority date of the application. Invention Overview

[0015] This invention provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent.

[0016] Preferably, the high concentration of the inhaled chelating agent is higher than 37.5 mg / dose.

[0017] Preferably, the high concentration of the inhaled chelating agent is higher than 50 mg / dose.

[0018] In one form of the invention, the high-concentration chelating agent is provided in a dosage form comprising at least 50 mg / dose or from 50 mg / dose to 300 mg / dose. The chelating agent may be administered once to four times daily, with a total dose up to about 1,200 mg / day, preferably at least 150 mg / day.

[0019] In one form of the invention, the high-concentration chelating agent is provided in a dosage form comprising at least 37.5 mg / dose or from 37.5 mg / dose to 300 mg / dose. The chelating agent may be administered once to four times daily, with a total dose up to about 1,200 mg / day, preferably at least 150 mg / day.

[0020] The preferred dosage is a chelating agent ranging from 37.5 mg / day to 1,200 mg / day. Preferably, a chelating agent of at least 50 mg / day is administered. The chelating agent may be administered once to four times daily, with a maximum total dose of about 1,200 mg / day.

[0021] Preferably, each dose of the chelating agent is administered over a period of no more than 8 hours. Preferably, the chelating agent and / or antibiotic are administered over a period of no more than 1 hour.

[0022] Preferably, the chelating agent is CaEDTA.

[0023] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation results in an increase in forced expiratory volume (FEV).

[0024] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is associated with a decrease in matrix metalloproteinase (MMP) activity.

[0025] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is associated with a reduction in the generation of hydroxyl radicals.

[0026] The present invention provides an inhalable formulation containing a high concentration of chelating agents.

[0027] The present invention provides an inhalable formulation comprising a high concentration of a chelating agent and capable of delivering a high concentration of the inhalable chelating agent in a single dose.

[0028] The present invention provides a kit for treating or preventing pneumonia, comprising (i) an inhalable formulation containing a high concentration of chelating agent; and (ii) instructions for use.

[0029] The present invention provides a kit for treating or preventing pneumonia, comprising (i) an inhalable formulation capable of delivering a high concentration of an inhalable chelating agent in a single dose; and (ii) instructions for use.

[0030] Use of high concentrations of chelating agents in the manufacture of inhalable formulations for the treatment or prevention of lung inflammation.

[0031] Use of inhalable chelating agents in pharmaceutical manufacturing for the delivery of high concentrations of inhalable chelating agents for the treatment or prevention of lung infections in a single dose. Overview of the attached figures

[0032] Other features of the invention will be described more fully in the following description of several non-limiting embodiments of the invention. This description is included for illustrative purposes only and should not be construed as a broad generalization, disclosure, or limitation of the invention as described above. The description will be taken with reference to the accompanying drawings, in which:

[0033] Figure 1 shows the in vitro action of EDTA submicron particles on Pseudomonas aeruginosa biofilms and their synergistic effect with tobramycin in CF mucus treated with atomized EDTA particles and / or tobramycin. The final concentration of tobramycin in the droplets was 325 μg / ml. 1A) Confocal microscopy image of the biofilm stained with BacLight LIVE / DEAD. 1B) Bacterial counts showing the quantitative effect of treatment.

[0034] Figure 2 The results showed that CaEDTA reduced the bacterial load in the lungs of patients with CF (cancerous pulmonary fibrosis) faster than antibiotic treatment alone. CF subjects were treated with nebulized CaEDTA (EDTA) or saline (placebo), and the bacterial load (colon-forming units per gram of mucus) in their sputum was monitored.

[0035] Figure 3A Shows the mean change (% points) in FEV1 in patients treated with CaEDTA or placebo from the start of treatment to week 10 (4 weeks post-treatment). Figure 3BThe study showed the relationship between FEV1 improvement (0-2 weeks) and body weight.

[0036] Figure 4 The figures show the EDTA concentrations achieved in the sputum of three CF subjects at 5 minutes and 2 hours after treatment with 75 mg nebulized CaEDTA.

[0037] Figure 5-7 This indicates that administering high concentrations of the chelating agent to the lungs of mice in the absence of infection reduced inflammation.

[0038] Figure 5 This shows the total white blood cell count in bronchoalveolar lavage fluid (BALF) of mice exposed to air or cigarette smoke (CS) and treated with intranasal administration of a medium or deferoxamine (DFO). Although cigarette smoke induces white blood cell counts as expected, treatment with DFO significantly reduces this effect.

[0039] Figure 6 The lung weight of mice exposed to air or CS and treated with the medium or DFO as described above is shown. Lung weight can be used as a proxy for inflammation; an increase in weight indicates more inflammation. In CS-treated mice, lung weight increased significantly as expected, while DFO treatment reduced the average weight, indicating reduced inflammation.

[0040] Figure 7 The results show that while CS increased the iron content in BALF, treatment with deferoxamine reduced this effect. Left: Mean iron content of mice in each group; Right: Scatter plot of the same data.

[0041] Invention Description Invention Details

[0042] Treatment or prevention methods

[0043] This invention provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent.

[0044] Preferably, the high concentration of the inhaled chelating agent is higher than 37.5 mg / dose.

[0045] Preferably, the high concentration of the inhaled chelating agent is higher than 50 mg / dose.

[0046] Previously, it has been demonstrated that inhaled EDTA alone cannot treat bacterial infections (Brown et al. (Am J DisChild. 1985 139(8):836-9); Hassett (Front Microbiol. 2016 7:291)). Brown et al. (1985) treated 10 children with chronic Pseudomonas aeruginosa infection with sodium EDTA for three months and observed no change in lung function. Others have reported that EDTA causes concentration-dependent bronchoconstriction (Beasley et al. (Br Med J (ClinRes Ed. 1987 294(6581):1197-8)); EDTA has no effect on FEV1 (Asmus et al. (J AllergyClin Immunol. 2001 107(1):68-72)). Therefore, there is no reason to believe that chelators would have a positive effect on subjects with cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), or other lung conditions that cause or are associated with inflammation. However, the present invention has surprisingly discovered that inhaled chelating agents can treat or prevent lung inflammation.

[0047] The lung environment of cystic fibrosis (CF) is generally considered to be acidic. However, it has recently been found that the pH of CF lungs is the same as that of normal lungs (Schultz et al., “Airway surface liquid pH in children with cystic fibrosis”. Nature Communications 2017 8(1):1409). Existing technologies use acidified nitrites, as described in US20160263151, so this formulation is unlikely to be effective for CF clinically, as it would not maintain acidification but would immediately restore the normal lung pH of 7.4.

[0048] Despite normal lung acidity in CF patients, iron levels have been found to differ significantly from those in normal lungs. Results from Stites et al. (Am J Respir Crit Care Med. 1999 160(3):796-80) showed that iron levels were significantly higher in the lungs of CF patients and smokers compared to healthy individuals. They also showed that most of this iron was in the ferrous form, Fe(II), and was significantly correlated with disease severity (Hunter et al., MBio. 2013 4(4):1-8). Ferrous iron can participate in the Fenton reaction, generating highly reactive oxygen free radicals that can severely damage tissues and DNA (Jomova et al. Toxicology. 2011 283(2-3):65-87; MacNee, Eur J Pharmacol. 2001 429(1-3):195-207).

[0049] Not wishing to be bound by theory, the method of the present invention is believed to alleviate inflammation by: (i) inactivating matrix metalloproteinases (MMPs) through zinc chelation; (ii) reducing the production of reactive oxygen species (ROS) through iron chelation; and / or (iii) reducing the bacterial load in the lungs by depriving bacteria of essential ions (e.g., iron and zinc). The effect on the individual lungs is one of, or a combination of, theoretical approaches to alleviating inflammation.

[0050] Inhalation is a localized method of drug delivery, thus reaching the target area, namely the lungs, more effectively and providing a high local concentration of the inhaled chelate. Inhalation avoids the undesirable side effects caused by systemic exposure to the active substance and reduces the risk of patients developing drug resistance.

[0051] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of the inflammation results in an increase in FEV1.

[0052] This invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is associated with reduced MMP activity. Matrix metalloproteinases (MMPs) are known to cause lung injury (Garratt et al., Eur Respir J. 2015 46(2):384-94) and MMP activity is Zn2+ dependent (Hazra et al., Molecular Vision 2012; 18:1701-1711). However, previous attempts to target MMPs in the lungs have been unsuccessful. This invention uses an inhaled chelating agent to chelate zinc in the lungs, thereby reducing MMP-induced lung injury and treating or preventing inflammation.

[0053] This invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is associated with a reduction in the generation of hydroxyl radicals. Iron is a key factor in lung injury (Stites et al. (Am J Respir Crit Care Med. 1999 160(3):796-80), as iron catalyzes the formation of hydroxyl radicals. However, to date, antioxidant trials have failed to produce significant improvements in lung function. This invention uses an inhaled chelating agent to chelate iron in the lungs, thereby reducing lung injury caused by hydroxyl radicals and treating or preventing inflammation.

[0054] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is caused by the removal or reduction of bacterial biofilms through the presence of the chelating agent. The reduction of biofilms increases the amount of bacteria and biofilms removed through coughing and expectoration.

[0055] The present invention also provides a method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation is achieved by removing or reducing bacterial proteases that can stimulate local inflammation, cause local tissue damage, and neutralize antibiotic activity. These enzymes are largely cationic, and removing cations from the environment is expected to inactivate these enzymes.

[0056] Preferably, the chelating agent is an iron chelating agent or a zinc chelating agent. More preferably, the chelating agent is an iron and zinc chelating agent (iron / zinc chelating agent). Alternatively, the chelating agent may be a mixture of two or more chelating agents, such as a mixture of an iron chelating agent and a zinc chelating agent, or a mixture of an iron chelating agent and an iron / zinc chelating agent.

[0057] The chelating agent is preferably selected from citric acid, phosphate, di, tri, and tetrasodium salts of ethylenediaminetetraacetic acid (EDTA), calcium salts of EDTA, ethylene glycol-bis-(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N'-diglycine (EDDA); 2,2'-(vinylimino)-dibutyric acid (EBDA); lauroyl EDTA. ; dilauroyl EDTA, triethylenetetramine dihydrochloride (TRIEN), diethylenetriaminepentaacetic acid (DPTA), triethylenetetraminehexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicillamine dimercaptosuccinic acid, etidronic acid, sodium hexametaphosphate, D-penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, and chloroquine and combinations thereof.

[0058] Preferably, the chelating agent is a pharmaceutically acceptable chelating agent.

[0059] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In another embodiment, the chelating agent is deferoxamine (DFO). In yet another embodiment, the chelating agent is deferasirox (DSX).

[0060] Preferably, the chelating agent has approximately the same iron affinity as EDTA and / or approximately the same zinc affinity as EDTA. The formation constant or stability constant (log K1) of EDTA at 25°C and 0.1 M is: 14.3 for Fe2+, 25.1 for Fe3+, and 16.5 for zinc.

[0061] In one embodiment, the chelating agent is a calcium salt of the chelating agent. Preferably, the chelating agent is CaEDTA.

[0062] In one embodiment, the chelating agent is provided in an inhalation formulation comprising 37.5 mg / dose to 300 mg / dose, 50 mg / dose to 300 mg / dose, about 75 mg / dose to 200 mg / dose, about 75 mg / dose to 100 mg / dose, about 37.5 mg / dose to 200 mg / dose, or about 50 mg / dose to 200 mg / dose; preferably about 37.5 mg / dose, 50 mg / dose, 75 mg / dose, 100 mg / dose, 200 mg / dose, or 300 mg / dose. The chelating agent is preferably provided in an inhalation formulation comprising at least 37.5 mg / dose. The chelating agent is also preferably provided in an inhalation formulation comprising at least 50 mg / dose.

[0063] The preferred total daily intake of the chelating agent is about 37.5 mg / day to 1,200 mg / day, about 50 mg / day to 1,200 mg / day, about 100 mg / day to 1,000 mg / day, about 300 mg / day to 900 mg / day, or about 400 mg / day to 800 mg / day; more preferably about 150 mg / day, 300 mg / day, 500 mg / day, or 600 mg / day.

[0064] The preferred total daily inhalation dose of chelating agent is approximately 0.1 mg chelating agent / kg body weight to 15 mg chelating agent / kg body weight, approximately 0.5 mg chelating agent / kg body weight to 10 mg chelating agent / kg body weight, approximately 1.0 mg chelating agent / kg body weight to 5 mg chelating agent / kg body weight; approximately 1.0 mg chelating agent / kg body weight to 3.5 mg chelating agent / kg body weight; preferably approximately 1.0 mg chelating agent / kg body weight, 1.5 mg chelating agent / kg body weight, 2.0 mg chelating agent / kg body weight, 2.5 mg chelating agent / kg body weight, 3.0 mg chelating agent / kg body weight, 3.5 mg chelating agent / kg body weight, 4.0 mg chelating agent / kg body weight, 4.5 mg chelating agent / kg body weight, 5.0 mg chelating agent / kg body weight, 10 mg chelating agent / kg body weight, and 15 mg chelating agent / kg body weight.

[0065] It has been established that if 75 mg of a chelating agent (such as CaEDTA) is inhaled, approximately 0.4 mM to 1.34 mM of the chelating agent can be detected in sputum from the lungs after 5 minutes.

[0066] Inhaled chelates are preferably delivered over time intervals of no more than 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes. If administered via dry powder, the inhaled chelate can be delivered in seconds, for example, one "spray" per second in the aerosol device or dry powder inhaler, with one or more sprays administered at each time point.

[0067] Preferably, the inhaled chelating agent is administered for at least 28 consecutive days. The inhaled chelating agent may be delivered for 2 days or longer, 3 days, 4 days, 5 days, 6 days, or 7 days. The inhaled chelating agent may be delivered for 2 to 28 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0068] Some subjects may benefit from being “loaded” with antibiotics and / or chelators for a period of time, administered at higher doses or more frequently over days or weeks, followed by a reduced dose or maintenance dose.

[0069] Therefore, this invention:

[0070] The total dose of inhaled chelating agent delivered ranged from 37.5 mg / day to 1,200 mg / day;

[0071] Administer the medication at least once a day, up to six times a day, preferably up to four times a day;

[0072] Dosage should be administered within an 8-hour period.

[0073] Preferably, the present invention:

[0074] The total dose of inhaled chelating agent delivered ranged from 37.5 mg / day to 1,200 mg / day;

[0075] Administer once or twice daily;

[0076] Administer the medication in cycles not exceeding one hour each time;

[0077] It contains CaEDTA as a chelating agent.

[0078] The preferred amount of any chelating agent can be calculated by comparing the chelating ability of the reagent with CaEDTA and then multiplying the given dosage range above by that amount. The resulting chelation level should be approximately equal to the preferred chelation level provided by the preferred amount of EDTA.

[0079] Preferably, the inflammation is caused by, resulting from, or related to the following lung conditions: cystic fibrosis (CF); asthma; chronic obstructive pulmonary disease (COPD); pulmonary hypertension; lung cancer; pulmonary fibrosis; bronchiectasis; bronchitis; bronchiolitis; acute respiratory distress syndrome; tuberculosis; nontuberculous mycobacterial (NTM) lung infections; pneumonia including but not limited to ventilator-associated pneumonia, community-acquired pneumonia, bronchopneumonia, lobar pneumonia; infections caused by bacteria such as Pseudomonas spp., Streptococcus pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, Staphylococcus spp., Klebsiella spp., Escherichia coli, Stenotrophomonas spp., and fungi, including Aspergillus, Hypnosporium, and Candida; conditions where infection may occur, such as intubated or ventilated patients; infections in lung transplant patients; bronchitis; pertussis; inner ear infections; streptococcal laryngitis; inhaled anthrax; prophylactic treatment or prevention of tularemia or sinusitis.

[0080] Preferably, the formulation is administered to subjects in need about once a day to about six times a day, more preferably about four times a day.

[0081] Alternatively, the formulation can be administered to the desired subject via continuous inhalation through a nebulizer. The nebulized formulation can be delivered for 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour, and each delivery (except for 24 hours and 12 hours) can be repeated several times within 24 hours.

[0082] Subjects typically receive a chelating agent at a dose of approximately 0.01 to 15 mg / kg / day. + 20% or + 10%. This dose is typically administered via nebulization or through an aerosol device that delivers at least one, preferably multiple, "sprays". For example, a subject may receive a single dose or multiple doses throughout the day of the chelate from 0.1 mg / kg to 15 mg / kg.

[0083] The total daily dose is preferably administered at least once daily, but can be divided into two or more daily doses. Some subjects may benefit from a period of "loading" the subject with antibiotics and / or chelators, administered at higher doses or more frequently for several days or weeks, followed by a reduced or maintenance dose. Because cystic fibrosis, COPD, etc., are typically chronic conditions, subjects may expect to receive the therapy for an extended period.

[0084] Regardless of the form of the drug formulation, the preferred range of the generated droplets or particles for inhalation is approximately 0.1 μm to 12 μm, or approximately 0.25 μm to 6 μm, preferably 1 μm to 6 μm, and more preferably approximately 2 μm to 4 μm. Alternatively, the particles may be 0.1 μm to 1.0 μm, 0.2 μm to 0.9 μm, 0.3 μm to 0.8 μm, 0.4 μm to 0.7 μm, or 0.5 μm. By generating inhaled particles with a narrow size range, the efficiency of the drug delivery system can be further improved and the repeatability of the dose can be enhanced. Therefore, it is preferred that the particles not only have a size range of 0.1 μm to 12 μm, or 2 μm to 6 μm, or approximately 3 to 4 μm, but also have an average particle size within a narrow range, so that 80% or more of the particles delivered to the subject have a particle size within ±20% of the average particle size, preferably ±10% of the average particle size, and more preferably ±5%.

[0085] "Particle size" is a concept introduced to compare the dimensions of solid particles and liquid particles (droplets). For droplets and aerosols, terms such as "aerodynamic diameter" and "mass median aerodynamic diameter (MMAD)" are used. Their definitions are given below.

[0086] The "aerodynamic diameter" is the diameter of a sphere of unit density that has the same final settling velocity as the particle in question. It is used to predict where such particles will deposit in the respiratory tract.

[0087] The "mass median aerodynamic diameter" is the geometrically average aerodynamic diameter. By weight, 50 percent of particles will be smaller than the MMAD, and 50 percent will be larger than the MMAD.

[0088] In particle size analysis, the suspension contains numerous moving particles of varying sizes. When a particle size analyzer analyzes these particles, it generates a particle size distribution curve covering the entire particle size range, from the smallest particle (up to 1 nm) to the largest (up to 100 μm). The cumulative frequency of the particles is calculated from this particle size distribution curve. 10 It refers to a specific particle size, in which 10% of the particles in the suspension have a diameter less than or equal to the specific particle size.

[0089] D 50 : with D 10 Similar, D 50 It is the cutoff diameter of 50% of the particle population in the formulation, and refers to a specific particle size, wherein 50% of the particles in the suspension have a diameter less than or equal to the specific particle size.

[0090] D 90 :D 90 It is the cutoff diameter of 90% of the particle population in the formulation, and refers to a specific particle size, in which 90% of the particles in the suspension have a diameter less than or equal to the specific particle size.

[0091] The term "respiratory tract" should be understood as the system of cells and organs that function in respiration. In particular, the organs, tissues and cells of the respiratory tract include: lungs, nose, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, lung cells (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelial cells, mast cells, goblet cells and intraepithelial dendritic cells.

[0092] In one form of the invention, the method of treating or preventing lung inflammation in a subject comprises administering a therapeutically or preventively effective concentration of an inhaled chelator in the form of a single or multiple doses of at least 37.5 mg / dose, wherein the dose or each dose of chelator is administered over a period not exceeding 8 hours.

[0093] In one form of the invention, the method of treating lung inflammation in a subject comprises administering a therapeutically effective concentration of an inhaled chelating agent in the form of a single or multiple doses of at least 37.5 mg / dose, wherein the dose or each dose of chelating agent is administered over a period of no more than 8 hours.

[0094] In one form of the invention, the method of preventing lung inflammation in a subject comprises administering an effective concentration of an inhaled chelator in the form of a single or multiple doses of at least 37.5 mg / dose, wherein the dose or each dose of chelator is administered over a period not exceeding 8 hours.

[0095] In one form of the invention, the method of treating or preventing lung inflammation in a subject includes treating or preventing lung inflammation in a subject who requires the treatment.

[0096] The term “therapeutic effective amount” as used in this article refers to an amount of preparation which, when administered according to the desired dosing regimen, is sufficient to partially achieve the desired therapeutic effect, or delay the onset of inflammation, or inhibit the development of inflammation, or partially or completely stop the onset or development of inflammation.

[0097] The term “preventative effective amount” as used in this article refers to an amount of preparation which, when administered according to the desired dosing regimen, is sufficient to at least partially prevent or delay the onset of inflammation.

[0098] As used herein, “treatment” refers to the suppression of a disease or symptom, i.e., preventing or reducing its development or at least one of its clinical or subclinical symptoms. “Treatment” also refers to the relief of a disease or symptom, i.e., resulting in the resolution of the disease or symptom or at least one of its clinical or subclinical symptoms. The benefit to the subject to be treated is statistically significant, or at least perceptible to the subject and / or physician. In the case of treating inflammation, the term treatment includes reducing or eliminating leukocyte infiltration (including macrophages, polymorphonuclear neutrophils, lymphocytes, and other immune cells); immunoglobulins; pro-inflammatory cytokines and inflammatory chemokines and their receptors; harmful mediators such as reactive oxygen species and proteolytic enzymes; the abundance and activity of MMPs; markers of oxidative stress; and one or more of bronchial hyperresponsiveness and exacerbation. The term treatment further includes one or more of increased anti-inflammatory cytokines and increased lung function (FEV1).

[0099] Based on the foregoing, those skilled in the art will understand that a variety of different treatment methods and administration routes can be used to treat individual subjects. Therefore, subjects already receiving medications such as intravenous ciprofloxacin or antibiotics may benefit from inhaling the formulations of the present invention. Some subjects may receive the high-concentration chelating agent formulations of the present invention solely by inhalation. Such subjects may have symptoms of cystic fibrosis, be diagnosed with a lung infection, or have symptoms of a medical condition that may benefit from administration of a high concentration of the chelating agent. The formulations of the present invention can also be used for diagnosis. In embodiments, for example, as part of a diagnostic procedure for a lung infection, a subject may receive a dose of the formulation of the present invention, wherein one or more of the subject's symptoms improve in response to the formulation.

[0100] Dosage form

[0101] The present invention provides an inhalable formulation containing a high concentration of chelating agents.

[0102] The inhalable formulation may be in the form of a dry powder for inhalation or in the form of a nebulizer for inhalation. Preferably, the formulation is suitable for inhalation to treat or prevent lung inflammation.

[0103] In one embodiment, the chelating agent is a calcium salt of the chelating agent. Preferably, the chelating agent is CaEDTA.

[0104] Preferably, the high concentration of the inhaled chelating agent is greater than 37.5 mg / dose. Preferably, the high concentration of the inhaled chelating agent is greater than 50 mg / dose. Preferably, the high-concentration chelating agent is provided in dosage forms containing 37.5 mg / dose to 300 mg / dose, 50 mg / dose to 300 mg / dose, about 75 mg / dose to 200 mg / dose, about 75 mg / dose to 100 mg / dose, or about 50 mg / dose to 200 mg / dose; more preferably, about 50 mg / dose, 75 mg / dose, 100 mg / dose, 200 mg / dose, or 300 mg / dose. The chelating agent is preferably provided in an inhaled dosage form containing at least 37.5 mg / dose. The chelating agent is preferably provided in an inhaled dosage form containing at least 50 mg / dose.

[0105] The total daily inhaled chelating agent is preferably from about 37.5 mg / day to 1,200 mg / day, 50 mg / day to 1,200 mg / day, about 100 mg / day to 1,000 mg / day, about 300 mg / day to 900 mg / day, or about 400 mg / day to 800 mg / day; more preferably about 300 mg / day, 500 mg / day, or 600 mg / day. The chelating agent can be administered at a maximum of about 1,200 mg / day, preferably at a total dose of at least 150 mg / day.

[0106] The total daily intake of the chelating agent is preferably from about 37.5 mg / day to 1,200 mg / day, about 50 mg / day to about 1,200 mg / day, about 100 mg / day to about 1,000 mg / day, about 300 mg / day to about 900 mg / day, about 400 mg / day to about 800 mg / day; more preferably about 150 mg / day, 300 mg / day, 500 mg / day or 600 mg / day.

[0107] The preferred total daily intake of chelating agent is approximately 0.1 mg chelating agent / kg body weight to 15 mg chelating agent / kg body weight, approximately 0.5 mg chelating agent / kg body weight to 10 mg chelating agent / kg body weight, approximately 1.0 mg chelating agent / kg body weight to 5 mg chelating agent / kg body weight; approximately 1.0 mg chelating agent / kg body weight to 3.5 mg chelating agent / kg body weight; preferably approximately 1.0 mg chelating agent / kg body weight, 1.5 mg chelating agent / kg body weight, 2.0 mg chelating agent / kg body weight, 2.5 mg chelating agent / kg body weight, 3.0 mg chelating agent / kg body weight, 3.5 mg chelating agent / kg body weight, 4.0 mg chelating agent / kg body weight, 4.5 mg chelating agent / kg body weight, 5.0 mg chelating agent / kg body weight, 10 mg chelating agent / kg body weight, and 15 mg chelating agent / kg body weight.

[0108] For example, a 50 mg dose of CaEDTA can be administered via a 4 ml nebulized solution of 33 mM (the molecular weight of C10H12CaN2Na2O8 is 274.27 g / mol). Similarly, a 75 mg dose can be administered via a 4 ml nebulized solution of 50 mM or a 100 mg dose can be administered via a 4 ml nebulized solution of 66 mM.

[0109] Preferably, the formulation is administered to the subject in need about once a day to about six times a day, more preferably four times a day.

[0110] Alternatively, the formulation can be administered to the desired subject via continuous inhalation or through a nebulizer. The nebulized formulation can be delivered for 24 hours, 12 hours, preferably 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour, and each delivery (except for 24 and 12 hours) can be repeated several times within 24 hours.

[0111] The formulations of the present invention can be administered to subjects using disposable packaging and portable, handheld, battery-powered devices, such as the AERx device (US Patent No. 5,823,178, Aradigm, Hayward, Calif.). Alternatively, the formulations of the present invention can be obtained using mechanical (non-electronic) devices. Other inhalation devices that can be used to deliver the formulations include conventional jet nebulizers, ultrasonic nebulizers, soft fog inhalers, dry powder inhalers (DPIs), metered-dose inhalers (MDIs), condensate aerosol generators, and other systems.

[0112] For use as an aerosol, the compounds of the present invention in solution or suspension, along with a suitable propellant, such as a hydrocarbon propellant like propane, butane, or isobutane, can be packaged together with conventional adjuvants in a pressurized aerosol container. A dry powder inhaler is a system that can be operated with a compressed air source to produce dry powder particles of a pharmaceutical formulation compressed into very small volumes. For inhalation, the system has multiple chambers or blister packs, each containing a single dose of the pharmaceutical formulation and a selection element for releasing that single dose.

[0113] An aerosol can be generated by forcing a drug through pores in a membrane, the pore size ranging from approximately 0.25 to 6 μm (US Patent No. 5,823,178). When the pores have this size, the particles passing through the pores to form an aerosol will have a diameter ranging from 0.5 to 12 µm. The drug particles can be released with the airflow to maintain the particles within this size range. The generation of small particles can be facilitated by using a vibrating device that provides a vibration frequency in the range of approximately 800 to approximately 4000 kHz. Those skilled in the art will recognize that some adjustments can be made to parameters such as the size of the pore from which the drug is released, the vibration frequency, the pressure, and other parameters based on the density and viscosity of the formulation. It should be noted that some embodiments aim to provide atomized particles with a diameter ranging from approximately 0.5 to 12 μm.

[0114] excipient

[0115] The exemplary forms of the formulations described herein can be manufactured using methods well known to those skilled in the art of formulation science. Additionally, the formulations described herein may include other optional excipients to aid in the manufacture and / or administration of the formulations described herein. Non-limiting examples of such excipients are well known in the art and include flavoring agents, coloring agents, palatants, antioxidants, viscosity modifiers, tension agents, drug carriers, sustained-release agents, comfort enhancers, emulsifiers, solubilizers, lubricants, binders, and other stabilizers to aid in the manufacture and / or administration of the formulation.

[0116] Preferably, the formulation is sterile. In another embodiment, the formulation of the present invention is stable.

[0117] In addition, a buffer can be added to adjust the pH of the formulation. Preferably, the formulation of the present invention contains tris(hydroxymethyl)aminomethane (TRIS, also known as THAM or aminobutanetriol) as a buffer. TRIS can further enhance the bactericidal effect of EDTA. Preferably, TRIS is added to the formulation of the present invention to buffer the formulation and increase the effectiveness of EDTA and / or antibiotics in treating or preventing bacterial infections.

[0118] Furthermore, the formulations of the present invention may contain antimicrobial preservatives.

[0119] Preferably, the pH of the formulation of the present invention is about 6.5 to 8.0, more preferably about 7.0 to 7.4. It has previously been found that the greater the decrease in pH, the higher the bacterial resistance to antimicrobial treatment. The preferred pH helps to avoid bacterial resistance to the formulation in the absence of acidified nitrites, said formulation comprising a combination of a high concentration of inhaled chelating agent and antibiotic.

[0120] In an alternative embodiment, the formulations of the present invention may comprise preservatives, suspending agents, wetting agents, tensioning agents, and / or diluents. The formulations provided herein may comprise one or more pharmacologically suitable suspensions, which are physiologically acceptable after inhalation administration, at about 0.01% to about 90%, or about 0.01% to about 50%, or about 0.01% to about 25%, or about 0.01% to about 10%, or about 0.01% to about 5%. Pharmacologically suitable fluids used herein include, but are not limited to, polar solvents, including, but not limited to, compounds containing hydroxyl or other polar groups. Solvents include, but are not limited to, water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, and polyoxyethylene alcohol. Polar solvents also include proton solvents, including, but not limited to, water, aqueous solutions of salts having one or more pharmaceutically acceptable salts, alcohols, glycols, or mixtures thereof. In an alternative embodiment, the water used in this formulation shall meet or exceed applicable regulatory requirements for inhaled medications.

[0121] In one embodiment, the formulation described herein may be aqueous and contain 0-90% water. In other embodiments, the aqueous formulation described herein may contain 20-80% water. In other embodiments, the aqueous formulation may contain 50-70% water. The water may further comprise purified, distilled, sterile, softened, or deionized water.

[0122] Alternatively, the formulation may be non-aqueous, anhydrous, or have negligible water content (e.g., less than 1%, less than 0.1%, less than 0.01%).

[0123] In one embodiment, the formulation further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0124] In addition to sterilization or as an alternative to sterilization, the formulations of the present invention may contain pharmaceutically acceptable preservatives to minimize the possibility of microbial contamination. Additionally, pharmaceutically acceptable preservatives may be used in the formulations of the present invention to increase the stability of the formulation. However, it should be noted that the selection of any preservative must take into account inhalation safety, as the treated tissue may be sensitive to irritants. Preservatives suitable for use herein include, but are not limited to, those that protect solutions from pathogenic particulate contamination, including phenethyl alcohol, benzalkonium chloride, or benzoic acid or benzoates such as sodium benzoate and phenethyl alcohol. In some embodiments, the formulations herein contain about 0.001% to about 10.0% w / w benzalkonium chloride, or about 0.01% v / w phenethyl alcohol. Preservatives may also be present in amounts of about 0.001% to about 1%, preferably about 0.002% to about 0.02%, more preferably 0.02% w / w.

[0125] The formulations provided herein may also contain one or more emulsifiers, wetting agents or suspending agents in amounts of about 0.001% to about 90%, about 0.001% to about 50%, about 0.001% to about 25%, about 0.001% to about 10%, or about 0.001% to about 1%. Such reagents used in this article include, but are not limited to, polyoxyethylene sorbitol fatty acid esters or polysorbates, including but not limited to, polyethylene sorbitol monooleate (polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitol monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitol tristearate), polyoxyethylene (20) sorbitol monooleate, polyoxyethylene (20) sorbitol monopalmitate, polyoxyethylene (20) sorbitol monostearate; lecithin; agar; carrageenan; bean gum; guar gum; tragacanth gum; gum arabic; xanthan gum; ebony gum; pectin; amidated pectin; ammonium phosphatidylcholine; microcrystalline cellulose; methylcellulose; hydroxypropyl cellulose; hydroxypropyl methylcellulose; ethyl methylcellulose; carboxymethyl cellulose; sodium, potassium and calcium salts of fatty acids; monoglycerides of fatty acids and Diglycerides; acetates of fatty acid monoglycerides and diglycerides; lactates of fatty acid monoglycerides and diglycerides; citrates of fatty acid monoglycerides and diglycerides; tartrates of fatty acid monoglycerides and diglycerides; mono- and diacetyl tartrates of fatty acid mono- and diglycerides; mixed acetates and tartrates of fatty acid monoglycerides and diglycerides; sucrose esters of fatty acids; sucrose glycerides; polyglycerides of fatty acids; polyglycerides of castor oil condensed fatty acids; 1,2-propanediol esters of fatty acids; sodium stearoyl-21 acrylate; calcium stearoyl-2-lactate; stearoyl tartrate; sorbitol monostearate; sorbitol tristearate; sorbitol monolaurate; sorbitol monooleate; sorbitol monopalmitate; soap bark extract; soybean oil dimeric fatty acid polyglycerides; oxidized polymerized soybean oil; and pectin extract.

[0126] The formulations of the present invention may contain about 0.001% to about 5% by weight of a humectant to inhibit mucosal dryness and prevent irritation. Any of a variety of pharmaceutically acceptable humectants may be used, including, for example, sorbitol, propylene glycol, polyethylene glycol, glycerin, or mixtures thereof.

[0127] The formulations of the present invention may further include adjuvants, such as bronchodilators, another anti-inflammatory drug, surfactants, aspirin, or ethanol.

[0128] The bronchodilators optionally used in the formulations of this invention include, but are not limited to, β2-adrenergic receptor agonists (e.g., albuterol, bambuterol, salbutamol, salmeterol, formoterol, aforterol, levosalbutanol, procaterol, indacaterol, carmoterol, milveterol, procaterol, terbutaline, etc.) and antimuscarinic agents (e.g., trospium, ipratropium, gluconate, acridineium, etc.). Combinations of drugs may be used.

[0129] Other anti-inflammatory drugs that may optionally be used in the formulations of this invention include, but are not limited to, inhaled corticosteroids (e.g., beclomethasone, budesonide, cicsolone, fluticasone, etiprednol, mometasone, etc.), leukotriene receptor antagonists and leukotriene synthesis inhibitors (e.g., montelukast, ziloxetine, isotetrast, zafirlukast, progesterone, ameruban, tylosone, etc.), and cyclooxygenase inhibitors (e.g., ibuprofen, ketoprofen, ketorolac, indomethacin, naproxen, zaltoprofen, lornoxicam, meloxicam, celecoxib, rumicoxib, etoricoxib, piroxicam, ampixicam, sinoxicam, diclofenac, biphenylacetic acid, lornoxicam, mesalazine, trifluralin, tenoridone, ellamod, pamigre, etc.). Combinations of drugs may be used. Aspirin may also be added as an anti-inflammatory drug.

[0130] The surfactants covered by this invention include, but are not limited to, synthetic surfactants ( Dipalmitoylphosphatidylcholine and oleic acid. A combination of these drugs can be used.

[0131] Antioxidants such as glutathione and vitamin E, zinc, and zinc salts of EDTA can be added.

[0132] Ethanol vapor can act as an antifoaming agent in the lungs, making sputum more liquefied, which can help breathing and reduce pulmonary edema. Ethanol can be added to the formulations of the present invention in proportions of 0.5% to 60%, more preferably 1 to 40%, 1 to 20%, or 1 to 10%. Ethanol can be added in proportions of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0133] The present invention also relates to the use of high concentrations of chelating agents administered by inhalation in combination with other drugs. These other drugs may include nucleotide sequences that may be incorporated into suitable delivery vectors such as plasmids or viral vectors. Other drugs may include therapeutic nucleotide sequences (DNA, RNA, siRNA), enzymes that reduce mucus viscoelasticity (such as dnase and other mucolytics), chemicals that positively regulate chloride ion channels or increase ion flow across cells, nicotine, P2Y2 agonists, elastase inhibitors, including α-1 antitrypsin (AAT), N-acetylcysteine, antibiotics and cationic peptides, such as lanthanide antibiotics, particularly nifedipine, short-acting bronchodilators (e.g., β2-adrenergic receptor agonists, such as salbutamol or indacaterol), M3 muscarinic antagonists (e.g., ipratropium bromide), K+-channel openers, long-acting bronchodilators (e.g., formoterol, salmeterol), and steroids (e.g., budesonide, fluticasone, triamcinolone, beclomethasone, cistanoedone, etc.). Xanthine, leukotriene antagonists (e.g., montelukast sodium), phosphodiesterase 4 inhibitors, adenosine receptor antagonists, various other anti-inflammatory drugs (e.g., Syk kinase inhibitors (AVE-0950), trypsin inhibitors (AVE-8923 & AVE-5638), tachykinin antagonists (AVE-5883), inducible nitric oxide synthase inhibitors (GW-274150), and others), transcription factor decoys, TLR-9 agonists, antisense oligonucleotides, siRNA, DNA, CGRP, lidocaine, inverse β2-agonists, anti-infective oxidative therapy, cytokine modulators (e.g., CCR3 receptor antagonists (GSK-766994, DPC-168, AZD-3778), TNF-α production inhibitors (LMP-160 and YS-TH2), and IL-4) Antagonists (AVE-0309), small molecule inhibitors of IgE, cell adhesion molecule (CAM) inhibitors, small molecules targeting VLA4 receptor or integrin α4β1 (e.g., R-411, PS-460644, DW-908e, and CDP-323), immunomodulators, including those that block T cell signaling by inhibiting calcineurin (tarolim), heparin neutralizers (Talactoferrin α), cytosol PLA2 inhibitors (Efipladib), or combinations thereof. If desired, subjects with CF may also be administered standard medicines, such as ivacartocin, alfa-chain enzyme, mannitol, or other approved medicines, in combination with the formulations of this invention, according to standard operating procedures.

[0134] The delivery of the combination product can be achieved by combining the drugs into a stable formulation, or by combining them when the drugs are delivered in separate containers and administered, or alternatively by delivering the product sequentially.

[0135] Preferably, the formulations of the present invention are stable. As used herein, the stability of a formulation provided herein refers to the length of time at a given temperature that more than 80%, 85%, 90%, or 95% of the initial amount of pharmaceutical substance (e.g., chelating agents and antibiotics) remains in the formulation. For example, the formulations provided herein can be stored between about 15°C and about 30°C and remain stable for at least 1, 2, 12, 18, 24, or 36 months. Similarly, the formulation may be suitable for administration to a subject in need after being stored at 25°C for more than 1, 2, 12, 18, 24, or 36 months. Alternatively, in another alternative embodiment, Arrhenius kinetics is used to determine that after the formulation has been stored at about 15°C to about 30°C for more than 1, 2, 12, 18, 24, or 36 months, more than 80%, more than 85%, more than 90%, or more than 95% of the initial amount of pharmaceutical substance remains.

[0136] As used herein, the statement "the formulation is stable during 'long-term storage'" means that the formulation is suitable for administration to subjects in need of it when the formulation has an estimated shelf life of: more than 1, 2, or 3 months of use at 25°C and more than 1, 2, or 3 years of storage at 5°C. In some embodiments herein, using Arrhenius kinetics, it is estimated that >80% or >85% or >90% or >95% of the chelating agent and antibiotic remain after this storage.

[0137] As used herein, the term “inflammation” refers to one or more signs of the body’s response to an attack, such as infection, environmental attack (including cigarette smoke), trauma, or hypersensitivity. Inflammation can be acute or chronic, and signs include tissue swelling, recruitment of different types of inflammatory cells, release of cytokines and mediators, and bronchial hyperresponsiveness. Inflammation can be localized, subclinical, or transient, and can also be more widespread and chronic. Inflammation can include humoral and cellular immune responses and may persist after the attack that triggered the inflammation has subsided. Signs of inflammation include, but are not limited to, elevated levels of inflammatory cells (e.g., dendritic cells, macrophages, neutrophils, lymphocytes, eosinophils, and mast cells), elevated levels of pro-inflammatory cytokines (e.g., TNFα, IL-1β, IL-6, IL-8, and IFNγ) and their receptors, excess proteases, including MMPs, ROS, and other modulators, and inflammatory markers such as C-reactive protein (CFP) in sputum and serum calprotectin. Short-term (“acute”) inflammation can cause airway swelling, changes in lung compliance, airway responsiveness and excessive mucus secretion, with clinical symptoms including increased respiratory rate and dyspnea, wheezing, cough and decreased FEV1. If it persists (“chronic”), it can lead to fibrosis, cystic changes and bronchiectasis, destruction of the airway wall and lung parenchyma structure.

[0138] Methods for preparing drugs

[0139] Use of high concentrations of chelating agents in the preparation of inhalable formulations for the treatment or prevention of lung inflammation.

[0140] Use of the inhalable chelating agent in the preparation of a medicament for the single-dose delivery of high concentrations of the inhalable chelating agent for the treatment or prevention of lung inflammation.

[0141] Preferably, the high-concentration chelating agent is provided in a dosage form containing at least 37.5 mg / dose, at least 50 mg / dose, or from 50 mg / dose to 300 mg / dose, or from 37.5 mg / dose to 300 mg / dose. The chelating agent is administered one to four times daily, up to a maximum of about 1,200 mg / day, preferably at a total dose of at least 150 mg / day. The chelating agent is preferably CaEDTA.

[0142] Reagent test kit

[0143] The present invention provides a kit for treating or preventing lung inflammation, comprising (i) an inhalable formulation containing a high concentration of chelating agent; and (ii) instructions for use.

[0144] The present invention provides a kit for treating or preventing lung inflammation, comprising (i) an inhalable formulation capable of delivering a high concentration of an inhalable chelating agent in a single dose; and (ii) instructions for use.

[0145] Preferably, the high-concentration chelating agent is provided in a dosage form containing at least 37.5 mg / dose, at least 50 mg / dose, or from 37.5 mg / dose to 300 mg / dose, or from 50 mg / dose to 300 mg / dose. The chelating agent is administered one to four times daily, up to a maximum of about 1,200 mg / day, preferably at a total dose of at least 150 mg / day. The chelating agent is preferably CaEDTA.

[0146] In one embodiment, the kit of the present invention comprises a formulation containing a therapeutically effective amount of a high concentration of inhaled chelating agent. In an alternative embodiment, the formulation is pre-measured, pre-mixed, and / or pre-packaged. Preferably, the inhalation solution is sterile.

[0147] The kit of the present invention also includes instructions for use designed to promote user compliance. As used herein, instructions for use refers to any label, insert, etc., and may be located on one or more surfaces of the packaging material, or may be provided on a separate diagram or any combination thereof. For example, in an embodiment, the kit of the present invention includes instructions for administering the formulation of the present invention. In one embodiment, the instructions indicate that the formulation of the present invention is suitable for treating lung inflammation. Such instructions may also include dosage instructions, as well as instructions for administration via a nebulizer or dry powder inhaler.

[0148] The inhaled chelating agent and any other active agent may be packaged individually so that practitioners or users can formulate them into pharmaceutical preparations as needed. Alternatively, pharmaceutical preparations containing the inhaled chelating agent and any other active agent may be packaged together to form the minimum dosage required by the practitioner or user. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient.

[0149] General Rules

[0150] Those skilled in the art will understand that variations and modifications can be made to the invention described herein, in addition to those specifically described. This invention includes all such variations and modifications. It also includes all steps, features, formulations, and compounds individually or collectively mentioned or pointed out in the specification, as well as any or all combinations of any two or more steps or features.

[0151] Every document, reference, patent application, or patent cited in this article is explicitly incorporated herein by full quotation, meaning that the reader should read and consider it as part of this article. The documents, references, patent applications, or patents cited herein are not repeated solely for the sake of brevity.

[0152] Descriptions, specifications and product lists of any products mentioned herein or in any document incorporated herein by reference are incorporated herein by reference and may be used in the practice of this invention.

[0153] The scope of this invention is not limited to any of the specific embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention described herein.

[0154] The invention described herein may include a range of one or more values ​​(e.g., dimensions, displacements, and field strengths). A range of values ​​will be understood to include all values ​​within that range, including the values ​​defining the range and values ​​adjacent to the range that result in the same or substantially the same result as the value immediately adjacent to the boundary of the defined range. Therefore, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximate values ​​that may vary according to the desired characteristics sought to be obtained according to the invention. Thus, "about 80%" means "about 80%" and also "80%". At a minimum, each numerical parameter should be interpreted according to the number of significant figures and common approximation methods.

[0155] Throughout this specification, unless the context otherwise requires, the word “comprising” or variations such as “including” or “containing” will be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes. It should also be noted that in this invention, particularly in the claims and / or paragraphs, terms such as “comprising,” “including,” and “containing” can have the meanings given to them under U.S. patent law. For example, “they” can mean “includes,” “included,” “including,” and the like; and terms such as “consistently of” have the meanings given to them under U.S. patent law, for example, they allow elements not explicitly stated but exclude elements found in the prior art or affecting the essential or novel features of the invention.

[0156] Other definitions of the selected terms used herein can be found in the detailed description of the invention and are used throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The term "activator" may refer to one activator or may encompass two or more activators.

[0157] The following embodiments are provided to more fully describe the ways in which the invention is used and to illustrate the best mode contemplated for implementing various aspects of the invention. It should be understood that these methods are not intended to limit the true scope of the invention, but are presented for illustrative purposes. Example

[0158] Other features of the invention are described more fully in the following non-limiting embodiments. This description is included for illustrative purposes only and should not be construed as limiting the broad description of the invention as described above.

[0159] Example 1: A method for treating or preventing lung infections by administering high concentrations of inhaled chelating agents.

[0160] Biofilms were grown in a simulated in vitro model using suspensions of cystic fibrosis mucus harvested from epithelial cell lines (Haley et al., BMC Microbiol 2012 12:181). Cultures of clinical strains of Pseudomonas aeruginosa (MIC tobramycin >256 µg / ml) were grown in carbon-free M63 to late stationary phase to simulate nutrient limitation.

[0161] Suspend a droplet of mucus (5 µl) on an inverted IBIDI coverslip and inoculate 10 3 Each colony-forming unit (CFU) was incubated at 35°C in a humid environment for 72 hours to allow biofilm development. The droplets were then treated with nebulized tobramycin (20 mg / ml), nebulized CaEDTA particles (10 mg / ml), or both simultaneously for 5 minutes. A control was treated with a nebulized 50 / 50 solution of 0.9% saline / water. After treatment, the droplets were incubated for 16 hours, stained with BacLight LIVE / DEAD (1 µl), and fixed in paraformaldehyde vapor for 30 minutes. The biofilm was observed using a confocal microscope.

[0162] Figure 1A The biofilm is thick and robust, and after treatment with aerosolized saline, most of the cells remain viable (green). As expected for drug-resistant strains, tobramycin treatment alone has little impact on viability. EDTA alone causes some killing (red cells). Surprisingly, the combination of tobramycin and EDTA kills the vast majority of biofilm cells. Figure 1B show Figure 1A Quantitative representation of microscopic images. The control biofilm was 1x10⁻⁶. 8 CFU / ml, while EDTA-tobramycin-treated biofilms showed a reduction of > 6 orders of magnitude, down to <10. 2 CFU / ml.

[0163] Patients aged ≥6 years with worsening CF upon admission were randomly assigned to receive either EDTA or saline (placebo) in addition to routine intravenous antibiotics and nebulized tobramycin. EDTA was administered as a nebulizer solution along with tobramycin, consisting of 4 ml of 0.9% saline containing 50 mM CaNa2EDTA and 111 mM Tris at pH 7.1. Following randomization, subjects were hospitalized for two weeks, receiving four times daily (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). Patients were then discharged and continued treatment twice daily for four weeks. Patients were monitored for another four weeks, bringing the total study duration to 10 weeks.

[0164] Sputum was induced for ≥5 minutes using nebulized 3% hypertonic saline at a rate of 8-10 L / min. Samples were collected before treatment and at weeks 2, 6, and 10, processed according to the relevant protocol, and stored at -80°C. Mucus was separated from clear sputum mixed with Sputalysin (1 ml per gram of sputum), vortexed, and incubated for one hour, then placed in skim milk glycerin storage medium and stored at -80°C.

[0165] Sputum samples were obtained from subjects via expectoration during the initial screening, and then at the third visit (approximately 2 weeks later), the fifth visit (6 weeks later), and finally at the follow-up visit (10 weeks later). Mucus was separated from the clear sputum, treated with Sputalysin (1 ml per gram of sputum), and then placed in a nonfat lactic acid glycerol storage medium (1 ml / 100 mg mucus), vortexed, and stored at -80°C.

[0166] Thaw the sample on ice, and then dilute the serial dilutions from the original concentration to a maximum of 10⁻⁷. Place 20 µL of each dilution into three separate McConkey (McC) agar or Blood agar (BA) plates. Incubate the plates at 35 °C.

[0167] Pseudomonas is defined as a transparent or very pale pink lactose-negative colony on Mc agar plates. Rough-morph colonies have a metallic sheen and rough edges, smooth-morph colonies grow slowly and have regular colony boundaries on Mc agar, and slimy-morph colonies are surrounded by a large amount of alginate secreted by the bacteria.

[0168] At 24 hours, the number of coarse, smooth, and slimy colonies were counted on McCagar and BA plates, respectively. The plates were then incubated for another 24 hours, and a confirmatory count of each colony morphology was performed. Individual colonies of each morphology present in each sample were selected and streaked onto BA plates to obtain pure cultures.

[0169] Further identification was performed using Gram staining to confirm that the isolate consisted of Gram-negative rod cells, and an oxidase-positive state was confirmed by smearing a small portion of the colony onto an oxidase test strip. Rapid development of a deep blue color indicated the presence of an oxidase-positive isolate.

[0170] Identification of *Pseudomonas* spp. was performed by testing for resistance to the antibiotic C390. Antibiotic-impregnated discs were placed on nutrient agar (NA) plates containing a suspension of the pure isolate in phosphate-buffered saline (PBS) at a McFarland density of 0.5. After overnight incubation at 35°C, the absence of any inhibition zone around the disc indicated antibiotic resistance. Single colonies of the identified *Pseudomonas* spp. (likely *Pseudomonas aeruginosa*) of each morphological type present in each isolate were selected and resuspended in glycerol / serum storage medium and stored at -80°C. Figure 2 The changes in Pseudomonas aeruginosa (McC) colony counts at weeks 2 and 6 compared to the start of treatment were shown. Two weeks after treatment, the colony count in the EDTA group decreased by >400-fold, compared to 4.5-fold in the placebo group.

[0171] Example 2: Treatment of lung inflammation led to a dose-dependent increase in FEV1.

[0172] CF subjects aged ≥6 years with worsening condition admitted to the hospital were randomly assigned to receive either EDTA or saline (placebo) in addition to routine intravenous antibiotics and nebulized tobramycin. EDTA was administered as a nebulizer solution along with tobramycin, consisting of 4 ml of 0.9% saline containing 50 mM CaNa2EDTA, 111 mM Tris, and pH 7.1. After randomization, subjects were treated in the hospital for two weeks, receiving four times daily (300 mg EDTA / day). Treatment continued twice daily for four weeks after discharge. Patients were monitored for another four weeks, bringing the total study time to 10 weeks.

[0173] Lung function was measured using a spirometry method at each study visit. Data was recorded as the best of three attempts, and results are expressed as a percentage of prediction.

[0174] Figure 3A This chart shows the mean changes in FEV1 between the two groups at weeks 2, 6, and 10 after the start of treatment. The mean increase in FEV1 after 2 weeks was 16% in the EDTA group and 5% in the placebo group. This difference persisted for four weeks after the completion of treatment, with an increase of 7% in the EDTA group and 2% in the placebo group. This indicates a significant improvement in lung function in the EDTA group, while the placebo group showed little change. Figure 3BThe EDTA group showed a negative correlation between FEV1 improvement and body weight (R2=0.70), but no correlation was found in the placebo group treated with tobramycin alone (R2=0.01). This indicates that EDTA has a dose-dependent effect on lung function (mg EDTA / kg body weight).

[0175] Figure 4 The study showed that delivery of 75 mg CaEDTA into the lungs resulted in a peak EDTA concentration of 0.41–1.34 mM 5 minutes after administration.

[0176] Example 3: Lung inflammation caused by cigarette smoke can be treated by administering high doses of chelating agents to the lungs.

[0177] The effects of chelating agents on lung inflammation were tested in a mouse model of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is known to induce lung inflammation, which can be measured by increased white blood cell count and increased lung weight.

[0178] Male BALB / c mice (n=8 per group) were exposed to a dose of cigarette smoke (3 cigarettes, 3 times daily, Monday through Friday) or filtered indoor air for two weeks. During the experiment, mice were intranasally treated three times daily with the iron chelator deferoxamine (DFO, 3.8 mg, 50 µl) or a carrier 30–60 minutes before each cigarette smoke exposure. Mice were then sacrificed, and their airways and lungs were assessed to determine the effects of cigarette smoke-induced inflammation and elemental concentrations.

[0179] Bronchoalveolar lavage fluid (BALF) (approximately 1 ml / mouse) was collected, and the lung was surgically removed and weighed. The total number of viable cells in the BALF was determined by mixing an equal volume of trypan blue with the BALF and manually counting the cells using a standard Neubauer hematology counter under a Zeiss Axioscope fluorescence microscope. Iron was measured by elemental analysis and quantification with standards of known metal content using laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS).

[0180] Figure 5 This indicates that, as expected, cigarette smoke significantly increases the total number of BALF white blood cells. Treatment with the iron chelator DFO significantly reduces this effect.

[0181] In line with this, Figure 6 Studies have shown that treatment with cigarette smoke significantly increases average lung weight, but treatment with CFO can prevent this effect.

[0182] As mentioned earlier, Stites et al. (Am J Respir Crit Care Med. 1999 160(3):796-80) showed that, compared with healthy individuals, patients with CF and smokers had significantly higher levels of iron in their lungs. Figure 7 (Left) It was confirmed that the average iron level in the BALF of mice exposed to cigarette smoke was significantly increased, and that treatment with DFO reduced the average iron content in the BALF. Figure 7 (Right) The results showed that among the seven mice treated with DFO (one of which was lost for reasons unrelated to the treatment), the iron content of BALF in six of them was at the same level as that in mice exposed to air.

[0183] Predictive Example P1: In vivo study of the effects of high-dose dry powder chelating agents on infection, inflammation, and oxidative stress

[0184] CF patients requiring tobramycin dry powder treatment will be randomly assigned to four groups, receiving 112 mg of dry powder twice daily for 28 days. Additionally, Group 1 (patients >18 years old) will receive escalating doses of dry powder CaEDTA (37.5 mg twice daily for 1 week; 75 mg twice daily for 2 weeks; 150 mg twice daily for 1 week). Group 2 (patients >18 years old) will receive CaEDTA (37.5 mg twice daily for 1 week; 75 mg twice daily for 2 weeks; 75 mg four times daily for 1 week). Group 3 (patients 12–18 years old) will receive CaEDTA (37.5 mg twice daily for 1 week; 75 mg twice daily for 2 weeks; 150 mg twice daily for 1 week). Finally, the control group will receive tobramycin alone for 28 days.

[0185] Sputum samples were collected weekly to assess biomarkers of infection and inflammation. Bacteria were monitored by sputum colony counts. As previously described, levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were determined using gelatin zymography and immunoassay, respectively, as measures of structural damage (Gaggar et al., Eur Respir J. 2011 38(3): 721–727; Garratt et al., Eur Respir J. 2015 46(2):384–94). As previously described, iron content in sputum was quantified by ICP-MS (Hunter et al., MBio. 2013 4(4):1–8). The amount of iron-binding protein was assessed using immunoassays. As previously described, myeloperoxidase activity was also measured as a measure of neutrophilic inflammation (Gaggar et al., Eur Respir J. 2011 38(3): 721–727). 3-Chlorotyrosine was measured as a biomarker for the strong oxidant hypochlorous acid. Levels were measured using stable isotope dilution gas chromatography and mass spectrometry (Gaggar et al., Eur Respir J. 2011 38(3): 721–727). Protein carbonyl compounds were measured as indicators of reactive oxygen species (ROS) using commercial immunoassay kits (Gaggar et al., Eur Respir J. 2011 38(3): 721–727). Oxidative stress was assessed by measuring glutathione (GSSG and GSH) using immunoassays, as previously described (Kettle et al., Eur Respir J. 2014 44(1):122-9). Gene expression of markers of inflammation and oxidative stress (e.g., IL-8, IL-6, TNFα) was also monitored using Nanostring, and proteins were measured by ELISA. Oxidative stress was also measured by metabolites, such as malondialdehyde (colorimetric assay) or 8-isoprostaglandin (ELISA). Iron will be measured by elemental analysis using laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS).

[0186] It can be expected that, compared with the placebo group, this experiment will show a reduction in inflammatory markers and a decrease in iron levels in the EDTA group. It can be further expected that this experiment will show changes in the balance between MMPs and TIMPs, especially between MMP-9 and TIMP-1, which are associated with the progression of bronchiectasis.

[0187] Further, the experiment is expected to show that subjects treated with EDTA had increased FEV1 and reduced bacterial load in their sputum compared to control patients.

[0188] Predictive Example P2: In vitro study of the effects of high-dose chelating agents on inflammation, lung injury, and oxidative stress.

[0189] Lung epithelial cells were grown in tissue cultures and induced to produce inflammation by exposure to Fe(II) or excess oxygen. Cells were treated with CaEDTA (0, 1, 5, 10, 25, 50 mM) for 30 min, 1, 3, 24, and 48 h.

[0190] Immunoassays are used to monitor changes in inflammatory markers such as IL-6, IL-8, TNF-α, and neutrophil elastase. Oxidative stress and toxicity are measured by assessing decreased glutathione (GSH) levels and apoptosis using TUNEL assays, both employing commercially available kits such as ThermoFisher Scientific's Glutathione Fluorescence Detection Kit and BioVision Inc.'s TUNEL DNA Gragmentation Analysis Kit.

[0191] It is expected that this experiment will show a concentration-dependent reduction in inflammatory markers in EDTA-treated cells compared to the control; a decrease in GSH indicating a reduction in reactive oxygen species in EDTA-treated cells compared to the control; and a reduction in apoptosis in EDTA-treated cells compared to the control, as measured by TUNEL assay.

[0192] Predictive Example P3: In vivo study of the effects of high-dose nebulized chelating agents on inflammation, lung injury, and oxidative stress.

[0193] CF subjects aged ≥6 years with worsening condition admitted to the hospital were randomly assigned to receive either nebulized EDTA or saline (placebo) in addition to routine intravenous antibiotics and nebulized tobramycin. EDTA was administered as a nebulizing solution along with tobramycin, consisting of 4 ml of 0.9% saline containing 50 mM CaNa2EDTA and 111 mM Tris at pH 7.1.

[0194] Following randomization, subjects were hospitalized for two weeks, receiving treatment four times daily (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). Patients were then discharged and continued treatment twice daily for four weeks. Patients were monitored for another four weeks, bringing the total study time to 10 weeks. Sputum was collected by induction with nebulized 3% hypertonic saline at a rate of 8–10 L / min for ≥5 minutes. Samples were collected before treatment and at weeks 2, 6, and 10, processed according to the relevant protocol, and stored at -80°C.

[0195] Inflammatory marker expression

[0196] The phlegm coughed up is stored in (In China, Qiagen is used) (or similar extraction kits to extract total RNA, convert it to cDNA, and use qPCR to monitor inflammatory markers, as described by Sivaneson et al. (MolMicrobiol 79, 1353-1366), and quantify them relative to known housekeeping genes, such as actin and / or GAPDH.)

[0197] It is expected that this experiment will show that, compared with the placebo group, the expression of inflammatory marker genes in the EDTA group is reduced on average.

[0198] Cell damage, free iron and oxidative stress

[0199] Coughed sputum was frozen directly without processing, and inflammatory markers were detected as described above. As previously mentioned, the levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were measured using gelatin zymography and immunoassay, respectively, as a measure of structural damage (Gaggar et al., Eur Respir J. 2011 38(3): 721–727; Garratt et al., Eur Respir J. 2015 46(2):384-94). As previously mentioned, the iron content in sputum was quantified by ICP-MS (Hunter et al., MBio. 2013 4(4):1-8). The amount of iron-binding proteins was assessed using immunoassay. As previously mentioned, oxidative stress was assessed by measuring glutathione (GSSG and GSH) using immunoassay (Kettle et al., Eur Respir J. 2014 44(1):122-9).

[0200] It can be expected that this experiment will show a reduction in inflammatory markers in the EDTA group compared to the placebo group. It can be further expected that this experiment will show changes in the balance between MMPs and TIMPs, especially between MMP-9 and TIMP-1, which are associated with the progression of bronchiectasis.

[0201] Predictive Example P4: In vivo study of the effects of high-dose chelating agents on inflammation, lung injury, and oxidative stress.

[0202] A single-center, randomized, double-blind, crossover study was conducted on subjects with cystic fibrosis. Subjects were randomized to receive either inhaled CaEDTA or saline (placebo) for two weeks. This was followed by a flushing phase, and then another two weeks of treatment (EDTA or placebo).

[0203] Iron levels, inflammatory markers, MMP / TIMP, and FEV1 were monitored as described above. Myeloperoxidase activity, as a measure of neutrophilic inflammation, was also determined, as previously mentioned (Gaggar et al., Eur Respir J. 2011 38(3):721–727). 3-Chlorotyrosine was used as a biomarker for hypochlorous acid, a strong oxidant. Levels were measured using stable isotope dilution gas chromatography and mass spectrometry (Gaggar et al., Eur Respir J. 2011 38(3): 721–727). Protein carbonyl compounds were measured as an indicator of reactive oxygen species (ROS) using commercial immunoassay kits (Gaggar et al., Eur Respir J. 2011 38(3): 721–727).

[0204] It can be expected that, compared with placebo treatment, subjects treated with EDTA will have lower levels of iron and inflammatory markers, altered MMP / TIMP balance, and elevated mean FEV1. Further, this experiment can be expected to show reduced myeloperoxidase activity and lower average levels of chlorotyrosine and carbonyl groups.

[0205] Clinical data showed efficacy with 300 mg / day for two weeks. The same study also indicated that 150 mg / day (75 mg twice daily) was beneficial for lung function and infection control. Figure 2 To reduce bacterial count; Figure 3A (For improved lung function). As those skilled in the art will understand, depending on the significance of the improvement (average FEV1 of 16%), it is likely that a lower dose, i.e., 75 mg / day (37.5 mg twice daily), is effective, as envisioned in predictive example P1.

[0206] Figure 4 Studies have shown that a single dose of 75 mg CaEDTA can result in up to 1.34 mM of EDTA within the mucus plug after 30 minutes. It is well known that the penetration of drugs such as tobramycin into CF sputum is significantly impaired (Kuhn, RJ (2001). Formulation of aerosolized therapeutics. Chest 120, 94S-98S), therefore, the concentration of EDTA in the airway surface fluid is very likely to be much higher than in the center. Therefore, it is reasonable to expect that a daily dose of 37.5 mg (4 times lower than the lower doses that have clinical benefit) will demonstrate efficacy in a complete study. This is especially true in the case of younger patients who receive higher doses per unit of body weight and generally show greater responses in FEV1 ( Figure 3B ).

[0207] Based on the foregoing guidance relating to the disclosed invention, various variations and modifications of the above-described modes for implementing various embodiments of the invention will be apparent to those skilled in the art without departing from the basic inventive concept. The above embodiments of the invention are merely exemplary and should not be construed as limiting in any way, and all such variations and modifications should be considered within the scope of the invention, the nature of which should be determined from the foregoing description.

[0208] The work of this invention was supported by an award from the Cystic Fibrosis Foundation Therapeutics.

Claims

1. A method for treating or preventing lung inflammation by administering a high concentration of an inhaled chelating agent.

2. The method of claim 1, wherein the concentration of the chelating agent is greater than 37.5 mg / dose.

3. The method of claim 1 or 2, wherein the concentration of the chelating agent is greater than 50 mg / dose.

4. The method of any one of claims 1 to 3, wherein the chelating agent is provided in a dosage form comprising 37.5 mg / dose to 300 mg / dose.

5. The method of any one of claims 1 to 4, wherein the chelating agent is provided in a dosage form comprising 50 mg / dose to 300 mg / dose.

6. The method of any of the preceding claims, wherein the chelating agent is provided at a total dose of up to about 1,200 mg / day.

7. The method of any of the preceding claims, wherein the chelating agent is CaEDTA.

8. The method of any of the preceding claims, wherein treatment or prevention of inflammation results in an increase in FEV.

9. The method of any of the preceding claims, wherein the treatment or prevention of inflammation is associated with a reduction in MMP activity.

10. The method of any of the preceding claims, wherein the treatment or prevention of inflammation is associated with a reduction in the production of hydroxyl radicals.

11. The method of any of the preceding claims, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (TRIS).

12. An inhalable formulation containing a high concentration of a chelating agent.

13. The formulation of claim 12, wherein the concentration of the chelating agent is greater than 37.5 mg / dose.

14. The formulation of claim 12, wherein the concentration of the chelating agent is greater than 50 mg / dose.

15. The formulation of claim 13 or 14, wherein the concentration of the chelating agent is from 37.5 mg / dose to 300 mg / dose.

16. The formulation of claim 13 or 14, wherein the concentration of the chelating agent is from 50 mg / dose to 300 mg / dose.

17. The formulation of any one of claims 12 to 16, wherein the chelating agent is provided at a total dose of up to about 1,200 mg / day.

18. The formulation of any one of claims 12 to 17, wherein the chelating agent is CaEDTA.

19. The formulation of any one of claims 12 to 18, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (TRIS).

20. A kit for treating or preventing lung inflammation, comprising (i) an inhalable formulation containing a high concentration of a chelating agent; and (ii) instructions for use.

21. Use of high-concentration chelating agents in the preparation of inhalable formulations for the treatment or prevention of lung inflammation.

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