Method for reducing lung inflammation

Abstract

To provide a method of treating or preventing inflammation in the lung.SOLUTION: The present invention provides a method comprising administering a high concentration of an inhalable chelating agent. Preferably, the concentration of the chelating agent is greater than 37.5 mg / dose. More preferably, the chelating agent is CaEDTA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides a method for treating or preventing inflammation in the lungs by administering high concentrations of an inhalable chelating agent. In one embodiment of the present invention, the present invention relates to a method for preventing inflammation in the lungs, and a formulation for use in the method. The disease is associated with or caused by cystic fibrosis. [Background technology]

[0002] Cystic fibrosis is characterized by susceptibility to infections that cause inflammation and lung damage. However, inflammation and lung injury can also occur in the absence of bacterial infection (Sly et al., Am J. Respir Crit Care Med. 2009, 180(2):146~ 52 pages).

[0003] Inflammation is the body's response to insults, including infection, trauma, and hypersensitivity. The inflammatory response is complex and involves multiple mechanisms for defense against pathogens and tissue repair. Inflammation in the body is usually caused by pathogens or in response to toxins, pollutants, irritants, and allergens. Caused by exposure.

[0004] During inflammation, many types of inflammatory cells are activated. Each of them produces cytokines and signaling They release substances that modify the activity of other inflammatory cells. The organization of these cells and molecules This leads to the progression of inflammation. Clinically, acute inflammation can lead to pneumonia and acute respiratory distress syndrome (ARDS). ), whereas chronic inflammation is seen in diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. It appears in diseases such as COPD. Since the lungs are an important organ for gas exchange, Excessive inflammation can be life-threatening. A delicate balance between inflammation and anti-inflammation is essential for lung function. It is essential for homeostasis.

[0005] Immunity includes innate and adaptive systems. Innate immunity is non-specific and causes a rapid response including inflammation when facing pathogen invasion. Adaptive immunity is antigen-specific. This adaptive immunity first detects a specific antigen and then recruits inflammatory cells to target that specific antigen. The innate and adaptive systems share components and act cooperatively for defense against pathogens.

[0006] The airway epithelium secretes various substances such as mucin, defensin, lysozyme, lactoferrin, and nitric oxide, which non-specifically protect the respiratory tract from microbial attack. Epithelial cells also produce several mediators such as reactive oxygen radicals, cytokines (TNF-α, IL-1β, granulocyte / macrophage colony-stimulating factor [GM -CSF]), and platelet-activating factor, and gather inflammatory cells on the inflammation site. Cytokines stimulate the release of arachidonic acid from membrane lipids, leading to the production of eicosanoids, which further stimulate mucus secretion by goblet cells and tissue inflammation.

[0007] Surfactants are located on the alveolar surface and contain four surfactant proteins (SP A - D). It is important for reducing the tension on the lung surface that these proteins play a decisive role in surfactant absorption onto the alveolar surface. SP-A and SP-D also participate in host defense. They bind to bacterial surface molecules, modulate leukocyte activity, and result in pathogen opsonization.

[0008] IgA secreted by plasma cells forms an additional epithelial protective barrier, which prevents the attachment of microorganisms to the epithelial surface. IgA also binds to pathogenic bacteria, causing phagocytosis and antibody-dependent cell-mediated cytotoxicity. Immunoglobulin E (IgE) induces immediate-type hypersensitivity in the respiratory organs. IgE produces a strong reaction by binding to IgE receptors on the surface of mast cells, basophils, eosinophils, and B lymphocytes. Repeated exposure to the same antigen induces degranulation and the release of inflammatory mediators including histamine, prostaglandins, leukotrienes, and

[0009] tryptase. These increase vascular permeability, bronchoconstriction, and inflammatory cell infiltration. U.S. Patent Application Publication No. 2016 / 0263151 teaches treating bacterial infections by using an inhaled antibiotic in combination with an acidified nitrite and an iron chelating agent. The iron chelating agent present in the

[0010] formulation of U.S. Patent Application Publication No. 2016 / 0263151 acts to provide a synergistic effect when combined with acidified nitrite, enhancing the ability of the antibiotic to act.

[0011] There is a need for a method of treating or preventing inflammation in the lung, or at least supplementing or providing an alternative to known treatment methods

[0012] The present invention seeks to provide an improved method or alternative for treating or preventing inflammation in the lungs by administering a high-concentration chelating agent for inhalation.

[0013] The following discussion of the background art is intended only to facilitate an understanding of the present invention. This discussion does not approve or authorize that any of the materials mentioned were, or were part of, common general knowledge at the filing date of the application. SUMMARY OF THE INVENTION

[0014] The present invention provides a method for treating or preventing inflammation in the lungs by administering a high-concentration chelating agent for inhalation.

[0015] Preferably, the high-concentration chelating agent for inhalation exceeds 37.5 mg / dose.

[0016] Preferably, the high-concentration chelating agent for inhalation exceeds 50 mg / dose.

[0017] In one form of the present invention, the high-concentration chelating agent is provided by a dosage form containing at least 50 mg / dose, or between 5 0 mg / dose and 300 mg / dose. The chelating agent may be administered 1 to 4 times daily at a total dosage of up to about 1,200 mg / day, preferably at least 150 mg / day.

[0018] In one form of the present invention, the high-concentration chelating agent is provided by a dosage form containing at least 37.5 mg / dose or between 37.5 mg / dose and 300 mg / dose. The chelating agent may be administered 1 to 4 times daily at a total dosage of up to about 1,200 mg / day, preferably at least 150 mg / day.

[0019] Preferably, a chelating agent is administered at a dose of 37.5 mg / day to 1,200 mg / day. Preferably, at least 50 mg / day of the chelating agent is administered. The chelating agent can be administered at a total daily dose of up to about 1,200 mg / day between 1 and 4 times a day.

[0020] Preferably, each dose of the chelating agent is administered over a period of 8 hours or less. Preferably, the chelating agent and / or the antibiotic is administered over a period of 1 hour or less.

[0021] Preferably, the chelating agent is CaEDTA.

[0022] The present invention provides a method for treating or preventing inflammation in the lungs by administering a high concentration of an inhalable chelating agent, wherein the treatment or prevention of the inflammation results in an increase in forced expiratory volume (FEV).

[0023] The present invention also provides a method for treating or preventing inflammation in the lungs by administering a high concentration of an inhalable chelating agent, wherein the treatment or prevention of the inflammation is accompanied by a reduction in matrix metalloprotease (

[0024] MMP) activity. The present invention provides a method for treating or preventing inflammation in the lungs by administering a high concentration of an inhalable chelating agent,

[0025] wherein the treatment or prevention of the inflammation is accompanied by a reduction in the production of

[0026] hydroxyl radicals. It is possible to deliver the topical agent as a single dose.

[0027] The present invention provides a kit for treating or preventing inflammation in the lungs, comprising (i) an inhalation preparation containing a high concentration of a chelating agent and (ii) instructions for use. Instructions for use.

[0028] The present invention provides a kit for treating or preventing inflammation in the lungs, comprising (i) an inhalation preparation capable of delivering a high concentration of an inhalation chelating agent as a single dose and (ii) instructions for use. Instructions for use. Instructions for use.

[0029] Use of a high concentration of a chelating agent in the manufacture of an inhalation preparation for treating or preventing inflammation in the lungs. Instructions for use.

[0030] Use of an inhalation chelating agent in the manufacture of a medicament for delivering a high concentration of an inhalation chelating agent as a single dose for treating or preventing inflammation in the lungs. Instructions for use.

[0031] Further features of the present invention are more fully described in the following description of some of its non-limiting embodiments. This description is included for the sole purpose of illustrating the present invention. This description should not be understood as limiting the broad general outline, disclosure, or description of the present invention as presented above. Reference will be made to the accompanying drawings for explanation. Instructions for use. Instructions for use. Instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

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Mode for Carrying Out the Invention

[0033] [Method of Treatment or Prevention] The present invention provides a method for treating (treat ) or preventing inflammation in the lungs by administering a high concentration of an inhaled chelating agent.

[0034] Preferably, the high concentration of the inhaled chelating agent exceeds 37.5 mg / dose.

[0035] Preferably, the high concentration of the inhaled chelating agent exceeds 50 mg / dose.

[0036] It has previously been shown that inhaled EDTA alone does not treat bacterial infections (Bro wn et al. (Am J Dis Child. 1985, 139(8):836 - 9 ); Hassett (Front Microbiol. 2016, 7:291) ). Brown et al. (1985) treated CF children chronically infected with Pseudomonas aeruginosa with aerosolized sodium EDTA for 3 months, and no changes in lung function were observed. EDTA causes concentration-dependent bronchoconstriction (Beasley et al. (Br Med J (Clin Res E d). 1987, 294(6581): 1197-8)), E DTA has been reported by others to have no effect on FEV1 (Asmus et al., (J Allergy Clin Immunol. 2001, 107(1): 68- 72)). Thus, there is no basis for believing that chelating agents have any positive effect on subjects having, for example, 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 found that inhaled chelating agents can treat or prevent lung inflammation .

[0037] CF lung environment is generally believed to be acidic. However, it has recently been shown that CF lungs have the same p H as normal lungs (Schultz et al., "Airway sur face liquid pH in children with cystic fi brosis" Nature Communications 2017, 8(1 ): 1409). Thus, current techniques using acidified nitrite, for example, those discussed in US Patent Application Publication No. 2016 / 0263151, etc., the formulation does not remain in an acidified state and immediately returns to the pH 7.4 of normal lungs, so it is unlikely to work clinically in CF .

[0038] It has been found that the acidity of CF lungs is normal, but the iron levels are significantly different from those of normal lungs . Stites et al., (Am J Respir Crit Care Med. 19 1999, Vol. 160 (No. 3): pp. 796-80, 2003. Iron levels in the lungs of CF patients are The iron levels in the lungs of smokers and adults were significantly elevated compared to healthy individuals. Most of it is in the ferrous form, Fe(II), and has also been shown to significantly correlate with disease severity. (Hunter et al., MBio. 2013, 4(4):1-8). is a frequen- tial factor that produces highly reactive oxygen radicals that can severely damage tissue and DNA. It can participate in the phenanthrene reaction (Jomova et al., Toxicology., 20 11, Volume 283 (Issue 2-3): Pages 65-87; MacNee, Eur J Pharm acol., 2001, Volume 429 (Nos. 1-3): Pages 195-207).

[0039] Without wishing to be bound by theory, the method of the present invention is based on the following: (i) chelation of zinc (ii) inactivation of matrix metalloproteinases (MMPs) by iron chelation (iii) reducing the production of reactive oxygen species (ROS); and / or (iv) reducing the amount of iron and zinc, Reducing the amount of bacteria in the lungs by depriving bacteria of key ions reduces inflammation Actions within individual lungs are thought to be one of the theorized ways that inflammation may be reduced. or any combination.

[0040] Inhalation is a localized method of administration and therefore requires minimal effort to reach the targeted area, i.e., the lungs. and may be more effective at achieving high and localized concentrations of inhaled chelating agents. Inhalation avoids the unwanted side effects of systemic exposure to the active substance and is reduces the risk of developing resistance.

[0041] The present invention further provides a method for treating or preventing inflammation in the lung by administering a high-concentration inhalable chelating agent, wherein the treatment or prevention of inflammation results in an increase in FEV.

[0042] The present invention further provides a method for treating or preventing inflammation in the lung by administering a high-concentration inhalable chelating agent, wherein the treatment or prevention of inflammation is accompanied by a decrease in MMP activity. It is known that matrix metalloproteinase (MMP) causes lung injury (Garratt et al., Eur Respir J. 2015, 46(2): 384-394) and that MMP activity is dependent on Zn (Hazra et al., Molecular Vision, 2012; 18: 1701-1711). However, past attempts to target MMP in the lung have been unsuccessful. The present invention uses an inhalable chelating agent that chelates zinc in the lung, thus reducing MMP-induced lung injury and treating or preventing inflammation. 2+

[0043] The present invention further provides a method for treating or preventing inflammation in the lung by administering a high-concentration inhalable chelating agent, wherein the treatment or prevention of inflammation is accompanied by a decrease in the production of hydroxyl radicals. Since Fe catalyzes the formation of hydroxyl radicals, iron is a major factor in lung injury (Stites et al., (Am J Respir Crit Care Med., 1999, 160(3): 796-80). However, antioxidant ​​​​​​​​​​​​​​Reduce Zika virus-induced lung injury and treat or prevent inflammation.

[0044] The present invention provides a method for treating or preventing infectious diseases in the lungs by administering a high-concentration inhalable chelating agent, wherein the treatment or prevention of inflammation is achieved by the presence of the chelating agent through the removal or reduction of biofilms produced by bacteria in the lungs. The reduction of biofilms enables an increase in the removal of bacteria and biofilms by coughing and expectoration.

[0045] The present invention provides a method for treating or preventing inflammation in the lungs by administering a high-concentration inhalable chelating agent, wherein the treatment or prevention of inflammation is achieved by stimulating local inflammation, causing local tissue damage, and removing or reducing protease enzymes produced by bacteria that can neutralize antibiotic activity. These enzymes are mainly cation-dependent, and it is expected that removing cations from the environment will inactivate these enzymes.

[0046] Preferably, the chelating agent is an iron chelating agent or a zinc chelating agent. More preferably, the chelating agent is a chelating agent for both iron and zinc (iron / zinc chelating agent). Alternatively, the chelating agent may be a mixture of two or more chelating agents, such as an iron chelating agent and a zinc chelating agent, or an iron / zinc chelating agent and a zinc chelating agent, or a mixture of an iron chelating agent and an iron / zinc chelating agent.

[0047] The chelating agent is preferably citric acid, phosphate, disodium salt, trisodium salt, and tetrasodium salt of ethylenediaminetetraacetic acid (EDTA ), calcium salt of EDTA Magnesium salt, ethylene glycol-bis-(β-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’ -(ethylenediimino)-dibutyric acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylenetetramine dihydrochloride (dihydrochloride)(TRIEN), diethylenetriaminepentaacetic acid (D PTA), triethylenetetraminehexaacetic acid (TTG), deferoxamine (DFO), de ferasirox (DSX), dimercaprol, zinc citrate, penicillamine (peni cilamine), succimer, edetronate, sodium hexametaphosphate, ede tate calcium disodium, D-penicillamine, polyphenol, gallol, catecho l, dimercaprol, tetrathiomolybdate, lactoferrin, and curioquinol l and combinations thereof.

[0048] Preferably, the chelating agent is a pharmaceutically acceptable chelating agent.

[0049] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA). In another embodiment the chelating agent is deferoxamine (DFO). In another embodiment, the chelating agent is deferasirox (DSX).

[0050] Preferably, the chelating agent has approximately the same iron affinity and / or approximately the same zinc affinity as EDTA. The formation constant for EDTA at 25 °C and 0.1 M with respect to or the stability constant (log K1) is Fe 2+ 14.3 for Fe 3+ To For iron it is 25.1, and for zinc it is 16.5.

[0051] In one embodiment, the chelating agent is a calcium salt of a chelating agent. The inhibitor is CaEDTA.

[0052] In one embodiment, the chelating agent is administered at a concentration of between 37.5 mg / dose and 300 mg / dose for 50 min. Between 75mg / dose and 300mg / dose, between 75mg / dose and 200mg / dose, about 75mg Between 100 mg / dose and 37.5 mg / dose, and between 200 mg / dose and 5 Between 0 mg / dose and 200 mg / dose; preferably about 37.5 mg / dose, 50 mg / dose dose, 75 mg / dose, 100 mg / dose, 200 mg / dose, or 300 mg / dose The chelating agent is provided in an inhalation dosage form having at least 37.5 mg / dose. The chelating agent is preferably provided in an inhalation dosage form having at least 50 mg / dose. The compound is preferably provided in an inhalable dosage form containing the compound.

[0053] The total amount of chelating agent inhaled per day is preferably about 37.5 mg / day to 1,20 Between about 50mg / day and 1,200mg / day, between about 100mg / day and 1,0 Between about 300mg / day and 900mg / day, Between about 400mg / day and 800mg / day between about 150 mg / day, 300 mg / day, 500 mg / day or 6 mg / day; 00mg / day.

[0054] The total amount of drug inhaled per day is preferably about 0.1 mg chelating agent / kg (body weight) ) to between about 0.5 mg chelating agent / kg (body weight) and 15 mg chelating agent / kg (body weight), between about 1.0 mg chelating agent / kg (body weight) and 10 mg chelating agent / kg (body weight), between about 1.0 mg chelating agent / kg (body weight) and 5 m g chelating agent / kg (body weight); between about 1.0 mg chelating agent / kg (body weight) and 3.5 mg chelating agent / kg (body weight); preferably about 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), 15 mg chelating agent / kg (body weight).

[0055] When about 75 mg of a chelating agent, such as CaEDTA, is inhaled, it is determined that about 0.4 mM to 1.34 mM of the chelating agent can be detected in the sputum from the lungs after 5 minutes.

[0056] The inhaled chelating agent is preferably delivered over a period of 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 or less. When the administration is by delivery of a dry powder, the inhaled chelating agent can be delivered over a period of seconds, for example, 1 second per "puff" of an aerosol device or a dry powder inhaler, and one or more puffs are administered at each time point.

[0057] Preferably, the inhaled chelating agent is administered for at least 28 consecutive days. The inhaled chelating agent is delivered for 2 days or more, 3 days, 4 days, 5 days, 6 days, or 7 days It can be done. The inhaled chelating agent can be delivered for 2 to 28 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0058] Some subjects can benefit from a period of "loading" the subject with an antibiotic and / or chelating agent with higher doses or more frequent administrations over a period of several days or weeks, followed by lower doses or maintenance doses of administration. Thus, the present invention provides

[0059] · delivering a total amount of inhaled chelating agent between 37.5 mg / day and 1,200 mg / day, · administered at least once a day to up to six times a day, preferably up to four times a day, · administered over a period of 8 hours or less.

[0060] Preferably, the present invention provides · delivering a total amount of inhaled chelating agent between 37.5 mg / day and 1,200 mg / day, · administered once or twice a day, · administered over a period of 1 hour or less per administration, · containing CaEDTA as the chelating agent.

[0061] The preferred amount of any chelating agent can be calculated by comparing the chelating ability of the agent to that of CaEDTA and then multiplying that number by the dosage ranges given above. The result should provide a level of chelation approximately equal to the preferred level of chelation provided by the preferred amount of EDTA.

[0062] Preferably, the infectious disease is cystic fibrosis (CF); asthma; chronic obstructive pulmonary disease (COPD); pulmonary hypertension; lung cancer; pulmonary fibrosis; bronchiectasis; acute respiratory distress syndrome; tuberculosis; nontuberculous mycobacteria​​​​ Nontuberculous mycobacteria (NTM) pulmonary infection; including, but not limited to, ventilator-associated pneumonia, community-acquired infectious pneumonia, bronchopneumonia, lobar pneumonia; infections caused by bacteria such as species of Pseudomonas, Streptococcus pneumoniae, Chlamydia, Mycoplasma pneumonia, species of Staphylococcus, species of Klebsiella spp, Escherichia coli, Stenotrophomonas spp, and fungi including Aspergillus, Scedosporium, and species of Candida; preventive treatment or prophylaxis against conditions in which the infection can occur, for example, in intubated or ventilated patients; infections in lung transplant patients; bronchitis; whooping cough (pertussis); inner ear infections; streptococcal throat infections; anthrax; tularemia; or sinusitis, caused by, causing, or associated with such pulmonary conditions. Preferably, the formulation is administered to the subject in need thereof about once daily to about six times daily, more preferably about four times daily. Alternatively, the formulation can be administered to the subject in need thereof via continuous inhalation via a

[0063] nebulizer. The nebulized formulation can be delivered over 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour, and each of these deliveries (apart from 24 and 12 hours) can be repeated several times within a

[0064] 24-hour period.

[0065] ​​​The subject will typically receive a chelating agent at about 0.01 - 15 mg / kg / day, ±20% or ±10% and will be administered this way, either by spraying or by at least one, preferably several, "puffs" from an aerosol device. For example, the subject can receive a single dose between 0.1 mg / kg chelating agent and 15 mg / kg per day, or multiple doses.

[0066] The total daily dose is preferably administered at least once per day, but can be divided into two or more administrations per day. Some subjects can benefit from a period of "loading" the subject with a chelating agent with higher doses or more frequent administrations over several days or weeks, followed by lower doses or maintenance doses for a period of time. Since cystic fibrosis, COPD, etc. are usually chronic conditions, subjects are expected to receive such treatment over a long period of time.

[0067] Regardless of the form of the pharmaceutical preparation, it is preferred to create inhaled droplets or particles in the range of about 0.1 μm - 12 μm, or about 0.25 μm - 6 μm, preferably 1 μm - 6 μm, more preferably about 2 μm - 4 μm. Alternatively, the particles can be 0.1 μm - 1.0 μm, 0.2 μ m - 0.9 μm, 0.3 μm - 0.8 μm, 0.4 μm - 0.7 μm, or 0.5 μm By creating inhaled particles with a relatively narrow range of sizes, it is possible to further increase the efficiency of the drug delivery system and improve the reproducibility of dosing. Thus, not only do the particles have a size in the range of 0.1 μm - 12 μm or 2 μm - 6 μm or about 3 - 4 μ m, but also by having an average particle size within a narrow range, the subject ​ 80% or more of the particles delivered thereto have a particle diameter within ±20% of the average particle diameter, preferably within ±10% of the average particle diameter, more preferably within ±5% of the average particle diameter. .

[0068] "Particle diameter" is a concept introduced for comparing the dimensions of solid particles and liquid particles (droplets). For droplets and aerosol agents, terms such as "aerodynamic diameter" and "mass median aerodynamic particle diameter (MMAD)" are used. Definitions are given below.

[0069] The "aerodynamic diameter" is the diameter of a unit-density sphere having the same terminal settling velocity as the particle in question. It is used to predict where such particles will deposit within the respiratory tract.

[0070] The "mass median aerodynamic particle diameter" is the geometric mean aerodynamic diameter. 50 weight percent of the particles are smaller than the MMAD and 50 weight percent of the particles are larger.

[0071] During the experiment for measuring the particle diameter, the suspension contains an infinite number of particles of different sizes that are in motion. When a particle size measuring instrument analyzes these particles, the particle size measuring instrument forms a particle distribution curve, and this curve encompasses the entire particle diameter range from the smallest particle that can be 1 nm to the largest particle that can be 100 μm. In the particle diameter distribution curve, the cumulative frequency is calculated for the particles. D refers to a specific particle diameter such that 10% of the number of particles in the suspension have a diameter smaller than or equal to this specific particle diameter. 10

[0072] D 50 :D 10 Similarly, D 50 is the cut-off diameter for 50% of the particle population in the formulation​​ is the diameter such that 50% of the particles in the suspension have a diameter smaller than or equal to this specific particle diameter refers to this specific particle diameter.

[0073] D 90 :D 90 is the cut-off diameter for 90% of the particle population in the formulation, such that 90% of the particles in the suspension have a diameter smaller than or equal to this specific particle diameter refers to this specific particle diameter.

[0074] The term "respiratory tract" is taken to mean the system of cells and organs that function in respiration, and in particular, the organs, tissues and cells of the respiratory tract are the lungs, nose, nasal passages, paranasal sinuses, upper pharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pulmonary alveoli (type 1 and type 2), ciliated mucosal epithelium, mucosal epithelium, squamous epithelial cells, mast cells, goblet cells, and intraepithelial dendritic cells.

[0075] In one form of the invention, a method of treating or preventing inflammation in the lungs of a subject comprises administering an inhalable chelating agent in a therapeutically effective or prophylactically effective concentration in a form of one or more administrations of at least 37.5 mg / dose, wherein the administration or each administration of the chelating agent is administered over a period of 8 hours or less.

[0076] In one form of the invention, a method of treating inflammation in the lungs of a subject comprises administering an inhalable chelating agent in a therapeutically effective concentration in a form of one or more administrations of at least 37.5 mg / dose wherein the administration or each administration of the chelating agent is administered over a period of 8 hours or less.

[0077] In one form of the invention, an inhaled chelating agent at a prophylactically effective concentration is administered in the form of one or more administrations of at least 37 .5 mg / dose to prevent inflammation in the lungs of a subject, where the administration or each administration of the chelating agent occurs over a period of 8 hours or less .

[0078] In one form of the invention, a method of treating or preventing inflammation in the lungs of a subject includes treating or preventing inflammation in the lungs of a subject in need of such treatment.

[0079] As used herein, the term "therapeutically effective amount" means an amount of a formulation that, when administered according to a desired dosing regimen, achieves at least partially the desired therapeutic effect, or delays the onset of, or inhibits the progression of, or partially or completely stops the onset or progression of inflammation such that the desired therapeutic effect is achieved at least in part, or the onset of inflammation is delayed, or the progression is inhibited, or the onset or progression is partially or completely stopped .

[0080] As used herein, the term "prophylactically effective amount" means an amount of a formulation that, when administered according to a desired dosing regimen, at least partially prevents inflammation or delays its onset .

[0081] As used herein, "treating" or "treatment" refers to inhibiting a disease or condition, i.e., stopping or reducing at least one of its onset or its clinical or asymptomatic symptoms . "Treating" or "treatment" further refers to alleviating a disease or condition, i.e., causing regression of at least one of the disease or condition or its clinical or asymptomatic symptoms . The benefits to the subject to be treated . . ​​​is statistically significant or at least perceptible to the subject and / or the physician In the context of treating inflammation, the term "treatment" includes leukocyte infiltration (including macrophages, polymorphonuclear neutrophils, lymphocytes and other immune cells); immunoglobulins; inflammation-induced cytokines and chemokines and their receptors; invasive mediators such as ROS and proteolytic enzymes; the abundance and activity of MMPs; markers of oxidative stress; and reducing or eliminating one or more of bronchial hypersensitivity and exacerbation. The term "treatment" further includes one or more of an increase in anti-inflammatory cytokines and an increase in lung function (FEV1).

[0082] Based on the above, it is understood by those skilled in the art that a single subject can be treated using multiple different treatment and administration means. Thus, a subject who has already received administration of such a drug, such as intravenous ciprofloxacin or an antibiotic, can benefit from inhalation of the agent of the present invention. Some subjects can receive by inhalation only the administration of a high-concentration chelating agent which is a formulation of the present invention. Such subjects may have symptoms of cystic fibrosis, may be diagnosed with a lung infection, or may have a medical condition with symptoms that can benefit from the administration of a high-concentration chelating agent to the subject. The formulation of the present invention may also be used for diagnosis. In one embodiment, for example, a subject can receive administration of the formulation of the present invention as part of a procedure for diagnosing a lung infection, and one or more of the subject's symptoms improve in response to the formulation.

[0083] [Formulation] ​​​​​​​​​​​​​​​The present invention provides an inhalation preparation containing a high-concentration chelating agent.

[0084] The inhalation preparation may be in the form of a dry powder for inhalation or a spray for inhalation. Preferably the preparation is adapted for inhalation for treating or preventing inflammation in the lungs.

[0085] In one embodiment, the chelating agent is a calcium salt of the chelating agent. Preferably, the chelating agent is CaEDTA.

[0086] Preferably, the high-concentration inhalation chelating agent exceeds 37.5 mg / dose. Preferably the high-concentration inhalation chelating agent exceeds 50 mg / dose. Preferably, the high-concentration chelating agent is between 37.5 mg / dose and 300 mg / dose, between 50 mg / dose and 300 mg / dose, between about 75 mg / dose and 200 mg / dose, between about 75 mg / dose and 100 mg / dose, between about 50 mg / dose and 200 mg / dose; preferably preparations containing about 50 mg / dose, 75 mg / dose, 100 mg / dose, 200 mg / dose or 300 mg / dose are provided. The chelating agent is preferably provided in an inhalation dosage form containing at least 37.5 mg / dose and preferably provided in an inhalation dosage form containing at least 50 mg / dose. The chelating agent is preferably provided in an inhalation dosage form containing at least 50 mg / dose.

[0087] The total amount of the chelating agent inhaled per day is preferably between about 37.5 mg / day and 1,200 mg / day, 50 mg / day and 1,200 mg / day, between about 100 mg / day and 1,000 mg / day, between about 300 mg / day and 900 mg / day, between about 400 mg / day and 800 mg / day; preferably about 300 mg / day, 500 mg / day or 600 mg / day. The chelating agent can be administered in a total amount of up to about 1,200 mg / day, preferably at least 150 mg / day. The total amount can be administered.

[0088] The total amount of the chelating agent inhaled per day is preferably between about 37.5 mg / day and 1,20 0 mg / day, between about 50 mg / day and 1,200 mg / day, between about 100 mg / day and 1,0 00 mg / day, between about 300 mg / day and 900 mg / day, between about 400 mg / day and 800 mg / day; preferably about 150 mg / day, 300 mg / day, 500 mg / day or 6 00 mg / day.

[0089] The total amount of the chelating agent inhaled per day is preferably between about 0.1 mg of chelating agent / kg (body weight) and 15 mg of chelating agent / kg (body weight), between about 0.5 mg of chelating agent / kg (body weight) and 10 mg of chelating agent / kg (body weight), between about 1.0 mg of chelating agent / kg (body weight) and 5 mg of chelating agent / kg (body weight); between about 1.0 mg of chelating agent / kg (body weight) and 3. 5 mg of chelating agent / kg (body weight); preferably about 1.0 mg of chelating agent / kg (body weight ), 1.5 mg of chelating agent / kg (body weight), 2.0 mg of chelating agent / kg (body weight), 2. 5 mg of chelating agent / kg (body weight), 3.0 mg of chelating agent / kg (body weight), 3.5 mg of che lating agent / kg (body weight), 4.0 mg of chelating agent / kg (body weight), 4.5 mg of chelating agent / kg (body weight), 5.0 mg of chelating agent / kg (body weight), 10 mg of chelating agent / kg (body weight), 15 mg of chelating agent / kg (body weight).

[0090] For example, 50 mg of CaEDTA administered can be administered as a 4 mL solution of 33 mM for spraying (molecular mass C 10 H 12CaN2Na2O8 is 274.27 g / mol ) Similarly, a dose of 75 mg may be administered at 50 mM in 4 ml, or a dose of 100 mg may be administered at 66 mM in 4 ml.

[0091] Preferably, the formulation is administered to the subject in need thereof between about once a day and about six times a day More preferably, it is administered about four times a day.

[0092] Alternatively, the formulation can be administered to the subject in need thereof via a nebulizer via continuous inhalation The aerosol formulation can be delivered for 24 hours, 12 hours, preferably 8 hours, 6 hours , 4 hours, 2 hours or 1 hour, and each of these deliveries can be repeated several times within a 24-hour period (separate from 24 and 12 hours).

[0093] The formulations of the present invention can be administered to the subject using disposable packages and portable, hand-held, battery-operated devices, such as the AERx device (U.S. Patent No. 5,823,178, Aradigm, Hayward, Calif.). Alternatively, the formulations of the present invention can also be performed using mechanical (non-electronic) devices. Conventional jet nebulizers , ultrasonic nebulizers, soft mist inhalers, dry powder inhalers (DPI) , metered dose inhalers (MDI), condensed aerosol generators, and other inhalation devices including other systems can be used to deliver the formulation. For use as an aerosol, the compounds of the present invention are combined with a suitable propellant, such as propane, in a pressurized aerosol container together with conventional adjuvants in solution or suspension

[0094] ​​It can be packaged together with a hydrocarbon propellant such as butane or isobutane. A dry powder inhaler is a system operable using a pressurized air source that produces dry powder particles of a pharmaceutical formulation compressed to a very small volume. For inhalation, the system comprises a plurality of chambers each having a single dose of the pharmaceutical formulation and a selection element for releasing a single dose. Or has a blister.

[0095] An aerosol agent can be created by forcing the drug through the pores of a membrane, and these pores have a size in the range of about 0.25 to 6 μm (U.S. Patent No. 5,823,178). When the pores have this size, the particles that escape through the pores to create the aerosol agent will have a diameter in the range of 0 .5 to 12 μm. The drug particles can be released with an air stream intended to keep the particles within this size range. The generation of small particles can be facilitated by the use of a vibrating device that provides a vibration frequency in the range of about 800 to about 4000 kilohertz. It will be recognized by those skilled in the art that some adjustments can be made in parameters such as the size of the pores through which the drug is released, the vibration frequency, pressure, and other parameters based on the density and viscosity of the formulation, etc., but it should be noted that the purpose of some embodiments is to provide aerosolized particles having a diameter in the range of about 0. 5 to 12 μm. It should be noted that some adjustments can be made in parameters such as the size of the pores through which the drug is released, the vibration frequency, pressure, and other parameters based on the density and viscosity of the formulation, etc., but the purpose of some embodiments is to provide aerosolized particles having a diameter in the range of about 0. 5 to 12 μm. It should be noted that the purpose of some embodiments is to provide aerosolized particles having a diameter in the range of about 0. 5 to 12 μm.

[0096] [Excipient] The above-exemplified forms of the formulations described herein can be manufactured by methods well known to those skilled in pharmaceutical science. Further, the formulations described herein can include any optional excipients useful in the manufacture and / or administration of the formulations described herein. The above-exemplified forms of the formulations described herein can be manufactured by methods well known to those skilled in pharmaceutical science. Further, the formulations described herein can include any optional excipients useful in the manufacture and / or administration of the formulations described herein. It can include other optional excipients useful for the manufacture and / or administration of the formulations described herein. It is possible. Non-limiting examples of such excipients are well-known in the art and are useful for the manufacture and / or administration, including flavoring agents, coloring agents, parabens, antioxidants, viscosity modifiers, isotonic agents, drug carriers, sustained release agents, comfort promoters, emulsifying agents, solubilizing aids, lubricants, binders and other stabilizers.

[0097] Preferably, the formulations of the present invention are sterile. In another embodiment, the formulations of the present invention are stable over time.

[0098] Furthermore, a buffer can be added to adjust the pH level of the formulation. Preferably, the formulations of the present invention contain tris(hydroxymethyl)aminomethane (TRIS, which is also known as THAM or tromethamine) as a buffer. TRIS may have an additional effect of increasing the bactericidal action of EDTA. Preferably, TRIS is added to the formulations of the present invention for both buffering and increasing the efficacy of EDTA and / or antibiotics in the treatment or prevention of bacterial infections.

[0099] Furthermore, the formulations of the present invention can contain antibacterial preservatives.

[0100] Preferably, the pH of the formulations of the present invention is between about 6.5 and 8.0, more preferably between about 7.0 and 7.4. It has previously been found that bacteria become more resistant to antibacterial therapy as the pH drops. The preferred pH helps to avoid bacterial resistance to formulations containing high concentrations of inhaled chelating agents in combination with antibiotics in the absence of acidified nitrite.

[0101] ​​​​​In one alternative embodiment, the formulations of the present invention may include preservatives, suspending agents, wetting agents, isotonic agents, and / or diluents. The formulations provided herein are physiologically acceptable when administered by inhalation, 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% of one or more pharmaceutically suitable suspending fluids. Pharmaceutically suitable fluids for use herein include, but are not limited to, polar solvents including compounds containing hydroxyl groups or other polar groups. Solvents include, but are not limited to, water or alcohols such as ethanol, isopropanol, as well as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, and glycols including polyoxyethylene alcohols. Polar solvents also include, but are not limited to, protic solvents including water, aqueous saline having one or more pharmaceutically acceptable salt(s), alcohols, glycols, or mixtures thereof. In one alternative embodiment, the water for use in the formulations of the present invention should meet or exceed the legal requirements applicable for use in inhalation drugs. In one embodiment, the formulations described herein are aqueous and may contain 0 to 90% water. In other embodiments, the aqueous formulations described herein may contain 20 to 80% water. In yet other embodiments, the aqueous formulation may contain 50 to 70% water. The water is fresh water and may be distilled, sterilized, demineralized, or deionized water. Polar solvents also include, but are not limited to, water, aqueous saline having one or more pharmaceutically acceptable salt(s), alcohols, glycols, or mixtures thereof. In one alternative embodiment, the water for use in the formulations of the present invention should meet or exceed the legal requirements applicable for use in inhalation drugs. In one alternative embodiment, the formulations of the present invention may include preservatives, suspending agents, wetting agents, isotonic agents, and / or diluents. The formulations provided herein are physiologically acceptable when administered by inhalation, 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%

[0102] of one or more pharmaceutically suitable suspending fluids. Pharmaceutically suitable fluids for use herein include, but are not limited to, polar solvents including compounds containing hydroxyl groups or other polar groups. Solvents include, but are not limited to, water or alcohols such as ethanol, isopropanol, as well as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, and glycols including polyoxyethylene alcohols. Polar solvents also include, but are not limited to, protic solvents including water, aqueous saline having one or more pharmaceutically acceptable salt(s), alcohols, glycols, or mixtures thereof. It may further contain.

[0103] Alternatively, the formulation is non-aqueous and contains no water or only a very small amount of water (e.g., less than 1%, less than 0.1%, less than 0.01%).

[0104] In one embodiment, the formulation further comprises one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0105] In addition to or instead of sterilization, the formulations of the present invention can contain pharmaceutically acceptable preservatives to minimize the possibility of microbial contamination. Further, pharmaceutically acceptable preservatives can be used in the formulations of the present invention to increase the stability of the formulations. However, it should be noted that since the treated tissue can be sensitive to stimulants, any preservative must be selected for inhalation safety. Preservatives suitable for use herein include, but are not limited to, those that protect the solution from contamination with pathogen particles, including phenylethyl alcohol, benzalkonium chloride or benzoic acid, or benzoates, such as sodium benzoate and the like and phenylethyl alcohol. In certain embodiments, the formulations herein contain from about 0.001% to about 10.0% w / w benzalkonium chloride, or from about 0.01% v / w phenylethyl alcohol. The preservative can also be present in an amount of from about 0.001% to about 1%, preferably from about 0.002% to about 0.02%, more preferably 0.02% w / w. The formulations provided herein can also be from about 0.001% to about 90%, or from about 0.00 ... ... ...

[0106] ... 1% to about 50%, or about 0.001% to about 25%, or about 0.001% to about 10%, and may contain one or more emulsifiers, wetting agents, or suspending agents in an amount of about 0.001% to about 1%. Such agents for use herein include, but are not limited to, polyoxyethylene sorbitan fatty acid esters or polysorbates, for example, but not limited to, sorbitan monooleate polyethylene (polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate; lecithin; agar; carrageenan; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; indian gum; pectin; amidated pectin; ammonium phosphate; microcrystalline cellulose; methylcellulose; hydroxypropylcellulose; hydroxypropylmethylcellulose; ethylmethylcellulose; carboxymethylcellulose; sodium, potassium, and calcium salts of fatty acids; monoglycerides and diglycerides of fatty acids; acetates of monoglycerides and diglycerides of fatty acids; lactates of monoglycerides and diglycerides of fatty acids; citrates of monoglycerides and diglycerides of fatty acids; tartrates of monoglycerides and diglycerides of fatty acids; monoacetyltartrates and diacetyltartrates of monoglycerides and diglycerides of fatty acids; mixed acetates of monoglycerides and diglycerides of fatty acids and esters of monoglycerides and diglycerides of fatty acids with other organic acids. Such agents for use herein include, but are not limited to, esters of monoglycerides and diglycerides of fatty acids with acetic acid and Call tartrate esters; sucrose esters of fatty acids; sucroglycerides; polygly cerol esters; polyglycerol esters of polycondensed fatty acids of castor oil; fatty acids of propane-1,2-diol esters; sodium stearoyl-21 acrylate (a ctylate); calcium stearoyl-2-lactylate; stearoyl tartrate ; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate ; sorbitan monooleate; sorbitan monopalmitate; quillaia extract; soybean oil of dimeric fatty acid polyglycerol esters; oxidized polymerized soybean oil; and pectin extract including.

[0107] The formulation of the present invention may contain about 0.001% to about 5% by weight of a humectant to inhibit mucosal dryness and prevent irritation. Sorbitol, propylene glycol, polyethylene gly col, glycerol or mixtures thereof, any of a variety of pharmaceutically acceptable humectants can be used.

[0108] The formulation of the present invention may further contain adjuvants such as bronchodilators, other anti-inflammatory agents, surfactants, aspirin, or ethyl alcohol.

[0109] The bronchodilators optionally used in the formulation of the present invention include, but are not limited to β2-adrenergic receptor agonists (e.g., albuterol, bambuterol, sa lbutamol, salmeterol, formoterol, arformoterol, levalbutamol ol, procaterol, indacaterol, carmoterol, milbeterol, procate rolls, terbutaline, etc.), and antimuscarinics (e.g., trospium, ipratropium ium, glycopyrronium, acridinium, etc.). Combinations of drugs can be used .

[0110] Additional anti-inflammatory agents that may optionally be used in the formulations of the present invention include, but are not limited to inhaled corticosteroids (e.g., beclomethasone, budesonide, ciclesonide , fluticasone, etiprednol, mometasone, etc.), leukotriene receptor antagonists and leukotriene synthesis inhibitors (e.g., montelukast, zileuton, ibudilast , zafirlukast, pranlukast, amlexanox, tepoxalin, etc.), cyclo oxygenase inhibitors (e.g., ibuprofen, ketoprofen, ketorolac, indomethacin , naproxen, zaltoprofen, lornoxicam, meloxicam, celecoxib , lumiracoxib, etoricoxib, piroxicam, ampiroxicam, cinoxicam , diclofenac, felbinac, lornoxicam, mesalazine, triflusal , tinoridine, iguratimod, prasugrel, etc.). Combinations of drugs can be used . Aspirin, which acts as an anti-inflammatory agent, can also be added

[0111] Surfactants included by the present invention include, but are not limited to, synthetic surfactants (E xosurf (registered trademark)), dipalmitoyl phosphatidylcholine and oleic acid . Combinations of drugs can be used

[0112] Antioxidants, such as glutathione and vitamin E, zinc salts of zinc and EDTA, etc can be added

[0113] Ethyl alcohol vapor acts as an anti-foaming agent in the lungs, making sputum more liquefied, and thereby assisting respiration and reducing pulmonary edema. Ethanol can be added to the preparation of the present invention in an amount between 0.5% and 60%, more preferably between 1% and 40%, between 1% and 20%, or between 1% and 1 0%. Ethanol can be added to be 1%, 2%, 3%, 4%, 5 %, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 55% or 60%.

[0114] The present invention also relates to the use of a high-concentration chelating agent in combination with other drugs administered via inhalation. These other drugs can include nucleotide sequences that may be incorporated into a suitable delivery vector, such as a plasmid or viral vector. Other drugs can include therapeutic nucleotide sequences (DNA, RNA, siRNA), enzymes that reduce the viscoelasticity of mucus, such as DNase and other mucolytics, chemicals that upregulate chloride ion channels or increase the flow of ions across cells, nicotine, P2Y2 agonists, such as elastase inhibitors including α-1 antitrypsin (AAT), N- acetylcysteine, antibiotics, and cationic peptides, such as lantibiotics, specifically, duramycin, short-acting bronchodilators (such as β2-adrenergic receptor agonist-like albuterol or indacaterol), M3 muscarinic antagonists (such as ipratropium bromide), K -channel openers, long-acting bronchodilators (such as formoterol, salmeterol), steroids (such as budesonide ), and + K-channel openers, long-acting bronchodilators (such as formoterol, salmeterol), steroids (such as budesonide Steroids (such as mometasone, fluticasone, triamcinolone, beclomethasone, ciclesonide, etc.), xanthines, leukotriene antagonists (e.g., montelukast sodium), phosphodiesterase 4 inhibitors, adenosine receptor antagonists, various other anti-inflammatory agents (e.g., Syk kinase inhibitor (AVE-0950), tryptase inhibitors (AVE-8923 and AVE-5638), tachykinin antagonist (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-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 the VLA4 receptor or integrin α4β1 (e.g., R-411, PS-460644, DW-908e and CDP-323), immunomodulatory substances (tacrolimus) that block T cell signaling by inhibiting calcineurin, heparin neutralizing agents (talactoferrin α), cytosolic PLA2 inhibitors (efipladib), or combinations thereof. If the subject has CF, the subject may also be administered standard drugs, e.g., ivacaftor, pulmozyme, mannitol, etc., or other approved drugs, in combination with the formulations of the present invention, according to standard practice. xanthines, leukotriene antagonists (e.g., montelukast sodium), phosphodiesterase 4 inhibitors, adenosine receptor antagonists, various other anti-inflammatory agents (e.g., such as Syk kinase inhibitor (AVE-0950), tryptase inhibitors (AVE-892 3 and AVE-5638), tachykinin antagonist (AVE-5883), inducible nitric oxide synthase inhibitors (GW-274150) and others), transcription factor decoys, TLR-9 agonists, antisense oligonucleotides, siRNA, DNA, CGR P, lidocaine, inverse β2-agonists, anti-oxidative therapy, cytokine modulators (e.g., CCR3 receptor antagonists (GSK-766994, DPC-168, A ZD-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 the VLA4 receptor or integrin. al pha.4.beta.1 (e.g., R-411, PS-460644, DW-908e and C DP-323), immunomodulatory substances (tacrolimus) that block T cell signaling by inhibiting calcineurin, heparin neutralizing agents (talactoferrin α), cytosolic PLA2 inhibitors (efipladib), or combinations thereof. If the subject has CF, the subject may also be administered standard drugs, e.g., ivacaftor, pulmozyme, mannitol, etc., or other approved drugs, in combination with the formulations of the present invention, according to standard practice. If the subject has CF, the subject may also be administered standard drugs, e.g., ivacaftor, pulmozyme, mannitol, etc., or other approved drugs, in combination with the formulations of the present invention, according to standard practice. approved drugs, in combination with the formulations of the present invention, according to standard practice.

[0115] Delivery of the combination product can be achieved by combining the drugs into one stable formulation, or providing the drugs in separate containers that will be combined at the time of administration, or alternatively by sequentially delivering the products.

[0116] Preferably, the formulations of the present invention are stable. As used herein, the stability of the formulations provided herein refers to the length of time that more than 80%, 85%, 90% or 95% of the initial amount of the drug substance, e.g., chelating agent and antibiotic, is present in the formulation at a given temperature. For example, the formulations provided herein are well-stable when stored between about 15°C and about 30°C and remain stable for at least 1, 2, 12, 18, 24 or 36 months. Also, after storage at 25°C for more than 1, 2, 12, 18, 24 or 36 months, the formulation may be suitable for administration to the subject in need. In another alternative embodiment, using Arrhenius kinetics, after storage of the formulation between about 15°C and about 30°C for more than 1, 2, 12, 18, 24 or 36 months, more than 80%, or more than 85%, or more than 90%, or more than 95% of the initial amount of the drug substance (e.g., chelating agent and antibiotic) remains.

[0117] As used herein, the description that a formulation is stable during "long-term storage" means that the formulation has a predicted shelf life of more than 1, 2 or 3 months at 25°C and a storage time of more than or equal to 1, 2 or 3 years at 5°C and is suitable for administration to the subject in need. In certain embodiments herein, using Arrhenius kinetics, after such storage, >80% or >85 ​​​​​​​​​​​​% or >90% or >95% of the chelating agent and antibiotic are expected to remain .

[0118] The term "inflammation" as used herein refers to inflammation caused by an invasion, such as an infection, an environmental One or more of the body's responses to insults (including tobacco smoke), trauma, or hypersensitivity Inflammation can be either acute or chronic, and symptoms include swelling of tissues. , recruitment of different types of inflammatory cells, release of cytokines and mediators, and bronchial inflammation. Inflammation may be localized, asymptomatic or temporary, or the inflammation may be more widespread. Inflammation can affect a wide range of tissues and become chronic. It stimulates both humoral and cellular immune responsiveness. Signs of inflammation may include inflammation of the lining of the stomach and may persist even after the insult that provoked it has been removed. Examples of such antibodies include, but are not limited to, inflammatory cells (e.g., dendritic cells, macrophages, neutrophils, etc.) Increased levels of inflammatory cytokines (e.g., leukocytes, lymphocytes, eosinophils, and mast cells), (e.g., TNFα, IL-1β, IL-6, IL-8, and IFNγ) and their receptors Increased levels of IL-1, excessive proteases including MMPs, ROS and other mediators, and Markers of inflammation, such as C-reactive protein (CFP) and sputum and serum carbohydrates Short-term inflammation ("acute") causes increased breathing rate and difficulty in breathing. Symptoms include airway swelling, pulmonary compression, and nausea, wheezing, coughing, and reduced FEV1. This can result in changes in the immune system, airway reactivity and mucus hypersecretion, and if sustained (see The structure of the airway wall and lung parenchyma in the form of fibrosis, cystic changes, and bronchiectasis (chronic lung disease). This can result in structural damage.

[0119] [Medicinal manufacturing method] Use of a high concentration chelating agent in the manufacture of an inhalation preparation for treating or preventing inflammation in the lungs thereof.

[0120] Use of an inhalation chelating agent in the manufacture of a medicament for delivering a high concentration of an inhalation chelating agent as a single dose for treating or preventing inflammation in the lungs thereof.

[0121] Preferably, the high concentration chelating agent is provided in a preparation containing at least 37.5 mg / dose, at least 50 m g / dose, or between 50 mg / dose and 300 mg / dose, or between 37.5 mg / dose and 300 mg / dose. The chelating agent is administered between 1 and 4 times daily up to a total dose of about 1,200 mg / day, preferably up to at least 150 mg / day. Preferably, the chelating agent is CaEDTA.

[0122] [Kit] The present invention provides a kit for treating or preventing inflammation in the lungs comprising (i) an inhalation preparation containing a high concentration chelating agent and (ii) instructions for use. thereof.

[0123] The present invention provides a kit for treating or preventing inflammation in the lungs comprising (i) an inhalation preparation capable of delivering a high concentration of an inhalation chelating agent as a single dose and (ii) instructions for use. thereof. thereof.

[0124] Preferably, the high concentration chelating agent is provided in a preparation containing at least 37.5 mg / dose, at least 50 m g / dose, or between 37.5 mg / dose and 300 mg / dose, or between 50 mg / dose and 300 mg / dose. The chelating agent is administered between 1 and 4 times daily up to a total dose of about 1,200 mg / day, preferably up to at least 150 mg / day. It can be obtained. Preferably, the chelating agent is CaEDTA.

[0125] In one embodiment, the kit of the present invention comprises a preparation containing a therapeutically effective amount of a high-concentration inhalable chelating agent. In an alternative embodiment, the preparation is pre-measured, pre-mixed, and / or pre-packaged. Preferably, the inhalation solution is sterilized. The kit of the present invention may also include instructions designed to promote user compliance. As used herein, "instructions" refers to any label, accompanying document, etc., which may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet, or any combination thereof. For example, in one embodiment, the kit of the present invention includes instructions for administering the preparation of the present invention. In one embodiment, the instructions indicate that the preparation of the present invention is suitable for the treatment of lung inflammation. Such instructions may also include instructions regarding the preparation, as well as instructions regarding administration via a nebulizer or dry powder inhaler.

[0126] The inhalable chelating agent and any additional active agent can be individually packaged so that a physician or user can formulate them into a pharmaceutical preparation as needed. Alternatively, a pharmaceutical preparation containing the inhalable chelating agent and any additional active agent can be packaged together, thus requiring a minimum formulation by a physician or user. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient. It can include. "Instructions", as used herein, refers to any label, accompanying document, etc., which may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet, or any combination thereof. It may be placed on one or more surfaces of the packaging material, or the instructions may be provided on a separate sheet, or any combination thereof. For example, in one embodiment, the kit of the present invention includes instructions for administering the preparation of the present invention. In one embodiment, the instructions indicate that the preparation of the present invention is suitable for the treatment of lung inflammation. Such instructions may also include instructions regarding the preparation, as well as instructions regarding administration via a nebulizer or dry powder inhaler. The kit of the present invention includes instructions for administering the preparation of the present invention. In one embodiment, the instructions indicate that the preparation of the present invention is suitable for the treatment of lung inflammation. Such instructions may also include instructions regarding the preparation, as well as instructions regarding administration via a nebulizer or dry powder inhaler. For the preparation, and also for administration via a nebulizer or dry powder inhaler. It may also include.

[0127] The inhalable chelating agent and any additional active agent can be individually packaged so that a physician or user can formulate them into a pharmaceutical preparation as needed. Alternatively, a pharmaceutical preparation containing the inhalable chelating agent and any additional active agent can be packaged together, thus requiring a minimum formulation by a physician or user. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient. Instead, a pharmaceutical preparation containing the inhalable chelating agent and any additional active agent can be packaged together. Thus, a minimum formulation by a physician or user is required. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredient.

[0128] [General Rules] A person skilled in the art will recognize that the present invention described herein may have modifications and variations other than those specifically described. The present invention includes all such modifications and variations. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features. All such modifications and variations are included in the present invention. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features. All such modifications and variations are included in the present invention. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features. All such modifications and variations are included in the present invention. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features. All such modifications and variations are included in the present invention. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features. All such modifications and variations are included in the present invention. The present invention also includes all steps, features, formulations, and compounds mentioned or shown in the specification, individually or in combination, and any and all combinations or any two or more steps or features.

[0129] Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be read and considered as part of this text by the reader. The fact that a document, reference, patent application, or patent cited in this text is not repeated in this text is for reasons of brevity only. Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be read and considered as part of this text by the reader. The fact that a document, reference, patent application, or patent cited in this text is not repeated in this text is for reasons of brevity only. Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be read and considered as part of this text by the reader. The fact that a document, reference, patent application, or patent cited in this text is not repeated in this text is for reasons of brevity only. Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be read and considered as part of this text by the reader. The fact that a document, reference, patent application, or patent cited in this text is not repeated in this text is for reasons of brevity only. Each document, reference, patent application, or patent cited in this text is hereby expressly incorporated by reference in its entirety, which means that it should be read and considered as part of this text by the reader. The fact that a document, reference, patent application, or patent cited in this text is not repeated in this text is for reasons of brevity only.

[0130] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this text or in any document incorporated by reference herein are hereby incorporated by reference into this text and can be used in the practice of the present invention. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this text or in any document incorporated by reference herein are hereby incorporated by reference into this text and can be used in the practice of the present invention. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this text or in any document incorporated by reference herein are hereby incorporated by reference into this text and can be used in the practice of the present invention. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this text or in any document incorporated by reference herein are hereby incorporated by reference into this text and can be used in the practice of the present invention.

[0131] The scope of the present invention is not limited by 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 present invention as described herein. The scope of the present invention is not limited by 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 present invention as described herein. The scope of the present invention is not limited by 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 present invention as described herein. The scope of the present invention is not limited by 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 present invention as described herein.

[0132] The invention described in this specification can include one or more ranges of values (e.g., size, replacement, and magnetic field strength, etc.). A range of values is considered to include all values within the range, which includes the values that define the range and the values that are immediately adjacent to the values that define the boundaries of the range and that result in the same or substantially the same result as the values immediately adjacent to the range. Thus, unless otherwise indicated specifically to the contrary, the numerical parameters described in the specification and the claims are approximate values that can vary depending on the desired characteristics to be obtained by the invention. Therefore, "about 80%" also means "about 80%" and further "80%". At least, each numerical parameter should be interpreted in light of the significant digits and ordinary rounding techniques. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations thereof, such as "comprises" or "comprising", is considered to mean the inclusion of the stated integers or groups of integers, but not the exclusion of any other integers or groups of integers. In this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. can have the meaning attributed to them under US patent law. For example, these can mean "includes", "included", "including", etc., and terms such as "consisting essentially of" and "consists essentially of"

[0133] ​​​​​​​​​​​​​​​​ have a meaning attributable to the United States Patent Law and, for example, these allow elements that are not explicitly recited, but it should also be noted that they exclude elements found in the prior art or that affect the basic or novel features of the invention.

[0134] Other definitions for selected terms used herein can be found in the detailed description of the invention and apply 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 belongs. The term "active agent" may mean one active agent or may include two or more active agents.

[0135] The following examples are intended to more fully describe how to use the invention described above and to describe the best mode contemplated for practicing various aspects of the invention. It should be understood that these methods are not intended to limit the true scope of the invention in any way, but rather are presented for illustrative purposes.

Examples

[0136] Further features of the invention are more fully described in the following non-limiting examples. This description is included for illustrative purposes only and should not be understood as a limitation on the broad description of the invention presented above.

[0137] [Example 1] Method for treating or preventing infectious diseases in the lungs by administering a high-concentration inhalable chelating agent Using cystic fibrosis mucus suspension droplets collected from epithelial cell lines, biofilms were grown in a realistic in vi tro model (Haley et al., BMC Micro biol, 2012, 12:181). Cultures of Pseudomonas aeruginosa clinical strains (MIC tobramycin > 2 56 μg / ml) were grown in M63 without a carbon source to mimic nutrient limitation and transferred to the late stationary phase.

[0138] Several drops of mucus (5 μl) were suspended from an inverted IBIDI coverslip and inoculated with 10 3 colony-forming units (CFU), and then incubated at 35 °C for 72 h in a humidified environment to generate a biofilm. The droplets were then treated with either aerosolized tobramycin (2 0 mg / ml), aerosolized CaEDTA particles (10 mg / ml), or both for 5 min. Controls were treated with a 50 / 50 solution of aerosolized 0.9% saline / water. After treatment, the droplets were incubated for 16 h and then stained with BacLight LIVE / DEAD (1 μl) and fixed with paraformaldehyde vapor for 30 min. Confocal microscopy was used to visualize the biofilm.

[0139] Figure 1A shows a thick and robust biofilm with mostly live cells (green) after treatment with aerosolized saline. As expected for resistant strains, treatment with tobramycin alone has little effect on viability. Treatment with EDTA alone causes some killing (red blood cells). The combination of tobramycin and EDTA kills most of the biofilm cells markedly. Figure 1B shows a quantitative representation of the microscopic images in Figure 1A. The control biofilm is 1×10 8was CFU / ml, but EDTA-tobramycin treated biofilms were reduced by >6 digits and <10 2 CFU / ml.

[0140] Patients aged ≧6 years with CF who were hospitalized due to worsening symptoms were randomly selected and received intravenous antibiotics and in addition to these usual treatments with nebulized tobramycin, EDTA or physiological saline (placebo) was administered. EDTA was administered as a 4 ml solution of 50 mM CaNa2EDTA, 111 mM Tris in 0.9% saline, pH 7.1 for nebulization along with tobramycin. After random selection, the subjects were treated in the hospital for 2 weeks, during which time the patients were treated 4 times a day (300 mg of EDTA / day, or 3.3 mg EDTA / kg / day). Then the patients were discharged and the treatment was continued twice a day for 4 weeks. The patients were monitored for an additional 4 weeks (300 mg of EDTA / day, or 3.3 mg EDTA / kg / day). Then the patients were discharged and the treatment was continued twice a day for 4 weeks. The patients were monitored for an additional 4 weeks and the total experimental time was 10 weeks. and the total experimental time was 10 weeks.

[0141] Sputum was induced using 8 - 10 L / min of 3% hypertonic saline for nebulization for ≧5 minutes. Samples were collected before treatment and processed according to the relevant protocol at 2, 6, and 10 weeks and stored at -80°C . Mucus was dissociated from clear sputum, mixed with Sputalysin (1 ml per gram of sputum ), vortexed, incubated for 1 hour, and then placed in Skim Milk G lycerol storage medium and stored at -80°C.

[0142] Sputum samples were obtained from the subjects' sputum at the first screening, then clinical examination 3 (around 2 weeks later), clinical examination 5 (6 weeks), and finally follow-up investigation (10 weeks). Mucus was dissociated from clear sputum and treated with Sputalysin (1 ml per gram of sputum) and Skim Milk Placed in a glycerol storage medium (1 ml / 100 mg of mucus), mixed by vortexing, and stored at -80 °C.

[0143] The samples were thawed on ice, serially diluted to a maximum of 10-7 of the original concentration, and 20 μL of each dilution was placed on each of three culture plates of McConkey (McC) agar or Blood agar (BA). The plates were incubated at 35 °C.

[0144] Species of the genus Pseudomonas were defined as transparent or very pale pink lactose-negative colonies on McC agar plates. Coarse colonies had a metallic sheen and a rough colony edge, smooth colonies had a regular colony border on McC agar, grew slowly, and mucoid colonies were surrounded by large amounts of alginate secreted by the bacteria.

[0145] The numbers of coarse, smooth, and mucoid colonies were counted at 24 hours on both McC agar and BA plates, the plates were incubated for a further 24 hours, and a confirmatory count of each colony type was performed. A single colony of each type present in each sample was picked up, streaked on a BA plate, and a pure culture was obtained.

[0146] Gram staining was performed to confirm that the isolates consisted of Gram-negative, rod-shaped cells, and further identification was carried out by confirming an oxidase-positive state by rubbing a very small part of the colony onto an oxidase test strip. A rapid development of dark blue indicates an oxidative positive isolate.

[0147] ​​​​​​​​​​​​​Confirmation of Pseudomonas species was performed by testing resistance to C390 antibiotics. Antibiotic-impregnated discs were placed on nutrient agar (NA) plates diffused with a suspension of pure isolates up to a McFarland density of 0.5 in phosphate-buffered saline (PBS). After overnight incubation at 35 °C, the absence of any inhibition zone around the disc indicated resistance to the antibiotics. Single colonies of each morphological type of the identified Pseudomonas species (presumably P. aeruginosa) present in each isolate were picked up, resuspended in a glycerol / serum storage medium, and stored at -80 °C. Figure 2 shows the change in colony counts of P. aeruginosa (McC) at 2 and 6 weeks compared to the start of treatment. Two weeks after treatment, the reduction in colony counts was >400-fold in the EDTA group

[0148] [Example 2] Treatment of lung inflammation results in a dose-dependent increase in FEV1 Subjects aged ≥6 years with CF hospitalized due to symptom exacerbation were randomly selected and administered EDTA or saline (placebo) in addition to these usual treatments with intravenous antibiotics and inhaled tobramycin. EDTA was administered together with tobramycin as a spray solution of 4 ml of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1. After random selection, the subjects were treated in the hospital for 2 weeks, during which the patients were treated 4 times a day (300 mg of

[0149] In the clinical trials of each experiment, lung function was measured by spirometry. The best of three attempts was recorded as data and the results were expressed as a percentage of the predicted value.

[0150] Figure 3A shows the average change in FEV1 for both groups at 2, 6, and 10 weeks after the start of treatment. The average increase in FEV1 after 2 weeks was 16% points in the EDTA group, compared with 5% points in the placebo group. This difference persisted for 4 weeks after the completion of treatment, with a 7% point increase in the EDTA group and a 2% point increase in the placebo group. This demonstrates a clear improvement in lung function in the EDTA group, while only a slight change was demonstrated in the placebo group. Figure 3B shows an inverse correlation between the improvement in FEV1 and body weight in the EDTA group (R = 0.70), while showing no correlation in the placebo group treated with tobramycin alone (R = 0.01). 2 =0 2 =0.01). This indicates that EDTA has a dose-dependent effect (mg EDTA / kg body weight) on lung function.

[0151] Figure 4 shows that the delivery of 75 mg of CaEDTA to the lungs results in peak EDTA concentrations of 0.41 - 1.34 mM 5 minutes after administration.

[0152] [Example 3] Tobacco smoke-induced lung inflammation can be treated by administering a high dose of a chelating agent to the lungs. The effect of chelating agents on lung inflammation was tested in a mouse model of chronic obstructive pulmonary disease (COPD). Tobacco smoke (CS) is known to induce lung inflammation, and lung inflammation is characterized by leukocytes​​​​​​​​​​​ It can be measured by an increase in the number of balls counted and an increase in lung weight.

[0153] Male BALB / c mice (8 mice per group) were exposed to a defined dose of cigarette smoke (3 cigarettes, 3 times a day, Monday to Friday), or filtered room air for a period of 2 weeks. During the experimental period, 30 - 60 minutes before each exposure to cigarette smoke, the mice were treated intranasally once a day with the iron chelating agent deferoxamine (DFO, 3.8 mg in 50 μl) or vehicle. Subsequently, the mice were sacrificed and the airways and lungs were evaluated for their effects on cigarette smoke - induced inflammation and element concentrations. Bronchoalveolar lavage fluid (BALF) was collected (approx. 1 ml / mouse), and the lungs were surgically removed and weighed. Equal amounts of trypan blue were mixed into the BALF, and the total number of viable cells in the BALF was determined by manual counting using a standard Neubauer hemocytometer with a Zeiss Axioscope fluorescence microscope. Iron was measured by elemental analysis using laser ablation inductively coupled plasma mass spectrometry (LA - ICP - MS) and quantified by comparison with a known standard of metal content. As expected, Figure 5 shows that cigarette smoke significantly increases the total number of BALF leukocytes. Treatment with the iron chelating agent DFO significantly reduces this effect. Consistent with this, Figure 6 shows that the mean lung weight is significantly increased by treatment with cigarette smoke, but treatment with CFO prevents this effect.

[0154]

[0155]

[0156]

[0157] ​​​​​​​​​​As previously described, Stites et al. (Am J Respir Crit Car e Med. 1999, 160(3):796 - 80) showed that iron levels are significantly increased in the lungs of CF patients as well as in the lungs of smokers compared to healthy individuals. Figure 7 (left) confirms that the mean iron level is significantly increased in the BALF of mice exposed to cigarette smoke and that treatment with DFO reduces the mean BALF iron content. Figure 7 (right) shows that in 6 out of 7 mice treated with DFO (1 mouse died for reasons unrelated to treatment), the BALF iron content was at the same level as that of mice exposed to air.

[0158] [Virtual Example P1: In Vivo Experiment on the Action of High - Dose Dry - Powder Chelating Agents against Infectious Diseases, Inflammation, and Oxidative Stress] Subjects with CF who require treatment with dry - powder tobramycin are assigned to 4 cohorts and administered 112 mg of dry - powder twice daily for 28 days. In addition, Cohort 1 (patients > 18 years old) is administered increasing doses of dry - powder CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks, 150 mg BID for 1 week). Cohort 2 (patients > 18 years old) is administered CaEDTA (37.5 mg BID for 1 week; 75 BI D for 2 weeks; 75 mg QID for 1 week). Cohort 3 (patients 12 - 18 years old) is administered CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks, 150 mg BID for 1 week). Finally, the observation cohort is administered tobramycin alone for 2 8 days.

[0159] Sputum samples are collected weekly and evaluated for markers of infection and inflammation. Bacteria are monitored by sputum colony -count. As a measure of structural damage, levels of matrix metallopro teinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) are measured as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-727; Garratt et al., Eur Respir J. 2015, 46(2):384-94) using zymography and immunoassay respectively. The amount of iron in sputum is quantified by ICP -MS as previously described (Hunter et al., Mbio. 2013, 4(4):1-8 ). The amount of iron-binding protein is evaluated using an immunoassay. Myeloperoxidase activity is also assayed as previously described (G aggar et al., Eur Respir J. 2011, 38(3):721-72 7) as a measure of neutrophil inflammation. 3-Chlorotyrosine is measured as a biomarker of hypochlorous acid, a strong oxidizing agent. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., Eur Respir J. 2011, 38(3): 721-727). Protein carbonyl is measured as an indicator of reactive oxygen species (ROS) using a commercially available immunoassay kit (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). Oxidative stress is evaluated by measuring glutathione (GSSG and GSH) as previously described (Kettle et al., Eur Respir J. 2014, using an immunoassay. aggar et al., Eur Respir J. 2011, 38(3):721-72 7). Determine. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., Eur Respir J. 2011, 38(3): 721-727). 721-727). Protein carbonyl is measured as an indicator of reactive oxygen species (ROS) using a commercially available immunoassay kit (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). Oxidative stress is evaluated by measuring glutathione (GSSG and GSH) as previously described (Kettle et al., Eur Respir J. 2014, using an immunoassay. Measure glutathione (GSSG and GSH) as previously described to evaluate oxidative stress (Kettle et al., Eur Respir J. 2014, Volume 44 (Issue 1): 122-9 pages). The gene expression of inflammatory and oxidative stress markers (e.g., IL-8 , IL-6, TNFα) is also monitored by Nanostring, and the protein is measured by ELISA. Oxidative stress is also measured via metabolites, e.g., malon dialdehyde (molondialdehyde) (colorimetric assay) or 8-iso prostaglandin (ELISA), etc. Iron is measured by elemental analysis using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS).

[0160] This experiment is expected to show a reduction in inflammatory markers and a decrease in iron levels in the EDTA group compared to the placebo group. This experiment is expected to show a change in the balance between MMP and TIMP, especially between M MP-9 and TIMP-1, which is associated with the progression of bronchiectasis .

[0161] This experiment is further expected to show a reduction in the amount of bacteria in sputum and an increase in FEV1 in subjects treated with EDTA compared to control patients.

[0162] [Virtual Example P2: In vitro experiment on the effect of high-dose chelating agents on inflammation, lung injury, and oxidative stress] Grow lung epithelial cells in tissue culture and induce inflammation by exposure to Fe(II) or excess oxygen . Treat the cells with CaEDTA (0, 1, 5, 10, 25, 50 mM) for 30 minutes at 1, 3, 24, and 48 hours.

[0163] Use immunoassays to measure inflammatory markers, e.g., IL-6, IL-8, TNF- ​Monitor α, neutrophil elastase, and other changes. Oxidative stress and toxicity are measured by evaluating the level of reduced glutathione (GSH ) and apoptosis based on the TUNEL assay, in which both are measured using commercially available assay kits, for example, The rmoFisher Scientific's Glutathione Fluores cent Detection Kit and BioVision Inc's TUNEL DNA Gragmentation Assay Kit, etc.

[0164] This experiment is expected to show that, compared to the control, inflammatory markers are concentration-dependently reduced in EDTA-treated cells; GSH is reduced (which indicates a reduction in reactive oxygen species in EDTA-treated cells compared to the control); and apoptosis is reduced in EDTA-treated cells compared to the control as measured by the TUNEL assay.

[0165] [Virtual Example P3: In Vivo Experiment on the Action of High-Dose Spray Chelating Agent against Inflammation, Lung Injury, and Oxidative Stress] Randomly select subjects aged ≥ 6 years with CF who were hospitalized due to worsening symptoms, and in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin, administer nebulized EDTA or saline (placebo). EDTA is administered as a nebulized solution of 50 mM CaEDTA in 4 ml, 111 mM tris in 0.9% saline, pH 7.1, together with tobramycin.

[0166] After random selection, the subjects are treated in the hospital for 2 weeks, during which the subjects receive treatment 4 times a day. 300 mg of EDTA per day, or up to 3.3 mg of EDTA / kg / day). Then, the subject is discharged from the hospital, and the treatment is continued twice a day for 4 weeks. The subject is further monitored for 4 weeks, and the total experimental time is set to 10 weeks. Induce sputum collection by spraying 3% hypertonic saline for nebulization at 8 - 10 L / min for ≥5 minutes . Collect samples before treatment, and process them according to the relevant protocol at the 2, 6, and 10 - week time points, and store them at -80 °C.

[0167] · Expression of inflammatory markers Store the expectorated sputum in RNAlater® and extract total RNA using Qiagen RNEasy (registered trademark) or a similar extraction kit, convert it to cDNA , and monitor the inflammatory markers using qPCR as described by Sivaneson et al. (Mol Microbiol, Vol. 79, pp. 1353 - 1366 ) and quantify it relative to known housekeeping genes, such as actin and / or GAPDH, etc.

[0168] This experiment is expected to show an average reduction in gene expression of inflammatory markers in the EDTA group compared to the placebo group.

[0169] · Cell damage, free iron, and oxidative stress Freeze the expectorated sputum without processing and assay for inflammatory markers as described above. As a measure of structural damage, measure the levels of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) using gelatin zymography and immunoassay respectively, as previously described (Gaggar et al., Eur Respir J. 2011, Vol. 38(3): 721 - 727; G ​ Arratt et al., Eur Respir J., 2015, Vol. 46(2): 384- 94). Quantify the amount of iron in sputum by ICP-MS as previously described (Hu nter et al., MBio. 2013, Vol. 4(4): 1-8). Use immunoassays to assess the amount of iron-binding proteins. Use immunoassays to measure glutathione (GSSG and GSH) as previously described to evaluate oxidative stress (Kettle et al., Eur Respir J. 2014, Vol. 44(1): 122-9).

[0170] This experiment is expected to show a reduction in inflammatory markers in the EDTA group compared to the placebo group. This experiment is also expected to show a change in the balance between MMP and TIMP, particularly between MMP-9 and TIMP-1 (which is associated with the progression of bronchiectasis).

[0171] [In vivo experiment on the effect of high-dose chelating agents on inflammation, lung injury, and oxidative stress] Conduct a single-site, randomized, double-blind, crossover experiment on subjects with cystic fibrosis. Randomly assign subjects to 2 weeks of treatment with inhaled CaEDTA or saline (placebo). After that, a washout period and then 2 weeks of the other treatment (EDTA or placebo) follow.

[0172] Monitor iron levels, inflammatory markers, MMP / TIMP, and FEV1 as described above. As previously described, myeloperoxidase as a measure of neutrophil inflammation ​​​Activity is also assayed (Gaggar et al., Eur Respir J. 2011, 3 8, no. 3: 721-727). Measure 3 -chlorotyrosine as a biomarker for the strong oxidant hypochlorous acid. Levels are measured using stable isotope dilution gas chromatography -mass spectrometry (Gaggar et al., Eur Respir J. 2011 , 38, no. 3: 721-727). Using a commercially available immunoassay kit, measure protein carbonyl as an indicator of reactive oxygen species (ROS) (Gaggar et al., Eur Respir J. 2011, 38, no. 3: 721-727). This experiment is expected to show a reduction in the levels of iron and inflammatory

[0173] markers, a change in the MMP / TIMP balance, and an increase in mean FEV1 in subjects treated with EDTA compared to placebo. This experiment is further expected to show a reduction in myeloperoxidase activity and a decrease in the mean levels of chlorotyrosine and carbonyl. Clinical data have demonstrated efficacy at 300 mg / day for 2 weeks. The same experiment has demonstrated that 1

[0174] 50 mg / day (75 mg BID) is beneficial for lung function and infections (Figure 2 for the decrease in bacterial counts; Figure 3A for the improvement in lung function). Given the significant magnitude of the improvement (mean FEV1 of 16 percentage points), as would be understood by a person skilled in the art, it is highly likely that much lower doses, i.e., 75 mg / day (37.5 mg BID), are effective, as envisioned by Virtual Example P1.

[0175] ​​​​​Figure 4 shows that a single administration of 75 mg of CaEDTA results in up to 1.34 mM of EDTA inside the mucus plug 30 minutes later. The penetration of drugs such as tobramycin into CF sputum is significantly delayed (Kuhn, R.J. (2001). Formulation of aerosolized therapeutics. Chest 120, 94S-98S), and thus it is known that the concentration of EDTA in the airway surface liquid is most likely substantially higher than in the center. Therefore, it is reasonable to expect that a daily dose of 37.5 mg (four times lower than the low dose with clinical merit) will show efficacy in a fully powered experiment. This is particularly applicable to young patients who are administered a higher dose per body weight and generally show a greater therapeutic effect in FEV1 (Figure 3B). Without departing from the basic concept of the present invention, based on the above teachings related to the disclosed invention, many variations and modifications of the above-described modes for carrying out various embodiments of the present invention will be apparent to those skilled in the art. The above embodiments of the present invention are merely illustrative and should in no way be construed as limiting, and all such variations and modifications are considered to be within the scope of the present invention, the nature of which is determined from the foregoing description. The research of the present invention was aided by a grant from the Cystic Fibrosis Foundation Therapeutics.

[0176]

[0177] ​​​​​​​​​​​​​

Claims

1. Methods for treating or preventing inflammation in the lungs by administering high concentrations of inhaled chelating agents Law.

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

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

4. The chelating agent is provided in a formulation containing between 37.5 mg / dose and 300 mg / dose. The method according to any one of claims 1 to 3,

5. The chelating agent is provided in a formulation containing between 50 mg / dose and 300 mg / dose; 5. The method according to any one of claims 1 to 4.

6. 6. The method of claim 1, wherein the chelating agent is provided in a total dose of up to about 1,200 mg / day. The method according to any one of claims 1 to 5.

7. 7. The method of claim 1, wherein the chelating agent is CaEDTA.

8. 8. Any one of claims 1 to 7, wherein the treatment or prevention of inflammation results in an increase in FEV The method described above.

9. 9. The method according to claim 1, wherein the treatment or prevention of inflammation is accompanied by a reduction in MMP activity. Method of posting.

10. 10. The method according to claim 1, wherein the treatment or prevention of inflammation is accompanied by a reduction in the production of hydroxyl radicals. The method according to any one of claims 1 to 5.

11. The chelating agent is combined with tris(hydroxymethyl)aminomethane (TRIS). The method according to any one of claims 1 to 10, wherein

12. Inhalation formulations containing high concentrations of chelating agents.

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

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

15. 13. The method according to claim 12, wherein the concentration of the chelating agent is between 37.5 mg / dose and 300 mg / dose.

15. The formulation according to claim 14.

16. 13. The method according to claim 12, wherein the concentration of the chelating agent is between 50 mg / dose and 300 mg / dose.

15. The formulation described in 14.

17. 17. The method of claim 12, wherein the chelating agent is provided in a total dose of up to about 1,200 mg / day. The formulation according to any one of claims 1 to 4.

18. 18. The method of claim 12, wherein the chelating agent is CaEDTA. Agent.

19. The chelating agent is combined with tris(hydroxymethyl)aminomethane (Tris).

19. The formulation according to any one of claims 12 to 18.

20. (i) an inhalation formulation containing a high concentration of a chelating agent; and (ii) instructions for use. A kit for treating or preventing inflammation in the lungs.

21. High Concentrations of Chelating Agents in the Preparation of Inhalation Formulations for Treating or Preventing Inflammation in the Lungs - Patent application Use of.

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