Methods for reducing pulmonary inflammation

Abstract

A method for treating or preventing inflammation in the lungs by administering high concentrations of inhalable chelating agents, particularly CaEDTA.

Description

[Technical Field]

[0001] The present invention relates to a method for treating or preventing inflammation in the lungs by administering high concentrations of inhaled chelating agents, and formulations for use in the method. In one embodiment of the present invention, the inflammation in the lungs 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, but inflammation and lung damage can also occur in the absence of bacterial infection (Sly et al., Am J Respir Crit Care Med. 2009, 180(2):146-52).

[0003] Inflammation is the body's response to insults, including infection, trauma, and hypersensitivity. The inflammatory response is complex and includes various mechanisms for defense against pathogens and tissue repair. In the lungs, inflammation is commonly caused by pathogens or by exposure to toxins, pollutants, irritants, and allergens.

[0004] During inflammation, many types of inflammatory cells are activated. Each releases cytokines and mediators to modify the activity of other inflammatory cells. The orchestration of these cells and molecules leads to the progression of inflammation. Clinically, acute inflammation is seen in diseases such as pneumonia and acute respiratory distress syndrome (ARDS), whereas chronic inflammation is manifested in diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Because the lung is a critical organ for gas exchange, excessive inflammation can be life-threatening. A delicate balance between pro- and anti-inflammatory mechanisms is essential for lung homeostasis.

[0005] Immunity comprises the innate and adaptive systems. Innate immunity is nonspecific and triggers a rapid response, including inflammation, in the face of pathogen invasion. Adaptive immunity is antigen-specific; it first detects a specific antigen and then recruits inflammatory cells to target that specific antigen. The innate and adaptive systems share components and work in concert to protect against pathogens.

[0006] Airway epithelia secrete various substances, such as mucins, defensins, lysozyme, lactoferrin, and nitric oxide, which nonspecifically 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 factors, to recruit inflammatory cells to the site of inflammation. Cytokines stimulate the release of arachidonic acid from membrane lipids, resulting in the production of eicosanoids, which further stimulate mucus secretion by goblet cells and tissue inflammation.

[0007] Surfactant is located on the alveolar surface and contains four surfactant proteins (SPs A-D). These proteins play a crucial role in surfactant absorption onto the alveolar surface, which is important for reducing lung surface tension. SPs-A and SP-D also participate in host defense; they bind to bacterial surface molecules, modulating leukocyte activity and leading to pathogen opsonization.

[0008] IgA secreted by plasma cells forms an additional protective epithelial barrier, preventing the attachment of microorganisms to the epithelial surface. IgA also binds to pathogens, triggering phagocytosis and antibody-dependent cell-mediated cytotoxicity. Immunoglobulin E (IgE) induces immediate-type hypersensitivity in the respiratory tract. IgE generates an intense response 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 proinflammatory mediators, including histamine, prostaglandins, leukotrienes, and tryptase. These increase vascular permeability, bronchoconstriction, and inflammatory cell infiltration.

[0009] U.S. Patent Application Publication No. 2016 / 0263151 teaches treating bacterial infections by using an inhaled antibiotic in combination with acidified nitrous acid and an iron chelator. The iron chelator present in the formulation of U.S. Patent Application Publication No. 2016 / 0263151 serves to provide a synergistic effect when combined with acidified nitrous acid, enhancing the ability of the antibiotic to work.

[0010] Current treatments for pulmonary inflammation include oral or inhaled steroids and nonsteroidal drugs that target the host's inflammatory response, but they are short-lived, require continuous treatment, and have significant side effects.

[0011] There is a need for methods to treat or prevent inflammation in the lungs, or at least to complement or provide an alternative to previously known treatment methods.

[0012] The present invention seeks to provide an improved or alternative method for treating or preventing inflammation in the lungs by administering high concentrations of inhaled chelating agents.

[0013] The preceding discussion of the background art is intended only to facilitate an understanding of the present invention. This discussion is not an admission or admission that any of the material mentioned is or was part of the common general knowledge at the priority 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 high concentrations of inhaled chelating agents.

[0015] Preferably, the high concentration inhaled chelating agent is greater than 37.5 mg / dose.

[0016] Preferably, the high concentration inhaled chelating agent is greater than 50 mg / dose.

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

[0018] In one form of the invention, the high concentration chelating agent is provided in a formulation 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 for a total dose of up to about 1,200 mg / day, preferably at least 150 mg / day.

[0019] Preferably, 37.5 mg to 1,200 mg of chelating agent is administered per day. Preferably, at least 50 mg of chelating agent is administered per day. The chelating agent can be administered one to four times daily for a total daily dose of up to about 1,200 mg per day.

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

[0021] Preferably, the chelating agent is CaEDTA.

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

[0023] The present invention also provides a method for treating or preventing inflammation in the lungs by administering high concentrations of an inhaled chelating agent, wherein the treatment or prevention of inflammation involves a reduction in matrix metalloproteinase (MMP) activity.

[0024] The present invention further provides a method of treating or preventing inflammation in the lungs by administering high concentrations of an inhalable chelating agent, wherein the treatment or prevention of inflammation involves a reduction in the production of hydroxyl radicals.

[0025] The present invention provides inhalation formulations containing high concentrations of chelating agents.

[0026] The present invention provides inhalable formulations containing high concentrations of chelating agents, and is capable of delivering high concentrations of inhalable chelating agents in a single dose.

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

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

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

[0030] 1. Use of an inhalable chelating agent in the manufacture of a medicament for delivering a high concentration of the inhalable chelating agent as a single dose to treat or prevent inflammation in the lungs.

[0031] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention. It should not be understood as limiting the broad overview, disclosure, or description of the invention as presented above. Reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows that submicron particles of EDTA kill P. aeruginosa biofilms and act synergistically with tobramycin in vitro. P. aeruginosa biofilms in CF mucus were treated with aerosolized EDTA particles and / or tobramycin. The final concentration of tobramycin in the droplets was 325 μg / ml. Figure 1A) Confocal microscopy images of biofilms stained with BacLight LIVE / DEAD; Figure 1B) Bacterial counts showing the quantitative effect of treatment. [Figure 2] CaEDTA reduces bacterial burden in CF lungs more rapidly than antibiotic treatment alone. CF subjects were treated with nebulized CaEDTA (EDTA) or saline (placebo) and the bacterial load in expectorated mucus was monitored (colony forming units per gram of mucus). [Figure 3] Figure 3A shows the mean change in FEV1 (% points) in patients treated with CaEDTA or placebo from the start of treatment over the entire 10-week period (4 weeks post-treatment). Figure 3B shows the relationship between improvement in FEV1 (0-2 weeks) and body weight. [Figure 4] FIG. 1 shows the EDTA concentrations achieved in the sputum of three CF subjects 5 minutes and 2 hours after treatment with 75 mg nebulized CaEDTA. [Figure 5]We demonstrate that administration of high concentrations of chelators to mouse lungs reduces inflammation in the absence of infection. Total leukocyte counts in bronchoalveolar lavage fluid (BALF) from mice exposed to air or cigarette smoke (CS) and treated with either vehicle or intranasal deferoxamine (DFO) are shown. As expected, cigarette smoke induces an increase in leukocyte counts, but treatment with DFO significantly reduces this effect. [Figure 6] We demonstrate that administration of high concentrations of chelators to mouse lungs reduces inflammation in the absence of infection. Lung weights are shown for mice exposed to air or CS and treated with vehicle or DFO as described above. Lung weight can be used as a proxy for inflammation, as increased weight indicates more inflammation. In CS-treated mice, lung weight significantly increased, as expected, and DFO treatment reduced mean weight, suggesting reduced inflammation. [Figure 7] These results demonstrate that administration of high concentrations of chelators to mouse lungs reduces inflammation in the absence of infection. CS increases BALF iron content, while treatment with deferoxamine attenuates this effect. Left: Mean iron content for each group of mice; Right: Scatter plot of the same data. DETAILED DESCRIPTION OF THE INVENTION

[0033] [Method of treatment or prevention] The present invention provides a method for treating or preventing inflammation in the lungs by administering high concentrations of inhalable chelating agents.

[0034] Preferably, the high concentration inhaled chelating agent is greater than 37.5 mg / dose.

[0035] Preferably, the high concentration inhaled chelating agent is greater than 50 mg / dose.

[0036] It has previously been shown that inhaled EDTA alone does not treat bacterial infections (Brown 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 nebulized sodium EDTA for 3 months and observed no changes in lung function. Others have reported that EDTA causes concentration-dependent bronchoconstriction (Beasley et al., Br Med J (Clin Res Ed). 1987, 294(6581):1197-8), and that EDTA has no effect on FEV1 (Asmus et al., J Allergy Clin Immunol. 2001, 107(1):68-72). Thus, there is no reason to believe that chelating agents have any positive effect on subjects, such as those with cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), or other lung conditions that cause or are associated with inflammation. However, the present inventors have surprisingly found that inhaled chelating agents can treat or prevent pulmonary inflammation.

[0037] It is commonly believed that the CF lung environment is acidic. However, it has recently been shown that CF lungs have the same pH as normal lungs (Schultz et al., "Airway surface liquid pH in children with cystic fibrosis," Nature Communications 2017, Vol. 8(1):1409). Therefore, current technologies using acidified nitrite, such as those discussed in U.S. Patent Application Publication No. 2016 / 0263151, are unlikely to work clinically in CF because the formulations do not remain acidified but quickly return to the normal lung pH of 7.4.

[0038] Although CF lungs have normal acidity, iron levels have been found to be significantly different from those of normal lungs. Stites et al. (Am J Respir Crit Care Med. 1999, 160(3):796-80) showed that iron levels are significantly elevated in the lungs of CF patients, as well as in the lungs of smokers, compared with healthy individuals. Most of this iron is in the ferrous form, Fe(II), which has also been shown to significantly correlate with disease severity (Hunter et al., MBio. 2013, 4(4):1-8). Ferrous iron can participate in the Fenton reaction, generating highly reactive oxygen radicals that can severely damage tissue and DNA (Jomova et al., Toxicology. 2011, 283(2-3):65-87; MacNee, Eur J Pharmacol. 2001, 429(1-3):195-207).

[0039] Without wishing to be bound by theory, the methods of the present invention are believed to reduce inflammation by (i) inactivating matrix metalloproteinases (MMPs) through zinc chelation, (ii) reducing reactive oxygen species (ROS) production through iron chelation, and / or (iii) reducing bacterial burden in the lungs by depriving bacteria of key ions such as iron and zinc. Each intrapulmonary effect may be one or any combination of the theorized ways of reducing inflammation.

[0040] Inhalation is a localized method of administration and therefore may be more effective in reaching the targeted area, i.e., the lungs, and can provide high and localized concentrations of the inhaled chelating agent. Inhalation avoids the undesirable side effects of systemic exposure to the active agent and reduces the risk of the patient developing tolerance.

[0041] The present invention further provides a method of treating or preventing inflammation in the lung by administering a high concentration of an inhaled 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 lungs by administering a high concentration of an inhaled chelating agent, wherein the treatment or prevention of inflammation involves a reduction in MMP activity. Matrix metalloproteinases (MMPs) have been shown to cause lung injury (Garratt et al., Eur Respir J. 2015, 46(2):384-94) and MMP activity is associated with the reduction of Zn 2+ It is known that MMPs are dependent on zinc (Hazra et al., Molecular Vision, 2012; 18:1701-1711). However, previous attempts to target MMPs in the lung have been unsuccessful. The present invention uses an inhalable chelating agent that chelates zinc in the lung, thereby reducing MMP-induced lung injury and treating or preventing inflammation.

[0043] The present invention further provides a method for treating or preventing inflammation in the lungs by administering high concentrations of an inhaled chelating agent, wherein the treatment or prevention of inflammation involves reducing the production of hydroxyl radicals. Iron is a major factor in lung injury because Fe catalyzes the formation of hydroxyl radicals (Stites et al., Am J Respir Crit Care Med., 1999, 160(3):796-80). However, antioxidant therapeutic trials have so far failed to produce significant improvements in lung function. The present invention uses an inhaled chelating agent to chelate iron in the lungs, thereby reducing hydroxyl radical-induced lung injury and treating or preventing inflammation.

[0044] The present invention further provides a method for treating or preventing infections in the lungs by administering high concentrations of an inhaled chelating agent, wherein the treatment or prevention of inflammation is achieved by the presence of the chelating agent removing or reducing biofilm produced by bacteria in the lungs, allowing for increased removal of bacteria and biofilm through coughing and expectoration.

[0045] The present invention further provides a method for treating or preventing inflammation in the lungs by administering high concentrations of inhaled chelating agents, where the treatment or prevention of inflammation is achieved by removing or reducing protease enzymes produced by bacteria that stimulate local inflammation, cause local tissue damage, and can neutralize antibiotic activity. These enzymes are primarily cation-dependent, and removing cations from the environment is expected to deactivate 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 (an 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 an iron chelating agent and an iron / zinc chelating agent.

[0047] The chelating agent is preferably selected from the group consisting of citric acid, phosphate, disodium, trisodium and tetrasodium salts of ethylenediaminetetraacetic acid (EDTA), calcium salt of EDTA, ethylene glycol-bis-(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N'-diglycine (EDDA); 2,2'-(ethylenediimino)-dibutanoic acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylenetetramine dihydrochioride (TRIEN), diethylenetriaminepentaacetic acid (DPTA), triethylenetetraminehexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicillamine, succimer, editronate, sodium hexametaphosphate, calcium disodium edetate, D-penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, and clioquinol, 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 about the same affinity for iron as EDTA, and / or about the same affinity for zinc as EDTA. The formation or stability constant (log K1) for EDTA at 25°C and 0.1M is 2+ 14.3 for Fe 3+ 25.1 for iron and 16.5 for zinc.

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

[0052] In one embodiment, the chelating agent is provided in an inhalable dosage form containing 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 37.5 mg / dose and 200 mg / dose, between about 50 mg / dose and 200 mg / dose; preferably about 37.5 mg / dose, 50 mg / dose, 75 mg / dose, 100 mg / dose, 200 mg / dose, or 300 mg / dose. The chelating agent is preferably provided in an inhalable dosage form containing at least 37.5 mg / dose. The chelating agent is preferably provided in an inhalable dosage form containing at least 50 mg / dose.

[0053] The total amount of chelating agent inhaled per day is preferably between about 37.5 mg / day and 1,200 mg / day, between about 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 150 mg / day, 300 mg / day, 500 mg / day, or 600 mg / day.

[0054] The total amount of drug inhaled per day is preferably between about 0.1 mg chelating agent / kg body weight and 15 mg chelating agent / kg body weight, between about 0.5 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 mg 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. The dosages are: 1.5mg chelating agent / kg body weight, 2.0mg chelating agent / kg body weight, 2.5mg chelating agent / kg body weight, 3.0mg chelating agent / kg body weight, 3.5mg chelating agent / kg body weight, 4.0mg chelating agent / kg body weight, 4.5mg chelating agent / kg body weight, 5.0mg chelating agent / kg body weight, 10mg chelating agent / kg body weight, and 15mg chelating agent / kg body weight.

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

[0056] The inhaled chelating agent is preferably delivered over a period of 8, 7, 6, 5, 4, 3, 2, 1, 45, 30, 20, 15, 10, or 5 minutes or less. If administration is by dry powder delivery, the inhaled chelating agent can be delivered over a period of several seconds, e.g., 1 second per "puff" of an aerosol device or dry powder inhaler, with one or more puffs administered at each time point.

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

[0058] Some subjects may benefit from a period of "loading" the subject with antibiotics and / or chelating agents, with higher or more frequent administration over a period of several days or weeks, followed by administration of lower or maintenance doses.

[0059] Therefore, the present invention provides Delivering a total dose of inhaled chelating agent between 37.5 mg / day and 1,200 mg / day, administered at least once per day and up to six times per day, preferably up to four times per day; Administered over a period of 8 hours or less.

[0060] Preferably, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis. Delivering a total dose of inhaled chelating agent between 37.5 mg / day and 1,200 mg / day, Administered once or twice daily, administered over a period of one hour or less per dose; Contains CaEDTA as a chelating agent.

[0061] The preferred amount of any chelating agent can be calculated by comparing the chelating capacity of the agent to that of CaEDTA and then multiplying that number by the dosage range 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 selected from the group consisting of cystic fibrosis (CF); asthma; chronic obstructive pulmonary disease (COPD); pulmonary hypertension; lung cancer; pulmonary fibrosis; bronchiectasis; acute respiratory distress syndrome; tuberculosis; non-tuberculous mycobacterial (NTM) lung infections; pneumonia, including but not limited to ventilator-associated pneumonia, community-acquired pneumonia, bronchopneumonia, lobar pneumonia; bacteria, such as Pseudomonas spp., Streptococcus pneumoniae, Chlamydia, Mycoplasma pneumonia, Staphylococcus spp., Klebsiella spp., E. coli, Stenotrophomonas spp., and Aspergillus, Scedosporium, and Candida spp. fungal infections, including those caused by fungi, including Pseudomonas sp.; prophylactic treatment or prevention of conditions in which infection may occur, for example, in intubated or ventilated patients; infections in lung transplant patients; caused by, causing, or associated with pulmonary conditions such as bronchitis; whooping cough (pertussis); inner ear infections; streptococcal throat infections; pulmonary anthrax; tularemia; or sinusitis.

[0063] Preferably, the formulation is administered to a subject in need thereof between about once per day and about six times per day, more preferably about four times per day.

[0064] Alternatively, the formulation can be administered to a subject in need thereof via a nebulizer via continuous inhalation. The nebulized formulation can be delivered for 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 24-hour period.

[0065] A subject will typically receive about 0.01 to 15 mg / kg / day of chelating agent, ±20% or ±10%. This dose is typically administered by nebulization or by at least one, and preferably several, "puffs" from an aerosol device. For example, a subject may receive a single dose of between 0.1 mg / kg and 15 mg / kg of chelating agent, or several doses over the course of a day.

[0066] The total daily dose is preferably administered at least once a day, but can be divided into two or more doses a day.Some patients can benefit from the period of "loading" the patient with chelating agent, which is administered at a higher dose or more frequently for a period of several days or weeks, followed by the administration of a lower dose or maintenance dose.Because cystic fibrosis, COPD, etc. are usually chronic conditions, patients are expected to undergo this therapy for a long time.

[0067] Regardless of the form of the drug formulation, it is preferable to produce droplets or particles for inhalation in the range of about 0.1 μm to 12 μm, or about 0.25 μm to 6 μm, preferably 1 μm to 6 μm, and more preferably about 2 μm to 4 μm. Alternatively, the particles may be 0.1 μm to 1.0 μm, 0.2 μm to 0.9 μm, 0.3 μm to 0.8 μm, 0.4 μm to 0.7 μm, or 0.5 μm. By producing particles for inhalation with a relatively narrow size range, it is possible to further increase the efficiency of the drug delivery system and improve dosing reproducibility. Thus, it is preferable that not only do the particles have a size range of 0.1 μm to 12 μm, 2 μm to 6 μm, or about 3 to 4 μm, but also that the mean particle size be within a narrow range so that 80% or more of the particles delivered to the subject have a particle diameter within ±20% of the mean particle size, preferably ±10%, and more preferably ±5% of the mean particle size.

[0068] "Particle size" is a concept introduced to compare the size of solid particles, liquid particles (droplets). For droplets and aerosols, terms such as "aerodynamic diameter" and "mass median aerodynamic diameter (MMAD)" are used. Definitions are given below.

[0069] "Aerodynamic diameter" is the diameter of a unit-density sphere that has the same critical settling velocity as the particle of interest. It is used to predict where such particles will deposit in the respiratory tract.

[0070] "Mass median aerodynamic diameter" is the geometric mean aerodynamic diameter. 50 percent of the particles, by weight, are smaller than the MMAD and 50% of the particles, by weight, are larger.

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

[0072] D 50 :D 10 Similarly, D 50 is the cutoff diameter for 50% of the particle population in a formulation and refers to a particular particle diameter such that 50% of the particles in suspension have a diameter less than or equal to this particular particle diameter.

[0073] D 90 :D 90 is the cutoff diameter for 90% of the particle population in a formulation and refers to a particular particle diameter such that 90% of the particles in suspension have a diameter less than or equal to this particular particle diameter.

[0074] The term "respiratory system" shall be taken to mean the system of cells and organs that function in respiration, and in particular respiratory organs, tissues and cells include the lungs, nose, nasal passages, paranasal sinuses, nasopharynx, larynx, trachea, bronchi, bronchioles, respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli, pneumocytes (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 a therapeutically or prophylactically effective concentration of an inhalable chelating agent in one or more doses of at least 37.5 mg per dose, wherein the or each dose 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 a therapeutically effective concentration of an inhalable chelating agent in one or more doses of at least 37.5 mg per dose, wherein the or each dose of the chelating agent is administered over a period of 8 hours or less.

[0077] In one form of the invention, a method for preventing inflammation in the lungs of a subject by administering a prophylactically effective concentration of an inhaled chelating agent in one or more doses of at least 37.5 mg per dose, wherein the or each dose of the chelating agent is administered 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 comprises treating or preventing inflammation in the lungs of a subject in need of such treatment.

[0079] The term "therapeutically effective amount," as used herein, means an amount of a formulation that, when administered according to a desired dosing regimen, is sufficient to at least partially achieve a desired therapeutic effect, or to delay the onset or inhibit the progression, or partially or completely halt the onset or progression of inflammation.

[0080] The term "prophylactically effective amount," as used herein, means an amount of a formulation sufficient to at least partially prevent or delay the onset of inflammation when administered according to a desired dosing regimen.

[0081] As used herein, "treating" or "treatment" refers to inhibiting a disease or condition, i.e., halting or reducing its onset or at least one of its clinical or asymptomatic symptoms. "Treatment" or "treatment" also refers to alleviating a disease or condition, i.e., causing regression of a disease or condition or at least one of its clinical or asymptomatic symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the subject and / or physician. In the context of treating inflammation, the term "treatment" includes reducing or eliminating one or more of the following: leukocyte infiltration (including macrophages, polymorphonuclear neutrophils, lymphocytes, and other immune cells); immunoglobulins; proinflammatory cytokines and chemokines and their receptors; nociceptive mediators, such as ROS and proteolytic enzymes; MMP abundance and activity; markers of oxidative stress; and bronchial hyperresponsiveness and exacerbation. The term "treatment" further includes one or more of increasing anti-inflammatory cytokines and increasing pulmonary function (FEV1).

[0082] Based on the above, those skilled in the art will understand that a single subject can be treated using multiple different treatments and administration methods. Thus, subjects already receiving such drugs, such as intravenous ciprofloxacin or antibiotics, can benefit from inhalation of the formulations of the present invention. Some subjects can receive only the high-concentration chelating agent formulation of the present invention via inhalation. Such subjects may have symptoms of cystic fibrosis, may have been diagnosed with a pulmonary infection, or may have symptoms of a medical condition that can benefit from the administration of a high-concentration chelating agent to the subject. The formulations of the present invention can also be used for diagnosis. In one embodiment, for example, a subject can receive the formulation of the present invention as part of a procedure to diagnose a pulmonary infection, and one or more of the subject's symptoms improve in response to the formulation.

[0083] [formulation] The present invention provides inhalation formulations containing high concentrations of chelating agents.

[0084] The inhalable formulation may be in the form of a dry powder for inhalation or in the form of a spray for inhalation. Preferably, the formulation is adapted for inhalation to treat or prevent inflammation in the lungs.

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

[0086] Preferably, the high concentration inhalant chelating agent is greater than 37.5 mg / dose. Preferably, the high concentration inhalant chelating agent is greater than 50 mg / dose. Preferably, the high concentration chelating agent is provided in a formulation containing 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 about 50 mg / dose, 75 mg / dose, 100 mg / dose, 200 mg / dose, or 300 mg / dose. The chelating agent is preferably provided in an inhalable dosage form containing at least 37.5 mg / dose. The chelating agent is preferably provided in an inhalable dosage form containing at least 50 mg / dose.

[0087] The total amount of chelating agent inhaled per day is preferably between about 37.5 mg / day and 1,200 mg / day, between 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 dose of up to about 1,200 mg / day, preferably at least 150 mg / day.

[0088] The total amount of chelating agent inhaled per day is preferably between about 37.5 mg / day and 1,200 mg / day, between about 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 150 mg / day, 300 mg / day, 500 mg / day, or 600 mg / day.

[0089] The total amount of chelating agent inhaled per day is preferably between about 0.1 mg chelating agent / kg body weight and 15 mg chelating agent / kg body weight, between about 0.5 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 mg 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 between about 1.0 mg chelating agent / kg body weight and 5 mg chelating agent / kg body weight. The dosages are: 1 mg chelating agent / kg body weight, 1.5 mg chelating agent / kg body weight, 2.0 mg chelating agent / kg body weight, 2.5 mg chelating agent / kg body weight, 3.0 mg chelating agent / kg body weight, 3.5 mg chelating agent / kg body weight, 4.0 mg chelating agent / kg body weight, 4.5 mg chelating agent / kg body weight, 5.0 mg chelating agent / kg body weight, 10 mg chelating agent / kg body weight, and 15 mg chelating agent / kg body weight.

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

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

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

[0093] The formulations of the present invention can be administered to a subject using disposable packaging and portable, handheld, battery-powered devices, such as the AERx device (U.S. Patent No. 5,823,178, Aradigm, Hayward, Calif.). Alternatively, the formulations of the present invention can be administered using mechanical (non-electronic) devices. Other inhalation devices can be used to deliver the formulations, including conventional jet nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powder inhalers (DPIs), metered dose inhalers (MDIs), condensation aerosol generators, and other systems.

[0094] For use as an aerosol, the compounds of the present invention can be packaged in a solution or suspension in a pressurized aerosol container together with conventional adjuvants and a suitable propellant, such as a hydrocarbon propellant such as propane, butane, or isobutane. Dry powder inhalers are systems that can be operated using a source of pressurized air to produce dry powder particles of the pharmaceutical formulation compressed into a very small volume. For inhalation, the system has multiple chambers or blisters, each containing a single dose of the pharmaceutical formulation and a selection element for releasing the single dose.

[0095] Aerosols can be created by forcing a drug through pores in a membrane, the pores having a size ranging from 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 will have diameters ranging from 0.5 to 12 μm. The drug particles can be released with an airflow intended to keep the particles within this size range. The production of small particles can be facilitated by the use of a vibration device that provides a vibration frequency ranging from about 800 to about 4000 kilohertz. While those skilled in the art will recognize that some adjustments can be made in parameters such as the size of the pores through which the drug is released, the vibration frequency, the pressure, and other parameters based on the density and viscosity of the formulation, it should be noted that the objective of some embodiments is to provide aerosolized particles having diameters ranging from about 0.5 to 12 μm.

[0096] [Excipients] The above-exemplified forms of the formulations described herein can be prepared by methods well known to those skilled in the art of formulation science. In addition, the formulations described herein can contain other optional excipients that are useful in the preparation and / or administration of the formulations described herein. Non-limiting examples of such excipients are well known in the art and include flavoring agents, coloring agents, paratants, antioxidants, viscosity modifiers, tonicity adjusters, drug carriers, sustained-release agents, comfort aids, emulsifiers, solubilization aids, lubricants, binders, and other stabilizers that are useful in the preparation and / or administration of the formulations.

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

[0098] Furthermore, a buffering agent can be added to adjust the pH level of the formulation. Preferably, the formulation of the present invention contains tris(hydroxymethyl)aminomethane (TRIS, also known as THAM, or tromethamine) as a buffering agent. TRIS may have the additional effect of increasing the bacterial killing effect of EDTA. Preferably, TRIS is added to the formulation of the present invention both to buffer the formulation and to increase the effectiveness of EDTA and / or antibiotics in treating or preventing bacterial infections.

[0099] Additionally, the formulations of the present invention may contain an antimicrobial preservative.

[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 shown that bacteria become more resistant to antimicrobial therapy as the pH drops. A preferred pH helps avoid bacterial resistance to formulations containing high concentrations of inhalable chelating agents in combination with antibiotics in the absence of acidifying nitrite.

[0101] In an alternative embodiment, the formulations of the present invention may contain preservatives, suspending agents, wetting agents, tonicity agents, and / or diluents. The formulations provided herein may contain from about 0.01% to about 90%, or from about 0.01% to about 50%, or from about 0.01% to about 25%, or from about 0.01% to about 10%, or from about 0.01% to about 5% of one or more pharmacologically suitable suspending fluids that are physiologically acceptable for administration by inhalation. Pharmacologically 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, and glycols, including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, and polyoxyethylene alcohols. Polar solvents also include protic solvents, including, but not limited to, water, aqueous saline with one or more pharmaceutically acceptable salt(s), alcohols, glycols, or mixtures thereof. In an alternative embodiment, water for use in the formulations of the present invention should meet or exceed applicable legal requirements for use in inhaled drugs.

[0102] In one embodiment, the formulations described herein may be aqueous and contain 0-90% water. In other embodiments, the aqueous formulations described herein may contain 20-80% water. In yet other embodiments, the aqueous formulations may contain 50-70% water. The water may be fresh water and may further include distilled, sterile, demineralized, or deionized water.

[0103] Alternatively, the formulation may be non-aqueous and contain no water or only trace amounts of water (eg, 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 a pharmaceutically acceptable preservative to minimize the possibility of microbial contamination. Furthermore, pharmaceutically acceptable preservatives can be used in the formulations of the present invention to increase the stability of the formulation. However, it should be noted that any preservative must be selected for inhalation safety, as the treated tissue may be sensitive to irritants. Suitable preservatives for use herein include, but are not limited to, those that protect the solution from contamination with pathogen particles, such as phenylethyl alcohol, benzalkonium chloride or benzoic acid, or benzoate salts such as sodium benzoate, and phenylethyl alcohol. In certain embodiments, the formulations herein contain about 0.001% to about 10.0% w / w benzalkonium chloride or about 0.01% v / w phenylethyl alcohol. Preservatives may also be present in amounts of about 0.001% to about 1%, preferably about 0.002% to about 0.02%, and more preferably 0.02% w / w.

[0106] The formulations provided herein may also contain from about 0.001% to about 90%, or from about 0.001% to about 50%, or from about 0.001% to about 25%, or from about 0.001% to about 10%, or from about 0.001% to about 1% of one or more emulsifying agents, wetting agents, or suspending agents.Such agents for use herein include, but are not limited to, polyoxyethylene sorbitan fatty acid esters or polysorbates, including, but not limited to, polyethylene sorbitan monooleate (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; carrageenum; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; gum asiatica; pectin; amidated pectin; ammonium phosphatides; microcrystalline cellulose; methylcellulose; hydroxypropyl cellulose; hydroxypropylmethylcellulose; ethylmethylcellulose; carboxymethylcellulose; sodium, potassium, and calcium salts of fatty acids; mono- and diglycerides of fatty acids. ;Acetate esters of mono- and diglycerides of fatty acids;Lactate esters of mono- and diglycerides of fatty acids;Citrate esters of mono- and diglycerides of fatty acids;Tartrate esters of mono- and diglycerides of fatty acids;Monoacetyltartaric and diacetyltartaric esters of mono- and diglycerides of fatty acids;Mixed acetate and tartaric acid esters of mono- and diglycerides of fatty acids;Sucrose esters of fatty acids;Sucroglycerides;Polyglycerol esters of fatty acids esters; polyglycerol esters of polycondensed fatty acids of castor oil; propane-1,2-diol esters of fatty acids; sodium stearoyl-21 actylate; calcium stearoyl-2-lactylate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopalmitate; Quillaja extract; polyglycerol esters of dimerized fatty acids of soybean oil; oxidatively polymerized soybean oil; and pectin extract.

[0107] The formulations of the present invention may contain about 0.001% to about 5% by weight of a humectant to inhibit drying of the mucous membrane and prevent irritation. Any of a variety of pharmaceutically acceptable humectants can be utilized, including sorbitol, propylene glycol, polyethylene glycol, glycerol, or mixtures thereof.

[0108] The formulations of the present invention may further include an adjuvant such as a bronchodilator, another anti-inflammatory agent, a surfactant, aspirin, or ethyl alcohol.

[0109] Bronchodilators optionally used in the formulations of the present invention include, but are not limited to, β2-adrenergic receptor agonists (e.g., albuterol, bambuterol, salbutamol, salmeterol, formoterol, arformoterol, levosalbutamol, procaterol, indacaterol, carmoterol, mirveterol, procaterol, terbutaline, etc.) and antimuscarinics (e.g., trospium, ipratropium, glycopyrronium, aclidinium, etc.). Combinations of drugs may 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, amelbant, tipelukast, etc.), cyclooxygenase inhibitors (e.g., ibuprofen, ketoprofen, ketorolac, indomethacin, naproxen, zaltoprofen, lornoxicam, meloxicam, celecoxib, lumiracoxib, etoricoxib, piroxicam, ampiroxicam, cinnoxicam, diclofenac, felbinac, lornoxicam, mesalazine, triflusal, tinoridine, iguratimod, pamicogrel, etc.). A combination of drugs can be used. Aspirin, which acts as an anti-inflammatory, can also be added.

[0111] Surfactants encompassed by the present invention include, but are not limited to, synthetic surfactant (Exosurf®), dipalmitoyl phosphatidylcholine, and oleic acid. Combinations of drugs may be used.

[0112] Antioxidants such as glutathione and vitamin E, zinc and the zinc salt of EDTA can be added.

[0113] Ethyl alcohol vapor acts as a defoaming agent in the lungs, making phlegm more liquefied, thereby assisting breathing and reducing pulmonary edema. Ethanol can be added to the formulations of the present invention at concentrations between 0.5% and 60%, more preferably between 1 and 40%, between 1 and 20%, or between 1 and 10%. Ethanol can be added at concentrations of 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 high concentrations of chelating agents in combination with other drugs administered via inhalation. These other drugs may include nucleotide sequences that may be incorporated into suitable delivery vectors, such as plasmids or viral vectors. Other drugs include therapeutic nucleotide sequences (DNA, RNA, siRNA), enzymes that reduce mucus viscoelasticity, such as DNase and other mucolytic agents, chemicals that upregulate chloride ion channels or increase the transcellular flux of ions, nicotine, P2Y2 agonists, elastase inhibitors including alpha-1 antitrypsin (AAT), N-acetylcysteine, antibiotics, and cationic peptides, such as lantibiotics, specifically duramycin, short-acting bronchodilators (e.g., beta-2-adrenergic receptor agonists like albuterol or indacaterol), M3 muscarinic antagonists (e.g., ipratropium bromide), K +-channel openers, long-acting bronchodilators (e.g., formoterol, salmeterol), steroids (e.g., budesonide, 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 inhibitors (AVE-0950), tryptase inhibitors (AVE-8923 and AVE-5638), tachykinin antagonists (AVE-5883), inducible nitric oxide synthase inhibitors (GW-274150), and others), transcription factor decoys, TLR-9 agonists, antisense oligonucleotides, siRNA, DNA, CGRP, lidocaine, reverse The therapeutic agent may be a beta2-agonist, an anti-infective oxidative therapy, a cytokine modulator (e.g., CCR3 receptor antagonists (GSK-766994, DPC-168, AZD-3778), TNF-α production inhibitors (LMP-160 and YS-TH2), and an IL-4 antagonist (AVE-0309)), a small molecule inhibitor of IgE, a cell adhesion molecule (CAM) inhibitor, a small molecule targeting the VLA4 receptor or integrin alpha 4 beta 1 (e.g., R-411, PS-460644, DW-908e, and CDP-323), an immunomodulator including those that block T cell signaling by inhibition of calcineurin (tacrolimus), a heparin neutralizer (talactoferrin alpha), a cytosolic PLA2 inhibitor (Efipladib), or a combination thereof. If a subject in need has CF, the subject can also be administered standard drugs, such as ivacaftor, pulmozyme, mannitol, etc., or other approved drugs, in combination with the formulations of the present invention, according to standard practice.

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

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

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

[0118] The term "inflammation," as used herein, refers to one or more symptoms of the body's response to an insult, such as an infection, an environmental insult (including cigarette smoke), a trauma, or an allergic reaction. Inflammation can be either acute or chronic, and symptoms include tissue swelling, recruitment of different types of inflammatory cells, release of cytokines and mediators, and bronchial hyperresponsiveness. Inflammation can be localized and asymptomatic or transient, or it can be more widespread and chronic. Inflammation can involve both humoral and cellular immune responses and can persist even after the insult that triggered it has been removed. Signs of inflammation include, but are not limited to, elevated levels of inflammatory cells (e.g., dendritic cells, macrophages, neutrophils, lymphocytes, eosinophils, and mast cells), elevated levels of proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6, IL-8, and IFNγ) and their receptors, excess proteases including MMPs, ROS, and other mediators, and markers of inflammation such as C-reactive protein (CFP) and sputum and serum calprotectin. Short-term inflammation ("acute") leads to airway swelling, altered lung compliance, airway reactivity, and mucus hypersecretion, along with clinical symptoms that may include increased respiratory rate and dyspnea, wheezing, cough, and reduced FEV1, which, if sustained ("chronic"), leads to structural damage to the airway wall and lung parenchyma in the form of fibrosis, cystic changes, and bronchiectasis.

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

[0120] 1. Use of an inhalable chelating agent in the manufacture of a medicament for delivering a high concentration of the inhalable chelating agent as a single dose to treat or prevent inflammation in the lungs.

[0121] Preferably, the high-concentration chelating agent is provided in a formulation containing at least 37.5 mg / dose, at least 50 mg / dose, or between 50 mg / dose and 300 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 dose of up to about 1,200 mg / day, preferably 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 formulation containing a high concentration of a chelating agent, and (ii) instructions for use.

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

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

[0125] In one embodiment, the kit of the present invention includes a formulation comprising a therapeutically effective amount of a highly concentrated inhaled chelating agent. In an alternative embodiment, the formulation is pre-measured, pre-mixed, and / or pre-packaged. Preferably, the inhalation solution is sterile.

[0126] The kits of the present invention may also include instructions designed to promote user compliance. Instructions, as used herein, refer to any label, package insert, etc., and may be located 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 kits of the present invention include instructions for administering the formulations of the present invention. In one embodiment, the instructions indicate that the formulations of the present invention are suitable for treating pulmonary inflammation. Such instructions may also include instructions for the formulation as well as instructions for administration via a nebulizer or dry powder inhaler.

[0127] The inhalation chelating agent and any other active agent can be individually packaged, so that doctors or users can formulate each into pharmaceutical preparations as needed.Alternatively, the pharmaceutical preparations that include the inhalation chelating agent and any other active agent can be packaged together, and thus require doctors or users to prepare a minimum amount of formulation.In any case, the packaging should maintain the chemical, physical and aesthetic integrity of active ingredients.

[0128] [General rules] Those skilled in the art will recognize that the invention described herein may have variations and modifications other than those specifically described.The invention includes all such variations and modifications.The invention also includes all of the steps, features, preparations and compounds mentioned or shown in the specification, individually or collectively, 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 expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this text. It is solely for reasons of brevity that documents, references, patent applications, or patents cited in this text are not repeated in this text.

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

[0131] The present invention is not intended to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention as described herein.

[0132] The invention described herein may include one or more ranges of values ​​(e.g., size, displacement, and magnetic field strength). Ranges of values ​​are intended to include all values ​​within the range, including the values ​​defining the range and values ​​immediately adjacent to the values ​​defining the boundaries for the range that produce the same or substantially the same results. Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Thus, "about 80%" means "about 80%," as well as "80%." At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0133] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations thereof, such as "comprises" or "comprising," are intended to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Within this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like, can have the meaning ascribed to them in U.S. patent law. For example, they can mean "includes," "included," "including," and the like, and it should also be noted that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they permit elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0134] Other definitions for selected terms used herein can be found in the detailed description of the invention and are applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The term "active agent" may refer to one active agent or may include two or more active agents.

[0135] The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these methods in no way serve to limit the true scope of the invention, but rather are presented for illustrative purposes. [Example]

[0136] Further features of the present invention are more fully described in the following non-limiting examples, which are included solely for purposes of illustrating the invention and should not be understood as a limitation on the broad description of the invention as set forth above.

[0137] [Example 1] Methods for treating or preventing pulmonary infections by administering high concentrations of inhaled chelating agents Droplets of suspended cystic fibrosis mucus collected from an epithelial cell line were used to grow biofilms in a realistic in vitro model (Haley et al., BMC Microbiol 2012, 12:181). Cultures of a clinical strain of P. aeruginosa (MIC of tobramycin >256 μg / ml) were grown in M63 without a carbon source to mimic nutrient limitation and transition to late stationary phase.

[0138] A few drops of mucus (5 μl) were suspended from an inverted IBIDI coverslip and 3 The droplets were inoculated with colony-forming units (CFU) of 1000 kJ / ml and then incubated in a humidified environment at 35°C for 72 hours to allow biofilm development. The droplets were then treated with either nebulized tobramycin (20 mg / ml), aerosolized CaEDTA particles (10 mg / ml), or both for 5 minutes. Controls were treated with a 50 / 50 solution of nebulized 0.9% saline / water. After treatment, the droplets were incubated for 16 hours and then stained with BacLight LIVE / DEAD (1 μl) and fixed with paraformaldehyde vapor for 30 minutes. Biofilms were visualized using confocal microscopy.

[0139] Figure 1A shows a thick and strong biofilm with mostly viable cells (green) after treatment with nebulizing saline. As expected for the resistant strain, treatment with tobramycin alone has little effect on viability. EDTA alone causes some killing (red blood cells). The combination of tobramycin and EDTA significantly kills the majority of biofilm cells. Figure 1B shows a quantitative representation of the microscopy image of Figure 1A. The control biofilm contained 1 x 10 8 CFU / ml, whereas EDTA-tobramycin-treated biofilms reduced the number by >6 orders of magnitude and <10 2 CFU / ml.

[0140] Patients with CF aged ≥6 years who were hospitalized due to symptomatic exacerbation were randomized to receive EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA was administered along with the tobramycin as a nebulized solution of 4 ml of 50 mM CaNa2EDTA, 111 mM Tris in 0.9% saline, pH 7.1. After randomization, subjects were treated in the hospital for 2 weeks, during which time they received treatment four times daily (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). Patients were then discharged and treatment continued twice daily for 4 weeks. Patients were monitored for an additional 4 weeks, bringing the total study time to 10 weeks.

[0141] Sputum was induced with nebulized 3% hypertonic saline at 8-10 L / min 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 Glycerol storage medium and stored at -80°C.

[0142] Sputum samples were obtained by expectoration from subjects at initial screening, then at visit 3 (approximately 2 weeks later), visit 5 (week 6), and finally at follow-up (week 10). Mucus was dissociated from clear sputum, treated with Sputalysin (1 ml per gram of sputum), placed in Skim Milk Glycerol storage medium (1 ml / 100 mg of mucus), vortex mixed, and stored at -80°C.

[0143] Samples were thawed on ice, serial dilutions were made up to 10 of the original concentration, and 20 μL of each dilution was placed onto each of three culture plates of McConkey (McC) agar or Blood agar (BA). Plates were incubated at 35°C.

[0144] Pseudomonas species were defined as clear or very pale pink lactose-negative colonies on McC agar plates. Rough-shaped colonies had a metallic sheen and rough colony edges, smooth-shaped colonies had regular colony borders on McC agar and grew slowly, and slimy-shaped colonies were surrounded by large amounts of alginate secreted by the bacteria.

[0145] The numbers of rough, smooth, and slimy colonies were counted at 24 hours on both McC agar and BA plates, and the plates were reincubated for another 24 hours, followed by confirmatory counts of each colony morphology. Single colonies of each morphology present in each sample were picked and streaked onto BA plates to obtain pure cultures.

[0146] Further differentiation was achieved by performing a Gram stain to confirm that the isolates consisted of Gram-negative, rod-shaped cells, and by confirming oxidase-positive status by rubbing a very small portion of the colony onto an oxidase test strip. Rapid development of a deep blue color indicates an oxidase-positive isolate.

[0147] Pseudomonas species were confirmed by testing for resistance to the C390 antibiotic. Antibiotic-impregnated disks were placed on nutrient agar (NA) plates containing a suspension of pure isolates in phosphate-buffered saline (PBS) to a McFarland density of 0.5. After overnight incubation at 35°C, the absence of any zone of inhibition around the disk indicated antibiotic resistance. Single colonies of each morphological type of confirmed Pseudomonas species (presumably P. aeruginosa) present within each isolate were picked, resuspended in glycerol / serum storage medium, and stored at -80°C. Figure 2 shows the change in colony counts for P. aeruginosa (McC) at 2 and 6 weeks compared to the start of treatment. After 2 weeks of treatment, the reduction in colony counts was >400-fold in the EDTA group compared to 4.5-fold in the placebo group.

[0148] [Example 2] Treatment of pulmonary inflammation results in a dose-dependent increase in FEV1 Subjects with CF aged ≥6 years who were hospitalized due to symptomatic exacerbation were randomized to receive EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA was administered along with the tobramycin in 4 ml of a nebulized solution of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1. After randomization, subjects were treated in the hospital for 2 weeks, during which time they received treatment four times daily (300 mg EDTA / day). After discharge, treatment was continued twice daily for 4 weeks. Subjects were monitored for an additional 4 weeks, bringing the total study time to 10 weeks.

[0149] At each study visit, pulmonary function was measured by spirometry. Data were recorded as the best of three attempts, and results were expressed as % of predicted value.

[0150] Figure 3A shows the mean change in FEV1 for both groups at 2, 6, and 10 weeks after the start of treatment. After 2 weeks, the mean increase in FEV1 was 16 percentage points in the EDTA group compared to 5 percentage points in the placebo group. This difference persisted 4 weeks after the completion of treatment, with a 7 percentage point increase in the EDTA group compared to a 2 percentage point increase in the placebo group. This demonstrates a clear improvement in lung function in the EDTA group, but only a slight change in the placebo group. Figure 3B shows the mean change in FEV1 for the EDTA group (R 2 =0.70), but not in the placebo group treated with tobramycin alone (R 2 =0.01), which indicates that EDTA has a dose-dependent effect (mg EDTA / kg body weight) on lung function.

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

[0152] [Example 3] Cigarette smoke-induced pulmonary inflammation can be treated by pulmonary administration of high doses of chelating agents The effects of chelators on lung inflammation were tested in a mouse model of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is known to induce lung inflammation, which can be measured by increased white blood cell counts and increased lung weight.

[0153] Male BALB / c mice (8 per group) were exposed to a defined dose of cigarette smoke (3 cigarettes, 3 times per day, Monday through Friday) or filtered room air for a 2-week period. During the experimental period, mice were treated intranasally with the iron chelator deferoxamine (DFO, 3.8 mg in 50 μl) or vehicle 30-60 minutes before each cigarette smoke exposure, 3 times per day. Mice were then sacrificed, and airways and lungs were assessed for effects on cigarette smoke-induced inflammation and elemental concentrations.

[0154] Bronchoalveolar lavage fluid (BALF) was collected (approximately 1 ml / mouse), and lungs were surgically removed and weighed. An equal volume of trypan blue was mixed with the BALF, and the total number of viable cells in the BALF was determined by manual counting in a standard Neubauer hemocytometer using 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 standards of known metal content.

[0155] Figure 5 shows that, as expected, cigarette smoke significantly increases the total number of BALF leukocytes, and treatment with the iron chelator DFO significantly reduces this effect.

[0156] Consistent with this, Figure 6 shows that mean lung weight is significantly increased by treatment with cigarette smoke, but treatment with CFO prevents this effect.

[0157] As previously described, Stites et al. (Am J Respir Crit Care Med. 1999, 160(3):796-80) demonstrated that iron levels were significantly elevated in the lungs of CF patients and smokers compared with healthy individuals. Figure 7 (left) confirms that mean iron levels were significantly increased in the BALF of mice exposed to cigarette smoke and that treatment with DFO reduced mean BALF iron content. Figure 7 (right) shows that in six of seven DFO-treated mice (one died for reasons unrelated to treatment), BALF iron content was similar to that of mice exposed to air.

[0158] Hypothetical Example P1: In vivo study of the effects of high-dose dry powder chelating agents on infection, inflammation, and oxidative stress. Subjects with CF requiring treatment with dry powder tobramycin will be assigned to four cohorts and will receive 112 mg of dry powder twice daily for 28 days. In addition, Cohort 1 (patients >18 years) will receive escalating 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) will receive CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks; 75 mg QID for 1 week). Cohort 3 (patients 12-18 years) will receive CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks; 150 mg BID for 1 week). Finally, the observational cohort will receive tobramycin alone for 28 days.

[0159] Sputum samples are collected weekly and evaluated for markers of infection and inflammation. Bacteria are monitored by sputum colony counts. As a measure of structural damage, levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are measured using gelatin zymography and immunoassay, respectively, 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). Iron levels in sputum are quantified by ICP-MS as previously described (Hunter et al., Mbio. 2013, 4(4):1-8). Iron-binding protein levels are assessed using immunoassays. Myeloperoxidase activity is also assayed as a measure of neutrophil inflammation as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). 3-Chlorotyrosine is measured as a biomarker for hypochlorous acid, a potent oxidant. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). Protein carbonyls are 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 assessed by measuring glutathione (GSSG and GSH) using immunoassays as previously described (Kettle et al., Eur Respir J. 2014, 44(1):122-9). Gene expression of inflammatory and oxidative stress markers (e.g., IL-8, IL-6, TNFα) is also monitored with Nanostring, and proteins are measured by ELISA. Oxidative stress can also be measured via metabolites, such as malondialdehyde (colorimetric assay) or 8-isoprostane (ELISA). Iron is measured by elemental analysis using laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS).

[0160] The study is expected to show reduced inflammatory markers and reduced iron levels in the EDTA group compared to the placebo group. The study is further expected to show a change in the balance between MMPs and TIMPs, particularly MMP-9 and TIMP-1, that accompanies the progression of bronchiectasis.

[0161] The study is further expected to show a reduction in sputum bacterial load and an increase in FEV1 in subjects treated with EDTA compared to control patients.

[0162] Hypothetical Example P2: In vitro studies of the effects of high doses of chelating agents on inflammation, lung injury, and oxidative stress. Lung epithelial cells grown in tissue culture were exposed to Fe(II) or excess oxygen to induce inflammation. Cells were treated with CaEDTA (0, 1, 5, 10, 25, or 50 mM) for 30 minutes, 1, 3, 24, and 48 hours.

[0163] Immunoassays are used to monitor changes in inflammatory markers, such as IL-6, IL-8, TNF-α, neutrophil elastase, and others. Oxidative stress and toxicity are measured by assessing reduced glutathione (GSH) levels and apoptosis based on the TUNEL assay, both of which are commercially available assay kits, such as the Glutathione Fluorescent Detection Kit from ThermoFisher Scientific and the TUNEL DNA Fragmentation Assay Kit from BioVision Inc.

[0164] This experiment is expected to show a concentration-dependent reduction in inflammatory markers in EDTA-treated cells compared to controls; a reduction in GSH (indicating reduced reactive oxygen species in EDTA-treated cells compared to controls); and reduced apoptosis in EDTA-treated cells compared to controls, as measured by TUNEL assay.

[0165] Hypothetical Example P3: In vivo study of the effects of high-dose nebulized chelating agents on inflammation, lung injury, and oxidative stress. Subjects with CF aged ≥6 years who are hospitalized due to symptomatic exacerbation will be randomized to receive nebulized EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA will be administered together with tobramycin in 4 ml of a nebulized solution of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1.

[0166] After randomization, subjects are treated in the hospital for two weeks, during which they receive treatment four times daily (up to 300 mg EDTA / day, or 3.3 mg EDTA / kg / day). Subjects are then discharged and treatment continues twice daily for four weeks. Subjects are monitored for an additional four weeks, for a total study time of 10 weeks. Sputum is collected by induction with nebulized 3% hypertonic saline at 8-10 L / min for ≥5 minutes. Samples are collected before treatment and at 2, 6, and 10 weeks, processed according to the relevant protocol, and stored at -80°C.

[0167] - Expression of inflammatory markers Sputum is stored in RNAlater®, and total RNA is extracted using Qiagen RNEasy® or a similar extraction kit, converted to cDNA, and inflammatory markers are monitored and quantified relative to known housekeeping genes, such as actin and / or GAPDH, using qPCR as described by Sivaneson et al. (Mol Microbiol, 79:1353-1366).

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

[0169] Cellular damage, free iron and oxidative stress Sputum samples were frozen without further processing and assayed for inflammatory markers as described above. As a measure of structural damage, levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were measured using gelatin zymography and immunoassay, respectively, 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). Sputum iron was quantified by ICP-MS as previously described (Hunter et al., MBio. 2013, 4(4):1-8). Iron-binding protein levels were assessed using immunoassays. Immunoassays are used to assess oxidative stress by measuring glutathione (GSSG and GSH) as previously described (Kettle et al., Eur Respir J. 2014, 44(1):122-9).

[0170] This study is expected to show a reduction in inflammatory markers in the EDTA group compared to the placebo group. This study is further expected to show a change in the balance between MMPs and TIMPs, particularly between MMP-9 and TIMP-1, which accompanies the progression of bronchiectasis.

[0171] Hypothetical Example P4: In vivo study of the effects of high doses of chelating agents on inflammation, lung injury, and oxidative stress. A single-center, randomized, double-blind, crossover study of subjects with cystic fibrosis will be conducted. Subjects will be randomized to two weeks of treatment with inhaled CaEDTA or saline (placebo). This will be followed by a washout period and then two weeks of the other treatment (EDTA or placebo).

[0172] Iron levels, inflammatory markers, MMP / TIMP, and FEV1 are monitored as described above. Myeloperoxidase activity is also assayed as a measure of neutrophil inflammation, as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). 3-Chlorotyrosine is measured as a biomarker for the potent oxidant hypochlorous acid. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., Eur Respir J. 2011, 38(3):721-727). Protein carbonyls are 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).

[0173] The study is expected to show reduced levels of iron and inflammatory markers, changes in MMP / TIMP balance, and increases in mean FEV1 in subjects treated with EDTA compared to placebo. The study is further expected to show reduced myeloperoxidase activity and lowered mean levels of chlorotyrosine and carbonyl.

[0174] Clinical data demonstrate efficacy at 300 mg / day for two weeks. The same study demonstrates benefits for lung function and infections at 150 mg / day (75 mg BID) (Figure 2 for bacterial count reduction; Figure 3A for lung function improvement). Given the significant magnitude of improvement (FEV1 mean 16 percentage points), those skilled in the art will appreciate that a much lower dose, i.e., 75 mg / day (37.5 mg BID), as envisioned by hypothetical Example P1, is highly likely to be effective.

[0175] Figure 4 shows that a single dose of 75 mg of CaEDTA results in 1.34 mM EDTA inside the mucus plug after 30 minutes. It is known that penetration of drugs such as tobramycin into CF sputum is significantly delayed (Kuhn, RJ (2001). Formulation of aerosolized therapeutics. Chest 120, 94S-98S). Therefore, it is most likely that the concentration of EDTA in the airway surface fluid is substantially higher than in the core. Therefore, it is reasonable to expect that a daily dose of 37.5 mg (fourfold lower than the clinically beneficial low dose) would be effective in a fully potentiated study. This is particularly true for young patients, who receive higher doses per body weight, which generally show a greater therapeutic effect on FEV1 (Figure 3B).

[0176] Numerous variations and modifications of the above-described modes of carrying out various embodiments of the invention will be apparent to those skilled in the art based on the above teachings related to the disclosed invention without departing from the basic inventive concept. The above-described embodiments of the invention are merely illustrative and should not be construed as limiting in any way, and all such variations and modifications are to be considered within the scope of the invention, the nature of which is determined from the foregoing description.

[0177] Research into this invention was supported by a grant from Cystic Fibrosis Foundation Therapeutics.

Claims

1. 1. A kit for treating or preventing chronic inflammation in the lungs, comprising: (i) an inhalation formulation containing a chelating agent at a concentration of at least 50 mM; and (ii) instructions for use of the formulation to administer 37.5 mg / day to 1,200 mg / day of the chelating agent, wherein each dose of the chelating agent is administered over a period of no more than two hours.

2. The kit of claim 1 , wherein the chelating agent is EDTA or DFO.

3. 10. The kit of claim 1, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (Tris).

4. 1. Use of a chelating agent for the manufacture of an inhalation formulation for treating or preventing chronic inflammation in the lung, wherein the formulation comprises a chelating agent at a concentration of at least 50 mM in the formulation, the formulation is manufactured to deliver 37.5 mg / day to 1,200 mg / day of the chelating agent, and the formulation is manufactured to deliver a single dose of the chelating agent over a period of 2 hours or less.

5. The use according to claim 4, wherein the chelating agent is EDTA or DFO.

6. The use according to claim 4, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (tris).

Images