Methods for reducing lung infections

Abstract

To provide a method of treating or preventing a bacterial infection in the lung.SOLUTION: The present invention provides a method for treating or preventing a bacterial infection in the lung of a subject by administering an inhaled antibiotic, such as tobramycin, and at least 37.5 mg / day of an inhaled chelating agent, such as calcium EDTA, each in one or more doses, in which the doses of the chelating agent and / or antibiotic are administered over a period not exceeding 8 hours.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for treating or preventing bacterial infections in the lungs of a subject by administering high concentrations of an inhaled chelating agent in combination with an inhaled antibiotic, and to formulations for use in the method. [Background technology]

[0002] Eradication of microbial infections can be difficult, especially when host defenses are compromised. In addition, many microorganisms form highly organized structures called biofilms in which they are protected from killing by immune cells and antibiotics through several mechanisms. These mechanisms include reduced antibiotic penetration, low metabolic activity, physiological adaptation, antibiotic-degrading enzymes, and selection for genetically resistant mutants (Stewart & Costerton Lancet. 2001, 358(9276):135-138).

[0003] Pseudomonas aeruginosa is an example of a Gram-negative bacterium that causes acute and chronic infections in compromised hosts. P. aeruginosa is commonly found in intensive care units and has a strong biofilm-forming ability that is a well-known obstacle to antibiotic intervention. Diseases that have been implicated in P. aeruginosa infection include cystic fibrosis (CF), ventilator-associated pneumonia (VAP), and chronic obstructive pulmonary disease (COPD) (Moradali et al., Front Cell Infect Microbiol. 2017, 7:39; Hassett et al., J Microbiol. 2014, 52(3):211-226).

[0004] Once organisms such as P. aeruginosa establish a foothold in the CF respiratory tract, challenges with the CF lung environment and bacterial resistance mechanisms, including biofilm formation and antibiotic-neutralizing enzymes, make successful eradication impossible. P. aeruginosa infections eventually become chronic, with infecting strains becoming increasingly resistant to antimicrobial agents, and persistent inflammation gradually reducing lung function (Hassett et al., J Microbiol. 2014, 52(3):211-226). Although treatments are improving, existing drugs have limited efficacy and patients ultimately require transplant surgery or die from respiratory failure.

[0005] What is needed are methods for treating or preventing infections in the lungs, or at least to supplement previously known methods of treatment.

[0006] The present invention seeks to provide an improved or alternative method for treating or preventing infections in the lungs by administering at least 37.5 mg / day of an inhaled chelating agent in combination with an antibiotic.

[0007] 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

[0008] The present invention provides a method for treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less.

[0009] Preferably, each of the chelating agent and antibiotic is administered in 1 to 4 doses per day.

[0010] Preferably, 37.5 mg / day to 1,200 mg / day of the chelating agent is administered. Preferably, at least 50 mg / day of the chelating agent is administered. The chelating agent may be administered between 1 to 4 times daily for a total daily dose of up to about 1,200 mg / day. Preferably, the chelating agent and / or antibiotic is administered over a period of 1 hour or less. Preferably, the chelating agent is CaEDTA.

[0011] Preferably, at least 10 mg / day of antibiotic is administered, or between 10 mg / day and 1,250 mg / day of antibiotic is administered. The antibiotic may be administered between 1 to 4 times daily for a total daily dose of up to about 1,250 mg / day, preferably up to 1,000 mg / day. Preferably, the antibiotic is tobramycin.

[0012] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less, and wherein the treatment reduces inflammation.

[0013] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less, and wherein the treatment or prevention of the infection is accompanied by or results in a decrease or reduction in MMP activity.

[0014] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less, and wherein the treatment or prevention of the infection is accompanied by or results in a reduction or decrease in the production of hydroxyl radicals.

[0015] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less, and wherein the treatment or prevention of the infection results in an increase in forced expiratory volume (FEV).

[0016] The present invention provides a dosage form for treating an infection comprising an inhaled chelating agent in combination with an inhaled antibiotic, each administered in one or more doses to deliver at least 37.5 mg / day of the inhaled chelating agent, and wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0017] The present invention provides an inhalation formulation for treating an infection comprising an inhaled antibiotic in combination with an inhaled chelating agent, each administered in one or more doses to deliver at least 37.5 mg / day of the inhaled chelating agent, and wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0018] The present invention provides an inhalation formulation for treating an infection comprising an inhalation chelating agent and an inhalation antibiotic, each delivered in one or more doses of at least 37.5 mg / day of the inhalation chelating agent, and wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0019] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject comprising: (i) at least 37.5 mg of an inhalable chelating agent; and (ii) instructions for use, the instructions providing that at least 37.5 mg / day of the inhalable chelating agent is delivered in conjunction with an inhaled antibiotic, and that the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0020] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject comprising: (i) an inhaled antibiotic; and (ii) instructions for use, the instructions providing that the antibiotic is delivered in combination with at least 37.5 mg / day of an inhaled chelating agent, and that the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0021] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject comprising: (i) at least 37.5 mg of an inhaled chelating agent and an inhaled antibiotic; and (ii) instructions for use, the instructions providing that at least 37.5 mg / day of the inhaled chelating agent is delivered and that the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0022] The present invention provides for the use of an inhaled chelating agent for the manufacture of a medicament for the treatment of an infectious disease, wherein the inhaled chelating agent is used in combination with an inhaled antibiotic to deliver at least 37.5 mg / day of the inhaled chelating agent, and wherein the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0023] The present invention provides the use of an inhaled antibiotic for the manufacture of a medicament for the treatment of an infectious disease, wherein the inhaled antibiotic is used in combination with an inhaled chelating agent, wherein at least 37.5 mg / day of the inhaled chelating agent is delivered, and the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0024] The present invention provides the use of an inhaled chelating agent and an inhaled antibiotic for the manufacture of a medicament for the treatment of an infectious disease, wherein at least 37.5 mg / day of the inhaled chelating agent is delivered and the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0025] Further features of the present invention are more fully described in the following description of some non-limiting embodiments thereof. This description is included solely for the purpose of illustrating the present invention. This description should not be understood as limiting the broad overview, disclosure or description of the present invention as presented above. Description is made with reference to the accompanying drawings. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 demonstrates the synergy of tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Checkerboard planktonic growth (OD600nm) table under aerobic conditions. The data shows that there is synergy between tobramycin and CaEDTA (Figure 1A) and between tobramycin and Tris-buffered CaEDTA (Figure 1B). [Diagram 2] 2A-2B demonstrate the synergy of tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Table of checkerboard biofilm growth (CV staining) under aerobic conditions. The data show a synergy between tobramycin and CaEDTA (FIG. 2A), which is further enhanced with Tris-buffered CaEDTA (FIG. 2B). [Diagram 3]3A-3B demonstrate synergy between tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Checkerboard planktonic growth (OD600 nm) table under anaerobic conditions. Data shows synergy between tobramycin and CaEDTA (FIG. 3A) and between tobramycin and Tris-buffered CaEDTA (FIG. 3B). [Figure 4] 4A-4B demonstrate synergy between tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Checkerboard biofilm table under anaerobic conditions. Data shows synergy between tobramycin and CaEDTA (FIG. 4A) and between tobramycin and Tris-buffered CaEDTA (FIG. 4B). [Diagram 5] 10 demonstrates the synergistic effect of tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Effect of CaEDTA and buffered CaEDTA against planktonic cells and biofilms under aerobic and anaerobic conditions. Concentrations were tobramycin: 4 μg / ml, CaEDTA: 6.25 mM, and Tris buffer: 12.5 mM as indicated. [Figure 6] Figure 6: Demonstration of synergy of tobramycin and CaEDTA or buffered CaEDTA as shown against P. aeruginosa WACC91. Shown is the synergy of CaEDTA and tobramycin submicron particles as measured by metabolism of P. aeruginosa biofilms in vitro. P. aeruginosa biofilms were grown in 96-well microtiter plates and exposed to aerosolized antibiotic and EDTA particles. Figure 6A: CaEDTA substantially enhances killing at tobramycin concentrations <1 mg / ml. Figure 6B: Synergy is independent of delivery mode (solution or dry powder). [Figure 7] FIG. 13 demonstrates that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa. Checkerboard P. aeruginosa WACC91 planktonic cells versus methicillin and CaEDTA. [Figure 8] 8A-8B demonstrate that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa. Checkerboard comparison of P. aeruginosa 001-11R (a CF clinical strain) planktonic cells (FIG. 8A) and biofilms (FIG. 8B) with carbenicillin and CaEDTA. [Figure 9] Figure 9 demonstrates that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa WACC91 planktonic cells versus tetracycline and buffered CaEDTA as measured by OD600nm (Figure 9A) and colony forming units, CFU / ml (Figure 9B). [Figure 10] Figure 10 demonstrates that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa WACC91 versus Azactam and buffered CaEDTA as measured by OD600nm (Figure 10A) and CFU / ml (Figure 10B). [Figure 11] Figure 11 demonstrates that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa WACC91 versus erythromycin and buffered CaEDTA as measured by OD600nm (Figure 11A) and CFU / ml (Figure 11B). [Figure 12] FIG. 1 demonstrates that CaEDTA enhances the efficacy of various antibiotics against P. aeruginosa, as shown: P. aeruginosa WACC91 planktonic cells versus colistin and buffered CaEDTA. [Figure 13] FIG. 10: Demonstrating that other chelators also enhance antibiotic efficacy: Checkerboard P. aeruginosa WACC91 planktonic cells versus tobramycin and ethylene glycol tetraacetic acid (EGTA). [Figure 14]Figures 14A-C demonstrate that other chelators also enhance antibiotic efficacy. Shows synergy between tobramycin and diethylenetriaminepentaacetic acid (DTPA). Figure 14A: Checkerboard; Figure 14B-C: OD600nm and colony counts for selected concentrations of antibiotics and chelators as indicated. [Figure 15] Figure 21 demonstrates that buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. Burkholderia cepacia planktonic cells versus tobramycin and CaEDTA. Figure 21A: Checkerboard assay; Figure 21B: Colony counts at selected concentrations. [Figure 16] Figure 1. Buffered CaEDTA demonstrates enhanced efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. Checkerboard: B. cepacia planktonic cells versus ciprofloxacin and CaEDTA. [Figure 17] Figure 17: Buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative: Checkerboard comparison of Klebsiella pneumoniae planktonic cells (Figure 17A) and biofilms (Figure 17B) with colistin and CaEDTA. [Figure 18] Figure 10: Buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. Checkerboard: Streptococcus pyogenes planktonic cells versus tetracycline and CaEDTA. [Figure 19]Figure 10: Buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. Checkerboard: Staphylococcus aureus MRSA planktonic cells versus vancomycin and CaEDTA. [Figure 20] Figure 20 demonstrates that buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. As shown, S. aureus MSSA planktonic cells versus ciprofloxacin and CaEDTA. Figure 20A: OD600nm; Figure 20B: CFU / ml. [Figure 21] Figure 21 demonstrates that buffered CaEDTA enhances the efficacy of various clinically relevant antibiotics against different bacterial species, both Gram-positive and Gram-negative. As shown, Staphylococcus aureus MRSA planktonic cells versus Ciprofloxacin and CaEDTA. Figure 21A: OD600nm; Figure 21B: CFU / ml. [Figure 22] Figure 14 demonstrates that CaEDTA increases antibiotic bactericidal activity. Time-kill assay of Pseudomonas aeruginosa ATCC27853 and ceftazidime (128ug / ml) with or without buffered CaEDTA (20mM). [Figure 23] Figure 14. CaEDTA demonstrates that CaEDTA increases the bactericidal activity of antibiotics. Time-kill assay of Burkholderia cepacia ATCC25416 and meropenem (32ug / ml) with or without buffered CaEDTA (20mM). [Figure 24]Figure 24: Submicron particles of CaEDTA kill P. aeruginosa biofilms and act synergistically with tobramycin in vitro. P. aeruginosa biofilms were grown in hanging drops of CF mucus and treated with aerosolized EDTA particles and / or tobramycin. The final concentration of tobramycin in the droplets was 325 μg / ml. Figure 24A: Confocal microscopy images of biofilms stained with BacLight LIVE / DEAD; Figure 24B: Bacterial counts showing the quantitative effect of treatment. [Diagram 25] FIG. 1 demonstrates that treatment of CF subjects with buffered CaEDTA in addition to tobramycin is more effective than treatment with tobramycin alone. It shows that treatment of CF subjects with buffered CaEDTA in addition to tobramycin reduces bacterial burden in the CF lung faster than treatment with antibiotics alone. CF subjects were treated with nebulized CaEDTA (EDTA) or saline (placebo) and bacterial load in expectorated mucus was monitored (colony forming units per gram of mucus). [Figure 26] FIG. 1 demonstrates that treatment of CF subjects with buffered CaEDTA in addition to tobramycin is more effective than treatment with tobramycin alone. Shown is the mean change in FEV1 (percentage points) in patients treated with CaEDTA or placebo over the entire 10-week period (4 weeks post-treatment) from the start of treatment. [Figure 27] 1 demonstrates that treatment of CF subjects with buffered CaEDTA in addition to tobramycin is more effective than treatment with tobramycin alone.The relationship between improvement in FEV1 (0-2 weeks) and body weight is shown. [Figure 28] FIG. 1 demonstrates that treatment of CF subjects with buffered CaEDTA in addition to tobramycin is more effective than treatment with tobramycin alone. Shown are the EDTA concentrations achieved in the sputum of three CF subjects 5 minutes and 2 hours after treatment with 75 mg nebulized CaEDTA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] [Method of treatment or prevention] The present invention provides a method for treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less.

[0028] Preferably, the subject is an animal, preferably a mammal, and most preferably a human.

[0029] 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 P. aeruginosa with nebulized sodium EDTA and oral tetracycline for 3 months and observed no changes in lung function or infection status. 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 high doses of inhaled chelating agents will have any positive effect on subjects, such as those with cystic fibrosis (CF), asthma, chronic obstructive pulmonary disease (COPD), or other pulmonary conditions that cause or are associated with infection and inflammation. However, the present invention has surprisingly found that administration of certain inhaled chelating agents in combination with antibiotics can treat or prevent pulmonary infection and inflammation.

[0030] It is commonly believed that the CF lung environment is acidic. However, it has recently been shown that the CF lung has the same pH as the normal lung (Schultz et al., "Airway surface liquid pH inchildren with cystic fibrosis," Nature Communications, Vol. 8:1409 (2017)). Thus, current techniques using acidified nitrite, such as those discussed in U.S. Patent Application Publication No. 2016 / 0263151, are unlikely to work clinically in CF, as the formulations do not remain in an acidified state, but quickly revert to the normal lung pH of 7.4.

[0031] Although CF lungs have normal acidity, iron levels have been found to be significantly different from normal lungs. Stites et al. (Am J Respir Crit Care Med. 1999, 160(3):796-80) have shown that iron levels are greatly elevated in the lungs of CF patients, as well as in the lungs of smokers, compared to 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, which generates highly reactive oxygen radicals that can severely damage tissues and DNA (Jomova et al., Toxicology. 2011, 283(2-3):65-87; MacNee, Eur J Pharmacol. 2001, 429(1-3):195-207).

[0032] Without wishing to be bound by theory, it is believed that the methods of the present invention reduce infection in the lungs by depriving bacteria of key ions, such as iron and zinc. This can occur on several levels: (i) Iron is essential for bacterial growth and survival. This is especially true in the case of biofilm formation, where iron removal leads to bacterial starvation and death; (ii) Biofilm cells are surrounded by a hydrated polymeric matrix consisting of negatively charged polysaccharides and DNA linked with positively charged metal ions. Cation removal causes the biofilm to disperse, thereby allowing antibiotics and immune cells to reach individual bacteria. (iii) The outer membrane of P. aeruginosa and other Gram-negative bacteria consists of negatively charged lipopolysaccharides cross-linked with divalent cations (Clifton et al., Langmuir, 2015, 31:404-412). Removal of cations increases outer membrane permeability and antibiotic susceptibility, an effect that is particularly pronounced in Pseudomonas spp. (Vaara, M. Microbiol Rev, 1992, 56:395-411), and / or (iv) Key to P. aeruginosa survival in the host is the production of secreted virulence factors, some of which are metal ion dependent for their function, such as elastase (Zn-dependent, Olson and Ohman, J Bacteriol 1992, 174:4140-4147) and β-lactamase (Zn-dependent, Fajardo et al., J Antimicrob Chemother 2014, 69:2972-2978).

[0033] Inhalation is a method of local administration and therefore may be more effective in reaching the target area, i.e., the lungs, providing high and localized concentrations of the inhaled chelating agent and antibiotic, avoiding unwanted side effects from systemic exposure of the active agents and reducing the risk of antibiotic resistance.

[0034] The chelating agent and the antibiotic can be delivered in one single formulation, or in two separate formulations. If the formulations are separate, they can be taken together (simultaneously) or sequentially. There can be a time lag of a few seconds, minutes, hours or days between the delivery of the formulations of the two active agents.

[0035] The compositions of the present invention can be delivered by a puffer (pressurized metered dose inhaler, dry powder inhaler) or a nebulizer. The compositions can be provided in one device (i.e., both the chelating agent and the antibiotic in one device), or the two active agents can be delivered from two different devices. For example, the two active agents can be delivered in two separate puffers, two separate nebulizers, or a puffer and a nebulizer, with one active agent in each device.

[0036] A method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered over a period of 8 hours or less, may be provided in the absence of acidified nitrite.

[0037] The invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering an inhaled antibiotic, and at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and wherein treating or preventing the infection results in an increase in FEV.

[0038] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and wherein the treatment or prevention of the infection results from the removal or reduction of biofilm produced by bacteria in the lungs due to the presence of the chelating agent.

[0039] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and wherein the treatment or prevention of the infection is due to increased bacterial outer membrane permeability due to the presence of the chelating agent.

[0040] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and wherein the treatment or prevention of the infection results from a reduction in bacterial virulence factors by chelation of metal ions with the chelating agent.

[0041] The present invention further provides a method of treating or preventing a bacterial infection in the lungs of a subject by administering one or more doses of an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each of which is administered for a period of 8 hours or less, and wherein the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and wherein the treatment or prevention of inflammation is accompanied by a reduction in MMP activity. It has been shown that matrix metalloproteinases (MMPs) cause lung injury (Garratt et al., Eur Respir J. 2015, 46(2):384-94) and that MMP activity increases the Zn 2+ It is known that MMPs are dependent on pulmonary pulmonary inflammation (Hazra et al., Molecular Vision, 2012; 18:1701-1711). However, past attempts to target MMPs in the lung have been unsuccessful. The present invention uses an inhalable chelating agent to chelate zinc in the lung, thereby reducing MMP-induced lung injury and treating or preventing inflammation.

[0042] The present invention further provides a method for treating or preventing bacterial infection in the lungs of a subject by administering an inhaled antibiotic and at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, where the or each dose of the chelating agent and / or antibiotic is administered for a period of 8 hours or less, and the treatment or prevention of inflammation is accompanied by a reduction in the production of hydroxyl radicals. Iron is a major factor in lung injury, as Fe(II) catalyzes the formation of hydroxyl radicals (Stites et al., (Am J Respir Crit Care Med., 1999, 160(3):796-80)). Fe(II) is abundant in CF lungs and significantly correlates with disease severity (Hunter et al., MBio., 2013, 4(4):1-8), but antioxidant trials have so far failed to produce significant improvements in lung function. The present invention uses inhaled chelating agents to chelate iron in the lungs, thus reducing hydroxyl radical-induced lung injury and treating or preventing inflammation.

[0043] 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, for example, 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.

[0044] The chelating agent is preferably selected from the group consisting of citric acid, phosphates, disodium, trisodium and tetrasodium salts of ethylenediaminetetraacetic acid (EDTA), calcium salts of EDTA, ethylene glycol-bis-(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); ethylene-N,N'-diglycine (EDDA); 2,2'-(ethylenediimino)-dibutanoic acid (EBDA); lauroyl EDTA; dilauroyl EDTA, triethylenetetramine. dihydrochioride (TRIEN), diethylenetriaminepentaacetic acid (DPTA), triethylenetetraminehexaacetic acid (TTG), deferoxamine (DFO), deferasirox (DSX), dimercaprol, zinc citrate, penicilamine, succimer, editronate, sodium hexametaphosphate, calcium disodium edetate, D-penicillamine, polyphenols, gallol, catechol, dimercaprol, tetrathiomolybdate, lactoferrin, and clioquinol, and combinations thereof.

[0045] Preferably, the chelating agent is a pharma- ceutically acceptable chelating agent.

[0046] 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).

[0047] 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 0.1 M is 0.01 to 0.01% Fe. 2+ For Fe, it is 14.3. 3+ 25.1 for iron and 16.5 for zinc.

[0048] In one embodiment, the chelating agent is a calcium salt of a chelating agent. Preferably, the chelating agent is CaEDTA.

[0049] In one embodiment, the chelating agent is provided in an inhalable dosage form containing between 37.5 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 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.

[0050] 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 300 mg / day, 500 mg / day or 600 mg / day, or at least 600 mg / day. The chelating agent can be administered in a total dose of up to about 1,200 mg / day, preferably at least 37.5 mg / day.

[0051] 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 being administered at each time point.

[0052] Preferably, the inhaled chelating agent and the inhaled antibiotic are administered for at least 28 consecutive days. The inhaled chelating agent and the inhaled antibiotic can be delivered for 2 or more days, 3, 4, 5, 6 or 7 days. The inhaled chelating agent and the inhaled antibiotic can be delivered for 2-28 days, 1, 2, 3 or 4 weeks. 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 doses or maintenance doses.

[0053] The inhaled chelating agent and the inhaled antibiotic can be provided in the absence of acidified nitrite.

[0054] Thus, the present invention provides Deliver a total dose of inhaled chelator between 37.5 mg / day and 1,200 mg / day; Delivering a total dose of inhaled antibiotic between 10mg / day and 500mg / day; administered at least once per day and up to six times per day, preferably up to four times per day; administered either as a single formulation containing both active agents or in separate formulations; and / or -Administered over a period of 8 hours or less.

[0055] Preferably, the present invention relates to a method for producing ... composition comprising the steps of: Deliver a total dose of inhaled chelator between 37.5 mg / day and 1,200 mg / day; Delivering a total dose of inhaled antibiotic between 10mg / day and 500mg / day; administered as a single formulation containing both active agents, Administered once or twice daily, administered over a period of one hour or less per dose; Contains CaEDTA as a chelating agent, and / or Contains tobramycin as an antibiotic.

[0056] 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.

[0057] 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 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.

[0058] Preferably, the antibiotic is tobramycin. However, it is not desired to limit the invention to tobramycin. Other antibiotics or anti-infectives, such as aminoglycosides, tetracyclines, sulfonamides, p-aminobenzoic acid, diaminopyrimidines, quinolones, β-lactams, β-lactam and β-lactamase inhibitors, glycopeptides, chloraphenicol, macrolides, penicillins, cephalosporins, corticosteroids, prostaglandins, ciprofloxacin, linomycin, clindamycin, spectinomycin, polymyxin B, colistin, vancomycin, Bacitracin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin, sulfones, clofazimine, thalidomide, polyene antifungals, flucytosine, imidazoles, triazoles, griseofulvin, terconazole, butoconazole cyclopyrax, ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine, or any combination thereof may be used.

[0059] Antibiotics encompassed by the present invention include, but are not limited to, quinolones (e.g., nalidixic acid, cinoxacin, ciprofloxacin, and norfloxacin), sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and the like), aminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and the like), tetracyclines (e.g., chlortetracycline, , oxytetracycline, methacycline, doxycycline, minocycline, etc.), para-aminobenzoic acid, diaminopyrimidines (e.g., trimethoprim, often used in conjunction with sulfamethoxazole, pyrazinamide, etc.), penicillins (e.g., penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, piperacillin, etc.), penicillinase-resistant penicillins (e.g., methicillin, oxacillin, cloxacillin, cyclosporine ... cephalosporins (e.g., cefadroxil, cephalexin, cephradine, cephalothin, cephapirin, cefazolin, etc.), second-generation cephalosporins (e.g., cefaclor, cefamandole, cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil, cefinetazole, cefprozil, loracarbef, ceforanide, etc.), third-generation cephalosporins (e.g., cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, etc.), , ceftazidime, cefixime, cefpodoxime, ceftibuten, etc.), other β-lactams (e.g., imipenem, meropenem, aztreonam, clavulanic acid, sulbactam, tazobactam, etc.), beta-lactamase inhibitors (e.g., clavulanic acid, etc.), chloramphenicol, macrolides (e.g., erythromycin, azithromycin, clarithromycin, etc.), lincomycin, clindamycin, spectinomycin, polymyxin B, polymyxins (e.g., polymyxin A, B, C, D, E1 (colistin A), or E2,colistin (such as colistin B or C), vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin, sulfones (e.g., dapsone, sulfoxone sodium, etc.), clofazimine, thalidomide, or any other antibacterial agent that can be lipid-encapsulated. Anti-infective agents may include anti-fungal agents including polyene anti-fungals (e.g., amphotericin B, nystatin, natamycin, etc.), flucytosine, imidazoles (e.g., miconazole, clotrimazole, econazole, ketoconazole, etc.), triazoles (e.g., itraconazole, fluconazole, etc.), griseofulvin, terconazole, butoconazole cyclopyrax, ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine, or any other antibiotic and its pharmaceutically acceptable salts and combinations thereof. Although the discussion and examples are primarily directed to tobramycin, it is not intended that the scope of this application be limited to this antibiotic. Combinations of drugs may be used.

[0060] Suitable "pharmaceutical acceptable salts" include non-toxic salts that are commonly used, such as salts with inorganic bases, such as alkali metal salts (e.g., sodium salts and potassium salts), alkaline earth metal salts (e.g., calcium salts and magnesium salts), ammonium salts; or salts with organic bases, such as amine salts (e.g., methylamine salts, dimethylamine salts, cyclohexylamine salts, benzylamine salts, piperidine salts, ethylenediamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, tris(hydroxymethylamino)ethane salts, monomethyl-monoethanolamine salts, procaine salts and caffeine salts), basic amino acid salts (e.g., arginine salts and lysine salts), tetraalkylammonium salts, or other salt forms that allow the pulmonary blood pressure reducing agent to remain soluble in liquid media, or to be prepared and / or effectively administered in liquid media, preferably aqueous media. The above salts can be prepared by conventional processes, such as from the corresponding acids and bases, or by salt exchange.

[0061] For example, in one alternative embodiment, the antibiotic may be utilized in a free base form or in a salt form (eg, as a pharma- ceutically acceptable salt). Examples of suitable pharma- ceutically acceptable salts include inorganic acid addition salts, such as hydrochloride, hydrobromide, sulfate, phosphate, and nitrate; organic acid addition salts, such as acetate, propionate, succinate, lactate, glycolate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, and ascorbate; salts with acidic amino acids, such as aspartate and glutamate; alkali metal salts, such as sodium and potassium; alkaline earth metal salts, such as magnesium and calcium; ammonium salts; organic base salts, such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, and N,N'-dibenzylethylenediamine; and salts with basic amino acids, such as lysine and arginine. The salts may be optionally hydrated or ethanol solvated.

[0062] Examples of ethers include, but are not limited to, alkyl ethers, such as lower alkyl ethers, for example, methyl ether, ethyl ether, propyl ether, isopropyl ether, butyl ether, isobutyl ether, t-butyl ether, pentyl ether, and 1-cyclopropylethyl ether; and medium or higher alkyl ethers, for example, octyl ether, diethylhexyl ether, lauryl ether, and cetyl ether; unsaturated ethers, for example, oleyl ether and linolenyl ether; lower alkenyl ethers, for example, vinyl ether, allyl ether, and the like; lower alkynyl ethers, for example, ethynyl ether and propynyl ether, and the like; hydroxy(lower) alkyl ethers, for example, hydoaryl ether ... lower alkoxy(lower)alkyl ethers, such as, for example, methoxymethyl ether and 1-methoxyethyl ether; optionally substituted aryl ethers, such as, for example, phenyl ether, tosyl ether, t-butylphenyl ether, salicyl ether, 3,4-di-methoxyphenyl ether and benzamidophenyl ether; and aryl(lower)alkyl ethers, such as, for example, benzyl ether, trityl ether and benzhydryl ether, or other ether forms which enable the antibiotic to remain soluble in a liquid medium or which are prepared and / or effectively administered in a liquid medium, preferably an aqueous medium.

[0063] Examples of esters include, but are not limited to, aliphatic esters, such as lower alkyl esters, such as methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, t-butyl ester, pentyl ester, and 1-cyclopropylethyl ester; lower alkenyl esters, such as vinyl ester and allyl ester; lower alkynyl ester, such as ethynyl ester and propynyl ester; hydroxy(lower)alkyl esters, such as hydroxyethyl ester; lower alkoxy(lower)alkyl esters, such as methoxymethyl ester and 1-methoxyethyl ester; and optionally substituted aryl esters, such as phenyl ester, tosyl ester, t-butylphenyl ester, salicyl ester, 3,4-di-methoxyphenyl ester, and benzamidophenyl ester; and aryl(lower)alkyl esters, such as benzyl ester, trityl ester, and benzhydryl ester, or other ester types that allow the antibiotic to remain soluble in a liquid medium or are prepared and / or effectively administered in a liquid medium, preferably an aqueous medium.

[0064] Also, the antibiotics for use in the formulations and methods provided herein may contain one or more chiral centers. Such chiral centers may be in either the (R) or (S) configuration, or may be a mixture thereof. Thus, the antibiotics for use in the formulations provided herein may be enantiomerically pure, or may be stereoisomeric or diastereomeric mixtures. It should be understood that the chiral centers of the compounds provided herein may be epimerized in vivo. Thus, one skilled in the art recognizes that administration of a compound in its (R) form is equivalent to administration of the compound in its (S) form for compounds epimerized in vivo.

[0065] In one embodiment, the antibiotic is provided in an inhalable dosage form containing between 10 mg / dose and 500 mg / dose, between about 50 mg / dose and 400 mg / dose, between about 100 mg / dose and 400 mg / dose, between about 200 mg / dose and 300 mg / dose; preferably about 10 mg / dose, 20 mg / dose, 30 mg / dose, 50 mg / dose, 200 mg / dose, 250 mg / dose, 200 mg / dose, or 300 mg / dose.

[0066] The total amount of antibiotic inhaled per day is preferably between about 10 mg / day and 2,000 mg / day, between 10 mg / day and 1,000 mg / day, between 50 mg / day and 2,000 mg / day, between 250 mg / day and 2,000 mg / day, between about 300 mg / day and 1,100 mg / day, between about 500 mg / day and 1,000 mg / day, between about 800 mg / day and 1,000 mg / day; preferably about 200 mg / day, 300 mg / day, 500 mg / day, 1,000 mg / day or 1,250 mg / day.

[0067] The antibiotic 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 per delivery. If administration is by dry powder delivery, the inhaled antibiotic is preferably 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 being administered at each time point.

[0068] When the antibiotic is tobramycin, commonly prescribed inhaled tobramycin doses are TOBI® Podhaler® (dry powder) 4 puffs of 28 mg (112 mg dose) twice daily for a total of 224 mg / day and OR Nebulised TOBI® 300 mg in 5 ml twice daily.

[0069] If the chelating agent is delivered in a puff, preferably each puff delivers between 37.5mg / puff and 300mg / puff, between about 75mg / puff and 200mg / puff, between about 75mg / puff and 100mg / puff, between about 50mg / puff and 200mg / puff, preferably about 37.5mg / puff, 50mg / puff, 75mg / puff, 100mg / puff, 200mg / puff or 300mg / puff.

[0070] Alternatively, if the chelating agent is delivered in a puff, preferably every second puff delivers between 37.5mg / 2xpuff and 300mg / 2xpuff, between about 75mg / 2xpuff and 200mg / 2xpuff, between about 75mg / 2xpuff and 100mg / 2xpuff, between about 50mg / 2xpuff and 200mg / 2xpuff; preferably about 37.5mg / 2xpuff, 50mg / 2xpuff, 75mg / 2xpuff, 100mg / 2xpuff, 200mg / 2xpuff or 300mg / 2xpuff.

[0071] If the antibiotic is delivered by puff, preferably each puff delivers between 10 mg / puff and 500 mg / puff, between about 50 mg / puff and 400 mg / puff, between about 100 mg / puff and 400 mg / puff, between about 200 mg / puff and 300 mg / puff; preferably about 10 mg / puff, 20 mg / puff, 30 mg / puff, 50 mg / puff, 200 mg / puff, 250 mg / puff, 200 mg / puff or 300 mg / puff.

[0072] Alternatively, if the antibiotic is delivered by puff, preferably every second puff is between 10mg / 2xpuff and 500mg / 2xpuff, between about 50mg / 2xpuff and 400mg / 2xpuff, between about 100mg / 2xpuff and 400mg / 2xpuff, between about 200mg / 2xpuff and 300mg / 2xpuff; preferably delivering about 10mg / 2xpuff, 20mg / 2xpuff, 30mg / 2xpuff, 50mg / 2xpuff, 200mg / 2xpuff, 250mg / 2xpuff, 200mg / 2xpuff or 300mg / 2xpuff.

[0073] When the composition contains both a chelating agent and an antibiotic, preferably the ratio of chelating agent:antibiotic is about 3:1, 1:10, 30:1, 3:5 or 1:1.5.

[0074] Preferably, the infectious disease is an infectious disease caused by cystic fibrosis (CF); asthma; chronic obstructive pulmonary disease (COPD); pulmonary hypertension; lung cancer; pulmonary fibrosis; bronchiectasis; acute respiratory distress syndrome; tuberculosis; nontuberculous mycobacterial (NTM) lung infections; pneumonia, including but not limited to ventilator-associated pneumonia, community-acquired pneumonia, bronchopneumonia, lobar pneumonia; 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 against 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.

[0075] Preferably, the chelating agent and / or antibiotic is administered to a subject in need thereof between about 1 time per day and about 6 times per day, more preferably about 2, 3 or 4 times per day.

[0076] Alternatively, the chelating agent and / or antibiotic can be administered to a subject in need thereof via continuous inhalation via a nebulizer. The nebulized chelating agent and / or antibiotic can be delivered for 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour, each of which can be repeated several times within a 24 hour period.

[0077] In one embodiment, the inhalation chelating agent and the antibiotic are administered one after the other (i.e., sequentially) once, two or three times a day, or are administered simultaneously together. When the drugs are administered simultaneously, the two compounds can be mixed immediately before administration, or the compounds can be mixed as one homogeneous mixture in an internal preparation, provided that physical and chemical stability is maintained. In one embodiment, the inhalation chelating agent and the antibiotic are mixed as one pharmaceutical preparation and administered to the subject by inhalation. A single pharmaceutical preparation and a single treatment provide ease of use and lead to better compliance by the subject.

[0078] The inhaled chelating agent and antibiotic doses are typically administered by spraying from an aerosol device or dry powder inhaler, or by at least one, and preferably several, "puffs." For example, a subject may receive a single dose, or several doses over the course of a day.

[0079] The total daily dose is preferably administered at least once a day or twice a day, but may be divided into three or more doses per day.Some subjects may benefit from a period of "loading" the subject with antibiotics and / or chelating agents, with higher or more frequent doses over a period of several days or weeks, followed by lower doses or maintenance doses.Because cystic fibrosis, COPD, etc. are usually chronic conditions, subjects are expected to undergo such therapy for a long period of time.

[0080] Regardless of the form of the drug formulation, it is preferred 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, 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 having 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, it is preferred that the particles not only have a size in the range of 0.1 μm to 12 μm or 2 μm to 6 μm or about 3 to 4 μm, but also that the average particle size is within a narrow range such that 80% or more of the particles delivered to the subject have a particle diameter that is within ±20% of the average particle size, preferably ±10% of the average particle size, more preferably ±5%.

[0081] "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.

[0082] "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.

[0083] "Mass median aerodynamic diameter" is the geometric mean aerodynamic diameter. Fifty percent of the particles, by weight, are smaller than the MMAD and 50% of the particles, by weight, are larger.

[0084] 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 that encompasses the entire size range starting from the smallest particles, which can be 1 nm, to the largest particles, which can be 100 μm. In the particle size distribution curve, a cumulative frequency is calculated for the particles. 10refers to a particular particle diameter such that 10% of the number of particles in a suspension have a diameter less than or equal to this particular particle diameter.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In one form of the invention, a method of treating or preventing a bacterial infection in the lungs of a subject by administering a therapeutically or prophylactically effective amount of an inhaled antibiotic and a therapeutically or prophylactically effective amount of at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0089] In one form of the invention, a method for treating a bacterial infection in the lungs of a subject by administering a therapeutically effective amount of an inhaled antibiotic and a therapeutically effective amount of at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0090] In one form of the invention, a method for preventing a bacterial infection in the lungs of a subject by administering a prophylactically effective amount of an inhaled antibiotic and a prophylactically effective amount of at least 37.5 mg / day of an inhaled chelating agent, each in one or more doses, wherein the or each dose of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0091] In one form of the invention, a method of treating or preventing a bacterial infection in the lungs of a subject comprises treating or preventing a bacterial infection in the lungs of a subject in need of such treatment.

[0092] 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, partially or completely halt the onset or progression of an infection and / or inflammation associated therewith. A therapeutically effective amount may further encompass delaying the onset or inhibiting the progression, partially or completely halting the onset or progression of an infection, or reversing or partially reversing the antimicrobial susceptibility of a pathogenic microorganism(s).

[0093] The term "prophylactically 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 prevent or delay the onset of microbial infection and / or associated inflammation.

[0094] As used herein, "treat" or "treatment" refers to inhibiting a disease or condition, i.e., arresting or reducing its onset or at least one of its clinical or asymptomatic symptoms. "Treat" or "treatment" further refers to alleviating a disease or condition, i.e., causing the regression of a disease or condition or at least one of its clinical or asymptomatic symptoms. The benefit to the subject to be treated is statistically significant or at least perceptible to the subject and / or physician. In the context of treating bacterial infections, the term treatment includes reducing or eliminating colonization by bacteria and / or bacterial growth, including reducing biofilm formation or destroying existing biofilms.

[0095] Based on the above, those skilled in the art will understand that a single subject can be treated using multiple different treatments and administration means. Thus, subjects who are already receiving such drugs, such as intravenous ciprofloxacin or antibiotics, can benefit from inhalation of the formulation of the present invention. Some subjects can receive at least 37.5 mg / day of inhaled chelating agent in combination with antibiotics by 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 are symptoms that can benefit from administering at least 37.5 mg / day of inhaled chelating agent in combination with antibiotics to the subject. The present invention can also be used for diagnosis. In one embodiment, for example, a subject can receive a chelating agent and / or an antibiotic as part of a procedure for diagnosing a pulmonary infection, where one or more of the subject's symptoms improve in response to the chelating agent and / or the antibiotic.

[0096] [formulation] The inhalation formulation may be in the form of an aerosol or dry powder for inhalation, or in the form of a mist for inhalation. Preferably, the formulation is adapted for inhalation to treat or prevent infections in the lungs.

[0097] Each of the chelating agent and the antibiotic can be delivered in one single formulation, or in two or more separate formulations. Each of the chelating agent and the antibiotic can be delivered, for example, in several puffs of a dry powder inhaler.

[0098] The chelating agent and the antibiotic can be delivered in one single formulation, or in two separate formulations. If the formulations are separate, they can be taken together (simultaneously) or sequentially. There can be a time lag of a few seconds, minutes, hours or days between the delivery of the formulations of the two active agents.

[0099] The compositions of the present invention can be delivered by a puffer (pressurized metered dose inhaler, dry powder inhaler) or a nebulizer. The compositions can be provided in one device (i.e., both the chelating agent and the antibiotic in one device), or the two active agents can be delivered from two different devices. For example, the two active agents can be delivered in two separate puffers, two separate nebulizers, or a puffer and a nebulizer, with one active agent in each device.

[0100] The inhaled chelating agent and the inhaled antibiotic can be provided in the absence of acidified nitrite.

[0101] In one embodiment, the chelating agent is a calcium salt of a chelating agent. Preferably, the chelating agent is CaEDTA.

[0102] Preferably, the chelating agent is provided in an inhalable dosage form containing between 37.5 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 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.

[0103] 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 300 mg / day, 500 mg / day or 600 mg / day, or at least 600 mg / day. The chelating agent can be administered in a total dose of up to about 1,200 mg / day, preferably at least 37.5 mg / day.

[0104] The inhalable 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 per delivery. If administration is by dry powder administration, the inhalable chelating agent is preferably 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 being administered at each time point.

[0105] For example, a 50 mg dose of CaEDTA can be administered as 4 mL of a 33 mM nebulizer 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.

[0106] Preferably, the antibiotic is tobramycin.

[0107] In one embodiment, the antibiotic is provided in an inhalable dosage form containing between 10 mg / dose and 500 mg / dose, between about 50 mg / dose and 400 mg / dose, between about 100 mg / dose and 400 mg / dose, between about 200 mg / dose and 300 mg / dose; preferably about 10 mg / dose, 20 mg / dose, 30 mg / dose, 50 mg / dose, 200 mg / dose, 250 mg / dose, 200 mg / dose or 300 mg / dose.

[0108] The total amount of antibiotic inhaled per day is preferably between about 10 mg / day and 2,000 mg / day, between 10 mg / day and 1,000 mg / day, between 50 mg / day and 2,000 mg / day, between 250 mg / day and 2,000 mg / day, between about 300 mg / day and 1,100 mg / day, between about 500 mg / day and 1,000 mg / day, between about 800 mg / day and 1,000 mg / day; preferably about 200 mg / day, 300 mg / day, 500 mg / day, 1,000 mg / day or 1,250 mg / day.

[0109] The inhaled antibiotic is preferably administered over a period of 8, 7, 6, 5, 4, 3, 2, 1, 45, 30, 20, 15, 10, or 5 minutes or less per administration. If administration is by dry powder delivery, the inhaled antibiotic is preferably administered 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 being administered at each time point.

[0110] Preferably, the chelating agent and / or antibiotic is administered to a subject in need thereof between about once per day and about six times per day, more preferably about twice per day.

[0111] Alternatively, the chelating agent and / or antibiotic can be administered to a subject in need via continuous inhalation via a nebulizer. The nebulized formulation can be delivered for 8 hours, 6 hours, 4 hours, 2 hours, or 1 hour, each of which can be repeated several times within a 24 hour period.

[0112] The formulation of the present invention can be administered to a subject using disposable packaging and portable, handheld, battery-powered devices, such as AERx devices (US Pat. No. 5,823,178, Aradigm, Hayward, Calif.). Alternatively, the formulation of the present invention can be administered using mechanical (non-electronic) devices. The formulation can be delivered using other inhalation devices, including conventional jet nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powder inhalers (DPIs), metered dose inhalers (MDIs), condensation aerosol generators, and other systems.

[0113] 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 with a suitable propellant, such as a hydrocarbon propellant such as propane, butane, or isobutane. Dry powder inhalers are systems that can be operated with a source of pressurized air that produces dry powder particles of the pharmaceutical formulation compressed into a very small volume. For inhalation, the system has multiple chambers or blisters, each of which contains a single dose of the pharmaceutical formulation and a selection element for releasing the single dose.

[0114] Aerosols can be created by forcing the drug through pores in a membrane, the pores having a size in the range of about 0.25-6 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 a diameter in the range of 0.5-12 μm. The drug particles can be released with an airflow intended to keep the particles within this size range. The generation of small particles can be facilitated by the use of a vibration device that provides a vibration frequency in the range of about 800 to about 4000 kilohertz. It should be noted that the objective of some embodiments is to provide aerosolized particles with a diameter in the range of about 0.5-12 μm, although one of ordinary skill 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.

[0115] [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 for 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 adjusting agents, tonicity adjusting agents, drug carriers, sustained release agents, comfort aids, emulsifiers, solubilizing aids, lubricants, binders and other stabilizers that are useful for the preparation and / or administration of the formulations.

[0116] Preferably, the formulations of the invention are sterile. In another embodiment, the formulations of the invention are stable.

[0117] In addition, 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.

[0118] Additionally, the formulations of the present invention may contain an antimicrobial preservative.

[0119] 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. A favorable pH helps to avoid bacterial resistance to the formulation, the inhaled chelating agent and / or the antibiotic.

[0120] The inhaled chelating agent and the inhaled antibiotic can be provided in the absence of acidified nitrite.

[0121] In an alternative embodiment, the formulations of the present invention may include preservatives, suspending agents, wetting agents, isotonicity agents and / or diluents. The formulations provided herein may include 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 pharmacologically suitable suspending fluids that are physiologically acceptable upon inhalation. Pharmacologically suitable fluids for use herein include, but are not limited to, polar solvents, including, but are not limited to, 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 pharma- ceutically 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.

[0122] 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.

[0123] 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%).

[0124] In one embodiment, the formulation further comprises one or more pharma- ceutically or physiologically acceptable carriers, diluents or excipients.

[0125] In addition to or instead of sterilization, the formulations of the present invention may contain a pharma- ceutically acceptable preservative to minimize the possibility of microbial contamination. Additionally, pharma-ceutically acceptable preservatives may be used in the formulations of the present invention to increase the stability of the formulation. However, it should be noted that any preservative must be selected for inhalation safety, since 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, and include phenylethyl alcohol, benzalkonium chloride or benzoic acid, or benzoate salts, such as sodium benzoate, and phenylethyl alcohol. In certain embodiments, the formulations of the present invention 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 an amount of about 0.001% to about 1%, preferably about 0.002% to about 0.02%, more preferably 0.02% w / w.

[0126] 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; hydroxypropylcellulose; 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;Tartaric acid esters of mono- and diglycerides of fatty acids;Monoacetyltartaric acid and diacetyltartaric acid 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.

[0127] The formulations of the present invention may contain about 0.001% to about 5% by weight of a humectant to inhibit drying of mucous membranes and prevent irritation. Any of a variety of pharma- ceutically acceptable humectants may be utilized, including sorbitol, propylene glycol, polyethylene glycol, glycerol, or mixtures thereof.

[0128] The formulations of the invention may further include an adjuvant, such as a bronchodilator, another anti-inflammatory agent, a surfactant, aspirin, or ethyl alcohol.

[0129] Bronchodilators that are 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.

[0130] 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 agent, can also be added.

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

[0132] Surfactants encompassed by the present invention include, but are not limited to, a synthetic surfactant (Exosurf®), dipalmitoyl phosphatidylcholine and oleic acid. Combinations of drugs may be used.

[0133] Ethyl alcohol vapor acts as a defoamer in the lungs, making phlegm more liquefied, thereby aiding breathing and reducing pulmonary edema. Ethanol can be added to the formulations of the invention at 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 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0134] The present invention also relates to the use of an inhaled chelating agent in combination with an inhaled antibiotic, where at least 37.5 mg / day of the inhaled chelating agent is delivered, and where the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less in further combination with other drugs administered via inhalation, such as other drugs that may comprise nucleotide sequences that may be incorporated into a suitable delivery vector, such as a plasmid or viral vector. Other drugs include therapeutic nucleotide sequences (DNA, RNA, siRNA), enzymes that reduce the viscoelasticity of mucus, such as DNase and other mucolytic agents, chemicals that upregulate chloride ion channels or increase the flux of ions across cells, nicotine, P2Y2 agonists, such as elastase inhibitors including alpha-1 antitrypsin (AAT), N-acetylcysteine, antibiotics and cationic peptides, such as lantibiotics, specifically duramycin, short-acting bronchodilators (such as β2-adrenergic receptor agonists like albuterol or indacaterol), M3 muscarinic antagonists (such as 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 transcription factor decoys, 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 invention, in accordance with standard practice.

[0135] Delivery of a combination product can be accomplished 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.

[0136] 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 at a given temperature, at least 80%, 85%, 90% or 95% of the original amount of drug substance, e.g., chelating agent and antibiotic, is present 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 more than 1, 2, 12, 18, 24 or 36 months of storage at 25°C. In another alternative embodiment, using Arrhenius kinetics, more than 80%, or more than 85%, or more than 90%, or more than 95% of the original amount of drug substance (e.g., chelating agent and antibiotic) remains after more than 1, 2, 12, 18, 24 or 36 months of storage of the formulation between about 15°C and about 30°C.

[0137] 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 at least 1, 2 or 3 months of useful life at 25° C. and a storage time of at least or equal to 1, 2 or 3 years 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 agent remain after such storage.

[0138] The term "infection" as used herein means colonization by a microorganism and / or the growth of a microorganism, particularly a bacterium. The bacterium may be gram-negative, such as a member of the genus Pseudomonas, or gram-positive, such as Staphylococcus aureus. The infection may not be obvious or may result in localized cell damage. The infection may be localized and asymptomatic, or may become acute or chronic clinical infections by spreading temporarily or alternatively. The infection may also be a past infection in which residual antigens from proteins associated with the anaerobic growth of P. aeruginosa remain in the host, or alternatively reactive host antibodies that bind to isolate proteins of P. aeruginosa proteins or peptides therefrom. The infection may also be a latent infection in which a microorganism is present in the subject, but the subject does not show symptoms of disease associated with the organism. Preferably, the infection is a respiratory infection, preferably a respiratory infection caused by P. aeruginosa.

[0139] [Method for producing a medicine] The present invention provides for the use of an inhaled chelating agent for the manufacture of a medicament for the treatment of an infectious disease, wherein the inhaled chelating agent is used in combination with an inhaled antibiotic to deliver at least 37.5 mg / day of the inhaled chelating agent, and wherein the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0140] The present invention provides the use of an inhaled antibiotic for the manufacture of a medicament for the treatment of an infectious disease, wherein the inhaled antibiotic is used in combination with an inhaled chelating agent, wherein at least 37.5 mg / day of the inhaled chelating agent is delivered, and wherein the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less.

[0141] The present invention provides the use of an inhaled chelating agent and an inhaled antibiotic to manufacture a medicament for the treatment of an infectious disease, wherein at least 37.5 mg / day of the inhaled chelating agent is delivered and the or each administration of the chelating agent and / or antibiotic is administered over a period of 8 hours or less. Preferably, the medicament does not contain acidified nitrite.

[0142] Preferably, the chelating agent is administered 1-4 times daily at a total dose of between 37.5 mg / day and 1,200 mg / day. Preferably, the chelating agent is administered over a period of 1 hour or less. Preferably, the chelating agent is CaEDTA.

[0143] Preferably, the antibiotic is administered at a total dose of between about 10 mg / day and 2,000 mg / day, preferably up to 1,000 mg / day, 1 to 4 times daily. Preferably, the antibiotic-containing formulation is administered over a period of 1 hour or less. Preferably, the antibiotic is tobramycin.

[0144] [kit] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject, comprising: (i) at least 37.5 mg of an inhalable chelating agent; and (ii) instructions for use, providing that at least 37.5 mg / day of the inhalable chelating agent is delivered in conjunction with an inhaled antibiotic, and that the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0145] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject comprising (i) an inhaled antibiotic and (ii) instructions for use, the instructions providing that the antibiotic is delivered in conjunction with at least 37.5 mg / day of an inhaled chelating agent, and that the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0146] The present invention provides a kit for treating or preventing a bacterial infection in the lungs of a subject, comprising: (i) at least 37.5 mg of an inhaled chelating agent and an inhaled antibiotic; and (ii) instructions for use, wherein at least 37.5 mg / day of the inhaled chelating agent is delivered and wherein the or each administration of the chelating agent and / or antibiotic is administered for a period of 8 hours or less.

[0147] Preferably, the inhaled chelating agent and / or the inhaled antibiotic is provided in the absence of acidifying nitrite.

[0148] Preferably, the chelating agent is administered 1-4 times daily at a total dose of between 37.5 mg / day and 1,200 mg / day. Preferably, the chelating agent is administered over a period of 1 hour or less. Preferably, the chelating agent is CaEDTA.

[0149] Preferably, the antibiotic is administered 1-4 times daily at a total dose of between about 10 mg / day and 2,000 mg / day, preferably up to 1,000 mg / day. Preferably, the antibiotic-containing formulation is administered over a period of 1 hour or less. Preferably, the antibiotic is tobramycin.

[0150] The chelating agent and / or antibiotic may be pre-measured, pre-mixed and / or pre-packaged. Preferably, the inhalation solution is sterile.

[0151] The kit of the present invention may also include instructions designed to promote user compliance. Instructions, as used herein, refer to any label, insert, etc., and 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 formulation of the present invention. In one embodiment, the instructions indicate that the formulation of the present invention is suitable for treating pulmonary infection. Such instructions may also include instructions for the formulation, as well as instructions for administration via a nebulizer or dry powder inhaler.

[0152] The inhalation chelating agent and the antibiotic can be packaged separately so that the physician or user can formulate each into a pharmaceutical formulation as needed. Alternatively, the pharmaceutical formulation containing the inhalation chelating agent and the antibiotic can be packaged together, thus requiring deminimum formulation by the physician or user. In any case, the packaging should maintain the chemical, physical, and aesthetic integrity of the active ingredients.

[0153] [General rules] Those skilled in the art will recognize that the present invention described herein may have variations and modifications other than those specifically described.The present invention includes all such variations and modifications.The present 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.

[0154] 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 for reasons of brevity only that documents, references, patent applications or patents cited in this text are not repeated in this text.

[0155] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products 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.

[0156] 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.

[0157] The invention described herein may include one or more ranges of values ​​(e.g., size, displacement, and magnetic field strength). A range of values ​​is considered 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. Thus, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on 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.

[0158] Throughout this specification, unless otherwise required by context, the word "comprise" or variations 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. In 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 terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent Law, for example, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0159] 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 the present invention belongs.The term "active agent" may refer to one active agent or may include two or more active agents.

[0160] The following examples serve to more fully describe how to use the above-described invention, as well as 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. EXAMPLES

[0161] 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 construed as a limitation on the broad description of the invention as set forth above.

[0162] [Microplate testing of antibiotics and adjuvants] Growth method 1: Cells were grown in M9 medium supplemented with arginine and MgSO4 in polystyrene 96-well plates (Nunc) by inoculating each well with 200 μL of an overnight culture of P. aeruginosa strain WACC91 and incubated at 37° C. for 24 h.

[0163] Growth method 2: Overnight cultures of related strains of P. aeruginosa were grown with shaking at 35°C in M63 medium supplemented with glucose (0.5% w / v), casamino acids (0.2% w / v) and MgSO4 (1 mM). 96-well microplates containing M63, along with serial dilutions of antibiotics and adjuvants as indicated, were cultured at OD in a final volume of 200 μl. 600nm The plates were incubated overnight at 35° C. without shaking.

[0164] Anaerobic cultures were incubated in a closed chamber with anaerobic sachets to remove oxygen.

[0165] Planktonic growth was monitored by OD reading or plate counting. Biofilms were quantified by staining with 40 μl of crystal violet dye (1% w / v in water) for 10 min at room temperature, washing twice with 250 μl of water, dissolving the dye in 250 μl of 50% ethanol, and reading the optical density at 595 nm.

[0166] Cells were grown as described and biofilm disruption or death was monitored by removing planktonic cells by pipetting. Attached biofilms were carefully washed three times with sterile PBS and fresh medium containing the therapeutic agent (tobramycin alone or with CaNa2EDTA in solution, Tris buffer or as dry powder particles) was added. Plates were incubated for another 24 hours and metabolic activity was measured. Biofilms were washed as described above and then fresh medium containing 0.02% w / v resazurin was added. Plates were mixed by rapid shaking and then incubated for 2 hours at 37°C. Metabolic activity, reflected by the conversion of resazurin to resafrin, was assessed by measuring the increase in fluorescence at an excitation wavelength of 530 nm and detection at 590 nm.

[0167] Checkerboard assays for other species were performed using Growth Method 1 with the following modifications: Burkholderia cepacia was incubated at 30°C and other bacteria were incubated at 37°C. Streptococcus pyogenes and Streptococcus pneumoniae were grown in Todd Hewitt Broth and Staphylococcus aureus was grown in Veal Infusion Broth.

[0168] [Example 1] High concentrations of chelating agents enhance the efficacy of tobramycin against Pseudomonas aeruginosa planktonic cells and biofilms EDTA can render bacteria susceptible to much lower concentrations of antibiotics. P. aeruginosa WACC91 cells were grown according to method 1 and tested in a checkerboard assay against different concentrations of tobramycin and CaEDTA.

[0169] Figure 1 shows that CaEDTA lowers the MIC of tobramycin for P. aeruginosa planktonic cells grown under aerobic conditions, and this effect is enhanced by Tris buffering. Figure 2 shows that CaEDTA lowers the concentration of tobramycin required to block P. aeruginosa biofilm formation. Many antibiotics work very poorly under anaerobic conditions due to slower metabolism. This is a particular challenge in chronic lung infections in patients with cystic fibrosis, where bacteria form biofilms inside anaerobic pockets of trapped airway mucus. Figures 3 and 4 demonstrate that the effect seen in Figures 1 and 2 is maintained under anaerobic conditions, particularly for buffered CaEDTA.

[0170] It is known from previous publications that at sub-MIC concentrations, tobramycin and other antibiotics do indeed increase biofilm formation in P. aeruginosa (Hoffman, LR, D'Argenio, DA, MacCoss, MJ, Zhang, Z., Jones, RA, and Miller, SI (2005). "Aminoglycoside antibiotics induce bacterial biofilm formation." Nature, 436, 1171-1175; Jones, C., Allsopp, L., Horlick, J., Kulasekara, H., and Filloux, A. (2013). "Subinhibitory concentration of kanamycin induces the Pseudomonas aeruginosa type VI secretion system." PLoS One 8, e81132.). Figure 5 shows that, indeed this is the case for tobramycin alone, the addition of CaEDTA reverses the effect, reducing planktonic and biofilm growth under both aerobic and anaerobic conditions. In addition to preventing the formation of new biofilms, Figure 6 further shows that CaEDTA increases the efficacy of tobramycin against existing P. aeruginosa biofilms, as measured by metabolic activity.

[0171] [Example 2] High concentrations of chelating agents enhance the antibacterial efficacy of a series of different antibiotics against Pseudomonas aeruginosa The effects seen in the previous examples are not specific to the antibiotic tobramycin, which belongs to the aminoglycoside class of antibiotics. Cells were grown according to method 2 and challenged with buffered CaEDTA and a series of clinically relevant antibiotics of different classes with different modes of action. Figures 7-12 show that buffered CaEDTA increases the susceptibility of P. aeruginosa planktonic cells and biofilms, as indicated, to the antibiotics methicillin (beta-lactam, Figure 7), carbenicillin (carboxypenicillin, Figure 8), tetracycline (tetracycline, Figure 9), aztreonam (monobactam; product Azactam, Figure 10), erythromycin (macrolide, Figure 11), and colistin (polymyxin, Figure 12).

[0172] [Example 3] Different chelating agents enhance the efficacy of tobramycin against Pseudomonas aeruginosa. As observed in previous examples, the effect of chelation on antibiotic susceptibility is not limited to the chelator EDTA. Cells were grown according to method 2 and challenged with different concentrations of tobramycin. Figures 13 and 14 show that ethylene glycol tetraacetic acid (EGTA) and diethylenetriaminepentaacetic acid (DTPA) also reduce the MIC of tobramycin against P. aeruginosa in a concentration-dependent manner similar to that observed for EDTA.

[0173] [Example 4] High concentrations of chelating agents enhance the efficacy of antibiotics against a variety of bacterial species The susceptibility of bacteria to antibiotics by chelators is not specific to P. aeruginosa but has been demonstrated for a variety of other bacterial species. Figures 15-21 show that different species of bacteria known to cause pulmonary infections in humans are susceptible to clinically relevant antibiotics in the presence of buffered CaEDTA. This effect is seen in both gram-negative and gram-positive bacteria, which often respond to very different antimicrobial agents.

[0174] Burkholderia cepacia is a gram-negative bacterium that can cause severe respiratory infections in patients with cystic fibrosis (Silva Filho, LV, Ferreira Fde, A., Reis, FJ, Britto, MC, Levy, CE, Clark, O. and Ribeiro, JD (2013) "Pseudomonas aeruginosa infection in patients with cystic fibrosis: scientific evidence regarding clinical impact, diagnosis, and treatment." J Bras Pneumol, 39, 495-512). Figures 15 and 16 show that the MICs of Burkholderia cepacia for two different classes of antibiotics, namely tobramycin (an aminoglycoside) and ciprofloxacin (a fluoroquinolone), are reduced in the presence of buffered CaEDTA. FIG. 17 shows that planktonic cells (FIG. 17A) and biofilms (FIG. 17B) of Klebsiella pneumoniae, another Gram-negative pulmonary pathogen, become susceptible to the antibiotic colistin in the presence of buffered CaEDTA.

[0175] For Gram-positive bacteria, Figures 18-21 show the reduction in MIC values ​​for planktonic cells and biofilms of S. pyogenes against tetracycline (Figure 18), S. aureus MRSA against vancomycin (Figure 19), and MSSA (Figure 20) and MRSA (Figure 21) against ciprofloxacin in the presence of buffered CaEDTA.

[0176] [Example 5] High concentrations of chelating agents increase bacterial killing by antibiotics Previous examples have demonstrated that chelating agents can inhibit the growth of planktonic cells and biofilms at concentrations of antibiotics lower than those of the antibiotics alone, likely as a result of increased killing by the antibiotics. Challenge microorganisms were grown on solid agar and suspended in 0.9% sodium chloride (SCI) to create a challenge suspension. Approximately 1 x 10 8 A 0.1 mL aliquot of the challenge suspension containing colony forming units was inoculated into 10 mL of Cation-Adjusted Mueller Hinton Broth (CaMHB) with the appropriate antibiotic or antibiotic and chelator as indicated and mixed thoroughly. The challenge suspension / antibiotic mixture was then incubated at 35° C. for the exposure time. At each time point, a 0.1 mL aliquot was removed and placed into 9.9 mL of Butterfield's phosphate buffer with product neutralizer, mixed thoroughly by vortexing, and plated using the appropriate agar. Recovered colonies were counted using a manual counter.

[0177] Figure 21 shows that killing of P. aeruginosa by ceftazidime is much more rapid in the presence of buffered CaEDTA, and Figure 22 shows a similar effect of meropenem against B. cepacia.

[0178] [Example 6] High concentrations of sprayable or dry powder chelating agents enhance tobramycin killing of Pseudomonas aeruginosa biofilms grown under realistic conditions Formulations of inhaled therapeutic agents include nebulized solutions and dry powders. CaEDTA dry powder was prepared by dry milling to produce submicron sized particles that can be dispersed by standard delivery devices.

[0179] Suspension droplets of cystic fibrosis mucus harvested from an epithelial cell line were used to grow biofilms in a realistic in vitro model (Haley, CL, Colmer-Hamood, JA and Hamood, AN (2012) "Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium" BMC Microbiol 12:181). Cultures of a clinical strain of P. aeruginosa (MIC tobramycin >256 μg / ml) were grown in M63 to late stationary phase without a carbon source to mimic nutrient limitation.

[0180] A few drops of mucus (5 μl) were suspended from an inverted IBIDI coverslip and then transferred to a 10 3 cfu were inoculated and then incubated for 72 hours at 35°C in a humidified environment to allow biofilm development. Droplets were then treated for 5 minutes with either nebulized tobramycin (20mg / ml), aerosolized EDTA particles (10mg / ml), or both. Controls were treated with a 50 / 50 solution of nebulized 0.9% saline / water. After treatment, droplets were incubated for 16 hours and then stained with BacLight LIVE / DEAD (1μl) and fixed with paraformaldehyde vapor for 30 minutes. Biofilms were visualized using confocal microscopy.

[0181] FIG. 24A shows a thick and strong biofilm with mostly viable cells (green) after treatment with nebulizing saline. As expected for a resistant strain, treatment with tobramycin alone has little effect on viability. CaEDTA alone causes some death (red blood cells). The combination of tobramycin and CaEDTA significantly kills the majority of the biofilm cells. FIG. 241B shows a quantitative display of the microscopy images of FIG. 24A. The control biofilm had 1×10 8 The colony forming units (CFU) / ml of EDTA-tobramycin-treated biofilms were reduced by >6 orders of magnitude and <10 2CFU / ml.

[0182] Aminoglycoside antibiotics have a narrow therapeutic index, meaning that there is a narrow therapeutic window between therapeutic and toxic effects, which in the case of tobramycin include nephrotoxicity and ototoxicity. Direct drug delivery to the lungs to achieve high peak concentrations can also be difficult, especially in patients with reduced pulmonary function. This often leads to treatment with sub-inhibitory concentrations of antibiotics, which selects for resistant strains of the infecting bacteria.

[0183] [Example 7] High-dose inhaled chelating agent reduces Pseudomonas aeruginosa infection in CF lungs Patients with CF age >6 years who were hospitalized due to symptom exacerbation were randomized to receive EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA was administered as 4 ml of nebulized solution of 50 mM CaNa2EDTA, 111 mM Tris in 0.9% saline, pH 7.1, along with the tobramycin. After randomization, subjects were treated in the hospital for 2 weeks, during which time patients were treated 4 times a day (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). Patients were then discharged and treatment continued twice a day for 4 weeks. Patients were monitored for an additional 4 weeks, making the total study time 10 weeks.

[0184] Sputum was induced with nebulized 3% hypertonic saline at 8-10 L / min for ≥5 min. Samples were collected pre-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 h, then placed in Skim Milk Glycerol storage medium and stored at -80°C.

[0185] 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 agar for Pseudomonas species. Plates were incubated at 35°C for 48 hours, and bacteria were enumerated by colony counts. Identification of P. aeruginosa was further confirmed by Gram dye (negative), oxidase test (negative), and resistance to C390 antibiotic. Data were expressed as colony forming units per milliliter (CFU / ml).

[0186] Figure 25 shows the change in colony counts for P. aeruginsosa (McC) at weeks 2 and 6 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.

[0187] [Example 8] High doses of inhaled chelators produce a concentration-dependent increase in FEV1 Patients with CF age ≥6 years who were hospitalized due to symptom exacerbation were randomized to receive EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA was administered as 4 ml of nebulized solution of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1, together with the tobramycin. After randomization, subjects were treated in the hospital for 2 weeks, during which they were treated 4 times a day (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). Subjects were then discharged and treatment continued twice a day for 4 weeks. Patients were monitored for an additional 4 weeks, making the total study time 10 weeks. At each study visit, pulmonary function was measured by spirometry. Data were recorded as the best of 3 attempts, and results were expressed as % of predicted values.

[0188] Figure 26 shows the mean change in FEV1 for both groups at 2, 6 and 10 weeks after the start of treatment, demonstrating a clear improvement in lung function in the EDTA group, but only a slight change in the placebo group.

[0189] FIG. 27 shows the EDTA group (R 2 = 0.70), but not in the placebo group treated with tobramycin alone (R 2 = 0.01), indicating that EDTA has a dose-dependent effect (mg EDTA / kg body weight) on pulmonary function.

[0190] The concentration of EDTA achieved in expectorated sputum was measured by LC-MS / MS. To confirm that EDTA penetrates the CF mucus to reach the most difficult to treat bacteria, sputum was cut from the mucus plug and a known mass was placed in a smoked glass HPLC vial. 13 LC-MS grade methanol (1 ml) containing labeled EDTA (10 μM) was added to each vial, mixed by vortexing for 5 min, and chilled at −20° C. for 20 min. The vials were then centrifuged at 100 g for 5 min to sediment precipitated proteins, and 754 μL of methanol extract was taken for derivatization. Concentrated hydrochloric acid (246 μl) was added, the vials were vortex mixed, and incubated at 65° C. for 6 h to methyl-esterify the EDTA. The derivatized extract (1 μl) was injected into a Waters microflow HPLC equipped with a triple quadrupole mass spectrometer detector. The amount of extracted derivatized EDTA present in the samples was quantified using the counts of the quantification ions at m / z 190.1 for labeled EDTA and m / z 188.0 for unlabeled EDTA at a fragmentation energy of 135.0 V.

[0191] Figure 28 shows that EDTA concentrations in the mucus plug 5 minutes after treatment are in the millimolar range, demonstrating efficient delivery and good penetration through the mucus. Peak concentrations in airway surface liquid are likely higher. EDTA is still detectable in the mucus after 2 hours, albeit at much lower levels, likely due to a combination of distribution and clearance.

[0192] [Hypothetical Example P1: Chelating agents act synergistically with antibiotics to enhance antibiotic efficacy] A method to test synergy between antibiotics and chelators is the checkerboard method. A series of different bacterial species are grown into biofilms in 96-well plates and then challenged with different concentrations of chelators and antibiotics. Colorimetric tests are used to indicate biofilm levels and metabolic activity (Bueno, "Anti-Biofilm Drug Susceptibility Testing Methods: Looking for New Strategies against Resistance Mechanism," J Microbial Biochem Technol 2014, S3; Orhan et al., "Synergy test sby E test and checkerboard methods of antimicrobial combinations against Brucella melitensis," J Clin Microbiol, January 2005; vol. 43(1): pp. 140-3).

[0193] This experiment is expected to show a concentration-dependent reduction in biofilm with increasing concentrations of either the antibiotic or the chelator alone, with a further reduction in biofilm levels when both are combined.

[0194] [Hypothetical Example P2: Buffering enhances the ability of chelators to multiply biofilm killing by antibiotics] A series of different bacterial species are grown into biofilms in 96-well plates and then challenged with different concentrations of chelating agents and buffers, such as Tris. Colorimetric tests are used to indicate biofilm levels and metabolic activity. The bacterial biofilms are further challenged with chelating agents and antibiotics while changing the acidity of the environment.

[0195] It is known that aminoglycoside antibiotics work better against planktonic cells and biofilms at high pH in vitro and in vivo (Lebeaux et al., J Infect Dis. 2014, 210(9):1357-66), and the affinity of chelating agents such as EDTA increases with pH. Thus, the present experiments are expected to show that the efficacy of chelating agents as antibiotic adjuvants is highest at or above physiological pH.

[0196] [Hypothetical Example P3: In vivo study of the effects of high doses of nebulized chelating agents on inflammation, lung injury, and oxidative stress] Subjects with CF age ≥ 6 years who are hospitalized due to symptom exacerbation are randomized to receive nebulized EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA is administered in 4 ml of nebulized solution of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1 along with the tobramycin.

[0197] After randomization, subjects are treated in the hospital for 2 weeks, during which they receive treatment 4 times a day (300 mg EDTA / day, or up to 3.3 mg EDTA / kg / day). They are then discharged and treatment continues twice a day for 4 weeks. Subjects are monitored for an additional 4 weeks, making the total study time 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.

[0198] -Inflammatory marker expression Expected sputum is stored in RNAlater® and total RNA is extracted using Qiagen RNEasy® or 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 in Sivaneson et al. (Sivaneson, M., Mikkelsen, H., Ventre, I., Bordi, C. and Filloux, A. (2011) "Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression" Mol Microbiol 79:1353-1366).

[0199] This study is expected to show a mean reduction in gene expression of inflammatory markers in the EDTA group compared to the placebo group.

[0200] Cellular damage, free iron and oxidative stress Expected sputum is frozen as is without 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) are measured using gelatin zymography and immunoassays, respectively, as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-27; Garratt et al., Eur Respir J. 2015, 46(2):384-94). The amount of iron in the sputum is quantified by ICP-MS as previously described (Hunter et al., MBio. 2013, 4(4):1-8). The amount of iron-binding proteins is 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).

[0201] The study is expected to show a reduction in inflammatory markers 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 between MMP-9 and TIMP-1, which accompanies the progression of bronchiectasis.

[0202] [Hypothetical Example P4: In vivo study of the effects of high dose dry powder chelating agents on infection, inflammation, and oxidative stress] Subjects with CF who need to be treated 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 old) will receive ascending doses of dry powder CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks; 150 mg BID for 1 week). Patients in cohort 2 (>18 years old) will receive CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks; 75 mg QID for 1 week). Patients in cohort 3 (12-18 years old) will receive CaEDTA (37.5 mg BID for 1 week; 75 mg BID for 2 weeks; 150 mg BID for 1 week). Finally, the control cohort will receive tobramycin alone for 28 days.

[0203] 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 immunoassays, respectively, as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-27; Garratt et al., Eur Respir J. 2015, 46(2):384-94). The amount of iron in sputum is quantified by ICP-MS as previously described (Hunter et al., 2013). Immunoassays are used to assess the amount of iron-binding proteins. Myeloperoxidase activity is also assayed as a measure of neutrophil inflammation, as previously described (Gaggar et al., 2011). 3-Chlorotyrosine is measured as a biomarker for hypochlorous acid, a strong oxidant. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., 2011). Commercially available immunoassay kits are used to measure protein carbonyls as indicators of reactive oxygen species (ROS) (Gaggar et al., 2011). Immunoassays are used to assess oxidative stress by measuring glutathione (GSSG and GSH) as previously described (Kettle et al., 2014). Gene expression of inflammatory and oxidative stress markers (e.g. IL-8, IL-6, TNFa) is also monitored with Nanostring and proteins are measured with ELISA. Oxidative stress can also be measured via metabolites, for example, malondialdehyde (colorimetric assay) or 8-isoprostane (ELISA). Iron is measured by elemental analysis using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS).

[0204] The 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. The experiment 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.

[0205] 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.

[0206] [Hypothetical Example P5: In vivo study of the effects of high doses of dry powder chelating agents on inflammation, lung injury, and oxidative stress] Subjects with CF age ≥ 6 years who are hospitalized due to symptom exacerbation are randomized to receive dry powder EDTA or saline (placebo) in addition to their usual treatment with intravenous antibiotics and nebulized tobramycin. EDTA is administered in 4 ml of nebulized solution of 50 mM CaEDTA, 111 mM Tris in 0.9% saline, pH 7.1 along with the tobramycin.

[0207] After randomization, subjects are treated in the hospital for 2 weeks, during which they receive treatment 4 times a day (up to 300 mg EDTA / day, or 3.3 mg EDTA / kg / day). They are then discharged and treatment continues twice a day for 4 weeks. Subjects are monitored for an additional 4 weeks, making the total study time 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.

[0208] -Inflammatory marker expression Expected sputum is stored in RNAlater and total RNA is extracted using Qiagen RNEasy® or 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 in Sivaneson et al. (Sivaneson, M, Mikkelsen, H., Ventre, I., Bordi, C. and Filloux, A. (2011) "Two-component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression" Mol Microbiol 79:1353-1366).

[0209] This study is expected to show a mean reduction in gene expression of inflammatory markers in the EDTA group compared to the placebo group.

[0210] Cellular damage, free iron and oxidative stress Expected sputum is frozen as is without 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) are measured using gelatin zymography and immunoassays, respectively, as previously described (Gaggar et al., Eur Respir J. 2011, 38(3):721-27; Garratt et al., Eur Respir J. 2015, 46(2):384-94). 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 proteins is assessed using immunoassays. Oxidative stress is assessed by measuring glutathione (GSSG and GSH) as previously described (Kettle et al., Eur Respir J. 2014, 44(1):122-9).

[0211] The 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. The experiment 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.

[0212] This experiment is further expected to show an increase in FEV1 in subjects treated with EDTA compared to placebo (vehicle only).

[0213] This is demonstrated in Example 4 above.

[0214] [Hypothetical Example P6: 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 treatment with inhaled CaEDTA or saline (placebo) 2-4 times daily for 2 weeks. This will be followed by a washout period and then 2 weeks of the other treatment (EDTA or placebo).

[0215] Iron levels, inflammatory markers, MMP / TIMP and FEV1 are monitored as above. Myeloperoxidase activity is also assayed as a measure of neutrophil inflammation as previously described (Gaggar et al., 2011). 3-Chlorotyrosine is measured as a biomarker of the potent oxidant hypochlorous acid. Levels are measured using stable isotope dilution gas chromatography with mass spectrometry (Gaggar et al., 2011). Protein carbonyls are measured as an indicator of ROS using a commercially available immunoassay kit (Gaggar et al., 2011).

[0216] The study is expected to show reduced levels of iron and inflammatory markers, changes in MMP / TIMP balance, and increased 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.

[0217] Clinical data demonstrate efficacy at 300 mg / day (75 mg QID) for 2 weeks (reduction in bacterial counts, FIG. 25; improvement in lung function, FIG. 26) and sustained effect for an additional 4 weeks at 150 mg / day (75 mg BID). Given the significant magnitude of improvement (mean 16 percentage points), it is highly likely that a much lower dose would be effective, i.e., 75 mg / day (37.5 mg BID), as envisioned by hypothetical Example P4, as would be understood by one of skill in the art.

[0218] Figure 28 shows that a single dose of 75 mg CaEDTA results in up to 1.34 mM EDTA inside the mucus plug after 30 minutes. Thus, concentrations in airway surface liquids are most likely substantially higher. In vitro data (Figures 1B and 2B) demonstrate that CaEDTA is effective at concentrations as low as 0.078 mM EDTA, or 17-fold lower than sputum concentrations. Thus, it would be reasonable to expect that a daily dose of 37.5 (4-fold lower than the lower dose with clinical benefit) would show efficacy in a fully potentiated experiment. This is especially true for young patients who receive higher doses per body weight, which generally show a greater therapeutic effect in FEV1 (Figure 27).

[0219] 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 relating 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 deemed to be within the scope of the invention, the nature of which is determined from the foregoing description.

[0220] Research into this invention was supported by an award from Cystic Fibrosis Foundation Therapeutics.

Claims

1. 1. An inhalable composition for treating a bacterial infection in the lungs, comprising an antibiotic that is tobramycin and a chelating agent that is ethylenediaminetetraacetic acid (EDTA), wherein the chelating agent is present in a concentration of at least 50 mM in the inhalable composition, the inhalable composition being used to deliver 37.5 mg / day to 300 mg / day of the chelating agent and 10 mg / day to 1000 mg / day of the antibiotic, each dose of the chelating agent and the antibiotic being delivered over a period of 2 hours or less, each dose of the inhalable composition comprising droplets or particles having a particle size of 0.1 μm to 12 μm, and the inhalable composition being free of acidified nitrite.

2. 2. The inhalation composition of claim 1, wherein the chelating agent is calcium ethylenediaminetetraacetic acid (CaEDTA).

3. 3. The inhalation composition of claim 1, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (TRIS).

4. An inhalation composition described in any one of claims 1 to 3, wherein the antibiotic and chelating agent are administered 1 to 4 times daily.

5. 1. Use of a chelating agent which is ethylenediaminetetraacetic acid (EDTA) at a concentration of at least 50 mM for the manufacture of an inhalation composition for the treatment of a bacterial infection in the lungs, wherein the inhalation composition is adapted to deliver 37.5 mg / day to 300 mg / day of the chelating agent, and the inhalation composition comprising the chelating agent is adapted to deliver 10 mg / day to 1000 mg / day of the inhalation antibiotic which is tobramycin when used with an inhalation antibiotic composition, wherein each dose of the inhalation composition comprises the chelating agent, the inhalation composition comprises the antibiotic adapted to be administered for a period of not more than 2 hours, each dose of the inhalation composition comprises droplets or particles having a particle size of 0.1 μm to 12 μm, and the inhalation composition does not comprise acidified nitrite.

6. 1. Use of an antibiotic which is tobramycin for the manufacture of an inhalation composition for the treatment of a bacterial infection in the lungs, wherein the inhalation composition is adapted to deliver 10 mg / day to 1000 mg / day of the antibiotic, and the inhalation composition comprising the antibiotic is adapted to deliver 37.5 mg / day to 300 mg / day of the inhalation chelating agent which is ethylenediaminetetraacetic acid (EDTA) at a concentration of at least 50 mM when used with an inhalation chelating agent composition, wherein each dose of the inhalation composition comprises the chelating agent, each dose of the inhalation composition comprises the antibiotic administered over a period of 2 hours or less, each dose of the inhalation composition comprises droplets or particles having a particle size of 0.1 μm to 12 μm, and the inhalation composition does not comprise acidified nitrite.

7. 7. The use according to claim 5 or 6, wherein the chelating agent is calcium ethylenediaminetetraacetic acid (CaEDTA).

8. 8. The use according to any one of claims 5 to 7, wherein the chelating agent is combined with tris(hydroxymethyl)aminomethane (TRIS).

9. i) the chelating agent is used for administration 1 to 4 times daily; and / or ii) the antibiotic is administered 1 to 4 times daily; 9. Use according to any one of claims 5 to 8.

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