Stable ascorbic acid compositions and methods of using same - Patents.com

Abstract

To provide compositions comprising organic acid, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, and methods of using the same.SOLUTION: A composition comprises: (a) glutathione, a glutathione derivative, a glutathione conjugate, or a pharmaceutically acceptable salt thereof; and (b) an organic acid; where the molar ratio of (a) to (b) is about 0.5-1:1. Preferably, the organic acid is ascorbic acid. Preferably, the composition further comprises (c) a bicarbonate salt.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 588,300, filed November 17, 2017, and No. 62 / 684,700, filed June 13, 2018, each of which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to compositions comprising an organic acid, glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof, and uses thereof. [Background technology]

[0003] background Glutathione or "GSH" has the formula: TIFF0007617179000001.tif18128, or a zwitterionic form thereof, e.g., a compound having the formula: Refers to the compound having TIFF0007617179000002.tif23128.

[0004] Healthy individuals have high concentrations of GSH in their airways. However, glutathione levels are depleted in individuals with chronic inflammatory airway diseases, such as lung transplant patients. Both inflammation and infection are associated with lung transplant rejection / dysfunction and failure. Current lung transplant treatments, including high doses of immunosuppressants and antibacterial agents, have systemic side effects and toxicity that can lead to further inflammation and infection. In addition, repeated use of high doses of antibiotics has been shown to cause multidrug-resistant infections.

[0005] Lower respiratory tract infections from chronic inflammatory airway diseases present in a variety of airway disorders and diseases, including, for example, lung transplant patients and patients with cystic fibrosis (CF) and bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis). Summary of the Invention

[0006] overview The present disclosure provides a composition comprising (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof, and (b) an organic acid, wherein the molar ratio of (a) to (b) is about 0.5 to 1:1, and the pH of the formulation is at least 5.5. In some embodiments, the organic acid is ascorbic acid. In some embodiments, the composition further comprises (c) a bicarbonate salt (e.g., sodium bicarbonate). In some embodiments, the composition does not comprise a bicarbonate salt.

[0007] In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1. In some embodiments, the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5-1:1. In some embodiments, the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5:1 or about 0.4-0.5:1:1.

[0008] In some embodiments, the reduced glutathione in the composition is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the composition after storage of the composition at about 5° C. for 4 weeks.

[0009] In some embodiments, the oxidized glutathione in the composition is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% by weight of the total glutathione in the composition after storage of the composition for 4 weeks (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0010] In some embodiments, reduced ascorbic acid is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of the total ascorbic acid in the composition after storage of the composition at about 5° C. for 4 weeks.

[0011] In some embodiments, the oxidized ascorbic acid in the composition is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the composition after storage of the composition at about 5° C. for 4 weeks.

[0012] In some embodiments, the composition is stored under ambient conditions, e.g., without nitrogen sparging. In some embodiments, the composition is stored with nitrogen sparging.

[0013] In some embodiments, the pH of the composition is about 5.5 to about 10, about 5.5 to about 8, about 6 to about 10, or about 6 to about 8. In some embodiments, the pH is about 5.5, about 6.5, about 7.0, or about 7.5. In some embodiments, the pH of the composition is 7±1.5. In some embodiments, the pH of the composition is about 6.

[0014] In some embodiments, the composition is an aqueous solution, a dry powder, or a lyophilizate.

[0015] Certain aspects of the disclosure are directed to methods of inhibiting or reducing the growth of a clinical isolate of bacteria, comprising contacting the clinical isolate with a disclosed composition.

[0016] Certain aspects of the present disclosure are directed to a method of inhibiting or reducing the formation of a clinically isolated bacterial biofilm, comprising contacting the clinical isolate with a composition comprising a disclosed composition.

[0017] Certain aspects of the present disclosure are directed to methods of treating or alleviating symptoms in a subject suffering from or at risk of a clinically isolated bacterial infection, comprising contacting the clinical isolate with a disclosed composition.

[0018] In some embodiments, the subject has a lung or airway disease or disorder, hi some embodiments, the lung or airway disease or disorder is cystic fibrosis or non-cystic fibrosis bronchiectasis.

[0019] Certain aspects of the disclosure are directed to methods of upregulating mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering a disclosed composition to the subject. In some embodiments, administering the composition reduces mucus viscosity in the patient. In some embodiments, administering the composition increases ciliary beat frequency of airway epithelial cells in the patient. In some embodiments, administering the composition increases mucociliary transport rate of airway epithelial cells in the patient. In some embodiments, administering the composition increases airway surface liquid height in the patient.

[0020] In some embodiments, the patient's airway epithelium is not colonized with bacteria. In some embodiments, the patient's airway epithelium is colonized with bacteria. In some embodiments, the patient has an active bacterial infection. In some embodiments, the patient does not have an active bacterial infection.

[0021] Certain aspects of the disclosure are directed to methods of reducing airway inflammation in a subject suffering from or at risk of airway inflammation, comprising administering to the subject a disclosed composition.

[0022] In some embodiments, administering the composition inhibits myeloperoxidase activity in the patient's neutrophils. In some embodiments, administering the composition reduces the formation of neutrophil extracellular traps. In some embodiments, administering the composition downregulates nitric oxide production from the patient's neutrophils. In some embodiments, administering the composition reduces exhaled nitric oxide (FeNO) in the patient by at least 20%. In some embodiments, the composition is administered twice daily. In some embodiments, the composition is administered by a nebulizer. In some embodiments, administering the composition reduces exhaled nitric oxide (FeNO) in the patient by at least 20% after one month of administration of the composition (e.g., twice daily).

[0023] In some embodiments, administering the composition downregulates production of at least one proinflammatory cytokine (e.g., TNF-α, IL-6, and / or IL-8) and / or at least one neutrophil- and macrophage-associated cytokine (e.g., MIP1α and / or MIP1β).

[0024] Certain aspects of the disclosure are directed to a method of reducing mucus viscosity in a subject suffering from or at risk of suffering from reduced mucus viscosity, comprising administering to the subject a disclosed composition.

[0025] Certain aspects of the disclosure are directed to a method of activating cystic fibrosis transmembrane receptor (CFTR) function in a subject suffering from reduced CFTR function, comprising administering to the subject a disclosed composition.

[0026] Certain aspects of the disclosure are directed to a method of increasing expression of the Cystic Fibrosis Transmembrane Receptor (CFTR) in a subject suffering from decreased expression of CFTR, comprising administering to the subject a disclosed composition.

[0027] Certain aspects of the disclosure are directed to methods comprising administering a disclosed composition in combination with a CFTR therapy selected from the group consisting of a CFTR amplifier (e.g., PTI-428), a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or a combination thereof), a CFTR enhancer / modulator (e.g., VX-770 (ivacaftor), QBW251, VX-561, PT1-808, or a combination thereof), a CFTR RNA regulator (e.g., QR-100, MRT5005, or a combination thereof), or any combination thereof. In some embodiments, the administration of the CFTR treatment is simultaneous with administration of the disclosed composition to the subject, or sequential in any order (e.g., the composition can be administered before or after the CFTR treatment). In some embodiments, the CFTR treatment is administered orally and the disclosed composition is administered by inhalation. In some embodiments, both the cystic fibrosis treatment and the disclosed composition are administered by inhalation.

[0028] In some embodiments, the methods include administering to the subject a composition of the present disclosure in combination with VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination thereof.

[0029] In some embodiments, administering a disclosed composition in combination with administering VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770 / VX-809 to a subject reduces mucus viscosity in the airways of the subject more than administration of the disclosed composition or VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770 / VX-809 alone.

[0030] In some embodiments, administering a disclosed composition in combination with administering VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770 / VX-809 to a subject increases the ciliary beat frequency of airway epithelial cells in the subject more than administration of the composition alone.

[0031] In some embodiments, administering a disclosed composition in combination with administering to a subject VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770 / VX-809 increases the mucociliary transport rate of airway epithelial cells in the subject more than administration of the composition alone.

[0032] In some embodiments, administering a composition of the present application in combination with administering VX-770, VX-809, or a combination of VX-770 / VX-809 to a subject increases the airway surface liquid height in the subject more than administering the composition alone.

[0033] In some embodiments, the disclosed compositions are administered in combination with VX-770, VX-809, or a combination of VX-770 / VX-809, and administration is simultaneous to a subject or sequential in any order (e.g., the compositions can be administered before or after a combination of VX-770 / VX-809).

[0034] In some embodiments, the composition is stable for at least 72 hours at 2-8° C. In some embodiments, the composition after storage for at least 72 hours at 2-8° C. comprises any one or more of: (a) essentially no precipitation, (b) contains less than 4% impurities, (c) has a pH of 6.0-7.0, and (d) shows minimal loss in solubility. [The present invention 1001] (a) glutathione, glutathione derivatives, glutathione conjugates, or pharma- ceutically acceptable salts thereof; (b) Organic acids and wherein the molar ratio of (a) to (b) is about 0.5 to 1:1. [The present invention 1002] The composition of claim 10, wherein the organic acid is ascorbic acid. [The present invention 1003] (c) The composition of any one of claims 1001 and 1002, further comprising a bicarbonate salt. [The present invention 1004] The composition of claim 10, wherein the bicarbonate is sodium bicarbonate. [The present invention 1005] Any of the compositions according to claims 1001 to 1004, comprising (a) glutathione, (b) ascorbic acid, and (c) sodium bicarbonate. [The present invention 1006] Any of compositions 1001 to 1005 of the present invention, wherein the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1. [The present invention 1007] Any of compositions 1001 to 1006 of the present invention, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5-1:1. [The present invention 1008] Any of compositions 1001-1007 of the present invention, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5:1 or about 0.4-0.5:1:1. [The present invention 1009] Any of the compositions according to claims 1001 to 1008, wherein the pH of the composition is at least about 5.5. [The present invention 1010] The composition according to any one of claims 1001 to 1009, wherein the pH of the composition is about 5.5 to about 10, about 5.5 to about 8, about 6 to about 10, or about 6 to about 8. [The present invention 1011] Any of the compositions of 1001 to 1010, wherein the pH is about 5.5, about 6.5, about 7.0, or about 7.5. [The present invention 1012] The composition of claim 10, wherein the pH of the composition is 7±1.5. [The present invention 1013] The composition of claim 10, wherein the pH of the composition is about 6. [The present invention 1014] Any of compositions 1001 to 1013 of the present invention, wherein the reduced glutathione in the composition is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the composition after storage of the composition at about 5°C for 4 weeks. [The present invention 1015] Any of compositions 1001 to 1014 of the present invention, wherein the oxidized glutathione in the composition is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% by weight of the total glutathione in the composition after storage of the composition for 4 weeks (e.g., at 5°C in an N2 atmosphere and / or ambient atmosphere). [The present invention 1016] Any of compositions 1001 to 1015 of the present invention, wherein reduced ascorbic acid is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of the total ascorbic acid in the composition after storage of the composition at about 5°C for 4 weeks. [The present invention 1017] Any of compositions 1001 to 1016 of the present invention, wherein the oxidized ascorbic acid in the composition is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the composition after storage of the composition at about 5°C for 4 weeks. [The present invention 1018] Any of the compositions according to claims 1001 to 1017, suitable for pharmaceutical use. [The present invention 1019] The composition according to any one of claims 1001 to 1018, which is an aqueous solution. [The present invention 1020] The composition of any one of claims 1001 to 1018, which is a dry powder. [The present invention 1021] Any of the compositions of claims 1001 to 1018, which are suitable for administration by inhalation. [The present invention 1022] A method for inhibiting or reducing the growth of a clinically isolated bacterium, the method comprising the step of contacting the clinically isolated bacterium with any one of the compositions of present inventions 1001 to 1021. [The present invention 1023] A method for inhibiting or reducing the formation of a clinically isolated bacterial biofilm, the method comprising the step of contacting the clinically isolated bacteria with a composition comprising any one of the compositions of present inventions 1001 to 1021. [The present invention 1024] A method for treating or alleviating symptoms in a subject suffering from or at risk of a clinically isolated bacterial infection, comprising contacting the clinical isolate with any of the compositions of the present inventions 1001 to 1021. [The present invention 1025] The method of claim 1024, wherein the subject has a disease or disorder of the lungs or airways. [The present invention 1026] The method of the present invention, wherein the lung or airway disorder is selected from the group consisting of chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute lung rejection or lung rejection and / or pulmonary dysfunction, pulmonary arterial hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bronchiectasis, bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD), bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation-related or chemical-related airway disease or disease, and any combination thereof. [The present invention 1027] The method of the present invention 1025, wherein the lung or airway disease or disorder is selected from the group consisting of chronic inflammatory lung disease, inflammation and / or infection associated with lung transplantation, acute or chronic lung rejection or pulmonary dysfunction, asthma, cystic fibrosis, bronchiectasis, or chronic obstructive pulmonary disease (COPD), or any combination thereof. [The present invention 1028] 1027. The method of claim 1027, wherein said lung or airway disease or disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, or cystic fibrosis bronchiectasis. [The present invention 1029] The method of claim 1027, wherein the subject is a lung transplant patient. [The present invention 1030] Any of the methods of claims 1024 to 1029, wherein the subject has a biofilm infection. [The present invention 1031] The method of any one of claims 1024 to 1030, further comprising the step of administering an additional therapeutic substance to the subject. [The present invention 1032] The method of claim 1031, wherein said additional therapeutic agent is an antibiotic. [The present invention 1033] The method of any of claims 1022 to 1032, wherein the clinical isolates include two or more clinical isolates. [The present invention 1034] The method of any one of claims 1022 to 1033, wherein the clinically isolated bacterium is gram-negative or gram-positive. [The present invention 1035] The method of any one of claims 10 to 34, wherein the two or more clinical isolates comprise a combination of Gram-negative and Gram-positive clinical isolates. [The present invention 1036] The method according to any one of claims 1022 to 1035, wherein the clinically isolated bacteria is mucoid or non-mucoid. [The present invention 1037] The method according to any one of claims 1022 to 1036, wherein the clinically isolated bacteria is aerobic, anaerobic, or aerotolerant. [The present invention 1038] The method of claim 1037, wherein the aerobic, anaerobic, or aerotolerant conditions involve nitrate. [The present invention 1039] The method according to any one of claims 1023 to 1038, wherein the clinically isolated bacterium is multidrug resistant. [The present invention 1040] A method for upregulating mucociliary clearance in a subject suffering from or at risk of reduced mucociliary clearance, comprising administering to the subject any of the compositions of present inventions 1001 to 1021. [The present invention 1041] The method of claim 1040, wherein administering said composition reduces mucus viscosity in said subject. [The present invention 1042] The method of claim 1040, wherein administering said composition increases the ciliary beat frequency of airway epithelial cells of said subject. [The present invention 1043] The method of claim 1040, wherein administering said composition increases the mucociliary transport rate of airway epithelial cells of said subject. [The present invention 1044] The method of claim 1040, wherein administering said composition increases airway surface liquid height in said subject. [The present invention 1045] The method of any of claims 1040 to 1044, wherein the subject is suffering from or at risk of suffering from a lung or airway disease or disorder. [The present invention 1046] The method of the present invention, wherein the lung or airway disorder is selected from the group consisting of chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute lung rejection or lung rejection and / or pulmonary dysfunction, pulmonary arterial hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bronchiectasis, bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD), bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation-related or chemical-related airway disease or disease, and any combination thereof. [The present invention 1047] The method of claim 1046, wherein said lung or airway disease or disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, or cystic fibrosis bronchiectasis. [The present invention 1048] The method according to any one of claims 1045 to 1047, wherein airway epithelial cells of the subject lack mucociliary clearance ability before administration of the composition. [The present invention 1049] The method of claim 1048, wherein the subject's airway epithelial cells lack mucociliary clearance capacity due to a genetic defect. [The present invention 1050] The method of claim 1045, wherein the subject has had a lung transplant. [The present invention 1051] The method of any of claims 1045 to 1050, wherein the airway epithelium of said subject is not colonized by bacteria. [The present invention 1052] The method of any of claims 1045 to 1050, wherein the subject's airway epithelium is colonized with bacteria. [The present invention 1053] The method of claim 1052, wherein the subject has an active bacterial infection. [The present invention 1054] The method of claim 1052, wherein the subject is not suffering from an active bacterial infection. [The present invention 1055] The method of any one of claims 1051 to 1054, wherein the subject is a pediatric patient. [The present invention 1056] The method of any one of claims 1051 to 1054, wherein the subject is an adult patient. [The present invention 1057] A method for reducing airway inflammation in a subject suffering from or at risk of having airway inflammation, the method comprising the step of administering to the subject any one of the compositions of present inventions 1001 to 1021. [The present invention 1058] The method of claim 1057, wherein administering the composition inhibits myeloperoxidase activity in neutrophils of the subject. [The present invention 1059] The method of any one of claims 1057 to 1058, wherein administering the composition reduces the formation of neutrophil extracellular traps. [The present invention 1060] The method of any one of claims 1057 to 1059, wherein the step of administering the composition downregulates production of nitric oxide from neutrophils of the subject. [The present invention 1061] The method of claim 1060, wherein administering said composition reduces exhaled nitric oxide in said subject by at least 20%. [The present invention 1062] The method according to any one of claims 1057 to 1061, wherein the production of at least one inflammatory cytokine is downregulated by administering said composition. [The present invention 1063] The method of claim 1062, wherein said at least one inflammatory cytokine comprises a cytokine associated with macrophage activation and / or with the recruitment of neutrophils and T cells. [The present invention 1064] The method of claim 1063, wherein said at least one inflammatory cytokine comprises TNF-α. [The present invention 1065] The method of claim 1063, wherein said at least one inflammatory cytokine comprises IL-6. [The present invention 1066] The method of claim 1063, wherein said at least one inflammatory cytokine comprises IL-8. [The present invention 1067] The method of any of claims 1057 to 1066, wherein the subject is suffering from or at risk of suffering from a lung or airway disease or disorder. [The present invention 1068] The method of the present invention, wherein the lung or airway disorder is selected from the group consisting of chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute lung rejection or lung rejection and / or pulmonary dysfunction, pulmonary arterial hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bronchiectasis, bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD), bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation-related or chemical-related airway disease or disease, and any combination thereof. [The present invention 1069] The method of claim 1068, wherein said lung or airway disease or disorder is cystic fibrosis, non-cystic fibrosis bronchiectasis, or cystic fibrosis bronchiectasis. [The present invention 1070] The method of claim 1067, wherein the subject has had a lung transplant. [The present invention 1071] The method of any of claims 1067 to 1070, wherein the airway epithelium of said subject is not colonized by bacteria. [The present invention 1072] The method of any of claims 1067 to 1071, wherein the patient's airway epithelium is colonized with bacteria. [The present invention 1073] The method of claim 1072, wherein the subject has an active bacterial infection. [The present invention 1074] The method of claim 1072, wherein the subject is not suffering from an active bacterial infection. [The present invention 1075] The method of any one of claims 1067 to 1074, wherein the subject is a pediatric patient. [The present invention 1076] The method of any one of claims 1067 to 1074, wherein the subject is an adult patient. [The present invention 1077] The method of any of claims 1040 to 1076, which does not significantly alter the pH of the airway endothelium of said subject. [The present invention 1078] A method for reducing mucus viscosity in a subject suffering from or at risk of suffering from reduced mucus viscosity, the method comprising administering to the subject any of the compositions of present inventions 1001 to 1021. [The present invention 1079] A method for activating cystic fibrosis transmembrane receptor (CFTR) function in a subject suffering from reduced CFTR function, the method comprising the step of administering any one of the compositions of the present inventions 1001 to 1021 to the subject. [The present invention 1080] A method for improving expression of the cystic fibrosis transmembrane receptor (CFTR) in a subject suffering from decreased expression of CFTR, the method comprising the step of administering any one of the compositions of present inventions 1001 to 1021 to the subject. [The present invention 1081] Any of the methods of claims 1024 to 1080, further comprising administering a CFTR therapy selected from the group consisting of a CFTR amplifier, a CFTR corrector, a CFTR enhancer / modulator, a CFTR RNA regulator, or any combination thereof. The method of claim 1081, wherein said CFTR corrector is selected from the group consisting of VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, and combinations thereof. [The present invention 1082] 1083. The method of claim 1081 or 1082, wherein said CFTR enhancer / modulator is selected from the group consisting of VX-770 (ivacaftor), QBW251, VX-561, PT1-808, and combinations thereof. [The present invention 1083] The method of any one of claims 1081 to 1083, wherein the CFTR amplifier is PTI-428. [The present invention 1084] The method of any one of claims 1081 to 1084, wherein the CFTR RNA modulator is QR-100, MRT5005, or a combination thereof. [The present invention 1085] The method of claim 1081, wherein said CFTR treatment comprises said CFTR corrector and said CFTR enhancer / modulator. [The present invention 1086] 1086. The method of claim 1086, wherein said CFTR treatment comprises VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination thereof. [The present invention 1087] The method according to any one of claims 1081 to 1087, wherein the mucus viscosity in the airways of the subject is reduced more than by administration of the composition alone. [The present invention 1088] The method according to any one of claims 1081 to 1088, wherein the ciliary beat frequency of airway epithelial cells of the subject is increased more than administration of the composition alone. [The present invention 1089] The method according to any one of claims 1081 to 1089, wherein the mucociliary transport rate of airway epithelial cells of the subject is increased more than administration of the composition alone. [The present invention 1090] The method of any of claims 1081 to 1090, wherein the composition and the CFTR are administered to the subject simultaneously or sequentially in any order. [The present invention 1091] Any of the methods of claims 1024 to 1091, wherein the subject's exhaled nitric oxide (FeNO) is reduced by at least 20% after administration of the composition for one month. [The present invention 1092] The method of claim 1092, wherein the subject is a lung transplant patient. [The present invention 1093] The method of any of claims 1024 to 1093, wherein the composition is administered by spraying twice daily. [The present invention 1094] Any of compositions of inventions 1001 to 1024 or any of methods of inventions 1024 to 1094, wherein the composition after storage for at least 72 hours at 2 to 8°C comprises any one or more of: (a) essentially no precipitation; (b) containing less than 4% impurities; (c) maintaining a pH of 6.0 to 7.0; and (d) experiencing minimal loss of solubility. [Brief description of the drawings]

[0035] [Figure 1] 1 shows the mucociliary transport rate of primary human bronchial epithelial cells (df508- / -) isolated from the airways of cystic fibrosis patients following administration of Composition 1. [Diagram 2] FIG. 1 shows mucociliary transport rates of primary human bronchial epithelial cells (df508− / −) isolated from the airways of cystic fibrosis patients following administration of Composition 1, bicarbonate, glutathione, bicarbonate+glutathione, or bicarbonate+ascorbate. [Diagram 3] NETosis staining of human neutrophils isolated from healthy volunteers following administration of phorbol myristate acetate (PMA) and Composition 2. [Figure 4] 1 shows DAF-FM diacetate staining of nitric oxide in human neutrophils following administration of lipopolysaccharide (LPS) and Composition 1. [Figure 5A]1 shows the concentration of the pro-inflammatory cytokine TNF-α secreted by human neutrophils following administration of lipopolysaccharide (LPS) and Composition 1. [Figure 5B] 1 shows the concentration of the pro-inflammatory cytokine IL-6 secreted by human neutrophils following administration of lipopolysaccharide (LPS) and Composition 1. [Figure 5C] 1 shows the concentration of the pro-inflammatory cytokine IL-8 secreted by human neutrophils following administration of lipopolysaccharide (LPS) and Composition 1. [Figure 6] FIG. 1 shows a cystic fibrosis transmembrane conductance receptor (CFTR) functional assay performed on wild-type cystic fibrosis bronchial epithelial cells (WT CFBE). [Figure 7] Images from a video showing the effect of Composition 1 (right panel) compared to PBS (left panel) on in vitro mucociliary transport (MCT) rate of primary human bronchial epithelial cells isolated from a cystic fibrosis patient (dF508- / - cells) at 0 hours (top) and 6 hours (bottom) after administration. [Figure 8] 8A-8B show slope analysis of MCT for airway surface liquid (ASL) (FIG. 8A) and ciliary beat frequency (CBF) (FIG. 8B) 6 hours after administration of Composition 1 compared to PBS in primary human bronchial epithelial cells (dF508- / - cells) isolated from cystic fibrosis patients. The correlation analysis shows that mucus viscosity is reduced by Composition 1. [Figure 9] 1 shows metalloprotease-9 (MMP-9) release from LPS-stimulated neutrophils following 1 hour treatment with Composition 1 (Ar) at dilutions of 1:100 and 1:10 compared to media (Med) and LPS only controls. MMP-9 is the 75-100 kDa band on the gel. [Figure 10]Figures 10A-10B show the release levels of neutrophil and macrophage associated cytokines: macrophage inflammatory protein 1 alpha (MIP1α) (Figure 10A) and macrophage inflammatory protein 1 beta (MIP1β) (Figure 10B) from LPS-activated neutrophils following administration of Composition 1 (Ar) at dilutions of 1:100 and 1:10 compared to media (M) and LPS only controls. #ANOVA<0.01 for 1 hour. *p<0.05 via Tukey post-hoc test LPS v. AR 1:10 + LPS 100. [Figure 11] Shown is the effect of the combination of Composition 1 and VX-770 / 809 (Composition 1 + VX-770 / 809, right panel) compared to PBS (left panel) and VX-770 / 809 alone (middle panel) on in vitro mucociliary transport (MCT) rate of primary human bronchial epithelial cells isolated from cystic fibrosis patients (dF508- / - cells). [Figure 12A] 1 shows sample quantification of airway surface liquid (ASL), ciliary beat frequency (CBF), and mucociliary transport (MCT) rate time course for dF508 CF HBE cells following administration of Composition 1 in combination with VX-770 / 809 (Composition 1 + VX-770 / 809), Composition 1 alone (Composition 1), VX-770 / 809 alone, or PBS control. Results show pooled data (N=20 HBE monolayers across 3 CF donors homozygous for F508del). **P<0.01, P<0.001, ****P<0.0001. [Figure 12B] Quantification of mucociliary transport rate change from baseline by Composition 1, VX-770 / 809, and the combination of Composition 1 and VX-770 / 809 compared to PBS (control). Results show pooled data (N=20 HBE monolayers across 3 CF donors homozygous for F508del). Mean±SEM ****P<0.0001. [Figure 13]

[0023] Figure 1 shows the mean percent change in spirometry measurements in lung transplant patients administered Composition 1 by nebulization twice daily for one month. Spirometry measurements included forced expiratory volume exhaled in one second (FEV1), forced vital capacity (volume) (FVC), and measurements of small and medium airway function (FEF 25-75%). These results are after one month of treatment. [Figure 14] Figure 1 shows the percent change in exhaled nitric oxide (FENO) in four lung transplant patients who received Composition 1 by nebulization twice daily for one month. CF, ILD, and A1AT refer to the patient's background disease (the etiology for which they underwent transplantation). These results are after one month of treatment. [Figure 15] Figure 1 shows stability data for formulations 1-9 having low, medium, or high concentrations of ascorbic acid and / or buffering agent after storage at 2-8°C for approximately 72 hours. Glutathione (GSH) was at the same concentration in each formulation. Composition 1 is designated as formulation 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description definition To facilitate the understanding of the present invention, a number of terms and phrases are defined below.

[0037] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0038] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both "A and B," "A or B," "A," and "B." Similarly, the term "and / or" in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0039] The term "about" as used herein means approximately ±10%. When the term "about" is used in conjunction with a numerical value or range, it modifies the value or range by expanding the upper and lower limits of the numerical values ​​set forth. In general, the term "about" is used herein to modify a numerical value above and below (higher or lower) the stated value by a variance of 10 percent, i.e., ±10%, unless a different variance (e.g., ±30%, ±20%, ±5%, ±1%, etc.) is indicated.

[0040] As used herein, "clinical bacterial isolate" refers to a bacterial strain isolated from a human subject or from a tissue sample taken from a human subject.

[0041] As used herein, "minimum inhibitory concentration" or "MIC" refers to the lowest concentration of an agent (eg, an antibiotic and / or a composition of the present application) that inhibits the visible growth of a bacterial species.

[0042] As used herein, "minimum bactericidal concentration" or "MBC" refers to the lowest concentration of an agent (e.g., an antibiotic and / or a composition of the present application) that kills a bacterial species.

[0043] As used herein, "minimum biofilm eradicating concentration" or "MBEC" refers to the lowest concentration of an agent (eg, an antibiotic and / or a composition of the present application) that inhibits the visible growth of a biofilm.

[0044] As used herein, "pharmaceutically acceptable" means safe and effective for use in humans. For example, as used herein, "pharmaceutically acceptable salt" refers to a salt of a compound disclosed herein that is safe and effective for use in a subject and possesses the desired biological activity of the compound.

[0045] As used herein, "biofilm" refers to a group of microorganisms, e.g., clinical bacterial isolates, in which the cells of the microorganisms adhere to each other and often to a surface. In some embodiments, these attached cells are embedded in a self-produced matrix of extracellular polymeric substances (EPS). In some embodiments, the biofilm comprises a single bacterial species. In other embodiments, the biofilm is a mixture of two or more bacterial species.

[0046] As used herein, "mucoid bacteria" refers to alginate-producing bacteria.

[0047] As used herein, "non-mucoid bacteria" refers to bacteria that do not produce alginate.

[0048] As used herein, "aerobic" refers to organisms, such as bacteria, that can live and grow in an oxygen-containing environment.

[0049] As used herein, "anaerobic" or "anaerobe" refers to an organism, e.g., a bacterium, that does not require oxygen to grow. In some embodiments, the organism is an "obligate anaerobe," meaning that it is damaged by the presence of oxygen; an "aerotolerant organism," meaning that it cannot use oxygen to grow but can tolerate its presence; or a "facultative anaerobe," meaning that it can grow without oxygen but will use oxygen when it is present.

[0050] As used herein, "extracellular" means outside a cell.

[0051] "Antibiotic resistance" refers to bacteria that are equipped with mechanisms that render antibiotics ineffective in killing the bacteria. Exemplary mechanisms include, for example, antibiotic destruction, antibiotic target modification, and restriction of antibiotic penetration and / or efflux. In some embodiments, bacteria become resistant to antibiotics by mutation.

[0052] As used herein, "multidrug resistance" (also referred to as "multiple drug resistance", "MDR", "multi-resistance") refers to antibacterial resistance exhibited by a type of microorganism, e.g., a type of bacteria, to multiple antibacterial drugs, e.g., antibiotics.

[0053] "Antibiotic tolerant" refers to certain viable (or persister) cells within a population of bacterial cells that are phenotypic variants (not mutants) of non-growing dormant cells. Persisters are killed slowly, if at all, by antibiotics and resume growth when antibiotic concentrations are reduced or stopped.

[0054] As used herein, "sensitized" refers to the susceptibility of a microorganism, e.g., a bacterium, to an antimicrobial agent, e.g., an antibiotic.

[0055] As used herein, "synergistic effect" refers to an effect that occurs between two or more therapeutic agents, e.g., a composition disclosed herein and an antibiotic, that produces an effect that is greater than the sum of the individual effects of the two or more therapeutic agents.

[0056] As used herein, "inhibit" means to prevent or stop, for example, to stop bacterial growth.

[0057] As used herein, "reduce" means to decrease or lower the amount, e.g., to reduce the amount of bacterial growth (e.g., compared to a starting point or compared between two or more groups).

[0058] As used herein, "treating" or "treatment" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more signs, symptoms or characteristics of a disease.

[0059] By "subject" or "patient" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In certain embodiments, the subject is a human patient in need of treatment.

[0060] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) or consecutive administration in any order.

[0061] Combination therapy can provide "synergism" and demonstrate "synergistic effect", i.e., the effect achieved when active ingredients are used together is greater than the sum of the effects resulting from using the compounds separately. Synergistic effect is achieved when active ingredients are (1) formulated together and administered or delivered simultaneously in a combined unit dosage formulation; (2) delivered sequentially, alternatingly, or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternating therapy, synergistic effect can be achieved when compounds are administered or delivered sequentially, for example, by different injections in separate syringes, or by inhalation of the first therapy and oral administration of the second therapy, or vice versa.

[0062] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of an active ingredient or ingredients to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. The formulation may be sterile.

[0063] An "effective amount" of a composition or active agent disclosed herein is an amount sufficient to achieve a specifically described purpose. An "effective amount" can be determined empirically and in a routine manner in relation to the described purpose.

[0064] The term "therapeutically effective amount" refers to an amount of a composition or active agent disclosed herein effective to "treat" a disease or disorder in a subject.

[0065] Glutathione and Glutathione Conjugate Compositions In certain aspects, the compositions of the present application comprise glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof.

[0066] In some embodiments, the composition comprises glutathione or a pharma- ceutically acceptable salt thereof.

[0067] Glutathione or "GSH" has the formula A: TIFF0007617179000003.tif18128, or a zwitterionic form thereof, e.g., a compound having the formula B: Refers to the compound having TIFF0007617179000004.tif23128.

[0068] Glutathione is the most abundant non-protein thiol in mammalian cells. It plays a role in detoxifying xenobiotic compounds and in the antioxidant action of reactive oxygen species and free radicals. See, e.g., Bray and Taylor, Canadian Journal of Physiology and Pharmacology 71:746-751 (1993).

[0069] High concentrations of GSH are present in the airways of healthy individuals, however, glutathione levels are depleted in individuals with chronic inflammatory airway diseases, such as lung transplant patients.

[0070] In some embodiments, the composition comprises a glutathione-containing conjugate or a pharma- ceutically acceptable salt thereof. In certain embodiments, the glutathione-containing conjugate, upon administration to a subject, is metabolized to release glutathione or a derivative thereof. In some embodiments, the glutathione-containing conjugate is referred to herein as a "disclosed conjugate compound" or a "glutathione conjugate."

[0071] In one embodiment, the disclosed conjugate compound has formula I: TIFF0007617179000005.tif28128, and pharma- ceutically acceptable salts and solvates thereof, wherein A 1 -OR 1 A 2 is Z 1 A 3 is hydrogen; and A 4 is R 3a or A 1 is Z 1 A 2 -OR 2 and A 3 is hydrogen; and A 4 is R 3a or A 1 -OR 1 A 2 -OR 2 and A 3 is Z 3 and A 4 is R 3a or A 1 is Z 2 A 2 -OR 2 and A 3 is hydrogen; and A 4 is R 3a or A 1 -OR 1 A 2 is Z 2 and A 3 is hydrogen; and A 4 is R 3a or A 1 -OR 1 A 2 -OR 2 A 3 is hydrogen; and A 4 is Z 3 or A 1 and A 2 are Z 1And A 3 is hydrogen; Z 1 teeth Selected from the group consisting of TIFF0007617179000006.tif20128; Z 2 teeth Selected from the group consisting of TIFF0007617179000007.tif20147; Z 3 teeth TIFF0007617179000008.tif20128; R 1 is selected from the group consisting of hydrogen and optionally substituted alkyl; R 2 is selected from the group consisting of hydrogen and optionally substituted alkyl; R 3a , R 3b , and R 3c are each independently selected from the group consisting of hydrogen and a protecting group; X is -O-; -O(CH2) m O-; -OCH2CH(R 4 )O-; -OCH(R 4 )CH2O-; and -O(CH2CH2O) n - selected from the group consisting of; R 4 teeth TIFF0007617179000009.tif20128; m is 1, 2, 3, 4, 5, 6, 7, or 8; n is 2, 3, 4, 5, 6, 7, or 8; and R 5 is selected from the group consisting of hydrogen and optionally substituted alkyl.

[0072] In another embodiment, the disclosed conjugate compounds are compounds having formula I, and pharma- ceutically acceptable salts and solvates thereof, wherein m is 2, 3, 4, 5, 6, 7, or 8.

[0073] In another embodiment, the disclosed conjugate compound has formula II: or a pharma- ceutically acceptable salt or solvate thereof, wherein R1, R3a, R3b, R3c, and X are as defined in relation to formula I.

[0074] In another embodiment, the disclosed conjugate compounds are enantiomerically enriched compounds having any one or more of the formulas in Table 1, and pharma- ceutically acceptable salts and solvates thereof, wherein R 1 , R 3a , R 3b , R 3c and X is as defined in relation to formula I.

[0075] Disclosed Compositions Glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, organic acids, or any combination thereof may be mixed with a pharmaceutical carrier, such as water, and optionally other ingredients, to obtain the "disclosed compositions." In some embodiments, the amount of glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof, such as reduced glutathione, in the disclosed compositions is about 30-90% by weight, about 30-85% by weight, about 30-80% by weight, about 30-75% by weight, about 30-70% by weight, about 30-65% by weight, about 30-60% by weight, about 30-55% by weight, or about 30-50% by weight. In some embodiments, the amount of glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof, e.g., reduced glutathione, in the disclosed compositions is 30-50% by weight.

[0076] In some embodiments, the disclosed compositions further comprise an organic acid, which may be selected from the group consisting of ascorbic acid, acetic acid, adipic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, camsylic acid, carbonic acid, chlorobenzoic acid, cholic acid, citric acid, edetic acid, edisylic acid, estolic acid, ethanesulfonic acid, formic acid, fumaric acid, gluceptic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, glycolylarsanilic acid, hippuric acid, 1-hydroxy-2-naphthoic acid, isethionic acid, isobutyric acid, isonicotinic acid, lactic ... The acid is selected from the group of acids consisting of lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, muconic acid, naphthalenesulfonic acid, nicotinic acid, oxalic acid, oleic acid, orotic acid, p-nitromethanesulfonic acid, pamoic acid, pantothenic acid, phthalic acid, polygalacturonic acid, propionic acid, saccharic acid, salicylic acid, stearic acid, suberic acid, succinic acid, sulfanilic acid, tannic acid, tartaric acid, p-toluenesulfonic acid, and any combination thereof. In some embodiments, the amount of organic acid, e.g., reduced ascorbic acid, in the disclosed compositions is about 10-90% by weight, about 10-85% by weight, about 10-80% by weight, about 10-75% by weight, about 10-70% by weight, about 10-65% by weight, about 10-60% by weight, about 10-55% by weight, about 10-50% by weight, about 10-45% by weight, about 10-40% by weight, about 10-35% by weight, about 10-30% by weight, about 1-30% by weight, about 1-20% by weight, or about 1-10% by weight. In some embodiments, the amount of organic acid, e.g., reduced ascorbic acid, in the disclosed compositions is 25-40% by weight.

[0077] In some embodiments, the disclosed compositions further comprise a bicarbonate. In some embodiments, the bicarbonate is sodium bicarbonate. In some embodiments, the amount of bicarbonate, e.g., sodium bicarbonate, in the disclosed compositions is about 10-90% by weight, about 10-85% by weight, about 10-80% by weight, about 10-75% by weight, about 10-70% by weight, about 10-65% by weight, about 10-60% by weight, about 10-55% by weight, about 10-50% by weight, about 10-45% by weight, about 10-40% by weight, about 10-35% by weight, about 10-30% by weight, about 1-30% by weight, about 1-20% by weight, or about 1-10% by weight. In some embodiments, the amount of bicarbonate, e.g., sodium bicarbonate, in the disclosed compositions is about 20-30% by weight. In some embodiments, the disclosed compositions do not comprise bicarbonate.

[0078] In some embodiments, the pH of the disclosed compositions is from about 6.0 to about 8. In some embodiments, the pH of the disclosed compositions is greater than 5.5 or at least 6.0 (e.g., 5.6-14, 5.7-14, 5.8-14, 5.9-14, 6-14, 5.6-12, 5.7-12, 5.8-12, 5.9-12, 6-12, 5.6-10, 5.7-10, 5.8-10, 5.9-10). , 6-10, 5.6-9, 5.7-9, 5.8-9, 5.9-9, 6-9, 5.6-8, 5.7-8, 5.8-8, 5.9-8, 6-8, 5.6-7.5, 5.7-7.5, 5.8-7.5, 5.9-7.5, 6-7.5, 5.6-7, 5.7-7, 5.8-7, 5.9-7, or 6-7).

[0079] In some embodiments, the disclosed compositions are formulated to maximize formulation stability and minimize oxidation of glutathione. Oxidized glutathione is associated with the generation of protein-carbonyls via glutathionylation. Glutathionylation occurs when oxidized glutathione dissociates and binds to proteins. Maintaining glutathione in a reduced state in solution prior to administration can reduce the risk of glutathionylation products that can lead to clinical complications such as bronchiectasis. In some embodiments, the oxidized glutathione (e.g., %GSSG) in the disclosed compositions is less than about 20% by weight, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% of the total glutathione in the disclosed compositions after 4 weeks of storage of the disclosed compositions (e.g., at 5°C in N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of oxidized glutathione in the disclosed compositions (e.g., %GSSG) is or does not exceed about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, about 2% to about 16%, about 2% to about 10%, or about 2% to 8% by weight of the total glutathione in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of oxidized glutathione in the disclosed compositions (e.g., %GSSG) is less than about 20%, less than about 18%, less than about 16%, or less than about 10% by weight of the total glutathione in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0080] In some embodiments, the reduced glutathione in the disclosed compositions is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., in N2 or ambient atmosphere) at about 5° C. for 4 weeks. In some embodiments, the percentage of reduced glutathione in the disclosed compositions is between about 80% and about 100%, between about 80% and about 98%, between about 82% and about 98%, between about 84% and about 98%, between about 86% and about 98%, between about 88% and about 98%, between about 90% and about 98%, or between about 92% and about 98% by weight of the total glutathione in the disclosed compositions after 4 weeks (e.g., in N2 or ambient atmosphere) at 5° C. In some embodiments, the percentage of reduced glutathione in the disclosed compositions is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, or at least 90% by weight of the total glutathione in the disclosed compositions after 4 weeks of storage at 5° C. in N2 or ambient atmosphere.

[0081] In some embodiments, the disclosed compositions are further formulated to maximize formulation stability and minimize oxidation of organic acids, such as ascorbic acid. In addition, when ascorbic acid is oxidized to dehydroascorbic acid (DHA), DHA can degrade and lead to the formation of protein adducts in a process called ascorbylation. Maintaining organic acids, such as ascorbic acid, in a reduced state in solution prior to administration can reduce the risk of ascorbylation from the decomposition products of dehydroascorbic acid. In some embodiments, reduced ascorbic acid (e.g., %ASC) is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of ascorbic acid in the disclosed compositions after 4 weeks of storage of the disclosed compositions (e.g., at 5°C in N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of reduced ascorbic acid in the disclosed compositions (e.g., %ASC) is between about 82% and about 100% or between about 85% and about 95% by weight of the total ascorbic acid in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of reduced ascorbic acid in the disclosed compositions (e.g., %ASC) is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90% by weight of the total ascorbic acid in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0082] In some embodiments, the oxidized ascorbic acid in the disclosed compositions is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the disclosed compositions after 4 weeks of storage of the disclosed compositions (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of oxidized ascorbic acid in the disclosed compositions is about 5% to about 20%, about 5% to about 18%, about 5% to about 10%, or about 5% to 9% by weight or less of the total ascorbic acid in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere). In some embodiments, the percentage of oxidized ascorbic acid in the disclosed compositions is less than about 20%, less than about 18%, less than about 16%, or less than about 10% by weight of the total ascorbic acid in the disclosed compositions after 4 weeks of storage (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere).

[0083] In certain aspects, the disclosed composition components in the ratios are formulated to maximize the stability of the formulation and minimize the oxidation of glutathione and organic acid, such as ascorbic acid. In some embodiments, glutathione and organic acid (e.g., ascorbic acid) are formulated to comprise molar equivalents in solution, such as a glutathione to ascorbic acid molar ratio of about 0.5-1:1, about 0.6-1:1, 0.7-1:1, 0.8-1:1, 0.9-1:1, or about 1:1. In some embodiments, glutathione and organic acid (e.g., ascorbic acid) are formulated to comprise a molar excess of organic acid (e.g., ascorbic acid) to glutathione in solution, such as a glutathione to ascorbic acid molar ratio of about 1:1.1, about 1:1.2, about 1:3, about 1:4, about 1:5.

[0084] In some embodiments, the disclosed compositions further comprise a bicarbonate (e.g., sodium bicarbonate). In some embodiments, the glutathione, organic acid (e.g., ascorbic acid), and bicarbonate (e.g., sodium bicarbonate) are combined in a molar ratio of glutathione to bicarbonate (e.g., sodium bicarbonate) to organic acid (e.g., ascorbic acid) of about 0.1-0.5:0.5-1:1, about 0.2-0.5:0.5-1:1, about 0.3-0.5:0.5-1:1, about 0.4-0.5:0. 5~1:1, approx. 0.49:0.5~1:1, approx. 0.5:0.5~1:1, approx. 0.1~0.5:0.6~1:1, approx. 0.2~0.5:0.6~1:1, approx. 0.3~0.5:0.6~1:1, approx. 0.4~0.5:0.6~1:1, approx. 0.49:0.6~1:1, approx. 0.5:0.6~1:1, approx. 0.1~0.5:0.7~1:1, approx. 0.2~0.5:0.7~1:1, approx. 0.3~ 0.5:0.7~1:1, approx. 0.4~0.5:0.7~1:1, approx. 0.49:0.7~1:1, approx. 0.5:0.7~1:1, approx. 0.1~0.5:0.8~1:1, approx. 0.2~0.5:0.8~1:1, approx. 0.3~0.5:0.8~1:1, approx. 0.4~0.5:0.8~1:1, approx. 0.49:0.8~1:1, approx. 0.5:0.8~1:1, approx. 0.1~0.5:0.9~1: 1, about 0.2-0.5:0.9-1:1, about 0.3-0.5:0.9-1:1, about 0.4-0.5:0.9-1:1, about 0.49:0.9-1:1, about 0.5:0.9-1:1, about 0.1-0.5:1:1, about 0.2-0.5:1:1, about 0.3-0.5:1:1, about 0.4-0.5:1:1, about 0.49:1:1, about 0.5:1:1. In some embodiments, the molar ratio of glutathione, organic acid (e.g., ascorbic acid), and bicarbonate (e.g., sodium bicarbonate) is 0.1-0.5:0.5-1:1, 0.4-0.5:0.5-1:1, 0.1-0.5:0.5:1, 0.1-0.5:1:1, or 0.4-0.5:1:1. In some embodiments, the molar ratio of glutathione, organic acid (e.g., ascorbic acid), and bicarbonate (e.g., sodium bicarbonate) is 0.49:0.5:1, 0.5:0.5:1, 0.49:1:1, or 0.5:1:1.

[0085] In some embodiments, the bicarbonate (e.g., sodium bicarbonate) is in a molar ratio less than the combined molar ratio of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof, and (b) an organic acid (e.g., ascorbic acid). In some embodiments, the molar ratio of glutathione, an organic acid (e.g., ascorbic acid), and a bicarbonate (e.g., sodium bicarbonate) is 0.1-0.49:0.5:1, 0.2-0.49:0.5:1, 0.3-0.49:0.5:1, or 0.4-0.49:0.5:1.

[0086] In some embodiments, the disclosed compositions comprise or consist essentially of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof, and (b) an organic acid, wherein the molar ratio of (a) to (b) is about 0.5-1:1, about 0.6-1:1, 0.7-1:1, 0.8-1:1, 0.9-1:1, or about 1:1, and the pH of the composition is about 5.5-14, about 6 to about 8, 7±1.5, 6±0.5, or about 6.

[0087] In some embodiments, the disclosed compositions comprise or consist essentially of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof; (b) an organic acid; and (c) a bicarbonate salt, wherein the molar ratio of (a) to (b) to (c) is about 0.1-0.5:0.5-1:1, about 0.4-0.5:0, about 0.5-0.6:1, about 0.6-0.8:1, about 0.8-0.9:1, about 0.9 ... 0.5 to 1:1, about 0.1 to 0.5:0.5:1, about 0.1 to 0.5:1:1, about 0.4 to 0.5:1:1, about 0.1 to 0.49:0.5:1, about 0.2 to 0.49:0.5:1, about 0.3 to 0.49:0.5:1, or about 0.4 to 0.49:0.5:1, and the pH of the composition is about 5.5 to 14, about 6 to about 8, 7±1.5, 6±0.5, or about 6.

[0088] The pharmaceutical composition for use in the present disclosure can be formulated with one or more physiologically acceptable carriers and / or excipients that facilitate the administration of organic acid, glutathione, glutathione derivatives, glutathione conjugates, their pharma- ceutically acceptable salts, or any combination thereof to a subject by intended route, for example, delivery by inhalation.In some embodiments, the pharmaceutical composition is an aqueous solution.In some embodiments, the pharmaceutical composition is a dry powder.

[0089] The disclosed composition can be prepared by conventional mixing, dissolving, granulating, dragee making, emulsifying, encapsulating, encapsulating, spray drying or lyophilization process known in the art.Specific formulations vary according to the selected route of administration.In one embodiment, glutathione, glutathione derivative, glutathione conjugate, their pharmaceutically acceptable salt, or any combination thereof is dissolved in a solvent, for example, water, for administration to the airway of a subject (e.g., intranasal administration).

[0090] The term "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid (e.g., water) or solid filler, diluent, excipient, solvent, or encapsulating material. A carrier is "pharmaceutically acceptable" in the sense of being suitable for use in humans, commensurate with a reasonable benefit / risk ratio, compatible with the other ingredients of the pharmaceutical formulation, and without toxicity, irritation, allergic response, immunogenicity, or other complications. Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, Fla., 2004).

[0091] In some embodiments, the disclosed compositions include an excipient. In some embodiments, the excipient is selected from the group consisting of a pH adjusting agent, a preservative, a chelating agent, and any combination thereof.

[0092] In certain embodiments, the disclosed compositions may include pH adjusters.pH adjusters are known in the art.See, for example, Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company (1990) and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000). Suitable examples of pharma- ceutically acceptable pH adjusting agents include, but are not limited to, ascorbic acid, citric acid, sodium citrate, sodium bicarbonate, potassium bicarbonate, dibasic sodium phosphate, magnesium oxide, calcium carbonate, magnesium hydroxide, buffers (e.g., acetate buffer, citrate buffer, phosphate buffer, lactate buffer, and borate buffer, and any combination thereof), fat-soluble fatty acid esters of ascorbic acid (vitamin C) (e.g., alone or in combination with a-hydroxy acids), oxidation-resistant saturated fatty acid esters of ascorbic acid (e.g., ascorbyl laurate, ascorbyl myristate, ascorbyl palmitate, ascorbyl stearate, and ascorbyl behenate, and any combination thereof), and any combination thereof. In some embodiments, the esters may be prepared using hydrogenated fats and oils, or fractions thereof, and may contain small amounts of other esters. Ascorbyl stearate prepared using canola, for example, may generally contain about 4% ascorbyl palmitate.

[0093] In one embodiment, the pH adjuster, e.g., ascorbic acid, is present in the disclosed compositions in an amount of about 0.01-50% by weight, about 10-90% by weight, about 10-85% by weight, about 10-80% by weight, about 10-75% by weight, about 10-70% by weight, about 10-65% by weight, about 10-60% by weight, about 10-55% by weight, about 10-50% by weight, about 10-45% by weight, about 10-40% by weight, about 10-35% by weight, about 10-30% by weight, about 1-30% by weight, about 1-20% by weight, or about 1-10% by weight. In some embodiments, the pH adjuster is present in the disclosed compositions in an amount of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by weight.

[0094] In certain embodiments, the disclosed compositions may contain preservatives.Pharmaceutically acceptable preservatives include, but are not limited to, various antibacterial and antifungal agents, solvents (e.g., ethanol, propylene glycol, benzyl alcohol and chlorobutanol, and any combination thereof), quaternary ammonium salts (e.g., cetylpyridinium chloride, benzalkonium chloride, and parabens, including but not limited to methylparaben, ethylparaben and propylparaben), chlorhexidine, benzoic acid and its salts, parahydroxybenzoic acid and its salts, alkyl esters of parahydroxybenzoic acid and its salts, nitrates, chlorides, acetates, phenylmercury salts such as borates, antioxidants, EDTA, sorbitol, phenol, boric acid and its salts, sorbic acid and its salts, thimerosal and nitromersol, and any combination thereof.

[0095] In one embodiment, the preservative is present in the disclosed compositions at about 0.01-50% by weight of the composition, e.g., about 1-30% by weight, about 1-20% by weight, or about 1-10% by weight, e.g., about 1% by weight, about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight, about 30% by weight, about 35% by weight, about 40% by weight, about 45% by weight, or about 50% by weight.

[0096] In certain embodiments, the disclosed compositions may include a chelating agent.Non-limiting examples of chelating agents include lactic acid, acetic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, aconitic acid, pimelic acid, sebacic acid, alimaronic acid, ethylmalonic acid, citric acid, malic acid, glyceric acid, tartaric acid, mevalonic acid, oxyglutaric acid, oxaloacetic acid, a-ketoglutaric acid, a-ketomalonic acid, glucuronic acid, galacturonic acid, mannuronic acid, aspartic acid, glutamic acid, glycine, alanine, lysine, histidine, arginine, cysteine, s-aminocaproic acid, phenylalanine, phenylglycine, p-hydroxyphenylglycine, p-aminophenylalanine, y-carboxyglutamic acid, iminodiacetic acid, hydroxyethyliminodiacetic acid, ethylenediaminediacetic acid, ethylenediaminetetraacetic acid, trans-cyclohexane-diaminetetraacetic acid, Examples of suitable chelating agents include diethylenediaminepentaacetic acid, alanine diacetic acid, diaminopimelic acid, phthalic acid, terephthalic acid, homophthalic acid, phenylsuccinic acid, phenylmalonic acid, oxanilic acid-o-carboxylic acid, anthralininoacetic acid, 2,4-dihydroxybenzoic acid, p-aminosalicylic acid, phthalglutamic acid, kynurenine, 1,2-hydroxybenzene-3,5-disulfonic acid, 4-amino-phenol-2-sulfonic acid, cysteic acid, 2-phosphoglyceric acid, glycero-3-phosphate, glucose-1,6-diphosphate, fructose-1,6-diphosphate, and phosphates (e.g., sodium phosphate, sodium aluminum phosphate, sodium acid phosphate, dipotassium phosphate, disodium phosphate, monobasic and sodium hexametaphosphate), and any combination thereof. Chelators may be included in the pharmaceutical compositions of the present disclosure either as parent molecules or, where appropriate, in salt form. For example, compounds containing acid functional groups may be used in protonated form or as a pharma- ceutically acceptable inorganic or organic salt that retains the chelating activity of the parent compound.

[0097] In one embodiment, the chelating agent is present in the disclosed compositions at about 0.01-50% by weight of the composition, e.g., about 1-30% by weight, about 1-20% by weight, or about 1-10% by weight, e.g., about 1% by weight, about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight, about 30% by weight, about 35% by weight, about 40% by weight, about 45% by weight, or about 50% by weight.

[0098] In certain embodiments, the disclosed compositions comprise glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof, bicarbonate (e.g., sodium bicarbonate or potassium bicarbonate), and / or a pH adjuster (e.g., ascorbic acid). In some embodiments, the disclosed compositions comprise glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof, and / or a pH adjuster such as an organic acid (e.g., ascorbic acid). In some embodiments, the compositions are claimed as being present in an amount of each component such that the amount of ascorbic acid is approximately molar equivalent to or in molar excess of the amount of glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof.

[0099] In some embodiments, the disclosed compositions include (a) glutathione, a glutathione derivative, a glutathione conjugate, or a pharma- ceutically acceptable salt thereof, (b) an organic acid, and (c) a bicarbonate. In further embodiments, the molar ratio of (a):(b):(c) in the disclosed compositions is 0.1-0.5:0.5-1:1 (e.g., 0.4-0.5:0.5-1:1, 0.1-0.5:0.5:1, 0.1-0.5:1:1, 0.4-0.5:1:1, 0.1-0.49:0.5:1, 0.2-0.49:0.5:1, 0.3-0.49:0.5:1, or 0.4-0.49:0.5:1).

[0100] In some embodiments, the organic acid in the disclosed compositions is ascorbic acid. In some embodiments, the bicarbonate in the disclosed compositions is sodium bicarbonate. In some embodiments, the compositions include (a) glutathione, (b) ascorbic acid, and (c) sodium bicarbonate. In certain embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (e.g., about 0.49:about 0.50:about 1). In other embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (e.g., about 0.49:about 1:about 1).

[0101] In some embodiments, the pH of the disclosed compositions is about 5.5 to about 14 (e.g., 5.5 to 7.5). In some embodiments, the pH of the disclosed compositions is about 6 to about 14 (e.g., 6 to 7.5). In some embodiments, the pH of the compositions is 7±1.5, 7±1.4, 7±1.3, 7±1.2, 7±1.1, 6±0.5, 6±0.4, 6±0.3, 6±0.2, 6±0.5, 6±0.1, or about 6.

[0102] In some embodiments, the disclosed compositions are storage stable for at least 72 hours at 2-8° C. In some embodiments, the disclosed compositions (a) remain essentially free of precipitation after at least 72 hours of storage at 2-8° C., (b) contain less than 7%, less than 6%, less than 5%, or less than 4% impurities after at least 72 hours of storage at 2-8° C., (c) have or maintain a pH of about 6-7.5 (e.g., 6.0-7.0) after at least 72 hours of storage at 2-8° C., and / or (d) may experience minimal loss in solubility after at least 72 hours of storage at 2-8° C.

[0103] How to use In some embodiments, the disclosed compositions are useful for treating, alleviating symptoms, or preventing pulmonary diseases, conditions, or disorders. In particular, the disclosed compositions can be used to inhibit or reduce the growth of clinically isolated bacteria and / or inhibit or reduce the formation of clinically isolated bacterial biofilms. The disclosed compositions can be used to treat or alleviate symptoms in subjects suffering from or at risk of clinically isolated bacterial infections. In another embodiment, the disclosed compositions are useful for upregulating mucociliary clearance in subjects suffering from or at risk of reduced mucociliary clearance, e.g., in subjects with or without active bacterial infection in the lungs. In another embodiment, the disclosed compositions are useful for reducing airway inflammation, e.g., in subjects suffering from or at risk of airway inflammation, with or without active bacterial infection in the lungs.

[0104] In some embodiments, the subject has a lung or airway disease or disorder.In some embodiments, the lung or airway disorder is chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, inflammation and / or infection associated with lung transplantation, acute or chronic lung rejection and / or pulmonary dysfunction, pulmonary arterial hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD), The disease is selected from the group consisting of bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), pulmonary alveolar proteinosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), inflammation and / or infection associated with mechanical ventilation, ventilator-associated pneumonia, asbestos-related airway disorder or disease, dust-related airway disorder or disease, silicosis, and radiation-related or chemical-related airway disease or disease, and any combination thereof.

[0105] In some embodiments, the lung or airway disease or disorder is selected from the group consisting of chronic inflammatory lung disease, inflammation and / or infection associated with lung transplantation, acute or chronic lung rejection or pulmonary dysfunction, asthma, cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), or chronic obstructive pulmonary disease (COPD), or any combination thereof. In another embodiment, the lung or airway disease or disorder is cystic fibrosis. In another embodiment, the subject is a lung transplant patient. In another embodiment, the subject is a non-cystic fibrosis bronchiectasis patient. In another embodiment, the subject is infected with a biofilm-producing bacteria.

[0106] In some embodiments, the disease or disorder of the lung or airway is bronchiectasis (BrE).Bronchiectasis can be diagnosed in patients with cystic fibrosis (CF bronchiectasis) or in patients without cystic fibrosis (non-CF bronchiectasis).To date, the treatments used in cystic fibrosis (CF) have been shown to be unsuccessful and potentially harmful in non-CF bronchiectasis.

[0107] CF disease, including non-CF bronchiectasis and CF-associated bronchiectasis, share some of the same characteristics but are clinically distinct. CF BrE primarily affects the upper lobes of the airways, whereas non-CF BrE is associated with greater involvement of the lower lobes. Non-CF BrE patients lack sufficient mucociliary transport mechanisms, so that treatments that only destroy mucus components (e.g., DNase) result in retention of sputum in the lower airways and lung parachyma rather than expulsion. Sputum in CF contains more DNA than BrE patients, clearly indicating an underlying physiological event. BrE patients generally have a higher prevalence in older women, whereas CF patients are diagnosed at a younger age, with equal male / female prevalence (O'Donnell et al., Chest 1998 Volume 113, Issue 5, Pages 1329-1334).

[0108] In some embodiments, the disclosed compositions are useful for treating, alleviating symptoms of, or preventing (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis).

[0109] In another aspect, the present disclosure provides a method for treating or preventing a clinically isolated bacterial infection in the respiratory tract of a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the disclosed composition. In another aspect, the disclosed composition is administered to the subject in combination with one or more antibiotics. The antibiotics may be administered locally to the lungs and / or systemically.

[0110] In another aspect, the disclosure provides a method of treating or preventing inflammation in the airways of a subject in need thereof, comprising administering to the subject a therapeutic amount of a disclosed composition.

[0111] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a mucosal tissue in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a disclosed composition. Non-limiting examples of mucosal tissues include the mouth, nose, eyes, ears, upper respiratory tract, lower respiratory tract, gastrointestinal tract, vagina, rectum, and urethra.

[0112] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder associated with a mucosa in a subject in need thereof, comprising administering to the subject a therapeutic amount of a disclosed composition to a suitable mucosa. In one aspect, the mucosa is the lung, such as the deep lung (alveolar region), and in another aspect, the mucosa is one or more of the eye, mouth, nose, rectum, and vagina.

[0113] In another embodiment, the disclosed compositions may be used to treat or prevent bronchiolitis obliterans and military-related lung injury, ie, lung injury in soldiers who have damaged their airways following unexplained exposure.

[0114] In another aspect, the present disclosure provides a use of the disclosed composition for the manufacture of a medicament for the treatment of a lung or airway disorder. In another aspect, the use further comprises administering to a subject one or more additional therapeutic agents.

[0115] The therapeutic method of the present disclosure comprises administering to a subject, e.g., a human patient, in need thereof, a therapeutically effective amount of the disclosed composition. The indication for such treatment varies according to the individual case and is subject to a medical evaluation (diagnosis) that takes into account the signs, symptoms and / or dysfunctions present, the risk of developing the particular signs, symptoms and / or dysfunctions, and other factors.

[0116] In one aspect, the disclosure provides a method for treating a lung or airway disorder or disease in a subject infected with or at risk of infection with a clinically isolated bacteria, comprising administering to the subject an effective amount of a disclosed composition.

[0117] In another embodiment, the lung or airway disorder or disease is selected from the group consisting of chronic inflammatory lung disease, inflammation and / or infection associated with lung transplantation, acute lung rejection, asthma, cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), and chronic obstructive pulmonary disease (COPD), and any combination thereof.

[0118] In another aspect, a lung or airway disorder or disease is treated by inhibiting the growth of a clinically isolated bacterium in the airway of a subject.

[0119] In another aspect, a lung or airway disorder or disease is treated by restoring homeostasis in and / or maintaining mucosal homeostasis in a subject in need thereof, the method comprising administering to the subject an effective amount of the disclosed composition.

[0120] In another aspect, the present disclosure provides a method of restoring and / or maintaining homeostasis in a mucosa in a subject in need thereof, comprising administering to the subject an effective amount of the disclosed composition.

[0121] In another aspect, the disclosure provides a method of restoring or maintaining homeostasis in a mucosa, comprising administering to a subject an effective amount of a disclosed composition.

[0122] In some embodiments, the disclosed method comprises administering to the airway of a subject an effective amount of a composition comprising a glutathione conjugate and / or glutathione, or a pharma- ceutically acceptable salt thereof. In another embodiment, the composition further comprises an organic acid, or a pharma- ceutically acceptable salt thereof. In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises a bicarbonate. In another embodiment, the bicarbonate is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid in the disclosed composition is ascorbic acid. In some embodiments, the bicarbonate in the disclosed composition is sodium bicarbonate. In some embodiments, the composition comprises (a) glutathione, (b) ascorbic acid, and (c) sodium bicarbonate. In certain embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (e.g., about 0.49:about 0.50:about 1). In another embodiment, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (eg, about 0.49:about 1:about 1).

[0123] In another embodiment, the amount of each of the glutathione conjugate and / or glutathione, or a pharma- ceutically acceptable salt thereof, the organic acid, or a pharma- ceutically acceptable salt thereof, and the bicarbonate of the composition is present such that the amount of bicarbonate results in a pH in the range of about 5.5 to about 14. In some embodiments, the pH of the disclosed compositions is about 5.5 to about 14 (e.g., 5.5 to 7.5). In some embodiments, the pH of the disclosed compositions is about 6 to about 14 (e.g., 6 to 7.5). In some embodiments, the pH of the compositions is 7±1.5, 7±1.4, 7±1.3, 7±1.2, 7±1.1, 6±0.5, 6±0.4, 6±0.3, 6±0.2, 6±0.5, 6±0.1, or about 6.

[0124] In another embodiment, the composition further comprises about 0.01% to about 5% by weight of a pharma- ceutically acceptable thiocyanate.

[0125] In another embodiment, the composition is in the form of particles, hi another embodiment, the particles are mixed with a gas or liquid propellant for use in inhalation therapy.

[0126] In another embodiment, the glutathione is reduced glutathione.

[0127] In another embodiment, administration of the composition to a subject provides a glutathione concentration in the airway surface liquid of the subject from about 0.1 mM to about 1.0 mM. In another embodiment, administration of the composition to a subject provides a thiocyanate concentration in the airway surface liquid of the subject from about 0.5 mM to about 3.0 mM.

[0128] In some embodiments, the oxidized glutathione in the disclosed compositions (e.g., % GSSG) is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0129] In some embodiments, the reduced glutathione in the disclosed compositions is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., in N2 or ambient atmosphere) at about 5° C. for 4 weeks.

[0130] In some embodiments, reduced ascorbic acid (e.g., % ASC) is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of the ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0131] In some embodiments, the oxidized ascorbic acid in the disclosed compositions is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0132] In some embodiments, the composition is an aqueous solution or a dry powder, hi other embodiments, the composition is administered to the subject by inhalation.

[0133] a. Antibacterial usage While most antibacterial agents (e.g., antibiotics) are active only against gram-negative bacteria or only against gram-positive bacteria, the disclosed compositions exhibit broad spectrum activity against both gram-negative and gram-positive bacteria. Such properties are unexpected in light of studies showing that ascorbic acid alone acts in a strain-dependent and highly concentration-dependent manner (see, e.g., Med J Aust. 1974 Feb 9;1(6):169-74; Hancock and Wong. Antimicrob agents and chemotherapy, July 1984: 48-52). Clinical conditions make the use of broad spectrum antibiotics difficult in chronic inflammatory airway diseases such as cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis) and lung transplantation. Furthermore, physicians often use antibiotics empirically and based on patient response. In contrast to the disclosed compositions, current antibiotic therapies do not show consistent efficacy against bacteria commonly found in chronic inflammatory airway diseases.

[0134] The disclosed compositions can actively inhibit multiple bacterial species without providing a bacterial protection mechanism.

[0135] Thus, in some embodiments, the disclosed compositions are useful for inhibiting or reducing the growth of clinically isolated bacteria. In some embodiments, the disclosed compositions are useful for inhibiting or reducing the formation of clinically isolated bacterial biofilms. In some embodiments, the disclosed compositions are useful for treating or reducing symptoms in subjects suffering from or at risk of clinically isolated bacterial infections.

[0136] In certain aspects, the clinical isolates of the present application may include a single strain or a combination of strains (i.e., two or more). In certain embodiments, the clinical isolates are selected from the group consisting of Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Actinobacter baumannii, Burkholderia pseudomallei, Burkholderia cepacia, or any combination thereof.

[0137] In some embodiments, the clinical isolate is gram negative. In certain embodiments, the gram negative clinical isolate is selected from the group consisting of Burkholderia cepacia, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia pseudomallei, or any combination thereof. In some embodiments, the Pseudomonas aeruginosa is mucoid or non-mucoid, or a combination thereof.

[0138] In some embodiments, the clinical isolate is Gram positive. In certain embodiments, the Gram positive clinical isolate is selected from the group consisting of Enterococcus, Streptococcus, Pneumococcus, Staphylococcus (e.g., Staphylococcus aureus or Methicillin-resistant Staphylococcus aureus (MRSA)), and any combination thereof. In some embodiments, the Gram positive clinical isolate is selected from the group consisting of Staphylococcus aureus or Methicillin-resistant Staphylococcus aureus (MRSA), or any combination thereof.

[0139] In some embodiments, the clinical isolate is mucoid. In certain embodiments, the mucoid clinical isolate is Pseudomonas aeruginosa, Burkholderia cepacia, and any combination thereof.

[0140] In some embodiments, the clinical isolate is non-mucoid. In certain embodiments, the clinical isolate is clinical non-mucoid Pseudomonas aeruginosa.

[0141] In some embodiments, the clinical isolates are aerobic. In certain embodiments, the aerobic clinical isolates are Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus, and Methicillin-resistant Staphylococcus aureus (MRSA), and any combination thereof. In certain embodiments, the aerobic conditions involve nitrate. In certain embodiments, the aerobic conditions do not involve nitrate.

[0142] In some embodiments, the clinical isolate is anaerobic or aerotolerant. In certain embodiments, the anaerobic or aerotolerant clinical isolate is selected from the group consisting of Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Burkholderia cepacia, Pseudomonas aeruginosa, Staphylococcus aureus, and any combination thereof. In certain embodiments, the anaerobic or aerotolerant conditions involve nitrate. In certain embodiments, the anaerobic or aerotolerant conditions do not involve nitrate.

[0143] In some embodiments, the clinical isolates are capable of survival extracellularly. In particular embodiments, the clinical isolates capable of survival extracellularly are Burkholderia cepacia, Pseudomonas aeruginosa, Staphylococcus aureus, and Methicillin-resistant Staphylococcus aureus (MRSA), and any combination thereof.

[0144] In some embodiments, the clinical isolate is multi-drug resistant. In certain embodiments, the multi-drug resistant clinical isolate is selected from the group consisting of Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Burkholderia cepacia, and any combination thereof.

[0145] In some embodiments, the clinical isolate is resistant to cationic protein therapy. In certain embodiments, the cationic protein therapy resistant clinical isolate is selected from the group consisting of Pseudomonas aeruginosa, Burkholderia cepacia, and any combination thereof.

[0146] Pseudomonas aeruginosa In one embodiment, the clinically isolated bacteria is Pseudomonas aeruginosa.

[0147] Pseudomonas aeruginosa is a gram-negative bacterium that can cause infections, especially in patients with weakened host defense mechanisms. It is the most common pathogen isolated from patients hospitalized for more than a week and is a frequent cause of hospital-acquired infections. Pseudomonas infections can be life-threatening.

[0148] P. aeruginosa is a metabolically variable bacterium that can cause a wide range of severe opportunistic infections in patients with reduced natural defenses. Predisposing conditions can include, for example, a disrupted epithelial barrier (as seen in burn patients), depletion of neutrophils (e.g., in cancer patients undergoing chemotherapy), the presence of foreign bodies (patients with central venous catheters), and altered mucociliary clearance (e.g., in individuals with cystic fibrosis). P. aeruginosa is inherently resistant to many antibiotics and can acquire resistance to others, making it difficult to treat. The tendency of P. aeruginosa to form biofilms further protects it from antibiotics and the host's immune system. In some embodiments, the clinical isolate is a mucoid P. aeruginosa. In some embodiments, the clinical isolate is a non-mucoid P. aeruginosa.

[0149] P. aeruginosa is commonly isolated from the airways of individuals with cystic fibrosis and is associated with an accelerated decline in lung function in these patients. Chronic lung colonization and infection also occurs in bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), a disease characterized by irreversible dilation of the bronchial tree, and chronic obstructive pulmonary disease, a disease characterized by airway narrowing and airflow abnormalities. In addition, P. aeruginosa is one of the most common causes of hospital-acquired pneumonia, especially in ventilated patients, which is associated with a particularly high mortality rate.

[0150] Exposure to different in vivo environments leads to the activation of different metabolic patterns in P. aeruginosa to defend itself against reactive nitrogen species, which may lead to host mucoid production.

[0151] Barkholideria cepacia In another embodiment, the clinically isolated bacterium is Burkholderia cepacia.

[0152] Burkholderia cepacia is an aerobic, Gram-negative bacillus found in a variety of aquatic environments. Burkholderia cepacia is a low pathogenic organism that is often present as a colonizer of fluids used in hospitals (e.g., irrigation and intravenous fluids). Burkholderia cepacia rarely causes infections in healthy hosts. Based on phenotypic and genotypic analyses, Burkholderia cepacia is one of nine genetic variants (genovar) that constitute the Burkholderia cepacia complex (BCC).

[0153] BCC are an important group of pathogens affecting patients with cystic fibrosis and chronic granulomatous disease, as well as immunocompromised and hospitalized patients. Avgeri et al. Int J Antimicrob Agents. 2009 May;33(5):394-404. BCC frequently mutate and acquire high-level resistance to many antimicrobial agents. These bacteria are associated with clinical deterioration and mortality in chronic inflammatory airway diseases and can even develop multidrug resistance. They are generally considered to be aerobic, although some exhibit fermentation and nitrate respiration. Sass et al. ISME J. 2013 Aug;7(8):1568-81.

[0154] Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) In another embodiment, the clinically isolated bacteria is Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA), both of which are gram-positive cocci.

[0155] Staphylococcus aureus is an important cause of pneumonia. These bacteria are particularly associated with the development of pleuropulmonary infections and typically coexist with Pseudomonas aeruginosa or Burkholderia cepacia in chronic inflammatory airway diseases. Tong et al. Clin Microbiol Rev. 2015 Jul;28(3):603-61. In one embodiment, Staphylococcus aureus is identified in lung transplant patients with acute and chronic rejection.

[0156] MRSA infections are caused by Staphylococcus aureus that has become resistant to many of the antibiotics used to treat common staph infections. HA-MRSA infections are typically associated with invasive procedures or devices such as surgery, intravenous tubes, or artificial joints. Community-acquired MRSA (CA-MRSA) is another type of MRSA infection that has occurred in the wider community, often among otherwise healthy people. This form often begins as a painful skin boil. It spreads by skin-to-skin contact. Risk populations include school wrestlers, childcare workers, and people living in crowded conditions.

[0157] b. Methods for increasing mucociliary clearance Mucociliary clearance is important for preventing infection and inflammation in the lungs. However, mucociliary clearance is often absent or reduced in chronic inflammatory airway diseases such as cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), lung transplantation, and chronic obstructive pulmonary disease. Furthermore, airway epithelial cells from cystic fibrosis patients are known to completely lack mucociliary clearance ability even in the absence of other inflammatory insults or bacterial colonization or infection. Although some studies have focused on modifying the airway microenvironment to restore the function of previously functional tissues, the disclosed compositions are capable of activating mucociliary clearance function in epithelial cells that lack such function.

[0158] Ascorbic acid can directly modulate CFTR activity (Proc Natl Acad Sci US A. 2004 Mar 9; 101(10): 3691-3696.). However, ascorbic acid has also been shown to have limited effects on mucociliary clearance (BMC Complement Altern Med. 2013; 13: 110.) and limited clinical efficacy (Sakasura Ann Otol 82; 1973). However, the disclosed compositions can restore mucociliary function in the absence of bacterial infection.

[0159] Glutathione, ascorbic acid, and bicarbonate administered individually have no clinical effect on mucociliary clearance or clinical outcome (Sakasura Ann Otol 82; 1973; Am J Respir Crit Care Med. 2013 Jul 1;188(1):83-9. doi: 10.1164 / rccm.201303-0427OC.). Furthermore, despite clinical data showing a direct effect on airway pH, the effect of bicarbonate was inconclusive.

[0160] The disclosed compositions can produce a synergistic effect between glutathione, ascorbic acid, and bicarbonate to activate mucociliary clearance to levels seen in normal cells (Am J Respir Crit Care Med. 2014 Aug 15; 190(4): 421-432.). Thus, in some embodiments, the disclosed compositions are useful for upregulating mucociliary clearance in subjects suffering from or at risk for reduced mucociliary clearance.

[0161] In some aspects, the disclosure includes a method of upregulating mucociliary clearance in a subject suffering from or at risk of reduced mucociliary clearance, comprising administering a composition comprising (a) glutathione, a glutathione derivative, a glutathione conjugate, or a pharma- ceutically acceptable salt thereof, and (b) an organic acid.

[0162] In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises (c) a bicarbonate. In another embodiment, the bicarbonate is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid in a is ascorbic acid. In some embodiments, the composition comprises (a) glutathione, (b) ascorbic acid, and (c) sodium bicarbonate. In certain embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (e.g., about 0.49:about 0.50:about 1). In another embodiment, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (e.g., about 0.49:about 1:about 1).

[0163] In another embodiment, the amount of each of the glutathione conjugate and / or glutathione, or a pharma- ceutically acceptable salt thereof, the organic acid, or a pharma- ceutically acceptable salt thereof, and the bicarbonate of the composition is present such that the amount of bicarbonate results in a pH in the range of about 5.5 to about 14. In some embodiments, the pH of the disclosed compositions is about 5.5 to about 14 (e.g., 5.5 to 7.5). In some embodiments, the pH of the disclosed compositions is about 6 to about 14 (e.g., 6 to 7.5). In some embodiments, the pH of the compositions is 7±1.5, 7±1.4, 7±1.3, 7±1.2, 7±1.1, 6±0.5, 6±0.4, 6±0.3, 6±0.2, 6±0.5, 6±0.1, or about 6.

[0164] In another embodiment, the composition further comprises about 0.01% to about 5% by weight of a pharma- ceutically acceptable thiocyanate.

[0165] In another embodiment, the composition is in the form of particles, hi another embodiment, the particles are mixed with a gas or liquid propellant for use in inhalation therapy.

[0166] In another embodiment, the glutathione is reduced glutathione.

[0167] In another embodiment, administration of the composition to a subject provides a glutathione concentration in the airway surface liquid of the subject from about 0.1 mM to about 1.0 mM. In another embodiment, administration of the composition to a subject provides a thiocyanate concentration in the airway surface liquid of the subject from about 0.5 mM to about 3.0 mM.

[0168] In some embodiments, the oxidized glutathione in the disclosed compositions (e.g., % GSSG) is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0169] In some embodiments, the reduced glutathione in the disclosed compositions is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., in N2 or ambient atmosphere) at about 5° C. for 4 weeks.

[0170] In some embodiments, reduced ascorbic acid (e.g., % ASC) is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of the ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0171] In some embodiments, the oxidized ascorbic acid in the disclosed compositions is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0172] In some embodiments, the composition is an aqueous solution or a dry powder, hi other embodiments, the composition is administered to the subject by inhalation.

[0173] In some aspects, administering the composition reduces mucus viscosity in the patient. In some aspects, administering the composition increases ciliary beat frequency in airway epithelial cells in the patient. In some aspects, administering the composition increases mucociliary transport rate in airway epithelial cells in the patient. In some aspects, administering the composition increases airway surface liquid height in the patient.

[0174] In some embodiments, the patient suffers from or is at risk of suffering from a chronic inflammatory airway disease. In some embodiments, the inflammatory airway disease is cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), asthma, chronic obstructive pulmonary disease, or pulmonary fibrosis. In some embodiments, the inflammatory airway disease is cystic fibrosis. In some embodiments, the airway epithelial cells of the patient lack mucociliary clearance capacity before administration of the composition. In some embodiments, the airway epithelial cells of the patient lack mucociliary clearance capacity due to a genetic defect.

[0175] In some embodiments, the patient has undergone a lung transplant.

[0176] In some embodiments, the patient's airway epithelium is not colonized with bacteria. In other embodiments, the patient's airway epithelium is colonized with bacteria. In some embodiments, the patient has an active bacterial infection. In other embodiments, the patient does not have an active bacterial infection.

[0177] In some embodiments, the patient is a pediatric patient, hi other embodiments, the patient is an adult patient.

[0178] c. Anti-inflammatory usage The hallmarks of inflammation in airway disease include the production of neutrophil extracellular traps (NETs), increased neutrophil myeloperoxidase activity, increased nitric oxide production, and increased proinflammatory cytokine production. Studies suggest that ascorbic acid specifically enhances myeloperoxidase (MPO) activity and promotes leukocyte recruitment, and that the presence of MPO is directly related to clinical complications and deterioration (Free Radic Biol Med. 2017 Oct 2;113:236-243). Furthermore, neutrophil extracellular trap formation caused by phorbol myristate acetate (PMA) is prevented under acidic conditions and induced under more basic conditions resulting from high concentrations of sodium bicarbonate.

[0179] The disclosed compositions directly inhibit inflammatory processes. In particular, the disclosed compositions not only inhibit MPO activity, but also downregulate cytokines related to NET formation, macrophage activation, and neutrophil and T cell recruitment, through a mechanism independent of reduced function. The disclosed compositions can downregulate pathological nitric oxide production levels, and thus do not destroy endogenous antibacterial properties. In some embodiments, administering the composition reduces the exhaled nitric oxide (FeNO) of the patient by at least 20%. Thus, in some embodiments, the disclosed compositions are useful for reducing airway inflammation in subjects suffering from or at risk of airway inflammation.

[0180] In some aspects, the disclosure includes a method of reducing airway inflammation in a subject suffering from or at risk of airway inflammation, the method comprising administering a composition comprising (a) glutathione, a glutathione derivative, a glutathione conjugate, or a pharma- ceutically acceptable salt thereof, and (b) an organic acid.

[0181] In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises (c) a bicarbonate. In another embodiment, the bicarbonate is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid in a is ascorbic acid. In some embodiments, the composition comprises (a) glutathione, (b) ascorbic acid, and (c) sodium bicarbonate. In certain embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (e.g., about 0.49:about 0.50:about 1). In another embodiment, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (e.g., about 0.49:about 1:about 1).

[0182] In another embodiment, the amount of each of the glutathione conjugate and / or glutathione, or a pharma- ceutically acceptable salt thereof, the organic acid, or a pharma- ceutically acceptable salt thereof, and the bicarbonate of the composition is present such that the amount of bicarbonate results in a pH in the range of about 5.5 to about 14. In some embodiments, the pH of the disclosed compositions is about 5.5 to about 14 (e.g., 5.5 to 7.5). In some embodiments, the pH of the disclosed compositions is about 6 to about 14 (e.g., 6 to 7.5). In some embodiments, the pH of the compositions is 7±1.5, 7±1.4, 7±1.3, 7±1.2, 7±1.1, 6±0.5, 6±0.4, 6±0.3, 6±0.2, 6±0.5, 6±0.1, or about 6.

[0183] In another embodiment, the composition further comprises about 0.01% to about 5% by weight of a pharma- ceutically acceptable thiocyanate.

[0184] In another embodiment, the composition is in the form of particles, hi another embodiment, the particles are mixed with a gas or liquid propellant for use in inhalation therapy.

[0185] In another embodiment, the glutathione is reduced glutathione.

[0186] In another embodiment, administration of the composition to a subject provides a glutathione concentration in the airway surface liquid of the subject from about 0.1 mM to about 1.0 mM. In another embodiment, administration of the composition to a subject provides a thiocyanate concentration in the airway surface liquid of the subject from about 0.5 mM to about 3.0 mM.

[0187] In some embodiments, the oxidized glutathione in the disclosed compositions (e.g., % GSSG) is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, or less than about 3% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in an N2 atmosphere and / or ambient atmosphere).

[0188] In some embodiments, the reduced glutathione in the disclosed compositions is greater than about 80%, greater than about 82%, greater than about 84%, greater than about 85%, greater than about 88%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, or greater than about 97% by weight of the total glutathione in the disclosed compositions after storage of the disclosed compositions (e.g., in N2 or ambient atmosphere) at about 5° C. for 4 weeks.

[0189] In some embodiments, reduced ascorbic acid (e.g., % ASC) is greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, or greater than about 90% by weight of the ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0190] In some embodiments, the oxidized ascorbic acid in the disclosed compositions is less than about 20%, less than about 18%, less than about 16%, less than about 15%, less than about 12%, less than about 10%, or less than about 9% by weight of the total ascorbic acid in the disclosed compositions after storage of the disclosed compositions (e.g., at 5° C. in a N2 atmosphere and / or ambient atmosphere) for 4 weeks.

[0191] In some embodiments, the composition is an aqueous solution or a dry powder, hi other embodiments, the composition is administered to the subject by inhalation.

[0192] In some embodiments, administering the composition inhibits myeloperoxidase activity in the patient's neutrophils. In some embodiments, administering the composition reduces the formation of neutrophil extracellular traps. In some embodiments, administering the composition downregulates nitric oxide production from the patient's neutrophils. In some embodiments, administering the composition reduces the patient's exhaled nitric oxide by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25%. In some embodiments, administering the composition reduces the patient's baseline exhaled nitric oxide by at least 20%.

[0193] In some embodiments, administering the composition downregulates the production of at least one inflammatory cytokine. In some embodiments, the at least one inflammatory cytokine comprises a cytokine associated with macrophage activation and / or neutrophil and T cell recruitment. In some embodiments, the at least one inflammatory cytokine comprises TNF-α. In some embodiments, the at least one inflammatory cytokine comprises IL-6. In some embodiments, the at least one inflammatory cytokine comprises IL-8.

[0194] In some embodiments, the patient suffers from or is at risk of suffering from chronic inflammatory airway disease.In some embodiments, the inflammatory airway disease is cystic fibrosis, bronchiectasis (e.g., non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), asthma, chronic obstructive pulmonary disease, or pulmonary fibrosis.In some embodiments, the inflammatory airway disease is cystic fibrosis.

[0195] In some embodiments, the patient has undergone a lung transplant.

[0196] In some embodiments, the patient's airway epithelium is not colonized with bacteria. In some embodiments, the patient's airway epithelium is colonized with bacteria. In certain embodiments, the patient has an active bacterial infection. In other embodiments, the patient does not have an active bacterial infection.

[0197] In some embodiments, the patient is a pediatric patient, hi other embodiments, the patient is an adult patient.

[0198] In some embodiments, the step of administering the composition does not significantly alter the pH of the patient's airway endothelium.

[0199] Administration The therapeutic method of the present disclosure can be achieved by administering the disclosed composition to a subject. The administration of the disclosed composition can be performed before, during, or after the onset of the disease, condition, or disorder of interest. Typically, the pharmaceutical composition is sterile and does not contain any toxic, carcinogenic, or mutagenic compounds that would cause adverse reactions when administered to a subject.

[0200] The disclosed compositions are administered in an amount effective for desired results, in a manner compatible with the dosage form. In a specific embodiment, the disclosed compositions are administered to a subject in a therapeutically effective amount. The therapeutically effective amount of the disclosed compositions required for use in treatment varies according to the nature of the condition being treated, the period of activity desired, and the age and condition of the patient, and is ultimately determined by the attending physician. The dosage and interval can be individually adjusted to provide sufficient plasma glutathione levels to maintain the desired therapeutic effect. The desired dosage can be administered in a single dose, or as multiple doses administered at appropriate intervals, for example, as divided doses 1, 2, 3, 4 or more times a day. Multiple doses are often desired or necessary. For example, glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof may be administered at a frequency of 4 doses delivered once a day every 4 days (q4d x 4); 4 doses delivered once a day every 3 days (q3d x 4); delivered once a day every 5 days (qd x 5); once a week for 3 weeks (qwk3); 5 days on, 2 days off, and 5 more days on (5 / 2 / 5); or any dosing regimen deemed appropriate for the circumstances.

[0201] The disclosed compositions may be administered over an extended period of time (e.g., as a chronic, lifelong treatment), such as for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months, or longer.

[0202] Any suitable dosing schedule can be followed. For example, dosing frequency can be weekly dosing. Dosing frequency can be daily or multiple dosing. Dosing frequency can be more than weekly dosing. Dosing frequency can be more than daily dosing, for example, any one of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more dosings per day. Dosing frequency can be intermittent (e.g., multiple dosings daily for 7 days followed by 7 days off, and repeating for any 14-day period, for example, 2 months, 4 months, 6 months or more). Dosing frequency can be continuous (e.g., weekly dosing for consecutive weeks).

[0203] The glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof used in the therapeutic methods of the present disclosure may be administered in an amount of about 0.005 to about 1,000 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof may be administered in an amount of about 0.005, about 0.05, about 0.5, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 milligrams per dose, including all doses between 0.005 and 1,000 milligrams.

[0204] The dosage of glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, or any combination thereof, or compositions containing them, may be about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg. The dosage of the composition may be any dosage, including but not limited to about 1 μg / kg. The dosage of the composition may be about 1 μg / kg, about 10 μg / kg, about 25 μg / kg, about 50 μg / kg, about 75 μg / kg, about 100 μg / kg, about 125 μg / kg, about 150 μg / kg, about 175 μg / kg, about 200 μg / kg, about 225 μg / kg, about 250 μg / kg, about 275 μg / kg, about 300 μg / kg, about 325 μg / kg, about 35 μg / kg, about 40 μg / kg, about 50 μg / kg, about 60 μg / kg, about 70 μg / kg, about 80 μg / kg, about 90 ... 0μg / kg, about 375μg / kg, about 400μg / kg, about 425μg / kg, about 450μg / kg, about 475μg / kg, about 500μg / kg, about 525μg / kg, about 550μg / kg, approximately 575μg / kg, approximately 600μg / kg, approximately 625μg / kg, approximately 650μg / kg, approximately 675μg / kg, approximately 700μg / kg, approximately 725μg / kg , about 750μg / kg, about 775μg / kg, about 800μg / kg, about 825μg / kg, about 850μg / kg, about 875μg / kg, about 900μg / kg, about 925μg / kg kg, approximately 950μg / kg, approximately 975μg / kg, approximately 1mg / kg, approximately 5mg / kg, approximately 10mg / kg, approximately 15mg / kg, approximately 20mg / kg, approximately 25mg / kg, approximately 30 The dosage may be any dosage, including but not limited to about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, or more. The dosages above are examples of average cases, but there may be individual cases where higher or lower dosages are beneficial, and such are within the scope of the present disclosure. In practice, the physician will determine the actual dosage regimen that is most suitable for each individual patient, which may vary depending on the age, weight, and response of the particular patient.

[0205] In one embodiment, glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, or any combination thereof is delivered to the upper third of the nasal cavity, the superior meatus, the olfactory region, and / or the nasal region, which in humans is typically about 2-10 cm2, located in the upper third of the nasal cavity, for deposition and absorption by the olfactory epithelium and subsequent transport by olfactory receptor neurons. 2 The olfactory region, located at the top of the nasal cavity within the superior nasal meatus, is useful for delivery in some embodiments because it is the only known part of the body where an extension of the CNS comes into contact with the surrounding environment (Bois et al. Fundamentals of Otolaryngology, p. 184, WB Saunders Co., Philadelphia, 1989).

[0206] In some embodiments, the disclosed compositions may be administered in a single "shock" dose, for example, at the time of a bronchoscopy. In other embodiments, the disclosed methods may be performed on an as-needed basis by self-medication.

[0207] Any of the dosing frequencies may be used with any of the dosage amounts. Additionally, any of the dosing frequencies and / or dosage amounts may be used with any of the disclosed compositions.

[0208] The disclosed compositions may be delivered in any suitable dosage. In exemplary embodiments of the disclosure, dosages for intranasal delivery range from about 25 microliters to 200 microliters or about 50 to 150 microliters, or from about 50, about 100, about 250, or about 500 microliters to about 1, about 2, about 3, about 3.5, or about 4 milliliters in humans. Typically, dosages are selected to be large enough to allow for delivery of a therapeutic amount while taking into account dilution of ASL in the conditions maintained in relatively "normal" airways (10-30 ml ASL) and cystic fibrosis (CF) airways (40-50 ml ASL or more plus thick, viscous, severely infected mucus secretions).

[0209] The disclosed compositions can be utilized for both veterinary and / or medical purposes. Suitable subjects of the present disclosure include, but are not limited to, mammals. The term "mammal" as used herein includes, but is not limited to, primates (e.g., simians and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), cows, sheep, goats, ungulates, pigs, horses, cats, dogs, rabbits, pinnipeds, rodents (e.g., rats, hamsters, and mice), and the like. In some embodiments of the present disclosure, the subject is a human. Human subjects include both males and females, and subjects of all ages, including neonatal, infant, juvenile, adolescent, adult, and geriatric subjects.

[0210] In some embodiments, the disclosed compositions are administered one or more times per day (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day). In certain embodiments, the subject is a human.

[0211] In some aspects of the methods of the present disclosure, the disclosed compositions may be administered via inhalation, intranasally, via the eye, via the ear, via sinus irrigation, or via a bronchoscope, or any combination thereof.

[0212] Intranasal administration of the disclosed compositions may be accomplished by any known method. In specific embodiments, intranasal administration is by inhalation (e.g., using an inhaler, atomizer, or nebulizer device), or alternatively by spray, tube, catheter, syringe, dropper, packtail, pipette, cotton ball, or the like.

[0213] As further examples, the disclosed compositions may be administered intranasally by (1) nasal drops, (2) powder or liquid spray or aerosol, (3) liquid or semi-solid by syringe, (4) liquid or semi-solid by cotton bud, pledget or other similar means of application, (5) gel, cream or ointment, (6) infusion, or (7) injection, or by any means now known or later developed in the art. In specific embodiments, the delivery method is by nasal drops, spray or aerosol. As used herein, aerosol may be used to deliver powders, liquids or dispersions (solids in liquids).

[0214] In some embodiments, the pharmaceutical formulation is directed upwards during administration to enhance delivery to the upper third (e.g., olfactory epithelium in the olfactory region) and lateral walls (e.g., nasal epithelium) of the nasal cavity. Additionally, placing the subject's head in a tilted back position or placing the subject's body in the Mygind or Mecca prayer position may be used to facilitate delivery to the olfactory region.

[0215] The formulations may be provided in single or multi-dose form. In the latter case, a means for measuring the dose may be provided. In the case of a dropper or pipette, this may be achieved by the patient or caregiver administering an appropriate, predetermined amount of the composition. In the case of a spray, this may be achieved, for example, by a metering atomizing spray pump.

[0216] In a further aspect, the present disclosure provides an intranasal spray device comprising the disclosed composition.

[0217] Many devices are known in the art for nasal delivery. Exemplary devices include particle dispersion devices, bidirectional devices, and devices using tip-based inkjet technology.

[0218] When a therapeutically effective amount of the disclosed composition is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. Preparation of such a parenterally acceptable solution, taking into due consideration pH, isotonicity, stability, etc., is within the skill of a person skilled in the art. Compositions for intravenous, cutaneous, or subcutaneous injection typically contain an isotonic vehicle.

[0219] The disclosed compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparations for injection can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with preservatives added.The compositions can be in the form of emulsions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0220] Pharmaceutical compositions for parenteral administration include aqueous solutions of active agents in water-soluble form. In addition, suspensions of glutathione, glutathione derivatives, glutathione conjugates, their pharma- ceutical acceptable salts, or any combination thereof, can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds and allow the preparation of highly concentrated solutions. Alternatively, the composition can be in powder form for constitution with a suitable vehicle, for example, pyrogen-free sterile water, before use.

[0221] Glutathione, glutathione derivatives, glutathione conjugates, their pharmaceutically acceptable salts, or any combination thereof, can also be formulated into rectal compositions, such as suppositories or retention enemas, containing conventional suppository bases.In addition to the formulations described above, glutathione, glutathione derivatives, glutathione conjugates, their pharmaceutically acceptable salts, or any combination thereof, can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection.Thus, for example, glutathione, glutathione derivatives, glutathione conjugates, their pharmaceutically acceptable salts, or any combination thereof can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in acceptable oil) or ion exchange resins.

[0222] The disclosed compositions may be administered orally, bucally, or sublingually in the form of tablets containing excipients such as starch or lactose, or in capsules or suppositories alone or mixed with excipients, or in the form of elixirs or suspensions containing flavorings or colorings. Such liquid preparations may be prepared with pharma- ceutically acceptable additives such as suspensions. The disclosed compositions may also be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the disclosed compositions are typically used in the form of a sterile aqueous solution that may contain other substances to make the solution isotonic with blood, for example, salts or monosaccharides such as mannitol or glucose.

[0223] The disclosed compositions may be present as solid formulations, such as, for example, particle formulations, or as solutions. In the case of particle formulations, the particles may be mixed with gas or liquid propellants for use in inhalation therapy. Other solid formulations include formulations for oral, buccal or colonic administration, and suppositories for rectal or vaginal administration. Exemplary formulations include, but are not limited to, eye drops, nebulizers, topical gels and ointments, dry powders, particles, sprays, liquids, anesthetics or vaporizers, auto-injectors, intrauterine devices, respimats for inhalation therapy, liniments, liposomes, lotions, formulations for intramuscular, intrathecal or subcutaneous injection, irrigation solutions, drops, and face masks.

[0224] In solution form, the formulations may be in the form of sprays for intranasal administration, formulations for use in nebulizers, and formulations for rectal administration such as enemas and colonies. Solutions containing water-miscible organic solvents such as propylene glycol and / or glycerol, as well as other ingredients typically found in vaginal and rectal lubricants, may also be used. Regardless of the solvent used, the solvent is typically present in a weight ratio of about 15 to about 85 weight percent based on the weight of the solids, more typically about 50 to about 85 weight percent.

[0225] The compositions and / or formulations of the present invention may be used to treat disorders associated with mucosa by delivering the compositions and / or formulations to the mucosa to be treated. In some embodiments, the mucosa may be in or near the lung, such as the deep lung (alveolar region), while in other embodiments, the mucosa may be in or near one or more of the eye, mouth, nose, rectum, and / or vagina.

[0226] Additional Therapeutic Substances In another aspect, the disclosure provides a method comprising administering to a subject an effective amount of an additional therapeutic agent.

[0227] The compositions comprising glutathione, glutathione derivatives, glutathione conjugates, pharma- ceutically acceptable salts thereof, organic acids, or any combination thereof may also be combined with antibiotics to enhance the therapeutic effect.

[0228] In another aspect, it is an object of the invention to identify clinical isolates in patient samples and test the efficacy of in vitro treatment by treatment with a composition comprising glutathione, a glutathione derivative, a glutathione conjugate, a pharma- ceutically acceptable salt thereof, an organic acid, or any combination thereof.

[0229] Drugs administered to the lungs often have some side effects, sometimes because of dosage, and sometimes because they damage lung tissue.In some aspects, therapeutic substances combined with the compositions disclosed herein can be effective at lower doses, and such lower doses can reduce the incidence of side effects.In another aspect, when therapeutic substances interact unfavorably with lung tissue, the compositions described herein can help restore the homeostasis of lung tissue, thereby minimizing or eliminating the damage caused by therapeutic substances.

[0230] In another aspect, the disclosure provides fluticasone, budesonide, mometasone, ciclesonide, flunisolide, beclomethasone, albuterol, levalbuterol, ipratropium, tiotropium, formoterol, arformoterol, indacaterol, aclidinium and pirbuterol, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, cayston, dicloxacillin, flucloxacillin, The therapeutic agent is selected from the group consisting of penicillins such as mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin and ticarcillin, combinations of penicillins such as amoxicillin / clavulanic acid, ampicillin / sulbactam, piperacillin / tazobactam, ticarcillin / clavulanic acid with other therapeutic agents, and any combination thereof.

[0231] In one embodiment, the therapeutic agent is selected from the group consisting of fluticasone, budesonide, mometasone, ciclesonide, flunisolide, beclomethasone, albuterol, levalbuterol, ipratropium, tiotropium, formoterol, arformoterol, indacaterol, aclidinium, casen, pirbuterol, and any combination thereof.

[0232] Additional therapeutic agents that may be combined with the compositions and formulations of the invention include fluticasone (sold, for example, as Flovent Diskus 50 or Flonase, GlaxoSmithKline), budesonide (sold, for example, as Pulmicort Respul or Rhinocort by AstraZeneca ("AZ")), mometasone (sold as Nasonex as a spray by Merck / SP or as Azmanex Twisthaler), ciclesonide (sold as Alvesco or Onmalis by Takeda Pharmaceuticals), flunisolide (sold as Aerobid by Roche or Aerospan HFA by GSK), beclomethasone (sold as Qvar or Onasl by Teva Pharmaceuticals), albuterol (sold as Proair HFA by Teva and as GSMA HFA by GSK), and cyclosporine (sold as cyclosporine by GSK). These include, but are not limited to, levabuterol (sold by Sunovion as Ventolin HFA), levabuterol (sold by Sunovion as Xopenex), ipratropium (sold by BI as Atrovent), tiotropium (sold by BI as Spiriva), salmeterol (sold by GSK as Serevent), formoterol (sold by Novartis as Foradyl and by DayPharma as Perfomist), arformoterol (sold by Sunovion as Brovana), indacaterol (sold by Novartis as Arcapta), aclidinium (sold by Forest Laboratories as Tudorza), pirbuterol (sold by Medicis as Maxair), and any combination thereof.

[0233] The method of the present invention comprises administering to a subject one or more additional therapeutic substances in combination with the disclosed composition.In one embodiment, the disclosed composition further comprises additional therapeutic substances, or the composition is administered in combination with or alternating with additional therapeutic substances.That is, in some embodiments, the composition and the additional therapeutic substances are directed to the same site in the same formulation, and in another embodiment, the composition can be administered via one route and the additional therapeutic substances can be administered via a different route.

[0234] In some embodiments, the additional therapeutic agent comprises a cystic fibrosis transmembrane receptor (CFTR) therapy. In some embodiments, the CFTR therapy comprises a CFTR amplifier (e.g., PTI-428), a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or a combination thereof), a CFTR enhancer / modulator (e.g., VX-770 (ivacaftor, approved as Kalydeco), QBW251, VX-561, PT1-808, or a combination thereof), a CFTR RNA regulator (e.g., QR-100, MRT5005, or a combination thereof), or any combination thereof.

[0235] In some embodiments, the additional therapeutic agent comprises a CFTR amplifier. The CFTR amplifier increases the amount of CFTR protein in the cell. There are six mutation classes in cystic fibrosis that vary in the degree of CFTR dysfunction. The most severe cystic fibrosis mutation has a genetic defect, such as a premature stop codon, that prevents CFTR from becoming a protein. Therapies proposed to treat this mutation include amplifiers that increase the amount of CFTR RNA in the cell. These are also known as "read-through therapies." In some embodiments, the CFTR amplifier is PTI-428.

[0236] In some embodiments, the additional therapeutic agent comprises a CFTR corrector. The CFTR corrector carries the CFTR protein to the cell surface. The most common cystic fibrosis mutation class is class II, where CFTR is misfolded and poorly transported to the cell surface. Therapies used to treat class II mutations, such as dF508, include a combination of a corrector that aids in protein folding and / or protein transport to the cell surface, and an enhancer that increases CFTR gating or ion transport through the protein. In some embodiments, the CFTR corrector is selected from the group consisting of VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, and combinations thereof.

[0237] In some embodiments, the additional therapeutic agent comprises a CFTR enhancer / modulator. The CFTR enhancer / modulator increases ion transport through CFTR at the cell surface. Class III and IV mutations include G551D, a mutation in which CFTR has reduced gating or ion transport. Enhancers / modulators can be used in patients with these mutations to widen the channel opening and allow sufficient ion transport. In some embodiments, the CFTR enhancer / modulator is selected from the group consisting of VX-770 (ivacaftor), QBW251, VX-561, PT1-808, and combinations thereof.

[0238] In some embodiments, the additional therapeutic agent comprises an RNA modulator. The RNA modulator can be used to allow delivery of genetically corrected CFTR to the cell surface. Class V-VI mutations result in a decrease in CFTR protein on the cell surface, either through a decrease in the number of CFTR transcripts or an increase in turnover. Amplification agents and RNA modulators can be used to treat these mutations. In some embodiments, the RNA modulator is selected from the group consisting of QR-100, MRT5005, or a combination thereof.

[0239] In some embodiments, CFTR treatment includes a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or a combination thereof) and a CFTR enhancer / modulator (e.g., VX-770 (ivacaftor), QBW251, VX-561, PT1-808, or a combination thereof). In some embodiments, the CFTR therapy includes the combination of VX-770 (ivacaftor) and VX-809 (lumacaftor), approved as Orkambi, the combination of VX-770 (ivacaftor) and VX-661 (tezacaftor), approved as Simdeco, the combination of VX-770 (ivacaftor), VX-661 (tezacaftor) and VX-445, the combination of VX-770 (ivacaftor), VX-661 (tezacaftor) and VX-659, the combination of VX-770 (ivacaftor), VX-661 (tezacaftor) and VX-152, or the combination of VX-770 (ivacaftor) and GLPG2222.

[0240] In some embodiments, the CFTR treatment includes a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or a combination thereof) and a CFTR amplifier (e.g., PTI-428). In some embodiments, the CFTR treatment includes the combination of VX-770 (ivacaftor) and VX-809 (lumacaftor), approved as Orkambi, and PTI-428.

[0241] In some embodiments, CFTR treatments include CFTR correctors (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or combinations thereof), CFTR potentiators / modulators (e.g., VX-770 (ivacaftor), QBW 251, VX-561, PT1-808, or combinations thereof), and CFTR amplifiers (e.g., PTI-428).

[0242] In some embodiments, CFTR therapy includes a CFTR enhancer / modulator and a CFTR corrector to improve CFTR gating / ion transport.

[0243] In some embodiments, the administration of CFTR therapy is simultaneous with the administration of the disclosed composition to the subject, or sequential in any order (e.g., the composition can be administered before or after CFTR therapy). In some embodiments, the CFTR therapy is administered orally and the disclosed composition is administered by inhalation. In some embodiments, both the cystic fibrosis therapy and the disclosed composition are administered by inhalation.

[0244] In some embodiments, the disclosed compositions are administered in combination with VX-770, VX-809, or a combination of VX-770 / VX-809, where the administration is simultaneous to a subject or sequential in any order (e.g., the compositions can be administered before or after the combination of VX-770 / VX-809).

[0245] The additional therapeutic agent is typically selected from drugs known to be useful for treating the disease, condition, or disorder suffered by the subject in need thereof. The selection of the additional therapeutic agent will depend on the disease, condition, or disorder to be treated or prevented in the subject. This determination is within the capabilities of the skilled artisan, especially in light of the present disclosure.

[0246] In one embodiment, one additional therapeutic agent is administered to the subject. In another embodiment, two additional therapeutic agents are administered to the subject. In another embodiment, three additional therapeutic agents are administered to the subject. In another embodiment, four additional therapeutic agents are administered to the subject. In another embodiment, five additional therapeutic agents are administered to the subject. In another embodiment, five or more additional therapeutic agents are administered to the subject. Non-limiting exemplary therapeutic agents are antifungal agents, antiviral agents, antibacterial agents, anti-inflammatory agents, immunosuppressants, bronchodilators, airway modulators, alpha lipoic acid, alpha tocopherol, docosahexaenoic acid, proline, glycine, curcumin, arginine, thiocyanate, glutathione, oxidized glutathione, reduced glutathione, cysteine, hypothiocyanate, lactoferrin, and lactoperoxidase, and any combination thereof. In another embodiment, the one or more therapeutic agents is glutathione, oxidized glutathione, or reduced glutathione.

[0247] The disclosed compositions and the additional therapeutic agent may be administered together as a single unit dose or separately as multiple unit doses, where the disclosed compositions are administered before, after, or simultaneously with the additional therapeutic agent. One or more doses of the disclosed compositions and / or one or more doses of the additional therapeutic agent may be administered to the subject.

[0248] In another embodiment, the additional therapeutic agent treats the desired disorder for which it is administered, but may cause some side effects, such as dryness of the mucosa resulting in discomfort and / or injury that can be addressed by administering the disclosed compositions.

[0249] In another embodiment, the disclosed compositions treat the desired disorder for which they are administered, but may cause some side effects, such as dryness of the mucosa resulting in discomfort and / or injury that can be addressed by administering additional therapeutic substances.

[0250] In some embodiments, one or more additional therapeutic agents and the disclosed compositions both treat the underlying disease, albeit through different means, so that additive or synergistic effects can be achieved.As a result, in some aspects of this embodiment, lower doses of the additional therapeutic agent can be effective, which lower doses can cause fewer side effects or provide another benefit to the subject.

[0251] It is understood that when the disclosed composition and one or more additional therapeutic substances are not administered simultaneously, they can be administered to the subject in need of them in any order.For example, the disclosed composition can be administered before, for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before, at the same time, or after, for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after the administration of one or more additional therapeutic agents to the subject in need of them. In some embodiments, the disclosed compositions and one or more additional therapeutic agents are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1-2 hours apart, 2-3 hours apart, 3-4 hours apart, 4-5 hours apart, 5-6 hours apart, 6-7 hours apart, 7-8 hours apart, 8-9 hours apart, 9-10 hours apart, 10-11 hours apart, 11-12 hours apart, 24 hours or less apart, or 48 hours or less apart. In one embodiment, the components of the combination therapy are administered from about 1 minute to about 24 hours apart.

[0252] In another embodiment, the additional therapeutic agent is an inhaled corticosteroid (ICS) or a bronchodilator.

[0253] prevention In addition to providing a method of treatment, the disclosed compositions may also be used to reduce symptoms or provide prophylaxis of various diseases and disorders associated with bacterial clinical isolate infections, impaired mucociliary clearance and / or airway inflammation.

[0254] Aspects of the present disclosure can be further defined by reference to the following non-limiting examples which detail the preparation of certain compositions of the present disclosure and methods for using the compositions of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

[0255] In vitro analysis of clinically isolated bacteria Clinically isolated samples from subjects include, but are not limited to, sputum, airway samples, surgical tissue samples, autopsy samples, and blood. Samples may be fixed and / or stored at -20°C or -80°C.

[0256] Isolation of CI species of clinically isolated bacteria can be achieved by any technique known in the art. CI species can be isolated from pure cultures (i.e., only one CI species) or from samples containing multiple bacteria. Common methods for isolating CI species include, but are not limited to, serial dilution of the initial clinical sample, centrifugation, plate streaking with or without an initial growth step in liquid medium, and growth of individual colonies of bacteria on solid or liquid media. CI can be grown using any medium known in the art, including, but not limited to, plankton and solid media.

[0257] Clinically isolated bacteria may be identified using any technique known in the art, including, but not limited to, morphological techniques (e.g., Gram staining, acid-fast staining); biochemical, serological, or protein expression tests (e.g., antibody interaction, slide agglutination, ELISA, Western blotting, immunohistochemistry, immunofluorescence, flow cytometry); phage typing; and DNA and / or RNA-based methods (e.g., DNA sequencing, G+C comparison, gPCR, RT-PCR, qPCR, rRNA sequencing, DNA fingerprinting by restriction fragment length polymorphism (RFLP), nucleic acid hybridization (e.g., Northern blotting, Southern blotting), microarrays, in situ hybridization including FISH).

[0258] The growth of clinical isolates in the presence of drugs (e.g., antibiotics) with or without the disclosed compositions, in the absence of drugs with or without the disclosed compositions, or placebos with or without the disclosed compositions, can be tested using any technique known in the art, including, but not limited to, dilution and time-dependent growth of CIs; identification of minimum inhibitory concentration (MIC); determination of minimum biofilm-eliminating concentration (MBC); and other methods known in the art (e.g., disk diffusion, E-test (AB Biodisc, Solna, Sweden)), any commercially available automated susceptibility testing system (e.g., Vitec System (bioMérieux, France), Walk-Away System (Dade International, Sacramento, California), enzymes (e.g., beta-lactamase)). The growth of CIs is tested in plankton or biofilm cultures.

[0259] In some aspects, the disclosure includes measuring the minimum inhibitory concentration (MIC). MICs may be determined on Muller-Hinton (MH) agar plates or in planktonic cultures by broth microdilution in MH. MICs may be determined for clinical isolates and / or laboratory type reference strain PAO1. In some embodiments, the clinically effective MIC range of the composition is about 1%-50%, about 1%-40%, about 5%-50%, about 5%-40%, about 5%-35%, about 10%-50%, about 10%-40%, about 10%-35%, or about 12%-32%.

[0260] In some aspects, the disclosure includes the measurement of in vitro biofilm formation for clinical isolates. In some embodiments, overnight cultures of bacteria are grown in LB with or without the compositions of the disclosure with shaking. The cultures are then diluted 1:100 and 100 L aliquots are added to 96-well plates, which are incubated at 37° C. for 72 hours to allow sufficient growth of the clinical isolates. After 2-3 washes with water, crystal violet is added for 15 minutes, followed by 3 rinses with water and then 95% ethanol. The material is then transferred to a new 96-well plate and the absorbance at 540 nm is determined.

[0261] In some aspects, the disclosure includes measuring the minimum biofilm elimination concentration (MBC) of the CI. For the minimum biofilm concentration (MBC), a protocol similar to the MIC protocol can be used, except that the bacteria are seeded on the plate used for bacterial attachment. In some embodiments, the optical density is measured to determine the MBC for the bacterial isolate. In some embodiments, the assay involves the formation of 96 identical biofilms on the plastic pegs of the lid of the MBC instrument. The biofilms are then exposed to the test antibiotic for a defined period of time, and then placed in fresh bacteriological medium in a second 96-well plate and incubated overnight. The MBC value is the lowest dilution that prevents regrowth of bacteria from the treated biofilm.

[0262] The present disclosure enables any of the above techniques in the presence or absence of nitrate.

[0263] The present disclosure enables any of the above techniques in the presence or absence of oxygen.

[0264] The present disclosure enables any of the above techniques in the presence or absence of one or more antibiotics. EXAMPLES

[0265] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are within the spirit and scope of the present application.

[0266] Example 1: Stability: Measuring reduced glutathione and ascorbic acid levels Composition 1 was prepared with 150 mg / ml glutathione, 88 mg / ml ascorbic acid, and 84 mg / ml sodium bicarbonate, corresponding to about 46.6%, about 27.3%, and about 26.1% of the total weight, respectively, for a total weight of 322 mg / ml. The molar ratios of the components in Composition 1 were 0.49 M glutathione, 0.50 M ascorbic acid, and 1 M HCO3.

[0267] Composition 2 was prepared with 150 mg / ml glutathione, 126 mg / ml ascorbic acid, and 84 mg / ml sodium bicarbonate, corresponding to about 33%, about 38.9%, and about 28.9% of the total weight, respectively, for a total weight of 360 mg / ml. The molar ratios of the components in Composition 1 were 0.49 M glutathione, 1 M ascorbic acid, and 1 M HCO3.

[0268] The amount of oxidation of glutathione and ascorbic acid was measured for composition 1 at pH 5.5, 6.0, and 6.5. Oxidized glutathione is related to the generation of protein-carbonyls via glutathionylation, which occurs when oxidized glutathione dissociates and binds to proteins. Oxidized glutathione (%GSSG) was measured after 4 weeks of storage of composition 1 at pH 5.5, 6.0, and 6.5 under N2 sparging and ambient conditions. In addition, when ascorbic acid is oxidized to dehydroascorbic acid (DHA), DHA can degrade and lead to the formation of protein adducts in a process called ascorbylation (Simpson et al., Biochim biophys Acta 2000;1501:12-24). Reduced ascorbic acid (%ASC) retained in composition 1 was measured after 4 weeks of storage of composition 1 at pH 5.5, 6.0, and 6.5 under N2 sparging (anaerobic) and ambient conditions. Specifically, the nitrogen sparged samples were mixed under anaerobic conditions sparged with nitrogen and the samples were packaged using nitrogen bubbling. Ambient samples were mixed under room air conditions and no nitrogen bubbling was used. The formulations were stored at 5°C for 4 weeks and the percentage of oxidized glutathione (GSSG) and ascorbic acid (ASC) was measured for each sample according to standard procedures.

[0269] As shown in Table 2, the oxidation of glutathione and ascorbic acid is pH and oxygen dependent, and the presence of glutathione increases the concentration of reduced ascorbic acid after 4 weeks of storage at 5°C. Furthermore, when oxygen is present in the solution, reduced glutathione (GSH) helped maintain the ascorbic acid concentration, but GSH itself is oxidized to GSSG at higher concentrations. These results demonstrate that the pH of Composition 1 is important for maintaining glutathione and ascorbic acid in their reduced states, and that the molar ratio of glutathione to ascorbic acid in Composition 1 further stabilizes the oxidation rates of both glutathione and ascorbic acid.

[0270] [Table 1]

[0271] Example 2: In vitro minimum inhibitory concentrations (MICs) of glutathione compositions against bacterial clinical isolates Composition 1 was prepared with 150 mg / ml glutathione, 88 mg / ml ascorbic acid, and 84 mg / ml sodium bicarbonate, corresponding to approximately 46.6%, 27.3%, and 26.1% of the total weight, respectively, for a total weight of 322 mg / ml. The molar ratios of the components in Composition 1 were 0.49 M glutathione, 0.50 M ascorbic acid, and 1 M HCO3.

[0272] The MICs of Composition 1 (glutathione, ascorbic acid, and HCO3); glutathione and ascorbic acid; and HCO3 were tested against clinical isolates including Burkholderia cepacia, Pseudomonas aeruginosa (Schroeter) Migula (ATCC 27853), Pseudomonas aeruginosa (Schroeter) Migula (ATCC 9027), Pseudomonas aeruginosa (Schroeter) Migula (ATCC BAA-2114), Klebsiella pneumoniae, and methicillin-resistant Staphylococcus aureus; laboratory strains of Moraxella catarrhalis, Stenotrophomonas maltophilia, and Acinetobacter baumannii; and eight samples of mucoid and nonmucoid clinical isolates of Pseudomonas aeruginosa from human lung. MICs for the laboratory clinical isolate Pseudomonas aeruginosa (Schroeter) Migula (ATCC BAA-47(PAO1)) were also tested. MICs were determined under anaerobic conditions in medium supplemented with nitrate, aerobic conditions supplemented with nitrate, or under ambient conditions.

[0273] Clinical isolates were plated in duplicate on agar plates overnight under anaerobic conditions at 36°C ± 1.0°C in 96-well plates. Laboratory isolates of Burkholderia cepacia were also grown in duplicate on agar plates overnight under aerobic conditions at 36°C ± 1.0°C in 96-well plates. Colonies from each clinical isolate grown on agar plates were then selected and plated in duplicate on 96-well plates and tested under three test solution conditions: Composition 1 (glutathione, ascorbic acid, and HCO3); glutathione and ascorbic acid; and HCO3 were diluted and added to the medium. Ten dilutions of each test solution were investigated: 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57%, 0.78%, 0.39%, 0.2%, and 0.1%. Colony isolates were cultured under anaerobic conditions in medium containing Luria Agar / Broth and nitrate with one of the three test solutions at 36°C ± 1.0°C for 12 or 24 hours. In additional tests, Burkholderia cepacia and Klebsiella pneumoniae were grown in aerobic medium containing only Luria Agar / Broth with Composition 1. The MIC of Composition 1 was also determined for certain Pseudomonas aeruginosa laboratory and clinical isolates, Moraxella catarrhalis, Stenotrophomonas maltophilia, Acinetobacter baumannii, and Burkholderia cepacia, grown under aerobic conditions with 1% nitrate. MICs were determined by visual inspection and OD 620 The MICs at 12, 24, and / or 48 h for the clinical isolates tested are shown in Table 2.

[0274] [Table 2] Bc: Burkholderia cepacia Kp: Klebsiella pneumoniae Pa: Pseudomonas aeruginosa Pa UAB: Pseudomonas aeruginosa clinical isolates from the lung MRSA: Methicillin-resistant Staphylococcus aureus Mc: Moraxella catarrhalis Sm: Stenotrophomonas maltophilia Ab: Acinetobacter baumannii NM: Nonmucoid type M: Mucoid type

[0275] In vitro assays for MIC, i.e., the lowest concentration of a test substance or drug that inhibits bacterial growth, can be measured by optical density or by visualization for the absence of growth. MIC is often used to indicate the effectiveness of antibacterial drugs. Generally, a lower MIC corresponds to a more potent drug.

[0276] The results show that bicarbonate in solution reduced the efficacy of glutathione in vitro, but Composition 1 inhibited bacteria. The MICs of bicarbonate alone were measured for representative bacterial isolates to determine the contribution of bicarbonate to in vitro inhibition.

[0277] The results show that Composition 1 was clinically effective against laboratory and clinical isolates of Pseudomonas aeruginosa, Burkholderia cepacia, Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus, Moraxella catarrhalis, Stenotrophomonas maltophilia, and Acinetobacter baumannii. Notably, laboratory and clinical isolate P. aeruginosa phenotypes can differ dramatically based on the conditions in which they are maintained, their passage number, and the environment in which they were isolated. Therefore, PAO1 is not predictive of how other laboratory or clinical isolates will be inhibited.

[0278] The anaerobic and aerobic effects against B. cepacia and K. pneumoniae were also evaluated in vitro. Both of these bacteria are particularly tenacious and difficult to inhibit, even with high concentrations of antimicrobial agents. They are both associated with clinical deterioration in chronic inflammatory airway diseases. For B. cepacia, the inhibitory effect lasted for more than 48 hours.

[0279] Composition 1 was also able to effectively inhibit MRSA in vitro, which was unexpected with the high salt concentration of Composition 1, since MRSA generally thrives under high salt concentrations.

[0280] Composition 1 was also able to effectively inhibit a mucoid clinical isolate of P. aeruginosa (Pa UAB-M-4). The mucoid phenotype is associated with multidrug resistance and clinical deterioration in patients with chronic inflammatory airway diseases.

[0281] Composition 1 was also able to effectively inhibit Burkholderia cepacia, Moraxella catarrhalis, Stenotrophomonas maltophilia, and Acinetobacter baumannii under aerobic conditions containing 1% nitrate, each of which is recognized as an emerging pathogen.

[0282] Example 3: In vitro analysis of mucociliary transport rates in dF508- / - primary human bronchial epithelial cells The ability of Composition 1 (prepared in Example 1) to affect the mucociliary transport (MCT) rate of primary human bronchial epithelial cells isolated from cystic fibrosis patients was evaluated in vitro. Mucociliary transport rate provides one measure of mucociliary clearance, the mechanism by which epithelial cells clear mucus from the airways.

[0283] Briefly, primary human bronchial epithelial cells were isolated from the airways of cystic fibrosis patients and terminally differentiated. All cells isolated from cystic fibrosis patients were represented by the dF508- / - genotype. Cells were cultured with phosphate-buffered saline (PBS), high (25%) concentration of composition 1, or low (12.5%) concentration of composition 1. Mucociliary transport velocity was assessed using micro-optical coherence tomography (uOCT) according to the method described in Liu, L., et al., PLoS One. 2013; 8(1): e54473. The ratio of glutathione to ascorbic acid to bicarbonate in composition 1 was 150mg / ml glutathione:88mg / ml ascorbic acid:84mg / ml bicarbonate, which represents a molar ratio of 0.5mol glutathione:0.5mol ascorbic acid:1mol bicarbonate.

[0284] As shown in Figure 1, incubation with Composition 1 increased mucociliary transport of dF508- / - primary human bronchial epithelial cells compared to incubation with a PBS control. In addition, at all time points tested, incubation with high concentrations of Composition 1 resulted in increased mucociliary transport rates compared to incubation with low concentrations of Composition 1. These data demonstrate that the Compositions enhance mucociliary transport of primary human bronchial epithelial cells in a dose-dependent manner.

[0285] Example 4: In vitro analysis of mucociliary transport rates in dF508- / - primary human bronchial epithelial cells Mucociliary transport rates of dF508- / - primary human bronchial epithelial cells were evaluated in vitro following administration of isolated components of Composition 1 (prepared in Example 1). Primary human bronchial epithelial cells were isolated from the airways of cystic fibrosis patients as described above in Example 2, and mucociliary transport rates were evaluated using uOCT according to the methods described in Liu, L., et al., PLoS One. 2013; 8(1): e5447. Cells were cultured with Composition 1, bicarbonate, glutathione, bicarbonate + glutathione, or bicarbonate + ascorbate prior to analysis via uOCT.

[0286] As shown in Figure 2, culturing dF508- / - primary human bronchial epithelial cells with Composition 1 increased the mucociliary transport rate compared to culturing with a PBS control, a solution containing only bicarbonate, a solution containing only glutathione, or a solution containing glutathione and bicarbonate. Culturing the cells with a solution containing only glutathione, only bicarbonate, or a combination of glutathione and bicarbonate did not change the mucociliary transport rate compared to culturing with a PBS control. These data demonstrate that Composition 1 improved mucociliary transport of primary human bronchial epithelial cells isolated from cystic fibrosis patients, while a solution containing only glutathione and bicarbonate (alone or in combination) had no significant effect on the mucociliary transport rate.

[0287] Example 5: In vitro analysis of neutrophil extracellular trap (NET) formation in human neutrophils Composition 2 was prepared with 150 mg / ml glutathione, 126 mg / ml ascorbic acid, and 84 mg / ml sodium bicarbonate, corresponding to approximately 33%, 38.9%, and 28.9% of the total weight, respectively, for a total weight of 360 mg / ml. The molar ratios of the components in Composition 1 were 0.49 M glutathione, 1 M ascorbic acid, and 1 M HCO3.

[0288] The ability of Composition 2 to control the formation of neutrophil extracellular traps (NETs) from human neutrophils was evaluated in vitro. NETs are defined as the release of myeloperoxidase (MPO), DNA strands, and other proinflammatory components (e.g., neutrophil elastase) from neutrophils, and NETs contribute to inflammation and infection in chronic inflammatory airway diseases (see, e.g., Martinez-Aleman, Front Cell Infect Microbiol. 2017; 7:104).

[0289] Briefly, neutrophils were isolated from healthy volunteers and cultured with phorbol myristate acetate (PMA) (50 mM) or control solution for 3 hours. The cells were then cultured with dilutions of Composition 1 or Composition 2 and stained with DAPI (4',6-diamidino-2-phenylindole) to detect extracellular DNA release and MPO. Images of NET formation were obtained by fluorescence microscopy.

[0290] As shown in Figure 3, neutrophils cultured with PMA alone exhibited extensive NET formation, while neutrophils cultured with Composition 2 exhibited a significant reduction in NET formation. Notably, neutrophils cultured with a solution containing a 50% dilution of Composition 2 exhibited the lowest amount of NET formation after stimulation with PMA, while neutrophils cultured with increasingly dilute solutions of Composition 2 (25%, 12.5%, 6.25%, and 3.125%) exhibited less of a reduction in NET formation. These data demonstrate that Composition 2 reduced NET formation from human neutrophils in a dose-dependent manner.

[0291] Example 6: In vitro analysis of nitric oxide (NO) release from human neutrophils The release of nitric oxide (NO) from human neutrophils after administration of Composition 1 (prepared in Example 1) was evaluated in vitro. NO is released from neutrophils in chronic inflammatory airway disease and is associated with the conversion of NO to nitrate and increased pulmonary infection and inflammation by nitrate (see, e.g., Francoeur, C. and Denis, M. Inflammation (1995) 19: 587).

[0292] Briefly, neutrophils were isolated from healthy volunteers and incubated with 10 μg / ml lipopolysaccharide (LPS) or a control solution, followed by incubation with various dilutions of Composition 1. Cells were stained with DAF-FM diacetate to detect NO, and images of NO release were captured by fluorescence microscopy.

[0293] As shown in Figure 4, neutrophils cultured with LPS alone exhibited extensive NO release, while neutrophils cultured with Composition 1 exhibited a significant decrease in NO release. In particular, neutrophils cultured with a solution containing a 50% dilution of Composition 1 exhibited the lowest NO release following stimulation with LPS, while neutrophils cultured with increasingly dilute solutions of Composition 1 (25%, 12.5%, 6.25%, and 3.125%) exhibited less of a decrease in NO release. These data demonstrate that Composition 1 reduces NO release from human neutrophils in a dose-dependent manner.

[0294] Example 7: Clinical Study of Exhaled Nitric Oxide (NO) from Bilateral Lung Transplant Patients The effect of Composition 1 (prepared in Example 1) on pathological exhaled NO was evaluated in human patients in vivo. Briefly, exhaled nitric oxide (FeNO) measurements were obtained from bilateral lung transplant patients before administration of Composition 1 and immediately after administration of Composition 1 using standard techniques (see, for example, Fisher, Thorax 1998; 53:454-458; Gabbay, Am J Respir Crit Care Med 2000; 162: 2182-2187; Kharitonov, The Lancet 1994; 343 (8890): 133-135; and Hubert, European Respiratory Journal 2009 34: 117-124). The patient showed an FeNO measurement of 119 ppb before administration of Composition 1, and the patient's FeNO measurement decreased to 24 ppb immediately after administration of Composition 1. In some patients, normal levels of NO are considered to be 20-50 ppb, and NO levels above 50 ppb are associated with infection and inflammation. Accordingly, patients showed a reduction from pathological levels of NO to normal levels of NO after a single treatment with Composition 1. These results were reproducible (data not shown). These data demonstrate the acute positive impact of Composition 1 on reducing exhaled NO levels in lung transplant patients.

[0295] Example 8: In vitro inflammatory cytokine analysis of human neutrophils Inflammatory cytokine assays were performed in vitro with human neutrophils after administration of dilutions of Composition 1. Briefly, 4×10 6 Adjust the PMN concentration to a density of 1 × 10 / ml and aliquot 0.25 ml of the resulting solution into a 24-well plate (1 × 10 6 PMN / 2-cm 2The addition of 0.25 ml of 3.125%, 6.25%, 12.5%, 25%, and 50% dilutions of 2× dose of Composition 1 (88 mg / ml vitamin C) was repeated twice. Cells were then pretreated with 0.25 ml of 3.125%, 6.25%, 12.5%, 25%, and 50% dilutions of 2× dose of Composition 1 (88 mg / ml vitamin C) for 1 hour at 37° C. Half of the wells were then added with 5 μl / well of 100× E. coli O111:B4 LPS (Sigma L4391) and incubated for 4 hours at 37° C. Conditioned media was collected from the wells and frozen for batch cytokine analysis. Quantification of human IL-6, human IL-8 / CXCL8, and human TNF-α in the conditioned media was performed under standard conditions using R&D Systems Quantikine ELISA kits.

[0296] As shown in Table 3 and Figures 5A-5C, human neutrophils stimulated with LPS and cultured with dilutions of Composition 1 secreted significantly less proinflammatory cytokines compared to those cultured with the control (medium only). In particular, neutrophils stimulated with LPS and cultured with medium only exhibited a TNF-α concentration of 23.8 pg / ml, while neutrophils cultured with 12.5% ​​Composition 1, 25% Composition 1, or 50% Composition 1 did not produce any detectable TNF-α (Table 3 and Figure 5A). Neutrophils cultured with 6.25% Composition 1 exhibited a TNF-α concentration of 11.7 pg / ml, approximately half that of neutrophils cultured with medium only (Table 3 and Figure 5A). Similarly, neutrophils stimulated with LPS and cultured with 50% composition 1, 25% composition 1, or 12.5% ​​composition 1 did not produce any detectable levels of IL-6, while neutrophils cultured with 6.25% composition 1 exhibited approximately 6-fold reduced IL-6 levels compared to neutrophils cultured with media alone (Table 3 and Figure 5B). Furthermore, neutrophils stimulated with LPS and cultured with 50% composition 1, 25% composition 1, or 12.5% ​​composition 1 did not produce any detectable levels of IL-8, while neutrophils cultured with 6.25% composition 1 exhibited approximately 3-fold reduced IL-8 levels compared to neutrophils cultured with media alone (Table 3 and Figure 5C). These data demonstrate that administration of composition 1 results in dose-dependent downregulation of proinflammatory cytokines involved in chronic inflammatory diseases such as cystic fibrosis, lung transplantation, and chronic obstructive pulmonary disease.

[0297] [Table 3]

[0298] Example 9: Cystic Fibrosis Transmembrane Conductance Receptor (CFTR) Functional Assay As shown in FIG. 6, Composition 1 (prepared in Example 1) reduced Isc (μA / cm) in wild-type cystic fibrosis bronchial epithelial cells in a cystic fibrosis transmembrane conductance receptor (CFTR) assay. 2 ) to improve.

[0299] Example 10: In vitro analysis of MCT rate, ASL, and CBF in dF508- / - primary human bronchial epithelial cells MCT rates were assessed in a mucociliary clearance assay according to the method in Example 3 above. Images from a video showing the functional microanatomical effects of Composition 1 on primary human bronchial epithelial cells isolated from cystic fibrosis patients (dF508- / - cells) at 0 and 6 hours post-administration are shown in Figure 7. These results showed that Composition 1 improved MCT rates compared to the PBS control at 6 hours post-administration.

[0300] Mucus viscosity is the primary mechanism in increasing MCT rather than increasing airway surface liquid (ASL) and ciliary beat frequency (CBF) (Liu et al., Am J Prespir Cell Mol Biol. 2014, 51(4):485-93). Slope analysis of MCT against airway surface liquid (ASL) and ciliary beat frequency (CBF) 6 hours after administration of Composition 1 compared to PBS in primary human bronchial epithelial cells (dF508- / - cells) isolated from cystic fibrosis patients is shown in Figure 8A and Figure 8B, respectively. Composition 1 improved MCT and functionally and proportionally increased ASL and CBF. These results indicate that Composition 1 can restore dF508 CF human bronchial epithelial (HBE) cells to normal MCT levels without increasing ASL and CBF to abnormal levels that may interfere with MCT function by oversaturating the mucus layer or by causing rapid but overly irregular ciliary beat patterns that impede mucus movement. These results support that Composition 1 has an effect on MCT by reducing mucus viscosity.

[0301] Example 11: Composition 1 reduces MMP-9 activity released from activated neutrophils Metalloprotease-9 (MMP-9) release from LPS-stimulated neutrophils was evaluated after administration of Composition 1 at dilutions of 1:100 and 1:10. Peripheral neutrophils were isolated from blood drawn from healthy donors. Neutrophils were treated with one of two dilutions of Composition 1 (1:10 or 1:100) or with medium (Med) alone for 1 hour. They were then stimulated with 10 or 100 μg of LPS for 1 hour. Supernatants from the cultures were collected and evaluated via MMP-9 zymograms.

[0302] Figure 9 shows the release of metalloprotease-9 (MMP-9) from LPS-stimulated neutrophils after 1 hour treatment with Composition 1 (Ar) at dilutions of 1:100 and 1:10 compared to media (Med) and LPS only controls. MMP-9 is a 75-100 kDa band on the gel.

[0303] Matrix metalloproteinases (MMPs) are released during neutrophil extracellular trap formation, i.e., degranulation, and are associated with the deterioration of chronic inflammatory airway diseases by destroying the airway epithelium. MMP-9 is released from LPS-stimulated neutrophils and is associated with clinical deterioration in cystic fibrosis patients (Gaggar et al., Eur Respir J., 2011 38(3):721-727).

[0304] Composition 1 significantly reduces the release of metalloprotease 9 from LPS-stimulated neutrophils. At a dilution of 1:100, MMP-9 expression induced by LPS100 is reduced by approximately 50%. At a dilution of 1:10, the same stimulation is reduced by approximately 75%.

[0305] These results demonstrate that Composition 1 reduces neutrophil release of MMP-9 in vitro and acts as an anti-inflammatory agent.

[0306] Example 12: Composition 1 reduces neutrophil- and macrophage-associated cytokines MIP1α and MIP1β Peripheral neutrophils were isolated from blood collected from healthy donors. Neutrophils were treated with one of two dilutions of Composition 1 (1:10 or 1:100) or with medium alone for 1 hour. They were then stimulated with 10 or 100 μg LPS for 1 hour. Supernatants from the cultures were collected and evaluated via multiplex analysis at 15 minutes and 1 hour after stimulation.

[0307] Neutrophil and Macrophage Associated Cytokines: Macrophage inflammatory protein 1 alpha (MIP1α) (FIG. 10A) and macrophage inflammatory protein 1 beta (MIP1β) (FIG. 10B) release levels from LPS-activated neutrophils following administration of Composition 1 at dilutions of 1:100 and 1:10 were reduced at 1 hour post-stimulation compared to media (M) and LPS only controls.

[0308] Example 13: Combination of Composition 1 and VX-770 / 809 The effect of the combination of composition 1 and VX-770 / 809 on MCT rate, ASL, and CBF was tested. VX-770 / 809 is a CFTR-specific modulator / potentiator / corrector that allows the expression and gating of cystic fibrosis transmembrane receptor (CFTR) on the surface of dF508 HBE. VX-770, also known as ivacaftor, is a CFTR potentiator. VX-809, also known as lumacaftor, is a CFTR corrector. However, the use of VX-770 / 809 alone does not alleviate all respiratory symptoms.

[0309] MCT rates were evaluated in a mucociliary clearance assay according to the method in Example 3 above. Images from a video showing the functional microanatomical effects of Composition 1 in combination with VX-770 / 809 compared to VX-770 / 809 alone and PBS controls on primary human bronchial epithelial cells isolated from cystic fibrosis patients (dF508- / - cells) are shown in Figure 11. Results from VX-770-809 treated cells showed that, despite a slight increase in MCT, a mucus bilayer formed, preventing adequate MCT operation (Figure 11, center panel). Combining Composition 1 with VX-770 / 809 not only resulted in a significant increase in MCT, but also disruption of the mucus bilayer, allowing better operation of MCT compared to VX-770 / 809 alone.

[0310] These results demonstrated that the combination of Composition 1 with VX-770 / 809 improved the MCT rate compared to the PBS control and reduced the mucus bilayer associated with the use of VX-770 / 809 in the mucociliary clearance assay.

[0311] Quantification of ASL, CBF and MCT changes by composition 1 alone, VX-770 / 809 and the combination of the two on dF508 CF HBE cells is shown in Figure 12A. The combination of composition 1 and VX-770 / 809 produces the most significant increase in MCT, CBF and ASL. These data demonstrate the improving effect of composition 1 when used in combination with CFTR VX-770 / 809.

[0312] As shown in FIG. 12B, incubation of Composition 1 or Composition 1 with VX-770 and VX-809 increased mucociliary transport of dF508- / - primary human bronchial epithelial cells compared to incubation with PBS control or compared to VX-770 and VX-809 alone. These data demonstrate that Composition 1 enhances mucociliary transport of primary human bronchial epithelial cells in a dose-dependent manner. Results represent pooled data obtained from 20 samples of HBE monolayers across three CF donors homozygous for F508del.

[0313] Example 14: Clinical study of administration of Composition 1 to bilateral lung transplant patients The effect of Composition 1 (prepared in Example 1) in bilateral lung transplant patients on pulmonary function (assessed by change in FEV1), change in exhaled nitric oxide (FeNO), and change in quality of life were evaluated. Briefly, Composition 1 (4 ml) was nebulized twice daily to four lung transplant patients using a PARI eFlow nebulizer.

[0314] Changes in lung function for each patient were assessed using the linear slope of change in FEV1 at enrollment (baseline) and after one month. Lung transplant patients had increases in clinic and home spirometry after one month of using Composition 1. This change correlated with a positive change in quality of life as measured by the St. George's Respiratory Questionnaire. These lung function results after one month are shown in Figure 13.

[0315] The change in FENO for each patient was assessed using the change in the mean baseline FENO measurement relative to the mean measurement after one month of using Composition 1. After one month of using Composition 1, patients had a decrease in FENO measurements. A decrease in FENO from baseline represents the resolution of inflammation / infection in post-lung transplant patients (see, e.g., Fisher, A., Thorax 1998;53:454-458). This change correlated with a positive change in quality of life as measured by the St. George's Respiratory Questionnaire. These FENO results after one month are shown in Figure 14.

[0316] Example 15: Storage stability of Composition 1 The effect of storage on ascorbic acid compositions with various amounts of buffer at a constant glutathione concentration was tested. For preparation of formulations 1-9 (formulation 9 corresponds to composition 1 (shown in Example 1)), the solutions were mixed with sterile water for injection and stored in a closed container protected from light. During testing, formulations 1-9 were evaluated to determine ascorbic acid and reduced and oxidized glutathione concentrations. A standard RP-HPLC method was used for these analytes. The method was validated to include specificity, precision, reproducibility, LOQ, mini linearity, in-vial NPLC sample stability, and stability-indicating check with informal forced degradation samples. The pH of each formulation was determined using a pH probe placed in a sample of each solution. The results are shown in Figure 15. Ascorbic acid (ACS) alone was unstable under the conditions tested (-20°C, 5°C, ambient) (data not shown).

[0317] Formulation stability is important for clinical applications to ensure consistent product and efficacy over time. Acidic conditions are present in and contribute to chronic inflammatory airway diseases (Morice, Breathe 2013 9: 256-266; Tang et al., J Clin Invest. 2016;126(3):879-891; Tate et al., Thorax 2002;57:926-929). Furthermore, innate immune functions such as adequate mucus clearance, optimal mucus viscosity, and innate bactericidal activity are optimal at a slightly acidic pH of 6-7. Thus, acidic conditions can impair immune function and cause airway epithelial death and dysfunction.

[0318] After about 72 hours of storage at about 2-8° C., only Composition 1 (Formulation 9) achieved good solubility without detectable precipitate formation and maintained a pH of about 6-7. Furthermore, impurities were less than 4% for Composition 1, i.e., 3.8%, after about 72 hours of storage at about 2-8° C.

[0319] The present invention has been described above using functional components to illustrate the implementation of specified functions and their relationships. The boundaries of these functional components are arbitrarily defined in this specification for the convenience of description. Alternative boundaries may be defined as long as the specified functions and their relationships are appropriately implemented.

[0320] The above description of the specific embodiments will sufficiently clarify the basic nature of the invention so that others may easily modify and / or adapt such specific embodiments for various applications without departing from the basic concept of the invention by applying knowledge within the scope of the art without undue experimentation. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the teaching and guidance provided herein. It should be understood that the expressions or terms in this specification are for the purpose of explanation and not limitation, as the terms or terms in this specification will be interpreted by those skilled in the art in light of the teaching and guidance.

[0321] The scope and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. (a) glutathione, or a pharma- ceutically acceptable salt thereof; (b) ascorbic acid; and (c) Bicarbonate wherein the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:

1.

2. 2. The pharmaceutical composition of claim 1, wherein the bicarbonate is sodium bicarbonate.

3. 2. The pharmaceutical composition of claim 1, wherein the amount of bicarbonate is about 1-30% by weight.

4. 2. The pharmaceutical composition of claim 1, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5-1:

1.

5. 2. The pharmaceutical composition of claim 1, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5:1 or about 0.4-0.5:1:

1.

6. 10. The pharmaceutical composition of claim 1, comprising about 0.49 M glutathione, about 0.50 M ascorbic acid, and about 1 M sodium bicarbonate.

7. 10. The pharmaceutical composition of claim 1, wherein the pH of the composition is about 5.5 to about 10, about 5.5 to about 8, about 6 to about 10, or about 6 to about 8.

8. 10. The pharmaceutical composition of claim 1, which is an aqueous solution.

9. 10. The pharmaceutical composition of claim 1, which is a dry powder.

10. 13. The pharmaceutical composition of claim 1, which is administered by inhalation.

11. 10. The pharmaceutical composition of claim 1, which reduces airway inflammation in a subject suffering from a viral infection of the lungs or airways.

12. (a) glutathione, or a pharma- ceutically acceptable salt thereof; (b) ascorbic acid; and (c) Bicarbonate wherein the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:

1.

13. 13. The pharmaceutical composition of claim 12, wherein the bicarbonate salt is sodium bicarbonate.

14. 13. The pharmaceutical composition of claim 12, wherein the amount of bicarbonate is about 1-30% by weight.

15. 13. The pharmaceutical composition of claim 12, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5-1:

1.

16. 13. The pharmaceutical composition of claim 12, wherein the molar ratio of (a):(b):(c) is about 0.4-0.5:0.5:1 or about 0.4-0.5:1:

1.

17. 13. The pharmaceutical composition of claim 12, comprising about 0.49 M glutathione, about 0.50 M ascorbic acid, and about 1 M sodium bicarbonate.

18. 13. The pharmaceutical composition of claim 12, wherein the pH of the composition is about 5.5 to about 10, about 5.5 to about 8, about 6 to about 10, or about 6 to about 8.

19. 13. The pharmaceutical composition of claim 12, which is an aqueous solution.

20. 13. The pharmaceutical composition of claim 12, which is a dry powder.

21. 13. The pharmaceutical composition of claim 12, which is administered by inhalation.

22. 13. The pharmaceutical composition of claim 12, which reduces mucus viscosity in the subject.

23. The pharmaceutical composition of claim 12, which increases the ciliary beat frequency of airway epithelial cells of the subject.

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