RU2799046C2 - Stable compositions of ascorbic acid and methods of their use - Google Patents

Abstract

FIELD: medicine, pharmaceuticals and therapy.

SUBSTANCE: group of inventions can be used in the treatment of respiratory diseases. A composition suitable for administration by inhalation comprises (a) glutathione or a pharmaceutically acceptable salt thereof; (b) ascorbic acid; and (c) a bicarbonate salt. The molar ratio (a):(b):(c) is approximately 0.1–0.5:0.5–1:1. Also a method of inhibiting or reducing the growth of clinical isolate bacteria or biofilm of clinical isolate bacteria; a method of treating or alleviating symptoms in a subject suffering from or at risk of infection with the bacteria of the clinical isolate; a method of enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance; a method of reducing airway inflammation in a subject suffering from or at risk of airway inflammation; a method for reducing mucus viscosity in a subject suffering from or at risk of impaired mucociliary clearance; a method for increasing the expression of a cystic fibrosis transmembrane receptor (CFTR) in a subject suffering from reduced expression of CFTR, the said methods comprising administering the said composition to the subject are provided.

EFFECT: increasing the effectiveness of the treatment of respiratory diseases ensured due to the synergistic effect between glutathione, ascorbic acid and bicarbonate.

35 cl, 15 dwg, 3 tbl, 15 ex

Description

Cross-reference to related applications

[0001] This application claims priority from U.S. Provisional Application No. 62/588,300, filed Nov. 17, 2017, and U.S. Provisional Application No. 62/684,700, filed June 13, 2018, the contents of which are incorporated herein by reference in their entirety.

Technical field

[0002] The present invention relates to compositions containing an organic acid, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, and their use.

State of the art

[0003] Glutathione or "GSH" refers to a compound of the formula:

,

,

or its zwitterionic form, for example, a compound of the formula:

.

.

[0004] In healthy individuals, high concentrations of GSH are present in the airways. However, in individuals with chronic inflammatory airway disease, such as lung transplant patients, glutathione stores are depleted. Both inflammation and infection are associated with lung transplant rejection/dysfunction and failure. Current lung transplant therapy, which includes high doses of immunosuppressants and antimicrobials, has systemic side effects and toxicity that can lead to further inflammation and infections. In addition, repeated use of high doses of antibiotics has been shown to lead to multidrug-resistant infections.

[0005] Lower respiratory tract infections due to chronic inflammatory airway diseases are present in multiple disorders and diseases of the respiratory tract, including, for example, patients with lung transplantation and patients with cystic fibrosis (CF) and bronchiectasis (for example, bronchiectasis caused by a disease other than cystic fibrosis , or bronchiectasis caused by cystic fibrosis).

Brief description of the invention

[0006] The present invention provides compositions comprising: (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically 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 and the pH of the composition 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 (eg, sodium bicarbonate). In some embodiments, the composition does not include 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 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%, based on the total weight of glutathione in the composition after storage of the composition for 4 weeks at about 5°C.

[0009] In some embodiments, 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%, based on the total weight of glutathione in the composition after storage of the composition for 4 weeks (e.g., at 5°C in an atmosphere N ₂ and/or in ambient atmosphere).

[0010] In some embodiments, the 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%, based on total weight. ascorbic acid in the composition after storage of the composition for 4 weeks at about 5°C.

[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%, based on the total weight of ascorbic acid in the composition after storage of the composition for 4 weeks at about 5°C.

[0012] In some embodiments, the composition is stored under ambient conditions, such as without nitrogen sparging. In some embodiments, the composition is stored using nitrogen sparging.

[0013] In some embodiments, the pH of the composition is from about 5.5 to about 10, from about 5.5 to about 8, from about 6 to about 10, or from 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 lyophilized powder.

[0015] Certain aspects of the invention relate to a method for inhibiting or reducing bacterial growth of a clinical isolate, comprising contacting the clinical isolate with a composition of the present invention.

[0016] Certain aspects of the invention relate to a method for inhibiting or reducing bacterial biofilm formation of a clinical isolate, comprising contacting the clinical isolate with a composition comprising a composition of the present invention.

[0017] Certain aspects of the invention relate to a method of treating or alleviating symptoms in a subject suffering from or at risk of infection with the bacteria of a clinical isolate, comprising contacting the clinical isolate with a composition of the present invention.

[0018] In some embodiments, the subject has a disease or disorder of the lungs or airways. In some embodiments, the lung or airway disease or disorder is bronchiectasis caused by cystic fibrosis or a disease other than cystic fibrosis.

[0019] Certain aspects of the invention relate to a method of enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering to the subject a composition according to the present invention. In some embodiments, administration of the composition reduces the viscosity of the patient's mucus. In some embodiments, administering the composition increases the ciliary beat frequency of the patient's airway epithelial cells. In some embodiments, the implementation of the introduction of the composition increases the rate of mucociliary transport of epithelial cells of the respiratory tract of the patient. In some embodiments, the implementation of the specified composition increases the height of the liquid covering the surface of the respiratory tract of the patient.

[0020] In some embodiments, the epithelium of the patient's respiratory tract is not colonized by bacteria. In some embodiments, the respiratory tract epithelium of the patient is colonized with bacteria. In some embodiments, the patient is suffering from an active bacterial infection. In some embodiments, the patient is not suffering from an active bacterial infection.

[0021] Certain aspects of the invention relate to a method of reducing airway inflammation in a subject suffering from or at risk of airway inflammation, comprising administering to the subject a composition according to the present invention.

[0022] In some embodiments, administration of said composition inhibits the myeloperoxidase activity of the patient's neutrophils. In some embodiments, the implementation of the specified composition reduces the formation of neutrophil extracellular traps. In some embodiments, the administration of said composition suppresses the production of nitric oxide (II) by the patient's neutrophils. In some embodiments, administration of said composition reduces the exhaled nitric oxide (II) fraction (FeNO) of the patient by at least 20%. In some embodiments, the composition is administered twice a day. In some embodiments, the composition is administered via a nebulizer. In some embodiments, administration of the composition reduces the patient's exhaled nitric oxide (II) fraction (FeNO) by at least 20% after 1 month of administration (eg, twice daily) of the composition.

[0023] In some embodiments, the implementation of the specified composition suppresses the production of at least one pro-inflammatory cytokine (for example, TNF-α, IL-6 and/or IL-8) and/or at least one cytokine associated with neutrophils and macrophages ( e.g. MIP1α and/or MIP1β).

[0024] Certain aspects of the invention relate to a method for reducing the viscosity of mucus in a subject suffering from or at risk of reducing the viscosity of mucus, comprising administering to the subject a composition according to the present invention.

[0025] Some aspects of the invention relate to a method for activating cystic fibrosis transmembrane receptor (CFTR) function in a subject suffering from reduced CFTR function, comprising administering to the subject a composition of the present invention.

[0026] Certain aspects of the invention relate to a method for increasing expression of the cystic fibrosis transmembrane receptor (CFTR) in a subject suffering from reduced expression of CFTR, comprising administering to the subject a composition of the present invention.

[0027] Some aspects of the invention relate to a method comprising administering a composition of the present invention 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 combinations thereof), CFTR potentiator/modulator (e.g. VX-770 (ivacaftor), QBW 251, VX- 561, PT1-808, or combinations thereof), a CFTR RNA modifier (eg, QR-100, MRT5005, or combinations thereof), or any combination thereof. In some embodiments, administration of CFTR therapy occurs simultaneously or sequentially with administration of a composition of the present invention to a subject and in any order (eg, the composition may be administered before or after CFTR therapy). In some embodiments, the CFTR therapy is administered orally and the composition of the present invention is administered via inhalation. In some embodiments, both the cystic fibrosis therapy and the composition of the present invention are administered via inhalation.

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

[0029] In some embodiments, administering a composition of the present invention in combination with administering to a subject VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770/VX-809 reduces the viscosity of mucus in the subject's airways to a greater extent, than administering the pure composition of the present invention or pure VX-770 (ivacaftor), pure VX-809 (lumacaftor), or pure VX-770/VX-809 combination.

[0030] In some embodiments, administering a composition of the present invention in combination with administering to a subject VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770/VX-809 increases the ciliary beat frequency of airway epithelial cells in the subject in to a greater extent than the introduction of a pure composition.

[0031] In some embodiments, administering a composition of the present invention in combination with administering to a subject VX-770 (ivacaftor), VX-809 (lumacaftor), or a combination of VX-770/VX-809 increases the rate of mucociliary transport of airway epithelial cells in the subject into to a greater extent than the introduction of a pure composition.

[0032] In some embodiments, administering a composition of the present application in combination with administering to a subject VX-770, VX-809, or a combination of VX-770/VX-809 increases the height of the fluid covering the surface of the subject's respiratory tract to a greater extent than administering pure compositions.

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

[0034] In some embodiments, the composition is stable at 2-8°C for at least 72 hours. In some embodiments, the composition, after storage at 2-8° C. for at least 72 hours, is any one or more of the following: (a) substantially free of sediment, (b) contains less than 4% impurities, (c) has pH 6.0-7.0 and (d) has minimal loss of solubility.

Brief description of drawings/figures

[0035] FIG. 1 shows the rate of mucociliary transport of primary human bronchial epithelial cells (df508-/-) isolated from the respiratory tract of patients with cystic fibrosis after administration of Composition 1.

[0036] FIG. 2 shows the rate of mucociliary transport of primary human bronchial epithelial cells (df508-/-) isolated from the respiratory tract of patients with cystic fibrosis after administration of Composition 1, bicarbonate, glutathione, bicarbonate + glutathione, or bicarbonate + ascorbate.

[0037] FIG. 3 shows staining of netized human neutrophils isolated from healthy volunteers after administration of phorbol myristate acetate (PMA) and Composition 2.

[0038] FIG. 4 shows DAF-FM staining with diacetate to detect nitric oxide in human neutrophils after administration of lipopolysaccharide (LPS) and Composition 1.

[0039] FIG. 5A-5C show the concentration of pro-inflammatory cytokines TNF-α (FIG. 5A), IL-6 (FIG. 5B) and IL-8 (FIG. 5C) secreted by human neutrophils after administration of lipopolysaccharide (LPS) and Composition 1.

[0040] FIG. 6 shows a cystic fibrosis transmembrane receptor (CFTR) functional assay performed using wild-type bronchial epithelial cells in cystic fibrosis (WT CFBE).

[0041] FIG. 7 is a frame from the video showing the effect of Composition 1 (right panels) versus PBS (left panels) on the in vitro mucociliary transport (MCT) rate of human primary bronchial epithelial cells isolated from cystic fibrosis patients (dF508 -/- cells) via 0 hours (top) and 6 hours (bottom) after administration.

[0042] FIG. 8A-8B show analysis of MCT slope with respect to airway surface fluid (ASL) height (FIG. 8A) and ciliary beat frequency (CBF) (FIG. 8B) 6 hours after Composition 1 administration compared to PBS in primary epithelial cells. human bronchial cells isolated from patients with cystic fibrosis (dF508 -/- cells). This correlation analysis indicates that the viscosity of the mucus decreases with the use of Composition 1.

[0043] FIG. 9 shows the release of metalloprotease-9 (MMP-9) from LPS-stimulated neutrophils after one hour of treatment with Composition 1 (Ar) at 1:100 and 1:10 dilutions compared to a control of pure medium (Med) and LPS. MMP-9 is a 75-100 kDa band on the gel.

[0044] FIG. 10A-10B show the levels of release of neutrophil and macrophage associated cytokines: macrophage inflammatory protein 1α (MIP1α) (FIG. 10A) and macrophage inflammatory protein 1β (MIP1β) (FIG. 10B) from LPS-activated neutrophils after administration of Composition 1 ( Ar) in dilutions of 1:100 and 1:10 compared to the control, which is pure medium (M) and LPS. #ANOVA < 0.01 over 1 hour. *p < 0.05 LPSv. AR 1:10 + LPS 100 via Tukey's posterior test.

[0045] FIG. 11 shows the effect of the combination of Composition 1 and VX-770/809 (COMP 1 + VX-770/809, right panel) compared to PBS (left panel) and pure VX-770/809 (center panel) on the rate of mucociliary transport (MCT). ) in vitro primary human bronchial epithelial cells isolated from patients with cystic fibrosis (dF508 -/- cells).

[0046] FIG. 12A shows quantification of change in airway surface fluid (ASL), ciliary beat frequency (CBF) and mucociliary transport rate (MCT) over time in dF508 CF HBE cells following administration of the combination of Composition 1 and VX-770/809 (COMP 1 + VX-770/809), pure Composition 1 (COMP 1), pure VX-770/809 or PBS control. The results show pooled data (N = 20 HBE monolayers in 3 CF donors homozygous for F508del). **P<0.01, P<0.001, ****P<0.0001.

[0047] FIG. 12B shows the quantitative assessment of the change in mucociliary transport rate from baseline compared to PBS (control) with Composition 1, VX-770/809 and the combination of Composition 1 with VX-770/809. The results show pooled data (N = 20 HBE monolayers in 3 CF donors homozygous for F508del). Mean ± SEM **** P<0.0001.

[0048] FIG. 13 shows the mean percentage change in spirometric measurements in lung transplant patients who were nebulized with Composition 1 for one month twice daily. Spirometry measurements include forced expiratory volume exhaled in one second (FEV1), forced vital capacity (volume) (FVC), and an index of small to medium airway function (FEF25-75%). These results represent the results after one month of treatment.

[0049] FIG. 14 shows the percent change in exhaled nitrogen fraction (FENO) in 4 lung transplant patients who were nebulized with Composition 1 for one month twice daily. CF, ILD, and A1AT are among the patients' underlying diseases (the etiology of the reason for transplantation). These results represent the results after one month of treatment.

[0050] FIG. 15 shows stability data after storage for about 72 hours at 2-8°C for Formulations 1-9 having low, medium or high concentrations of ascorbic acid and/or buffer. Glutathione (GSH) was at the same concentration in each formulation. Composition 1 is shown as Composition 9.

Detailed description of the invention

Definitions

[0051] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0052] In the present invention and the claims, singular and definite articles include the plural unless the context clearly dictates otherwise.

[0053] The term "and/or" used in a phrase such as "A and/or B" is intended to include both "A and B" and "A or B", "A" and "B". Similarly, the term "and/or" used in a phrase such as "A, B and/or C" covers each of the following implementations: 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 (pure); B (pure); and C (pure).

[0054] As used herein, the term "about" means approximately ±10%. When the term "about" is used in conjunction with a numeric value or range, it modifies that value or range by extending the boundaries above and below the specified numeric values. Generally, the term "about" is used in this document to change a numerical value above and below the stated value by a 10 percent deviation, up or down (above or below), i.e. ± 10%, unless another deviation is specified (for example, ± 30 %, ±20%, ±5%, ±1%, etc.).

[0055] The term "clinical isolate bacteria" as used herein refers to a bacterial strain that has been isolated from a human or from a tissue sample taken from a human.

[0056] The term "minimum inhibitory concentration" or "MIC" as used herein refers to the lowest concentration of an agent (eg, antibiotic and/or composition of the present application) that will inhibit apparent bacterial growth.

[0057] The term "minimum bactericidal concentration" or "MBC" as used herein refers to the lowest concentration at which an agent (eg, antibiotic and/or composition of the present application) kills bacteria.

[0058] As used herein, the term "minimum biofilm kill concentration" or "MBEC" refers to the lowest concentration of an agent (eg, antibiotic and/or composition of the present application) that will inhibit visible biofilm growth.

[0059] "Pharmaceutical acceptable" as used herein means safe and effective for use in humans. For example, "pharmaceutically acceptable salt" as used herein refers to those salts of the compounds disclosed herein that are safe and effective for use in a subject and that have the desired biological activity of the compound.

[0060] "Biofilm", as used herein, refers to a group of microorganisms, eg, clinical isolate bacteria, in which microbial cells adhere to each other, and often these cells adhere to a surface. In some embodiments, these adherent cells are included in a self-replicating matrix of an extracellular polymeric substance (EPS). In some embodiments, the biofilm contains a single species of bacteria. In other embodiments, the biofilm is a mixture of two or more bacterial species.

[0061] "Mucid bacteria" as used herein refers to alginate-producing bacteria.

[0062] "Non-mucilaginous bacteria" as used herein refers to bacteria that do not produce alginate.

[0063] "Aerobic" as used herein refers to an organism, such as bacteria, that can survive and grow in an oxygen-containing environment.

[0064] The term "anaerobic" or "anaerobic" as used herein refers to an organism, such as bacteria, that does not require oxygen for growth. In some embodiments, the organism is "obligate anaerobe", meaning that it is harmed by the presence of oxygen; "aerotolerant organism", meaning that it cannot use oxygen for growth, but is tolerant of its presence; or "facultative anaerobe", meaning that it can grow without oxygen, but will use oxygen if present.

[0065] "Extracellular" as used herein means outside of a cell.

[0066] "Antibiotic resistant" refers to bacteria that have a mechanism that ensures that the antibiotic is not effective in killing the bacteria. Examples of mechanisms include, for example, degradation of the antibiotic, modification of the target antibiotic, and limited entry and/or clearance of the antibiotic. In some embodiments, bacteria become resistant to antibiotics due to a mutation.

[0067] The term "multidrug resistance" (also referred to as "multidrug resistance", "MDR", "multiple resistance") in the present invention refers to antimicrobial resistance exhibited by a microorganism species, e.g., a bacterial species, to multiple antimicrobial medicines, such as antibiotics.

[0068] "Antibiotic tolerance" refers to specialized surviving (or persistent) cells in a population of bacterial cells that are phenotypic variants (not mutants) that are non-growing cells at rest. Antibiotics kill persistent cells very slowly if they do, and cells resume growth when antibiotic concentrations decrease or antibiotic exposure is stopped.

[0069] The term "sensitized" as used herein refers to the susceptibility of a microorganism, such as a bacterium, to an antimicrobial drug, such as an antibiotic.

[0070] "Synergistic effect" as used herein means an effect that occurs between two or more therapeutic agents, for example, a composition described herein, and an antibiotic, which provides an effect that is greater than the sum of the individual effects of the two or more therapeutic agents.

[0071] "Inhibiting" as used herein means blocking or stopping, for example, stopping the growth of bacteria.

[0072] The term "reduction" as used herein refers to a decrease or decrease in the amount, for example, a decrease in the amount of bacterial growth (eg, compared to a starting point or when two or more groups are compared).

[0073] The term "treat" or "treatment" as used herein refers to the partial or complete relief, elimination, amelioration, alleviation, delay of onset, inhibition of progression, reduction in severity and/or reduction in the incidence of one or more manifestations, symptoms, or signs of a disease .

[0074] By "subject" or "patient" is meant any subject, in particular a mammal, for which diagnosis, prognosis, or therapy is required. In certain embodiments, the mammal is a human. In other embodiments, the subject is a human patient. In a particular embodiment, the subject is a human patient in need of treatment.

[0075] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (parallel) or sequential administration in any order.

[0076] Combination therapy can provide "synergism" and show a "synergistic" effect, that is, when the effect achieved when the active ingredients are used together is greater than the sum of the effects resulting from the use of the compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in the form of a combination dosage formulation; (2) delivered serially, alternately or in parallel in the form of separate formulations; or (3) some other scheme. When delivered in the case of alternative therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example, through different injections in separate syringes or by inhalation of one drug and oral administration of a second drug, or vice versa.

[0077] The term "pharmaceutical composition" refers to a medicinal product that is in such a form as to provide the biological activity of the active ingredient or active ingredients, and which does not contain additional components that have unacceptable toxicity to the subject to whom the composition will be administered. The composition may be sterile.

[0078] An "effective amount" of a composition or active agent, as described herein, is an amount sufficient to achieve a particular stated objective. An "effective amount" may be determined empirically and in a routine manner, depending on the stated purpose.

[0079] The term "therapeutically effective amount" refers to an amount of a composition or active agent, as described herein, effective to "treat" a disease or disorder in a subject.

Compositions containing glutathione and glutathione conjugate

[0080] In certain aspects, the compositions of the present application include glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof.

[0081] In some embodiments, the composition contains glutathione or a pharmaceutically acceptable salt thereof.

[0082] Glutathione or "GSH" refers to a compound of Formula A:

[0083] or its zwitterionic form, such as a compound of Formula B:

[0084] Glutathione is the most abundant non-protein thiol in mammalian cells. It plays a role in the detoxification of xenobiotic compounds and in the oxidation of reactive oxygen species and free radicals. See, for example, Bray and Taylor, Canadian Journal of Physiology and Pharmacology 71:746-751 (1993).

[0085] In healthy individuals, high concentrations of GSH are present in the airways. However, in individuals with chronic inflammatory airway disease, such as lung transplant patients, glutathione stores are depleted.

[0086] In some embodiments, the composition comprises a glutathione-containing conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the glutathione-containing conjugate is metabolized to release glutathione or a derivative thereof upon administration to a subject. In some embodiments, the glutathione-containing conjugate is referred to herein as the "conjugate of the present invention" or "glutathione conjugate".

[0087] In one embodiment, the conjugate compound of the present invention is a compound of Formula I:

I,

I ,

[0088] and pharmaceutically acceptable salts and solvates thereof, wherein

[0089] A ¹ is -OR ¹ ; A ² is Z ¹ ; A ³ is hydrogen; and A ⁴ is R ^3a ; or

[0090] A ¹ is Z ¹ ; A ² is -OR ² ; and A ³ is hydrogen; and A ⁴ is R ^3a ; or

[0091] A ¹ is -OR ¹ ; A ² is -OR ² ; and A ³ is Z ³ ; and A ⁴ is R ^3a ; or

[0092] A ¹ is Z ² ; A ² is -OR ² ; and A ³ is hydrogen; and A ⁴ is R ^3a ; or

[0093] A ¹ is -OR ¹ ; A ² is Z ² ; and A ³ is hydrogen; and A ⁴ is R ^3a ; or

[0094] A ¹ is -OR ¹ ; A ² is -OR ² ; A ³ is hydrogen; and A ⁴ is Z ³ ; or

[0095] A ¹ and A ² are each Z ¹ and A ³ is hydrogen;

[0096] Z ¹ is selected from the group consisting of:

и

;

And

;

[0097] Z ² is selected from the group consisting of:

,

и

;

,

And

;

[0098] Z ³ is selected from the group consisting of:

и

;

And

;

[0099] R ¹ is selected from the group consisting of hydrogen and optionally substituted alkyl;

[0100] R ² is selected from the group consisting of hydrogen and optionally substituted alkyl;

[0101] Each of R ^3a , R ^3b and R ^3c is independently selected from the group consisting of hydrogen and a protective group;

[0102] X is selected from the group consisting of:

[0103] -O-;

[0104] -O(CH ₂ ) _m O-;

[0105] -OCH ₂ CH(R ⁴ )O-;

[0106] -OCH(R ⁴ )CH ₂ O-; And

[0107] -O(CH ₂ CH ₂ O) _n -;

[0108] R ⁴ is:

;

;

[0109] m is 1, 2, 3, 4, 5, 6, 7, or 8;

[0110] n is 2, 3, 4, 5, 6, 7, or 8; And

[0111] R ⁵ is selected from the group consisting of hydrogen and optionally substituted alkyl.

[0112] In another embodiment, the conjugate compound of the present invention is a compound of Formula I and pharmaceutically acceptable salts and solvates thereof, wherein m is 2, 3, 4, 5, 6, 7, or 8.

[0113] In another embodiment, the conjugate compound of the present invention is a compound of Formula II:

II,

II ,

or a pharmaceutically acceptable salt or solvate thereof, wherein R ¹ , R ^3a , R ^3b , R ^3c and X are as defined for Formula I.

[0114] In another embodiment, the implementation of the conjugate compound according to the present invention is an enantiomerically enriched compound having any one or more formulas from Table 1, and its pharmaceutically acceptable salts and solvates, where R ¹ , R ^3a , R ^3b , R ^3c and X defined for Formula I.

Compositions according to the present invention

[0115] Glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, an organic acid, or any combination thereof, may be mixed with a pharmaceutical carrier, such as water, and optionally other components, to form a "composition of the present invention." In some embodiments, the amount of glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, such as reduced glutathione, in a composition of the present invention is about 30-90% by weight, about 30-85% by weight, about 30-80 wt%, about 30-75 wt%, about 30-70 wt%, about 30-65 wt%, about 30-60 wt%, about 30-55 wt%, about 30- 50% wt. In some embodiments, the amount of glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, such as reduced glutathione, in a composition according to the present invention is 30-50% by weight.

[0116] In some embodiments, the composition of the present invention further comprises an organic acid. In some embodiments, the organic acid is selected from the group of acids consisting of ascorbic, acetic, adipic, aspartic, benzenesulfonic, benzoic, butyric, camphorsulfonic, camsilic, carbonic, chlorobenzoic, cholic, citric, ethylenediaminetetraacetic, 1,2-ethanedisulfonic (edisylic), laurylsulfuric (estolic), ethanesulfonic, formic, fumaric, glucoheptonic (gluceptic), gluconic, glucuronic, glutamic, glycolic, glycolylarsanilic (glycollylarsanilic), hippuric, 1-hydroxy-2-naphthoic, isethionic, isobutyric, isonicotinic, lactic, lactobionic, maleic , malic, malonic, almond, methanesulfonic, mucinic, muconic, naphthalenesulfonic, nicotinic, oxalic, oleic, orotic, p-nitromethanesulfonic, pamoic, pantothenic, phthalic, polygalacturonic, propionic, sugar, salicylic, stearic, suberic, succinic, sulfanilic, tannic , tartaric, p-toluenesulfonic and any combination thereof. In some embodiments, the amount of organic acid, such as reduced ascorbic acid, in the composition of the present invention is about 10-90 wt%, about 10-85 wt%, about 10-80 wt%, about 10-75 wt%. ., about 10-70 wt%, about 10-65 wt%, about 10-60 wt%, about 10-55 wt%, about 10-50 wt%, about 10-45 wt%, about 10-40 wt%, about 10-35 wt%, about 10-30 wt%, about 1-30 wt%, about 1-20 wt%. or about 1-10% of the mass. In some embodiments, the amount of organic acid, for example, reduced ascorbic acid, in the composition according to the present invention is 25-40% of the mass.

[0117] In some embodiments, the composition of the present invention further comprises a bicarbonate salt. In some embodiments, the bicarbonate salt is sodium bicarbonate. In some embodiments, the amount of a bicarbonate salt, such as sodium bicarbonate, in the composition of the present invention is about 10-90% by weight, about 10-85% by weight, about 10-80% by weight, about 10-75% by weight. , about 10-70 wt%, about 10-65 wt%, about 10-60 wt%, about 10-55 wt%, about 10-50 wt%, about 10-45 wt%, about 10-40 wt%, about 10-35 wt%, about 10-30 wt%, about 1-30 wt%, about 1-20 wt%. or about 1-10% of the mass. In some embodiments, the amount of bicarbonate salt, for example, sodium bicarbonate, in the composition according to the present invention is about 20-30% of the mass. In some embodiments, the composition of the present invention does not contain a bicarbonate salt.

[0118] In some embodiments, the pH of the composition according to the present invention is from about 6.0 to about 8. In some embodiments, the pH of the composition according to the present invention is greater than 5.5 or at least 6.0 (for example, from 5.6 up to 14, from 5.7 to 14, from 5.8 to 14, from 5.9 to 14, from 6 to 14, from 5.6 to 12, from 5.7 to 12, from 5.8 to 12, 5.9 to 12, 6 to 12, 5.6 to 10, 5.7 to 10, 5.8 to 10, 5.9 to 10, 6 to 10, 5.6 to 9, 5.7 to 9, 5.8 to 9, 5.9 to 9, 6 to 9, 5.6 to 8, 5.7 to 8, 5.8 to 8, 5.9 to 8, 6 to 8, 5.6 to 7.5, 5.7 to 7.5, 5.8 to 7.5, 5.9 to 7.5, 6 to 7.5, 5.6 to 7, 5.7 to 7, 5.8 to 7, 5.9 to 7, or 6 to 7).

[0119] In some embodiments, the composition of the present invention is formulated to maximize formulation stability and minimize glutathione oxidation. Oxidized glutathione is associated with the formation of protein carbonyls via glutathionylation. Glutathionylation occurs when oxidized glutathione dissociates and attaches to proteins. Maintaining glutathione in a reduced state in solution prior to administration may reduce the risk of glutathionylation products, which can lead to clinical complications such as bronchiectasis. In some embodiments, the oxidized glutathione (e.g., %GSSG) in the composition of the present invention 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% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5°C in an atmosphere of N ₂ and/or ambient atmosphere). In some embodiments, the percentage of oxidized glutathione (e.g., %GSSG) in the composition of the present invention is no more than about 2% to about 20%, about 2% to about 18%, about 2% to about 16%, from about 2% to about 16%, from about 2% to about 10%, or from about 2% to 8% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere). In some embodiments, the percentage of oxidized glutathione (e.g., % GSSG) in the composition of the present invention is less than about 20%, less than about 18%, less than about 16%, or less than about 10% by weight. based on the total weight of glutathione in the composition according to the present invention after storage for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0120] In some embodiments, the reduced glutathione in the composition of the present invention 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% based on the total weight of glutathione in the composition of the present invention after storage of the composition of the present invention for 4 weeks at about 5° C (eg N ₂ atmosphere or ambient atmosphere). In some embodiments, the percentage of reduced glutathione in a composition of the present invention is from about 80% to about 100%, from about 80% to about 98%, from about 82% to about 98%, from about 84% to about 98%, from about 86% to about 98%, from about 88% to about 98%, from about 90% to about 98%, or from about 92% to about 98% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage for 4 weeks at 5°C (eg, N ₂ atmosphere or ambient atmosphere). In some embodiments, the percentage of reduced glutathione in the composition of the present invention is at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, or at least 90% by weight. based on the total weight of glutathione in the composition according to the present invention after storage for 4 weeks at 5° C. in an atmosphere of N ₂ or ambient atmosphere.

[0121] In some embodiments, the composition of the present invention is also formulated to maximize formulation stability and minimize organic acid oxidation, such as ascorbic acid. In addition, when ascorbic acid is oxidized to dehydroascorbate (DHA), DHA can be broken down and lead to the formation of protein adducts in a process called ascorbylation. The presence of an organic acid, such as ascorbic acid, in a reduced state in solution prior to administration may reduce the risk of ascorbic acid addition from dehydroascorbate degradation products. 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%, based on on the weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (at 5°C in an atmosphere of N ₂ and/or in an ambient atmosphere). In some embodiments, the percentage of reduced ascorbic acid (eg, %ASC) in the composition of the present invention is from about 82% to about 100%, or from about 85% to about 95% by weight. calculated on the total weight of ascorbic acid in the composition according to the present invention after storage for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere). In some embodiments, the percentage of reduced ascorbic acid (e.g., %ASC) in the composition of the present invention is at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89% or at least 90% of the mass. based on the total weight of ascorbic acid in the composition according to the present invention after storage for 4 weeks (for example, at °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0122] In some embodiments, the oxidized ascorbic acid in the composition of the present invention 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%. wt. based on the total weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere). In some embodiments, the percentage of oxidized ascorbic acid in a composition of the present invention is no more than about 5% to about 20%, about 5% to about 18%, about 5% to about 10%, or about 5% to 9% wt. based on the total weight of ascorbic acid in the composition according to the present invention after storage for 4 weeks (for example, at 5°C in an atmosphere of N ₂ and/or ambient atmosphere). In some embodiments, the percentage of oxidized ascorbic acid in the composition of the present invention is less than about 20%, less than about 18%, less than about 16%, or less than about 10% by weight. calculated on the total weight of ascorbic acid in the composition according to the present invention after storage for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0123] In certain aspects, the composition ratios of the present invention are formulated to maximize formulation stability and minimize oxidation of glutathione and an organic acid, such as ascorbic acid. In some embodiments, glutathione and an organic acid (e.g., ascorbic acid) are formulated to contain molar equivalents in solution, e.g., the molar ratio of glutathione to ascorbic acid is about 0.5-1:1, about 0.6-1:1: 1, 0.7-1:1, 0.8-1:1. 0.9-1:1, or about 1:1. In some embodiments, glutathione and an organic acid (e.g., ascorbic acid) are formulated such that they contained a molar excess of organic acid (e.g. ascorbic acid) relative to glutathione in solution, e.g. the molar ratio of glutathione to ascorbic acid is about 1:1.1, about 1:1.2, about 1:3, about 1:4 , approximately 1:5.

[0124] In some embodiments, the composition of the present invention further comprises a bicarbonate salt (eg, sodium bicarbonate). In some embodiments, the glutathione, organic acid (e.g., ascorbic acid) and bicarbonate salt (e.g., sodium bicarbonate) are formulated to be in a molar ratio of glutathione to organic acid (e.g., ascorbic acid) to bicarbonate salt (e.g., bicarbonate sodium) 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, about 0.49: 0.5-1:1, about 0.5: 0.5-1:1, about 0.1 -0.5:0.6-1:1, about 0.2-0.5:0.6-1:1, about 0.3-0.5:0.6-1:1, about 0, 4-0.5:0.6-1:1, about 0.49:0.6-1:1, about 0.5:0.6-1:1, about 0.1-0.5:0 ,7-1:1, about 0.2-0.5:0.7-1:1, about 0.3-0.5:0.7-1:1, about 0.4-0.5: 0.7-1:1, about 0.49:0.7-1:1, about 0.5:0.7-1:1, about 0.1-0.5:0.8-1:1 , about 0.2-0.5:0.8-1:1, about 0.3-0.5:0.8-1:1, about 0.4-0.5:0.8-1: 1, about 0.49:0.8-1:1, about 0.5:0.8-1:1, about 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 (for example, ascorbic acid) and a bicarbonate salt (for example, 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 a bicarbonate salt (e.g. sodium bicarbonate) is 0.49:0.5:1, 0.5:0.5:1, 0.49:1:1, or 0.5:1:1.

[0125] In some embodiments, the content of a bicarbonate salt (e.g., sodium bicarbonate) is less than the combined molar ratio of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, and (b) an organic acid (e.g., ascorbic acid ). In some embodiments, the molar ratio of glutathione, organic acid (e.g., ascorbic acid), and bicarbonate salt (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.

[0126] In some embodiments, a composition of the present invention comprises or consists essentially of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, and (b) an organic acid, where 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 from about 5.5 to 14, from about 6 to about 8, 7 ± 1.5, 6 ± 0.5, or about 6.

[0127] In some embodiments, a composition of the present invention comprises or consists essentially of (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, (b) an organic acid, (c) a bicarbonate salt, in which the molar ratio of (a) to (b) to (c) is about 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, 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, and the pH of the composition is from about 5, 5 to 14, about 6 to about 8, 7±1.5, 6±0.5, or about 6.

[0128] Pharmaceutical compositions for use in the present invention may be formulated using one or more physiologically acceptable carriers and/or excipients that facilitate administration to a subject of an organic acid, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof. the 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.

[0129] The compositions of the present invention can be prepared by conventional mixing, dissolving, granulating, drageeing, emulsifying, encapsulating, trapping, spray drying, or lyophilizing processes known in the art. The specific composition depends on the chosen route of administration. In one embodiment, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, is dissolved in a solvent, such as water, for administration to the respiratory tract of a subject (eg, for intranasal administration).

[0130] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier such as a liquid (eg, water) or solid excipient, diluent, excipient, solvent, or encapsulating material. The carrier is "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of the pharmaceutical composition and suitable for use in humans without causing toxicity, irritation, allergic response, immunogenicity, or other complications commensurate with a reasonable benefit/risk ratio. See , 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.

[0131] In some embodiments, the compositions of the present invention contain 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.

[0132] In certain embodiments, the compositions of the present invention may contain a pH adjusting agent. pH adjusting agents are known in the art. See, eg , 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 pharmaceutically 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 buffers , citrate buffers, phosphate buffers, lactic acid buffers, and borate buffers, and any combination thereof), fat-soluble fatty acid esters of ascorbic acid (vitamin C) (e.g., alone or in combination with α-hydroxy acids), oxidation-stable esters of saturated fatty acids and ascorbic acid (eg ascorbyl laurate, ascorbyl myristate, ascorbyl palmitate, ascorbyl stearate and ascorbyl behenate and any combination thereof) and any combination thereof. In some embodiments, esters can be made using hydrogenated oils or fats, or fractions thereof, and contain small amounts of another ester. For example, canola-derived ascorbyl stearate can typically contain about 4% ascorbyl palmitate.

[0133] In one embodiment, a pH adjusting agent, such as ascorbic acid, is present in the composition of the present invention in an amount of about 0.01-50 wt%, 10-90 wt%, about 10-85 wt%, about 10-80 wt%, about 10-75 wt%, about 10-70 wt%, about 10-65 wt%, about 10-60 wt%, about 10-55 wt%, about 10 -50 wt%, about 10-45 wt%, about 10-40 wt%, about 10-35 wt%, about 10-30 wt%, about 1-30 wt%, about 1-20 % wt. or about 1-10% of the mass. In some embodiments, the pH adjusting agent is present in the composition of the present invention in an amount of about 1% by weight, about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% wt., about 30% wt., about 35% wt., about 40% wt., about 45% wt. or about 50% of the mass. from composition.

[0134] In specific embodiments, the compositions of the present invention 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., cetilipridinium chloride, benzalkonium chloride, and parabens, including, but not limited to methylparaben, ethylparaben and propylparaben), chlorhexidine, benzoic acid and its salts, parahydroxybenzoic acids and their salts, parahydroxybenzoic acid alkyl esters and their salts, phenylmercurates such as nitrate, chloride, acetate and borate, antioxidants, EDTA , sorbitol, phenol, boric acid and its salts, sorbic acid and its salts, thimerosal and nitromersol, and any combinations thereof.

[0135] In one embodiment, the implementation of the preservative is present in the composition according to the present invention in an amount of about 0.01-50 wt. -%, for example, about 1-30 wt. -%, about 1-20 wt.%. or about 1-10% wt., for example, about 1% wt., about 5% wt., about 10% wt., about 15% wt., about 20% wt., about 25% wt., about 30% wt. ., about 35% wt., about 40% wt., about 45% wt. or about 50% of the mass. from composition.

[0136] In specific embodiments, the compositions of the present invention may contain chelating agents. 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, allylmalonic acid, ethylmalonic acid, citric acid, malic acid, glyceric acid, tartaric acid, mevalonic acid, hydroxyglutaric acid, oxaloacetic acid, α-ketoglutaric acid, α-ketomalonic acid, glucuronic acid, galacturonic acid (galaceturonic acid), mannuronic acid, aspartic acid, glutamic acid, glycine, alanine, lysine, histidine, alginine, cysteine , ε-aminocaproic acid, phenylalanine, phenylglycine, p-hydroxyphenylglycine, p-aminophenylalanine, γ-carboxyglutamic acid, iminodiacetic acid, hydroxyethyliminodiacetic acid, ethylenediaminedioacetic acid, ethylenediaminetetraacetic acid, trans-cyclohexane-diaminetetraacetic acid, diethylenediaminep entaacetic acid, alanine diacetic acid, diaminopimelic acid , phthalic acid, terephthalic acid, homophthalic acid, phenylsuccinic acid, phenylmalonic acid, oxanilic acid-o-carboxylic acid, anthralininoacetic acid (anthralininoacetic acid), 2,4-dihydroxybenzoic acid, p-aminosalicylic acid, phthalylglutamic acid, kynurenine, 1, 2-hydroxybenzene-3,5-disulfonic acid, 4-aminophenol-2-sulfonic acid, cysteic acid, 2-phosphoglyceric acid, glycero-3-phosphoric acid, glucose-1,6-diphosphoric acid, fructose-1,6- diphosphoric acid, and phosphates (eg, sodium phosphate, sodium aluminum phosphate, sodium hydrogen phosphate, dipotassium phosphate, disodium phosphate, sodium phosphate monobasic, and sodium hexametaphosphate), and any combinations thereof. Chelating agents may be included in the pharmaceutical compositions of the present invention, either as a parent molecule or as a salt, where appropriate. For example, compounds containing an acidic functional group can be used in protonated form or in the form of a pharmaceutically acceptable inorganic or organic salt that retains the chelating activity of the parent compound.

[0137] In one embodiment, the implementation of the chelating agent is present in the composition according to the present invention in an amount of about 0.01-50 wt. -%, for example, about 1-30 wt. -%, about 1-20 wt.%. or about 1-10% wt., for example, about 1% wt., about 5% wt., about 10% wt., about 15% wt., about 20% wt., about 25% wt., about 30% wt., about 35% wt., about 40% wt., about 45% wt. or about 50% of the mass. from the mass of the composition.

[0138] In some embodiments, the composition of the present invention comprises glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof; a bicarbonate (eg sodium bicarbonate or potassium bicarbonate) and/or a pH modifier (eg ascorbic acid). In some embodiments, the composition of the present invention comprises glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, and/or a pH modifier such as an organic acid (eg, ascorbic acid). In some embodiments, a composition is claimed in which an amount of each component is present such that the amount of ascorbic acid is approximately a molar equivalent or molar excess of glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof.

[0139] In some embodiments, the composition of the present invention comprises (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof; (b) an organic acid; and (c) a bicarbonate salt. In other embodiments, the molar ratio of (a):(b):(c) in the composition of the present invention 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).

[0140] In some embodiments, the organic acid in the composition of the present invention is ascorbic acid. In some embodiments, the bicarbonate salt in the composition of the present invention is sodium bicarbonate. In some embodiments, the composition contains: (a) glutathione; (b) ascorbic acid; and (c) sodium bicarbonate. In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (eg, about 0.49:about 0.50:about 1). In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (eg, about 0.49:about 1:about 1).

[0141] In some embodiments, the pH of the composition of the present invention is from about 5.5 to about 14 (eg, from 5.5 to 7.5). In some embodiments, the pH of the composition of the present invention is from about 6 to about 14 (eg, from 6 to 7.5). In some embodiments, the pH of the composition 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.

[0142] In some embodiments, the composition according to the present invention is stable when stored at 2-8°C for at least 72 hours. In some embodiments, the composition according to the present invention may (a) not form precipitates after storage at 2-8°C for at least 72 hours, (b) contain less than 7%, less than 6%, less than 5%, or less than 4% impurities after storage at 2-8°C for at least 72 hours, (c) have or maintain a pH of about 6 to 7.5 (e.g. 6.0-7.0) after storage at 2-8°C for at least 72 hours, and/or (d) have minimal loss of solubility after storage at 2-8° C. for at least 72 hours.

Methods of application

[0143] In some embodiments, the compositions of the present invention are suitable for treating, alleviating symptoms, or preventing a lung disease, condition, or disorder. In particular, the compositions of the present invention can be used to inhibit or reduce the growth of clinical isolate bacteria and/or to inhibit or reduce biofilm formation of clinical isolate bacteria. Compositions of the present invention can be used to treat or relieve symptoms in a subject suffering from or at risk of infection with the bacteria of the clinical isolate. In other embodiments, compositions of the present invention are suitable for enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, such as a subject with or without an active bacterial infection in the lungs. In other embodiments, the compositions of the present invention are suitable for reducing airway inflammation in a subject suffering from or at risk for airway inflammation, eg, with or without active bacterial infection in the lungs.

[0144] In some embodiments, the subject has a disease or disorder of the lungs or airways. In some embodiments, the lung or airway disorder is selected from the group consisting of chronic inflammatory lung disease, pulmonary fibrosis, pulmonary vasculitis, pulmonary sarcoidosis, lung transplant inflammation and/or infection, acute or chronic lung rejection and/or dysfunction, pulmonary arterial hypertension, bronchitis, sinusitis, asthma, cystic fibrosis, bronchiectasis (eg, bronchiectasis due to a disease other than cystic fibrosis or bronchiectasis due to cystic fibrosis), bacterial infection, fungal infection, parasitic infection, viral infection, chronic obstructive pulmonary disease (COPD ), bronchiolitis obliterans syndrome (BOS), primary ciliary dyskinesia (PCD), alveolar prothienosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), ventilator-associated inflammation and/or infection, ventilator-associated pneumonia, asbestos-associated respiratory disorder or disease, dust-associated respiratory disorder or disease, silicosis, and radiation- or chemical agent-associated respiratory disease or disorder, and any combination thereof.

[0145] In some embodiments, the lung or airway disease or disorder is selected from the group consisting of chronic inflammatory lung disease, lung transplant inflammation and/or infection, acute or chronic lung rejection or dysfunction, asthma, cystic fibrosis, bronchiectasis ( for example, bronchiectasis caused by a disease other than cystic fibrosis or bronchiectasis caused by cystic fibrosis) 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 patient with bronchiectasis caused by a disease other than cystic fibrosis. In another embodiment, the subject is infected with biofilm producing bacteria.

[0146] In some embodiments, the lung or airway disease or disorder 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, treatments that have been used in cystic fibrosis (CF) have not worked and have been shown to be potentially harmful in non-CF bronchiectasis.

[0147] Non-CF bronchiectasis and CF disease, including CF-associated bronchiectasis, are clinically distinct, although they share some similarities. CF BrE primarily affects the upper parts of the respiratory tract, while non-CF BrE is associated with involvement of the lower parts. Because patients with non-CF BrE do not have appropriate mucociliary transport mechanisms, therapy that only destroys mucus components (eg, DNase) results in sputum production in the lower airways and lung parenchyma rather than sputum clearance. CF sputum contains more DNA than BrE patients, suggesting a different underlying physiology. BrE tends to occur in older patients and has a higher prevalence in women, while CF is diagnosed in younger patients and CF has a similar prevalence in men and women (O'Donnell et al., Chest 1998 Volume 113, Issue 5 , Pages 1329-1334).

[0148] In some embodiments, the compositions of the present invention are suitable for treating, alleviating symptoms, or preventing a disease (eg, bronchiectasis caused by a disease other than cystic fibrosis or bronchiectasis caused by cystic fibrosis).

[0149] In another embodiment, the present invention provides methods for treating or preventing bacterial infection of a clinical isolate in the respiratory tract of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present invention. In another embodiment, a composition of the present invention is administered to a subject in combination with one or more antibiotics. Antibiotics can be administered locally to the lungs and/or systemically.

[0150] In another embodiment, the present disclosure relates to methods for treating or preventing inflammation in the airways of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present invention.

[0151] In another embodiment, the present disclosure relates to methods for treating or preventing a mucosal tissue disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present invention. Non-limiting examples of mucosal tissue include the mouth, nose, eye, ear, upper respiratory tract, lower respiratory tract, gastrointestinal tract, vagina, rectum, and urethra.

[0152] In another embodiment, the present disclosure relates to methods for treating or preventing a mucosal-associated disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present invention to appropriate mucosal membranes. In one embodiment, the mucous membranes are the lungs, such as the deep lung (alveolar region), and in other embodiments, the mucous membranes are one or more of the eyes, mouth, nose, rectum, and vagina.

[0153] In another embodiment, the composition of the present invention can be used to treat or prevent bronchiolitis obliterans and war-related lung injury, that is, lung injury in military personnel who have damaged the airways due to exposures of an unknown nature.

[0154] In another embodiment, the present invention provides the use of a composition of the present invention for the manufacture of a medicament for the treatment of a lung or airway disorder. In another embodiment, the use further comprises administering to the subject one or more additional therapeutic agents.

[0155] Therapeutic methods of the present invention comprise administering a therapeutically effective amount of a composition of the present invention to a subject in need thereof, eg, a human patient. The indication for such treatment depends on the individual case and is subject to a medical examination (diagnosis), which takes into account the existing signs, symptoms and / or disorders, the risks of developing specific signs, symptoms and / or disorders, and other factors.

[0156] In one embodiment, the present invention provides a method of treating a disorder or disease of the lung or respiratory tract in a subject infected with the bacteria of the clinical isolate, or at risk of infection with the bacteria of the clinical isolate, the method comprising administering to the subject an effective amount of a composition of the present invention.

[0157] 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., bronchiectasis, caused by a disease other than cystic fibrosis, or bronchiectasis caused by cystic fibrosis) and chronic obstructive pulmonary disease (COPD), and any combination thereof.

In another embodiment, the disorder or disease of the lung or respiratory tract is treated by inhibiting the growth of the bacteria of the clinical isolate in the respiratory tract of the subject.

[0159] In another embodiment, the lung or airway disorder or disease is treated by restoring homeostasis and/or maintaining homeostasis in the mucosa of a subject in need thereof, the method comprising administering to the subject an effective amount of a composition of the present invention.

[0160] In another embodiment, the present invention provides a method of restoring homeostasis and/or maintaining homeostasis in the mucosa of a subject in need thereof, the method comprising administering to the subject an effective amount of a composition of the present invention.

[0161] In another embodiment, the present invention provides a method of restoring or maintaining homeostasis in a mucosa, comprising administering to a subject an effective amount of a composition of the present invention.

[0162] In some embodiments, the methods of the present invention comprise administering to the subject's respiratory tract an effective amount of a composition comprising a glutathione conjugate and/or glutathione or a pharmaceutically acceptable salt thereof. In another embodiment, the composition further comprises: an organic acid or a pharmaceutically acceptable salt thereof. In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises a bicarbonate salt. In another embodiment, the bicarbonate salt is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid in the composition of the present invention is ascorbic acid. In some embodiments, the bicarbonate salt in the composition of the present invention is sodium bicarbonate. In some embodiments, the composition contains: (a) glutathione; (b) ascorbic acid; and (c) sodium bicarbonate. In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (eg, 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 (eg, about 0.49:about 1:about 1).

[0163] In another embodiment, each of the components such as a glutathione conjugate and/or glutathione or a pharmaceutically acceptable salt thereof, an organic acid or a pharmaceutically acceptable salt thereof, and a bicarbonate salt is present in the composition such that a given amount of the bicarbonate salt results in a pH of a range of from about 5.5 to about 14. In some embodiments, the pH of the composition of the present invention is from about 5.5 to about 14 (eg, from 5.5 to 7.5). In some embodiments, the pH of the composition of the present invention is from about 6 to about 14 (eg, from 6 to 7.5). In some embodiments, the pH of the composition 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 implementation of the composition additionally contains from about 0.01% to about 5% of the mass. pharmaceutically acceptable thiocyanate salt.

[0165] In another embodiment, the composition is in particulate form. In 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, administering the composition to a subject provides a concentration of about 0.1 mM to about 1.0 mM glutathione in the fluid covering the subject's respiratory tract. In another embodiment, administering the composition to a subject provides a concentration of about 0.5 mM to about 3.0 mM thiocyanate in the fluid covering the subject's respiratory tract.

[0168] In some embodiments, oxidized glutathione (e.g., %GSSG) in the composition of the present invention 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% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5°C in an atmosphere of N ₂ and/or ambient atmosphere).

[0169] In some embodiments, the reduced glutathione in the composition of the present invention 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% based on the total weight of glutathione in the composition of the present invention after storage of the composition of the present invention for 4 weeks at approx. 5 °C (eg N ₂ atmosphere or ambient atmosphere).

[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 % based on the weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (at 5°C in an atmosphere of N ₂ and/or in an ambient atmosphere).

[0171] In some embodiments, the oxidized ascorbic acid in the composition of the present invention 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%. wt. based on the total weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0172] In some embodiments, the composition is an aqueous solution or a dry powder. In another embodiment, the composition is administered to a subject via inhalation.

a. Antibacterial uses

[0173] Although most antibacterial agents (eg, antibiotics) have activity exclusively against Gram-negative bacteria or exclusively against Gram-positive bacteria, the composition of the present invention shows a broad spectrum of activity against both Gram-negative and Gram-positive bacteria. Such properties are unexpected in view of studies showing that ascorbic acid itself acts in a strain dependent and highly concentration dependent manner (see, for example, Med J Aust. 1974 Feb 9;1(6):169- 74; Hancock and Wong Antimicrob agents and chemotherapy, July 1984: 48-52). Clinical conditions complicate the use of broad-spectrum antibiotics for chronic inflammatory airway diseases such as cystic fibrosis, bronchiectasis (eg, non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis)) and lung transplantation. In addition, doctors often prescribe antibiotics based on experience and patient response. Unlike the compositions of the present invention, current antibiotic therapies do not demonstrate adequate efficacy against bacteria commonly found in chronic inflammatory airway diseases.

[0174] The compositions of the present invention can actively inhibit a variety of bacterial species without providing the bacteria with a protective mechanism.

[0175] Accordingly, in some embodiments, the compositions of the present invention are suitable for inhibiting or reducing bacterial growth of a clinical isolate. In some embodiments, the compositions of the present invention are suitable for inhibiting or reducing biofilm formation of clinical isolate bacteria. In some embodiments, the compositions of the present invention are suitable for treating or alleviating symptoms in a subject suffering from or at risk of infection with the bacteria of the clinical isolate.

[0176] In certain aspects, the clinical isolate according to the present application may include a single bacterial strain or a combination (ie, two or more) bacterial strains. In some embodiments, the clinical isolate is 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.

[0177] In some embodiments, the clinical isolate is Gram-negative. In some embodiments, the gram-negative clinical isolate is selected from the group consisting of Burkholderia cepacia , Pseudomonas aeruginosa , Actinobacter baumannii , Burkholderia pseudomallei , or any combination thereof. In some embodiments, Pseudomonas aeruginosa is a mucus-forming or non-mucus-forming bacterium, or a combination thereof.

[0178] In some embodiments, the clinical isolate is Gram positive. In some embodiments, the Gram-positive clinical isolate is selected from the group consisting of Enterococcus , Streptococcus , Pneumococcus , Staphylococcus ( eg , Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA)) or any combination thereof. In some embodiments, the gram-positive clinical isolate is Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA), or a combination thereof.

[0179] In some embodiments, the clinical isolate is mucus-forming. In some embodiments, the mucus-forming clinical isolate is Pseudomonas aeruginosa , Burkholderia cepacia , and any combination thereof.

[0180] In some embodiments, the clinical isolate is not mucus-forming. In certain embodiments, the clinical isolate is a clinical nonmucus producing Pseudomonas aeruginosa .

[0181] In some embodiments, the clinical isolate is aerobic. In some embodiments, the aerobic clinical isolate is Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus , and methicillin-resistant Staphylococcus aureus (MRSA), and any combination thereof. In certain embodiments, aerobic conditions are nitrate conditions. In certain embodiments, aerobic conditions are conditions in the absence of nitrates.

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, anaerobic or aerotolerant conditions are conditions with the presence of nitrates. In certain embodiments, anaerobic or aerotolerant conditions are conditions in the absence of nitrates.

[0183] In some embodiments, the clinical isolate may live extracellularly. In some embodiments, the clinical isolate that can live extracellularly is Burkholderia cepacia , Pseudomonas aeruginosa , Staphylococcus aureus , and methicillin-resistant Staphylococcus aureus (MRSA), and any combination thereof.

[0184] In some embodiments, the clinical isolate is multidrug resistant. In some embodiments, the multidrug-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.

[0185] In some embodiments, the clinical isolate is resistant to treatment with a cationic protein. In some embodiments, the cationic protein-resistant clinical isolate is selected from the group consisting of Pseudomonas aeruginosa , Burkholderia cepacia , and any combination thereof.

Pseudomonas aeruginosa

[0186] In one embodiment, the bacterium of the clinical isolate is Pseudomonas aeruginosa .

[0187] Pseudomonas aeruginosa is a gram-negative bacterium that can cause infection, especially in patients with compromised host defense mechanisms. It is the most common pathogen isolated from patients who have been hospitalized for more than 1 week and is a common cause of nosocomial infections. Pseudomonas infections can be life-threatening.

[0188] Pseudomonas aeruginosa is a metabolically volatile bacterium that can cause a wide range of severe opportunistic infections in patients with compromised natural defenses. Provoking conditions may include, for example, a compromised epithelial barrier (as in patients with a burn wound), a lack of neutrophils (for example, in cancer patients receiving chemotherapy), the presence of a foreign body (in a patient with a central venous catheter), and altered mucociliary clearance (for example, in individuals with cystic fibrosis). Pseudomonas aeruginosa is inherently resistant to many antibiotics and can become resistant to others, making treatment difficult. The propensity of Pseudomonas aeruginosa to form biofilms further protects it from antibiotics and the host's immune system. In some embodiments, the clinical isolate is a mucus-forming Pseudomonas aeruginosa . In some embodiments, the clinical isolate is Pseudomonas aeruginosa , which is not a mucus former.

[0189] Pseudomonas aeruginosa is commonly isolated from the respiratory tract of individuals with cystic fibrosis and is associated with an accelerated decline in lung function in these patients. Chronic lung colonization and infection also occur in bronchiectasis (bronchiectasis caused by a disease other than cystic fibrosis or cystic fibrosis bronchiectasis), a disease characterized by irreversible dilatation of the bronchial tree, and chronic obstructive pulmonary disease, a disease characterized by narrowing of the airways and airway obstruction. flow. In addition, Pseudomonas aeruginosa is one of the most common causes of nosocomial pneumonia, especially in ventilated patients; this is associated with a particularly high mortality rate.

[0190] Exposure to various environments in vivo leads to the activation of various metabolic patterns in Pseudomonas aeruginosa to protect against reactive nitrogen species. This can lead to mucus production.

Burkholderia cepacia

[0191] In another embodiment, the bacterium of the clinical isolate is Burkholderia cepacia .

[0192] Burkholderia cepacia is an aerobic Gram-negative bacillus found in a variety of aquatic environments. Burkholderia cepacia is a low virulence organism and is a frequent colonizer of hospital fluids (eg, irrigation solutions, intravenous fluids). Burkholderia cepacia rarely causes infection in healthy hosts. Based on phenotypic and genotypic analysis, Burkholderia cepacia is one of the 9 genovars that make up the Burkholderia cepacia complex (BCC).

[0193] BCC is 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 Antimicrobial Agents. 2009 May;33(5):394-404. BCC mutates frequently and acquires a high level of resistance to many antimicrobial agents. These bacteria are associated with deterioration and mortality in chronic inflammatory airway diseases and may even develop resistance to multiple drugs. Although generally thought to be aerobic, some exhibit fermentation and nitrate respiration. Sass et al . ISME J. 2013 Aug;7(8):1568-81.

Staphylococcus aureus and methicillin resistant Staphylococcus aureus (MRSA)

[0194] In another embodiment, the clinical isolate bacteria are Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (MRSA), both of which are Gram-positive cocci.

[0195] Staphylococcus aureus is an important cause of pneumonia. These bacteria have been implicated in pleuropulmonary infections in some way and are commonly co-localized with Pseudomonas aeruginosa or Burkholderia cepacia in chronic inflammatory airway disease. Tong et al . Clin Microbiol Rev. 2015 Jul;28(3):603-61. In one embodiment, Staphylococcus aureus has been identified in patients with acute and chronic lung transplant rejection.

[0196] MRSA infection is caused by Staphylococcus aureus , which has become resistant to many of the antibiotics used to treat common staph infections. HA-MRSA infections are usually associated with invasive procedures or devices such as surgery, intravenous tubes, or artificial joints. Highly populated area MRSA (CA-MRSA) is another type of MRSA infection that occurs in the wider community, often among healthy people. This form often begins with painful furunculosis of the skin. It is spread by skin-to-skin contact. Risk groups include groups such as high school wrestlers, child care workers, and people who live in densely populated areas.

b. Ways to increase mucociliary clearance

[0197] Mucociliary clearance is critical to preventing infections and inflammation in the lungs. However, mucociliary clearance is often absent or impaired in chronic inflammatory airway diseases such as cystic fibrosis, bronchiectasis (eg, non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), lung transplantation, and chronic obstructive pulmonary disease. Moreover, it is known that airway epithelial cells in patients with cystic fibrosis completely lack mucociliary clearance capacity without other inflammatory damage, bacterial colonization or infection. Although some research has focused on modifying the airway microenvironment to restore the function of previously functioning tissue, the compositions of the present invention are capable of activating mucociliary clearance functions in epithelial cells that are otherwise devoid of such functions.

[0198] Ascorbic acid can directly modulate CFTR activity (Proc Natl Acad Sci U S A. 2004 Mar 9; 101(10): 3691-3696). However, ascorbic acid has also been shown to have a limited effect on mucociliary clearance (BMC Complement Altern Med. 2013; 13:110) and has limited clinical efficacy (Sakasura Ann Otol 82; 1973). However, the compositions of the present invention can restore mucociliary function in the absence of bacterial infection.

[0199] When used alone, glutathione, ascorbic acid, and bicarbonate do not have any clinical effect on mucociliary clearance or clinical outcomes (Sakasura Ann Otol 82; 1973; Am J Respir Crit Care Med. 2013 Jul 1;188(1):83 -9. doi: 10.1164/rccm.201303-0427OC). In addition, the effect of bicarbonate has been mixed, despite clinical evidence of a direct effect on respiratory pH.

[0200] Compositions of the present invention provide a synergistic effect between glutathione, ascorbic acid and bicarbonate and can activate mucociliary clearance to levels seen in normal cells (Am J Respir Crit Care Med. 2014 Aug 15; 190(4): 421-432) . Accordingly, in some embodiments, the composition of the present invention is suitable for enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance.

[0201] In some embodiments, the present invention includes a method of enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering to the subject a composition comprising: (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, and (b ) organic acid.

[0202] In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises: (c) a bicarbonate salt. In another embodiment, the bicarbonate salt is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid is ascorbic acid. In some embodiments, the composition contains: (a) glutathione; (b) ascorbic acid; and (c) sodium bicarbonate. In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (eg, 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 (eg, about 0.49:about 1:about 1).

[0203] In another embodiment, each of the components such as a glutathione conjugate and/or glutathione or a pharmaceutically acceptable salt thereof, an organic acid or a pharmaceutically acceptable salt thereof, and a bicarbonate salt is present in the composition such that a given amount of the bicarbonate salt results in a pH of a range of from about 5.5 to about 14. In some embodiments, the pH of the composition of the present invention is from about 5.5 to about 14 (eg, from 5.5 to 7.5). In some embodiments, the pH of the composition of the present invention is from about 6 to about 14 (eg, from 6 to 7.5). In some embodiments, the pH of the composition 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.

[0204] In another embodiment, the implementation of the composition additionally contains from about 0.01% to about 5% of the mass. pharmaceutically acceptable thiocyanate salt.

[0205] In another embodiment, the composition is in particulate form. In another embodiment, the particles are mixed with a gas or liquid propellant for use in inhalation therapy.

[0206] In another embodiment, the glutathione is reduced glutathione.

[0207] In another embodiment, administering the composition to a subject provides a concentration of about 0.1 mM to about 1.0 mM glutathione in the fluid covering the subject's respiratory tract. In another embodiment, administering the composition to a subject provides a concentration of about 0.5 mM to about 3.0 mM thiocyanate in the fluid covering the subject's respiratory tract.

[0208] In some embodiments, the oxidized glutathione (e.g., %GSSG) in the composition of the present invention 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% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5°C in an atmosphere of N ₂ and/or ambient atmosphere).

[0209] In some embodiments, the reduced glutathione in the composition of the present invention 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%, based on the total weight of glutathione in the composition of the present invention after storage of the composition of the present invention for 4 weeks at about 5°C (for example, in an atmosphere of N ₂ or ambient atmosphere).

[0210] 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 % based on the weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (at 5°C in an atmosphere of N ₂ and/or in an ambient atmosphere).

[0211] In some embodiments, the oxidized ascorbic acid in the composition of the present invention 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%. wt. based on the total weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0212] In some embodiments, the composition is an aqueous solution or a dry powder. In another embodiment, the composition is administered to a subject via inhalation.

[0213] In some embodiments, administering the composition reduces the viscosity of the patient's mucus. In some embodiments, administering the composition increases the ciliary beat frequency of the patient's airway epithelial cells. In some embodiments, the implementation of the introduction of the composition increases the rate of mucociliary transport of epithelial cells of the respiratory tract of the patient. In some embodiments, the implementation of the specified composition increases the height of the liquid covering the surface of the respiratory tract of the patient.

[0214] In some embodiments, the patient suffers from a chronic inflammatory disease of the respiratory tract or is at risk of developing this disease. In some embodiments, the inflammatory airway disease is cystic fibrosis, bronchiectasis (eg, non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), asthma, chronic obstructive pulmonary disease, or pulmonary fibrosis. In another embodiment, the lung or airway disease or disorder is cystic fibrosis. In some embodiments, the patient's airway epithelial cells do not have mucociliary clearance capability prior to administration of said composition. In some embodiments, the airway epithelial cells of said patient do not have mucociliary clearance capability due to a genetic deficiency.

[0215] In other embodiments, the patient has undergone a lung transplant.

[0216] In some embodiments, the epithelium of the patient's respiratory tract is not colonized by bacteria. In other embodiments, the respiratory tract epithelium of the patient is colonized with bacteria. In some embodiments, the patient is suffering from an active bacterial infection. In other embodiments, the patient does not suffer from an active bacterial infection.

[0217] In some embodiments, the patient is a pediatric patient. In other embodiments, the patient is an adult patient.

c. Anti-inflammatory uses

[0218] Signs of inflammation in diseases of the respiratory tract include the production of neutrophil extracellular traps (NET), increased activity of neutrophil myeloperoxidase, increased production of nitric oxide (II) and increased production of pro-inflammatory cytokines. Studies show that ascorbic acid specifically enhances myeloperoxidase (MPO) activity and promotes leukocyte recruitment, and that the presence of MPO is directly associated with disease complication and deterioration (Free Radic Biol Med. 2017 Oct 2;113:236-243). In addition, neutrophil extracellular trap formation induced by phorbol myristate acetate (PMA) can be prevented by acidic conditions and initiated under more basic conditions resulting from high concentrations of sodium bicarbonate.

[0219] Compositions according to the present invention directly suppress the inflammatory process. In particular, the compositions of the present invention inhibit MPO activity as well as inhibit cytokines associated with NET formation, macrophage activation, and recruitment of neutrophils and T cells in a mechanism independent of repair functions. Compositions according to the present invention can reduce the level of abnormal production of nitric oxide (II) and, therefore, do not cause a violation of endogenous antibacterial properties. In some embodiments, administration of said composition reduces the exhaled nitric oxide (II) fraction (FeNO) of the patient by at least 20%. Accordingly, in some embodiments, the compositions of the present invention are suitable for reducing airway inflammation in a subject suffering from or at risk for airway inflammation.

[0220] In some embodiments, the present invention includes a method of enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering to the subject a composition comprising: (a) glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, and (b ) organic acid.

[0221] In another embodiment, the organic acid is ascorbic acid. In another embodiment, the composition further comprises: (c) a bicarbonate salt. In another embodiment, the bicarbonate salt is sodium bicarbonate or potassium bicarbonate. In some embodiments, the organic acid is ascorbic acid. In some embodiments, the composition contains: (a) glutathione; (b) ascorbic acid; and (c) sodium bicarbonate. In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:0.5-1:1 (eg, about 0.49:about 0.50:about 1). In some embodiments, the molar ratio of (a):(b):(c) is about 0.1-0.5:1:1 (eg, about 0.49:about 1:about 1).

[0222] In another embodiment, each of the components such as a glutathione conjugate and/or glutathione or a pharmaceutically acceptable salt thereof, an organic acid or a pharmaceutically acceptable salt thereof, and a bicarbonate salt is present in the composition such that a given amount of the bicarbonate salt results in a pH of a range of from about 5.5 to about 14. In some embodiments, the pH of the composition of the present invention is from about 5.5 to about 14 (eg, from 5.5 to 7.5). In some embodiments, the pH of the composition of the present invention is from about 6 to about 14 (eg, from 6 to 7.5). In some embodiments, the pH of the composition 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.

[0223] In another embodiment, the implementation of the composition additionally contains from about 0.01% to about 5% of the mass. pharmaceutically acceptable thiocyanate salt.

[0224] In another embodiment, the composition is in particulate form. In another embodiment, the particles are mixed with a gas or liquid propellant for use in inhalation therapy.

[0225] In another embodiment, the glutathione is reduced glutathione.

[0226] In another embodiment, administering the composition to a subject provides a concentration of about 0.1 mM to about 1.0 mM glutathione in the fluid covering the subject's respiratory tract. In another embodiment, administering the composition to a subject provides a concentration of about 0.5 mM to about 3.0 mM thiocyanate in the fluid covering the subject's respiratory tract.

[0227] In some embodiments, the oxidized glutathione (e.g., %GSSG) in the composition of the present invention 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% of the mass. based on the total weight of glutathione in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5°C in an atmosphere of N ₂ and/or ambient atmosphere).

[0228] In some embodiments, the reduced glutathione in the composition of the present invention 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% based on the total weight of glutathione in the composition of the present invention after storage of the composition of the present invention for 4 weeks at about 5° C (eg N ₂ atmosphere or ambient atmosphere).

[0229] 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 % based on the weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (at 5°C in an atmosphere of N ₂ and/or in an ambient atmosphere).

[0230] In some embodiments, the oxidized ascorbic acid in the composition of the present invention 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%. wt. based on the total weight of ascorbic acid in the composition according to the present invention after storage of the composition according to the present invention for 4 weeks (for example, at 5 °C in an atmosphere of N ₂ and/or ambient atmosphere).

[0231] In some embodiments, the composition is an aqueous solution or a dry powder. In another embodiment, the composition is administered to a subject via inhalation.

[0232] In some embodiments, administration of the composition inhibits the myeloperoxidase activity of the patient's neutrophils. In some embodiments, administration of the composition reduces the formation of neutrophil extracellular traps. In some embodiments, the administration of the composition suppresses the production of nitric oxide (II) by the patient's neutrophils. In some embodiments, administration of the composition reduces the exhaled nitric oxide fraction of the patient by at least 20%, at least 21%, at least 22%, at least 23%, at least 24% or at least 25%. In some embodiments, the implementation of the specified composition reduces the initial fraction of nitric oxide in the exhaled air of the patient by at least 20%.

[0233] In some embodiments, administration of the composition suppresses the production of at least one inflammatory cytokine. In some embodiments, at least one inflammatory cytokine includes a cytokine associated with macrophage activation and/or neutrophil and T cell recruitment. In some embodiments, at least one inflammatory cytokine comprises TNF-α. In some embodiments, at least one inflammatory cytokine comprises IL-6. In some embodiments, at least one inflammatory cytokine comprises IL-8.

[0234] In some embodiments, the patient suffers from or is at risk of developing a chronic inflammatory airway disease. In some embodiments, the inflammatory airway disease is cystic fibrosis, bronchiectasis (eg, non-cystic fibrosis bronchiectasis or cystic fibrosis bronchiectasis), asthma, chronic obstructive pulmonary disease, or pulmonary fibrosis. In another embodiment, the lung or airway disease or disorder is cystic fibrosis.

[0235] In other embodiments, the patient has undergone a lung transplant.

[0236] In some embodiments, the epithelium of the patient's respiratory tract is not colonized by bacteria. In some embodiments, the respiratory tract epithelium of the patient is colonized with bacteria. In some embodiments, the patient is suffering from an active bacterial infection. In other embodiments, the patient does not suffer from an active bacterial infection.

[0237] In some embodiments, the patient is a pediatric patient. In other embodiments, the patient is an adult patient.

[0238] In some embodiments, administration of the composition does not significantly alter the pH of the patient's airway endothelium.

Introduction

[0239] Therapeutic methods of the present invention may be carried out by administering to a subject a composition of the present invention. Administration of the composition of the present invention may be before, during or after the onset of the disease, condition or disorder of interest. Typically, pharmaceutical compositions are sterile and do not contain toxic, carcinogenic, or mutagenic compounds that would cause an adverse reaction when administered to a subject.

[0240] Compositions of the present invention are administered in a manner compatible with the dosage formulation, in an amount that will be effective for the desired result. In specific embodiments, the compositions of the present invention are administered to a subject in a therapeutically effective amount. The therapeutically effective amount of the compositions of the present invention required for use in treatment will vary depending on the nature of the condition being treated, the period of time for which the activity is needed, and the age and condition of the patient, and is ultimately determined by the attending physician. Dose sizes and intervals can be adjusted individually to provide plasma levels of glutathione sufficient to maintain the desired therapeutic effects. The desired dose may be administered as a single dose or as multiple doses administered at appropriate intervals, for example as one, two, three, four or more sub-doses per day. Multiple doses are often desirable or necessary. For example, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof may be administered at the following frequency: four doses delivered as a single dose per day at four day intervals (q4d x 4); four doses delivered as a single dose per day at three-day intervals (q3d x 4); one dose delivered per day at five-day intervals (qd x 5); one dose per week for three weeks (qwk3); five daily doses with two days rest, and five more daily doses (5/2/5); or any dosage regimen determined to be appropriate under the circumstances.

[0241] Compositions of the present invention may be administered over an extended period, such as at least about one month, at least about 2 months, at least about 3 months, at least about 6 months, or at least about 12 months. or longer (eg, as permanent lifelong treatment).

[0242] Any suitable dosing schedule can be followed. For example, the dosing frequency may be once a week. The dosing frequency may be once a day or several times a day. The dosing frequency may be more than once per week. The frequency of dosing may be more than once a day, for example, any of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 daily doses. Dosing frequency may be intermittent (eg, multiple daily dosing for 7 days followed by no dosing for 7 days, repeated for any 14 day time period such as 2 months, 4 months, 6 months or more). The dosing frequency may be continuous (eg, once a week for several weeks continuously).

[0243] Glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof used in the therapeutic method of the present invention may be administered in an amount of from about 0.005 to about 1000 milligrams per dose, from about 0.05 to about 250 milligrams per dose, or from about 0.5 to about 100 milligrams per dose. For example, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof may be administered at a dose of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150 , 200, 250, 300, 350, 400, 450 or 500 milligrams, including all doses from 0.005 to 1000 milligrams.

[0244] The dosage of glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination or composition thereof, may be from about 1 ng/kg to about 200 mg/kg, about 1 μg/kg to about 100 mg /kg or from 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 can be any dosage including, but not limited to, 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 mcg/kg, about 150 mcg/kg, about 175 mcg/kg, about 200 mcg/kg, about 225 mcg/kg, about 250 mcg/kg, about 275 mcg/kg, about 300 mcg/kg, about 325 µg/kg, about 350 µg/kg, about 375 µg/kg, about 400 µg/kg, about 425 µg/kg, about 450 µg/kg, about 475 µg/kg, about 500 µg/kg, about 525 µg/kg kg, about 550 µg/kg, about 575 µg/kg, about 600 µg/kg, about 625 µg/kg, about 650 µg/kg, about 675 µg/kg, about 700 µg/kg, about 725 µg/kg, about 750 mcg/kg, about 775 mcg/kg, about 800 mcg/kg, about 825 mcg/kg, about 850 mcg/kg, about 875 mcg/kg, about 900 mcg/kg, about 925 mcg/kg, about 950 µg/kg, about 975 µg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg kg, 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 above dosages are given as examples for the average case, but there may be individual cases in which higher or lower dosages are needed, and such dosages are within the scope of the present invention. In practice, the physician will determine the actual dosage regimen most appropriate for a particular patient, which may vary depending on the age, body weight and response of the particular patient.

[0245] In one embodiment, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof is delivered to the upper third of the nasal cavity, the upper part of the nasal passage, the olfactory region, and/or the sinus region. The olfactory region is a small area, typically about 2-10 cm ² in humans, located in the upper third of the nasal cavity to allow deposition and absorption by the olfactory epithelium and subsequent neuronal transport of olfactory receptors. Being located on the roof of the nasal cavity, in the upper part of the nasal passage, the olfactory region is suitable for delivery in some implementations, as it is the only known part of the body in which the site of the CNS comes into contact with the environment (Bois et al . Fundamentals of Otolaryngology , p.184, WB Saunders Co., Philadelphia, 1989).

[0246] In some embodiments, the composition of the present invention may be administered as a single "loading" dose, such as during bronchoscopy. In other embodiments, the methods of the present invention may be performed as needed by self-treatment.

[0247] Any dosing frequency can be used with any dosing amount. In addition, any dosing frequency and/or amount of dosing can be applied to any of the compositions according to the present invention.

[0248] The composition according to the present invention can be delivered in any suitable volume of administration. In exemplary embodiments of the present invention, the intranasal delivery volume is from about 25 microliters to 200 microliters, or from about 50 to 150 microliters, or from about 50, 100, 250, or 500 microliters to 1, 2, 3, 3.5 or 4 milliliters for a person. Typically, the volume of administration is chosen to be large enough to allow delivery of therapeutic amounts when considering dilution in ASL under maintenance conditions in the relatively "normal" airways (10-30 ml ASL) and in the cystic fibrosis (CF) airways (40-50 ml ASL or more and secretion of thick, persistent and highly infected mucus).

[0249] Compositions of the present invention may find use in veterinary and/or human medicine. Suitable subjects according to the present invention include, but are not limited to, mammals. As used herein, the term "mammal" includes, but is not limited to, primates (e.g. monkeys and humans), non-human primates (e.g. monkeys, baboons, chimpanzees, gorillas), cattle, sheep, goats, ungulates, pigs, horses, cats, dogs, lagomorphs, pinnipeds, rodents (eg rats, hamsters and mice), etc. In some embodiments of the present invention, the subject is a human. Human subjects include both males and females, as well as subjects of all ages, including neonates, infants, adolescents, young adults, adults, and the elderly.

[0250] In some embodiments, the composition of the present invention is administered one or more times per day (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day). In particular embodiments, the subject is a human.

[0251] In some embodiments of the methods of the present invention, the composition of the present invention may be administered by inhalation, intranasally, through the eye, through the ear, through sinus irrigation, or through a bronchoscope, or any combination thereof.

[0252] Intranasal administration of the composition according to the present invention can be achieved by any known method. In specific embodiments, intranasal administration is via inhalation (eg, using an inhaler, nebulizer, or nebulizer), alternatively, using a spray, tube, catheter, syringe, dropper, bag, pipette, compress, and the like.

[0253] By way of further illustration, the composition of the present invention can be administered intranasally in the form of (1) nasal drops, (2) powder or liquid sprays or aerosols, (3) liquids or semi-solids via syringe, (4) liquids or semi-solids substances by swab, compress or other similar means of application, (5) gel, cream or ointment, (6) infusion, or (7) by injection, or by any method currently known or developed in the art. In specific embodiments, the delivery method is nasal drops, spray, or aerosol. As used herein, aerosols can be used to deliver powders, liquids, or dispersions (solids in liquids).

[0254] In some embodiments, the pharmaceutical composition is administered in an upward direction to improve delivery to the upper third (eg, olfactory epithelium in the olfactory region) and side walls (eg, nasal epithelium) of the nasal cavity. In addition, to facilitate delivery to the olfactory region, the orientation of the subject's head in a tilted position or the orientation of the subject's body in the Mygind's position or in the head-down position (praying-to-Mecca position) can be used.

[0255] Formulations can be prepared in single or multi-dose form. In the latter case, dosing means may be provided. In the case of a dropper or pipette, it may be administered by the patient or caregiver by administering an appropriate predetermined volume of the composition. In the case of spraying, this can be carried out, for example, with a dosing spray pump.

[0256] In a further aspect, the present invention provides a nasal spray device comprising a composition of the present invention.

[0257] Many devices are known in the art for nasal delivery. Exemplary devices include particle dispersing devices, bi-directional devices, and devices that use chip-based inkjet technologies.

[0258] When a therapeutically effective amount of a composition of the present invention is administered by intravenous, dermal, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, taking into account pH, isotonicity, stability, and the like, is within the skill of the person skilled in the art. The composition for intravenous, dermal or subcutaneous administration usually contains an isotonic carrier.

[0259] the Compositions according to the present invention can be prepared for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain adjuvants such as suspending, stabilizing and/or dispersing agents.

[0260] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. In addition, suspensions of glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, may be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or carriers include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds and allow highly concentrated solutions to be obtained. Alternatively, the present composition may be in the form of a powder for reconstitution prior to use with a suitable vehicle, for example, sterile, pyrogen-free water.

[0261] Glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, may also be formulated for rectal administration, such as suppositories or retention enemas, for example, containing conventional suppository bases. In addition to the formulations previously described, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof, may also be formulated as a depot formulation. Such long acting formulations may be administered by implantation (eg subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, or any combination thereof can be prepared using suitable polymeric or hydrophobic materials (eg, in the form of an emulsion in a suitable oil) or ion exchange resin.

[0262] The compositions of the present invention may be administered orally, buccally or sublingually in the form of tablets containing excipients such as starch or lactose, or in the form of capsules or vaginal suppositories, either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavors or colorants. Such liquid formulations may be formulated with pharmaceutically acceptable additives such as suspending agents. Compositions according to the present invention can also be administered parenterally, for example, intravenously, intramuscularly, subcutaneously or intracoronary. For parenteral administration, the compositions of the present invention are generally administered in the form of a sterile aqueous solution which may contain other substances, for example salts or monosaccharides such as mannitol or glucose, to render the solution isotonic with blood.

[0263] The compositions of the present invention may be present, for example, as a solid formulation, such as a particulate formulation, or in the form of a solution. In a particulate formulation, the particles may be mixed with gases 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, the following formulations: eye drops, nebulizer formulations, topical gels and ointments, dry powders, particles, sprays, liquids, anesthesia machine or vaporizer formulations, autoinjectors, intrauterine devices, respimates, liniments, liposomes, lotions, formulations for intramuscular, intrathecal or subcutaneous injections, douches, infusions and face masks.

[0264] Solution formulations can be in the form of nasal sprays, formulations for use in nebulizers, and formulations for rectal administration, such as enemas and colonic administration. Solutions can also be used that include water-miscible organic solvents such as propylene glycol and/or glycerin and other ingredients commonly found in vaginal and rectal lubricants. Regardless of the solvents used, the solvent is typically present in a weight ratio of from about 15 to about 85 weight percent based on the weight of the solids, and more often from about 50 to about 85 weight percent.

[0265] The compositions and/or formulations of the present invention can be used to treat mucosal disorders by delivering the compositions and/or formulations to the mucosal(s) to be treated. In some embodiments, the mucosa may be in or near the lungs, for example, in the deep regions of the lungs (alveolar region), and in other embodiments, the mucosa(s) may be in or near one or more of : eyes, mouth, nose, rectum and/or vagina.

Additional Therapeutic Agents

[0266] In another embodiment, the present invention provides a method comprising administering to a subject an effective amount of an additional therapeutic agent.

[0267] Compositions containing glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, an organic acid, or any combination thereof, can also be combined with antibiotics to increase the effectiveness of treatment.

[0268] In another embodiment, one of the objectives of the present invention is to identify clinical isolates in a patient sample and test the effectiveness of treatment in vitro by treatment with a composition containing glutathione, a glutathione derivative, a glutathione conjugate, a pharmaceutically acceptable salt thereof, an organic acid, or any combination thereof.

[0269] Drugs administered to the lungs are often associated with certain side effects, in some cases due to dosage, and in other cases due to damage to lung tissue. In some embodiments, the therapeutic agents in combination with the compositions described herein are effective at lower doses, and at such lower doses, the incidence of side effects may be reduced. In other embodiments where the therapeutic agent interacts adversely with lung tissue, the compositions described herein can help restore homeostasis to lung tissue and thus help minimize or eliminate damage caused by therapeutic agents.

[0270] In another embodiment, the present invention provides a therapeutic agent selected from the group consisting of fluticasone, budesonide, mometasone, ciclesonide, flunisolide, beclomethasone, albuterol, levalbuterol, ipratropium, tiotropium, formoterol, arformoterol, indacaterol, aclidinium, and pirbuterol, penicillins such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, caiston, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin and ticarcillin, and combinations of penicillins with other therapeutic agents such as amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, and any combination thereof.

[0271] 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, caiston, pirbuterol, and any combination thereof. .

[0272] Additional therapeutic agents that can be combined with the compositions and compositions of the present invention include, but are not limited to, fluticasone (for example, sold as Flovent diskus 50 or as Flonase, GlaxoSmithKline), budesonide (for example, sold as Pulmicort respules or Rhinocort by Astra Zeneca ("AZ"), mometasone (sold as Nasonex spray or Asmanex Twisthaler by Merck/SP), ciclesonide (sold as Alvesco or Onmaris by Takeda Pharmaceuticals), flunisolide (sold as Aerobid by Roche Palo or Aerospan HFA by GSK), beclomethasone (sold as Qvar or Onasl by Teva Pharmaceuticals), albuterol (sold as ProAir HFA by Teva and Ventolin HFA by GSK), levalbuterol (sold as Xopenex by Sunovion), ipratropium (sold as Atrovent by BI), tiotropium (marketed as Spiriva by BI), salmeterol (marketed as Serevent by GSK), formoterol (marketed as Foradil by Novartis and Perforomist by Dey Pharma), armoterol (marketed as Brovana by Sunovion), indacaterol (marketed as Arcapta by Novartis), aclidinium (sold as Tudorza by Forest Labs), pirbuterol (sold as Maxair by Medicis), and any combination thereof.

[0273] The present methods encompass administering to a subject one or more additional therapeutic agents in combination with a composition of the present invention. In one embodiment, the composition of the present invention further comprises or the composition is administered in combination or alternately with an additional therapeutic agent. That is, in some embodiments, the composition and additional therapeutic agents are directed to the same site in the same formulation, and in other embodiments, the composition may be administered by one route and the additional therapeutic agent(s) may be administered by another route.

[0274] In some embodiments, the additional therapeutic agent comprises cystic fibrosis transmembrane receptor (CFTR) therapy. In some embodiments, the CFTR therapy includes 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 combinations thereof), CFTR potentiator/modulator (e.g., VX-770 (ivacaftor, licensed under the brand name Kalydeco), QBW 251, VX-561, PT1-808, or combinations thereof), CFTR RNA modifier (eg QR-100, MRT5005 or combinations thereof) or any combination thereof.

[0275] In some embodiments, the additional therapeutic agent comprises a CFTR amplifier. CFTR amplifiers increase the amount of CFTR protein in the cell. In cystic fibrosis, there are six classes of mutations that provide varying degrees of CFTR dysfunction. The most serious mutations in cystic fibrosis have a genetic defect, such as a premature stop codon, that prevents CFTR from being converted to a protein. Therapies proposed to treat this mutation include amplifiers that increase the amount of CFTR RNA in the cell. They are also known as "read-through therapies". In some embodiments, the CFTR cycler is PTI-428.

[0276] In some embodiments, the additional therapeutic agent includes a CFTR corrector. CFTR correctors transfer the CFTR protein to the cell surface. The most common class of mutations in cystic fibrosis is class II, in which CFTR is misfolded and not transported properly to the cell surface. Therapy used to treat class II mutations, such as dF508, includes a combination of correctors that promote protein folding and/or protein transfer to the cell surface and potentiators that increase CFTR membrane channel opening 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.

[0277] In some embodiments, the additional therapeutic agent comprises a CFTR potentiator/modulator. CFTR potentiators/modulators increase ion transport through CFTR at the cell surface. Class III and IV mutations include G551D and are mutations in which CFTR is impaired in membrane channel opening or ion transport. Potentiators/modulators can be used in patients with these mutations to increase channel opening and ensure proper ion transport. In some embodiments, the CFTR potentiator/modulator is selected from the group consisting of VX-770 (ivacaftor), QBW 251, VX-561, PT1-808, and combinations thereof.

[0278] In some embodiments, the additional therapeutic agent contains an RNA modifier. RNA modifiers can be used to ensure that the genetically engineered CFTR is delivered to the cell surface. Mutations of classes V-VI lead to a decrease in CFTR proteins on the cell surface, either by reducing the number of CFTR transcripts or by increasing the turnover rate. RNA amplifiers and modifiers can be used to treat these mutations. In some embodiments, the RNA modifier is selected from the group consisting of QR-100, MRT5005, or a combination thereof.

[0279] In some embodiments, the CFTR therapy comprises a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or their combinations) and a CFTR potentiator/modulator (e.g. VX-770 (ivacaftor), QBW 251, VX-561, PT1-808 or combinations thereof). In some embodiments, the CFTR therapy includes: a combination of VX-770 (ivacaftor) and VX-809 (lumacaftor), which is licensed under the brand name Orkambi; a combination of VX-770 (ivacaftor) and VX-661 (tezacaftor), which is approved for use under the trademark Symdeko; a combination of VX-770 (ivacaftor), VX-661 (tezacaftor), and VX-445; a combination of VX-770 (ivacaftor), VX-661 (tezacaftor), and VX-659; a combination of VX-770 (ivacaftor), VX-661 (tezacaftor), and VX-152; or a combination of VX-770 (ivacaftor) and GLPG2222.

[0280] In some embodiments, the CFTR therapy comprises a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or their combinations) and a CFTR cycler (eg PTI-428). In some embodiments, the CFTR therapy includes: a combination of VX-770 (ivacaftor) and VX-809 (lumacaftor), which is licensed under the trademark Orkambi, and PTI-428.

[0281] In some embodiments, the CFTR therapy comprises a CFTR corrector (e.g., VX-809 (lumacaftor), VX-661 (tezacaftor), VX-445, VX-659, VX-152, FDL169, GLPG2222, PT-801, or their combinations), a CFTR potentiator/modulator (eg VX-770 (ivacaftor), QBW 251, VX-561, PT1-808 or combinations thereof) and a CFTR amplifier (eg PTI-428).

In some embodiments, the CFTR therapy includes a CFTR potentiator/modulator and a CFTR corrector to enhance CFTR membrane channel opening/ion transport.

[0283] In some embodiments, administration of CFTR therapy occurs simultaneously or sequentially with administration of a composition of the present invention to a subject and in any order (eg, the composition may be administered before or after CFTR therapy). In some embodiments, the CFTR therapy is administered orally and the composition of the present invention is administered via inhalation. In some embodiments, both the cystic fibrosis therapy and the composition of the present invention are administered via inhalation.

[0284] In some embodiments, the composition of the present invention is administered in combination with VX-770, VX-809, or a combination of VX-770/VX-809, and the administration to the subject is simultaneous or sequential and in any order (for example, the composition can be administered before or after the VX-770/VX-809 combination).

[0285] The additional therapeutic agent is usually selected from drugs known to be suitable for the treatment of the disease, condition or disorder affecting the subject in need thereof. The choice of additional(s) therapeutic(s) agent(s) will depend on the disease, condition or disorder that is being treated or prevented in the subject. This definition is within the purview of those skilled in the art, especially in light of the present invention.

[0286] In one embodiment, the subject is administered one additional therapeutic agent. 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. Examples of non-limiting therapeutic agents are antifungal agents, antiviral agents, antibacterial agents, anti-inflammatory agents, immunosuppressants, bronchodilators, airway modulators, alpha lipoic acid, alpha tocopherol, docosahexanoic acid, proline, glycine, curcumin, arginine. , thiocyanate, glutathione, oxidized glutathione, reduced glutathione, cysteine, hypothiocyanate, lactoferrin, and lactoperoxidase, and any combinations thereof. In another embodiment, the one or more therapeutic agents are glutathione, oxidized glutathione, or reduced glutathione.

[0287] The composition of the present invention and the additional therapeutic agent may be administered together as a single dose or separately in multiple doses, wherein the composition of the present invention is administered before the additional therapeutic agent, after the additional therapeutic agent, or simultaneously with the additional therapeutic agent. The subject may be administered one or more doses of a composition of the present invention and/or one or more doses of an additional therapeutic agent.

[0288] In another embodiment, the additional therapeutic agent treats the desired disorder for which it is administered, but may cause certain side effects, such as dryness of the mucous membranes, resulting in discomfort and / or damage, which can be eliminated by administration of the composition according to the present invention .

[0289] In another embodiment, the composition according to the present invention treats the desired disorder for which it is administered, but may cause certain side effects, for example, dryness of the mucous membranes, which leads to discomfort and / or damage, which can be eliminated by the introduction of an additional therapeutic agent .

[0290] In some embodiments, one or more additional therapeutic agents and the composition of the present invention both treat the underlying disorder, albeit in different ways, thus an additive or synergistic effect can be achieved. As a result, in some aspects of this embodiment, lower doses of the additional therapeutic agent may be more effective, and lower doses may result in fewer side effects or provide other benefits to the subject.

[0291] When the composition of the present invention and one or more additional therapeutic agents are not administered simultaneously, it should be understood that they can be administered in any order to a subject in need thereof. For example, the composition according to the present invention can be administered, 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 administration of one or more additional therapeutic agents to the subject in need thereof. In some embodiments, the composition of the present invention and one or more additional therapeutic agents are administered at 1 minute intervals, at 10 minutes intervals, at 30 minutes intervals, at less than 1 hour intervals, at 1 hour intervals, at 1 hour to 2 hour intervals. hours, with an interval of 2 hours to 3 hours, with an interval of 3 hours to 4 hours, with an interval of 4 hours to 5 hours, with an interval of 5 hours to 6 hours, with an interval of 6 hours to 7 hours, with an interval of 7 hours to 8 hours, with an interval of 8 hours to 9 hours, with an interval of 9 hours to 10 hours, with an interval of 10 hours to 11 hours, with an interval of 11 hours to 12 hours, with an interval of not more than 24 hours or from no more than 48 hours apart. In one embodiment, the components of the combination therapy are administered at intervals of from about 1 minute to about 24 hours.

[0292] In another embodiment, the additional therapeutic agent is a corticosteroid (ICS) or inhaled bronchodilator.

Prevention

[0293] In addition to providing methods of treatment, the compositions of the present invention can also be used to alleviate symptoms or provide prevention of various diseases and disorders associated with clinical isolate bacterial infection, impaired mucociliary clearance, and/or airway inflammation.

[0294] Embodiments of the present invention may be further defined with reference to the following non-limiting examples, which describe in detail the preparation of specific compositions according to the present invention and methods of using the compositions according to the present invention. Those skilled in the art will appreciate that many modifications, both to materials and methods, can be made without departing from the scope of the present invention.

Analysis of a clinical isolate bacterium in vitro

[0295] Clinical isolate (CI) samples from a subject may include, but are not limited to, sputum, respiratory samples, surgical tissue samples, autopsy samples, and blood. Samples can be fixed and/or stored at -20°C or -80°C.

[0296] Isolation of clinical isolate (CI) bacterial species can be carried out by any method known in the art. CI can be isolated from pure cultures (that is, having only one species of CI) or from a sample with several bacteria. Common methods for isolating CI include, but are not limited to, serial dilution of the original clinical specimen, centrifugation, plate marking with or without an initial stage of growth in liquid media, and growth of single bacterial colonies on solid or liquid media. CI is grown using any media known in the art, including but not limited to planktonic and solid media.

[0297] Clinical isolate bacteria can be identified using any technique known in the art, including, but not limited to, morphological techniques (eg, Gram stain, rapid acid stain); biochemical, serological, or protein expression tests (eg, antibody interaction, slide agglutination, ELISA, Western blotting, immunohistochemistry, immunofluorescence, flow cytometry); phage typing; and DNA and/or RNA based techniques (e.g., DNA sequencing, G+C comparisons, gPCR, RT-PCR, qPCR, rRNA sequencing, restriction fragment length polymorphism (RFLP) DNA typing), nucleic acid hybridization (e.g., Northern blot, Southern blot), microarray, in situ hybridization including FISH).

[0298] Growth of clinical isolates in the presence of a drug (e.g. antibiotic) with or without a composition of the present invention, in the absence of a drug with or without a composition of the present invention, or a placebo with or without a composition of the present invention can be tested using any techniques known in the art, including, but not limited to, dilution and slow growth of CI; determination of minimum inhibitory concentrations (MIC); determination of the minimum concentration that eliminates biofilm (MBC); and other methods known in the art (e.g., disk diffusion method, E-test (AB Biodisk, Solna, Sweden), any commercially available automated susceptibility testing system (e.g., Vitek System (bioMerieux, France), Walk-Away System (Dade International, Sacramento, CA), an enzyme (eg, beta-lactamase)).CI growth was tested in planktonic or biofilm cultures.

[0299] In some aspects, the present invention includes the measurement of minimum inhibitory concentrations (MICs). MIC can be determined on Mueller-Hinton (MH) agar plates or in planktonic cultures by broth microdilutions in MH. MICs can be determined for clinical isolate strains and/or laboratory control PAO1 strains. 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%.

[0300] In some aspects, the present invention includes measuring in vitro biofilm formation for clinical isolate bacteria. In some embodiments, an overnight culture of bacteria is grown in LB with and without a composition of the present invention with shaking. The culture is then diluted 1:100 and 100 µl aliquots are added to a 96-well plate which is incubated for 72 hours at 37° C. to ensure proper growth of the clinical isolates. After two to three washes with water, crystal violet is added for 15 minutes, then washed three times with water, then 95% ethanol is added. The materials are then transferred to a fresh 96-well plate and absorbance is determined at 540 nm.

[0301] In some aspects, the present invention includes measuring the minimum biofilm-eliminating concentration (MBC) of CI. For Biofilm Eliminating Minimum Concentrations (MBC), a protocol similar to the MIC protocol can be used, except that the bacteria are plated on a plate used for bacterial adhesion. In some embodiments, optical densities are measured to determine MBC for a bacterial isolate. In some embodiments, the assay involves forming 96 identical biofilms on plastic pins on the lid of the MBC device. The biofilms are then exposed to antibiotics for a certain period of time, then placed in fresh bacteriological medium in a second 96-well plate and incubated overnight. The MBC value represents the lowest dilution that prevents bacterial re-growth from the treated biofilm.

[0302] The present invention allows any of the above methods in the presence or absence of nitrate.

[0303] The present invention allows for any of the above techniques in the presence or absence of oxygen.

[0304] The present invention allows for any of the above techniques in the presence or absence of one or more antibiotics.

Examples

[0305] It should 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 made to those skilled in the art and should be included within the spirit and scope of this application. .

Example 1: Stability: Determination of Reduced Glutathione and Ascorbic Acid Levels

[0306] Composition 1 was prepared from 150 mg/ml glutathione, 88 mg/ml ascorbic acid and 84 mg/ml sodium bicarbonate based on a total weight of 322 mg/ml, which corresponds to a % of the total weight of about 46.6%, 27.3% and 26.1% respectively. The molar ratio of components in Composition 1 was 0.49 M glutathione, 0.50 M ascorbic acid and 1 M HCO ₃ .

[0307] Composition 2 was prepared from 150 mg/ml glutathione, 126 mg/ml ascorbic acid, and 84 mg/ml sodium bicarbonate based on a total weight of 360 mg/ml, which corresponds to a % of the total weight of about 33%, 38, 9% and 28.9% respectively. The molar ratio of the components in Composition 2 was 0.49 M glutathione, 1 M ascorbic acid and 1 M HCO ₃ .

[0308] The amount of oxidized glutathione and ascorbic acid was determined for Composition 1 at pH 5.5, 6.0 and 6.5. Oxidized glutathione is associated with the formation of protein carbonyls via glutathionylation, which occurs when oxidized glutathione dissociates and attaches to proteins. % Oxidized glutathione (%GSSG) was determined after 4 weeks of storage of Composition 1 at pH 5.5, 6.0 and 6.5 under N ₂ sparging and ambient conditions. In addition, when ascorbic acid is oxidized to dehydroascorbate (DHA), DHA can be broken down and lead to the formation of protein adducts in a process called ascorbylation (Simpson et al., Biochim biophys Acta 2000; 1501:12-24). The % Reduced Ascorbic Acid maintained in Composition 1 (%ASC) was determined after 4 weeks of storage of Composition 1 at pH 5.5, 6.0 and 6.5 under N ₂ sparging conditions (anaerobic conditions) and at ambient conditions. In particular, samples under nitrogen sparge were mixed under anaerobic conditions under nitrogen sparge, and the samples were packaged under nitrogen sparge. Samples at ambient conditions were mixed in room air without nitrogen sparging. 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.

[0309] 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. Moreover, when oxygen is present in the solution, reduced glutathione (GSH) helps maintain the concentration of ascorbic acid, but GSH itself is oxidized to GSSG at higher concentrations. These results demonstrate that the pH of Formulation 1 is important in maintaining glutathione and ascorbic acid in their reduced state and that the molar ratios of glutathione and ascorbic acid in Formulation 1 further stabilize the oxidation rates of both glutathione and ascorbic acid.

Table 1 :

Compound pH Atmosphere %GSSG %ASC Composition 1 5.5 bubbling N ₂ 2.86 89.47 Composition 2 6.0 bubbling N ₂ 6.54 91.20 Composition 3 6.5 bubbling N ₂ 9.11 91.27 Composition 4 5.5 Environment 6.44 90.66 Composition 5 6.0 Environment 15.97 90.25 Composition 6 6.5 Environment 19.94 91.25 Pure ascorbic acid Environment 77.88

Example 2 Minimum Inhibitory Concentration (MIC) of Glutathione Containing Compositions Against Clinical Isolate Bacteria in vitro

[0310] Composition 1 was prepared from 150 mg/ml glutathione, 88 mg/ml ascorbic acid and 84 mg/ml sodium bicarbonate based on a total weight of 322 mg/ml, which corresponds to a % of the total weight of about 46.6%, 27.3% and 26.1% respectively. The molar ratio of components in Composition 1 was 0.49 M glutathione, 0.50 M ascorbic acid and 1 M HCO ₃ .

[0311] MIC for Composition 1 (glutathione, ascorbic acid and HCO₃); glutathione and ascorbic acid; and HCO₃ have been tested against clinical isolates, includingBurkholderia cepacia,Pseudomonas aeruginosa (Schroeter) Migula (ATCC 27853),Pseudomonas aeruginosa (Schroeter) Migula (ATCC 9027),Pseudomonas aeruginosa (Schroeter) Migula (ATCC BAA-2114),Klebsiella pneumoniamethicillin resistantStaphylococcus aureus; laboratory strainsMoraxella catarrhalis,Stenotrophomonas maltophilia,Acinetobacter baumannii; and eight samples of mucus-forming and non-mucus-formingPseudomonas aeruginosa from human lung clinical isolates. Also tested MIC for laboratory clinical isolatePseudomonas aeruginosa (Schroeter) Migula (ATCC BAA-47 (PAO1)). MICs were determined under anaerobic conditions with nitrate supplementation, aerobic conditions with nitrate supplementation, or ambient conditions.

[0312] Clinical isolates were inoculated in duplicate on agar plates overnight under anaerobic conditions at 36 °C ± 1.0 °C in a 96-well plate. Laboratory isolates of Burkholderia cepacia were also grown in duplicate overnight under aerobic conditions at 36°C ± 1.0°C in a 96-well plate. Colonies were then selected from each clinical isolate grown on agar plates to inoculate 96-well plates in duplicate and tested under the conditions of the three test solutions: Composition 1 (glutathione, ascorbic acid and HCO ₃ ); glutathione and ascorbic acid; and HCO ₃ were diluted and added to the medium. Ten dilutions were tested for each test solution: 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57%, 0.78%, 0.39%, 0.2% and 0 ,1%. Colony isolates were incubated for 12 hours or 24 hours at 36 °C ± 1.0 °C under anaerobic conditions in a medium containing Luria agar/broth and nitrate with one of the three test solutions. In an additional test, Burkholderia cepacia and Klebsiella pneumoniae were grown in aerobic media containing pure Luria agar/broth with Composition 1. The MIC for Composition 1 was also determined for specific laboratory and clinical isolates of Pseudomonas aeruginosa , Moraxella catarrhalis , Stenotrophomonas maltophilia , Acinetobacter baumannii and Burkholderia cepacia , grown under aerobic conditions with a nitrate content of 1%. MIC was determined by visual inspection and OD ₆₂₀ . MICs at 12, 24 and/or 48 hours in the clinical isolates tested are shown in Table 2.

Table 2 :

organism Anaerobic (+ nitrate) Aerobic (+ nitrate) Aerobic (environment) Composition 1 GSH+ASC HCO3 Composition 1 Composition 1 BC 25416 25% (24 h); 6.25% (48 hours) 6.25% (12 hours) 3.13% (12 hours); 25% (24h) Kp 4352 25% (12 hours); 25% (24h) 50% (12 hours); 50% (12 hours) Pa 27853 12.5% (12 hours); 12.5% (24h) Pa 9027 6.25% (12 hours); 25% (24h) 6.25% (12 hours) Pa BAA-2114 3.13% (12 hours) 0.39% (12 hours) Pa BAA-47 (PAO1) 6.25% (12 hours) 0.39% (12 hours) 12.5% (12 hours) 6.25% (12 hours) Pa UAB-NM-1 12.5% (12 hours) 0.78% (12 hours) 12.5% (12 hours) 1.56% (12 hours) Pa UAB-M-2 6.25% (12 hours) 0.39% (12 hours) 12.5% (12 hours) 3.13% (12 hours) Pa UAB-NM-3 6.25% (12 hours) 0.78% (12 hours) 12.5% (12 hours) Pa UAB-4 6.25% (12 hours) Pa UAB-NM-5 6.25% (12 hours) 0.78% (12 hours) 12.5% (12 hours) MRSA UAB-1 12.5% (12 hours) 0.39% (12 hours) 25% (12 hours) MRSA UAB-2 25% (12 hours) 0.39% (12 hours) MRSA UAB-3 6.25% (12 hours) 0.39% (12 hours) 25% (12 hours) MRSA UAB-4 25% (12 hours) 0.39% (12 hours) Mc ATCC 25238 1.56% (12 hours) SM ATCC 13636 6.25% (12 hours) Ab ATCC 19606 6.25% (12 hours)

[0313] Bc: Burkholderia cepacia

[0314] Kp: Klebsiella pneumonia

[0315] Pa: Pseudomonas aeruginosa

[0316] Pa UAB: P. aeruginosa clinical lung isolate

[0317] MRSA: Methicillin-resistant Staphylococcus aureus

[0318] Mc: Moraxella catarrhalis

[0319] Sm: Stenotrophomonas maltophilia

[0320] Ab: Acinetobacter baumannii

[0321] NM: Non-mucus forming

[0322] M: Mucus

[0323] An in vitro MIC analysis, that is, the lowest concentration of a test substance or drug that inhibits bacterial growth, can be performed by optical density or visualization of no growth. MIC is often used to determine the effectiveness of an antibacterial drug. A lower MIC usually corresponds to a more active drug.

[0324] The results show that Composition 1 inhibits bacteria even though bicarbonate in solution reduced the in vitro efficacy of glutathione. The MIC for bicarbonate was separately measured for representative bacterial isolates to determine the contribution of bicarbonate to in vitro inhibition.

[0325] 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 . It is noteworthy that the phenotypes of laboratory and clinical isolates of Pseudomonas aeruginosa can differ significantly depending on the conditions in which they are kept, the number of passages and the environment from which they were isolated. Therefore, PAO1 does not predict inhibition of other laboratory or clinical isolates.

[0326] Anaerobic and aerobic effects on B. cepacia and K. pneumoniae were also evaluated in vitro . Both of these bacteria are particularly resistant and difficult to inhibit even at high concentrations of antibacterial agents. They are both associated with clinical worsening of chronic inflammatory airway diseases. For B. cepacia, the inhibitory effect lasted for 48 hours.

[0327] Composition 1 was also able to effectively inhibit MRSA in vitro . This was unexpected due to the high salt concentration in Composition 1, since MRSA generally grows well at high salt concentrations.

[0328] Composition 1 was also able to effectively inhibit mucus-forming Pseudomonas aeruginosa from a clinical isolate (Pa UAB-M-4). The mucus-forming phenotype is associated with multidrug resistance and clinical deterioration in patients with chronic inflammatory airway disease.

[0329] Composition 1 was also able to effectively inhibit Burkholderia cepacia , Moraxella catarrhalis , Stenotrophomonas maltophilia and Acinetobacter baumannii under aerobic conditions at 1% nitrate. Each of these bacteria has been recognized as a resurgent pathogen.

Example 3: In vitro analysis of mucociliary transport rate in dF508-/- primary human bronchial epithelial cells

[0330] The ability of Composition 1 (prepared in Example 1) to influence the rate of mucociliary transport (MCT) of primary human bronchial epithelial cells isolated from patients with cystic fibrosis was evaluated in vitro . The mucociliary transport rate provides a measure of mucociliary clearance, which is the mechanism by which epithelial cells clear mucus from the airways.

[0331] Briefly, primary human bronchial epithelial cells were isolated from the respiratory tract of patients with cystic fibrosis and were finally differentiated. All cells isolated from patients with cystic fibrosis were represented by the dF508-/- genotype. Cells were incubated with phosphate buffered saline (PBS), Composition 1 at high concentration (25%) or Composition 1 at low concentration (12.5%). Micro-optical coherence tomography (uOCT) was used to assess the rate of mucociliary transport according to the methods described in Liu, L., et al. , PLoS One. 2013; 8(1): e54473. The ratio of glutathione and ascorbic acid to bicarbonate salt in Composition 1 was 150 mg/ml glutathione: 88 mg/ml ascorbic acid: 84 mg/ml bicarbonate salt, which is represented by the molar ratio of 0.5 mol glutathione: 0.5 mol ascorbic acid: 1 mole of bicarbonate.

[0332] As shown in FIG. 1 , incubation with Composition 1 increased mucociliary transport of primary human bronchial epithelial cells dF508-/- compared to incubation with PBS control. In addition, at all time points tested, incubation with Composition 1 at a high concentration resulted in an increase in the rate of mucociliary transport compared to incubation with Composition 1 at a low concentration. These data demonstrate that the composition enhances mucociliary transport of primary human bronchial epithelial cells in a dose-dependent manner.

Example 4: In vitro analysis of mucociliary transport rate in dF508-/- primary human bronchial epithelial cells

[0333] The rate of mucociliary transport of primary human bronchial epithelial cells dF508 -/- after administration of the individual components of Composition 1 (obtained in Example 1) was evaluated in vitro . As described above in Example 2, primary human bronchial epithelial cells were isolated from the respiratory tract of patients with cystic fibrosis, and uOCT was used to assess the rate of mucociliary transport according to the methods described in Liu, L., et al. , PLoS One. 2013; 8(1): e54473. Cells were incubated with Composition 1, bicarbonate, glutathione, bicarbonate+glutathione, or bicarbonate+ascorbate prior to uOCT analysis.

[0334] As shown in FIG. 2 , incubation of dF508 -/- human primary bronchial epithelial cells with Composition 1 increased their mucociliary transport rate compared to incubation with PBS control, pure bicarbonate solution, pure glutathione solution, or glutathione and bicarbonate solution. Incubation of cells with solutions containing pure glutathione, pure bicarbonate, or a combination of glutathione and bicarbonate did not change the rate of mucociliary transport compared to incubation with PBS control. These data demonstrate that solutions containing pure glutathione and bicarbonate (alone or in combination) do not significantly affect the rate of mucociliary transport, while Composition 1 enhances mucociliary transport of primary human bronchial epithelial cells isolated from patients with cystic fibrosis.

Example 5: In vitro analysis of neutrophil extracellular trap (NET) formation in human neutrophils

[0335] Composition 2 was prepared from 150 mg/ml glutathione, 126 mg/ml ascorbic acid, and 84 mg/ml sodium bicarbonate based on a total weight of 360 mg/ml, corresponding to a % of the total weight of about 33%, 38, 9% and 28.9% respectively. The molar ratio of the components in Composition 2 was 0.49 M glutathione, 1 M ascorbic acid and 1 M HCO ₃ .

[0336] The ability of Composition 2 to control the formation of neutrophil extracellular traps (NET) in human neutrophils was evaluated in vitro . NET is defined as the release of myeloperoxidase (MPO), DNA strands, and other pro-inflammatory components (e.g., neutrophil elastase) from neutrophils, and NET promotes inflammation and infection in chronic inflammatory airway diseases (see, e.g., Martinez-Aleman, Front Cell Infect Microbiol. 2017; 7:104).

[0337] Briefly, neutrophils were isolated from healthy volunteers and incubated with phorbol myristate acetate (PMA) (50 mm) or control solution for three hours. Cells were then incubated with dilutions of Composition 1 or Composition 2 and stained with DAPI (4',6-diamidino-2-phenylindole) to detect extracellular DNA release and MPO was detected. Images of NET formation were obtained using fluorescence microscopy.

[0338] As shown in FIG. 3 , neutrophils incubated with pure PMA showed significant NET production, while neutrophils incubated with Composition 2 showed a marked decrease in NET production. In particular, neutrophils incubated with a solution containing 50% dilution of Composition 2 showed the least amount of NET formation after PMA stimulation, while neutrophils incubated with significantly more dilute solutions of Composition 2 (25%, 12.5%, 6. 25% and 3.125%) showed a less intense decrease in NET formation. These data demonstrate that Composition 2 reduced NET production in human neutrophils in a dose dependent manner.

Example 6: In vitro analysis of the release of nitric oxide (II), NO, from human neutrophils

[0339] The release of nitric oxide (II), NO, from human neutrophils after administration of Composition 1 (as prepared in Example 1) was evaluated in vitro . NO is released from neutrophils in chronic inflammatory airway disease and is associated with increased lung infections and inflammation due to the conversion of NO to nitrate and nitrate (see, for example, Francoeur, C. and Denis, M. Inflammation (1995) 19: 587).

Briefly, neutrophils were isolated from healthy volunteers, incubated with 10 μg/ml lipopolysaccharide (LPS) or control solution, and incubated with various dilutions of Formulation 1. Cells were stained with DAF-FM diacetate for NO detection, and images of NO release were obtained using fluorescence microscopy.

[0341] As shown in FIG. 4 , neutrophils incubated with pure LPS showed a significant NO release, while neutrophils incubated with Composition 1 showed a marked decrease in NO release. In particular, neutrophils incubated with a solution containing 50% dilution of Composition 1 showed the least amount of NO release after LPS stimulation, while neutrophils incubated with significantly more dilute solutions of Composition 1 (25%, 12.5%, 6. 25% and 3.125%) showed a less intense decrease in NO release. These data demonstrate that Composition 1 reduced NO release in human neutrophils in a dose dependent manner.

Example 7 Clinical Study of Exhaled Nitric Oxide, NO, in a Bilateral Lung Transplant Patient

[0342] The effect of Composition 1 (as prepared in Example 1) on pathological exhaled NO was evaluated in a human patient in vivo . Briefly, exhaled nitric oxide (II) fraction (FeNO) measurements were performed on a bilateral lung transplant patient before Composition 1 administration and again immediately after Composition 1 administration using standard methods (see, e.g., 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). While the patient had a FeNO value of 119 ppb before Composition 1, the patient's FeNO decreased to 24 ppb immediately after Composition 1. In some patients, normal NO levels are considered to be 20-50 ppb, and NO levels above 50 ppb are associated with infection and inflammation. Accordingly, the patient experienced a decrease in NO levels from pathological to normal NO levels after a single treatment with Composition 1. These results were reproducible (data not shown). These data demonstrate the strong beneficial effect of Composition 1 in reducing exhaled NO levels in lung transplant patients.

Example 8: In vitro analysis of inflammatory cytokines of human neutrophils

[0343] In vitro inflammatory cytokine assays were performed with human neutrophils after administration of dilutions of Composition 1. Briefly, polymorphonuclear leukocytes (PMN) were adjusted to a density of 4×10 ⁶ /ml in DMEM (Gibco 11995-065) without the addition of fetal bovine serum (FBS ), and 0.25 ml of the resulting solution was added to the wells of a 24-well plate in duplicate (1x10 ⁶ PMN/2-cm ² per well). The cells were then pre-treated with 0.25 ml dilutions of 3.125%, 6.25%, 12.5%, 25% and 50% of the 2-fold dose of Composition 1 (88 mg/ml vitamin C) for 1 hour at 37°C. . Then, 100X LPS E. coli O111:B4 LPS (Sigma L4391) at 5 µl/well was added to half of the wells and incubated for 4 hours at 37°C. Conditioned media were collected from the wells and frozen for batch analysis of cytokines. Quantification of human IL-6, human IL-8/CXCL8 and human TNF-α in conditioned media was performed under standard conditions using R&D Systems Quantikine ELISA kits.

[0344] As shown in Table 3 and FIG. 5A-5C , human neutrophils stimulated with LPS and incubated with Composition 1 dilutions secreted significantly lower amounts of pro-inflammatory cytokines compared to those incubated with control (pure medium). In particular, neutrophils stimulated with LPS and incubated with pure medium showed a TNF-α concentration of 23.8 pg/ml, while neutrophils incubated with 12.5% Composition 1, 25% Composition 1, or 50% Composition 1, showed no detectable TNF-α (Table 3 and FIG. 5A ). Neutrophils incubated with 6.25% of Composition 1 showed a TNF-α concentration of 11.7 pg/ml, about half that of neutrophils incubated with pure medium (Table 3 and FIG. 5A ). Similarly, neutrophils stimulated with LPS and incubated with 50% Composition 1, 25% Composition 1, or 12.5% Composition 1 did not produce any detectable levels of IL-6, while neutrophils incubated with 6.25% Composition 1 showed an approximately 6-fold reduction in IL-6 levels compared to neutrophils incubated with pure medium (Table 3 and FIG. 5B ). Moreover, neutrophils stimulated with LPS and incubated with 50% Composition 1, 25% Composition 1, or 12.5% Composition 1 did not produce any detectable levels of IL-8, while neutrophils incubated with 6.25% Composition 1 showed an approximately 3-fold reduction in IL-8 levels compared to neutrophils incubated with pure medium (Table 3 and FIG. 5C ). These data demonstrate that the administration of Composition 1 results in dose-dependent inhibition of pro-inflammatory cytokines involved in chronic inflammatory diseases such as cystic fibrosis, lung transplantation, and chronic obstructive pulmonary disease.

Table 3:

LPS processing Treatment with Composition 1 IL-6 (pg/ml) IL-8 (pg/ml) TNF-α (mg/ml) Control Wednesday 0.0 289.0 0.0 Control 50% Composition 1 0.0 1.0 0.0 Control 25% Composition 1 0.0 0.0 0.0 Control 12.5% Composition 1 0.1 0.0 0.0 Control 6.25% Composition 1 0.0 130.0 0.0 Control 3.125% Composition 1 0.4 315.0 0.0 LPS Wednesday 12.1 1993.0 23.8 LPS 50% Composition 1 0.0 0.0 0.0 LPS 25% Composition 1 0.0 0.0 0.0 LPS 12.5% Composition 1 0.0 0.0 0.0 LPS 6.25% Composition 1 2.2 643.0 11.7 LPS 3.125% Composition 1 13.3 1808.0 37.5

Example 9 Cystic Fibrosis Transmembrane Receptor (CFTR) Functional Assay

[0345] As shown in FIG. 6 , Composition 1 (prepared in Example 1) enhances short circuit current (Isc) (µA/cm ² ) in bronchial epithelial cells in wild type cystic fibrosis in the cystic fibrosis transmembrane receptor (CFTR) assay.

Example 10: In vitro rate analysis of MCT, ASL and CBF in dF508-/- primary human bronchial epithelial cells

[0346] The MCT rate was assessed by mucociliary clearance analysis according to the methods described in Example 3 above. Frames from the video, which demonstrates the effect of Composition 1 on the functional microanatomy of primary human bronchial epithelial cells isolated from patients with cystic fibrosis (dF508 -/- cells) at 0 and 6 hours after administration, are shown in FIG. 7 . These results indicated that composition 1 increased the rate of MCT compared to the PBS control 6 hours after administration.

[0347] The predominant mechanism for the increase in MCT is mucus viscosity (Liu et al., Am J Prespir Cell Mol Biol. 2014, 51(4):485-93), and not an increase in the height of the airway surface fluid (ASL) and not ciliary beat frequency (CBF). Analysis of MCT slope with respect to airway surface fluid height (ASL) and ciliary beat frequency (CBF) 6 hours after Formulation 1 administration compared to PBS in primary human bronchial epithelial cells isolated from cystic fibrosis patients (dF508 -/- cells ) is shown in FIG. 8A and FIG. 8B , respectively. Composition 1 enhanced MCT and functionally and proportionately increased ASL and CBF. These results indicate that Composition 1 can restore human bronchial epithelial cells (HBE) dF508 CF to normal MCT levels without increasing ASL and CBF to excessive levels, which can impair MCT function by oversaturating the mucus layer or leading to a fast but too erratic beating pattern. cilia, which interferes with the movement of mucus. These results confirm that Composition 1 has an effect on MCTs by reducing the viscosity of the mucus.

Example 11 Composition 1 Reduces the Activity of Released MMP-9 from Activated Neutrophils

[0348] The release of metalloprotease-9 (MMP-9) from LPS-stimulated neutrophils was assessed after administration of Composition 1 at 1:100 and 1:10 dilutions. Peripheral neutrophils were isolated from blood taken from a healthy donor. Neutrophils were treated with one of two dilutions of Composition 1 (1:10 or 1:100) or pure medium (Med) for 1 hour. They were then stimulated with 10 or 100 μg LPS for 1 hour. Supernatants from cultures were collected and evaluated using the MMP-9 zymogram.

[0349] FIG. 9 shows the release of metalloprotease-9 (MMP-9) from LPS-stimulated neutrophils after 1 hour treatment with composition 1 (Ar) at 1:100 and 1:10 dilutions compared to a control of pure medium (Med) and LPS. MMP-9 is a 75-100 kDa band on the gel.

[0350] During neutrophil extracellular trap formation or degranulation, matrix metalloproteinases (MMPs) are released and this is associated with exacerbation of chronic inflammatory airway diseases by destroying the airway epithelium. MMP-9 is released from LPS-stimulated neutrophils and this is associated with a clinical decline in patients with cystic fibrosis (Gaggar et al., Eur Respir J., 2011 38(3):721-727).

[0351] Composition 1 significantly reduces the release of metalloprotease-9 from LPS-stimulated neutrophils. A 1:100 dilution reduces LPS 100-induced MMP-9 expression by approximately 50%. A 1:10 dilution reduces the same stimulation by about 75%.

[0352] These results show that Composition 1 reduces the release of MMP-9 neutrophils in vitro and acts as an anti-inflammatory agent.

Example 12 Composition 1 Reduces Neutrophil and Macrophage Associated Cytokines, MIP1 α and MIP1β

[0353] Peripheral neutrophils were isolated from blood taken from a healthy donor. Neutrophils were treated with one of two dilutions of Composition 1 (1:10 or 1:100) or pure medium for 1 hour. They were then stimulated with 10 or 100 μg LPS for 1 hour. Culture supernatants were collected and evaluated by multiple analysis at 15 minutes and 1 hour after stimulation.

[0354] Release levels of neutrophil and macrophage associated cytokines macrophage inflammatory protein 1α (MIP1α) ( FIG. 10A ) and macrophage inflammatory protein 1β (MIP1β) ( FIG. 10B ) from LPS-activated neutrophils after administration of composition 1 at dilutions 1:100 and 1:10 decreased 1 hour after stimulation compared to the control, which is pure medium (M) and LPS.

Example 13 Combination of Composition 1 and VX-770/809

[0355] The effect of the combination of Composition 1 and VX-770/809 on the speed of MCT, ASL and CBF was tested. VX-770/809 is a CFTR-specific modulator/potentiator/corrector that mediates the expression of the transmembrane receptor (CFTR) in cystic fibrosis and the opening of a membrane channel on the surface of dB508 HBE. VX-770, also known as ivacaftor, is a CFTR potentiator. VX-809, also known as lumacaftor, is a CFTR corrector. However, pure VX-770/809 does not relieve all respiratory symptoms.

[0356] The MCT rate was assessed by mucociliary clearance analysis according to the methods described in Example 3 above. Stills from the video showing the effect of Composition 1 in combination with VX-770/809 compared to pure VX-770/809 and PBS control on the functional microanatomy of primary human bronchial epithelial cells isolated from cystic fibrosis patients (dF508-/- cells), shown in FIG. 11 . The results for cells treated with VX-770-809 showed that, despite a slight increase in MCT, a mucus bilayer was formed preventing proper MCT movement ( FIG. 11 , center panel). The combination of Formulation 1 with VX-770/809 resulted not only in a significant increase in MCT, but also in the destruction of the mucus bilayer, which allowed for better MCT movement compared to pure VX-770/809.

[0357] These results indicated that the combination of Composition 1 and VX-770/809 increased the MCT rate compared to the PBS control and reduced the mucus bilayer, which is associated with the use of VX-770/809 in the mucociliary clearance assay.

[0358] Quantification of changes in ASL, CBF and MCT using pure Composition 1, VX-770/809 and their combination in dF508 CF HBE cells is shown in FIG. 12A . The combination of Formulation 1 and VX-770/809 resulted in the largest increase in MCT, CBF and ASL. These data demonstrate the improved effect of Composition 1 when used in combination with CFTR VX-770/809.

[0359] As shown in FIG. 12B , incubation with Composition 1 or Composition 1 supplemented with VX-770 and VX-809 increased mucociliary transport of primary human bronchial epithelial cells dF508-/- compared to incubation with PBS control or compared to pure combination of VX-770 with VX- 809. These data demonstrate that Composition 1 enhances mucociliary transport of primary human bronchial epithelial cells in a dose dependent manner. The results show pooled data taken from 20 HBE monolayer samples from 3 CF donors homozygous for F508del.

Example 14: Clinical study of the administration of Composition 1 to patients with bilateral lung transplantation

[0360] The effect of Composition 1 (as prepared in Example 1) in lung transplant patients on lung function (as measured by change in FEV1), changes in exhaled nitric oxide (II) fraction (FeNO) and changes in quality of life was evaluated. Briefly, Composition 1 (4 ml) was administered by nebulizer twice daily to four (4) lung transplant patients using a PARI eFlow nebulizer.

[0361] The change in lung function of each patient was assessed using a linear slope of FEV1 change at enrollment (baseline) and 1 month later. Lung transplant patients had elevated spirometry measurements in the clinic and at home after using Composition 1 for one month. This change correlated with a positive change in quality of life, which was determined using the St. George's Respiratory Questionnaire. These lung function results at 1 month are shown in FIG. 13 .

[0362] Changes in FENO in each patient were assessed using the change in mean measurements of FENO at baseline relative to mean measurements after application of Composition 1 at 1 month. Patients showed reduced FENO measurements after using Composition 1 for one month. A decrease in FENO from baseline indicates resolution of inflammation/infection in lung transplant patients (see, eg, Fisher, A., Thorax 1998;53:454-458). This change correlated with a positive change in quality of life, which was determined using the St. George's Respiratory Questionnaire. These FENO results at 1 month are shown in FIG. 14 .

Example 15: Storage stability of Composition 1

[0363] Tested the effect of storage on compositions with ascorbic acid, including various amounts of buffer with a constant concentration of glutathione. For the preparation of Formulations 1-9 (Formulation 9 corresponds to Formulation 1 (as shown in Example 1)) the solutions were mixed using sterile water for injection and stored in airtight containers that were protected from light. In Testing Formulations 1-9 were evaluated for ascorbic acid and reduced and oxidized glutathione concentrations. For these analyzed samples, a standard RP-HPLC method was used. The method was validated including specificity, accuracy, repeatability, LOQ, linearity, NPLC sample vial stability, and stability control using arbitrary forced digestion samples. The pH of each formulation was determined using a pH probe placed in a sample of each solution. According to the results shown in FIG. 15 , ascorbic acid (ACS) alone was unstable under test conditions (-20°C, 5°C, ambient temperature) (data not shown).

[0364] Formulation stability is critical for clinical use to ensure product uniformity and efficacy over time. Acidic conditions are present in chronic inflammatory airway diseases and contribute to the disease (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). In addition, innate immune functions such as proper mucociliary clearance, optimal mucus viscosity, and innate bactericidal activity are optimal at a slightly acidic pH of 6 to 7 (Fisher et al., J Membr Biol. 2006; 211(3): 139- 150). Thus, acidic conditions can impair immune function and lead to death and dysfunction of the airway epithelium.

[0365] After storage for about 72 hours at a temperature of about 2-8°C, only Composition 1 (Formulation 9) showed good solubility, no observable precipitation, and maintenance of a pH of about 6-7. In addition, impurities after storage for about 72 hours at a temperature of about 2-8°C were less than 4%, i.e. 3.8%, for Composition 1.

[0366]

* * *

[0367] The present invention has been described above in terms of functional blocks illustrating the implementation of certain functions and their relationship. The boundaries of these functional blocks have been arbitrarily defined herein for the convenience of the invention. Alternative boundaries may be defined, provided that the specified functions and their relationships are properly performed.

[0368] The foregoing description of specific embodiments so fully discloses the general nature of the present invention that others may, by applying the knowledge of the art, easily modify and/or adapt such specific implementations for various applications, without additional experimentation, without going beyond the general concept of the present invention. Therefore, such adaptations and modifications are intended to be consistent with the meaning and range of equivalents of the described embodiments based on the description and guidance provided herein. It should be understood that the phrases or terms in this document are for the purposes of the invention and not limitations, thus, the terms or phrases of the present invention should be interpreted by a person skilled in the art in light of this description and guidance.

[0369] The scope and scope of the present invention should not be limited by any of the above exemplary embodiments, but should only be determined in accordance with the following claims and their equivalents.

Claims (57)

1. Composition suitable for administration by inhalation, containing: (a) glutathione or a pharmaceutically acceptable salt thereof; (b) ascorbic acid; And (c) bicarbonate salt; wherein the molar ratio (a):(b):(c) is about 0.1-0.5:0.5-1:1. 2. Composition according to claim 1, characterized in that the bicarbonate salt is sodium bicarbonate. 3. Composition according to claim. 1 or 2, characterized in that the composition contains: (a) glutathione; (b) ascorbic acid; and (c) sodium bicarbonate. 4. Composition according to claim 2 or 3, characterized in that the molar ratio (a):(b):(c) is about 0.4-0.5:0.5-1:1, about 0.4- 0.5:0.5:1 or about 0.4-0.5:1:1. 5. The composition according to any one of paragraphs. 1-4, characterized in that the pH of the composition is at least about 5.5; from about 5.5 to about 10; about 5.5 to about 8; from about 6 to about 10; or from about 6 to about 8. 6. Composition according to any one of paragraphs. 1-5, characterized in that the pH of the composition is about 5.5, about 6, about 6.5, about 7.0 or about 7.5. 7. The composition according to any one of paragraphs. 1-6, characterized in that: (a) 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%, based on the total weight of glutathione in the composition after storage of the composition for 4 weeks at about 5°C; (b) 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%, based on the total weight of glutathione in the composition after storage of the composition for 4 weeks; (c) the 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%, based on the total weight of ascorbic acid in the composition after storage of the composition for 4 weeks at about 5°C; or (d) 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%, based on the total weight of ascorbic acid in the composition after storage of the composition for 4 weeks at about 5°C. 8. The composition according to any one of paragraphs. 1-7, which is suitable for pharmaceutical use. 9. The composition according to any one of paragraphs. 1-8, characterized in that the composition is an aqueous solution or dry powder. 10. A method of inhibiting or reducing the growth of bacteria of a clinical isolate or a biofilm of bacteria of a clinical isolate, including the introduction of a composition according to any one of paragraphs. 1-9. 11. A method of treating or alleviating symptoms in a subject suffering from or at risk of infection with bacteria of a clinical isolate, comprising administering a composition according to any one of paragraphs. 1-9. 12. The method of claim 11, further comprising administering to the subject an additional therapeutic agent. 13. The method of claim 12 wherein the additional therapeutic agent is an antibiotic. 14. The method according to any one of paragraphs. 10-13, characterized in that the clinical isolate contains two or more clinical isolates. 15. The method according to any one of paragraphs. 10-14, characterized in that the clinical isolate is resistant to multiple drugs. 16. A method of enhancing mucociliary clearance in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering to the subject a composition according to any one of paragraphs. 1-9. 17. The method according to p. 16, characterized in that the introduction of the specified composition: (a) reduces the viscosity of the mucus in the subject; (b) increases the ciliary beat frequency of the subject's airway epithelial cells; (c) increases the rate of mucociliary transport of epithelial cells in the respiratory tract of the subject; and/or (d) increases the height of the fluid covering the surface of the respiratory tract in the subject. 18. The method according to any one of paragraphs. 11-17, characterized in that the epithelial cells of the respiratory tract of the specified subject do not have the ability of mucociliary clearance prior to the introduction of the specified composition. 19. The method according to claim 18, characterized in that the epithelial cells of the respiratory tract of the specified subject do not have the ability of mucociliary clearance due to a genetically determined deficiency. 20. A method of reducing airway inflammation in a subject suffering from or at risk of airway inflammation, comprising administering to the subject a composition according to any one of paragraphs. 1-9. 21. The method of claim 20 wherein administration of said composition inhibits the myeloperoxidase activity of the subject's neutrophils. 22. The method according to p. 20 or 21, characterized in that the introduction of the specified composition: (a) reduces the formation of neutrophil extracellular traps; (b) inhibits nitric oxide(II) production by the subject's neutrophils; (c) reduces the exhaled nitric oxide (II) fraction of the subject by at least 20%; and/or (d) suppresses the production of at least one inflammatory cytokine. 23. The method according to p. 22, characterized in that at least one specified inflammatory cytokine includes: (a) a cytokine associated with macrophage activation and/or neutrophil and T cell recruitment; (b) TNF-α; (c) IL-6; and/or (d) IL-8. 24. The method according to any one of paragraphs. 11-23, characterized in that the subject suffers from or is at risk of a disease or disorder of the lungs or airways. 25. The method according to p. 24, characterized in that the disorder of the lungs or airways: (a) 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 or chronic rejection and/or lung 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), alveolar prothienosis, idiopathic pulmonary fibrosis, eosinophilic pneumonia, eosinophilic bronchitis, acute respiratory distress syndrome (ARDS), ventilator-associated inflammation and/or infection, ventilator-associated pneumonia, asbestos-related airway disorder or disease, respiratory tract disorder or disease, associated with dust, silicosis and disease or disorder of the respiratory tract associated with radiation or chemical agent, and any combination thereof; or (b) is cystic fibrosis, bronchiectasis caused by a disease other than cystic fibrosis, or bronchiectasis caused by cystic fibrosis. 26. The method according to any one of paragraphs. 11-25, characterized in that the subject has undergone a lung transplant. 27. The method according to any one of paragraphs. 11-25, characterized in that it does not significantly change the pH of the endothelium of the respiratory tract of the subject. 28. A method for reducing the viscosity of mucus in a subject suffering from or at risk of impaired mucociliary clearance, comprising administering to the subject a composition according to any one of paragraphs. 1-9. 29. A method for increasing the expression of the cystic fibrosis transmembrane receptor (CFTR) in a subject suffering from reduced expression of CFTR, comprising administering to the subject a composition according to any one of paragraphs. 1-9. 30. The method according to any one of paragraphs. 11-29 further comprising administering a CFTR therapy selected from the group consisting of a CFTR amplifier, a CFTR corrector, a CFTR potentiator/modulator, a CFTR RNA modifier, or any combination thereof. 31. The method according to any one of paragraphs. 12-15, 18, 19 and 30, characterized in that the viscosity of mucus in the subject's airways decreases, the ciliary beat frequency of the subject's airway epithelial cells increases, and/or the rate of mucociliary transport of the subject's airway epithelial cells increases to a greater extent than the viscosity of the mucus in the subject's airways is reduced, the ciliary beat frequency of the subject's airway epithelial cells is increased, and/or the rate of mucociliary transport of the subject's airway epithelial cells is increased by administration of a composition containing only glutathione or a pharmaceutically acceptable salt thereof, ascorbic acid, and a bicarbonate salt. 32. The method of claim 30 or 31, wherein the composition and CFTR are administered to the subject simultaneously or sequentially in any order. 33. The method according to any one of paragraphs. 30-32, characterized in that the fraction of nitric oxide (II) in the exhaled air of the subject (FeNO) is reduced by at least 20% after 1 month of administration of the composition. 34. The method according to any one of paragraphs. 30-33, characterized in that the composition is administered using a nebulizer twice a day. 35. The composition according to any one of paragraphs. 1-9 or the method according to any one of paragraphs. 10-34, wherein said composition, after storage at 2-8°C for at least 72 hours, is one or more of the following: (a) no observable precipitate, (b) contains less than 4% impurities, and (c) maintains a pH of 6.0-7.0.

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