Methods and compositions for producing nitrogen oxides and their use for delivering nitrogen oxides via the airway

Abstract

[0003] Provided are combinations, kits, or compositions comprising: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyols. When the one or more nitrites are reacted with the proton source in the presence of the one or more organic polyols, the combinations, kits, or compositions provide reaction products comprising nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof. These are useful for treating various disorders by delivering the combinations or compositions, or the nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, to a subject via the respiratory tract.

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for producing nitric oxide, optionally other nitric oxides, and / or optionally their precursors, and their use for delivering nitric oxide, optionally other nitric oxides, and / or optionally their precursors to human and animal subjects via the respiratory tract, for example, to treat disorders responsive to nitric oxide. [Background technology]

[0002] Nitric oxide (NO) and nitric oxide precursors have been widely studied as potential pharmaceutical agents. Nitric oxide is a potent vasodilator synthesized and released by vascular endothelial cells, playing an important role in regulating vascular local resistance and blood flow, among other things. In mammalian cells, nitric oxide is primarily produced by the enzymatic oxidation of L-arginine along with L-citrulline. Nitric oxide is also released from the skin by a mechanism that appears to be independent of the NO synthase enzyme. Nitric oxide is also involved in the inhibition of both platelet and leukocyte aggregation and adhesion, cell proliferation, scavenging superoxide radicals, and regulating endothelial permeability. The role of nitric oxide in cancer therapy is discussed in Biochemistry (Moscow), 63(7), 802-809 (1998), the disclosure of which is incorporated herein by reference. Nitric oxide has been shown to have antibacterial properties, as reviewed by F.C. Fang in J. Clin. Invest. 99(12), 2818-2825 (1997) and described in WO 95 / 22335 and WO 02 / 20026 (University of Aberdeen), the disclosures of which are incorporated herein by reference. Other known uses and applications of systems for the production of nitric oxide, other nitrogen oxides and their precursors are provided below in the description of the present invention.

[0003] Substantial problems remain associated with the efficient production and delivery of nitric oxide, other nitrogen oxides, and their precursors to organisms and cells for processing. A widely adopted system for producing nitric oxide relies on the acidification of nitrite with inorganic acid to initially produce equimolar amounts of nitrous acid (HNO2) relative to the starting nitrite, which then readily decomposes to nitric oxide and nitrate along with hydrogen ions and water. This decomposition is represented by the following equation (1): 3HNO2 → 2NO+NO3 - +H + +H2O (1)

[0004] To maximize the yield of NO, conventional methods have continued to perform nitrite acidification at pHs below about 4, where nitrite formation is generally favored. However, using a pH <4 is not suitable for in vivo use, where the acid is in contact with animal tissue. While higher pHs are safer for cells and biological systems, conventional systems have not produced satisfactory yields of NO at pHs above 4. Increasing the amount of NO produced above pH 4 requires large amounts of nitrite, which is impractical and uneconomical for therapeutic applications. Furthermore, due to the short half-life of nitrite, the conversion represented by Equation (1) cannot be easily controlled, making controlled release of therapeutic nitric oxide difficult. The reaction between one or more nitrites and a proton source produces nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, and is referred to herein as a "NOx-forming reaction" or a "NOx-producing reaction," or the like, where "NOx" is used to refer to the products of acidification of nitrites, particularly nitric oxide, other nitrogen oxides, and their precursors, individually and collectively, in any combination. Each component of the NOx produced may be released as a gas or may be released into the reaction mixture. It is understood that the gas may pass into solution in the mixture, or may first pass into solution and then be released as a gas, or any combination thereof.

[0005] WO 00 / 53193 (the disclosure of which is incorporated herein by reference) describes a cream or ointment for treating skin ischemia and promoting wound healing, in which the proton source is ascorbic acid. TM A gel based on ascorbic acid has been described, and in Example 7, the gel was tested both when in direct contact with the skin and when the skin was protected with a film. It was claimed that the use of ascorbic acid avoided significant skin irritation (WO 00 / 53193, p. 2). However, in reality, when the gel was in direct contact with the skin, the low pH of the gel caused insufficient skin irritation, and when a film was present, the skin protective film weakened the gel's effectiveness. As a result, the gel is not commercially available. The composition of WO 00 / 53193 does not contain polyol.

[0006] WO 02 / 20026 (the disclosure of which is incorporated herein by reference) describes a skin formulation for treating drug-resistant skin infections, in which the proton source is citric acid or salicylic acid. A nitrite-containing composition and an acid-containing composition are dispensed from a twin-barrel dispenser, and then the compositions are mixed to allow the acid to react with the nitrite, before being spread on the skin. Propylene glycol and polyethylene glycol are taught as optional preservatives, and glycerin (glycerol) is taught as an optional thixotropic agent for use with the nitrite composition. Propylene glycol was used in a pair of creams, citric acid and nitrite, respectively, which were mixed in situ to initiate the reaction between the acid and nitrite (e.g., WO 02 / 20026, Example 3, Formulation 1). Glycerol, along with cetostearyl alcohol, was used in a pair of lotions containing citric acid and nitrite, which were mixed in situ to initiate the reaction between the acid and nitrite (e.g., WO 02 / 20026, Example 3, Formulation 3). The preferred pH of the reaction mixture is 5 or less, particularly 4 or less, which is expected to cause undesirable skin irritation. Nasal sprays have also been taught, which can use reduced acids such as ascorbate or ascorbyl palmitate, so as to avoid using higher pHs that could irritate the sensitive nasal mucosa. However, it has been observed that a higher pH slows the reaction (WO 02 / 20026, page 16, paragraph 2).

[0007] U.S. Patent No. 6,103,275 (issued August 15, 2000) (the disclosure of which is incorporated herein by reference) describes the use of a reducing agent, such as ascorbic acid, in conjunction with an organic acid with a pKa of 1 to 4, such as maleic acid, to acidify nitrite. Viscous (gel) compositions are used to delay the release of the reaction product for topical use. The acid and nitrite remain separate until nitric oxide production is initiated, and the reducing agent is contained in at least one of the first and second gels. The pH range in which this method should be used is not specified. However, the fact that the buffer components are referred to as acids may indicate that these compounds exist primarily in their protonated form; therefore, the pH of the composition should be substantially below 4. While the presence of an acid with a pKa of 1 to 4 ensures good buffering capacity of the formulation at that pH, the incorporation of such an acid is a convenient way to ensure that the pH is maintained at a level such that the continued efficiency of converting nitrite to nitric oxide is maintained; low pHs are expected to cause substantial, undesirable skin irritation upon contact with the skin. The compositions of US Pat. No. 6,103,275 do not contain polyols.

[0008] In WO 2003 / 013489 (the disclosure of which is incorporated herein by reference), 3% polyvinyl alcohol (PA) was proposed as a gel base for each of citric acid and nitrite, which were to be mixed together in situ (WO 2003 / 013489). (WO 2003 / 013489, Example 7). However, test data (WO 2003 / 013489, Tables 11 and 12) show that a stable gel could not be formed without mixing or using PA and PA compositions together. Apart from the above suggestions which did not result in a final composition, the composition of WO 2003 / 013489 does not contain a polyol.

[0009] U.S. Patent Application Publication No. 2005 / 0037093 (the disclosure of which is incorporated herein by reference) describes nitric oxide generating compositions based on nitrite reactions and mentions optional excipients including polyvinyl alcohol, propylene glycol, and polyethylene glycol.

[0010] Chinese Patent Application Publication No. 101028229 (the disclosure of which is incorporated herein by reference) describes a cosmetic product that generates nitric oxide by reacting nitrite with acid. It teaches the optional use of, among other additives, glycerin, propylene glycol, and glycerin monostearate. Trihydroxyethylamine is further mentioned as an ingredient in certain examples.

[0011] Chinese Patent Application Publication No. 101062050 (the disclosure of which is incorporated herein by reference) describes a hair growth promoter that generates nitric oxide by reacting nitrite with an acid. It teaches the optional use of, among others, glycerin, propylene glycol, and glycerin monostearate as additives. D-pantothenyl alcohol and a combination of panthenol and inositol are mentioned as ingredients in specific examples.

[0012] WO 2008 / 110872 (the disclosure of which is incorporated herein by reference) describes, for example, a foamable nitric oxide donor composition optionally containing a polar solvent selected from a polyol and polyethylene glycol (paragraphs

[0055] and

[0056] ). Specific polyols are described as propylene glycol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, glycerin, butane-1,2,3-triol, butane-1,2,4-triol, and hexane-1,2,6-triol. Polyvinyl alcohol, polyethylene glycol 1000 (PEG1000), PEG4000, PEG6000, and PEG8000 are listed as optional additional ingredients in the list of many polymeric agents (paragraph

[0062] ). Polyols such as glycerol (glycerin), propylene glycol, hexylene glycol, diethylene glycol, and propylene glycol, as well as ethylene glycol, hexylene glycol, other glycols, and polyethylene glycol are also mentioned as optional penetration enhancers in paragraphs

[0190] and

[0191] .

[0013] WO 2009 / 019498 (the disclosure of which is incorporated herein by reference) describes the use of a non-thiol reducing agent having a pKa not between 1 and 4 as an additional component to the nitrite and proton source. Examples of non-thiol reducing agents are stated to be iodide anion, butylated hydroquinone, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and β-carotene. Apart from butylated hydroquinone, the compositions of WO 2009 / 019498 do not contain a polyol.

[0014] WO 2014 / 188174 and WO 2014 / 188175 (the disclosures of which are incorporated herein by reference) describe a method for preparing a proton source in a three-dimensional polymer matrix. A bandage system and transdermal delivery system for skin lesions has been described, which is a hydrogel containing pendant carboxylic acid and sulfonate groups covalently bonded to a nitrite. The skin-contacting primary layer is a polypropylene mesh into which nitrite is absorbed. When the mesh is placed on the skin and the hydrogel is layered on top of the mesh as the top layer, the reaction product of the acid and nitrite is successfully delivered to the skin without unacceptable skin irritation. WO 2014 / 188175 discloses an alternative skin-contacting primary layer, for example, a dissolvable film formed from polyvinyl alcohol and containing nitrite. Both references teach that the hydrogel may contain glycerol, but do not describe its purpose. However, it is well known that glycerol is added to this type of hydrogel as a plasticizer (see, e.g., WO 00 / 06215, p. 14, the disclosure of which is incorporated herein by reference). These references disclose that the absence of certain hydroxyl-containing components is preferred, particularly 1-thioglycerol, erythorbate, ascorbic acid and butylated hydroquinone.

[0015] U.S. Patent Application Publication No. 2014 / 0335207 (the disclosure of which is incorporated herein by reference) describes a topical mixture that generates nitric oxide upon mixing a "nitrite medium" with an "acidifying medium." Specific embodiments of the "nitrite medium" are individually described in paragraphs

[0050] through

[0055] , in which the nitrite is present with one or more polyol components. The typical nitrite medium described in paragraphs

[0054] and

[0055] contains a polyol selected from glycerin, glyceryl stearate, caprylyl glycol, ethylhexylglycerin, and hexylene glycol, while specific embodiments described in other paragraphs contain some of the above plus butylene glycol. These polyols are also components of the "acidifying medium" embodiments described in paragraphs

[0056] through

[0062] .

[0016] U.S. Patent Application Publication No. 2015 / 0030702 (the disclosure of which is incorporated herein by reference) describes a skin dressing based on a nitrite-acid reaction. The skin dressing contains a non-thiol reducing agent such as hydroquinone or butylated hydroquinone. The skin dressing may contain a hydrogel containing a hydrophilic polymer such as polyvinyl alcohol or polyethylene glycol.

[0017] U.S. Patent Application Publication No. 2017 / 0209485 (the disclosure of which is incorporated herein by reference) describes devices and methods for topically applying nitric oxide in a foam or serum carrier. The use of glycerol and (unspecified) "glycerol-like components" as optional additives to increase surface tension and / or lower vapor pressure is described in paragraph

[0070] .

[0018] U.S. Patent Application Publication No. 2019 / 0134080 (the disclosure of which is incorporated herein by reference) describes compositions and methods for topically applying a nitric oxide-generating system to the skin as a foam formed from a multi-part combination including a first solution containing at least one nitrite reactant and a second solution containing at least one acidic reactant. A device for retaining, aerating, and dispensing the components of the combination as a foam is also described. The use of glycerol as an optional additive to increase surface tension and / or lower vapor pressure is mentioned (paragraph

[0068] ). Summary of the Invention [Problem to be solved by the invention]

[0019] The present invention provides a method for producing nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, using a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids as nitrite acidifying agents in the presence of one or more organic polyols. This invention is based on the inventors' surprising discovery that nitrite gases (collectively referred to as NOx) can be produced more efficiently and with increased reaction power than previously possible. Furthermore, it has been found that the antimicrobially effective reaction products of such reaction systems using organic reducing acids as nitrite acidifying agents can be delivered at physiologically acceptable pHs, e.g., pHs between about 5 and about 8, with or without the use of one or more organic polyols, making reaction systems operating at such pHs available for direct delivery as compositions with beneficial physiological activity, such as in vivo antimicrobial activity. The nitric oxide-generating methods underlying the present invention have been found to produce physiologically effective amounts of nitric oxide, optionally other oxides of nitrogen, and / or optionally their precursors, for extended periods of time, e.g., greater than about 2 hours, e.g., greater than about 5 hours, e.g., greater than about 10 hours, optionally after an initial strong burst of NOx gas production, leading to potentially significant uses in medicine and other applications. If a strong initial burst is not required, administration of the reaction mixture to the subject can be performed at a period of time after the initiation of the NOx-forming reaction, for example, about 10 minutes, 30 minutes, or 1 hour or more after the initiation of the NOx-forming reaction. [Means for solving the problem]

[0020] The present invention is a specific embodiment of the more general inventive advances of the present invention as defined by the appended claims and disclosed in the following description. The present invention as defined by the appended claims relates to the application of the general inventive advances to combinations and compositions in which an NO-producing reaction takes place, and the delivery of the gas products of that reaction to a human or animal subject via the subject's nose, mouth, airways, or lungs. All aspects, examples, embodiments, and preferences described herein in connection with the disclosure are defined in the appended claims and are equally and independently applicable to the invention as defined by the appended claims.

[0021] The present disclosure provides systems, methods, combinations, kits, and compositions for producing nitric oxide and optionally other nitrogen oxides and / or their precursors. The systems, methods, combinations, kits, and compositions include, as reactants, one or more nitrite salts and a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids. The systems, methods, combinations, kits, and compositions further include one or more organic polyols. The use of reducing acids (i.e., carboxylic acid reducing acids and non-carboxylic acid reducing acids) allows for the production of nitric oxide and optionally other nitrogen oxides and / or their precursors at a pH somewhat higher than 4, for example, in the range of 5 to 8. The present disclosure further provides systems, methods, combinations, kits, and compositions for antimicrobial use, where one or more organic polyols are optional and the reaction is carried out at a starting pH of the proton source in the range of 5 to 8.

[0022] According to a first aspect, the present disclosure provides a method for producing nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, comprising reacting one or more nitrite salts with a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids under reaction conditions suitable to produce nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, wherein the reaction is carried out in the presence of one or more organic polyols; Characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not exclusively glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols are not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not exclusively polyvinyl alcohol; (h) in any one or more of (b) to (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol.

[0023] Nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof prepared by the method according to the first aspect of the present disclosure constitutes a second aspect of the present disclosure.

[0024] According to a third aspect, the present disclosure provides a method for enhancing the power output of a reaction of one or more nitrites with a proton source to produce nitric oxide, optionally other nitrogen oxides, and / or other oxides of their precursors, comprising using a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids, and conducting the reaction in the presence of a reaction power-enhancing amount of one or more organic polyols. The enhancement in the power output of the reaction is compared to a reaction carried out under the same conditions but without the one or more organic polyols.

[0025] According to a fourth aspect, the present disclosure provides for the use of one or more organic polyols in a reaction mixture of one or more nitrites and a proton source to enhance the power output of the reaction to produce nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, wherein the proton source comprises one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids. The enhanced power output of the reaction is compared to a reaction conducted under the same conditions but without the one or more organic polyols.

[0026] According to a fifth aspect, the present disclosure provides a combination, kit or composition for producing nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, by reacting one or more nitrites with a proton source. The kit or composition comprises: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; (iii) one or more organic polyols; Characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not exclusively glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols are not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not exclusively polyvinyl alcohol; (h) in any one or more of (b) to (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol.

[0027] When the proton source comprises a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix and the combination or kit comprises two or more separate compositions, it is preferred that one or more polyols are not present in the separate compositions in direct contact with or mixed with the hydrogel.

[0028] The chemical compounds of the combination, kit or composition of the fifth aspect of the present disclosure may be, for example, those described above. It may consist essentially of components (i), (ii), and (iii), and optionally water and / or a pH buffer. The term "consisting essentially of" may allow for the presence of small amounts of one or more additional components, for example, as long as the effects of the above components (i), (ii), and (iii), and optionally water and / or a pH buffer, are not adversely affected. The total amount of such one or more additional components may suitably be less than about 20% by weight or volume, e.g., less than about 15% by weight or volume, e.g., less than about 10% by weight or volume, e.g., less than about 5% by weight or volume, of the combination, chemical components of the kit, or composition.

[0029] The chemical components of the combination, kit or composition may, for example, consist of components (i), (ii) and (iii) above, optionally water and / or a pH buffer, and / or one or more additional components in an amount of less than about 20% by weight or volume, such as less than about 15% by weight or volume, for example less than about 10% by weight or volume, for example less than about 5% by weight or volume of the chemical components of the combination, kit or composition.

[0030] According to a sixth aspect, the present disclosure provides a method of preparing a combination, kit or composition comprising: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyols; This involves combining components (i), (ii) and (iii) in proximity to one another to form a combination or kit, or in admixture to form a composition; Characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not exclusively glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols are not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not exclusively polyvinyl alcohol; (h) in any one or more of (b) to (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols are propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethyl amine This product does not contain: ethanol, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol.

[0031] As used herein, the term "combination" refers to separate substances or compositions (referred to as "components") used together in close proximity. Bringing the components into close proximity can be accomplished in multiple stages, whereby some, but not all, of the components are first combined or combined together in a subcombination, and then combined with one or more additional components or other subcombinations or subcombinations. "Close proximity" can include an intimate mixture, solution, or suspension, or can mean physical proximity without being in an intimate mixture, solution, or suspension, such as being provided together in separate containers in a kit for later convenient use. For example, the nitrite component and proton source component each comprise one or more nitrites (or several thereof) and one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids (or several thereof), and can be stored separately or in separate containers in a kit and combined for use by mixing to initiate the NOx-producing reaction. One or more organic polyols may be provided in one or both of the nitrite component and the proton source component, or separately in an organic polyol component that is also mixed when the NOx-producing reaction is initiated. Any one or more of the components may themselves be present in multiple portions and multiple containers. For example, the NO nitrite and proton source may be in the same solution and therefore capable of reacting. x The combination can be in close proximity so that the production reaction is initiated immediately, or the combination can be in close proximity so that the NOx production reaction is not initiated immediately but rather one or more additional steps or actions occur before initiation, for example, because the nitrite and proton source are in a dry powder mixture or are present as encapsulated particles that require water (e.g., from a mucous membrane that the combination contacts) before the NOx production reaction can begin.

[0032] In embodiments, the first to sixth aspects of the disclosure may each independently include the above-mentioned feature (a) only, feature (b) only, feature (c) only, feature (d) only, feature (e) only, feature (f) only, feature (g) only, feature (h) referring to only (b), feature (h) referring to only (c), feature (h) referring to only (d), feature (h) referring to only (e), feature (h) referring to only (f), feature (h) referring to only (g), features (a) and (b) only, feature (h) referring to only features (a) and (b), features (a) and (c) only, feature (h) referring to only features (a) and (c), features (a) and (d), feature (h) referring to only features (a) and (d), feature (h) referring to only features (a) and (e), feature (h) referring to only features (a) and (e), feature ( The feature (h) may be characterized by only features (a) and (f), feature (h) referring to only features (a) and (f), feature (a) and (g), feature (h) referring to only features (a) and (g), feature (b) and (c), feature (h) referring to only features (b) and (c), feature (h) referring to only features (b) and (d), feature (h) referring to only features (b) and (d), feature (h) referring to only features (b) and (e), feature (h) referring to only features (b) and (e), feature (h) referring to only features (b) and (f), feature (h) referring to only features (a), (b), (c), and (f), feature (h) referring to only features (a), (b), (c), and (f), all of features (a) through (g), or feature (h) referring to features (a) and (b) and all of features (c) through (g).

[0033] In other embodiments, the first to sixth aspects of the invention may be characterized, independently of one another, by only the above-mentioned features (c), (f), and (i); only features (c), (f), and (j); feature (i) and feature (h) referring to features (c) and (f); feature (j) and feature (h) referring to features (c) and (f); features (d), (g), and (i) only; features (d), (g), and (j) only; feature (i) and feature (h) referring to features (d) and (g); feature (j) and feature (h) referring to features (d) and (g); features (e), (f), and (i) only; features (e), (f), and (j) only; feature (h) referring to features (i) and (e) and (f); or feature (h) referring to features (e) and (f).

[0034] The first to sixth aspects of the present disclosure include all of features (a) to (g), feature (h) referring to features (a), (b), and all of features (c) to (g), features (c), (f), and (i) only, features (c), (f), and (j) only, feature (h) referring to feature (i) and features (c) and (f), feature (h) referring to feature (j) and features (c) and (f), features (d), (g), and (i) only, and feature (d). and (g) and (j) only, feature (h) referring to feature (i) and features (d) and (g), feature (h) referring to feature (j) and features (d) and (g), features (e), (f) and (i) only, features (e), (f) and (j) only, feature (h) referring to feature (i) and features (e) and (f), feature (h) referring to feature (j) and features (e) and (f). If features (c) and (f) characterize the disclosure, features (d), (e) and (g) are unnecessary, in which case features (d), (e) and (g) (or feature (h) referring to features (d), (e) and (g)) may be omitted from the list and considered as an example featuring features (c) and (f) (or feature (h) referring to features (c) and (f)).

[0035] As used herein, "amount of one or more organic polyols that enhances the reaction output" means that the amount of one or more organic polyols enhances the amount and / or duration of output of nitric oxide, optionally other nitrogen oxides, and / or optionally at least one of their precursors from the NOx-producing reaction, compared to when the reaction is carried out under the same conditions but without the one or more organic polyols. "Amount" specifically refers to the total mass of gaseous nitric oxide released per gram of nitrite available for reaction in the starting reaction system. Experimental studies underlying the present invention measured and found that the amounts of gaseous nitric oxide and, optionally, other gases released were also enhanced. Therefore, since the total mass of NOx generated is believed to be enhanced by the present invention, "amount" is understood to include the total mass of nitric oxide that goes into solution in the reaction mixture as well as the total mass of NOx reaction products. "Output time" specifically refers to the length of time during which gaseous nitric oxide and, optionally, at least one other gas are released in the reaction before the reaction is complete. For the same reasons explained above in the discussion of the term "reaction power-enhancing amount of one or more organic polyols," the term "power period" is also considered to include the length of time during which nitric oxide passes through solution in the reaction mixture and the length of time during which NOx reaction products are produced. As is well known, eventually the nitrite is consumed by reaction with the proton source, the pH rise during the NOx-forming reaction reaches its maximum, and the reaction stops. The method of the first aspect of the present invention preferably improves the yield of the NOx-forming reaction, particularly, but not exclusively, the amount of NO produced, e.g., the amount of NO produced, by at least about 5%, e.g., at least about 10%, e.g., at least about 25%, e.g., at most about 150%, e.g., at most about 125%, e.g., at most about 100%, e.g., at most about 75%. Preferably, the method of the first aspect of the present invention enhances the length of time during which at least one nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, preferably nitric oxide, is generated during the reaction before the reaction is at least about 5%, such as at least about 10% complete.Using the present invention, at least one nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, preferably nitric oxide, and most preferably gaseous nitric oxide, is released, particularly in effective amounts, for a period of at least about 10 minutes. The enhancement can be up to 2 hours, e.g., at least about 5 hours, e.g., up to about 10 hours or more. This enhancement in nitric oxide generation can represent, for example, up to about 150% or more of the time period in which the same amount of nitric oxide is generated without the use of the polyol component, e.g., up to about 125% of the time period in which the same amount of nitric oxide is generated without the use of the polyol component, e.g., up to about 100% of the time period in which the same amount of nitric oxide is generated without the use of the polyol component, e.g., up to about 75% of the time period in which the same amount of nitric oxide is generated without the use of the polyol component.

[0036] The production of nitric oxide, optionally other nitrogen oxides, and / or optionally precursors may be for any purpose, both therapeutic and non-therapeutic, as exemplified and explained below.

[0037] According to a seventh aspect, the present disclosure provides a therapeutic or non-therapeutic method for delivering nitric oxide, optionally other nitric oxides and / or optionally their precursors, to a target location, such as any cell, organ, surface, structure, subject, or interior space thereof, comprising (a) administering to or near the target location a combination or composition according to the fifth aspect of the present disclosure; or (b) using a method according to the first or third aspect of the present disclosure, or carrying out a use according to the fourth aspect of the disclosure, or using a combination, kit, or composition according to the fifth aspect of the disclosure, to generate nitric oxide, optionally other nitric oxides and / or optionally their precursors, and delivering the thereby generated nitric oxide, optionally other nitric oxides and / or optionally their precursors to or near the target location; or (c) delivering nitric oxide, optionally other nitric oxides and / or their precursors according to the second aspect of the present disclosure to or near the target location.

[0038] The method of the seventh aspect of the present disclosure may be, for example, a method for treating a microbial infection in a subject in need thereof. The subject may be, for example, a human subject or other mammalian subject. The microbial infection may be, for example, a bacterial, viral, fungal, microbial, or any combination thereof.

[0039] The method of the seventh aspect of the present disclosure may be, for example, a method of vasodilation performed on a subject. The subject may be, for example, a human subject or other mammalian subject.

[0040] The method of the seventh aspect of the present disclosure may be, for example, an antimicrobial method. An antimicrobial method may be to reduce the number, prevent the growth, or limit the growth rate of microorganisms, such as bacteria, viruses, fungal cells, and / or microparasites, at a given location. The microorganisms targeted by such methods may be, for example, planktonic cells or particles, or may exist as a biofilm or other colony. Any population of microorganisms targeted by the present disclosure, whether planktonic or not, may consist of one microbial species or strain, or may include two or more species or strains.

[0041] According to an eighth aspect, the present disclosure provides a combination, kit or composition according to the fifth aspect of the disclosure, or nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, according to the second aspect of the disclosure, for use in therapy.

[0042] The combination, kit or composition, or nitric oxide, optionally other nitric oxides and / or optionally precursors thereof, for use according to the eighth aspect of the present disclosure may be for use in, for example, a method of treatment for delivering nitric oxide, optionally other nitric oxides and / or optionally precursors thereof to a subject or its interior space, the method comprising (a) administering to or near the subject or its interior space a combination or composition according to the fifth aspect of the present disclosure; or (b) using a method according to the first or third aspect of the present disclosure, or carrying out a use according to the fourth aspect of the disclosure. or (c) using a combination, kit or composition according to the fifth aspect of the disclosure to generate nitric oxide, optionally other nitrogen oxides and / or their precursors, and delivering the generated nitric oxide, optionally other nitrogen oxides and / or their precursors to or near a subject or an interior space thereof; or (d) using a combination, kit or composition according to the second aspect of the disclosure to generate nitric oxide, optionally other nitrogen oxides and / or their precursors to or near a subject or an interior space thereof.

[0043] According to the present disclosure, we have surprisingly found that good antibacterial activity in terms of bacteriostatic and biocidal effects is also provided when the proton source is citric acid (an organic carboxylic acid) or ascorbic acid (an organic non-carboxylic acid reducing acid) with an initial pH in the range of 5-8, as evidenced by up to 100% killing of M. abscessus after three days and / or kill of M. tuberculosis, H1N1 influenza virus, SARS-CoV virus, and SARS-CoV-2 virus. The term "initial pH" herein refers to the pH of the initially formed aqueous solution of the proton source, including any desired pH buffer, prior to the presence of other components of the reaction mixture that affect its initial pH. This antibacterial effect appears to be enhanced by the presence of one or more organic polyols, such as mannitol or sorbitol, but is not dependent on the presence of the one or more organic polyols. The discovery of potent antibacterial effects from NOx-generating reaction products in acids (e.g., citric acid or ascorbic acid) with an initial pH in the range of 5-8 is particularly surprising and offers promising applications in the treatment of respiratory and pulmonary infections, including those difficult to treat and / or resistant to antibiotics, including tuberculosis, multidrug-resistant tuberculosis, and nontuberculous mycobacterial infections. Treatment of such infections can be proposed by inhaling an aerosolized aqueous composition containing the reaction mixture, or its components or precursors, at a pH in the range of 5-8. Treatment of infections involving multiple pathogens, potentially including pathogens from one or more of the bacterial, viral, fungal, and parasitic groups, known as "broad-spectrum" treatment (including therapeutic and / or prophylactic treatment, as well as in vitro treatment of animate and inanimate surfaces and spaces to prevent the spread of pathogens), is also enabled by the present invention.

[0044] According to a ninth aspect, the present disclosure provides a modification of the antimicrobial method according to the seventh aspect, comprising (a) administering to the targeted microorganism, or to its vicinity, or to a subject infected with a microorganism, or to the interior space of such a subject, a combination or composition according to the fifth aspect of the present disclosure; or (b) using a method according to the first or third aspect of the present disclosure, or carrying out a use according to the fourth aspect of the present disclosure, or using a combination, kit or composition according to the fifth aspect of the present disclosure to generate nitric oxide, optionally other nitrogen oxides and / or optionally their precursors, and delivering the thereby generated nitric oxide, optionally nitrogen oxides and / or optionally their precursors to the targeted microorganism, or to the vicinity, or to a subject infected with a microorganism, or to the interior space of such a subject; or (c) delivering nitric oxide, optionally nitrogen oxides and / or optionally their precursors according to the second aspect of the present disclosure to the targeted microorganism, or to its vicinity, or to a subject infected with a microorganism, or to the interior space of such a subject. However, the initial pH of the aqueous solution of the proton source, including any desired buffer, prior to the presence of other components of the NOx-producing reaction mixture that affect pH, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, is in the range of 5 to 8, and the one or more polyols are optional and may be omitted.

[0045] In carrying out the method according to the ninth aspect of the present disclosure, the combination, kit or composition according to the fifth or eighth aspect of the present disclosure may be used to produce nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof; However, the initial pH of the aqueous solution of the proton source, including any desired buffer, prior to the presence of other components of the NOx-producing reaction mixture that affect pH, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, is in the range of 5-8, and the one or more polyols are optional and may be omitted.

[0046] The method of the ninth aspect of the present disclosure may be, for example, a method for treating a microbial infection in a subject in need thereof. The subject may be, for example, a human subject or other mammalian subject. The microbial infection may be, for example, a bacterial, viral, fungal, or microparasitic infection, or any combination thereof. The microbial infection may be on the skin of the subject, including the mucous membrane. The microbial infection may be in an internal space of the subject, for example, in accordance with the present invention, the lining of the nose, mouth, airway, lung, or pulmonary pleura of the subject.

[0047] The components and mixtures used in all aspects of the present disclosure that are administered to the human or animal body, as well as any carriers and excipients that are administered to the human or animal body, are preferably biocompatible and / or pharmaceutically acceptable to minimize tissue irritation and inflammation upon administration.

[0048] Combinations, kits, and compositions according to the present disclosure can be stored and used with a variety of suitable apparatus and devices, as described in more detail below. Methods according to the present disclosure can be suitably carried out using such apparatus and devices, as described in more detail below.

[0049] It should be understood that all embodiments, examples, and preferences specifically described with respect to any one or more aspects of the present disclosure are applicable to any one or more other aspects of the present disclosure. Furthermore, any method or use according to one aspect of the present disclosure may, where appropriate, be carried out using a combination, kit, or composition according to any other aspect. [Brief explanation of the drawings]

[0050] [Figure 1] 1 shows a cumulative plot of nitric oxide released (nmol NO per mg nitrite) over time for different reaction conditions in Example 1. [Figure 2] 1 shows the results of various tests described in Example 2. [Figure 3] 1 shows the results of various tests described in Example 2. [Figure 4] 1 shows the results of various tests described in Example 2. [Figure 5] 1 shows the results of various tests described in Example 2. [Figure 6] 1 shows the results of various tests described in Example 2. [Figure 7] 1 shows the results of various tests described in Example 2. [Figure 8] 1 shows the results of various tests described in Example 2. [Figure 9] 1 shows the results of various tests described in Example 2. [Figure 10] 1 shows the results of various tests described in Example 2. [Figure 11] 1 shows the results of various tests described in Example 2. [Figure 12] 1 shows the results of various tests described in Example 2. [Figure 13] 1 shows the results of various tests described in Example 2. [Figure 14] 1 shows the results of various tests described in Example 2. [Figure 15] 1 shows the results of various tests described in Example 2. [Figure 16] 1 shows the results of various tests described in Example 2. [Figure 17] 1 shows a schematic diagram of the apparatus used for SIFT-MS measurements. [Figure 18] 1 shows the results of various tests described in Example 3 regarding the antibacterial activity of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solution combinations against M. abscessus. [Figure 19] 1 shows the results of various tests described in Example 3 regarding the antibacterial activity of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solution combinations against M. abscessus. [Figure 20]1 shows the results of various tests described in Example 3 regarding the antibacterial activity of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solution combinations against M. abscessus. [Figure 21] 1 shows the results of various tests described in Example 3 regarding the antibacterial activity of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solution combinations against M. abscessus. [Figure 22-1] 1 shows the results of the test described in Example 4 regarding the minimum inhibitory concentrations (MICs) of a number of clinical isolate cultures for a solution containing citric acid, sodium nitrite, and mannitol. [Figure 22-2] 1 shows the results of the test described in Example 4 regarding the minimum inhibitory concentrations (MICs) of a number of clinical isolate cultures for a solution containing citric acid, sodium nitrite, and mannitol. [Figure 23] 1 shows the results of the test described in Example 5 regarding the antibacterial activity against Pseudomonas aeruginosa for carboxylic acid-nitrite solutions with and without polyols. [Figure 24] 1 shows the results of the test described in Example 6 for antibacterial activity against M. tuberculosis HN 878 in THP-1 cells. [Figure 25] 1 shows the results of the test described in Example 6 for antibacterial activity against M. tuberculosis HN 878 in THP-1 cells. [Figure 26] 1 shows the results of the test described in Example 6 for antibacterial activity against M. tuberculosis HN 878 in THP-1 cells. [Figure 27] 1 shows the results of the test described in Example 6 for antibacterial activity against M. tuberculosis HN 878 in THP-1 cells. [Figure 28]The results of the test described in Example 7 for cytotoxicity (LDH cytotoxicity assay) and antibacterial activity against H1N1 influenza A virus in MDCK cells are shown. (a) Cytotoxicity is shown in gray, with the cytotoxicity scale to the right, for a dilution range (horizontal axis is nitrite molar concentration) of MOI=0.002 (●) and MOI=0.02 (■). (Cytotoxicity at measured nitrite concentrations up to 0.015 M was 1% or less of the LDH control.) (b) Plate photographs are shown for MOI=0.002 and nitrite concentrations of 0.15 M, 0.015 M, and 0.0015 M compared with oseltamivir (1 μM). The order of plates listed above corresponds to the left-to-right order of plates in the figure (there were two experiments, and plates from each corresponding experiment are shown above and below). The rightmost pair of plates, immediately to the right of the oseltamivir pair of plates, is the virus control. Cytotoxicity is shown below each pair of test plates as % of LDH control (mean of triplicate LDH assays at 24 hours post-infection). [Figure 29] 1 shows the results of a test of the effectiveness of an acidic solution of sodium nitrite, citric acid, and mannitol buffered to pH 5.8 with sodium hydroxide in killing M. abscessus compared to amikacin and a negative control under similar conditions (described in Example 3). [Figure 30] 1 shows in schematic form an embodiment of the invention described in Example 10 for use in treating a pulmonary infection in a human subject. [Figure 31] The contact points between a liquid NO-generating formulation according to the present invention and lung tissue (right side of FIG. 31) are shown compared to inhaled gaseous nitric oxide (left side of FIG. 31). [Figure 32]Figure 1 shows the results of the LDH cytotoxicity assay of Example 8 (Experiments 1 and 2). Data are expressed as the mean + standard deviation (SD) of two experiments. SD is shown as a gray error bar. Maximum LDH activity (cells + lysis buffer) was set to 100%, and all sample results were compared to this value. The LDH positive control was the positive control from the kit. The black bar (2-hour incubation) is the left-hand bar of each pair of bars in each case, and the red bar (24-hour incubation) is the right-hand bar of each pair of bars in each case. [Figure 33] The results of the antiviral test against SARS-CoV-2 in Example 8 (Experiment 1) at an MOI of 3.0 are shown. In Experiment 1, one virus yield reduction assay was performed using SARS-CoV-2 at four multiplicities of infection (MOIs) and confirmed using back titration of the inoculum virus. For cells inoculated at an MOI of 3, 2.1 log10 TCID50 / ml was detected in the virus control wells after titration. A reduction in SARS-CoV-2 yield was observed for some of the conditions tested. After 24 hours of incubation, almost no virus was detected at the three lowest MOIs (i.e., 0.3, 0.03, and 0.003). Presumably, 24 hours of replication on Vero E6 cells is not sufficient to obtain high levels of progeny virus. Data are presented as the mean + standard deviation (SD) of duplicate titrations. SD is shown as error bars. The horizontal dotted line level with chloroquine and cell control log10 TCID50 / ml values ​​is the limit of detection (LOD) of the assay. [Figure 34]Figure 8 shows the results of the antiviral test against SARS-CoV-2 (Experiment 2) in Example 8. (a) MOI 3.0, (b) MOI 0.3. This method corresponds to Experiment 1 at these MOIs, except that the formulation used was that of Experiment 2 and incubation was performed for 48 hours instead of 24 hours to increase the level of progeny virus. Data are presented as the mean + standard deviation (SD) of duplicate titrations. SD is shown as error bars. The horizontal dotted line level, with the chloroquine and cell control log10 TCID50 / ml values, is the limit of detection (LOD) of the assay. [Figure 35] Figure 1 shows the results of the antiviral test against SARS-CoV in Example 9 at an MOI of 3.0. Prior to staining the cell monolayer with crystal violet, two plates were checked microscopically and scored for cytopathic effect (CPE). CPE was detected in these plates in the form of cell debris on top of the underlying monolayer. Results are shown for two plates examined microscopically. Data are a single titration per condition. For the remaining plates, CPE could not be scored because the cell monolayer was too dense after crystal violet staining. The horizontal dotted line level with the cell control log10 TCID50 / ml value is the limit of detection (LOD) of the assay. DETAILED DESCRIPTION OF THE INVENTION

[0051] Aspects of the present disclosure will now be described in detail with reference to specific embodiments. The specific embodiments described below may be applied to any of the aspects of the present disclosure unless clearly incompatible with such aspects. A specific embodiment may also be combined with all other specific embodiments, except where this is not possible.

[0052] Nitrite and nitrite components Embodiments of the present disclosure include the use of one or more nitrites. Hereinafter, "nitrite component" encompasses one or more nitrites themselves, as well as any component of a reaction system for producing nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, including one or more nitrites.

[0053] The selection of nitrite salts is not particularly limited. Specific examples of nitrite salts that can be used in the compositions of the present disclosure include alkali metal nitrites or alkaline earth metal nitrites. In some embodiments, one or more nitrite salts are selected from LiNO 2 , NaNO 2 , KNO 2 , RbNO 2 , CsNO 2 , FrNO 2 , AgNO 2 , Be(NO 2 ) 2 , Mg(NO 2 ) 2 , Ca(NO 2 ) 2 , Sr(NO 2 ) 2 , Mn(NO 2 ) 2 , Ba(NO 2 ) 2 , Ra(NO 2 ) 2 , and any mixture thereof.

[0054] In certain embodiments, the nitrite is NaNO2 or KNO2. In one embodiment, the nitrite is NaNO2.

[0055] In one embodiment, the nitrite component is present in a dry form, optionally in particulate form such as a powder. The nitrite salt component may be provided for use in the disclosure. If desired, the nitrite salt component may be encapsulated or microencapsulated, for example, to control or delay the reaction between one or more nitrite salts and a proton source. The dry form and / or encapsulation may aid in the storage of the nitrite salt component, alone or in admixture with other components of the reaction to generate nitric oxide according to the present disclosure. Furthermore, the dry form and / or encapsulation may aid in the incorporation of the nitrite salt component, alone or in admixture with other components of the reaction to generate nitric oxide according to the present disclosure, into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, prosthetic valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles and inhalers (handheld and nebulizers). For more information, see the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components."

[0056] If desired, the nitrite component can be present as a dry powder or crystals, optionally encapsulated or microencapsulated, or in conjunction with a gel or other carrier system, e.g., an aqueous carrier, e.g., an aqueous gel, or a solution thereof. A dry or powdered nitrite component can conveniently be prepared into a solution by the addition of water before use. The molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly prior to (e.g., immediately before) acidification, can range from about 0.001 M to about 5 M. In some embodiments, the molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly prior to (e.g., immediately before) acidification, ranges from about 0.01 M to about 2 M. In some embodiments, the molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly prior to (e.g., immediately before) acidification, ranges from about 0.1 M to about 2 M. In more specific embodiments, the molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly prior to (e.g., immediately before) acidification, ranges from about 0.2 M to about 1.6 M. In embodiments, the molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, and particularly prior to (e.g., immediately before) acidification, can range from 0.8 to 1.2 M. For example, the molar concentration of nitrite ions in the nitrite solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, and particularly prior to (e.g., immediately before) combination with the organic carboxylic acid component, can be about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.5 M, or about 1.7 M.

[0057] It should be noted that combining two or more precursor solutions of a NOx-producing reaction mixture results in a dilution of the concentration of each solute in each solution or a combination of solutes, as is well known to those skilled in the art. For example, mixing equal volumes of two 1 M solutions of solutes A and B will change the concentration of A to 0.5 M and the concentration of B to 0.5 M. Unless otherwise specified, the concentrations of nitrites described herein are the concentrations of nitrites in the liquid, i.e., initial solution, before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture that are added as solutions. The actual concentrations in the NOx-producing reaction mixture can be readily derived by knowing the components of the reaction mixture and how it was prepared.

[0058] Optionally, the nitrite component can include one or more polyols or several such polyols, in dry form or in a carrier liquid.

[0059] If it is desired to store the nitrite component in a gel or other carrier system, such as an aqueous carrier, e.g., as an aqueous gel or solution, the nitrite-containing system is preferably buffered to an appropriate pH to prevent decomposition of the nitrite during storage, preferably a pH of about 6 to 9, e.g., about 7.

[0060] The nitrite component is preferably not contacted with a proton source until it is desired to produce nitric oxide, optionally other nitrogen oxides, and / or their optional precursors. For this reason, the nitrite component is preferably maintained in a reservoir or container of a kit, apparatus, or device. Alternatively, however, the dry components of the nitrite component, the proton source, and one or more polyols can be maintained as a dry composition, e.g., a particulate mixture, and the reaction initiated by simply adding water or other suitable solvent or liquid carrier.

[0061] The nitrite may be a pharmaceutically acceptable grade nitrite. In some embodiments, the nitrite is pharmacopoeial grade. In other words, the nitrite may comply with one or more current pharmacopoeial approval standards for nitrite. For example, the nitrite may comply with one or more of the approval standards for nitrite in the United States Pharmacopoeia (USP), the European Pharmacopoeia, or the Japanese Pharmacopoeia.

[0062] In certain embodiments, the nitrite used has one or more of the following limitations on its properties: (i) the nitrite contains no more than about 0.02%, about 0.01%, or about 0.001% by weight of sodium carbonate; (ii) the nitrite contains about 10 ppm (0.001% by weight) or less of an anti-caking agent, such as sodium alkylnaphthalene sulfonate; (iii) Nitrite is a white to off-white solid; (iv) nitrite has a positive identification for the cation measured according to the relevant method in the relevant United States Pharmacopoeia; (v) nitrite has a positive test for nitrite identity determined in accordance with the applicable method of the relevant United States Pharmacopoeia; (vi) the nitrite contains about 97% by weight or more or 98% by weight or more of nitrite, and / or 102% by weight or less or 101% by weight or less of nitrite, as optionally determined by a relevant USP calorimetric method, e.g., as determined by ion chromatography (e.g., ion chromatography combined with suppressed conductivity detection); (vii) the nitrite has a pH of about 7 to about 9, or about 8 to about 9, when measured in a 10% solution at 25°C, optionally measured in accordance with the relevant United States Pharmacopoeia and / or using a pH meter; (viii) the loss on drying of the nitrite is about 0.25% by weight or less or about 0.01% by weight or less; (ix) the nitrite has a water content of about 0.5% by weight or less, as determined by Karl Fischer; (x) the heavy metal content in the nitrite is about 10 ppm or less, optionally, the heavy metal content in the nitrite is about 10 ppm or less; (xi) the nitrite contains not more than about 0.4% by weight of nitrate, and if the nitrite is sodium nitrite, not more than about 0.4% by weight of sodium nitrate, and if the nitrite is potassium nitrite, not more than about 0.4% by weight of potassium nitrate; (xii) the nitrite contains about 0.005% by weight or less or about 0.001% by weight or less of insoluble matter; (xiii) the nitrite contains not more than about 0.005% by weight chloride; (xiv) the nitrite contains not more than about 0.01% by weight of sulfate; (xv) the nitrite contains not more than about 0.001% by weight of iron; (xvi) the nitrite contains not more than about 0.01% by weight of calcium; (xvii) If the nitrite is not potassium nitrite, the nitrite is not more than about 0.005% by weight. containing less than or about 0.001% by weight of potassium, and if the nitrite is not sodium nitrite, containing less than or about 0.005% by weight or less than or about 0.001% by weight of sodium; (xviii) the nitrite contains about 0.1% by weight or less, about 5000 ppm or less, about 1000 ppm or less, about 500 ppm or less, about 100 ppm or less, or about 10 ppm or less of organic volatile compounds; (xix) the nitrite contains about 0.1% by weight or less, about 5000 ppm or less, about 1000 ppm or less, about 500 ppm or less, about 100 ppm or less, or about 10 ppm or less of ethanol; (xx) the nitrite contains less than or equal to about 3000 ppm, less than or equal to about 1000 ppm, less than or equal to about 500 ppm, less than or equal to about 100 ppm, or less than or equal to about 10 ppm of methanol; (xxi) the nitrite contains about 50 ppm or less, about 25 ppm or less, about 20 ppm or less, about 10 ppm or less, about 7.9 ppm or less, about 8 ppm or less, about 6 ppm or less, about 5.6 ppm or less, or about 2.5 ppm or less of non-volatile organic carbon; (xxii) The nitrites contain not more than about 0.05 ppm of mercury; (xxiii) the nitrite contains about 2 ppm or less or about 0.2 ppm or less of aluminum; (xxiv) the nitrite contains about 3 ppm or less or about 1 ppm or less of arsenic; (xxv) the nitrite contains not more than about 0.003% by weight or not more than about 0.001% by weight of selenium; (xxvi) The total aerobic count of the microbial load in the nitrite is less than or equal to about 100 CFU / g; (xxvii) The total yeast and mold count in the nitrate is less than or equal to about 20 CFU / g; (xxviii) the nitrite contains less than about 0.25 EU / mg or less than 0.018 EU / mg bacterial endotoxin; and (xxix) The nitrite contains less than about 0.1 ppm of phosphate (e.g., sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate), and preferably the nitrite contains no detectable amount of phosphate.

[0063] In particular embodiments, the nitrite salt has two or more of the characteristics (i) through (xxix). In further embodiments, the nitrite salt has five or more of the characteristics (i) through (xxix). In still further embodiments, the nitrite salt has ten or more of the characteristics (i) through (xxix). In still further embodiments, the nitrite salt has fifteen or more of the characteristics (i) through (xxix). In some embodiments, the nitrite salt has twenty or more of the characteristics (i) through (xxix). In particular embodiments, the nitrite salt has all of the characteristics (i) through (xxix). In more particular embodiments, the nitrite salt is sodium nitrite, which has all of the characteristics (i) through (xxix).

[0064] In some embodiments, the nitrite salt optionally contains from about 97% to about 101% by weight of nitrite as determined by relevant USP calorimetry, e.g., ion chromatography, such as ion chromatography coupled with suppressed conductivity detection. In other embodiments, the nitrite salt optionally contains from about 98% to about 102% by weight of nitrite as determined by relevant USP calorimetry, e.g., ion chromatography, such as ion chromatography coupled with suppressed conductivity detection.

[0065] In certain embodiments, the nitrite has the following characteristics: (i) the nitrite contains not more than about 0.02% by weight of sodium carbonate; (ii) the nitrite contains about 10 ppm or less of an anti-caking agent; (vi) Nitrite is approximately 97 times the weight of nitrite as determined by USP calorimetry. % by weight or more and / or containing 101% by weight or less of nitrites; (viii) the loss on drying of the nitrite is about 0.25% by weight or less; (ix) the nitrite has a water content of about 0.5% by weight or less; (x) the heavy metal content in the nitrite is about 10 ppm or less; (xi) the nitrite contains not more than about 0.4% by weight of nitrate; (xii) the nitrite contains not more than about 0.005% by weight of insoluble matter; (xiii) the nitrite contains not more than about 0.005% by weight chloride; (xiv) the nitrite contains not more than about 0.01% by weight of sulfate; (xv) the nitrite contains not more than about 0.001% by weight of iron; (xvi) the nitrite contains not more than about 0.01% by weight of calcium; (xviii) the nitrite contains about 5000 ppm or less, about 1000 ppm or less, about 500 ppm or less, about 100 ppm or less, or about 10 ppm or less of organic volatile compounds; (xxi) the nitrite contains about 10 ppm or less or about 2.5 ppm or less of non-volatile organic carbon; (xxii) The nitrites contain not more than about 0.05 ppm of mercury; (xxiii) the nitrite contains not more than about 2 ppm aluminum; (xxiv) the nitrite contains about 3 ppm or less of arsenic; (xxv) the nitrite contains not more than about 0.003% by weight of selenium; (xxvi) The total aerobic count of the microbial load in the nitrite is less than or equal to about 100 CFU / g; (xxvii) The total yeast and mold count in the nitrate is not more than about 20 CFU / g; and (xxviii) The nitrites contain less than about 0.25 EU / mg of bacterial endotoxins.

[0066] In these embodiments, the nitrite may be sodium nitrite, containing about 0.005% or less by weight of potassium. Preferably, the sodium nitrite also has one or more of the following limitations: (iii) Sodium nitrite is a white to off-white solid; (iv) sodium nitrite has a positive identification for sodium determined in accordance with the relevant method of the relevant United States Pharmacopoeia; (v) sodium nitrite has a positive test for identification of nitrite determined in accordance with the applicable method of the relevant United States Pharmacopoeia; (vii) sodium nitrite, when measured in a 10% solution at 25°C, has a pH of about 7 to about 9, or about 8 to about 9, optionally measured in accordance with the relevant United States Pharmacopoeia and / or using a pH meter; (xix) sodium nitrite containing about 0.1% by weight or less, about 5000 ppm or less, about 1000 ppm or less, about 500 ppm or less, about 100 ppm or less, or about 10 ppm or less of ethanol; (xx) the nitrite contains about 3000 ppm or less, about 1000 ppm or less, about 500 ppm or less, about 100 ppm or less, or about 10 ppm or less of methanol; and (xxix) The nitrite contains less than about 0.1 ppm of phosphate (e.g., sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate), and preferably the nitrite does not contain detectable amounts of phosphate.

[0067] Characteristics (i) through (xxix) can be determined according to the relevant methods in United States Pharmacopoeia XXXII (2009). Methods for determining characteristics (i) through (xxix) are provided in WO 2010 / 093746, the disclosure of which is incorporated herein by reference in its entirety. Methods for preparing sodium nitrite having one or more of characteristics (i) through (xxix) are also described in WO 2010 / 093746.

[0068] a proton source comprising one or more organic carboxylic acids and a proton source component; Embodiments of the present disclosure include a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids. Hereinafter, the term "proton source component" encompasses the proton source itself and any component of a reaction system containing the proton source for producing nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors.

[0069] In this section, organic carboxylic acids are exemplified in more detail.

[0070] As used herein, "organic carboxylic acid" refers to any organic acid containing one or more -COOH groups in the molecule. The organic carboxylic acid may be straight-chain or branched. The carboxylic acid may be saturated or unsaturated. The carboxylic acid may be aliphatic or aromatic. The carboxylic acid may be acyclic or cyclic. The carboxylic acid may be a vinylic carboxylic acid.

[0071] The organic carboxylic acid may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids that can be used in the present disclosure include α-hydroxycarboxylic acids, β-hydroxycarboxylic acids, and γ-hydroxycarboxylic acids.

[0072] The one or more organic carboxylic acids, or each of them if there is more than one, should preferably have a pKa1 of less than about 7, more preferably less than 7.0.

[0073] The one or more carboxylic acids may include or consist of one or more reduced carboxylic acids.

[0074] The carboxylic acid may be an acid hydrogel containing pendant -COOH groups covalently bonded to the polymer molecules that form the three-dimensional polymer matrix of the hydrogel. Examples of such carboxylic acid-containing hydrogels are described, for example, in International Publication Nos. 2007 / 007115, 2008 / 087411, 2008 / 087408, 2014 / 188174, and 2014 / 188175, the disclosures of which are all incorporated herein by reference. Such hydrogels typically contain pendant carboxylic acids in acid or salt form and sulfonyl groups covalently bonded to the three-dimensional polymer matrix. For further details, see the section below entitled "Other Reservoirs for Components: Hydrogels."

[0075] However, it is generally preferred that at least one of the one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids is not covalently bonded to a polymer or macromolecule, such as a polymer or macromolecule that forms a three-dimensional polymer or macromolecule matrix of a hydrogel. Without wishing to be bound by theory, evidence, such as the dependence of the effect on the stereoisomerism of the polyol, detailed below in the section entitled "Organic Polyols," suggests that the effect of the present disclosure to enhance the output of the reaction of one or more nitrites with a proton source is achieved at least in part by the effect of the organic polyol molecules interacting with the nitrites and protons during the acidification reaction, which means that the mobility of the reactant molecules to orient and reposition during the reaction under the influence of the polyol molecules may be important. As in the eighth embodiment of the present disclosure, it is assumed that the same mobility between the reactants in the reaction of one or more nitrites with a proton source is important even if a polyol is not necessarily present.

[0076] The organic carboxylic acid can be selected from, for example, salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropionic acid, β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid (embonic acid), stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobionic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, their salts, and their combinations.In certain embodiments, the organic carboxylic acid is selected from citric acid, its salts, and their combinations.In a particular embodiment, the organic carboxylic acid is citric acid or its salts. The carboxylic acid may be or include a polymeric or polymerized carboxylic acid, such as, for example, polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid, or copolymers of lactic acid and glycolic acid. As used herein, the term "organic carboxylic acid" also encompasses partial or total esters of organic carboxylic acids, or partial or total salts thereof, which can act as a proton source when used in accordance with the present invention.

[0077] The pH of the proton source immediately prior to contacting one or more nitrites with the proton source is preferably buffered to control the pH within a known range and limit the rate of pH increase as the nitrites are consumed. See the section below entitled "pH Control; Optional Buffer System" for further details. It is specifically contemplated that at least one organic carboxylic acid that is a proton source may suitably be present along with its conjugate base. The acid and its conjugate base may suitably form a buffer in the aqueous carrier. The buffer may be selected to maintain a desired pH as the NOx-producing reaction proceeds, preferably in the range of about 3 to 9, e.g., about 4 to 8, and preferably about 5 to about 8 for physiological contact or contact with living cells and organisms. The conjugate base, if present, may be added separately or may be generated in situ from the proton source by adjusting the pH using an acid and / or base, preferably an inorganic acid and / or inorganic base.

[0078] The initial pH of an aqueous solution of a proton source, including any desired buffers, before (e.g., immediately before) other components of the NOx-producing reaction mixture that affect pH are added, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, is suitably in the range of about 3 to 9, e.g., about 4 to 8, e.g., about 5 to 8. As used herein with respect to a proton source, "initial pH" refers to the pH of an aqueous solution of a proton source, including any desired buffers, before (e.g., immediately before) other components of the NOx-producing reaction mixture that affect pH are added (some, but not all, of the components). The dry powder proton source material or other precursor to the aqueous solution of a proton source is used in an amount appropriate to result in an aqueous solution having the desired initial pH.

[0079] If it is desired to store the proton source component in a gel or other carrier system, such as an aqueous carrier, e.g., as an aqueous gel or solution, it is preferable to buffer the system containing the proton source to an appropriate pH to prevent sustained acidity and to prevent decomposition of the proton source during storage. A pH of about 3 to 6, e.g., about 3 to 5, is preferred. If necessary, the pH can be raised by adding a base immediately prior to use of the proton source component.

[0080] For example, some patients have an intolerance to citric acid. Patients should be tested for possible acid intolerance before treatment, and the acid component should be selected accordingly.

[0081] In one embodiment, the proton source component or portion thereof can be provided in dry form, optionally in particulate form such as a powder, for use in the present disclosure. If desired, the proton source component or portion thereof can be used to, for example, control or otherwise affect the reaction of one or more nitrites with the proton source. The proton source may be encapsulated or microencapsulated for the purpose of delaying release. Encapsulated forms may be particularly useful when the proton source normally has a liquid or gel state at room temperature. Dry forms and / or encapsulation may aid in the storage of the proton source, either alone or in admixture with other components of the reaction to produce nitric oxide according to the present disclosure. Furthermore, dry forms and / or encapsulation may aid in the incorporation of the proton source component into small objects, such as medical devices, either alone or in admixture with other components of the reaction to produce nitric oxide according to the present disclosure. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, prosthetic valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. For more information, see the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components."

[0082] The one or more organic carboxylic acids, optionally encapsulated or microencapsulated, can be present in the proton source component as a dry powder or crystals, or in conjunction with a gel or other carrier system, such as an aqueous carrier, e.g., an aqueous gel, or a solution thereof. A proton source component containing an organic carboxylic acid in dry or powder form can be conveniently prepared into a solution prior to use by adding water. The molar concentration of the total proton source (including any organic non-carboxylic reducing acids present) in such a solution, prior to (e.g., just before) the addition of any other components of the NOx-producing reaction mixture, and particularly prior to (e.g., just before) the initiation of reaction with nitrite, can range from about 0.001 M to about 5 M. In some embodiments, the molar concentration of the total proton source in such a solution, prior to (e.g., just before) the addition of any other components of the NOx-producing reaction mixture, and particularly prior to (e.g., just before) the initiation of reaction with nitrite, ranges from about 0.01 M to about 2 M. In some embodiments, the molar concentration of the total proton source in such a solution prior to initiation of the reaction with nitrite ranges from about 0.1 M to about 2 M. In more specific embodiments, the molar concentration of the total proton source in such a solution prior to initiation of the reaction with nitrite ranges from about 0.2 M to about 1.6 M. In embodiments, the molar concentration of the total proton source in such a solution prior to initiation of the reaction with nitrite may range from 0.8 to 1.2 M. For example, the molar concentration of the total proton source in such a solution prior to initiation of the reaction with nitrite may be about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.5 M, or about 1.7 M.

[0083] As used herein, "molar concentration of total proton sources," "concentration of total proton sources," and the like refer to the molar concentration of protons (H + ) donor moiety or proton (H +) should be understood to refer to the concentration of either the organic carboxylic acid and / or the organic non-carboxylic acid used as a proton source in accordance with the present invention at a pH where at least one of the donor moieties (if more than one is present) is predominantly protonated, i.e., greater than 50% protonated on a molar basis. In other words, if the pH is adjusted to a higher pH prior to initiation of the NOx-forming reaction, thereby reducing the degree of protonation, the molar concentration or concentration of the total proton source should not be considered to be reduced accordingly.

[0084] It should be noted that combining two or more precursor solutions of the NOx-producing reaction mixture will result in a dilution of the concentration of each solute or combination of solutes in each solution, as is well known to those skilled in the art. For example, mixing equal volumes of two 1 M solutions of solutes A and B will change the concentration of A to 0.5 M and the concentration of B to 0.5 M. Unless otherwise specified, the concentrations of the proton source described herein are the concentrations in the initial solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture that are added as liquids, e.g., solutions. The actual concentrations in the NOx-generating reaction mixture can be readily derived by knowing the components of the reaction mixture and how it was prepared.

[0085] The proton source component in dry or powder form can be conveniently diluted prior to use by adding water. It can be prepared in solution.

[0086] Optionally, one or more organic carboxylic acids may be present in dry form or in a carrier liquid, in a mixture or solution with one or more polyols or several of such polyols.

[0087] The nitrite salt component is preferably not brought into reactive contact with the proton source until it is desired to produce nitric oxide, optionally other nitrogen oxides and / or their optional precursors. For this reason, the proton source component, or a portion thereof, is preferably maintained in a reservoir or container of the kit, apparatus, or device. Alternatively, however, the dry components of one or more nitrite salts or nitrite salt components, the proton source, and one or more polyols can be maintained as a dry composition, e.g., a particulate mixture, and the reaction initiated simply by adding water or other suitable solvent or liquid carrier.

[0088] a proton source component comprising one or more organic non-carboxylic reducing acids; The above discussion of proton source components comprising or consisting of one or more organic carboxylic acids equally applies to proton source components comprising or consisting of one or more organic non-carboxylic reducing acids. In this section, organic non-carboxylic reducing acids are exemplified in more detail.

[0089] As used herein, the term "organic non-carboxylic acid reducing acid" refers to any organic reducing acid that does not contain a -COOH group in the molecule. The organic non-carboxylic acid reducing acid may be linear or branched. The non-carboxylic acid reducing acid may be saturated or unsaturated. The non-carboxylic acid reducing acid may be aliphatic or aromatic. The non-carboxylic acid reducing acid may be acyclic or cyclic. The non-carboxylic acid reducing acid may be vinylic.

[0090] The one or more organic non-carboxylic reducing acids, or each of them if there is more than one, should preferably have a pKa1 of less than about 7, more preferably less than 7.0.

[0091] For the reasons explained above, it is generally preferred that at least one of the one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids is not covalently bonded to polymer molecules, e.g., polymer molecules forming the three-dimensional polymer matrix of a hydrogel.

[0092] The organic non-carboxylic acid reducing acid may be selected from, for example, ascorbic acid; ascorbyl palmitate (ascorbyl palmitate); ascorbic acid derivatives such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid, and 6-O-dodecanedioyl ascorbic acid; acidic reductants such as reducing acids; erythorbic acid; oxalic acid; salts thereof; and combinations thereof. In a particular embodiment, the organic non-carboxylic acid reducing acid is ascorbic acid or a salt thereof.

[0093] The organic non-carboxylic acid reducing acid may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic acid reducing acids that can be used in the present disclosure include acidic reductants, such as the reducing acid 2,3-dihydroxy-2-cyclopentanone.

[0094] The pH of the proton source and / or reaction mixture after contacting one or more nitrites with the proton source is preferably buffered to control the pH within a known range and to control the increase in pH as the nitrites are consumed. For more information, see "pH Control; Optional Buffering" below. See the section entitled "Systems." It is particularly contemplated that at least one organic non-carboxylic acid reducing acid, which is a proton source, may suitably be present along with its conjugate base. The acid and its conjugate base may suitably form a buffer solution in the aqueous carrier. The buffer solution may be selected to maintain a desired pH as the NO-producing reaction proceeds, preferably in the range of about 3 to 9, e.g., about 4 to 8, and preferably in the range of about 5 to about 8 for physiological contact or for contact with living cells and organisms. The conjugate base, if present, may be added separately or may be generated in situ from the proton source by adjusting the pH using an acid and / or base, preferably an inorganic acid and / or inorganic base.

[0095] The initial pH of the aqueous solution of the proton source, including any desired buffers, before (e.g., immediately before) other components of the NOx-producing reaction mixture that affect pH are added, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, is suitably in the range of about 3 to 9, e.g., about 4 to 8, e.g., about 5 to 8. The dry powdered proton source material or other precursor to the aqueous solution of the proton source is used in an amount appropriate to result in an aqueous solution having the desired initial pH.

[0096] If it is desired to store the proton source component in a gel or other carrier system, such as an aqueous carrier, e.g., as an aqueous gel or solution, it is preferable to buffer the system containing the proton source to an appropriate pH to prevent sustained acidity and to prevent decomposition of the proton source during storage. A pH of about 3 to 6, e.g., about 3 to 5, is preferred. If necessary, the pH can be raised by adding a base immediately prior to use of the proton source component.

[0097] Some reducing acids, such as oxalic acid, are toxic. The acid component should be selected accordingly.

[0098] One or more organic non-carboxylic acid reducing acids can be used in the proton source component in addition to or instead of one or more organic carboxylic acids in the methods described above. See the section "Proton Source Comprising One or More Organic Carboxylic Acids and a Proton Source Component" for more information.

[0099] Organic polyols and organic polyol components Embodiments of the present disclosure include one or more organic polyols. Hereinafter, the terms "organic polyol component" or "polyol component" include the organic polyol itself and any component of a reaction system containing the organic polyol to produce nitric oxide, optionally other nitrogen oxides and / or optionally their precursors.

[0100] As used herein, "organic polyol" refers to an organic molecule having two or more hydroxyl groups that is not a proton source for the nitrite reaction and is not a sugar or polysaccharide. Here, the terms "sugar" and "polysaccharide" include oligosaccharides, glycans, and glycosaminoglycans. Thus, an organic polyol has a pKa of about 7 or greater, e.g., 7.0 or greater.

[0101] Here, "organic polyol" preferably excludes reducing agents. Thus, in one embodiment of the present invention among all aspects, organic polyol excludes reducing agents. Examples of reducing agents that have two or more hydroxyl groups and are organic molecules that are not sugars or polysaccharides are thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate, erythorbic acid, and erythorbate. Thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate, and erythorbate are reducing agents, and therefore are preferably excluded from "organic polyol." In any case, ascorbic acid and erythorbic acid, in particular, are nitrites. They are excluded from "organic polyols" because they are a source of protons for the reaction. For the avoidance of doubt, we note that reducing agents that are proton sources, e.g., ascorbic acid and / or erythorbic acid, are not excluded from the proton source of the present invention, or from the proton source components, combinations, kits, compositions, uses, methods, or any other part of the present invention, and from the procedures performed when they are present as a proton source.

[0102] The organic polyol may be cyclic, acyclic, or a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, the one or more organic polyols may be selected from one or more alkanes substituted with two or more OH groups, one or more cycloalkanes substituted with two or more OH groups, one or more cycloalkylalkanes substituted with two or more OH groups, and any combination thereof. Most preferably, the organic polyol does not have any substituents other than OH.

[0103] Preferably, the one or more organic polyols are one or more acyclic organic polyols.Preferably, the one or more acyclic organic polyols are selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms.Preferably, the one or more acyclic organic polyols are selected from alditols, such as alditols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms.Preferably, the one or more organic polyols do not contain saponins, sapogenins, steroids, or steroid glycosides.

[0104] Alternatively, the one or more organic polyols may be one or more cyclic organic polyols. In these embodiments, the one or more cyclic organic polyols may be cyclic sugar alcohols or cyclic alditols. For example, the one or more cyclic polyols may be cyclic sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, or cyclic alditols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. A specific example of a cyclic polyol is inositol.

[0105] In some embodiments, the one or more organic polyols have seven or more hydroxyl groups. In certain embodiments, the one or more organic polyols are sugar alcohols or alditols having seven or more hydroxyl groups. In more particular embodiments, the one or more organic polyols have nine or more hydroxyl groups. In further embodiments, the one or more organic polyols are sugar alcohols or alditols having nine or more hydroxyl groups. In some embodiments, the one or more organic polyols have 20 or fewer hydroxyl groups. In certain embodiments, the one or more organic polyols are sugar alcohols or alditols having 20 or fewer hydroxyl groups. In more particular embodiments, the one or more organic polyols have 15 or fewer hydroxyl groups. In further embodiments, the one or more organic polyols are sugar alcohols or alditols having 15 or fewer hydroxyl groups. The one or more organic polyols may have a number of hydroxyl groups in the range of 7 to 20, more particularly in the range of 9 to 15. In certain embodiments, the one or more organic polyols contain 9, 12, 15, or 18 hydroxyl groups.

[0106] Preferably, the one or more organic polyols are sugar alcohol compounds comprising (consisting of) one or more monosaccharide units and one or more acyclic sugar alcohol units, for example. The one or more organic polyols may be sugar alcohol compounds comprising (consisting of) one or more monosaccharide units and one or more acyclic sugar alcohol units in a linear chain, or one or more monosaccharide units and one or more acyclic sugar alcohol units in a branched chain.

[0107] As used herein, a monosaccharide unit refers to a monosaccharide that is covalently bound to at least one other unit (another monosaccharide unit or an acyclic sugar alcohol unit) in a compound. An acyclic sugar alcohol unit refers to an acyclic sugar alcohol covalently bonded to at least one other unit (a monosaccharide unit or another acyclic sugar alcohol unit) in a compound. The units in the compound may be linked via an ether bond. In some embodiments, one or more monosaccharide units are covalently bonded to other units of the compound via a glycosidic bond. In certain embodiments, each monosaccharide unit is covalently bonded to other units of the compound via a glycosidic bond. In certain embodiments, the sugar alcohol compound is a glycoside having a monosaccharide or oligosaccharide glycone and an acyclic sugar alcohol aglycone.

[0108] Preferred acyclic sugar alcohol units are sugar alcohol units having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. In certain embodiments, the acyclic sugar alcohol unit is selected from the group consisting of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and volemitol units.

[0109] In certain embodiments, one or more of the monosaccharide units are C5 or C6 monosaccharide units. In other words, one or more of the monosaccharide units are pentose or hexose units. In more particular embodiments, each monosaccharide unit is a C5 or C6 monosaccharide unit. In certain embodiments, one or more sugar alcohol units are a C5 or C6 sugar alcohol unit. In more particular embodiments, each sugar alcohol unit is a C5 or C6 sugar alcohol unit.

[0110] In certain embodiments, a sugar alcohol compound may comprise, for example, n monosaccharide units and m acyclic sugar alcohol units, where n is an integer greater than or equal to 1, m is an integer greater than or equal to 1, and (n + m) is 10 or less. In certain embodiments, a sugar alcohol compound may comprise, for example, a chain of n monosaccharide units terminated with one acyclic sugar alcohol unit, where n is an integer from 1 to 9. In these embodiments, the chain of monosaccharide units may be covalently linked by a glycosidic bond. In certain embodiments, each monosaccharide unit is covalently linked to another monosaccharide unit or to an acyclic sugar alcohol unit by a glycosidic bond. In certain embodiments, a sugar alcohol compound may comprise, for example, a chain of one, two, or three monosaccharide units terminated with one acyclic alcohol unit. One, two, three, or each monosaccharide unit may be a C5 or C6 monosaccharide unit. The acyclic alcohol unit may be a C5 or C6 sugar alcohol unit. Examples of sugar alcohol compounds include, but are not limited to, isomalt, maltitol, and lactitol (n=1); maltotriitol (n=2); and maltotetriitol (n=3).

[0111] Such sugar alcohol compounds can be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, oligosaccharide refers to a sugar consisting of 3 to 10 monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides can be synthesized (e.g., by hydrogenation) from disaccharides, oligosaccharides, or polysaccharides (e.g., by hydrolysis and hydrogenation), but are not limited to compounds synthesized from disaccharides, oligosaccharides, or polysaccharides. For example, sugar alcohols derived from disaccharides can be formed from the dehydration reaction of monosaccharides and sugar alcohols. The one or more organic polyols may be sugar alcohols derived from disaccharides, trisaccharides, or tetrasaccharides. Examples of sugar alcohols derived from disaccharides include, but are not limited to, isomalt, maltitol, and lactitol. Examples of sugar alcohols derived from trisaccharides include, but are not limited to, maltotriitol. Examples of sugar alcohols derived from tetrasaccharides include, but are not limited to, maltotetriitol.

[0112] Suitable organic polyols include erythritol, threitol, arabitol, xylitol, Examples of suitable organic polyols include erythritol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol, polyglycitol, and any combination thereof. Glycerol can be used, and when present, it is preferably present together with one or more other organic polyols, such as erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol, polyglycitol, or any combination thereof.

[0113] Many organic polyols have one or more chiral centers, so they exist in stereoisomeric forms.All stereoisomers and optical isomers and isomeric mixtures of organic polyols are intended to be included within the scope of this disclosure and patent.In particular, D- and / or L-forms of all chiral organic polyols and all mixtures thereof can be used.

[0114] Interestingly, the effectiveness of the use of polyols in the present disclosure has been found to be stereochemically dependent. Thus, the selection of the enantiomer form or mixture of enantiomers of one or more organic polyols for use in the present disclosure can affect the outcome of the reaction of nitrite with a proton source, at least with respect to the amount of NO produced.

[0115] For example, sorbitol is a stereoisomer of mannitol, differing from the other in the orientation of one hydroxyl group. As shown in Examples 2D and 2E (Figures 5 and 6) below, the effects of sorbitol and mannitol on the output of the reaction of nitrite with a proton source differ in otherwise identical reaction systems.

[0116] In certain embodiments, the organic polyol is selected from the group consisting of arabitol, xylitol, mannitol, sorbitol, and any combination thereof. The arabitol may be D- or L-arabitol or a mixture thereof. The xylitol may be D- or L-xylitol or a mixture thereof. The sorbitol may be D- or L-sorbitol or a mixture thereof. The mannitol may be D- or L-mannitol or a mixture thereof.

[0117] In certain embodiments, the one or more polyols are sugar alcohol compounds comprising (consisting of) one or more monosaccharide units and one or more acyclic sugar alcohol units (including sugar alcohols derived from disaccharides or oligosaccharides), e.g., as described herein, when the systems, methods, combinations, kits, and compositions described herein are used in the treatment of tuberculosis infection or antimicrobial therapy to reduce the number of tuberculosis bacteria.

[0118] In one embodiment, the organic polyol component may be provided for use in the present disclosure in a dry form, optionally in a granular form such as a powder. If desired, the organic polyol may be encapsulated or microencapsulated, for example, to control or delay the participation of the polyol in the reaction between one or more nitrites and a proton source. An encapsulated form may be particularly useful when the organic polyol normally has a liquid or gel state at room temperature. The dry form and / or encapsulation may aid in the storage of the organic polyol component, alone or in admixture with other components of the reaction to generate nitric oxide according to the present disclosure. Furthermore, the dry form and / or encapsulation may aid in the incorporation of the organic polyol component, alone or in admixture with other components of the reaction to generate nitric oxide according to the present disclosure, into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, cardiac catheters, and the like. These include organ assist devices, prosthetic valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. For more information, see the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components."

[0119] Alternatively, the organic polyol component may include a carrier medium, such as an aqueous carrier liquid or a gel carrier. If the organic polyol is normally liquid at room temperature, it may be used as is without additional carrier components, or may be used mixed with one or more carrier additives, such as water.

[0120] The one or more organic polyols, optionally encapsulated or microencapsulated, can be present in the polyol component as a dry powder or crystals, or in combination with a gel or other carrier system, such as an aqueous carrier, e.g., an aqueous gel, or a solution thereof. A polyol component containing an organic polyol in dry or powder form can be conveniently prepared into a solution by adding water before use. The total molar concentration of the one or more polyols in such a solution prior to the initiation of reaction with nitrite can be any concentration up to the saturation limit of each polyol in the solution. For example, the total molar concentration of the one or more polyols can range from about 0.001 M to about 5 M. In some embodiments, the total molar concentration of the one or more polyols in such a solution prior to the initiation of reaction with nitrite ranges from about 0.01 M to about 2 M. In some embodiments, the total molar concentration of the one or more polyols in such a solution prior to the initiation of reaction with nitrite ranges from about 0.1 M to about 2 M. In more specific embodiments, the total molar concentration of the one or more polyols in such a solution prior to the initiation of reaction with nitrite ranges from about 0.2 M to about 1.6 M. In embodiments, the total molar concentration of the one or more polyols in such a solution prior to initiation of reaction with the nitrite may range from 0.8 to 1.2 M. For example, the total molar concentration of the one or more polyols in such a solution prior to initiation of reaction with the nitrite may be about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.5 M, or about 1.7 M.

[0121] It should be noted that combining two or more precursor solutions of the NOx-producing reaction mixture will result in a dilution of the concentration of each solute in each solution or a combination of solutes, as is well known to those skilled in the art. For example, mixing equal volumes of two 1 M solutions of solutes A and B will change the concentration of A to 0.5 M and the concentration of B to 0.5 M. Unless otherwise specified, the concentrations of organic polyols described herein are the concentrations in the initial solution prior to (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture that are added as liquids, e.g., solutions. The actual concentrations in the NOx-generating reaction mixture can be readily derived by knowing the components of the reaction mixture and how it was prepared.

[0122] The polyol component in dry or powder form can be conveniently made into a solution before use by adding water.

[0123] Optionally, the polyol can be in dry form or in a carrier liquid, in a mixture or solution with one or more nitrites or proton sources or several such proton sources.

[0124] In certain embodiments in which the nitrite is kept separate from other components of the reaction prior to use to generate nitric oxide, the nitrite component can include one or more polyols. In these embodiments, the organic carboxylic acid component can be substantially free of polyols. In alternative embodiments, the organic carboxylic acid component includes one or more polyols. In these embodiments, the nitrite component can be substantially free of polyols. In further embodiments, the organic carboxylic acid component and the nitrite component can each include one or more polyols, which can be the same or different between the two components.

[0125] In another embodiment, the organic carboxylic acid component and the nitrous acid component may be substantially free of polyols, and one or more polyols may be included in a separate polyol component.

[0126] the relative concentrations of nitrite, proton source, and optional polyol in the reaction mixture; The total molar concentration of the optional one or more organic polyols in the polyol component or reaction solution at (or before) the initiation of the NOx-forming reaction may be about 0.05 to about 3 times the total molar concentration of nitrite ions, e.g., about 0.1 to about 2 times, e.g., about 0.25 to about 1.5 times, e.g., about 0.3 to about 1.2 times the total molar concentration of nitrite ions in the nitrite component or reaction solution. The same relative molar concentration between the one or more organic polyols and nitrite ions is suitably provided in a component of a combination or kit according to the invention, or in a composition according to the invention, prior to (e.g., immediately before) the initiation of the NOx-forming reaction.

[0127] The total molar concentration of the optional one or more organic polyols in the polyol component or reaction solution at (or before) the initiation of the NOx-producing reaction may suitably be about 0.05 to about 3 times the total molar concentration of the proton source, for example, about 0.1 to about 2 times the total molar concentration of the proton source in the proton source component or reaction solution. The same relative molar concentration between the one or more organic polyols and the proton source is suitably provided in a component of a combination or kit according to the invention, or in a composition according to the invention, prior to (e.g., immediately before) the initiation of the NOx-producing reaction.

[0128] Optional Additional Ingredients A combination, kit, or composition for use in the present disclosure may be incorporated into a range of diluents, carriers, and excipients, and / or may be provided with one or more additional components, particularly functional components intended to provide one or more particular benefits to the combination, kit, or composition it is used in. Such diluents, carriers, excipients, and / or additional components will generally be physiologically compatible if in vivo use is desired.

[0129] Examples of suitable physiologically compatible diluents, carriers and / or excipients include, but are not limited to, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talc, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, and the like.

[0130] Generally speaking, depending on the intended mode of administration, pharmaceutical formulations contain from about 0.005% to about 95% by weight, preferably from about 0.5% to about 50% by weight, of the combination or composition of the present invention or components thereof. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art.

[0131] The excipient may be selected from known excipients for delivery of nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, depending on the intended use or route of administration by which the reactants and / or reaction products will be delivered to the target site. For example, creams, lotions, and ointments may be formulated by incorporating the nitrite salt into excipients such as cream, lotion, and ointment bases or other thickeners and thickeners (e.g., Eudragit L100, carbopol, carboxymethylcellulose, or hydroxymethylcellulose). The proton source may be incorporated into an excipient selected from carbopol, carboxymethylcellulose, hydroxymethylcellulose, methylcellulose, or into an aqueous base. When film formation is desired, film-forming excipients, such as propylene glycol, polyvinylpyrrolidone (Polyvinylpyrrolidone), etc., may be used. Don), gelatin, guar gum and shellac may also be used.

[0132] Optional additional components can be selected from, for example, sweeteners, taste masking agents, thickeners, viscosifiers, wetting agents, lubricants, binders, film-forming agents, emulsifiers, solubilizers, stabilizers, colorants, odorants, salts, coating agents, antioxidants, active ingredients, and preservatives.Such components are well known in the art, and detailed descriptions thereof are not necessary for those skilled in the art.Examples of auxiliary substances such as wetting agents, emulsifiers, lubricants, binders, and solubilizers include, for example, sodium phosphate, potassium phosphate, acacia gum, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc. Sweeteners or taste-masking agents may include, for example, sugars, saccharin, aspartame, sucralose, neotame, or other compounds that beneficially affect the taste, aftertaste, perceived unpleasant saltiness, sourness, or bitterness, which reduces the tendency of oral or inhaled formulations to irritate the recipient (e.g., cough, sore throat, or other undesirable side effects that may, for example, reduce the delivered dose or adversely affect patient compliance with prescribed treatment regimens). Certain taste-masking agents may complex with one or more nitrites. Examples of thickening agents, thickeners, and film-forming agents are provided above.

[0133] The selection of active agents and other additional components, such as those serving as diluents, carriers, and excipients, can be determined by compatibility with the treatment regimen for the relevant disease or condition, as well as the desired route of administration of the combination or composition according to the present disclosure. Standard references such as the Formulary on line (https: / / bnf.nice.org.uk / ), Remington: "The Science & Practice of Pharmacy", 22nd Edition (2012), or Physician's Desk Reference, 71st Edition (2017) can be consulted.

[0134] Examples of routes of administration by which components and compositions according to the present disclosure may be administered to animal (including human) subjects for therapeutic purposes include topical (e.g., creams, lotions, gels, ointments, pastes, emollients, sprays), otic, nasal (e.g., sprays), vaginal, rectal (e.g., suppositories), oral (e.g., mists, sprays, mouthwashes, aerosols), enteral (e.g., tablets, pastilles, lozenges, capsules, syrups, elixirs) and parenteral (e.g., injectable solutions), ocular, otic, nasal or throat (e.g., intravenous drip), or via the respiratory tract or lungs (e.g., mists, aerosols, powder inhalation).

[0135] Examples of active agents that can be incorporated into or administered with components and compositions according to the present disclosure include antibiotics, steroids, anesthetics (e.g., lignocaine (lidocaine), amethocaine (tetracaine), xylocaine, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivacaine, cocaine, or any combination thereof), analgesics, anti-inflammatory agents (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs)), anti-infectives, vaccines, immunosuppressants, anticonvulsants, anti-dementia drugs, prostaglandins, antipyretics, antifungals, antivirals, vasodilators or vasoconstrictors, sunscreens (e.g., PABA), antihistamines, hormones such as estrogen, progesterone, or androgens, antiseborrheic agents, alpha or beta blockers, vitamins, emollients, enzymes, mast cell stabilizers, stabilizers), scabicides, pediculicides, scarring agents, keratolytic agents, lubricants, narcotics, shampoos, anti-acne products, burn treatments, cleansers, deodorants, bleaching agents, diaper rash treatment products, emollients, moisturizers, photosensitizers, poison ivy or poison oak or sumac products, sunburn treatments, proteins, peptides, proteoglycans, nucleotides, oligonucleotides (DNA, RNA, etc.), minerals, growth factors, tar-containing preparations, honey-containing formulations (e.g., formulations containing manuka honey), wart treatments, moist dressings, wound care products, or any combination thereof.

[0136] Specific examples include analgesics such as ibuprofen, indomethacin, diclofenac, acetylsalicylic acid, acetaminophen, propranolol, metoprolol, and oxycodone; thyroid-releasing hormones; sex hormones such as estrogen, progesterone, and testosterone; insulin; verapamil; vasopressin; hydrocortisone; scopolamine; nitroglycerin; isosorbide dinitrate; antihistamines such as terfenadine; clonidine; nicotine; nonsteroidal immunosuppressants such as cyclosporine, methotrexate, azathioprine, mycophenylalanine, cyclophosphamide, TNF-α antagonists, and anti-IL5, anti-IL4Ra, anti-IL6, anti-IL13, anti-IL17, and anti-IL23 cytokine monoclonal antibodies; anticonvulsants; and drugs for Alzheimer's disease, dementia, and / or Parkinson's disease, such as apamorphine and rivastigmine. If desired, any of the optional additional ingredients may be encapsulated or microencapsulated, e.g., to control or delay their release. See the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Ingredients" for more information.

[0137] Optional encapsulation of ingredients (e.g., microencapsulation) At least some of the components of the combinations, kits and compositions for use in the present disclosure may be encapsulated, for example microencapsulated.

[0138] The use of microencapsulated components for NO production is useful because it prolongs the production of relatively unstable compounds (e.g., NO) from precursors in a chemically stable form. Multiple microencapsulated reactants and / or one or more optional additional components can be easily mixed and contacted in a dry environment, and NO production can be initiated simply by adding a small amount of water to the precursor mixture. Alternatively, such a mixture of microencapsulated reactants and / or one or more optional additional components can be applied directly to a subject (e.g., skin, mucosal surface) or, in accordance with the present invention, to the subject's nose, mouth, airways, and / or lungs. Here, the physiological environment itself provides sufficient water to trigger the release of therapeutic amounts of NO. A further advantage is that the volume occupied by the microencapsulated reactants and / or one or more optional additional components is relatively small, allowing them to be easily incorporated into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, prosthetic valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles.

[0139] One example of a manufacturing method for encapsulating or microencapsulating reactants and / or one or more optional additional components is spray drying a melt or polymer solution of the reactants and / or one or more optional additional components to produce a finely divided powder of individual particles containing the material dispersed within a polymer matrix. Other encapsulation or microencapsulation methods, such as pan coating, air suspension coating, centrifugal extrusion, fiber spinning, fiber extrusion, nozzle vibration, ionotropic gelation, coacervation phase separation, interfacial crosslinking, in-situ polymerization, and matrix polymerization, can also be used. The encapsulating polymer is preferably biocompatible. Such polymers include natural polymers such as ethyl cellulose, zein (a prolamin seed storage protein found in certain grass species, including corn and cane), chitosan, hyaluronic acid, and alginic acid, or biodegradable polyesters, polyanhydrides, poly(orthoesters), polyphosphazenes, or polysaccharides (see Park et al., Molecules 10 (2005), pp. 141-161). As described above, Compositions in which two chemicals are microencapsulated are well known for the delivery of pharmaceuticals and other agents. See U.S. Patent No. 4,130,639 to Shalaby and Jamiolkowski et al.; U.S. Patent No. 6,491,748 to Buchholz and Meduski et al. However, in virtually all such compositions, it is the therapeutic agent that is microencapsulated, and the therapeutic agent is not produced by reaction of the microencapsulated reagents. However, appropriate modification of the teachings of the prior art is within the skill of those in the art. Nitric oxide-releasing polymers have been described as medical articles containing NO adducts / donors. See, for example, U.S. Patent No. 7,829,553 to Arnold (carbon-based diazeniumdiolates bound to hydrophobic polymers); U.S. Patent No. 7,135,189 to Knapp (nitrosothiol precursors and nitric oxide donors).

[0140] pH control; optional buffer system The composition may have a controlled pH. In particular, the composition may have a pH in the range of 3.0 to 8.0, more particularly in the range of 4.0 to 8.0. In more specific embodiments, the composition may have a pH in the range of 4.0 to 7.4. In even more specific embodiments, the composition may have a pH in the range of 4.0 to 6.0. In these embodiments, the composition may have a pH in the range of 4.5 to 6.0.

[0141] The pH of the composition may be controlled by any known method. In certain embodiments, the pH of the organic carboxylic acid component or organic reduced acid component is controlled before combining with the nitrite component. In some embodiments, the organic carboxylic acid component or organic reduced acid component comprises a buffer. The buffer may be a pharmacologically acceptable buffer (e.g., phosphate buffer).

[0142] In some embodiments, the buffer is formed by mixing an organic carboxylic acid or an organic non-carboxylic acid reducing acid with its salt counterpart. For example, the organic carboxylic acid component can include an organic carboxylic acid and a salt of an organic carboxylic acid. The organic non-carboxylic acid reducing acid component can include an organic non-carboxylic acid reducing acid and a salt of an organic non-carboxylic acid reducing acid. In certain embodiments, the organic carboxylic acid component includes citric acid and citrate. In other embodiments, the organic carboxylic acid component or the organic reducing acid component includes ascorbic acid and ascorbate. In some embodiments, the organic carboxylic acid component includes an organic carboxylic acid and a salt of an additional organic acid. For example, the organic carboxylic acid component can include citric acid and ascorbate. In yet further embodiments, the organic carboxylic acid component can further include an organic carboxylic acid, a salt of an organic carboxylic acid, and a salt of an organic carboxylic acid. For example, the organic carboxylic acid component can include citric acid, citrate, and ascorbate.

[0143] In other embodiments, the buffer solution is formed by adjusting the pH of an organic carboxylic acid or organic non-carboxylic acid reducing acid so that the acid (protonated form) coexists in admixture with its salt counterpart. This is suitably achieved by adding a strong inorganic base and, optionally, a strong inorganic acid to the organic carboxylic acid or organic non-carboxylic acid reducing acid in amounts to generate a buffer system in situ. Examples of suitable strong inorganic bases include sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, and cesium hydroxide. Examples of suitable strong inorganic acids include hydrochloric acid, sulfuric acid, hydrobromic acid, and nitric acid.

[0144] The buffer can include one or more physiological buffers, particularly when the combination or composition according to the present disclosure comes into contact with cells or the skin, mucosa, or other tissues of an animal (including a human), such as in the case of administration to the nose, mouth, airways, or lungs according to the present invention. Examples of suitable physiologically compatible buffers include buffers in the pH range of about 5 to about 9, such as 2-amino-2-methyl-1,3-propanediol, N-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)-iminodiacetic acid (ADA), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (BICINE), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS), 1,3-bis[tris] (hydroxymethyl)methylamino]-propane (BIS-TRIS propane), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), diglycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS ... (ethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic anhydride (POPSO), dibasic sodium phosphate, monobasic sodium phosphate, dibasic potassium phosphate, monobasic potassium phosphate, [trimethylsilyl]-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) ... Examples of suitable hydroxymethylamino compounds include 2-hydroxy-3-[tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO), 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), N-[tri(hydroxymethyl)-methyl]glycine (tricine), or 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA).

[0145] Osmolality of the composition In particular, the solute strength of any solution of nitrite, proton source, organic polyol, or any combination thereof delivered to a physiological system by a route that results in contact with the skin, mucous membranes, or nose, mouth, respiratory tract, or lungs of a human or animal subject in accordance with the present invention should be controlled to avoid undesirable dehydration of any of the subject's organs and tissues.

[0146] Osmolality (Osm), defined as the number of moles of solute dissolved in one kilogram of solvent, can be expressed as osmolality per kilogram (Osmol / kg). The osmolality of any solution administered to a human or animal subject in accordance with the present disclosure should generally be in the range of about 100 to about 5000 mOsmol / kg, e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 to about 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 mOsmol / kg.

[0147] Mixture of ingredients that initiates NOx formation It has been found that the order in which the components of a NOx-producing system are mixed to initiate NOx production can affect the results of using the NOx thereby produced. Evidence of this effect is provided in Example 6 below.

[0148] In this example, we demonstrate that the efficacy of compositions according to the invention for killing the bacterium M. tuberculosis HN878 in THP-1 cells differs depending on whether, on the one hand, the nitrite, proton source and organic polyol components are first mixed in the desired proportions at concentrations higher than desired in the composition as used, and then the concentrate is appropriately diluted with water to form the composition as used, or, on the other hand, the nitrite, proton source and organic polyol components are first mixed in the desired proportions at the concentrations desired for the composition as used.

[0149] Furthermore, it is not possible to predict which method of mixing the components will produce better results in terms of antimicrobial efficacy. In general, diluting a relatively concentrated premix to produce a used composition appears to produce better antimicrobial efficacy against M. tuberculosis HN878 in THP-1 cells, but in some cases produces results that are not as good as methods in which the components are first mixed at the desired concentrations for use.

[0150] Thus, in one embodiment of the present invention, a method for preparing a NOx-producing composition includes mixing nitrite, proton source, and organic polyol components in desired proportions at concentrations higher than desired in the composition to be used to form a concentrate premix, followed by diluting the concentrate premix appropriately with water to provide the composition to be used.

[0151] Thus, in another embodiment of the present invention, a method for preparing a NOx-producing composition comprises mixing a nitrite salt, a proton source, and an organic polyol component in the desired concentrations and proportions for the composition in the form in which it is to be used to provide the composition in use.

[0152] Preferred Embodiments Preferred embodiments of the first to eighth aspects of the present disclosure are those in which one or more of the following are present: the one or more nitrites include (e.g., contain, consist essentially of, or consist of) one or more alkali metal or alkaline earth metal nitrites, e.g., sodium nitrite, potassium nitrite, or any combination thereof; the proton source comprises (e.g., contains, consists essentially of, or consists of) ascorbic acid or an ascorbic acid / ascorbate buffer; citric acid or a citric acid / citrate buffer; or any combination of two or more thereof; - the ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citrate buffer, or any combination of two or more thereof, molecules are not covalently bound to a polymer or macromolecule; - the one or more organic polyols comprise (e.g., contain, consist essentially of, or consist of) a straight-chain sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule; for example, sorbitol; mannitol; arabitol; xylitol; or any combination of two or more thereof; - the one or more organic polyols are, for example, sugar alcohol compounds comprising (e.g., consisting of) a chain of one, two, or three monosaccharide units terminated with one acyclic alcohol unit, optionally wherein one, two, three, or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the acyclic alcohol unit is a C5 or C6 sugar alcohol unit, for example, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol; - the total molar concentration of one or more organic polyols in the polyol component or reaction solution at or before the start of the NOx-forming reaction is 0.05 to 3 times the total molar concentration of nitrite ions in the nitrite component or reaction solution; - the total molar concentration of one or more organic polyols in the polyol component or in the reaction solution at or before the start of the NOx-forming reaction is 0.05 to 3 times the total molar concentration of the proton source in the proton source component or in the reaction solution; - For applications that do not involve contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source prior to, and especially immediately prior to, the initiation of the NO-producing reaction is in the range of 3.0 to 9.0; - For applications involving contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source prior to, and especially immediately prior to, the initiation of the NO-producing reaction is in the range of 4.0 to 8.0; - in applications involving contact of the reaction mixture with the nose, mouth, airways or lungs of an animal (including human) subject in accordance with the present invention, the pH of the proton source prior to, and in particular immediately prior to, the initiation of the NO-producing reaction is in the range of 5.0 to 8.0; - The target microorganism is selected from the microorganisms listed under the section "Target for Antimicrobial Use", such as, but not limited to, influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

[0153] Preferred embodiments of the ninth aspect of the present disclosure are those in which one or more of the following are present: the one or more nitrites include (e.g., contain, consist essentially of, or consist of) one or more alkali metal or alkaline earth metal nitrites, e.g., sodium nitrite, potassium nitrite, or any combination thereof; the proton source comprises (e.g., contains, consists essentially of, or consists of) ascorbic acid or an ascorbic acid / ascorbate buffer; citric acid or a citric acid / citrate buffer; or any combination of two or more thereof; - the ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citrate buffer, or any combination of two or more thereof, molecules are not covalently bound to a polymer or macromolecule; - the one or more organic polyols comprise (e.g., contain, consist essentially of, or consist of) a straight-chain sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule; for example, sorbitol; mannitol; arabitol; xylitol; or any combination of two or more thereof; - the one or more organic polyols are, for example, sugar alcohol compounds comprising (e.g., consisting of) a chain of one, two, or three monosaccharide units terminated with one acyclic alcohol unit, optionally wherein one, two, three, or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the acyclic alcohol unit is a C5 or C6 sugar alcohol unit, for example, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol; - the total molar concentration of one or more organic polyols in the polyol component or reaction solution at or before the start of the NOx-forming reaction is 0.05 to 3 times the total molar concentration of nitrite ions in the nitrite component or reaction solution; - the total molar concentration of one or more organic polyols in the polyol component or in the reaction solution at or before the start of the NOx-forming reaction is 0.05 to 3 times the total molar concentration of the proton source in the proton source component or in the reaction solution; - For applications that do not involve contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source prior to, and especially immediately prior to, the initiation of the NO-producing reaction is in the range of 3.0 to 9.0; - For applications involving contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source prior to, and especially immediately prior to, the initiation of the NO-producing reaction is in the range of 4.0 to 8.0; - in applications involving contact of the reaction mixture with the nose, mouth, airways or lungs of an animal (including human) subject in accordance with the present invention, the pH of the proton source prior to, and in particular immediately prior to, the initiation of the NO-producing reaction is in the range of 5.0 to 8.0; - The target microorganism is selected from the microorganisms listed under the section "Target for Antimicrobial Use", such as, but not limited to, influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

[0154] Combinations and Compositions The NOx-forming reaction can be initiated in a number of ways, which generally involve contacting one or more nitrites with a proton source under conditions that allow the NOx-forming reaction to be initiated.

[0155] The reaction can be initiated by combining the separate components of the combination. This combination can be achieved in vitro, and the resulting composition can be administered to a subject or applied to any surface to be treated according to the present disclosure. Alternatively, the released gas can be administered to a subject or applied to any surface to be treated according to the present disclosure. Furthermore, the use of both resulting compositions can proceed at intervals in time, such that the composition is administered to a subject or applied to any surface to be treated after some generation of gas has occurred.

[0156] The combination may be stepwise, for example, with dry powder forms of the ingredients mixed first and then mixed with water or another liquid carrier medium to initiate the reaction. Alternatively, dry powder forms of the ingredients may be mixed separately with water or another liquid carrier medium first, and then the two or more liquids may be mixed to initiate the reaction.

[0157] Alternatively, at least some of the components of the NOx-forming reaction according to the present disclosure may be present as a mixture in a single composition, and the NOx-forming reaction is initiated on the composition. One possible way to initiate the NOx-forming reaction can be, for example, by adding a key component or additive that initiates the reaction, such as water if the components of the composition are in a dry or encapsulated form; or a proton source if the components of the composition lack a proton source.

[0158] A kit according to the present disclosure typically includes one or more components of a combination according to the present disclosure or a composition according to the present disclosure under conditions in which a NOx-forming reaction does not occur. Each component of the kit is typically held in a container, which may be separate or adapted to facilitate the mixing required to initiate the NOx-forming reaction. The key starting component for initiating the NOx-forming reaction, which must be introduced by the user of the kit into the other necessary components, may be, for example, one of the nitrite component, the proton source component, or the polyol component, or may be an additional component, typically a commonly available component such as water, which may be supplied by the user.

[0159] The combination and composition parameters defined and described in this patent typically include physical parameters such as pH, concentration, and osmolality. Whenever possible, these should be measured before the initiation of the NOx-forming reaction. Unless otherwise specified, the pH parameter refers to the pH of the proton source in deionized water at a concentration intended to initiate the NOx-forming reaction. Unless otherwise specified, the solution concentration refers to the concentration before mixing with other components to initiate the NOx-forming reaction. Typically, when a nitrite salt reacts with an organic carboxylic acid or organic reducing acid to produce nitric oxide gas upon mixing, such parameters cannot be easily measured while the NOx-forming reaction is in progress.

[0160] Furthermore, it should be noted that the concentrations of the components in the reaction mixture do not necessarily correspond to the concentrations of each component in the combination before mixing. For example, assume a composition for initiating a NOx-forming reaction according to the present disclosure is formed from approximately equal volumes of a nitrite component and a proton source component added together as pre-prepared solutions. In that embodiment, the reaction composition when mixed has a nitrite concentration that is half the concentration of the nitrite component and a proton source concentration that is half the concentration of the proton source component.

[0161] The components of the combination and composition can be in any suitable physical form depending on the intended use of the system during or after the NOx-producing reaction. For example, each component of the combination and composition can be in the form of a liquid, gel, or film, so that the NOx-producing reaction mixture is also in the form of a liquid, gel, or film. The liquid may be adapted to be sprayed for inhalation into the respiratory tract or lungs. If the NOx-producing mixture is intended to be applied to the mouth or throat, the components of the combination and composition may be in the form of a mouthwash or drink. Alternatively, if the NOx-producing reaction mixture is intended to be applied to the skin for topical administration, the components of the combination and composition may be in the form of an ointment, lotion, or cream.

[0162] Multi-component systems, kits and dispensers The multi-component systems described herein may include a nitrite component and a proton source component, optionally with a polyol component, as defined in accordance with the present disclosure and as described herein. The components in the multi-component system are adapted to contact each other, and the reaction mixture and / or evolved gases are dispensed by means of a suitable container or reservoir for holding the components prior to use, and by means for mixing the components, dispensing the reaction mixture and / or evolved gases, and generally controlling said mixing and dispensing. In one preferred embodiment, the reaction mixture may be dispensed in the form of a mist or aerosol of droplets entrained in an airflow.

[0163] The kits and dispensers of the present disclosure generally include at least some of the containers for holding the components prior to use, at least one device or other means for mixing the components, dispensing the reaction mixture and / or released gas, and generally controlling said mixing and dispensing, as well as the components (if any) contained in the kit or dispenser containers prior to use. Instructions for use, or instructions on where to find instructions for use (e.g., online instructions for use), may be present as appropriate. Such kits and dispensers constitute further aspects of the present disclosure.

[0164] Kits of the present disclosure may be relatively simple collections of containers and means for mixing components, dispensing reaction mixtures and / or evolved gases, and generally controlling said mixing and dispensing. Such kits may be appropriately provided for research purposes or where a wide range of variation in mixing and dispensing operations is expected and tolerated.

[0165] Other kits of the present disclosure may be more complex collections of one or more containers containing consumables (which are the combinations and / or compositions required by the user to initiate the NOx-producing reaction, optionally with water or other commonly available components supplied by the user) along with one or more dispensers of the present disclosure.

[0166] Dispensers of the present disclosure are generally adapted for repeated, similar actions of dispensing the reaction mixture, the carrier containing the reaction mixture, and / or the released gas. The dispenser may include a pump or injection system for conveying the NOx-producing reaction mixture or the composition containing the released gas from the dispenser and directing it toward the target. The injection system may use pressurized and / or liquefied gas, and for medical use, a pharmaceutically acceptable or biocompatible gas, such as pressurized air or pressurized / liquefied butane, is appropriate. Alternatively, aspiration from the user's lungs may be used to convey the NO-producing reaction mixture or the composition containing the released gas from the dispenser and direct it toward the target. Dispensers used in the present disclosure may suitably include an actuator device, such as a manually operable trigger or button, that allows a user to activate the dispenser. Such dispensers may be adapted for use by professionals, researchers, consumers, or patients, and correspondingly, may be adapted to accommodate the intended treatment pathway for the target. The device can be adapted to facilitate the passage of the fluid.

[0167] A wide range of kits and dispenser devices are known in principle, which can be used or readily adapted to hold the components prior to use, mix the components or facilitate said mixing, dispense the reaction mixture and / or composition including the evolved gas, and generally control or facilitate said mixing and dispensing.

[0168] example: - Syringes, e.g. twin barrel dispensing syringes. A container system, e.g., including two containers, e.g., a pumping, squeezing, or shaking container, for mixing at least a nitrite salt component and a proton source component and dispensing a composition comprising a NOx-forming reaction or evolved gas. Such a system is described in U.S. Patent Application Publication No. 2019 / 0134080, the disclosure of which is incorporated herein by reference. A device for holding and mixing components prior to use in an aqueous solution, nebulizing the liquid reaction mixture, dispensing the liquid reaction mixture for inhalation into the human lung, and generally controlling said mixing and dispensing. Examples include soft mist inhalers, jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Selection of nebulizers, droplet sizes, adjuvants, packaging forms, etc. suitable for inhalation of the atomized NOx-producing reaction medium by acidifying nitrite are described in WO 03 / 032928 and WO 2009 / 086470, the disclosures of which are incorporated herein by reference. The device may be arranged to nebulise a pre-mixed liquid reaction mixture after it has been loaded into the nebuliser and to dispense it for inhalation into a human lung, and generally control said mixing and dispensing. A device for holding and mixing components prior to use in an aqueous solution, aerosolizing and dispensing a liquid reaction mixture for inhalation into the human lung, and generally controlling said mixing and dispensing. Examples include metered-dose inhalers. Selection of droplet size, adjuvants, packaging formats, etc. suitable for inhalation of an aerosolized NOx-producing reaction medium by acidifying nitrite is described in WO 03 / 032928 and WO 2009 / 086470, the disclosures of which are incorporated herein by reference. Techniques and devices for spraying nitric oxide-releasing solutions into the upper airways are described in US Pat. No. 9,730,956, the disclosure of which is incorporated herein by reference. - A device for holding ingredients in dry powder form prior to use and dispensing them for inhalation into the human lung. Examples include dry powder inhalers (DPIs), which can be formulated as single-dose capsules or multi-dose dry powder inhalers, either as reservoir powders or as separate blisters of multiple doses. Selection of powder particle size, adjuvants, packaging formats, etc. suitable for inhalation of dry powder combinations to provide a reaction medium in the lungs for in situ generation of NO by acidification of nitrite is described in WO 2009 / 086470, the disclosure of which is incorporated herein by reference. - Dispensers for holding ingredients prior to use in solution form, aerating them, and dispensing them as a foam for use as a skin antiseptic or to treat skin disorders are described in U.S. Patent Application No. 2013 / 0200109, U.S. Patent No. 7,066,356, and U.S. Patent Application No. 2019 / 0134080, the disclosures of which are incorporated herein by reference; A transdermal patch assembly for holding ingredients and distributing them to the skin of a subject is described in WO 2014 / 188175, the disclosure of which is incorporated herein by reference.

[0169] The dose of the combinations and compositions or released gases of the present disclosure may be determined based on the disease, disorder, or condition being treated (in the case of medical treatment) or the desired effect (in the case of non-medical treatment), the amount of steroids needed, and the amount of steroids needed. According to the severity of treatment and the pathological condition, age and health of the subject being treated, or in the case of non-medical treatment, the nature of the target being treated, it can vary widely.In the case of medical treatment, the doctor will ultimately determine the appropriate dosage to be used.In the case of non-medical treatment, those skilled in the art can study the appropriate dosage and treatment method by reviewing the relevant literature through reasonable tests.

[0170] In some embodiments, the composition undergoing the NOx-producing reaction, or the gas released therefrom, can be administered to a target location, such as a microbial cell, biological tissue, organ, structure, or subject, within 600 seconds after combining the nitrite component and the proton source component, thereby exposing the target location to a large burst of nitric oxide.

[0171] In some embodiments, the composition undergoing the NOx-producing reaction can be formed in situ or at or near a target location, such as on, within, or near a microbial cell, biological tissue, organ, structure, or subject, including inanimate surfaces and spaces. In these embodiments, administration occurs substantially 0 seconds after combining the nitrite component and the proton source component. In other embodiments, the composition is administered to or near the target location between greater than 0 and less than 600 seconds after combining the nitrite component and the proton source component. In more specific embodiments, the composition is administered between 0 and 120 seconds. In yet further embodiments, the composition is administered between 0 and 60 seconds.

[0172] In other embodiments, the composition undergoing the NOx-forming reaction, or the gas released therefrom, can be administered to a target location, e.g., a microbial cell, biological tissue, organ, structure, or subject, for more than 600 seconds, e.g., more than 2000 seconds, e.g., more than 4000 seconds, e.g., more than 8000 seconds, after combining the nitrite component and the proton source component. In such cases, the target location, e.g., a microbial cell, biological tissue, organ, structure, or subject, may not necessarily be exposed to a large burst of nitric oxide, but may still experience beneficial properties, such as antimicrobial effects. In these embodiments, the composition undergoing the NOx-forming reaction, or the gas released therefrom, can be administered up to 48 hours after combining the nitrite component and the proton source component. In certain embodiments, the composition or gas released therefrom can be administered up to several weeks or months, for example, up to about 6 months, or up to about 2 months, or up to about 1 month, or up to about 3 weeks, or up to about 2 weeks, or up to about 1 week, or up to about 3 days, or up to 24 hours, after combining the nitrite component and the proton source component.

[0173] When properly stored, the composition undergoing the NOx-producing reaction, or the gas released therefrom, can be administered more than 48 hours after combining the nitrite and proton source components. For example, the composition may be stored in a sealed container, e.g., under vacuum. Storage in a sealed container typically occurs within 24 hours after combining the nitrite and organic carboxylic acid or organic reducing acid. The composition may be stored in a sealed container within 600 seconds after combining the nitrite and proton source components. In this manner, a proportion of nitric oxide gas can be retained. When the NOx-producing composition is stored at low temperatures, e.g., at temperatures ranging from about -30°C to about +10°C, e.g., from about 1°C to about 10°C, the rate of gas generation can be substantially slowed, allowing the composition to have a significantly longer shelf life.

[0174] In certain embodiments, the aerosol dispenser can include multiple reservoirs, with a first reservoir containing a nitrite component in liquid form (e.g., an aqueous solution) and a second reservoir containing a proton source component in liquid form (e.g., an aqueous solution), in which each component is present before, during, and after the nitrite and proton source components are mixed. may then be suitably mixed with the propellant.

[0175] In another specific embodiment, the dispenser may be a single-barrel syringe containing the composition of the present disclosure. The viscosity of the composition may be selected so that it can be dispensed from the syringe by manual operation or by power operation of the syringe. For example, the composition may be a liquid or a gel.

[0176] In another specific embodiment, the dispenser may be a multi-barrel syringe having a first barrel containing a nitrite salt component and a second barrel containing a proton source component. The viscosities of the components may be selected so that they can be dispensed from the syringe by manual operation or by power operation of the syringe. For example, each component may independently be a liquid or a gel.

[0177] Other reservoirs for ingredients: hydrogels In some embodiments of the present disclosure, molecular reservoirs, such as hydrogels, can be used. Hydrogels are highly hydrated, usually crosslinked, three-dimensional polymers (homopolymers or copolymers) or macromolecular networks capable of absorbing and retaining many times their dry weight of water, other aqueous liquids, or other non-aqueous hydrophilic liquids. Absorption of liquid is usually accompanied by swelling of the hydrogel. By appropriately selecting the chemical groups of the components covalently bonded to the polymer or macromolecule, acidic hydrogels or hydrogels with other special chemical properties can be prepared.

[0178] Hydrogels that can be used as proton source components in the present disclosure are known.Examples of such acidic -COOH group-containing hydrogels are described in, for example, WO 2007 / 007115, WO 2008 / 087411, WO 2008 / 087408, WO 2014 / 188174 and WO 2014 / 188175, and the documents referred to therein, the disclosures of which are all incorporated herein by reference.The use of such hydrogels in skin care using NOx generation, including transdermal delivery of medicines in conjunction with NOx generation, is particularly described in WO 2014 / 188174 and WO 2014 / 188175.Specific examples of such hydrogels include homopolymers and copolymers of acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (ATBS, available from Vinati Organics Ltd) and their salts. Polymers formed from monomers comprising or consisting of (meth)acrylic acid include pendant carboxylic acid groups for use as a proton source in accordance with the present disclosure.

[0179] Thus, for example, a multi-component system can include a first acidic hydrogel pad or layer component containing a proton source component and, optionally, an organic polyol, and another component can be a nitrite component. The nitrite component can be, for example, a liquid medium containing dissolved nitrite. In this manner, the surface of the hydrogel pad or layer can be contacted with the nitrite component to initiate the NOx-producing reaction. Alternatively, the nitrite component can be a solid support, such as a pad or layer, containing nitrite in a form that dissolves in the absorbed liquid of the hydrogel upon contact between the solid support and the hydrogel.

[0180] Typically, the solid carrier pad or layer is permeable (fully permeable or at least semi-permeable) to the diffusion of nitric oxide. Thus, the combination of the solid carrier pad or layer with a hydrogel, along with the nitrite and proton source components, allows nitric oxide to diffuse to the treatment area. The solid carrier pad or layer may be, for example, a mesh, a nonwoven batt, a film, a foam, an alginate layer, or a membrane.

[0181] In certain embodiments, the solid support layer is a mesh. The mesh may be a solid, typically flexible, multiplicity of connected strands that form a lattice of holes or gaps through which a particular substance passes. The mesh may be a woven or nonwoven fabric. In some embodiments, the mesh is a nonwoven fabric.

[0182] The solid carrier layer, for example, a mesh, can be made of a polymer material. Examples of suitable polymer materials include, but are not limited to, viscose, polyamide, polyester, polypropylene, or blends thereof. The polymer material may be treated, for example, to increase its hydrophilicity. In a specific embodiment, the solid carrier layer is a polypropylene mesh.

[0183] In certain embodiments, the solid support is absorbent and the nitrite component is at least partially absorbed, imbibed, or impregnated into the solid support. The absorbed, imbibed, or impregnated nitrite component may be a (dried) solid or may be in aqueous solution within the solid support.

[0184] In certain embodiments, the solid support comprises two or more layers, and the nitrite component is absorbed, imbibed, or impregnated into at least one layer, or coated onto at least one outer layer. For example, the solid support can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more layers (e.g., polypropylene mesh layers) that are absorbed, imbibed, impregnated, or coated with one or more nitrite salts in dry and / or solution form.

[0185] Acidic hydrogels have a natural buffering effect due to the large supply of pendant protonated acidic groups inside, from which H + Ions can migrate through the absorbed aqueous medium to maintain a relatively acidic pH at the surface of the hydrogel structure as pendant acidic moieties at the surface are deprotonated during the NOx-producing reaction.

[0186] Non-acidic (e.g., neutral or basic) hydrogels are also known and can be imbibed with and contain a nitrite and / or polyol component for use in the present disclosure. The proton source component can be contacted with such hydrogels by a proton source provided in a liquid medium contacted with the hydrogel and / or by a proton source absorbed, imbibed, impregnated, or coated on a solid support. In such hydrogels, it may be provided that none of the nitrite, proton source, or polyol components is covalently bound to the polymer or macromolecular network of the hydrogel; for example, all components required for the present disclosure may be imbibed in the hydrogel and contained in an aqueous medium within the hydrogel, but not covalently bound to the polymer or macromolecular network of the hydrogel, given that the nitrite and proton source components must not react together until initiation of the NOx-producing reaction is desired.

[0187] The thickness of the hydrogel pad or layer may range from 0.5 to 2 mm. In some embodiments, the thickness of the hydrogel pad or layer is in the range of 1 to 2 mm. In certain embodiments, the thickness of the hydrogel pad or layer is in the range of 1.0 to 1.6 mm.

[0188] The features described above with respect to the proton source component generally apply equally to any acidic hydrogel that acts as a proton source component. Thus, for example, the hydrogel may include a buffer to maintain the pH of the hydrogel in the range of 4.0 to 9.0, or 5.0 to 8.0. .

[0189] In some embodiments, the hydrogel may include a barrier layer. The barrier layer is typically a polymer film, such as a polyurethane film, disposed on the outer surface of the hydrogel. In use, the barrier layer is typically disposed on the surface of the hydrogel opposite, e.g., the subject's skin, to provide a barrier between the combined multi-component system and the atmosphere. The surface of the barrier film adjacent to the hydrogel typically has a larger surface area than the adjacent hydrogel surface. In this manner, the barrier layer can extend beyond the periphery of the hydrogel. In these embodiments, the barrier layer may have an adhesive around its peripheral edge to adhere the hydrogel, e.g., to the subject's skin, during use.

[0190] In certain embodiments, the present disclosure provides a two-component system comprising: a) one or more meshes absorbed, impregnated, or coated with one or more nitrites (e.g., NaNO2); and b) a hydrogel comprising a proton source containing one or more selected from an organic carboxylic acid and an organic non-carboxylic reducing acid; wherein component (a) is separate from component (b), and one or more of components (a) and (b) further comprise one or more organic polyols. (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not exclusively glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols are not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not exclusively polyvinyl alcohol; (h) in any one or more of (b) to (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) The one or more organic polyols are propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetrahydrofuran ... No. 1-(2-hydroxybenzoates), ...

[0191] For the avoidance of doubt, it is hereby acknowledged that the embodiments and preferences of features (a)-(h) discussed above in relation to aspects of the present disclosure apply equally to this embodiment.

[0192] Such a system may be used, for example, by combining components (a) and (b) to initiate a NOx-producing reaction. Such a combination may then be used in therapy or other treatment (e.g., by topical application) of the human or animal body. The use may be as described in WO 2014 / 188174 and WO 2014 / 188175, or as described below. The system may also be used in non-medical applications, as described below. When the system is used in a topical medical application in which it contacts the skin (including mucous membranes) of a subject, one or more meshes may be a skin-contacting layer.

[0193] Therapeutic or surgical use Compositions undergoing NOx-producing reactions according to the present disclosure, and the gases released therefrom, have many uses in therapy and surgery, including therapeutic and / or prophylactic therapy, surgery to correct diseases, disorders and conditions, cosmetic surgery, human, veterinary medicine, and reconstructive surgery, including surgery. Where a physical ailment or abnormality responsive to treatment with the composition or gases released therefrom causes or exacerbates anxiety, depression, or another psychiatric disease or disorder, treating, preventing, or alleviating the physical condition can correspondingly treat, prevent, or alleviate the mental condition, thereby extending the uses of the present disclosure to the field of mental health.

[0194] Many physiological effects of, and medical treatments based on, nitric oxide and nitric oxide-generating compositions have been reported in the literature, resulting in the development of numerous treatments. The following non-exhaustive list is provided by way of example. The listed uses, as well as other uses not listed, are encompassed by this disclosure and patent. Nitric oxide dilates blood vessels, increasing blood supply and / or decreasing blood pressure (see van Faassen et al, Med. Res. Rev., September 2009; 29(5), pp. 683-741); The acute effects of oral nitric oxide supplements to reduce blood pressure, improve vascular compliance, and restore epithelial function in hypertensive patients are described in Houston et al. in J. Clin. Hypertens. (Greenwich), July 2014, 16(7), 524-529; Nitric oxide protection of tissues from damage due to reduced blood supply (see van Faassen et al, Med. Res. Rev., September 2009; 29(5), pp. 683-741); Nitric oxide acts as a neurotransmitter in nitrergic neurons, such as those active on smooth muscle of the gastrointestinal tract and erectile tissue (see Toda et al, Pharmacol. Ther., May 2005; 106(2), pp. 233-266); Nitric oxide-mediated inhibition of vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium, supporting vascular homeostasis (see Dessey and Ferron, Current Medical Chemistry - Anti-inflammatory and Anti-allergy Agents in Medical Chemistry, 2004; 3(3), pp. 207-216); the action of nitric oxide to reduce cardiac contractility and heart rate (see Navin et al, J. Cardiovascular Pharmacology, 2002; 39(2), pp. 298-309); Critical neonatal care to promote capillary and pulmonary dilation, for example, in the treatment of primary pulmonary hypertension in neonatal patients and after meconium aspiration (Barrington et al, The Cochrane Database of Systematic Reviews, 2017;1, CD000399 (https: / / www.ncbi.nlm.nih.gov / pubmed / 17375630); see also Chotigeat et al., J. Med. Assoc. Thai, 2007;90(2), pp.266-271; see also Hayward et al., Cardiovascular Research, 1999;43(3), pp.628-638); Prevention of vascular damage, endothelial dysfunction, vascular inflammation, neuropathy, non-healing ulcers, and reduced risk of needing lower limb amputation in diabetic patients (see nfb University Studdies - “Nitric Oxide Holds Promise for Dibetes”, http: / / www.nfb.org / Images / nfb / Publications / vod / vod212 / vodspr0613.htm); Correction of hypoxemia in acute lung injury, acute respiratory distress syndrome, and severe pulmonary hypertension, treating reversible causes of hypoxemic respiratory distress (see Mark et al, N. Eng. J. Med, Dec. 22, 2005, 353(25), pp. 2683-2695); Administration of nitric oxide as rescue therapy in patients with acute right ventricular failure secondary to pulmonary embolism (see Summerfield et al, 2011; Respir. Care 57(3), pp. 444-448); Treatment of angina pectoris, paraquat poisoning and other cardiovascular effects (see Abrams, The American Journal of Cardiolody, 1996, 77(13), pp. 31C-37C); Treatment of bladder contractile dysfunction (Moro et al, Eur. J. Pharmacol, January 2012, pp. 445-449; see also Andersson et al, Br. J. Pharmacol, 2008, 153(7), pp. 1438-1444); Treatment of acute and chronic pulmonary infections and sepsis (Fang et al, Nature Reviews. Microbiology, October 2004; 2(10), pp. 820-832; see also Goldfarb et al, Critical Care Medicine, January 2007; 35(1), pp. 290-292); Toxic reactive nitrogen intermediates (RNIs), including nitric oxide, have been suggested as effector molecules in the antimycobacterial action of activated mouse macrophages against Mycobacterium tuberculosis (see Chan et al, J. Exp. Med, April 1992, pp. 1111-1122); Gaseous nitric oxide may be effective in treating antibiotic-resistant bacterial and fungal lung infections in patients with cystic fibrosis (see Deppisch et al., February 9, 2016; “Gaseous nitric oxide to treat antibiotic-resistant bacterial and fungal lung infections in patients with cystic fibrosis: a Phase I clinical study,” DOI 10.1007 / s15010-016-0879-x); Nitric oxide has been reported as a potential topical broad-spectrum antimicrobial agent for skin diseases, with a low likelihood of resistance development (see BL Adler and AJ Friedman, Future Sci. OA, 2015; 1(1), FSO37); Nitric oxide is a neurotransmitter that is involved in neural activity and a variety of functions, from avoidance learning in both sexes to genital erections (see Kim et al, J. Nutrition, 2004, 134, 28735); The use of nitric oxide to treat male impotence and erectile dysfunction is described in Sullican et al, Cardiovascular Research, August 1999, 43(3), pp. 658-665; The potential use of nitric oxide as a surgical adjuvant to aid wound healing, reduce ischemia-reperfusion injury, aid cardiac and pulmonary recovery from surgery, aid recovery from vascular surgery, and aid postoperative recovery from orthopedic surgery has been reported (see A Krausz and AJ Friedman, Future Sci. OA, 2015; 1(1), FSO56); The antibacterial and wound healing effects of NO are described in WO 95 / 22335 and Hardwick et al., 2001, Clin. Sci. 100, 395-400; European Patent No. 1411908 (University of Aberdeen) reports data purportedly showing that nitric oxide is effective in treating subungual infections involving Aspergillus niger; topical application of NOx live compositions to the skin for the treatment of fungal skin infections such as Tinea Pedis (Athlete's Foot) (see Weller et al, J. Am. Acad. Dermatol, April 1998, 38(4), pp. 559-563); topical application of NOx-generating compositions to the skin for the treatment of viral skin infections (see WO 99 / 44622); topical application of NOx-generating compositions to the skin for the treatment of conditions in which vasoconstriction is the underlying problem, such as Raynaud's syndrome (also known as Raynaud's phenomenon) (see Tucker et al, Lancet, November 13, 1999, 354, 9191, pp. 1670-1675); The use of acidified nitrites as agents to produce local production of nitric oxide at the skin surface for the treatment of peripheral ischemia and related conditions such as Raynaud's phenomenon, post-surgical wounds and wounds such as burns is described in WO 2000 / 053193; The use of liquid nitric oxide-releasing solution (NORS) to treat human wounds is claimed by U.S. Patent No. 9,730,956 (Stenzler et al.). NORS is also alleged to have antibacterial, antifungal, and / or antiviral properties, and data are provided that purportedly demonstrate antibacterial effects against Acetobacter baumannii, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Mannheimia haemolytica. Data are provided that purportedly demonstrate antiviral effects of NORS against H1N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, and bovine parainfluenza-3 virus. Data are provided that purportedly demonstrate antifungal effects of NORS against Trichophyton rubrum and Trichophyton mentagrophytes. Chou SH et al., Effect of band removal on pulmonary expression of ET-1, eNOS and cGMP in left ventricular pressure-overloaded rats, Exp. Biol. Med. 2005, 231, 954-959; Gladwin MT et al., Nitrite as a vascular endocrine nitric oxide reservoir contributing to hypoxic signaling, cytoprotection, and vasodilation, Am. J. Physiol. Heart Circ. Physiol. 2006, 291, pp. H2026-H2035; Hunter CJ, et al., Inhaled nebulized nitrite is a hypoxia-sensitive, NO-dependent selective pulmonary vasodilator, Nat. Med. 2004, 10, 1122-1127; Ozaki M et al, Endothelial nitric oxide reduces hypoxic pulmonary vascular remodeling, Hypertension, 2001, 37, 322-327; Rubin LJ, 2006, Pulmonary arterial hypertension, Proc. Am. Thorac. Soc. 3, pp. 111-115; Yellon DM et al, 2007, Myocardinal Reperfusion Injury, N. Engl. J. Med., 357, 1121-35; Duranski MR et al., Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver, J. Clin. Invest., 2005, 115, 1232-1240; Jung KH et al., Early intravenous infusion of sodium nitrite protects the brain from ischemia-reperfusion injury in vivo, Stroke, 2006, 37, 2744-2750; Esme H. et al., Beneficial effect of supplemental nitric oxide donor given during the reperfusion period in reperfusion-induced lung injury, Thorac. Cardiovase. Surg., 2006, 54, 477-483; The use of acidified nitrite to release NO as an agent for improving human skin quality is described in Chinese Patent Application No. 101028229; The use of acidified nitrites to release NO as an agent for promoting human hair growth and preventing or treating alopecia is described in Chinese Patent Application No. 101062050.

[0195] Other general details of the physiological effects of nitric oxide are described, for example, in Lancaster et al., Proc Natl Acad Sci, 1996, 91, 8137-8141; Ignarro et al., Proc Natl Acad Sci, 1987, 84, 9265-9269; and are reviewed in Brent, J Cell Science, 2003, 116, 9-15; Murad, N Engl J Med, 2006, 355, 2003-2011.

[0196] Published pharmacological forms for delivery of NO are reviewed in Butler and Feelisch, Circulation, 2008, 117, 2151-2159.

[0197] The disclosure of each of the above-cited publications is incorporated herein by reference.

[0198] This disclosure relates to all therapeutic and surgical uses of nitric oxide and nitric oxide generating systems. This disclosure is applicable to, including but not limited to, the specific therapeutic and surgical uses disclosed in the above references, as well as all other published therapeutic and surgical uses, and therapeutic and surgical uses based on the underlying knowledge of the physiological effects of nitric oxide and the products of the nitric oxide generating reaction.

[0199] vasodilation The property of nitric oxide to induce vasodilation characterizes many treatments using the disclosed combinations and compositions, and the gases released therefrom.

[0200] Specific examples of diseases, disorders and conditions that respond to vasodilation include, but are not limited to, conditions associated with ischemia and skin lesions.

[0201] Conditions associated with tissue ischemia include Raynaud's syndrome, severe primary vasospasm, and tissue ischemia caused by, for example, surgery, septic shock, radiation, or peripheral vascular disease (e.g., diabetes and other chronic systemic diseases).

[0202] When used in the treatment or prevention of conditions associated with tissue ischemia resulting from surgery, the combinations or compositions of the present disclosure, or nitric oxide released from NOx-producing reactions using the present disclosure, may be administered to a subject before, during, or after surgery. The combinations, compositions, or released gases can be administered at or near the surgical site. Examples of surgical procedures in which this treatment or prevention of tissue ischemia can be used include transplant surgery, tissue or organ transplant surgery, coronary artery surgery, carotid artery catheterization, surgery to provide an indwelling arterial or venous catheter for administering systemic medications such as chemical agents, cosmetic surgery including, but not limited to, pedicle or rotational flaps, revision surgery in which an incision is made in the same area as a previous surgical procedure, surgery performed in areas of inadequate perfusion of the skin and / or underlying tissues or when inadequate perfusion is expected as a result of a concomitant disease (such as arteriosclerosis or diabetes), surgery following injury or trauma to blood vessels, and surgery to remove or correct cutaneous or subcutaneous arteriovenous malformations.

[0203] For example, the combination, composition, or released gas can be used to treat or prevent ischemic reperfusion injury in an organ by administering the combination, composition, or released gas according to the present disclosure to the organ. The organ can be one or more selected from the heart (e.g., to prevent or treat myocardial ischemia), the brain (e.g., to treat or prevent cerebral ischemia and infarction (stroke)), the lung (e.g., to treat or prevent ischemic reperfusion injury in the lung), the kidney (e.g., to treat or prevent ischemic reperfusion injury in the kidney), and the liver (e.g., to treat or prevent ischemic reperfusion injury in the liver). The surgery can be organ transplantation. The administration of the combination, composition, or released gas can be performed after the onset of ischemia or can be prophylactic.

[0204] Transdermal Drug Delivery Applications The ability of nitric oxide to induce transdermal delivery of drugs represents another important utility of the disclosed combinations and compositions, and the gases released therefrom.

[0205] WO 02 / 17881 and WO 2014 / 188175 (the disclosures of which are incorporated herein by reference) describe the use of combinations and compositions for generating nitric oxide and gases released therefrom for transdermal drug delivery, and the same conditions, preferences, and examples described in these publications for such uses are applicable to the combinations and compositions of the present disclosure, and the gases released therefrom.

[0206] Typically, the combination and composition of the present disclosure comprises one or more pharmaceutically active agents that are delivered transdermally to the subject, and is provided in the form of a topical combination or composition that is applied to the skin of the subject.For examples of pharmaceutically active agents that can be used, see the section above titled "optional additional ingredients".

[0207] A suitable topical combination can include a nitrite-containing mesh and a separate proton source-containing hydrogel, the two adapted for use together on the skin of a subject, as described above in the section "Other Reservoirs for Compositions or Composition Systems; Hydrogels." The polyol and pharmaceutically active agent may be provided in one or more separate components of the combination, or may be incorporated into the hydrogel, or any combination of these options may be used for the polyol and pharmaceutically active agent, respectively.

[0208] Treating wounds, skin lesions, and burns The properties of nitric oxide to induce vasodilation and transdermal delivery of drugs, and to kill or prevent microbial growth, have led to another important utility of the disclosed combinations and compositions and the gases released therefrom in the treatment of wounds, skin lesions and burns.

[0209] Conditions treatable using the present disclosure include ulcers, skin donor sites, surgical wounds (post-operative), burns (such as thermal burns, superficial burns, partial-thickness burns, and full-thickness burns), lacerations, and abrasions. Wounds can be chronic or acute. Ulcers can be of various origins, such as arterial or venous origin. Examples of ulcers include leg ulcers, e.g., chronic or acute leg ulcers, pressure ulcers, e.g., chronic or acute pressure ulcers, venous ulcers, and ulcers associated with diabetes, such as diabetic foot ulcers.

[0210] WO 2014 / 188174 (the disclosure of which is incorporated herein by reference) describes the use of combinations and compositions for producing nitric oxide and the gas released therefrom to treat wounds, skin lesions, and burns, and the same conditions set forth in this publication are applicable to the combinations and compositions of the present disclosure and the gas released therefrom.

[0211] Typically, the combination and composition of the present disclosure comprises one or more pharmaceutically active agents and is provided in the form of a topical combination or composition for application to the skin of a subject.For examples of pharmaceutically active agents that can be used, please refer to the section entitled "Optional Additional Ingredients" above.For the treatment of wounds, skin lesions and burns, one or more pharmaceutically active agents can be suitably selected from analgesics and / or anesthetics (e.g., local anesthetics) (e.g., analgesics and / or anesthetics for relieving chronic pain, acute pain or neuropathic pain), antibacterial agents, disinfectants, anti-inflammatory agents and anti-scarring agents.

[0212] A suitable topical combination can include a nitrite-containing mesh and a separate proton source-containing hydrogel, the two adapted for use together on the skin of a subject, as described above in the section "Other Reservoirs for Compositions or Composition Systems; Hydrogels." The polyol and pharmaceutically active agent may be provided in one or more separate components of the combination, or may be incorporated into the hydrogel, or any combination of these options may be used for the polyol and pharmaceutically active agent, respectively.

[0213] Topical antibiotics For antimicrobial applications, therapeutically effective NO doses can be low, e.g., as low as several hundred ppm, e.g., 100-600 ppm (see, e.g., Ghaffari et al., Nitric Oxide Biol. 2004; ... Science and Chemistry, 2009, 14, 21-29, the disclosure of which is incorporated herein by reference), the effectiveness of nitric oxide is substantially dependent on the time that skin contact is maintained (Ormerod et al, BMC Research Notes, 2011, 4, 458-465, the disclosure of which is incorporated herein by reference).

[0214] Proposals for slow local release of nitric oxide have been published (see, e.g., U.S. Pat. No. 6,103,275). However, the resulting local NO dose lasts for less than one hour, providing poor local antibacterial activity. As discussed above in the section entitled "Multi-Component Systems, Kits, and Dispensers," and as shown in the Examples below, the present disclosure allows for much longer NO dosing periods in both topical and non-topical administration systems, resulting in substantial clinical benefits.

[0215] In particular, it has been found that the disclosed combinations and compositions are capable of providing a strong output of nitric oxide in the first approximately 200-500 seconds after the NOx-producing reaction begins (the "initial burst"), optionally followed by a slower release of nitric oxide (the "tail") that extends over an extended period of time before gas generation ceases or drops below effective levels. The NO doses released by the disclosed combinations and compositions exceed published minimum effective antibacterial doses, providing potentially effective topical antibacterial uses of the disclosed combinations and compositions and the gas released therefrom.

[0216] Formulations of NOx-generating combinations and compositions for topical antimicrobial applications are fully described in the prior art, e.g., U.S. Patent Application Publication No. 2014 / 0056957, the disclosure of which is incorporated herein by reference, and such formulations are also applicable to the combinations and compositions of the present disclosure. Another suitable topical combination can include a nitrite-containing mesh and a separate proton source-containing hydrogel, the two adapted for use together on a subject's skin, as described in the "Other Reservoirs for Compositions or Composition Systems; Hydrogels" section above. The polyol and pharmaceutically active agent may be provided in one or more separate components of the combination, or may be incorporated into the hydrogel, or any combination of these options may be used for the polyol and pharmaceutically active agent, respectively.

[0217] Other percutaneous or topical treatments Other topical applications of nitric oxide and nitric oxide-generating compositions include stimulating hair growth and treating impotence and erectile dysfunction.

[0218] The combinations and compositions of the present disclosure can be formulated for topical application for such treatments.

[0219] Topical dressings and dressing systems, e.g., wound dressings In topical treatments, it is often desirable to cover or protect the treated area of ​​skin while the treatment is being applied. This can help prevent contamination of the wound, help remove pus or debris from the healing process, prevent or limit loss of the therapeutic composition when bathing or showering, or through contact with clothing, or as a result of the subject's normal activities, and cushion the treated area against knocks or abrasions.

[0220] To this end, it is common to incorporate the treatment into a topical bandage or dressing system, such as a wound dressing or bandage system. The bandage, or at least one component part of the bandage system, typically includes a backing sheet, which may be water-impermeable or water-permeable, optionally with a skin-adhesive portion, and optionally with other layers, such as a gauze or padding layer.

[0221] In a further aspect, the present disclosure provides a topical dressing, e.g., a wound or skin dressing, or a dressing system, comprising the combination or composition according to the fifth aspect of the present disclosure, wherein at least one component of the dressing or dressing system comprises a backing sheet and, optionally, one or more other layers, e.g., a layer selected from a gauze and a padding layer. The combination or composition according to the fifth aspect of the present disclosure is suitably disposed on the side of the backing sheet facing the skin, positioned such that, when the dressing is applied to the skin and the NOx-producing reaction is initiated, the desired skin area is treated with the NOx-producing reaction mixture or gases released therefrom.

[0222] The dressing or dressing system may suitably be provided in a sealed sterile pack prior to use.

[0223] Use in the nose, mouth, airways and lungs The properties of nitric oxide to induce vasodilation and transdermal delivery of drugs and to kill or prevent the growth of microorganisms have led to another important utility of the combinations and compositions of the present invention and the gas generated therefrom in treating the mucous membranes and tissues of the nose, mouth, airways and lungs, and / or using the nose, mouth, airways and lungs as routes of administration for delivering the combinations and compositions of the present invention to human or animal subjects.

[0224] Conditions that can be treated using the present invention include, for example, viral infections such as influenza, SARS-CoV or SARS-CoV-2, pulmonary arterial hypertension, ischemia-reperfusion injury of the heart, brain and organs involved in transplantation, chronic obstructive pulmonary disease (COPD) (particularly emphysema, chronic bronchitis), severe asthma, asthma including viral and bacterial-induced exacerbations, refractory (irreversible) asthma, pneumonia, tuberculosis, non-tuberculous mycobacterial infections, other bacterial and viral pulmonary infections, for example secondary bacterial infections following viral infection of the respiratory tract.

[0225] WO 2009 / 086470 (the disclosure of which is incorporated herein by reference) describes the use of nebulized liquid combinations and compositions for generating nitric oxide and the gases emitted therefrom to treat diseases of the nose, mouth, airways and lungs, and / or the use of the nose, mouth, airways and lungs as routes of administration for delivering such combinations and compositions to human or animal subjects, and the same conditions, preferences and examples described in that publication for such uses are also applicable to the combinations and compositions and the gases emitted therefrom of the present invention.

[0226] Typically, the combinations and compositions of the present invention for delivery to the nose, mouth, airways and lungs comprise one or more pharmaceutically active agents.For examples of pharmaceutically active agents that can be used, see the section above entitled "Optional Additional Ingredients."

[0227] There are two principal delivery methods possible for practicing the present invention via the nasal, oral, respiratory tract, or pulmonary delivery route: 1) the combination or composition of the present invention is delivered directly to the nose, mouth, respiratory tract, or lungs; 2) the gas released from the NOx-producing reaction using the present invention is delivered to the nose, mouth, respiratory tract, or lungs without the combination or composition of the present invention entering the patient's body.

[0228] 1. Delivery of the combination or composition directly to the nose, mouth, airways, or lungs The combination or composition, or components thereof, may be delivered in dry solid form directly to the nose, mouth, airways, or lungs, whereby the fluids of the mucous membranes dissolve the solid component material and initiate the NOx-producing reaction.

[0229] The components of the combination may be administered separately or together. In the present invention, the proton source or at least one component thereof may be administered before the remaining components, so that a relatively acidic environment is established in the mucosa, which is why the nitrite component is in When contacted in situ with a proton source component, it aids in rapid initiation of the NOx-forming reaction.

[0230] Delivery of any dry component of the combination, or dry composition, directly to the nose, mouth, airways, or lungs may be suitably accomplished by dry powder inhalation using a dry powder inhaler, which delivers a therapeutically effective dose of one or more dry powder components (e.g., one or more nitrite, proton source, and polyol components) or the dry powder composition to a subject, and the dry powder inhaler delivers an aerosol containing particles with a volume average diameter of less than 6 microns to the subject. The dry powder inhaler may be configured to administer a single or multiple doses loaded with dry powder such that the dry powder inhaler delivers about 0.1 mg to about 100 mg of one or more dry powder components or dry powder composition per inhaled breath to the subject, in particles with a volume average diameter of less than 6 microns.

[0231] Additionally or alternatively, the combination or composition, or components thereof, may be delivered directly to the nose, mouth, airways, or lungs as a mist or spray of droplets of a solution of one or more of the nitrite component, proton source component, and polyol component.

[0232] The embodiments of the present invention described herein are generally applicable to direct delivery to the nose, mouth, airways, or lungs of a subject. For example, but not limited to, a combination or composition, or its components, may be administered directly to the nose, mouth, airways, or lungs of a subject with one or more physiologically compatible diluents, carriers, and / or excipients, and / or with one or more additional components, or specific functional components intended to provide one or more specific benefits. Examples of suitable physiologically compatible diluents, carriers, and / or excipients include, but are not limited to, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talc, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, etc. If desired, minor amounts of nontoxic auxiliary substances, such as wetting agents, emulsifiers, lubricants, binders, and solubilizers, for example, sodium phosphate, potassium phosphate, gum arabic, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like, may be present. Generally speaking, depending on the intended mode of administration, pharmaceutical preparations contain from about 0.005% to about 95% by weight, preferably from about 0.5% to about 50% by weight, of the combination or composition of the present invention or its components. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art. For example, Martindale, 39th edition (2017), the Merck Index, 15th edition (2013), Goodman & Gilman's "The Pharmacological Basis of Therapeutics", 13th edition (2017), the British National Formulary online (https: / / bnf.nice.org.uk / ), Remington: "The science & practice of Pharmacy", 22nd edition (2012), or the Physician's See Desk Reference, 71st Edition (2017).

[0233] In one preferred embodiment, the combination or composition for delivery to the nose, mouth, airways or lungs of a subject takes the form of a unit dosage form, such as a vial containing a liquid, a solid to be suspended, a dry powder, a lyophilisate or other composition, which may suitably include, along with the components of the NOx-producing reaction, a diluent (e.g., lactose, sucrose, dicalcium phosphate, etc.); a lubricant (e.g., magnesium stearate, etc.); a binder (e.g., starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives, etc.).

[0234] Inject any droplets containing the components of the combination, or the composition in droplet form, into the nose, mouth, respiratory tract or Direct delivery to the lung may be suitably accomplished by delivering a therapeutically effective dose of one or more liquid components (e.g., one or more of the nitrite component, proton source component, and polyol component) or a liquid form of the composition to a subject by inhalation using a nebulizer, wherein the nebulizer delivers an aerosol containing particles having a volume average diameter of less than 5 microns to the subject. The nebulizer may be adapted for single or multiple dose loading of the liquid components of the combination or liquid composition such that the nebulizer delivers from about 0.1 mg to about 100 mg per inhaled breath of one or more liquid components or a liquid form of the composition to the subject in droplets having a volume average diameter of less than 5 microns, preferably droplets having a size ranging from about 2 to about 5 μm.

[0235] In one embodiment, the nebulizer is selected based on enabling the formation of an aerosol of droplets containing the components of the combination, or the formation of a composition in the form of droplets having a predominantly aerodynamic diameter (MMAD) of about 2 to about 5 microns.

[0236] In one embodiment, a delivered amount of droplets containing the components of the combination, or a composition in droplet form, provides a therapeutic effect against pulmonary pathology, respiratory infections and / or extrapulmonary, systemic distribution to also treat extrapulmonary and systemic diseases.

[0237] Previously, two types of nebulizers, jet and ultrasonic, have been shown to be capable of generating and delivering aerosol particles between 2 and 4 μm in size. These particle sizes have been shown to be optimal for middle respiratory tract deposition and therefore for the treatment of pulmonary bacterial infections caused by Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa. However, unless specially formulated solutions are used, these nebulizers typically require larger volumes to administer sufficient amounts of drug for therapeutic efficacy. Jet nebulizers utilize air-pressure disruption of aqueous solutions into aerosol droplets. Ultrasonic nebulizers utilize shearing of aqueous solutions by piezoelectric crystals. However, jet nebulizers are typically only about 10% efficient under clinical conditions, while ultrasonic nebulizers are only about 5% efficient. Therefore, despite the large amount of drug placed in the nebulizer, only a fraction of 10% of the drug is deposited and absorbed in the lungs. Smaller particle sizes or slower inhalation rates allow for deep lung deposition. Depending on the indication, e.g., mid-airway deposition for antibacterial activity, or mid- and / or alveolar deposition for pulmonary arterial hypertension and systemic delivery, both mid-pulmonary and alveolar deposition may be desired for the present invention. Exemplary disclosures of compositions and methods for delivery of formulations using nebulizers can be found, for example, in U.S. Patent Application Publication No. 2006 / 0276483, including a description of the techniques, protocols, and characterization of aerosolized mist delivery using a vibrating mesh nebulizer. The disclosure of U.S. Patent Application Publication No. 2006 / 0276483 is incorporated herein by reference.

[0238] Thus, in one embodiment, a vibrating mesh nebulizer is used to deliver a liquid droplet aerosol or droplet-form composition containing the components of the combination in a preferred embodiment. The vibrating mesh nebulizer comprises a liquid reservoir in fluid contact with a diaphragm, and inhalation and exhalation valves. In one embodiment, about 1 to about 5 ml of the liquid formulation to be delivered is placed in the reservoir, and the aerosol generator is activated to generate an atomized aerosol having a particle size selected between about 1 and about 5 μm in volume average diameter.

[0239] Thus, for example, in a preferred embodiment, one or both of the nitrite component formulation or the proton source component, optionally including one or more organic polyols according to the present invention, are placed in a liquid nebulizer inhaler to produce a particle size of about 1 to about 5 μm volume mean diameter. The dosage is prepared in a volume of about 1 to about 5 ml to deliver about 7 to about 700 mg, preferably about 17.5 to about 700 mg in about 1 to about 5 ml, more preferably about 17.5 to about 350 mg in about 1 to about 5 ml, preferably about 0.1 to about 300 mg in about 1 to about 5 ml, more preferably about 0.25 to about 90 mg in about 1 to about 5 ml.

[0240] By way of non-limiting example, a spray liquid or droplet form composition containing the components of the combination may be administered at the recited respirable delivery dose in less than about 20 minutes, preferably less than about 10 minutes, more preferably less than about 7 minutes, more preferably less than about 5 minutes, more preferably less than about 3 minutes, and in some cases most preferably less than about 2 minutes.

[0241] By way of non-limiting example, in other situations, nebulized liquid or droplet-form compositions containing the components of the combination may achieve improved tolerability and / or exhibit shape-enhancing area under the curve (AUC) characteristics when administered over a longer period of time. Under these conditions, the respirable delivery dose described is greater than about 2 minutes, preferably greater than about 3 minutes, more preferably greater than about 5 minutes, more preferably greater than about 7 minutes, more preferably greater than about 10 minutes, and in some cases from about 10 to about 20 minutes.

[0242] An example of a separate component formulation may include: (i) a nitrite salt in an aqueous solution having a pH greater than about 6, e.g., in the range of about 6 to about 8, e.g., about 7; and (ii) a proton source component in an aqueous solution, wherein at least two separate liquid solution components (i) and (ii) can be mixed to form a NOx-generating composition, which can be used to load a nebulizer for delivery to a human patient or veterinary subject.

[0243] For aqueous and other non-pressurized liquid systems, various nebulizers (including small-volume nebulizers) are available to aerosolize the components of combination or composition.Compression-driven nebulizers incorporate jet technology and use compressed air to generate liquid aerosol.Such devices are commercially available from, for example, Healthdyne Technologies; Invacare; Mountain Medical Equipment; Pari Respiratory (Midlothian, VA); Mada Medical; Puritan-Bennet; Schuco, DeVilbiss Health Care, and Hospitalak.Ultrasonic nebulizers rely on mechanical energy in the form of vibration of piezoelectric crystals to generate respirable droplets, and are commercially available from, for example, Omron Healthcare and DeVilbiss Health Care.Vibrating mesh nebulizers rely on either piezoelectric pulses or mechanical pulses to generate respirable droplets.Other examples of nebulizers for use with the nitrites, nitrite salts, or nitrite- or nitric oxide-donating compounds described herein include U.S. Pat. Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; Nos.; U.S. Pat. Nos. 6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303, all of which are incorporated herein by reference in their entireties.

[0244] Commercially available examples of nebulizers that may be used with droplets containing the components of the droplet-form combinations or compositions described herein include Respirgard II®, Aeroneb®, Aeroneb® Pro, AeroEclipse XL®, and Aeroneb® Go manufactured by Aerogen (Aerogen Ltd., Galway, Ireland); AERx® and AERx Essence manufactured by Aradigm. TMPorta-neb®, Freeway Freedom, manufactured by Respironics, Inc. (Marysville, PA, USA) TM , SideStream, SideStream Plus, Ventstream, and I-neb, and PARI LC-Plus®, PARI LC-Star®, PARI LC-Sprint®, and e-Flow manufactured by PARI, GmbH (PARI Respiratory Equipment, Midlothian, Virginia, USA; PARI GmbH, Starnberg, Germany). TM Any of these nebulizers can be used with either a face mask or a mouthpiece, according to the manufacturer's specifications. By way of further non-limiting example, U.S. Patent No. 6,196,219 is incorporated herein by reference in its entirety.

[0245] In one embodiment, an aqueous formulation containing soluble or nanoparticle drug is provided. In aqueous aerosol formulations, the drug may be present at a concentration of about 0.67 mg / ml to about 700 mg / ml; in certain preferred embodiments, the nitrite salt is present at a concentration of about 0.667 mg nitrite anion per ml to about 100 mg nitrite anion per ml. Such formulations provide effective delivery to the appropriate areas of the lung, and more concentrated aerosol formulations have the additional advantage of allowing large amounts of drug to be delivered to the lung in a very short period of time. In one embodiment, the formulation is optimized to provide a well-tolerated formulation. Thus, certain preferred embodiments include a nitrite salt (such as sodium nitrite, potassium nitrite, or magnesium nitrite) and are formulated to have a good taste, a pH of about 4.7 to about 6.5, an osmolality of about 100 to about 3600 mOsmol / kg, and, in certain further embodiments, an osmotic ion (e.g., chloride, bromide) concentration of about 30 to about 300 mM.

[0246] In one embodiment, the solution or diluent used to prepare the aerosol formulation has a pH range of about 4.5 to about 9.0, preferably about 4.7 to about 6.5 (e.g., as an acidic mixture) or about 7.0 to about 9.0 as a single-vial configuration. This pH range, as well as the inclusion of a taste-masking agent according to certain embodiments as described elsewhere herein, improves tolerability. If the aerosol is either acidic or basic, it may cause bronchospasm and coughing. The safe pH range is relative; some patients can tolerate mildly acidic aerosols, while others experience bronchospasm. Any aerosol with a pH below about 4.5 typically induces bronchospasm. Aerosols with a pH between about 4.5 and about 5.5 occasionally cause bronchospasm. Any aerosol with a pH above about 8 may be poorly tolerated, as body tissues generally cannot buffer alkaline aerosols. Aerosols with controlled pH values ​​below about 4.5 and above about 8.0 typically produce lung irritation accompanied by severe bronchospasm, coughing, and inflammatory responses. For these reasons and to avoid bronchospasm, coughing, or irritation in patients, the optimal pH for aerosol formulations has been determined to be between about 5.5 and about 8.0.

[0247] As a result, in one embodiment, aerosol formulations for use as described herein are adjusted to a pH of about 4.5 to about 7.5, with the most preferred pH range for the acidic mixture being about 4.7 to about 6.5, and the most preferred pH range for the single vial configuration being about 7.0 to about 8.0. By way of non-limiting example, certain embodiments disclosed herein also include compositions containing a pH buffer or pH adjuster, typically a salt prepared from an organic acid or base, and, in preferred embodiments, an acidic excipient (e.g., citric acid) as described herein. The solution may include a buffer such as a non-reducing acid such as an acid or a citrate salt such as sodium citrate) or a citrate or other buffer described above and with reference to Table 1. Thus, these and other representative buffers may include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine, hydrochloride, or phosphate buffers.

[0248] Many patients have increased sensitivity to a variety of chemical tastes, including bitter, salty, sweet, and metallic. To create a well-tolerated drug product, taste masking can be achieved by adding taste-masking agents and excipients, osmolality adjusters, and sweeteners.

[0249] Many patients have high sensitivity to various chemicals and a high incidence of bronchospasm, asthma, or other cough-related complications. Their airways are particularly sensitive to hypotonic or hypertonic, acidic or alkaline conditions, and the presence of any permeant ions, such as chloride. An imbalance in these conditions or the presence of chloride above a certain concentration level can lead to bronchospasm or inflammatory events and / or cough, which significantly impairs treatment with inhalable formulations. Both of these conditions can prevent efficient delivery of aerosolized medication to the endobronchial space without the advantageous use of controlled pH, osmolality, and taste-masking agents, as disclosed in certain embodiments herein.

[0250] In some embodiments, the osmolality of aqueous solutions of the nitrite compounds disclosed herein (or, in separate embodiments, nitrite-donating compounds or nitric oxide-donating compounds) is adjusted by providing an excipient. In some cases, an amount of a permeant ion, such as chloride, bromide, or another anion, can facilitate successful and effective delivery of aerosolized nitrite. However, it has been discovered that for the nitrite components disclosed herein, the amount of such permeant ion may be lower than the amount typically used for aerosolized administration of other pharmaceutical compounds.

[0251] Bronchospasm or cough reflexes are not always improved by the use of a diluent for aerosolization with a given osmolality. However, these reflexes can often be adequately controlled and / or suppressed when the osmolality of the diluent is within a specific range. A preferred solution for aerosolization of safe and tolerable therapeutic compounds has a chloride concentration ranging from about 30 mM to about 300 mM, preferably about 50 mM to about 150 mM, and a total osmolality of about 100 to about 3600 mOsmol / kg. This osmolality controls bronchospasm, and the chloride concentration as a permeant anion controls coughing. Since both are permeant ions, bromide or iodide anions can be used in place of chloride. Additionally, bicarbonate may replace the chloride ion.

[0252] Nanoparticle drug dispersions can also be lyophilized to obtain powders suitable for nasal or pulmonary delivery. Such powders may contain aggregated nanodrug particles with surface modifiers. Such aggregates can have a size within the respirable range, e.g., MMAD of about 2 to about 5 microns.

[0253] 2. Delivery of gases released from the NO-producing reaction to the nose, mouth, airways, or lungs Inhalers for delivering metered amounts of nitric oxide to a patient's lungs are well known. Generally speaking, nitric oxide is generated off-site and delivered to a hospital or clinic in a pressurized cylinder connected to a specialized delivery device for use. The INOmax Therapy system is described, for example, at (BOC Healthcare, UK, https: / / www.bochealthcare.co.uk / en / products-and-services / products-and-services-by-category / medical-gases / inomax / inomax.html). The abbreviation INOmax (inhaled nitric oxide) is commonly used to refer to the serial number of the INOmax Therapy system. The INOmax Therapy system is used for under- and INOvent delivery devices. An evaluation of the INOmax Therapy system is published, for example, in Kimse et al., Chest, June 1998, 113(6), 1650-1657, the disclosure of which is incorporated herein by reference.

[0254] The method according to the first aspect of the invention may suitably be carried out in a dedicated NO production facility, and the gas product according to the second aspect of the invention provided to the user in a pressurized cylinder in the usual manner, which is then used in conjunction with dispensing, monitoring, dosing, mixing and delivery equipment in known manner.

[0255] Antibiotic targets As discussed above, the NOx-producing reactions of the present disclosure and the gases released therefrom potentially have bactericidal or biostatic effects against a wide range of microorganisms, leading to many antimicrobial applications.

[0256] The microorganisms can be, for example, any one or more selected from bacterial cells, virus particles, and / or fungal cells, or microparasites, and can be individual cells, organisms, or colonies. The bacterial cells, virus particles, and / or fungal cells or microparasites can be present on or in a host organism, for example, as intestinal microorganisms of humans or other animals, or in bacterial infections of humans or other animals. The bacteria, fungal cells, and / or virus particles, and / or microparasites can be in vitro, in vivo, or ex vivo.

[0257] The present disclosure can be particularly useful in treating or preventing microbial infections at the site of a skin lesion in a subject. The present disclosure can be particularly useful in the treatment of preventing microbial infections in immunosuppressed subjects.

[0258] Where the microorganism is present in a bacterial infection, a fungal infection, a viral or microparasitic infection of humans or other animals, the infection may be associated with a disease such as, for example, the common cold, influenza, tuberculosis, SARS, COVID-19, pneumonia or measles.

[0259] 1. Bacterial cells The bacterium may be a pathogenic bacterial species. The microbial infection may be an infection caused by a pathogenic bacterial species, including gram-positive and gram-negative, aerobic and anaerobic, antibiotic-susceptible and antibiotic-resistant bacteria.

[0260] Examples of bacterial species that can be targeted using the present invention include species of the genus Actinomyces, Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia, any combination thereof can also be targeted by the present invention.

[0261] In certain embodiments, the microorganism may be a pathogenic species of Corynebacterium, Mycobacterium, Streptococcus, Staphylococcus, Pseudomonas, or any combination thereof.

[0262] In more specific embodiments, the targeted microorganism is Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii; Borrelia afzelii; Borrelia recurrentis; Brucella abortus; Lucera canis; Brucella melitensis; Brucella suis; Campylobacter jejuni; Chlamydia pneumoniae; Chlamydia trachomatis; Chlamydia psittacosis; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheriae; Ehrlichia canis; Ehrlichia chaffeensis; Enterococcus faecalis; Enterococcus faecium; Escherichia coli, including E. coli O157, such as enterotoxigenic E. coli (ETEC), enteropathogenic E. coli, enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC); H7; Francisella tularensis; Haemophilus influenzae; Helicobacter pylori; Klebsiella pneumoniae; Legionella pneumophila; Leptospirosis The pathogen may be selected from Pira bacteria; Listeria monocytogenes; Mycobacterium leprae; Mycobacterium tuberculosis; Mycobacterium abscessus; Mycobacterium ulcerans; Mycoplasma pneumoniae; Neisseria gonorrhoeae; Neisseria meningitidis; Pseudomonas aeruginosa; Nocardia asteroides; Rickettsia rickettsii; Salmonella typhi; Salmonella typhimurium; Shigella sonnei; Shigella dysenteriae; Staphylococcus aureus; Staphylococcus epidermidis; Staphylococcus saprophyticus; Group B hemolytic streptococci; Streptococcus pneumoniae; Group A hemolytic streptococci; Streptococcus viridans; Treponema pallidum; Vibrio cholerae (Vibrio cholerae); Yersinia pestis; and combinations thereof.

[0263] In particular, the microorganism may be selected from Chlamydia pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or a combination thereof.

[0264] The microorganism may be an antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species, or an antibiotic-resistant or antibiotic-sensitive strain of a bacterial species. The use of nitric oxide to treat methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) is described, for example, in International Publication No. 02 / 20026, the disclosure of which is incorporated herein by reference. Examples of antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species that can be killed or treated using the present invention include methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

[0265] 2.Fungal cells The microorganism may be a pathogenic fungal species. The microbial infection may be an infection caused by a pathogenic fungal species, including pathogenic yeasts.

[0266] Examples of fungal species that may be targeted using the present invention include species of Aspergillus, Blastomyces, Candida (e.g., Candida auris), Coccidioides, Cryptococcus (particularly Cryptococcus neoformans or Cryptococcus gattii), Histoplasma, Mucormycetes, Pneumocystis (e.g., Pneumocystis jirovecii), Sporothrix, Talaromyces, or any combination thereof.

[0267] Examples of fungal infections include aspergillosis (including allergic bronchopulmonary aspergillosis), tinea pedis (athlete's foot), infections caused by pathogenic Candida species, vaginal yeast infections, fungal toenail infections and diaper rash, tinea cruris (jock itch), and tinea corporis (ringworm).

[0268] 3. Virus Particles The microorganism may be a virus particle. Infections may be caused by pathogenic viruses.

[0269] Examples of viruses that can be targeted using the present invention include influenza viruses, parasites, and viruses that can be targeted using the present invention. These include influenza virus, adenovirus, norovirus, rotavirus, rhinovirus, coronavirus, respiratory syncytial virus (RSV), astrovirus, and hepatitis virus. In particular, the compositions of the present invention may be used to treat or prevent infection caused by one of the group selected from the group consisting of H1N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine parainfluenza-3 virus, SARS-CoV, SARS-CoV-2, and any combination thereof.

[0270] In particular, the present invention can be applied to the treatment of diseases or disorders caused by viral infections. Examples of such diseases that can be targeted by the present invention include respiratory viral diseases, gastrointestinal viral diseases, exanthematous viral diseases, hepatic viral diseases, cutaneous viral diseases, hemorrhagic viral diseases, and neuroviral diseases.

[0271] Respiratory viral infections include influenza, rhinovirus (i.e., common cold virus), respiratory syncytial virus (RSV), adenovirus, coronavirus infections (e.g., COVID-19), and severe acute respiratory syndrome (SARS). Gastrointestinal viral infections include norovirus, rotavirus, adenovirus, and astrovirus. Exanthematous viral diseases include measles, rubella, chickenpox, shingles, roseola, smallpox, varicella, and chikungunya virus. Liver viral diseases include hepatitis A, B, C, D, and E. Skin viral diseases include genital warts, oral herpes, genital herpes, and molluscum contagiosum. Hemorrhagic viral diseases include Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, and Crimean-Congo hemorrhagic fever. Neuroviral diseases that can be targeted using the present invention include polio, viral meningitis, viral encephalitis and rabies.

[0272] 4. Parasitic microorganisms The microorganism may be a parasitic microorganism (microparasite). Infections may be caused by pathogenic parasitic microorganisms.

[0273] Examples of parasitic microorganisms that can be targeted using the present invention include protozoa.

[0274] In particular, the present invention can target the following protozoan groups: amoebas (e.g., amoebae, e.g., Entamoeba genus, such as Entamoeba histolytica or Entamoeba dispar), flagellates (e.g., flagellates, such as Giardia and Leishmania), ciliates (e.g., ciliates, such as Balantidium), sporozoans (e.g., Plasmodium and Cryptosporidium), and any combination thereof.

[0275] Parasitic infections that may be treated using the present invention include malaria, amoebic dysentery, and leishmaniasis (eg, cutaneous, mucocutaneous, or visceral leishmaniasis).

[0276] Human / Animal Host or Subject The subject can be an animal or a human subject. The term "animal" as used herein can generally include humans, although when the term "animal" appears in phrases such as "animal or human subject," it will be understood from the context to refer specifically to a non-human animal, or that the reference to "human" is simply to specify, for the avoidance of doubt, the option that the animal may be a human.

[0277] In certain embodiments, the subject is a human subject. The human subject may be an infant or an adult subject.

[0278] In certain embodiments, the subject is a vertebrate subject. The vertebrate may be any of Agnatha (jawless fish), Chondrichthyes (cartilaginous fish), Osteichthyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals). In certain embodiments, the subject is a mammalian or avian animal subject.

[0279] In certain embodiments, the subject is a domesticated species of animal. A domesticated species of animal is any of the following: - Commensal animals adapted to the human ecological niche (e.g., dogs, cats, guinea pigs) - Forage animals or livestock that are sought or raised for food (e.g., cattle, sheep, pigs, goats); and - Animals primarily used for draft purposes (e.g. horses, camels, donkeys)

[0280] Examples of livestock include, but are not limited to, alpacas, addax, bison, camels, canaries, capybaras, cats, cattle (including Bali cattle), chickens, collared peccaries, deer (including fallow deer, sika deer, white-lipped deer, and white-tailed deer), dogs, donkeys, doves, ducks, elands, elks, emus, ferrets, gayal, goats, geese, guinea fowl, guinea pigs, tragelan, horses, llamas, minks, moose, rats, mules, musk oxen, ostriches, parrots, pigs, pigeons, quails, rabbits, rats (including African reed mice), reindeer, siberian oryx, sheep, turkeys, buffalo, yaks, and zebu cattle.

[0281] Organs, structures and internal spaces of the animal / human host or subject The organs to which the compositions or multi-component systems of the present disclosure are administered are not limited, and include the skin and organs of the respiratory, urogenital, cardiovascular, digestive, endocrine, excretory, lymphatic, immune, integumentary, muscular, nervous, reproductive, and skeletal systems.

[0282] Examples of organs of the cardiovascular system include the heart, lungs, blood, and blood vessels. Examples of organs of the digestive system include the salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, and anus. Examples of organs of the endocrine system include the hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland, and adrenal glands. Examples of organs of the excretory system include the kidneys, ureters, bladder, and urethra. Examples of organs of the lymphatic system include lymph, lymph nodes, and blood vessels. Examples of organs of the immune system include the tonsils, adenoids, thymus, and spleen. Examples of organs of the integumentary system include the skin, hair, and nails of mammals, as well as the scales and feathers of fish, reptiles, and birds. Examples of organs of the nervous system include the brain, spinal cord, and nerves. Examples of organs of the reproductive system include the genitals, such as the ovaries, fallopian tubes, uterus, vulva, vagina, testes, vas deferens, seminal vesicles, prostate, and penis. Examples of organs of the skeletal system include bones, cartilage, ligaments, and tendons.

[0283] Cavities of a human subject include, but are not limited to, the mouth, nose, ears, throat, respiratory tract, lungs, gastrointestinal tract, dorsal body cavities such as the cranial or spinal cavity, or ventral body cavities such as the thoracic, abdominal or pelvic cavities. Nasal, oral, airway and pulmonary routes of administration are features of the present invention.

[0284] In vitro antibacterial treatment of surfaces The components and compositions of the present disclosure, as well as the released gases from NOx-producing reactions according to the present disclosure, may be used to apply antimicrobial treatments in vitro, where "in vitro" means that the surface being treated is not living, even if it is ultimately intended for medical use.

[0285] Examples of such utilities include methods for sterilizing surgical instruments, hypodermic needles, and other medical devices prior to use to reduce or prevent the spread of pathogens, as well as methods for sterilizing surgical instruments, hypodermic needles, and other medical devices prior to use in hospitals or This includes cleaning or treating surfaces in a doctor's office or elsewhere.

[0286] Other examples include methods for sterilizing prosthetic devices and implantable devices such as stents (e.g., coronary stents), surgical screws, rods, plates and splints, orthopedic implants, cardiac pacemakers, insulin infusion devices, catheters, ostomy appliances, intraocular lenses, cochlear implants, electrical pain relief implants, implantable contraceptives, neurostimulators, artificial heart valves, electrodes, intravenous drips and drug delivery devices prior to placing the device within a subject's body.

[0287] If desired, the components or compositions of the present disclosure can be coated onto the surface of a prosthesis or implantable device, so that the NO released in the NOx-producing reaction can perfuse into other tissues or organs or exert other physiological effects in the vicinity of the prosthesis or implantable device.

[0288] The techniques for making the surface of prosthetic or implantable device biocompatible, including incorporating functional coatings such as coatings containing the components or compositions of the present disclosure, are well known to those skilled in the art.See, for example, Gultepe et al., Advanced Drug Delivery Reviews, March 8, 2010, 62(3), pp. 305-315; and US Patent No. 5,702,754 and US Patent No. 6,270,788.The entire disclosures of the publications mentioned therein are incorporated herein by reference.

[0289] Compositions and methods for more general antimicrobial treatment of inanimate surfaces are well known in the art and do not require detailed description herein. Antimicrobial compositions are used, for example, in the healthcare industry, food service industry, meat processing industry, and the private sector by individual consumers. Antimicrobial cleansing compositions typically contain one or more active antimicrobial agents or components thereof, surfactants, and one or more other ingredients, such as dyes, fragrances, pH adjusters, thickeners, skin conditioners, etc., in a water and / or alcohol carrier. Broad-spectrum disinfectant or antimicrobial compositions are intended to reduce the pathogen burden of a range of pathogens on surfaces. Typically, the compositions are liquid (or are configured to be liquid from a solid premix before use), and the liquid is adjusted to any desired concentration, appropriately with the addition of water, and then spread or sprayed onto the surface to be treated, often with the aid of a cloth or other wiping device, and then may be left to dry or wiped away. Conventional compositions and surface treatment methods are generally applicable for use with the present invention, provided that the active antimicrobial agent is or includes the NOx-generating composition or a component thereof according to the present invention.

[0290] For further details and examples of known antimicrobial compositions and methods of use that may be used in connection with the present invention, see, e.g., U.S. Patent Nos. 6,110,908; 5,776,430; 5,635,462; 6,107,261; 6,034,133; 6,136,771; 8,034,844; EP 0 505 935; and WO 98 / 01110; WO 95 / 32705; WO 95 / 09605; and WO 98 / 55096, the contents of which are incorporated herein by reference in their entireties.

[0291] Uses to improve human and / or animal well-being In addition to the medical uses described above, the present disclosure can be used in non-therapeutic applications in human or animal subjects. Non-therapeutic applications are distinguished from therapeutic applications in that the subject is healthy or the application is not targeted to treat a diagnosed disease, disorder, or condition in the subject.

[0292] Non-therapeutic applications may include treatments aimed at improving a subject's sense of well-being or happiness, or increasing a subject's metabolic efficiency or immune activity, so that the subject is better able to function normally or fight off future infections. Non-therapeutic applications also encompass treatments that aid in a subject's cognitive function or engender a sense of confidence and control.

[0293] For use in such non-therapeutic applications, the combination and composition of the present disclosure can be formulated in a similar manner to pharmaceutical preparations or in a non-pharmaceutical manner.For further details of pharmaceutical preparations, please refer to the section entitled "Optional Additional Ingredients" above.Non-pharmaceutical preparations can suitably include food additives, dietary supplement preparations, food, beverage and drink additives.Preparations adapted to be added to food and beverages can suitably be in the form of liquid or powder.Nutraceutical preparations can suitably be in the form of tablets, capsules or orally ingestible liquids.

[0294] As noted above in the section entitled "Therapeutic or Surgical Uses," medical and / or surgical uses of the present disclosure may provide secondary benefits to the patient in terms of greater well-being or self-confidence.

[0295] Plant applications The beneficial effects of nitric oxide on living or dead plants are known, and the present disclosure includes methods, devices, combinations, kits, compositions, uses, and applications of the gas emitted therefrom to provide beneficial effects to living or dead plants.

[0296] Examples of known uses of nitric oxide and nitric oxide production systems in plants include: Prevention or delay of wilting of cut flowers and plants with nitric oxide (Siegel-Itzkovich, BMJ, 1999;319(7205), 274; Mur et al, 2013; “Nitric oxide in plants: an assessment of the current state of knowledge”, AoB PLANTS doi:10.1093 / aobpla / pls052 (see https: / / doi.org / 10.1093%2Faobpla%2Fpls052); Nitric oxide regulation of plant-pathogen interactions, promotion of plant hypersensitive responses, symbiosis with organisms in nitrogen-fixing root nodules, development of lateral and adventitious roots and root hairs, and control of stomatal opening (Mur et al., 2013; see above); The role of nitric oxide in antioxidant and reactive oxygen species responses in plants (Verma et al., 2013; “Nitric oxide (NO) counteracts cadmium-induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO, and antioxidant responses”; see BioMetals); The role of nitric oxide in the signaling pathways of auxin, cytokinin and other plant hormones (see Liu et al, Proceedings of the National Academy of Sciences, 2013;110(4), pp. 1548-1553).

[0297] The disclosure of each of the above-cited publications is incorporated herein by reference.

[0298] Additionally, the antimicrobial effects of the disclosed nitric oxide generating systems and gases released therefrom, as particularly described in the sections entitled "Therapeutic or Surgical Uses," "Topical Antimicrobial Use," "Use in the Nose, Mouth, Airways and Lungs," and "Targeting of Antimicrobial Use," are equally applicable to targeting microbial infections in plants, and the present disclosure also extends to such uses.

[0299] The above known and all other uses of nitric oxide and nitric oxide producing systems in plants, when used in conjunction with the present disclosure and / or nitric oxide producing reactions using nitric oxide, optionally other nitrogen oxides and / or their precursors optionally produced thereby, constitute further aspects of the present disclosure.

[0300] The plants to be treated may in particular be crops or domestic plants, ie plant species cultivated by humans.

[0301] Crops include, but are not limited to, food crops such as grains, vegetables, and fruits, crops for pharmaceutically active ingredients such as quinine, crops for fiber such as cotton or flax, crops for other materials such as rubber and wood, and crops for flowers such as roses and tulips.

[0302] Further examples of crops for human food consumption include, but are not limited to, crops for producing rice, wheat, sugarcane and other sugar crops, maize (corn), soybean oil, potato, palm oil, cassava, legume pulses, sunflower seed oil, rapeseed oil, mustard oil, sorghum, millet, groundnuts, beans, sweet potato, banana, soybean, cottonseed oil, peanut, groundnut oil, yams, tomatoes, grapes, onions, apples, coffee, mango, mangosteen, guava, chili, pepper, tea, cucumber, orange, walnut, almond, carrot, turnip, coconut, tangerine, lemon, lime, strawberry, and hazelnut crops.

[0303] Example The following non-limiting examples are provided to further illustrate the present invention.

[0304] Materials, equipment and methods used in Examples 1 and 2 solution 0.1 and 1 M citric acid (Health Supplies Limited, Thornton Heath, UK), 0.1 M sodium citrate (Fisher Scientific, Loughborough, UK), 1 M sodium nitrite (Sigma Aldrich, Dorset, UK), 0.5 and 1 M sorbitol (Special Ingredients, Chesterfield, UK), 0.5 and 1 M D-mannitol (Sigma Aldrich, Dorset, UK), 3 M sodium hydroxide (Fisher Scientific, Loughborough, UK), and 0.1 and 1 M L-ascorbic acid (ICN, Biomedicals Inc, Oshio, US) were prepared by dissolving the appropriate mass in deionized water. Deionized water (18.2 MΩ) was obtained from an Arium Mini lab water system (Sartorius, Germany).

[0305] Citric acid / citrate buffer was prepared by two methods: 1. Add stock solutions of 0.1 M citric acid and 0.1 M sodium citrate dropwise using the volumes listed in Sigma Aldrich, 2018 (https: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html); 2. Dissolve a known mass of citric acid for 0.1 M or 1 M preparation in a small amount of deionized water, then add a stock solution of 3 M sodium hydroxide and deionized water dropwise to achieve the desired buffer solution pH (pH 3 to pH 6.2).

[0306] An ascorbic acid / ascorbate buffer was prepared similarly to Method 1, substituting ascorbic acid and sodium ascorbate for citric acid and sodium citrate.

[0307] Polyol content is determined by adding a known mass of sodium nitrite to a stock solution of polyol (e.g., This was achieved by dissolving in ethanol (e.g., sorbitol or mannitol).

[0308] The order of addition of the components of the buffer solution and stock solution is not critical and any mixing order can be used.

[0309] All standard solutions were used within 48 hours of preparation. Calibration buffers were prepared using phthalate (pH 4) and phosphate (pH 7) tablets (Fisher Scientific UK Ltd, Leicestershire, UK) dissolved in deionized water.

[0310] Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) Start-Up and Validation A Voice200 selected ion flow tube mass spectrometer (SIFT-MS) (Syft Technologies Ltd, New Zealand) was used for all gas analyses described in this report. This instrument uses helium (BOC, Surrey, UK) as the carrier gas.

[0311] Prior to analysis, the SIFT-MS was prepared for use with a simple startup procedure. The instrument was taken out of standby mode and a series of pressure checks were performed to ensure capillary action was within operating tolerances. An automated verification procedure was then performed using the manufacturer's calibration gas standards (Syft Technologies Ltd, New Zealand) containing benzene, toluene, ethylbenzene, and xylene. Finally, an in-house performance check was performed using a 10 ppm nitrogen dioxide standard (Air Products PLC, Surrey, UK).

[0312] NO generation procedure The SIFT-MS instrument, reaction chamber and gas paths were set up as shown in FIG.

[0313] The temperature in the reaction chamber was continuously monitored using an HT1 temperature smart sensor (SensorPush, New York, US). The reaction chamber, a 670 mL plastic (bisphenol A-free (BPA-free)) clip-lock tab with a silicone seal (Tesco, Welwyn Garden City, UK), was attached to a pump, allowing humidified air to continuously circulate through the chamber and over the SIFT-MS inlet capillary. Humidification was achieved by pumping air through two Dreschel bottles containing deionized water, in a manner similar to that described in Vernon, W., and Whitby, L., 1931, "The quantitative humidification of air in laboratory experiments," Trans. Faraday Soc. 27, pp. 248–255. The system was allowed to equilibrate for 30 min before use. Continuous SIFT-MS scans were initiated for real-time detection and quantification of NO, NO2, and HONO. Once stable baseline readings (constant concentrations for >2 min) were observed for these compounds, samples were placed in the reaction chamber and monitored for 3 h.

[0314] After validation of the SIFT-MS, a capillary inlet extension heated to 120 °C was attached to the outlet of the reaction chamber via a T-junction, allowing the SIFT-MS to sample the gases exiting the reaction chamber in real time.

[0315] Samples were prepared by weighing approximately 0.3 cm x 0.3 cm of carded nonwoven 20 gsm (20 gsm) polypropylene mesh (manufactured by RKW-Group, Frankenthal, Germany) into a weigh boat (~3 mg). This was reweighed after adding a 10 μL droplet of test or control solution to the center of the mesh (ensuring that the droplet was submerged in the mesh). Finally, the loaded mesh in the weigh boat was placed in a reaction chamber, and a final 10 μL droplet of buffer solution was pipetted onto the center of the mesh. The reaction chamber was quickly sealed, and the generation of nitrogen species was observed instantaneously at the SIFT-MS interface.

[0316] Analysis of evolved gas The gases produced were analyzed using the selected ion mode of the SIFT-MS, with scans performed in successive batches lasting 1000 seconds each. The masses of the following products were repeatedly scanned: 30 m / z for nitrous acid, 48 m / z for nitrous acid, 46 m / z for nitrogen dioxide, and 30 m / z for nitric oxide. These measurements were performed using hydronium (HO) + ), nitrosium (NO + ), and dioxygenyl (O2 + Air was flowed through the chamber at 660 ml / min, and the SIFT-MS inlet sampled this air stream at a flow rate of 2.7 ml / min.

[0317] pH measurements in all examples A simple pH meter (Mettler Toledo, Switzerland) with a glass electrode and LE438 probe was used for all pH measurements. The accuracy of this electrode was verified using a second pH meter, a handheld 205 probe (Testo, Alton, US). Fresh calibration buffer was used for daily calibration of the pH meter.

[0318] Example 1 Nitric oxide generation using 1M / c, pH 3 citric acid in contact with a mesh containing orthorhombic 1M sodium nitrite, with and without 1M polyol The SIFT-MS instrument, reaction chamber, and gas paths were set up as shown in Figure 17, as described above.

[0319] Two test meshes were prepared by imbibing two test solutions of 1M sodium nitrite containing 1M mannitol and 1M sorbitol, respectively, onto the meshes as described above.

[0320] A control solution of 1 M sodium nitrite without polyol was imbibed onto the mesh as above to prepare a control mesh.

[0321] A buffer solution of 1 M citric acid / citrate buffer with a pH of approximately 3, prepared by either of the two methods 1 and 2 above, was added to the test mesh and control mesh in each test, respectively, and gas production was initiated as described above.

[0322] The results are shown in Table 1.

[0323] The data show that 1 M sodium nitrite-absorbed meshes in contact with 1 M / c, pH 3 citric acid produced significantly more nitric oxide when the mesh contained 1 M mannitol or 1 M sorbitol (mannitol having a greater effect than sorbitol) than when no polyol was present.

[0324] Example 2 A study on the effects of various carboxylic acids, acid concentrations, pH and polyols on the production of nitric oxide Samples were prepared as above, with the organic acid, pH and polyol varied as follows:

[0325] [Table 1] TIFF2023510662000002.tif255151TIFF2023510662000003.tif255150TIFF2023510662000004.tif34162

[0326] The SIFT-MS instrument, reaction chamber, and gas paths were set up as shown in Figure 17, as described above.

[0327] The test solution was absorbed into the mesh as described above to prepare the test mesh.

[0328] When used, a control mesh was prepared by imbibing a control solution of 1 M sodium nitrite without polyol onto the mesh as described above.

[0329] The buffer solution or solutions prepared by either of the two methods 1 and 2 above and having the pH values ​​described above were added to each test, and a control mesh, if used, was added to each test to initiate the production of the gas described above.

[0330] The results are shown in Figures 2 to 13. "Normal" in the figures indicates that no polyol is present.

[0331] Figure 2 compares the NO release rates produced by citric acid / citrate buffer or ascorbic acid / ascorbate buffer (pH ≈3) in the absence of polyol. The graph clearly shows that citric acid / citrate buffer produces a higher initial burst and release is sustained at higher levels for longer than ascorbic acid / ascorbate buffer. Citric acid / citrate buffer peaks at approximately 55,000 ppb, while ascorbic acid / ascorbate buffer peaks at approximately 28,000 ppb.

[0332] Figure 3 shows the citric acid / citrate buffer and nitrite systems with and without polyol. The polyol concentration is 1M. The presence of polyol changes the release rate, initial burst, and resulting release over time compared to the absence of polyol. Xylitol and mannitol produce the highest peaks, followed by sorbitol, no polyol, and arabitol. In the 500-1000 second region, xylitol and arabitol have the highest output, followed by mannitol, sorbitol, and no polyol. The burst peaks are: mannitol = xylitol (approximately 64,000 ppb) > sorbitol (approximately 53,000 ppb) > no polyol (approximately 50,000 ppb) > arabitol (approximately 40,000 ppb).

[0333] Figure 4 shows the ascorbic acid / ascorbate buffer and nitrite system with and without polyol. The polyol concentration is 1 M. The burst peaks are mannitol (approximately 40,000 ppb) > arabitol (approximately 35,000 ppb) > xylitol = no polyol (approximately 30,000 ppb) > sorbitol (approximately 23,000 ppb), a different order from the citrate / citric acid buffer system in Figure 3.

[0334] Figure 5 is for the citric acid / citrate buffer and nitrite system with and without polyol (the "no polyol" line, which has a burst peak roughly equivalent to the mannitol line, has been omitted for clarity). The polyol concentration is 0.5M. Burst peak: arabitol (approximately 76,000 ppb) > no polyol = mannitol xylitol (approximately 48,000 ppb) > xylitol = sorbitol (approximately 40,000 ppb). This is a different order compared to the similar 1 M polyol-citric acid / citrate buffer system (Figure 3), demonstrating that the polyol effect is polyol concentration dependent.

[0335] Figure 6 shows the results for the ascorbic acid / ascorbate buffer and nitrite systems with and without polyol (the "no polyol" line, which has a burst peak roughly equivalent to the sorbitol line, is omitted for clarity). The polyol concentration is 0.5 M. The burst peaks are: xylitol (approximately 50,000 ppb) > mannitol (approximately 38,000 ppb) > sorbitol = no polyol (approximately 30,000 ppb) > arabitol (approximately 23,000 ppb). A different sequence is again observed compared to the analogous citric acid / citrate buffer (0.5 M polyol) and ascorbic acid / ascorbate (1 M polyol) systems (Figures 5 and 4, respectively). Thus, the effect of polyols is demonstrated to be dependent on polyol chemistry / stereochemistry and polyol molar concentration.

[0336] Figures 7 and 8 compare the NO release rates from citric acid / citrate buffer or ascorbic acid / ascorbate buffer and the presence of polyol (0.5 M). These graphs highlight some of the differences observed in Figures 2-6. The citric acid / citrate buffer in Figure 7 peaks at approximately 76,000 ppb, while the ascorbic acid / ascorbate buffer peaks at approximately 22,000 ppb. The citric acid / citrate buffer in Figure 8 peaks at approximately 48,000 ppb, while the ascorbic acid / ascorbate buffer peaks at approximately 38,000 ppb.

[0337] Figure 9 compares cumulative output for 1M polyol concentrations. For ascorbic acid / ascorbate buffer, the difference at, say, 3000 seconds is small, with the order being mannitol > sorbitol = arabitol > xylitol. For citric acid / citrate buffer at 3000 seconds, the order is xylitol > arabitol > mannitol > sorbitol > no polyol. The data show that nitric oxide output can be increased by about 100% or more, for example, between no polyol (curve E, which gives a cumulative nitric oxide generation of about 10,000 nmol per mg of nitrite after 3000 seconds, which increases further) and xylitol (curve A, which gives a cumulative nitric oxide generation of about 20,000 nmol per mg of nitrite after the same time, which also increases).

[0338] Figure 10 compares the cumulative output for 0.5 M polyol concentration. For citric acid / citrate buffer at 3000 seconds, the order is arabitol > mannitol = xylitol > sorbitol > no polyol (the "no polyol" line for citric acid / citrate buffer, which is below the sorbitol line, has been omitted for clarity). For ascorbic acid / ascorbate buffer at 3000 seconds, the order is xylitol > mannitol > sorbitol > arabitol. Again, this order is different compared to 1 M polyol (Figure 9).

[0339] Figures 11-13 compare the cumulative plots for 1 M citric acid / citrate buffer, sodium nitrite (1 M), mannitol (0.5 M), and different pH values. As the pH increased, the differences became smaller, and at pH 6.2, the differences disappeared. Thus, these experiments show that the effect of polyols is also pH-dependent.

[0340] FIG. 14 shows the cumulative NO (nmol / cm) for citric acid / citrate buffer (1 M, pH ≈ 2) with and without glycerol (1 M and 2 M) present in a 1 M sodium nitrite solution. 2Over the first 2000 seconds, the NO output for 1M and 2M glycerol is slightly lower than that without polyol. At longer times, the glycerol-containing formulations have a greater output, with 2M glycerol having a greater output.

[0341] FIG. 15 shows the cumulative NO (nmol / cm) for citric acid / citrate buffer (1 M, pH ≈2) and 1 M sodium nitrite solution with and without polyol in the nitrite solution. 2 The plot shows the output of the mannitol / nitrite solution versus the mesh area. The plot shows that the inclusion of glycerol in the mannitol / nitrite solution reduces the output compared to the absence of glycerol. Surprisingly, however, unlike mannitol, the inclusion of glycerol in the sorbitol / nitrite solution increases the NO output compared to the output in the absence of glycerol.

[0342] When glycerol was used, a 1 M glycerol solution was first prepared and used to prepare a 1 M sorbitol or 1 M mannitol solution, which was then used to prepare a 1 M nitrite solution.

[0343] Figure 16 shows the cumulative NO output (mol / mg nitrite) for citric acid / citrate buffer (1 M, pH 5.8) with and without mannitol (0.5 M) present in sodium nitrite (1 M) solution. The plot shows that the inclusion of polyol results in a greater NO output after about 2000 seconds of reaction time.

[0344] FIG. 16 shows that at physiologically relevant pH levels above about 5, particularly above about 5.5, mannitol enhances nitric oxide production compared to a similar system without mannitol, providing cumulative levels of 1400 nmol of NO per mg of nitrite after 10,000 seconds (167 minutes).

[0345] Example 3 Activity against M. abscessus cultures using various organic acid and nitrite solutions with and without polyols. material 4.7 g of Middlebrook 7H9 broth base (Sigma-Aldrich) was reconstituted in 900 ml of distilled water and autoclaved for 15 minutes at 121°C. Middlebrook ADC growth supplement (Sigma-Aldrich) was added to the autoclaved 7H9 solution (50 ml per 450 ml, for a total of 100 ml).

[0346] 1M Sodium Nitrite (Emsure): In a clean screw-top glass bottle, dissolve 6.9 g of sodium nitrite powder in 100 ml of distilled water. Autoclave the mixture at 121°C for 15 minutes.

[0347] 1 M Citric Acid (Sigma-Aldrich): In a clean screw-top glass bottle, dissolve 19.2 g of citric acid powder in 100 ml of distilled water. Autoclave the mixture at 121° C. for 15 minutes.

[0348] 1M Ascorbic Acid (Sigma-Aldrich): Add 17.6 g of ascorbic acid powder to a sterile glass bottle. Dissolve completely in 100 ml of sterile distilled water. It was prepared daily using strict sterile techniques due to its short half-life. It was not autoclaved due to its inherent instability, but was filtered through a 0.2 μ filter before use.

[0349] 1M Sodium citrate tribasic dihydrate (Sigma-Aldrich): In a clean screw-top glass bottle, dissolve 29.4 g of sodium citrate powder in 100 ml of distilled water. Autoclave the mixture at 121 °C for 15 min.

[0350] 1M L-ascorbic acid sodium salt (Acros Organics): In a clean screw-top glass bottle, dissolve 19.8 g of sodium ascorbate powder in 100 ml of distilled water. Autoclave the mixture at 121° C. for 15 minutes.

[0351] For experiments with polyols, D-mannitol (Sigma-Aldrich) was used. Polyols were added to the sodium nitrite stock solutions described above to prepare the following stock solutions: Stock solution A - 1M sodium nitrite and 0.5M mannitol Stock solution B - 1.5 M sodium nitrite and 0.5 M mannitol

[0352] A stock solution of 1.5M citric acid was also prepared.

[0353] The molarity of each component was adjusted for dilution factors to ensure the correct final molarity of each experimental solution.

[0354] Mycobacterium abscessus (MAB) The laboratory reference strain, Mycobacterium abscessus ATCC 19977lux, was used for all experimental conditions in this example.

[0355] method 50 ml Falcon tubes were labeled Tube T (test suspension), Tube A (acid control), and Tube C (control).

[0356] Eight milliliters of 7H9 + ADC supplement was added to each tube. Then, 100 μl of MAB suspension (pre-grown to approximately 3-4 McFarland standards) was added. A baseline relative light unit (RLU) reading of the MAB suspension was taken. The contents were mixed by vortexing.

[0357] Tube contents in the absence of polyol (mannitol) Tube T 1 ml of sodium nitrite (1 M) solution was added to the tube, followed immediately by 1 ml of citric acid solution (1 M) or ascorbic acid solution (1 M) to give a final concentration of 0.1 M in 10 ml. The contents were mixed by gentle inversion and incubated at 37°C for 24 hours. Tube A1 ml of citric acid solution (1 M) or ascorbic acid solution (1 M) was added to the tube, and 1 ml of sterile distilled water was added to bring the final volume to 10 ml, testing a 0.1 M concentration for the acid. The contents were mixed by gentle inversion and incubated at 37°C for 24 hours. Tube C 2 ml of sterile distilled water was added to the tube to bring the total volume to 10 ml. This was a control to assess growth under optimal conditions. The contents were mixed by gentle inversion and incubated at 37°C for 24 hours.

[0358] Contents of Tube T in the presence of polyol (mannitol) When mannitol was present, the contents of tube T were as follows: 1. Tube T: 1 ml sodium nitrite (1 M) + mannitol (0.5 M) and 1 ml citric acid (1 M) 2. Tube T: 1 ml sodium nitrite (1.5 M) + mannitol (0.5 M) and 1 ml citric acid (1 M) 3. Tube T: 1 ml sodium nitrite (1 M) + mannitol (0.5 M) and 1 ml citric acid (1.5 M)

[0359] RLU was measured at 30 min, 60 min and 24 h incubation to assess the activity of T, A and C solutions.

[0360] After 24 hours of incubation, tubes C, A, and T were plated onto Columbia blood agar (VWR Chemicals). Plates were incubated at 37°C for 72 hours. Colony forming units (CFU) were read on days 3, 5, and 7 of incubation. All work was performed in a CL2 biological safety cabinet within the CL2 laboratory facility.

[0361] The results are shown in Figures 18 to 21.

[0362] Figure 18 shows that a solution of 0.1 M citric acid and 0.1 M nitrite (Tube T) was more effective at eradicating and reducing M. abscessus cultures after 7 days at pH 5 and 5.5 compared to a 0.1 M citric acid only solution (Tube A) at pH values ​​of 6.0, 6.5, 7.0, and 7.4. Figure 18 also shows that a solution of 0.1 M ascorbic acid and 0.1 M nitrite (Tube T) was more effective at eradicating and reducing M. abscessus cultures after 7 days at pH values ​​of 5.0, 5.5, and 6.0 compared to an ascorbic acid only solution (Tube A) at pH values ​​of 6.5, 7.0, and 7.4.

[0363] Figure 19a) shows that a solution of 0.1 M citric acid and 0.1 M nitrite was effective in reducing the CFU of a culture of M. abscessus after 3 days of incubation, and that a solution of 0.1 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in almost completely eradicating the culture of M. abscessus after 3 days of incubation. Figure 19b) shows that a solution of 0.1 M citric acid and 0.1 M nitrite without mannitol was effective in maintaining the reduced CFU of M. abscessus after 5 days of incubation. This figure also shows that a solution of 0.1 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in reducing the CFU of M. abscessus after 5 days of incubation.

[0364] Figure 20a) shows that a solution of 0.15 M citric acid and 0.1 M nitrite was effective in reducing the CFU of M. abscessus cultures after 3 days of incubation, and a solution of 0.15 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in eradicating M. abscessus cultures after 3 days of incubation. Figure 20b) shows that a solution of 0.15 M citric acid and 0.1 M nitrite without mannitol was effective in maintaining the reduced CFU of M. abscessus after 5 days of incubation. This figure also shows that a solution of 0.15 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in eradicating M. abscessus cultures after 5 days of incubation.

[0365] Figure 21 shows that a solution of 0.1 M citric acid and 0.15 M nitrite was effective in reducing the CFU of a culture of M. abscessus after 3 days of incubation and maintaining the reduction in CFU of a culture of M. abscessus after 5 days of incubation. The figure also shows that a solution of 0.1 M citric acid and 0.15 M nitrite containing 0.05 M mannitol was effective in eradicating a culture of M. abscessus after 3 and 5 days of incubation.

[0366] Example 4 Mycobacterium abscessus (Mabs) and Mycobacterium in cultures of a series of clinical isolates Minimum inhibitory concentrations (MICs) of carboxylic acid-nitrite-polyol solutions against Mycobacterium tuberculosis (Mtb) Healthy volunteers Peripheral blood samples were collected from healthy volunteers who provided written informed consent (ethical approval reference REC No. 12 / WA / 148).

[0367] Mycobacterial strains Both Mycobacterium abscessus (ATCC 19977) and Mycobacterium tuberculosis (H37RV) strains contained a bacterial luciferase (lux) gene cassette (luxCDABE) that allows for measurement of relative light units (RLU), as well as traditional colony-forming unit (CFU) measurements of bacterial survival.

[0368] [Table 2]

[0369] Treatment conditions Treatment 1: 0.15M citric acid, 0.1M sodium nitrite, and 0.05M mannitol. Treatment 2: 0.1M citric acid, 0.15M sodium nitrite, and 0.05M mannitol.

[0370] Broth microdilution minimum inhibitory concentration (MIC) The MICs of each treatment against M. abscessus and M. tuberculosis were determined according to the Clinical and Laboratory Standards Institute guidelines for antimicrobial susceptibility testing (M07-A9). Two-fold dilutions of each treatment were performed across plates, which were incubated at 37°C and read on days 3 and 7 for Mab and 14 and 21 for Mtb. Tests were performed in duplicate.

[0371] All work was performed in a CL2 biological safety cabinet within a CL2 laboratory facility.

[0372] The minimum inhibitory concentration of a solution of 1.5 M citric acid, 1 M sodium nitrite, and 0.5 M mannitol against M. abscessus was found to be 4.7 mM, and the minimum inhibitory concentration of a solution of 1.5 M citric acid, 1 M sodium nitrite, and 0.5 M mannitol against M. tuberculosis was found to be 2.3 mM.

[0373] The minimum inhibitory concentration of a solution of 1 M citric acid, 1.5 M sodium nitrite, and 0.5 M mannitol against M. abscessus was found to be 3.1 mM, and the minimum inhibitory concentration of a solution of 1 M citric acid, 1.5 M sodium nitrite, and 0.5 M mannitol against M. tuberculosis was found to be 1.6 mM.

[0374] The minimum inhibitory concentrations (MICs) were measured for isolates Nos. 570, 571, 573, 575, 578, 579, 580, 581, 582, 583, 584, 585, 589, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 616, 617, 619, 812, 825, 829, 839, 845, 848, 853, 857, 858, 873, 894, 895, 896, 897, 898, 899, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 830, 831, 832, 833, 834, 835, 836, 837, 838, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889 898, 909, 919, 928, 932, 942, 944, 955, 956, 959, 963, 964, 965, 968, 975, 980, 982, 985, 993, 995, 1000, 1001, 1007, 1011, 1017, 1023, 1024, 1026, 1027, 1042, 1043, 1044 Broth microdilution was also performed using 5, 1047, 1049, 1054, 1063, 1066, 1067, 1070, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1082, 1086, 1094, 1096, 1101, 1103, 1104, and 1106. Each individual isolate was evaluated in duplicate.

[0375] The results of testing the clinical isolate are shown in Figures 22a and 22b. These graphs show the MIC of nitric oxide against M. abscessus in duplicate, with readings obtained after 3, 4, and 5 days of incubation of the isolate. Plates were also read on day 7 of incubation, but no differences were observed compared to day 5. The laboratory strain ATCC 19977 lux was used as a control in both experiments, and comparisons with the clinical isolate are shown.

[0376] Figure 22 shows that the citric acid-nitrite-mannitol solution is effective across a wide range of clinical isolates. The minimum inhibitory concentrations for the majority of clinical isolates were within 0.02 M for the 0.1 M citric acid, 0.15 M nitrite, and 0.05 M mannitol solution (Figure 22a), and the minimum inhibitory concentrations for the majority of clinical isolates were within 0.04 M for the 0.15 M citric acid, 0.1 M nitrite, and 0.05 M mannitol solution (Figure 22b).

[0377] In both figures, the MICs of certain samples varied on different days. These are samples with multiple dots above the isolated sample's identification code. Generally speaking, in this situation, higher MICs were observed on later days of incubation than lower MICs. Overall, the combination of low citric acid (0.1 M) and high sodium nitrite (0.15 M) (Figure 22(a)) is more effective than the combination of high citric acid (0.15 M) and low sodium nitrite (0.1 M) (Figure 22(b)).

[0378] Additional data demonstrating the killing of M. abscessus in vitro by carboxylic acid-nitrite-polyol solutions is shown in Figure 29, which shows the M. abscessus killing efficacy of an aqueous formulation of sodium nitrite, citric acid buffered to pH 5.8 with sodium hydroxide solution, and mannitol compared to amikacin and a negative control over a 24-hour period under similar conditions.

[0379] Example 5 Antibacterial activity of carboxylic acid-nitrite solutions with and without polyols against Pseudomonas aeruginosa Equipment and media UKAS Calibrated Pipettes (100-1000μL Range) - Proline® Plus UKAS calibrated multichannel pipettes (P300 and P20) - Gilson®, UK Universal Tube - SLS, UK Calibrated Balance - HR-100A Microbial Incubator - Heratherm TM , ThermoFisher Scientific, UK Tryptone Soy Agar (TSA) – Southern Group Laboratories, UK Tryptone Soy Broth (TSB) - Acumedia®, SLS Ltd, UK Malt agar - Acumedia®, Acumedia®, SLS Ltd, UK Brain Heart Infusion Broth (BHIB) - Acumedia®, SLS Ltd, UK Sabouraud Dextrose Broth (SDB) - Acumedia®, SLS Ltd, UK Dey-Engley Neutralizer (DE-N) - Acumedia®, SLS Ltd, UK Citric acid – Sigma, UK Sodium nitrite – Sigma, UK Mannitol - Sigma, UK Sorbitol – Sigma, UK

[0380] Microbial testing Pseudomonas aeruginosa NCTC 13618 isolated from a patient with cystic fibrosis

[0381] [Table 3] Concentration 1 - 1M citric acid + 1M sodium nitrite (with or without 0.5M polyol) Concentration 2 - 0.5M citric acid + 1M sodium nitrite (with or without 0.5M polyol) Concentration 3 - 0.5M citric acid + 0.5M sodium nitrite (with or without 0.5M polyol)

[0382] Dey-Engley Neutralizer Verification A 24-h culture of Pseudomonas aeruginosa was harvested from tryptone soy agar (TSA) and 1 × 10 8 ±5×10 7 CFUmL -1 This was used to prepare a suspension, which was further diluted in Brain Heart Infusion Broth (BHIB) to a concentration of 1 x 10 5 ±5×10 4 CFUmL -1 A working suspension of was prepared.

[0383] The starting inoculum was confirmed by serial dilution and spread plating. Neutralizer validation was performed using control (9 mL phosphate-buffered saline (PBS) and 1 mL inoculum), toxicity (9 mL Dey-Engley neutralizer (DE-N) and 1 mL inoculum), and neutralizer efficacy (8 mL neutralizer, 1 mL test agent, and 1 mL inoculum) samples. After 5 minutes of treatment, 200 μL of suspension was removed from each tube, serially diluted, and 100 μL was plated on TSA. The agar plates were incubated at 37 ± 2°C for 18–24 hours.

[0384] Antibacterial effect against planktonic organisms A 24-h culture of P. aeruginosa was harvested from the TSA and 1 × 10 8 ±5×10 7 CFUmL -1 This was further diluted with BHIB to a concentration of 1 × 10 6 ±5×10 4 CFUmL -1 A working suspension of 8 mL was prepared by filling a universal tube with 8 mL of the bacterial solution.

[0385] One mL of citric acid solution and one mL of sodium nitrite solution were added to each test agent to obtain the required concentration, as described above. The solutions were incubated at 37 ± 2°C for 24 hours. After the incubation period, 1 mL was removed from each tube and transferred to a tube containing 9 mL of neutralizing agent. Serial dilutions and plate counts were used to quantitate viable organisms.

[0386] The results are shown in Figure 23.

[0387] Data show antibacterial efficacy against P. aeruginosa for: - citric acid (1M) mixed with nitrite (1M) with or without polyol (0.5M) ("concentration 1") - citric acid (0.5M) mixed with nitrite (1M) with or without polyol (0.5M) ("concentration 2") - citric acid (1M) mixed with nitrite (0.5M) with or without polyol (0.5M) ("concentration 3")

[0388] The citric acid solutions are pH 5.2 (formulations 1, 3 and 5) and 6.0 (formulations 2, 4 and 6). Formulations 1 and 2 contain no polyol; formulations 3 and 4 contain mannitol; formulations 5 and 6 contain sorbitol.

[0389] At pH 5.2, all formulations show good efficacy. At pH 6, the formulation containing mannitol is slightly more effective.

[0390] Example 6 The efficacy of formulations containing nitrite, organic acids, and polyols against M. tuberculosis HN 878 in THP-1 cells was evaluated.

[0391] formulation The formulations were prepared as shown in the following tables. When the preparation method is described as "concentrate," indicated by the suffix FC in the reference sample, this means that the formulation was first made as a concentrated premix containing all three components, sodium nitrite (0.75 M), polyol (0.25 M), and acid (0.5 M), and then diluted with distilled water to reach the desired concentrations of each, as listed in the table. When the preparation method is described as "dilution," indicated by the suffix FD in the reference sample, this means that the formulation was first made as a premix containing all three components at the desired concentrations, i.e., sodium nitrite (0.15 M), polyol (0.05 M), and acid (0.1 M), and then diluted with distilled water to reach the desired concentrations of each, as listed in the table.

[0392] Within each formulation, a range of concentrations of sodium nitrite, namely 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg / ml for in vitro bacterial inhibition assay against M. tuberculosis HN878, was prepared by serial dilution.

[0393] [Table 4]

[0394] MIC macrophage testing was performed using the THP-1 macrophage (1) compound screening assay.

[0395] Preparation and culture of macrophages: THP-1 cells were grown for 2 weeks. Then, 5 × 10 THP-1 cells were added to the culture medium. 5 The cells were suspended in complete DMEM medium for macrophages at a concentration of 1 × 10 cells / mL. 2 mL of cells were added per well of a 24-well tissue culture plate (1 × 10 cells per well). 6) in 24-well plates. One 24-well plate of cells allows for a range of seven drug concentrations plus an untreated control to be tested in triplicate. In addition to the drug assay plate, one additional plate (or at least three additional wells) was seeded to determine bacterial uptake on the day of infection. Cells were incubated at 37°C in 5% CO2 in a humidified chamber. DMEM antibiotic / antimycotic-free complete medium was not changed during the 3-day assay.

[0396] Complete DMEM medium for macrophages: Dulbecco's Modified Eagle's Medium (Cellgro 15-017-cv) supplemented with: Heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO, F-0500-A) (10%) L-929 conditioned medium (10%) L-glutamine (Sigma G-7513) (2mM) HEPES buffer (Sigma H-0887) (10mM) Antibiotic / Antifungal (Sigma A-9909) (1X) MEM non-essential amino acids (Sigma M-7145) (1X) 2-mercaptoethanol (Sigma M-6250) (50 nM)

[0397] L-929 conditioned medium: ATCC L-929 (CCL-1) cells were cultured at 75 cm 2 4.7 x 10 cells in 55 mL of DMEM + 10% fetal bovine serum in a flask 5 The cells were seeded with THP-1 cells. The cells were grown on THP-1 cells for 3 days. On day 3, the supernatant was collected, filtered through a 0.45 μm filter, aliquoted, and frozen at -20°C. The cell-free filtrate was used in DMEM medium for THP-1 infection.

[0398] Infection of THP-1 cells: On day 0, the medium was removed from the cells and replaced with 0.2 ml of antibiotic / antimycotic-free DMEM containing M. tuberculosis HN878 at an MOI of 1 macrophage to 10 bacteria. The tissue culture plate was placed in a closed Ziploc baggie for return to the incubator. Once in the incubator, the baggie was opened. The cells were incubated with the bacteria for 2 hours. After infection, bacteria attached to the outside of the cells were removed, and each well was washed once with phosphate-buffered saline (PBS), followed by the addition of 2 ml of antibiotic / antimycotic-free complete DMEM medium with various drug concentrations. To prepare drug concentrations, serial two-fold dilutions were performed by adding 10 ml of the previous suspension to 10 ml of the complete medium plus serum of the next tube. The tissue culture plate was incubated at 37°C + 5% CO. 2 (drug remained in the wells for 3 days.) Each drug concentration was tested in triplicate wells.

[0399] Cell lysate plating and cell viability assessment for THP-1 cells were performed 2 hours, 1 day, 2 days, and 5 days after infection. Tissue culture medium was removed from all wells, and cells were washed twice with 1 ml of PBS. Next, 1 ml of sterile double-distilled water + 0.05% Tween-80 was added to each well, and the cells were allowed to stand at room temperature for 5–10 minutes. Cell lysates were serially diluted 1:10 in sterile saline in 24-well tissue culture plates. Diluted cell lysates were plated on 7H11 / OADC agar in 1 / 1000 dilution steps. (Each 24-well TC plate of cells requires four 24-well TC plates for serial dilutions, and 24 agar "quad" plates.) Plates were incubated at 32°C for 30 days, and colonies were counted to determine CFU / ml.

[0400] result In vitro optical density results of THP-1 HN878 Minimum inhibitory concentration (MIC) reported as the most diluted composition that inhibits the bacteria (i.e., the highest dilution level of a particular formulation on a scale listed as 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / ml). [Table 5]

[0401] The results are shown in Figures 24 to 27.

[0402] Figure 24: Efficacy of 30RESP001FC and FD (concentrate and dilutions) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP001FC (concentrate) (A) and 30RESP001FD (dilution) (B) formulations was evaluated for intracellular killing of M. tuberculosis HN878 (□) in THP-1 macrophages at 2 hours (day 0), 1 day, 2 days, and 5 days post-infection, and after treatment with 16 μg / ml (△), 8 μg / ml (▽), 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (●), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each plot in Figure 24, the △ and ▽ plot lines for the 16 μg / ml and 8 μg / ml treatments are distinguishable from the ▲ and ▼ plot lines for the 0.25 μg / ml and 0.125 μg / ml treatments, respectively, because the 16 μg / ml and 8 μg / ml treatments are more effective. In other words, the 16 μg / ml and 8 μg / ml treatment plot lines show significantly lower CFU values ​​than the 0.25 μg / ml and 0.125 μg / ml treatments, especially on day 5. Similarly, the □ plot line for the 1 μg / ml treatment can be easily distinguished from the □ plot line for the no treatment, because the 1 μg / ml treatment is more effective. The CFU value for the □ plot line for the no treatment increases after 1 day, reaching 1 x 10 4 remains above.

[0403] The 30RESP001FC and FD compositions designated "16 μg / ml" in the MIC table above and in Figure 24 contain 0.15 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentrations after dilution), and the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions are 50% dilutions (i.e., halving the concentration) of the previous compositions, on the order of 16 to 0.125 μg / ml, respectively.

[0404] THP-1 macrophages were infected with M. tuberculosis at an MOI of 1:10, and the number of intracellular bacteria was measured using the bacterial colony counting method (CFU) 2 hours after infection (day 0), 1, 2, and 5 days later. The values ​​shown are the mean ± standard deviation from one independent experiment. Notably, treatment with 30RESP001FC and FD (concentrates and dilutions) at 16 μg / ml and 8 μg / ml against M. tuberculosis HN878 showed increased efficacy compared to untreated controls ( * , p<0.05).

[0405] Figure 25: Efficacy of 30RESP002FC and FD (concentrate and dilutions) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP002FC (concentrate) (A) and 30RESP002FD (dilution) (B) formulations was evaluated for intracellular killing of M. tuberculosis HN878 (□) in THP-1 macrophages at 2 hours post-infection (day 0), days 1, 2, and 5, and after treatment with 16 μg / ml (△), 8 μg / ml (▽), 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (●), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each plot in Figure 25, the △ and ▽ plot lines for the 16 μg / ml and 8 μg / ml treatments are distinguishable from the ▲ and ▼ plot lines for the 0.25 μg / ml and 0.125 μg / ml treatments, respectively, because the 16 μg / ml and 8 μg / ml treatments are more effective. In other words, the 16 μg / ml and 8 μg / ml treatment plot lines show significantly lower CFU values ​​than the 0.25 μg / ml and 0.125 μg / ml treatments, especially on day 5. Similarly, the □ plot line for the 1 μg / ml treatment can be easily distinguished from the □ plot line for the no treatment, because the 1 μg / ml treatment is more effective. The CFU value for the □ plot line for the no treatment increases after 1 day, reaching 1 x 10 4 remains above.

[0406] The 30RESP002FC and FD compositions, designated "16 μg / ml" in the MIC table above and in Figure 25, contained 0.15 M sodium nitrite, 0.05 M lactitol, and 0.1 M citric acid / citrate (final molar concentrations after dilution) and were diluted to 8, 4, 2, 1, 0.5, The 0.25 and 0.125 μg / ml compositions are 50% dilutions (ie, halving the concentration) of the previous compositions on the order of 16 to 0.125 μg / ml, respectively.

[0407] THP-1 macrophages were infected with M. tuberculosis at an MOI of 1:10, and the number of intracellular bacteria was measured using bacterial colony counting (CFU) at 2 hours, 1, 2, and 5 days post-infection. Values ​​shown are the mean ± standard deviation from one independent experiment. Compared to untreated controls, treatment with 30RESP002FC (concentrate) at 16 μg / ml and 30RESP002FD (dilutions) at 16 μg / ml and 8 μg / ml increased efficacy against M. tuberculosis HN878 ( * , p<0.05).

[0408] Figure 26: Efficacy of 30RESP003FC and FD (concentrate and dilutions) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP003FC (concentrate) (A) and 30RESP003FD (dilution) (B) formulations was evaluated for intracellular killing of M. tuberculosis HN878 (□) in THP-1 macrophages at 2 hours (day 0), 1 day, 2 days, and 5 days post-infection, and after treatment with 16 μg / ml (△), 8 μg / ml (▽), 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (●), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each plot in Figure 26, the △ and ▽ plot lines for the 16 μg / ml and 8 μg / ml treatments are distinguishable from the ▲ and ▼ plot lines for the 0.25 μg / ml and 0.125 μg / ml treatments, respectively, because the 16 μg / ml and 8 μg / ml treatments are more effective. In other words, the 16 μg / ml and 8 μg / ml treatment plot lines show significantly lower CFU values ​​than the 0.25 μg / ml and 0.125 μg / ml treatments, especially on day 5. Similarly, the □ plot line for the 1 μg / ml treatment can be easily distinguished from the □ plot line for the no treatment, because the 1 μg / ml treatment is more effective. The CFU value for the □ plot line for the no treatment increases after 1 day, reaching 1 x 10 4 remains above.

[0409] The 30RESP003FC and FD compositions designated "16 μg / ml" in the MIC table above and in Figure 26 contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentrations after dilution), and the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions are 50% dilutions (i.e., halving the concentration) of the previous compositions, on the order of 16 to 0.125 μg / ml, respectively.

[0410] THP-1 macrophages were infected with M. tuberculosis at an MOI of 1:10, and the number of intracellular bacteria was measured using bacterial colony counting (CFU) at 2 hours, 1, 2, and 5 days post-infection. Values ​​shown are the mean ± standard deviation from one independent experiment. Compared to untreated controls, treatment with 30RESP003FC (concentrate) at 16 μg / ml and 8 μg / ml, and 30RESP003FD at 16 μg / ml, showed increased efficacy against M. tuberculosis HN878 ( * , p<0.05).

[0411] Figure 27: Efficacy of 30RESP004FC and FD (concentrate and dilutions) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP004FC (concentrate) (A) and 30RESP004FD (dilution) (B) formulations was evaluated for intracellular killing of M. tuberculosis HN878 (□) in THP-1 macrophages at 2 hours post-infection (day 0), days 1, 2, and 5, and after treatment with 16 μg / ml (△), 8 μg / ml (▽), 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (●), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each plot in Figure 27, the △ and ▽ plot lines for the treatments of 16 μg / ml and 8 μg / ml can be distinguished from the ▲ and ▼ plot lines for the treatments of 0.25 μg / ml and 0.125 μg / ml, respectively. This is because the treatment is more effective. In other words, the plots for the 16 μg / ml and 8 μg / ml treatments show significantly lower CFU values ​​than the 0.25 μg / ml and 0.125 μg / ml treatments, especially on day 5. Similarly, the 1 μg / ml treatment plot (□) can be easily distinguished from the no treatment plot (□) because the 1 μg / ml treatment is more effective. The CFU value for the no treatment plot (□) increases after 1 day, reaching 1 x 10 4 remains above.

[0412] The 30RESP004FC and FD compositions designated "16 μg / ml" in the MIC table above and in Figure 27 contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M ascorbic acid / ascorbate (final molar concentrations after dilution), and the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions are 50% dilutions (i.e., halving the concentration) of the previous compositions, on the order of 16 to 0.125 μg / ml, respectively.

[0413] THP-1 macrophages were infected with M. tuberculosis at an MOI of 1:10, and the number of intracellular bacteria was measured using the bacterial colony counting method (CFU) 1, 2, and 5 days after infection. Values ​​shown are the mean ± standard deviation from one independent experiment. Compared to the untreated control, treatment with 30RESP004FC (concentrate) at 16 μg / ml and 8 μg / ml increased efficacy against M. tuberculosis HN878 ( * , p<0.05).

[0414] It is concluded that the formulation exhibits in vitro inhibition of M. tuberculosis HN878 at appropriate doses above the MIC.

[0415] It should also be noted that the method by which the formulations are manufactured affects their in vitro antibacterial efficacy against M. tuberculosis HN878 in the study of Example 6.

[0416] This is exemplified by comparing the potency of the 8 μg / ml concentration of Formulation 1 between the FC and FD versions (Figure 24A vs. Figure 24B). The potency of the FC version increases strongly for at least 5 days after incubation, while the potency of the FD version increases less strongly over the same period. This contrasts with the 16 μg / ml concentration, which shows very similar and excellent potency over the same period, as between the FC and FD versions.

[0417] A different behavior is observed for Formulation 2 (Figure 25A vs. Figure 25B). The potency of the FD version at a concentration of 16 μg / ml increases more strongly than the FC version for the first 2 days after incubation and remains unchanged thereafter, but by 5 days after incubation the potency is superior for the FD version and very superior for the FC. At an 8 μg / ml concentration, the potency of the FD version increases to a strong and excellent potency for at least 5 days after incubation, whereas the potency of the FC version increases less strongly over the same period.

[0418] Thus, at least at higher concentrations, the step in which water is added to arrive at the final inhibitor formulation can substantially affect the antimicrobial activity of the formulation, both in terms of initial antimicrobial activity and the extent of killing over a 5-day period. Generally speaking, although not universally, first preparing the formulation as a concentrated premix of the desired relative molar ratios of sodium nitrite, polyol, and acid components at a concentration higher than desired for use (e.g., at least 3-fold higher than desired for use, e.g., at least 5-fold higher than desired for use, e.g., about 3-fold to about 80-fold higher than desired for use), and then diluting the concentrate to obtain the formulation for use, results in superior antimicrobial activity over a 0- to 5-day period after incubation.

[0419] Example 7 Cytotoxicity and antiviral activity of carboxylic acid-nitrite-polyol solutions against H1N1 influenza A virus Test formulations designated F1C1, F1C2, and F1C3, corresponding to formulation 30RESP001FC in Example 6, their 10-fold dilutions, and their 100-fold dilutions, were used with oseltamivir solution (1 μM) and a virus control to obtain comparative cytotoxicity and H1N1 influenza A virus killing effects in MDCK cells after 24 hours. Cytotoxicity was evaluated by the LDH cytotoxicity assay as in Example 8. Antibacterial activity against H1N1 influenza A virus in MDCK cells was measured at an MOI of 0.002 (●) and an MOI of 0.02 (■) at various dilutions (horizontal axis represents nitrite molar concentration). Cytotoxicity is shown in gray, with a cytotoxicity scale on the right (cytotoxicity at measured nitrite concentrations up to 0.015 M was 1% or less of that of the LDH control). Plate photographs were obtained at an MOI of 0.002 and nitrite concentrations of 0.15M, 0.015M, and 0.0015M compared to oseltamivir (1 μM). The results are shown in Figure 28. The order of plates listed in the last sentence corresponds to the left-to-right order of plates in the figure (there were two experiments, with corresponding plates from each experiment shown above and below). The right-most pair of plates, immediately to the right of the oseltamivir pair of plates, is the virus control. Cytotoxicity is shown below each pair of test plates as a % of LDH control (average of 3 LDH assays at 24 hours post-infection).

[0420] The results show that at appropriate doses, the nitrite / citric acid / polyol formulation completely eradicated the virus, demonstrating its clear superiority over oseltamivir. Similar antiviral activity of the nitrite / citric acid / polyol formulation has also been demonstrated against rhinovirus and respiratory syncytial virus (RSV).

[0421] These results demonstrate that therapeutic and prophylactic treatment of respiratory viral infections in human and animal subjects is provided by the nitrite / acid / polyol formulations according to the present invention.

[0422] Example 8 Cytotoxicity and antiviral activity of carboxylic acid-nitrite-polyol solutions against coronavirus SARS-CoV-2 material Test formulation F1 (pH 5.8) Six test concentrations of Formulation 1 (F1) in aqueous solutions of sodium nitrite, citric acid, and mannitol (a polyol) at pH 5.8 were prepared from stock solutions of 1.5 M sodium nitrite, 0.91 M citric acid / citrate buffer at pH 5.8, and 0.5 M mannitol solution by the method described below to obtain the following test compositions:

[0423] [Table 6]

[0424] Controls used in F1 A pH 5.8 control formulation was prepared from 0.1 M citric acid + assay buffer + cells.

[0425] The negative control was assay buffer plus cells.

[0426] The positive control was chloroquine + cells.

[0427] Test formulation F2 (pH 5.4) Six test concentrations of Formulation 2 (F2), an aqueous solution of sodium nitrite, citric acid at pH 5.4, and mannitol (a polyol), were prepared from stock solutions of 1.5 M sodium nitrite, 0.91 M citric acid / citrate buffer (pH 5.4), and 0.5 M mannitol solution by the method described below to obtain the following test compositions:

[0428] [Table 7]

[0429] Controls used with F2 A pH 5.4 control formulation was prepared from 0.1 M citric acid + assay buffer + cells. Ta.

[0430] The negative control was assay buffer plus cells.

[0431] The positive control was chloroquine + cells.

[0432] Chemical Reagents Sodium nitrite: Grade: Sodium Nitrite Ultrapure Ph Eur, USP, Sodium Nitrite CAS No. 7632-00-0, EC No. 231-555-9, Ultrapure Ph Eur, USP, Sigma Aldrich, Product Code 1.065441000. Citric acid: Grade: Citric Acid Anhydrous Powder EMPROVE® ESSENTIAL Ph Eur, BP, JP, USP, E 330, FCC, Sigma Aldrich, product code 1.002425000. D-Mannitol: Grade: D-Mannitol meets EP, FCC, and USP conformity tests. Sigma Aldrich, product code M8429-100G. Chloroquine phosphate: Grade: Pharmaceutical secondary standard, Sigma Aldrich, product code PHR1258-1G.

[0433] Preparation of stock solutions To prepare citric acid solution, add 19.2 g of citric acid to 90 ml of distilled water, followed by 10 ml of 3 M sodium hydroxide, and dilute with distilled water to adjust the pH (160 ml for pH 5.4, 190 ml for pH 5.8). Alternatively, add 20 ml of distilled water to 19.2 g of citric acid, followed by 1.2 g of solid sodium hydroxide, then adjust the pH with 10 M sodium hydroxide and distilled water to 100 ml. The solution is sterilized by syringe filtration using a 0.22 μm filter.

[0434] To prepare a 1.0 M sodium nitrite solution, add 6.9 g of sodium nitrite to 100 mL of distilled water. To prepare a 1.5 M sodium nitrite solution, add 10.35 g of sodium nitrite to 100 mL of distilled water.

[0435] In certain cases, 9.1 g of mannitol was added to give a concentration of 0.5 M. The solution is sterilized by syringe filtration using a 0.22 μM filter.

[0436] Preparation of formulations The pH of the buffered citric acid solution is controlled to the desired value before mixing with the nitrite and mannitol solution. The pH stated for the formulation is the pH of the buffered citric acid solution prepared before mixing with the nitrite and mannitol solution.

[0437] One suitable method for constructing the formulation is as follows: Sodium nitrite (1.5M) containing 0.5M mannitol is added to a mixing vessel, followed immediately by the pH-controlled citric acid solution in a 1:1 mixture (nitrite + polyol:citric acid). The solution is mixed by gentle inversion. Once mixed, the mixture is held at ambient temperature for 5 minutes in a sealed container (e.g., a 50 ml Falcon tube). The resulting solution containing 0.75M nitrite, 0.25M mannitol, and citric acid is then diluted 5-fold with assay buffer (1.2x concentrated) to obtain final test concentrations in the assay of 0.15M nitrite, 0.05M mannitol, and, e.g., 0.1M citric acid. Serial dilutions of the 1:1 mixture (e.g., starting mixtures of 0.75M nitrite, 0.25M mannitol, and 0.5M citric acid) are prepared using distilled water and / or assay buffer. All formulation concentrations are stored at ambient temperature. Solutions are prepared fresh for each experiment.

[0438] Additional Controls As an additional control, we used a range of concentrations of S-nitroso-N-acetylpenicillamine (SNAP) known to be suitable for this purpose, designated SNAP50, SNAP100, SNAP200, SNAP300, and SNAP400. SNAP is a known NO donor that served as a positive NO-donating control in these studies to verify that NO is not cytotoxic in vitro. To control for potential effects of the N-acetylpenicillamine (NAP) moiety on the assay, corresponding concentrations of NAP were used as NO blank controls, designated NAP50, NAP100, NAP200, NAP300, and NAP400.

[0439] virus SARS-CoV-2 clinical isolates

[0440] cell line Vero E6

[0441] Assay LDH assay (cytotoxicity): CyQUANT™ LDH Cytotoxicity Assay Kit, Invitrogen; Cat No. C20300 and C20301. Tissue culture infectious dose (TCID50) was determined using cytopathic effect (CPE) scoring as readout (virus titration).

[0442] The cytotoxicity of nitrite formulations (all concentrations), pH 5.8 or pH 5.4 citrate controls, negative controls, and positive controls (chloroquine as described in Keyaerts, E., Biochem Biophys Res Commun, 323, 264-268 (2004), the contents of which are incorporated herein by reference) was tested on Vero E6 cells 2 and 24 hours after nitrite / control addition. LDH release was measured as readout at 2 and 24 hours. Each compound / formulation was tested in triplicate per experiment.

[0443] SARS-CoV-2 inhibition: At time 0, Vero E6 cells were infected with virus in the presence of formulation or control and incubated for 1 hour. After this incubation period, the inoculum was removed and the cells were washed. Fresh formulation or control was then added to the washed cells. At 24 hours post-infection, Vero E6 cell supernatants were harvested and titrated. Virus titrations were incubated for 6 days before being readout to measure the reduction in virus yield. Four separate tests were performed at MOIs, including 3.0 and 0.3; however, only these two MOIs were titrated. Readout was performed by crystal violet staining (cell monolayer) followed by CPE scoring.

[0444] result The results are shown in Figures 32 to 34.

[0445] Figure 32 shows the results of the LDH cytotoxicity assay (combined graphs from Experiments 1 and 2, using Test Formulations 1 and 2, respectively). Data are presented as the mean + standard deviation (SD) of two experiments. SD is shown as a gray error bar. Maximum LDH activity (cells + lysis buffer) was set to 100%, and all sample results were compared to this value. The LDH positive control was the kit's positive control. Black bars (2-hour incubation) are the left-hand bars of each pair of bars in each case, and red bars (24-hour incubation) are the right-hand bars of each pair of bars in each case. - is.

[0446] Figure 33 shows the results of the antiviral test against SARS-CoV-2 in Experiment 1 at an MOI of 3.0. In Experiment 1, one virus yield reduction assay was performed using SARS-CoV-2 at four multiplicities of infection (MOIs) and confirmed using back titration of the inoculated virus. For cells inoculated at an MOI of 3, 2.1 log10 TCID50 / ml was detected in the virus control wells after titration. A reduction in SARS-CoV-2 yield could be observed in some of the conditions tested. After 24 hours of incubation, almost no virus was detected at the three lowest MOIs (i.e., 0.3, 0.03, and 0.003). Presumably, 24 hours of replication in Vero E6 cells is not sufficient to obtain high levels of progeny virus. Data are presented as the mean + standard deviation (SD) of duplicate titrations. SD is shown as error bars. The horizontal dotted level of the chloroquine and cell control log10 TCID50 / ml values ​​is the limit of detection (LOD) of the assay.

[0447] Figure 34 shows the results of antiviral testing against SARS-CoV-2 in Experiment 2 at (a) an MOI of 3.0 and (b) an MOI of 0.3. The method corresponds to Experiment 1 at these MOIs, except that the formulation was the Experiment 2 formulation (Test Formulation 2 at various concentrations), and incubation was performed for 48 hours instead of 24 hours to increase the level of progeny virus. Data are presented as the mean + standard deviation (SD) of duplicate titrations. SD is shown as error bars. The horizontal dotted line levels for chloroquine and control log10 TCID50 / ml values ​​are the limit of detection (LOD) of the assay.

[0448] Consideration The NO-generating aqueous formulations show no cytotoxicity in the LDH assay (Figure 32). In particular, at high concentrations of nitrite, acid, and polyols, the in vitro antiviral activity against SARS-CoV-2 is impressive and comparable to that of chloroquine (Figures 33 and 34).

[0449] The NO-generating aqueous formulation is surprisingly effective at high pH. pHs of 5.4 and 5.8 have been tested, but it is expected that it will also be effective at lower pHs of 5.2 and below.

[0450] Furthermore, data reveal that organic carboxylic acids (e.g., citric acid buffered to pH 5.4 or 5.8) have surprisingly low cytotoxicity and high in vitro antiviral activity against SARS-CoV-2 in the absence of NO-generating formulations (Figures 32–34; “citric acid pH 5.8” and “citric acid pH 5.4” bars). The relatively high pH of carboxylic acid formulations makes them attractive as pulmonary active agents, as they are expected to be nontoxic to the airways and lung tissue surfaces. SARS-CoV-2 belongs to the same coronavirus family as SARS-CoV, and because of the similarities between the viruses, it is predictable that such organic carboxylic acids will also show corresponding efficacy against the SARS-CoV virus, the coronavirus that causes severe acute respiratory syndrome (SARS), which experienced well-documented outbreaks in 2002 and 2003.

[0451] Example 9 Antiviral activity of carboxylic acid-nitrite-polyol solutions against coronavirus SARS-CoV To investigate the similarity between the antiviral activity provided by the present invention against SARS-CoV-2 and the antiviral activity provided by the present invention against SARS-CoV, the following experiment was performed.

[0452] Formulations F1C1, F1C2, F1C3, and F1C4 were incubated with SARS-CoV-2 at an MOI of 3.0. The plates were tested for antiviral activity against V. The method is similar to the antiviral test described in Example 8. Prior to staining the cell monolayer with crystal violet, two plates were checked microscopically and scored for cytopathic effect (CPE). CPE in the form of cellular debris above the underlying monolayer was found to be present on these plates.

[0453] The results of two plates checked by microscope are shown in Figure 35. Data are for a single titration per condition. For the remaining plates, the cell monolayer was too dense to score for CPE after crystal violet staining. The horizontal dotted level of the cell control log10 TCID50 / ml value is the limit of detection (LOD) of the assay.

[0454] As shown in Figure 35, at least formulations F1C1 and F1C2 exhibited excellent in vitro antiviral activity against SARS-CoV.

[0455] Example 10 Human inhaler One embodiment of a human inhaler using a liquid composition according to the present invention is shown schematically in FIGS.

[0456] The inhaler is suitably powered by compressed gas and is configured to deliver, in a generally conventional manner, a single dose of entrained droplets of the nitrite / acid / polyol formulation from a reservoir within the inhaler in response to a single manual actuation of the inhaler. The subject typically actuates the inhaler and simultaneously inhales, as conventionally done by asthmatics when using an inhaler. As shown in Figure 30, a treatment time of approximately 3 minutes per dose is appropriate, providing a duration of effect of up to approximately 2 hours with an appropriate dose of the active composition.

[0457] The airborne droplets enter the subject's infected lungs, where they contact the infected (e.g., virally infected) lung membranes. The right side of Figure 31 shows the effect of the present invention when multiple drops of an aqueous nitric oxide (NO)-generating composition ("aqueous NO") are deposited on the lung mucosa. The left side of Figure 31 shows the corresponding effect when gaseous nitric oxide is inhaled by the subject ("inhaled nitric oxide") instead of the aqueous nitric oxide (NO)-generating composition.

[0458] As shown, when inhaled nitric oxide is used, efficacy can be significantly reduced. Not only is a portion of the inhaled nitric oxide exhaled by the subject before passing through the lung mucosa and entering the bloodstream, but another portion of the inhaled nitric oxide is oxidized to toxic nitrogen dioxide (NO) by oxygen in the inhaled air. In addition to depleting the availability of gaseous nitric oxide for treating the subject, nitrogen dioxide has adverse effects on the subject's lungs.

[0459] As a result, more efficient and effective delivery of nitric oxide to a patient's lungs and, via the lungs, into the patient's bloodstream is achieved by using a nitrite / acid / polyol formulation according to the present invention.

[0460] conclusion The present invention has been described broadly, but is not intended to be limited thereto. Variations and modifications that would be readily apparent to one skilled in the art are intended to be included within the scope of the appended claims. To the extent that the law of the particular jurisdiction in which this invention is patented provides for enforcement of the patent against the misuse of technology equivalent to the appended claims, the patentee intends that the patent cover such equivalent technology.

[0461] Equivalents to the scope of protection of the appended claims are also covered by the claims to the extent permitted by applicable law. For example, generally speaking, the NOx generating The order of mixing components or portions of components of a reaction is not important, so long as the NOx-forming reaction is not initiated prematurely. Any mixing order of essential and non-essential components of any combination, kit, or composition of the present invention is intended to be covered. If one or more components are used in liquid form, e.g., as a solution, the effect of that component or a mixture of those components on the concentration of solutes (including, but not limited to, that component or those components) in the reaction mixture or any portion of the reaction mixture may be different compared to when that one or more components are used in solid or liquid form at different volumes or concentrations. The use of all equivalent concentrations and / or physical forms (solid, liquid, solution) of components to form the combinations, kits, and compositions of the present invention, and all equivalent steps and sequences of steps to prepare said combinations, kits, and compositions, even if not described or specifically claimed herein, is within the scope of the present claims, to the extent permitted by applicable law.

Claims

1. 1. A therapeutic or non-therapeutic method of delivering nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, to a human or animal subject via the nose, mouth, airways or lungs of the subject, comprising: (A) administering to a subject via the subject's nose, mouth, airways or lungs a combination or composition for producing nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, by reaction of one or more nitrites with a proton source, The combination or composition comprises: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyols; and characterized by one or more of the following: (a) one or more organic polyols are present in a reaction power enhancing amount, said reaction power enhancement being compared to a reaction conducted under the same conditions but without the one or more organic polyols; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols is not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not polyvinyl alcohol only; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols are propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexyl hydroxybenzo ... The exclusion does not include ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol. or (B) administering nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, to the subject via the subject's nose, mouth, airways or lungs, (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids, in the presence of which the reaction is carried out, under reaction conditions suitable to produce nitric oxide, and optionally other nitrogen oxides and / or optionally precursors thereof; (iii) one or more organic polyols; and preparing the compound by a process comprising reacting Characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols is not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not polyvinyl alcohol only; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol. A method comprising:

2. 10. The method of claim 1, wherein the proton source comprises a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix, the combination or kit comprises two or more separate compositions, and the one or more polyols are not present in the separate compositions in direct contact with or mixed with the hydrogel.

3. 3. The method of claim 1 (A) or claim 2, wherein the combination or composition consists essentially of components (i), (ii) and (iii), and optionally water and / or a pH buffer.

4. 3. The method of claim 1 (A) or claim 2, wherein the combination or composition consists of components (i), (ii) and (iii), and optionally water and / or a pH buffer and / or one or more additional components in an amount of less than about 20% by weight or volume of the combination or composition.

5. 5. The method of any one of claims 1 to 4, which is a method for treating a microbial infection, e.g., a bacterial, viral, fungal, microbiological infection, or any combination thereof, in a subject in need thereof, e.g., a human subject or other mammalian subject.

6. 5. The method of any one of claims 1 to 4, which is a method of vasodilation performed on a subject, such as a human subject or other mammalian subject.

7. 5. The method of any one of claims 1 to 4, for example an antimicrobial method for reducing the number of, preventing the growth of, or limiting the rate of growth of microorganisms, such as bacteria, viruses, fungal cells and / or microparasites, at a location in a subject.

8. 6. The method of claim 5, wherein the microbial infection is in the skin of the subject, e.g., a mucous membrane, or an interior space of the subject, e.g., the nose, mouth, airways or lungs of the subject, or the lining of the lung pleura of the subject.

9. 8. The modification of claim 7, wherein the combination or composition administered to a subject has an initial pH of the aqueous solution of the proton source, including any desired buffer, in the range of 5 to 8, prior to the presence of any other components of the NOx-producing reaction mixture that affect the pH or reaction mixture at the start of reaction with one or more nitrites, and wherein the one or more polyols may optionally be omitted.

10. 10. The method of any one of claims 1 to 9, carried out in connection with a surgical method or a method comprising both therapy and surgery.

11. (A) A combination or composition for use in therapy and / or surgery to produce nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, by reacting one or more nitrites with a proton source, comprising: said combination or composition comprising: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyols; and characterized by one or more of the following: (a) one or more organic polyols are present in a reaction power enhancing amount, said reaction power enhancement being compared to a reaction conducted under the same conditions but without the one or more organic polyols; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols is not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not polyvinyl alcohol only; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol. wherein said treatment and / or surgery comprises administering said combination or composition to said subject via said subject's nose, mouth, airway or lungs; or (B) Nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, for use in therapy and / or surgery, (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids, in the presence of which the reaction is carried out, under reaction conditions suitable to produce nitric oxide, and optionally other nitrogen oxides and / or optionally precursors thereof; (iii) one or more organic polyols; and preparing the compound by a process comprising reacting Characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) The proton source is a pendant carboxylic acid covalently attached to a three-dimensional polymer matrix. Not only hydrogels containing acid groups; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols is not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not polyvinyl alcohol only; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol. wherein said treatment and / or surgery comprises administering said nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, to said subject via said subject's nose, mouth, airways or lungs; or (C) a combination or composition for administration to the nose, mouth, airways, or lungs of a subject to produce nitric oxide, and optionally other nitrogen oxides and / or optionally precursors thereof, by reaction of one or more nitrites with a proton source, comprising: said combination or composition comprising: (i) one or more nitrites; (ii) a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and (iii) one or more organic polyols; and characterized by one or more of the following: (a) one or more organic polyols are present in a reaction power enhancing amount, said reaction power enhancement being compared to a reaction conducted under the same conditions but without the one or more organic polyols; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols is not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not polyvinyl alcohol only; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol. A substance or composition which is

12. 12. The substance or composition according to claim 11, wherein the treatment and / or surgery comprises a method according to any one of claims 1 to 10.

13. The one or more nitrites may be LiNO ２ , NaNO ２ , KNO ２ , RbNO ２ , CsNO ２ , FrNO ２ , AgNO ２ , Be(NO ２ ) ２ , Mg(NO ２ ) ２ , Ca(NO ２ ) ２ , Sr(NO ２ ) ２ , Mn(NO ２ ) ２ , Ba(NO ２ ) ２ , Ra(NO ２ ) ２ 13. The method, substance or composition of any one of claims 1 to 12, wherein the compound is selected from the group consisting of:

14. The one or more nitrites may be NaNO ２ , KNO ２ 14. The method, substance or composition of claim 13, wherein the compound is a hydroxybenzoate, ...

15. 15. A method, material or composition according to any one of claims 1 to 14, wherein the one or more nitrites, or any component of a NOx producing reaction system containing one or more nitrites, is present in dry form, for example in particulate dry form.

16. 15. A method, material or composition according to any one of claims 1 to 14, wherein the one or more nitrites, or any component of a NOx generating reaction system containing one or more nitrites, is present in solution in an aqueous carrier, such as an aqueous liquid or gel.

17. 17. The method, or substance or composition, of claim 16, wherein the molar concentration of nitrite ions in said solution is in the range of about 0.001M to about 5M.

18. 18. A method, material or composition according to any one of claims 1 to 17, wherein the pH of the one or more nitrites, or any component of a NOx producing reaction system containing the one or more nitrites, is buffered, preferably to a pH of from about 6 to about 9.

19. The one or more organic carboxylic acids of the proton source may be selected from the group consisting of salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropanoic acid, β-hydroxypropanoic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid (embonic acid), stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobionic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, 19. The method, substance or composition of any one of claims 1 to 18, selected from: one or more polymeric or polymerized carboxylic acids, such as polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid; one or more acid hydrogels containing pendant -COOH groups covalently bonded to polymer molecules that form the three-dimensional polymer matrix of the hydrogel; partial or complete esters and partial or complete salts thereof that can act as proton sources; and any mixture or combination thereof.

20. 20. The method, substance, or composition of claim 19, wherein the one or more carboxylic acids are selected from citric acid, salts thereof, and combinations thereof.

21. 21. The method, substance, or composition of any one of claims 1 to 20, wherein the one or more non-carboxylic acid reducing acids of the proton source are selected from ascorbic acid; ascorbyl palmitate (ascorbyl palmitate); ascorbic acid derivatives such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid, and 6-O-dodecanedioyl ascorbic acid; acidic reductants such as reducing acids; erythorbic acid; oxalic acid; salts thereof; and combinations thereof.

22. 22. The method, substance or composition of claim 21, wherein the organic non-carboxylic reducing acid is ascorbic acid or a salt thereof. 。

23. 23. A method, material or composition according to any one of claims 1 to 22, wherein the proton source, or some of its components, or any component of a NOx producing reaction system containing the proton source, is present in dry form, for example in particulate dry form.

24. 23. A method, material or composition according to any one of claims 1 to 22, wherein the proton source, or some of its components, or any component of a NOx producing reaction system containing the proton source, is present in solution in an aqueous carrier, such as an aqueous liquid or gel.

25. 25. The method, or substance or composition, of claim 24, wherein the molar concentration of the proton source in the solution is in the range of about 0.001M to about 5M.

26. 26. A method, or substance or composition according to any one of claims 1 to 25, wherein the pH of the proton source is preferably buffered to a pH of from about 3 to about 9, such as from about 4 to about 8, for example from about 5 to about 8.

27. 27. A method, substance or composition according to any one of claims 1 to 26, wherein the one or more organic polyols are selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, such as alditols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.

28. 28. The method, substance, or composition of any one of claims 1 to 27, wherein the one or more organic polyols are selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol, polyglycitol, glycerol, and any combination thereof.

29. 29. The method, substance or composition of claim 27 or claim 28, wherein the one or more organic polyols are selected from arabitol, xylitol, mannitol, sorbitol, and any combination thereof.

30. 30. The method, material or composition of any one of claims 1 to 29, wherein the one or more organic polyols, or any component of a NOx producing reaction system containing the one or more organic polyols, is present in dry form, for example in particulate dry form.

31. 30. The method, material or composition of any one of claims 1 to 29, wherein the one or more organic polyols, or any component of a NOx-producing reaction system containing the one or more organic polyols, is present in solution in an aqueous carrier, such as an aqueous liquid or gel.

32. 32. The method, or substance or composition, of claim 31 , wherein the total molar concentration of the one or more organic polyols in the solution is in the range of about 0.001 M to about 5 M.

33. (a) the total molar concentration of any one or more organic polyols in the polyol component or in the reaction solution at or before the initiation of the NOx-forming reaction is from about 0.05 to about 3 times, e.g., from about 0.1 to about 2 times, e.g., from about 0.25 to about 1.5 times, the total molar concentration of said nitrite ions in said nitrite component or in said reaction solution; or (b) the total molar concentration of any one or more organic polyols in the polyol component or in the reaction solution at or before the initiation of the NOx-forming reaction is from about 0.05 to about 3 times, e.g., from about 0.1 to about 2 times, e.g., from about 0.25 to about 1.5 times, the total molar concentration of said proton source in said proton source component or in said reaction solution; 33. A method, or substance or composition according to any one of claims 1 to 32.

34. Nitric oxide, and optionally other nitrogen oxides, are produced by reaction of one or more nitrites with a proton source.

34. The method, or substance or composition of any one of claims 1 to 33, wherein the combination or composition for producing the compound and / or optionally a precursor thereof further comprises one or more additional ingredients selected from diluents, carriers, excipients, sweeteners, taste masking agents, thickeners, viscosifiers, humectants, film formers, lubricants, binders, emulsifiers, solubilizers, stabilizers, colorants, odorants, salts, coating agents, antioxidants, active agents, preservatives, and any combination thereof.

35. 35. A kit for use in a method, or for use in preparing and optionally delivering a substance or composition, according to any one of claims 1 to 34, comprising in addition to the (i), (ii) and, if present, (iii) chemical components at least one of: containers for holding said components prior to use; at least one device or other means for mixing said components, dispensing the reaction mixture and / or released gases and controlling said mixing and dispensing; instructions for use; and directions to where the instructions for use can be found, e.g., online instructions.

36. A dispenser for use in the method of any one of claims 1 to 10 and 13 to 34, comprising: the chemical components (i), (ii), and, if present, (iii); at least one container for holding said components prior to use; at least one device or other means for controlling the mixing of the components and distribution of the reaction mixture; the one or more ingredients and / or released gas from said dispenser; and directing it toward a target.

37. 37. The dispenser of claim 36, wherein the dispenser is adapted for repeated similar operations of dispensing the reaction mixture, one or more components thereof, a carrier containing the reaction mixture, a carrier containing one or more components of the reaction mixture, and / or the released gas.

38. 38. The dispenser of claim 36 or claim 37, wherein the dispenser comprises a pump or jetting system for conveying the NO-generating reaction mixture, one or more of its components, or a composition comprising released gas from the dispenser and directing it toward a target.

39. 39. The dispenser of any one of claims 36 to 38, wherein the dispenser is adapted to direct the reaction mixture, one or more components thereof, a carrier comprising the reaction mixture, a carrier comprising one or more components of the reaction mixture, and / or the released gas into the nose, mouth, airways or lungs of a human or animal subject.

40. a pressurized cylinder of nitric oxide gas; and a delivery device connectable to said pressurized cylinder and adapted to deliver said nitric oxide gas from said pressurized cylinder to the nose, mouth, airways or lungs of a human or animal subject; wherein the nitric oxide is produced by a process comprising reacting one or more nitrite salts with a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids under suitable reaction conditions to produce nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof; 1. A nitric oxide dispenser, wherein the reaction is carried out in the presence of one or more organic polyols, A dispenser characterized by one or more of the following: (a) the one or more organic polyols are present in a reaction power enhancing amount; (b) the proton source is not the only hydrogel containing pendant carboxylic acid groups covalently attached to a three-dimensional polymer matrix; (c) the one or more organic polyols are not solely glycerol; (d) if one or more thickeners are used, the one or more organic polyols are not solely glycerol; (e) if one or more plasticizers are used, the one or more organic polyols are not solely glycerol; (f) the one or more organic polyols are not exclusively polyvinyl alcohol; (g) if one or more thickeners are used, the one or more organic polyols are not exclusively polyvinyl alcohol; (h) in any one or more of (b) through (g) above, the word "not only" is replaced with "not including"; (i) the one or more organic polyols are not solely propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol; (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerin monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, glycols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the above with glycerol and / or polyvinyl alcohol.

41. 41. Nitric oxide, optionally other nitrogen oxides and / or precursors thereof, dispensed using a dispenser according to any one of claims 38 to 40.

42. 10. A method, substance or composition, or kit, or dispenser, or nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, when dispensed using a dispenser according to any one of the preceding claims, the one or more nitrites comprise (e.g., contain, consist essentially of, or consist of) one or more alkali metal or alkaline earth metal nitrites, e.g., sodium nitrite, potassium nitrite, or any combination thereof; the proton source comprises (e.g., contains, consists essentially of, or consists of) ascorbic acid or an ascorbic acid / ascorbate buffer; citric acid or a citric acid / citrate buffer; or any combination of two or more thereof; said molecule of ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citrate buffer, or any combination of two or more thereof, Not covalently attached to a polymer or macromolecule; - the one or more organic polyols comprise (e.g., contain, consist essentially of, or consist of) a straight-chain sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule; e.g., sorbitol; mannitol; arabitol; xylitol; or any combination of two or more thereof; - the total molar concentration of the one or more organic polyols in the polyol component or reaction solution at or before the start of the NOx-producing reaction is 0.05 to 3 times the total molar concentration of nitrite ions; - at or before the start of the NOx-forming reaction, the total molar concentration of the one or more organic polyols in the polyol component or in the reaction solution is 0.05 to 3 times the total molar concentration of the proton source in the proton source component or in the reaction solution; - for applications that do not involve contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source, particularly immediately prior to initiation of the NO-producing reaction, is in the range of 3.0 to 9.0; - for applications involving contact of the reaction mixture with cells or animal (including human) skin (including mucous membranes), organs or other tissues, the pH of the proton source, particularly immediately prior to initiation of the NO-producing reaction, is in the range of 4.0 to 8.0; - for applications involving contact of the reaction mixture with the nose, mouth, airways or lungs of an animal (including human) subject, the pH of the proton source prior to, and particularly immediately prior to, the initiation of the NO-producing reaction is in the range of 5.0 to 8.0; - Targeted microorganisms include Actinomyces, Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, Treponema, Ureaplasma, Vibrio, E. coli, and the viruses listed in the list of influenza virus, parainfluenza virus, adenovirus, norovirus, rotavirus, rhinovirus, coronavirus, respiratory syncytial virus (RSV), astrovirus, hepatitis virus, and combinations thereof; and the protozoan group listed in the list of sarcozoa, flagellates, ciliates, sporozoa, and any combination thereof; for example, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, non-tuberculosis mycobacteria including Mycobacterium abscessus, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

43. 10. A method, or substance or composition, or kit, or dispenser, or nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, when dispensed using a dispenser according to any one of the preceding claims, which does not contain (i.e. excludes) a reducing agent when one or more organic polyols are present.

44. 11. A method of treatment according to any one of claims 1 to 10, or any one of the preceding claims depending on any one of claims 1 to 10, wherein the treatment is treatment or prevention of rhinovirus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis or influenza infection in a subject.

45. 44. The substance, composition, kit, dispenser, nitric oxide or other nitrogen oxides and / or optionally precursors thereof of any one of claims 11 to 43 for use in therapy, wherein the therapy is treatment or prevention of rhinovirus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis or influenza infection in a subject.

46. 46. ​​The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of claim 44 or claim 45, wherein said treatment is treatment or prevention of a rhinovirus infection in a subject.

47. 46. ​​The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of claim 44 or claim 45, wherein said treatment is treatment or prevention of infection with SARS-CoV (SARS) in a subject.

48. 46. ​​The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of claim 44 or claim 45, wherein said treatment is treatment or prevention of infection with SARS-CoV-2 (COVID-19) in a subject.

49. 46. ​​The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of claim 44 or claim 45, wherein said treatment is treatment or prevention of infection with Mycobacterium tuberculosis in a subject.

50. 46. ​​The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of claim 44 or claim 45, wherein said treatment is treatment or prevention of influenza infection in a subject.

51. 51. The method, substance, composition, kit, dispenser, nitric oxide, or other nitrogen oxides and / or optionally precursors thereof of any one of claims 44 to 50, wherein the subject is a human.

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