Methods and compositions for treating and controlling tuberculosis

Abstract

The present invention provides one or more agents selected from nitric oxide (NO), nitric oxide-generating compositions, combinations or combinable associations of components for nitric oxide-generating compositions, and mixtures thereof, for use as antibacterial agents against tuberculosis and Mycobacterium tuberculosis.

Description

[Technical Field]

[0001] The present invention relates to methods and compositions for treating and controlling tuberculosis. [Background technology]

[0002] Tuberculosis, a respiratory disease, is caused by bacteria of the genus Mycobacterium. Currently, there is no completely satisfactory treatment for this disease, which causes significant casualties in many countries. In many cases, tuberculosis exists in association with other pathogenic infections, including viral infections. A drug effective against tuberculosis would be substantially beneficial if it possessed broad activity, including activity against viruses and other pathogens commonly present in tuberculosis patients.

[0003] The present invention is based on the remarkable discovery that one or more activators selected from nitric oxide (NO), nitric oxide-producing compositions, components or combinations or combinatable aggregates of components of nitric oxide-producing compositions, and mixtures thereof are effective in vitro antibacterial agents against M. tuberculosis, providing effective in vivo treatment (both therapeutic and prophylactic) for tuberculosis in humans and animals. Effective antibacterial treatment of surfaces (including non-living surfaces, as well as the hands, arms, and other outer surfaces of the body of humans or animals) and spaces to prevent the transmission of M. tuberculosis and the resulting contamination of surfaces is also provided by the present invention.

[0004] In a preferred embodiment of the present invention, nitric oxide can be produced by an NO-producing system comprising a nitrite and a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acids. The organic non-carboxylic acids may be organic non-carboxylic acid reducing acids. Such a system can be embodied in an NO-producing composition that can be administered to the lungs of a patient.

[0005] The NO-generating system may contain one or more organic polyols. If present, the one or more organic polyols may preferably contain sugar alcohols comprising one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0006] The activator, for example, an NO-generating composition, can be delivered to the patient's lungs in any suitable physical form, such as a liquid, or in the form of droplets contained in a carrier gas or air, such as an aerosol or mist.

[0007] According to the present invention, it has been further found that an acid that functions as a proton source for the production of nitric oxide may be effective when buffered to a relatively high pH, ​​for example, a pH of about 5 to about 8, for example, above about 5.2, for example, in the range of 5.2 to 5.8, i.e., a pH that is physiologically acceptable to the tissues of the patient's mouth, nasal passages, airways, and lungs.

[0008] Nitric oxide and NO-producing compositions, as discussed herein, possess a variety of antibacterial and other beneficial physiological activities, and as a result, the antibacterial activity against M. tuberculosis provided by the present invention may be accompanied by beneficial activity against other pathogens that may infect or to which the patient may be susceptible (including secondary bacterial infections, viral infections, parasitic infections, and fungal infections).

[0009] As reported herein, it has been found in vitro that the antibacterial effect against M. tuberculosis can be enhanced when NO-producing compositions are prepared in a particular manner, namely by one of the following methods: (a) A method for preparing a NOx-generating composition, comprising mixing components of a nitrite, a proton source, and an organic polyol in a desired proportion at a higher concentration than desired in the composition to be used to form a concentrated premixture, and then preferably diluting the concentrated premixture with water to provide the composition to be used. (b) A method for preparing a NOx-generating composition, comprising mixing a nitrite, a proton source, and components of an organic polyol in desired proportions at desired concentrations of the composition to be used, to provide the composition to be used.

[0010] These alternative methods constitute a particular aspect of the present invention.

[0011] Nitric oxide (NO) and its precursors have been widely studied as potential pharmaceutical agents. Nitric oxide is a potent vasodilator synthesized and released by vascular endothelial cells, playing a crucial role, among other things, in regulating local vascular resistance and blood flow. In mammalian cells, nitric oxide is produced primarily 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 inhibiting both platelet and leukocyte aggregation and adhesion, inhibiting cell proliferation, scavenging superoxide radicals, and regulating endothelial permeability. The role of nitric oxide in cancer treatment was discussed in Biochemistry (Moscow), 63(7), 802-809 (1998), whose disclosure is incorporated herein by reference. Nitric oxide has been shown to possess antibacterial properties, as outlined by FC Fang in J.Clin.Invest.99(12),2818-2825(1997), whose disclosures are incorporated herein by reference, and as described, for example, in WO95 / 22335 and WO02 / 20026 (Aberdeen University). Other known uses and applications of the system for producing nitric oxide, other oxides of nitrogen, and their precursors are provided below in the description of the present invention.

[0012] Substantial problems still exist regarding the efficient production of nitric oxide, other nitrogen oxides, and their precursors for therapeutic purposes, as well as their delivery to organisms and cells. The system widely employed for nitric oxide production relies on the acidification of nitrite using a mineral acid, which produces equimolar amounts of initial nitrite (HNO2) compared to the starting nitrite, and then the nitrite is readily decomposed into nitric oxide and nitrate by hydrogen ions and water. The decomposition can be expressed by the following equilibrium equation (1): 3HNO2 → 2NO + NO3 - +H + +H2O (1)

[0013] To maximize NO yield, it is conventional to perform nitrite acidification at a pH below approximately 4, where nitrite formation is generally preferable. However, the use of pH < 4 is unsuitable for in vivo use where the acid comes into contact with animal tissue. Higher pH levels are more beneficial to cells and biological systems, but at pH levels above 4, the previous system did not produce a satisfactory yield of NO. Increasing the amount of NO produced above pH 4 requires large amounts of nitrite, which is impractical and uneconomical for therapeutic applications. In addition, controlled release of nitric oxide for therapeutic use is difficult because the conversion represented by equation (1) is not easily controllable considering the short half-life of nitrite. Reactions between one or more nitrites and a proton source to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally their precursors are referred herein to as “NOx-producing reactions” or “reactions for producing NOx,” or similar wording, and “NOx” is used to refer to both the individual and any combination of collective acidification products of nitrites, particularly nitric oxide, other oxides of nitrogen, and their precursors. Each component of the NOx produced can be released as a gas, or can be in solution in the reaction mixture, or can be in solution first and then released as a gas. It will be understood that this could be either one of these, or any combination thereof.

[0014] WO00 / 53193, the disclosure of which is incorporated herein by reference, describes a cream or ointment for treating skin ischemia and promoting wound healing, wherein the proton source is ascorbic acid. Example 1 describes a gel based on KY Jelly (trademark), and in Example 7, both tests were conducted with the gel in direct contact with the skin and with the skin protected by a membrane. The use of ascorbic acid was claimed to avoid significant skin inflammation (WO00 / 53193, page 2). However, in practice, when the gel was in direct contact with the skin, the degree of skin inflammation was not satisfactory due to the low pH of the gel, and the skin protective membrane attenuated the effect of the gel when the membrane was present. As a result, the gel is not commercially available. The composition of WO00 / 53193 does not contain polyol.

[0015] WO02 / 20026, whose disclosure is incorporated herein by reference, describes skin preparations for treating drug-resistant infections of the skin, wherein the proton source is citric acid or salicylic acid. Nitrite-containing compositions and acid-containing compositions are dispensed from a twin-barrel dispenser, and the compositions are then mixed to induce a reaction of the acid 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 of citric acid and nitrite, respectively, which were mixed in situ to initiate the reaction between the acid and the nitrite (e.g., WO02 / 20026, Example 3, Formulation 1). Glycerol was used in combination with cetostearyl alcohol in a pair of lotions of citric acid and nitrite, respectively, which were mixed in situ to initiate the reaction between the acid and the nitrite (e.g., WO02 / 20026, Example 3, Formulation 3). The preferred pH of the reaction mixture is 5 or less, particularly 4 or less, as undesirable skin inflammation would be expected. Nasal sprays have also been taught, in which reducing acids such as ascorbic acid or ascorbic palmitate may be used to avoid irritation of the sensitive nasal mucosa by using a higher pH. However, it is recognized that the higher the pH, the slower the reaction will be (WO02 / 20026, p. 16, paragraph 2).

[0016] US6103275 (published on August 15, 2000), the disclosure of which is incorporated herein by reference, describes the use of reducing agents such as ascorbic acid together with organic acids such as maleic acid having a pKa of 1 to 4 to acidify nitrite. Viscous (gel) compositions are used to delay the release of reaction products for topical use. The acid and nitrite are separated and held until the production of nitric oxide is initiated, and the reducing agent is described as being included in at least one of the first and second gels. The pH range in which the method should be used is not specified. However, the fact that the buffer components are referred to as acids may indicate that these compounds are present mainly in their protonated form, and thus the pH of the composition needs to be substantially lower than 4. The presence of acids having a pKa of 1 to 4 ensures good buffering capacity of the formulation at that pH. Incorporation of such acids is a convenient way to ensure that the pH is maintained at a level such that the continuous efficiency of converting nitrite to nitric oxide is maintained, but the low pH is expected to cause substantially undesirable skin irritation upon contact with the skin. The composition of US6103275 does not contain polyols.

[0017] WO2003 / 013489, which is incorporated herein by reference, proposes 3% polyvinyl alcohol (PA) as a gel base for citric acid and nitrite, respectively, which are mixed together in situ (WO2003 / 013489, Example 7). However, the test data (WO2003 / 013489, Tables 11 and 12) indicate that PA was unable to form stable gels, and PA compositions were never mixed or used together. Apart from the above proposal which was not followed up to the final composition, the compositions of WO2003 / 013489 do not contain polyols.

[0018] ​U.S. Patent Application No. 2005 / 0037093, incorporated herein by reference, describes a nitric oxide-producing composition based on a nitrite-acid reaction and refers to optional excipients including polyvinyl alcohol, propylene glycol, and polyethylene glycol.

[0019] Chinese Patent Application No. CN101028229, whose disclosure is incorporated herein by reference, describes a cosmetic that produces nitric oxide by the reaction of a nitrite with an acid. In particular, it teaches the optional use of glycerin, propylene glycol, and glyceryl monostearate as additional ingredients. In certain examples, trihydroxyethylamine is further mentioned as an ingredient.

[0020] Chinese Patent Application No. CN101062050, whose disclosure is incorporated herein by reference, describes a hair growth promoting product that generates nitric oxide by the reaction of a nitrite with an acid. In particular, it teaches the optional use of glycerin, propylene glycol, and glyceryl monostearate as additional ingredients. In certain examples, D-pantothenyl alcohol and a combination of panthenol and inositol are mentioned as ingredients.

[0021] WO2008 / 110872, whose disclosure is incorporated herein by reference, describes effervescent nitric oxide donor compositions optionally containing a polar solvent selected from, for example, polyols and polyethylene glycol (paragraphs

[0055] and

[0056] ). Specific polyols described are 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. As optional additional components, polyvinyl alcohol, polyethylene glycol 1000 (PEG1000), PEG4000, PEG6000, and PEG8000 are mentioned in many lists of polymer agents (paragraph

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

[0190] and

[0191] .

[0022] WO2009 / 019498, whose disclosure is incorporated herein by reference, describes the use of non-thiol reducing agents having a pKa between 1 and 4 as additional components of nitrites and proton sources. Examples of non-thiol reducing agents described are iodide anions, butylated hydroquinone, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and beta-carotene. Apart from butylated hydroquinone, the compositions of WO2009 / 019498 do not contain polyols.

[0023] WO2014 / 188174 and WO2014 / 188175, whose disclosures are incorporated herein by reference, describe bandage and transdermal delivery systems for skin lesions in which the proton source is a hydrogel containing pendant carboxylic acids and sulfonic acid bases covalently bonded to a three-dimensional polymer matrix. The primary layer of skin contact is inhaled nitrite. This is a polypropylene mesh. When the mesh is placed on the skin and a hydrogel is overlaid on the mesh as the top layer, it has been found that the reaction products of the acid and nitrite are well delivered to the skin without unacceptable skin irritation. WO2014 / 18175 discloses, for example, a soluble film formed from polyvinyl alcohol and containing nitrite, which is a substitute skin in contact with the primary layer. Both references teach that the hydrogel may contain glycerol, but the purpose is not described. However, it is well known that glycerol is added to this type of hydrogel as a plasticizer (see, for example, WO00 / 06215, page 14, whose disclosure is incorporated herein by reference). The references disclose the preference for the absence of certain hydroxyl-containing components, in particular 1-thioglycerol, erythorbate, ascorbic acid, and butylated hydroquinone.

[0024] U.S. Patent Application No. 2014 / 0335207, whose disclosure is incorporated herein by reference, describes a local mixture that generates nitric oxide when mixed with a “nitrite medium” and an “acidifying medium.” Specific embodiments of the “nitrite medium” are described individually in paragraphs

[0050] to

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

[0054] and

[0055] contain 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 and butylene glycol. These polyols are also components of the embodiments of the “acidifying medium” described in paragraphs

[0056] to

[0062] .

[0025] U.S. Patent Application No. 2015 / 0030702, whose disclosure is incorporated herein by reference, describes a skin bandage based on a nitrite-acid reaction. The skin bandage comprises a non-thiol reducing agent such as hydroquinone or butylated hydroquinone. The skin bandage may comprise a hydrogel containing a hydrophilic polymer such as polyvinyl alcohol or polyethylene glycol.

[0026] U.S. Patent Application No. 2017 / 0209485, whose disclosure is incorporated herein by reference, describes an apparatus and method 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 decrease vapor pressure is described in paragraph

[0070] .

[0027] U.S. Patent Application No. 2019 / 0134080, whose disclosure 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 combination of multiple parts, including a first solution comprising at least one nitrite reactant and a second solution comprising at least one acidic reactant. Devices for holding, permeating, and distributing the components of the combination as a foam are also described. The use of glycerol as an optional additive to increase surface tension and / or decrease vapor pressure is mentioned (paragraph

[0068] ).

[0028] As mentioned above, the present invention is based on the remarkable discovery that one or more activators selected from nitric oxide (NO), nitric oxide-producing compositions, components or combinations or combinatable aggregates of components of nitric oxide-producing compositions, and mixtures thereof are effective in vitro antibacterial agents against M. tuberculosis, providing effective in vivo treatment (both therapeutic and prophylactic) for tuberculosis in humans and animals, and preventing the transmission of M. tuberculosis and the resulting contamination of surfaces (non-living surfaces, as well as the hands, arms, and other outer surfaces of the human or animal body). Effective antibacterial treatment of spaces (including) is also provided by the present invention.

[0029] In a preferred embodiment of the present invention, nitric oxide can be produced by an NO-producing system comprising a nitrite and a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acids. The organic non-carboxylic acids may be organic non-carboxylic acid reducing acids. Such a system can be embodied in an NO-producing composition that can be administered to the lungs of a patient.

[0030] The NO-generating system may contain one or more organic polyols. If present, the one or more organic polyols may preferably contain sugar alcohols comprising one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0031] The activator, for example, a component or combination of components of a NO-producing composition or a nitric oxide-producing composition, or a combinatorial aggregate thereof, can be delivered to the patient's lungs in any suitable physical form, for example, in liquid form, or in the form of droplets contained in a supported gas or air, for example, as an aerosol or mist.

[0032] According to the present invention, it has been further found that an acid that functions as a proton source for the production of nitric oxide may be effective when buffered to a relatively high pH, ​​for example, about 5 to about 8, for example, about 5.2 or higher, for example, in the range of 5.2 to 5.8, i.e., to a pH physiologically acceptable to the tissues of the patient's mouth, nasal passages, airways, and lungs.

[0033] Nitric oxide and NO-producing compositions, as discussed herein, possess a variety of antibacterial and other beneficial physiological activities, and as a result, the antibacterial activity against M. tuberculosis provided by the present invention may be accompanied by beneficial activity against other pathogens that may infect or to which the patient may be susceptible (including secondary bacterial infections, viral infections, parasitic infections, and fungal infections).

[0034] As reported herein, it has been found in vitro that the antibacterial effect against M. tuberculosis can be enhanced when NO-producing compositions are prepared in a particular manner, namely by one of the following methods: (a) A method for preparing a NOx-generating composition, comprising mixing components of a nitrite, a proton source, and an organic polyol in a desired proportion at a higher concentration than desired in the composition to be used to form a concentrated premixture, and then preferably diluting the concentrated premixture with water to provide the composition to be used. (b) A method for preparing a NOx-generating composition, comprising mixing a nitrite, a proton source, and components of an organic polyol in desired proportions at desired concentrations of the composition to be used, to provide the composition to be used.

[0035] These alternative methods constitute a particular aspect of the present invention.

[0036] Nitric oxide, optionally other oxides of nitrogen, and / or optionally its precursors (collectively referred to as NOx) can be produced more efficiently and with improved reaction power than ever before, using a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids as nitrite oxidizers, in the presence of one or more organic polyols. In addition, it has been found that the antibacterial effective reaction products of such reaction systems using organic reducing acids as nitrite oxidizers can be delivered with or without the use of one or more organic polyols at a physiologically acceptable pH, e.g., about 5 to about 8, making such reaction systems operating at such pH available for direct delivery as compositions with beneficial physiological activity, such as in vivo antibacterial activity. The nitric oxide production method underlying the present invention uses a physiologically effective amount of nitric oxide, optionally other oxides of nitrogen, and / or It has been found that the precursor can be selectively produced for a prolonged period, e.g., over approximately 2 hours, 5 hours, or 10 hours, after the initial strong ejection of NOx gas, leading to potentially significant uses in pharmaceuticals and other applications. If an initial strong ejection is not required, the administration of the reaction mixture to the target can be carried out for a certain period after the start of the NOx production reaction, e.g., for approximately 10 minutes, 30 minutes, or 1 hour or more after the start of the NOx production reaction. [Overview of the project]

[0037] The present invention is a particular embodiment of the more general advantages of the invention as defined in and disclosed in the appended claims. The present invention as defined in and disclosed in the appended claims relates to combinations and compositions in which NO production reactions are carried out, and to applications of the general advantages of the invention relating to the delivery of the gaseous products of the reaction to human or animal subjects via the nose, mouth, respiratory tract, or lungs of the subject. All aspects, examples, embodiments, and priorities described herein in connection with this disclosure are equally and independently applicable to the present invention as defined in and disclosed in the appended claims.

[0038] This disclosure provides systems, methods, combinations, kits and compositions for producing nitric oxide and, optionally, other oxides of nitrogen and / or, optionally, their precursors.

[0039] The systems, methods, combinations, kits, and compositions comprise, as reactants, one or more nitrites 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 comprise one or more organic polyols. The use of reducing acids (i.e., carboxylic acid reducing acids and non-carboxylic acid reducing acids) makes it possible to produce nitric oxide, and optionally other oxides of nitrogen, and / or optionally 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 antibacterial use, wherein one or more organic polyols are optionally used, and the reaction is carried out at a starting pH of the proton source in the range of 5 to 8.

[0040] According to a first aspect, the present disclosure provides a method for producing nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, comprising reacting one or more nitrites with a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, under reaction conditions suitable for producing nitric oxide, optionally other oxides of nitrogen, 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) One or more organic polyols are present in an amount that enhances the reaction output. (b) The proton source is not simply a hydrogel containing pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix. (c) One or more organic polyols are not simply glycerols. (d) If one or more viscosity increasing agents are used, one or more organic polyols must not simply be glycerols. (e) If one or more plasticizers are used, one or more organic polyols must not be simply glycerols. (f) One or more organic polyols are not simply polyvinyl alcohols. (g) If one or more viscosity increasing agents are used, one or more organic polyols must not be simply polyvinyl alcohols. (h) The word "simply not" is replaced by "does not include" in one or more of the above (b) to (g). (i) One or more organic polyols are simply propylene glycol, polyethylene glycol Coal, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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. (j) One or more organic polyols are not propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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 specified 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.

[0041] Nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, prepared by a method according to a first aspect of the present disclosure constitute a second aspect of the present disclosure.

[0042] According to a third aspect, the disclosure provides a method for improving the output of a reaction between one or more nitrites and a proton source to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally their precursors, comprising using a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, and carrying out the reaction in the presence of one or more organic polyols in an amount that improves the reaction output. The improvement in reaction output is compared to a reaction carried out under the same conditions but without the organic polyols.

[0043] According to a fourth aspect, the disclosure provides the use of one or more organic polyols in a reaction mixture to enhance the output of a reaction between one or more nitrites and a proton source in the reaction mixture for producing nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, wherein the proton source comprises one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids. The enhancement of the reaction output is compared to a reaction carried out under the same conditions but without the one or more organic polyols.

[0044] According to a fifth aspect, the present disclosure relates to a combination, kit, or composition for producing nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof by reaction of one or more nitrites with a proton source, wherein the combination, kit, or composition is (i) one or more nitrites, (ii) One or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids Includes a proton source, (iii) Provide a combination, kit, or composition comprising one or more organic polyols characterized by one or more of the following: (a) One or more organic polyols are present in an amount that enhances the reaction output. (b) The proton source is not simply a hydrogel containing pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix. (c) One or more organic polyols are not simply glycerols. (d) If one or more viscosity increasing agents are used, one or more organic polyols must not simply be glycerols. (e) If one or more plasticizers are used, one or more organic polyols must not be simply glycerols. (f) One or more organic polyols are not simply polyvinyl alcohols. (g) If one or more viscosity increasing agents are used, one or more organic polyols must not be simply polyvinyl alcohols. (h) The word "simply not" is replaced by "does not include" in one or more of the above (b) to (g). (i) One or more organic polyols are not simply propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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. (j) One or more organic polyols are not propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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 specified 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.

[0045] If the proton source comprises a hydrogel containing 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 preferable that one or more polyols are not present in the separate compositions in direct contact with or miscible with the hydrogel.

[0046] The chemicals of the combinations, kits, or compositions of the fifth aspect of this disclosure include, for example, the above-mentioned components (i), (ii), and (iii), as well as optionally water and / or p It may essentially consist of H buffer. The expression “essentially consists of” means, for example, that the presence of small amounts of one or more additional components may be permitted, provided that the effects of components (i), (ii) and (iii) described above, as well as optionally water and / or pH buffer, are not adversely affected. The total amount of such one or more additional components may preferably be less than about 20% by weight or volume of the combination, the chemical components of the kit, or the composition, for example 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.

[0047] The chemicals of a combination, kit, or composition may consist of the above-described components (i), (ii), and (iii), as well as optionally water and / or pH buffer and / or one or more additional components in amounts less than about 20% by weight or volume percent of the chemical components of the combination, kit, or composition, e.g., less than about 15% by weight or volume percent, e.g., less than about 10% by weight or volume percent, e.g., less than about 5% by weight or volume percent.

[0048] According to the sixth aspect, this disclosure is: (i) one or more nitrites, (ii) A proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, (iii) comprising one or more organic polyols, The present invention provides a method comprising combining or forming a kit of constituent components (i), (ii), and (iii) in close proximity to each other, or mixing them to form a composition. One or more organic polyols are characterized by one or more of the following: (a) One or more organic polyols are present in an amount that enhances the reaction output. (b) The proton source is not simply a hydrogel containing pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix. (c) One or more organic polyols are not simply glycerols. (d) If one or more viscosity increasing agents are used, one or more organic polyols must not simply be glycerols. (e) If one or more plasticizers are used, one or more organic polyols must not be simply glycerols. (f) One or more organic polyols are not simply polyvinyl alcohols. (g) If one or more viscosity increasing agents are used, one or more organic polyols must not be simply polyvinyl alcohols. (h) The word "simply not" is replaced by "does not include" in one or more of the above (b) to (g). (i) One or more organic polyols are not simply propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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. (j) One or more organic polyols, propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethyl ammonium It shall not contain D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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, caprylic glycol, glycols other than those described 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.

[0049] As used herein, the term “combination” refers to separate substances or compositions (referred to as “components”) that are brought into close proximity and used together. Bringing components into close proximity can be achieved in multiple steps, by which some, but not all, of the components are first combined into a sub-combination or partial combination, which is then brought into close proximity with one or more further components or other sub-combinations or partial combinations. “Proximity” may include close mixtures, solutions, or suspensions, or may indicate approximate physical proximity, not the total volume of a close mixture, solution, or suspension, for example, in separate containers of a kit in which the components are provided together for ease of later use. For example, nitrite components and proton source components, each containing one or more nitrites (or some of them) and one or more acids (or some of them) selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, may be stored separately or in separate containers of a kit and brought together for use by mixing to initiate a NOx production reaction. One or more organic polyols may be provided with one or both of the nitrite component and the proton source component, or separately with the organic polyol component which is also mixed when the NOx production reaction is initiated. One or more of the components themselves may be present in multiple parts and multiple containers. For example, the combination can be brought close together in such a way that the NOx production reaction is initiated immediately, since the nitrite and proton source are in the same solution and are therefore able to react. Alternatively, the combination can be brought close together in such a way that the NOx production reaction is not initiated immediately, but requires one or more further steps or actions before initiation, since, for example, the nitrite and proton source are in a dry powder mixture or exist as encapsulated particles that require water (e.g., from mucous membranes in contact with the combination) before the NOx production reaction is initiated.

[0050] In embodiments, the first to sixth aspects of the present disclosure may be independent of each other and refer to feature (a) only, or feature (b) only, or feature (c) only, or feature (d) only, or feature (e) only, or feature (f) only, or feature (g) only, or feature (h) if only (b) is referred to, or feature (c) only, or feature (h) if only (d) is referred to, or feature (h) if only (e) is referred to, or feature (h) if only (f) is referred to, or feature (h) if only (g) is referred to, or feature (a) and (b) only, or feature (a) and (b) if only (h) is referred to, or feature (h) if only (a) and (b) is referred to, or feature (h) if only (a) and (b) is referred to, or feature (h) If only (a) and (c) or if referring to feature (h), then feature (a) and (c), or if only feature (a) and (d), or if referring to feature (a) and (d), then feature (h), or if only feature (a) and (e), or if referring to feature (a) and (e), then feature (h), or if only feature (a) and (f), or if referring to feature (a) and (f), then feature (h), or if only feature (a) and (g), or if referring to feature (a) and (g), then feature (h), or if only feature (b) and (c), or if referring to feature (b) and (c), then feature (h), or if only feature (b) and (d), or if referring to feature (b) and (d) In cases where feature (h) is mentioned, or feature (b) and (e) alone may be featured, or in cases where feature (b) and (e) are mentioned, feature (h) may be mentioned, or feature (b) and (f) alone may be featured, or in cases where feature (b) and (f) are mentioned, feature (h) may be mentioned, or feature (a), (b), (c), and (f) alone may be featured, or feature (a), (b), (c), and (f) alone may be featured, or feature (h) may be mentioned, or all of feature (a) to (g), or all of feature (c) to (g) may be mentioned, feature (a) and (b) may be featured together with feature (h).

[0051] In other embodiments, the first to sixth aspects of the present invention may be independent of each other, either by referring only to features (c), (f), and (i) above, or only to features (c), (f), and (j), or by referring to features (i) and (h) when referring to features (c) and (f), or by referring to features (j) and (h), or by referring only to features (d), (g), and (i), or by referring only to features (d), (g), and (j) ) alone, or if referring to features (d) and (g), features (i) and (h), or if referring to features (d) and (g), features (j) and (h), or if referring to features (e), (f), and (i) alone, or if referring to features (e), (f), and (j) alone, or if referring to features (e) and (f), features (i) and (h), or if referring to features (e) and (f), features (j) and (h).

[0052] The first to sixth aspects of this disclosure refer to all of (a) to (g), or, if referring to features (c) to (g), features (a) and (b) together with feature (h), or features (c), (f), and (i) only, or features (c), (f), and (j) only, or if referring to features (i) and (h), features (c) and (f), or if referring to features (j) and (h), features (c) and (f), or if referring to features (d), (g), and (i), or features Characterized by (d), (g), and (j) only, or, if referring to features (d) and (g), features (i) and (h), or if referring to features (d) and (g), features (j) and (h), or, if referring to features (e), (f), and (i) only, or if referring to features (e), (f), and (j) only, or if referring to features (e) and (f), features (i) and (h), or if referring to features (e) and (f), features (j) and (h). Features (d), (e), and (g) overlap with features (c) and (f) if they characterize the disclosure, in which case features (d), (e), and (g) (or feature (h) if referring to features (d), (e), and (g)) may be omitted from the list and may be considered examples of the characterizing features (c) and (f) (or feature (h) if referring to features (c) and (f)).

[0053] As used herein, the expression "amount of one or more organic polyols that enhances the reaction output" means that the amount of one or more organic polyols that causes the amount of nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors from the NOx production reaction and / or the output period to be higher than when the reaction is carried out under the same conditions without the use of one or more organic polyols. The expression "amount" means, in particular, the total mass of gaseous nitric oxide produced per gram of nitrite available for reaction in the initial reaction system. Experimental work that forms the basis of the present invention has also measured the amounts of gaseous nitric oxide and optionally other gases produced and found that these are enhanced. Thus, since the total mass of NOx produced is considered to be enhanced by the present invention, the expression "amount" can also be understood to include the total mass of nitric oxide that becomes solution in the reaction mixture and the total mass of NOx reaction products. The expression "output period" means, in particular, the length of time that gaseous nitric oxide, optionally and at least one of the other gases, is produced in the reaction before the reaction is complete. For the same reasons explained above in the consideration of the phrase "an amount that enhances the reaction power of one or more organic polyols," the phrase "power period" is also considered to include the length of time that nitric oxide is dissolved in the reaction mixture, as well as the length of time that NOx reaction products are produced. As is well known, eventually the nitrite is depleted by reaction with the proton source, the pH which rises during the NOx production reaction reaches its maximum value, and the reaction stops. The method of the first aspect of the present invention provides a yield, limiting, NOx production reaction. It is preferable, however, to improve the amount of NO produced, for example, the amount of gaseous NO produced, by at least about 5%, for example, at least about 10%, for example, at least about 25%, for example, by an improvement of up to about 150%, for example, by an improvement of up to about 125%, for example, by an improvement of up to about 100%, for example, by an improvement of up to about 75%. The method of the first aspect of the present invention is preferable to improve the length of time over which at least one of nitric oxide, optionally other oxides of nitrogen, and / or optionally at least one of its precursors, preferably nitric oxide, is produced in the reaction by at least about 5%, for example, at least about 10%, before the reaction is complete. Using the present invention, the period over which nitric oxide, optionally other oxides of nitrogen, and / or optionally at least one of its precursors, preferably nitric oxide, and most preferably gaseous nitric oxide is produced, particularly in an effective amount, can be improved by at least about 2 hours, for example, at least about 5 hours, for example, up to or more than about 10 hours. This degree of improvement in the time required for nitric oxide generation may represent, for example, an improvement of more than 150% or up to approximately 125% of the time required to generate the same amount of nitric oxide without using polyol components, for example, an improvement of up to approximately 100%, for example, an improvement of up to approximately 75%.

[0054] The formation of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors may be for any purpose. Both therapeutic and non-therapeutic purposes are exemplified and discussed below.

[0055] According to the seventh aspect, the Disclosure provides a therapeutic or non-therapeutic method for delivering nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors to a target site, for example, any cell, organ, surface, structure, object, or internal space thereof, wherein the method comprises (a) administering a combination or composition according to the fifth aspect of the Disclosure to or near the target site, or (b) generating nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors using a method according to the first or third aspect of the Disclosure, or by employing the use according to the fourth aspect of the Disclosure, or by using a combination, kit, or composition according to the fifth aspect of the Disclosure, and delivering the nitric oxide, optionally other nitrogen oxides, and / or their precursors thus generated to or near the target site, or (c) delivering nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors to or near the target site according to the second aspect of the Disclosure.

[0056] A method of the seventh aspect of this disclosure could be, for example, a method for treating a bacterial infection in a subject requiring treatment. The subject could be, for example, a human subject or other mammalian subject. The bacterial infection could be, for example, a bacterial infection, a viral infection, a fungal infection, a microparasitic infection, or any combination thereof.

[0057] A method of the seventh aspect of this disclosure may be, for example, a method of vasodilation performed on a subject, which may be, for example, a human subject or other mammalian subject.

[0058] A method of the seventh aspect of this disclosure could be, for example, an antibacterial method. An antibacterial method could reduce the number of bacteria, such as bacteria, viruses, fungal cells, and / or microparasites, in a given location, thereby preventing their growth or limiting their growth rate. Bacteria targeted by such a method could be, for example, planktonic cells or particles, or they could exist as biofilms or other colonies. Any bacterial population targeted by this disclosure, whether planktonic or non-planktonic, could consist of one bacterial species or strain, or could include two or more species or strains.

[0059] According to the eighth aspect, the present disclosure provides for use in treatment, as described in the fifth aspect of the present disclosure. The present disclosure provides combinations, kits, or compositions, or nitric oxide, optionally other nitrogen oxides, and / or their precursors, according to a second aspect of the present disclosure.

[0060] For use according to the eighth aspect of this disclosure, a combination, kit, or composition, or nitric oxide, optionally another nitrogen oxide, and / or optionally its precursor, may be used, for example, in a therapeutic method for delivering nitric oxide, optionally another nitrogen oxide, and / or optionally its precursor to a subject or an internal space therein, wherein the method involves (a) administering a combination or composition according to the fifth aspect of this disclosure to a subject, an internal space therein, or (b) using a method according to the first or third aspect of this disclosure, or according to the fourth aspect of this disclosure. (c) Using the above, or using a combination, kit, or composition according to the fifth aspect of the present disclosure to produce nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, and to deliver the nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors produced thereby to a target, an internal space, or its vicinity, or (c) delivering nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors to a target, an internal space, or its vicinity according to the second aspect of the present disclosure.

[0061] According to this disclosure, it has been surprisingly found that good antibacterial activity in terms of biostatic and biocidal effects, demonstrated by killing up to 100% of M. abscessus after 3 days and / or killing M. tuberculosis, H1N1 influenza virus, SARS-CoV virus, and SARS-CoV-2 virus, is also provided when the proton source is citrate (organic carboxylic acid) or ascorbic acid (organic non-carboxylic acid reducing acid) having an initial pH in the range of 5–8. The expression “initial pH” as herein refers to the pH of the aqueous solution of the proton source initially formed, containing any desired pH buffer, before the presence of other components of the reaction mixture that would affect that initial pH. This antibacterial effect does not depend on the presence of one or more organic polyols, but appears to be enhanced by the presence of one or more organic polyols, e.g., mannitol or sorbitol. The discovery of the potent antibacterial effect of acids (e.g., citric acid or ascorbic acid) from NOx-producing reaction products with an initial pH in the range of 5–8 is particularly remarkable, offering promising applications in the treatment of respiratory and pulmonary infections, including those that are difficult to treat and / or resistant to antibiotics, such as tuberculosis, multidrug-resistant tuberculosis, and non-tuberculous Mycobacterium infections. Treatment of such infections can be proposed via inhalation of an aqueous composition administered by a nebulizer containing the reaction mixture, its components, or its precursors, at a pH in the range of 5–8. Treatment of multiple pathogen infections, potentially including pathogens from two or more of the groups of bacteria, viruses, fungi, and parasites, known as "broad-spectrum" treatments (including therapeutic and / or prophylactic treatments, as well as in vitro treatments of biological and non-biological surfaces and spaces to prevent the spread of pathogens), is also made possible by the present invention.

[0062] According to the ninth aspect, the Disclosure provides a modified version of the antibacterial method according to the seventh aspect, the method comprising: (a) administering a combination or composition according to the fifth aspect of the Disclosure to a target bacterium, its vicinity, an object infected with the bacterium, or the internal space of such an object; or (b) producing nitric oxide, optionally other oxides of nitrogen, and / or optionally its precursors by using a method according to the first or third aspect of the Disclosure, or by performing use according to the fourth aspect of the Disclosure, or by using a combination, kit, or composition according to the fifth aspect of the Disclosure, and delivering the nitric oxide, optionally other oxides of nitrogen, and / or optionally its precursors thus produced to a target bacterium, its vicinity, an object infected with the bacterium, or the internal space of such an object; or (c) nitric oxide, optionally according to the second aspect of the Disclosure The method comprises delivering other oxides of nitrogen, and / or optionally their precursors, to a target bacterium, or its vicinity, or an object infected with the bacterium, or to the internal space of such an object, provided that the initial pH of an aqueous solution of a proton source containing any desired buffer is in the range of 5 to 8, and one or more polyols are optionally and may be omitted, before the presence of other components of the NOx-producing reaction mixture that would affect the pH, or the pH of the reaction mixture at the start of the reaction with one or more nitrites.

[0063] When carrying out the method according to the ninth aspect of the present disclosure, combinations, kits, or compositions according to the fifth or eighth aspect of the present disclosure can be used to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, provided that the initial pH of the aqueous solution of a proton source containing any desired buffer is in the range of 5 to 8 before the presence of other components of the NOx-producing reaction mixture that would affect the pH, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, and that one or more polyols are optionally omitted.

[0064] A method of the ninth aspect of this disclosure may be, for example, a method for treating a bacterial infection in an object requiring treatment. The object may be, for example, a human object or other mammalian object. The bacterial infection may be, for example, a bacterial infection, a viral infection, a fungal infection, a microparasitic infection, or any combination thereof. The bacterial infection may be present on the skin of the object, including mucous membranes. The bacterial infection according to the present invention may be present in the internal space of the object, for example, the nose, mouth, respiratory tract, lungs, or the inner lining of the pulmonary pleura of the object.

[0065] The components and mixtures used in all embodiments of this disclosure, as well as any carriers and excipients administered to the human or animal body, are preferably biocompatible and / or pharmaceutically acceptable in order to minimize tissue irritation and inflammation at the time of administration.

[0066] The combinations, kits, and compositions relating to this disclosure may be stored and used with a variety of suitable apparatus and devices, which will be described in more detail below. The methods relating to this disclosure may preferably be carried out using such apparatus and devices, which will be described in more detail below.

[0067] All embodiments, examples, and priorities specifically described in relation to any one or more aspects of this disclosure are understood to be applicable to any one or more other aspects of this disclosure. In addition, where desired, any method or use in one aspect of this disclosure may be carried out using any combination, kit, or composition in any other aspect. [Modes for carrying out the invention]

[0068] Aspects of this disclosure will be described in detail with reference to specific embodiments. The specific embodiments described below may apply to any of the embodiments of this disclosure unless they are obviously incompatible with such embodiments. Each specific embodiment is also combinable with each other and with all other specific embodiments, unless they are incompatible.

[0069] Nitrites and nitrite components Aspects of this disclosure involve the use of one or more nitrites. Hereinafter, the term “nitrite component” encompasses one or more nitrites themselves, and any component of a reaction system for producing nitric oxide, optionally other oxides of nitrogen, and / or optionally, one or more nitrites as precursors.

[0070] The selection of nitrites is not particularly limited. Specific examples of nitrites that can be used in the compositions of this disclosure include alkali metal nitrites or alkaline earth metal nitrites. In some embodiments, one or more nitrites are selected from LiNO2, NaNO2, KNO2, RbNO2, CsNO2, FrNO2, AgNO2, Be(NO2)2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2, Ra(NO2)2, and any mixture thereof.

[0071] In certain embodiments, the nitrite is NaNO2 or KNO2. In one embodiment, the nitrite is NaNO2.

[0072] In one embodiment, the nitrite component may be provided for use in the Disclosure in a dry form, optionally in a particulate form such as a powder. Where desired, the nitrite component may be encapsulated or microencapsulated, for example, for the purpose of controlling or delaying the reaction between one or more nitrites and a proton source. The dry form and / or encapsulation can assist in the storage of the nitrite component, whether alone or in a mixture with other components of the reaction for producing nitric oxide according to the Disclosure. Furthermore, the dry form and / or encapsulation can assist in the incorporation of the nitrite component into small objects such as medical devices, whether alone or in a mixture with other components of the reaction for producing nitric oxide according to the Disclosure. Such objects include, for example, wound dressings, bandages, vascular stents and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic surgical screws and pins, and other thin medical articles and / or implantable articles, as well as inhalers (handheld and nebulizers). For further details, please refer to the section titled "Optional Encapsulation of Constituent Components (e.g., Microencapsulation)" below.

[0073] If desired, optionally encapsulated or microencapsulated nitrite components may exist as dry powders or crystals, or in association with gels or other carrier systems, such as aqueous gels or solutions thereof. Nitrite components in dry or powder form can be easily dissolved by adding water before use. The molar concentration of nitrite ions in such a nitrite solution before (e.g. immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g. immediately before) acidification, may be in the range of about 0.001 M to about 5 M. In some embodiments, the molar concentration of nitrite ions in the nitrite solution before (e.g. immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g. immediately before) acidification, is in the range of about 0.01 M to about 2 M. In some embodiments, the molar concentration of nitrite ions in the nitrite solution before (e.g. immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g. immediately before) acidification, is in the range of about 0.1 M to about 2 M. In more specific embodiments, the molar concentration of nitrite ions in the nitrite solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g., immediately before) acidification, is in the range of about 0.2 M to about 1.6 M. In embodiments, the molar concentration of nitrite ions in the nitrite solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g., immediately before) acidification, may be in the range of 0.8 to 1.2 M. For example, the molar concentration of nitrite ions in the nitrite solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g., immediately before) combination with organic carboxylic acid components, 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.

[0074] It should be noted that combining two or more precursor solutions of a NOx production reaction mixture will result in 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 two 1M solutions of equal volumes of solutes A and B will result in a change in the concentration of A to 0.5M and a change in the concentration of B to 0.5M. Unless otherwise stated or implied, the concentrations of nitrites described herein are those in the initial solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture, which are added as liquids, e.g., solutions. The actual concentrations in the NOx-producing reaction mixture can be readily derived by knowing the components of the reaction mixture and the method of its preparation.

[0075] If desired, the nitrite components may include one or more polyols, or some of such polyols, whether in a dry form or in a carrier liquid.

[0076] If it is desirable that the nitrite components be stored in a gel or other carrier system, such as an aqueous gel or solution, the system containing the nitrite is preferably buffered to a pH suitable for preventing the decomposition of the nitrite during storage. A pH of about 6 to 9, for example, about 7, is preferred.

[0077] It is preferable that the nitrite components not be brought into contact with the proton source until it is desired to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally their precursors. For this reason, the nitrite components are preferably held in a reservoir or container of the kit, apparatus, or device. However, alternatively, the nitrite components, proton source, and dry components of one or more polyols may be held as a dry composition, e.g., a particulate mixture, and the reaction may be initiated by simply adding water or another suitable solvent or liquid carrier.

[0078] The nitrite may be of a pharmaceutically acceptable grade. In some embodiments, the nitrite is pharmacopoeia grade. In other words, the nitrite may conform to one or more valid pharmacopoeia research papers on nitrites. For example, the nitrite may conform to one or more research papers on nitrites from the United States Pharmacopeia (USP), the European Pharmacopoeia, or the Japanese Pharmacopoeia.

[0079] In certain embodiments, the nitrite used has one or more of the following limitations regarding its characteristics: (i) The nitrite contains approximately 0.02% by weight, approximately 0.01% by weight, or less than approximately 0.001% by weight of sodium carbonate. (ii) The nitrite contains an anti-caking agent such as alkyl-naphthalene sulfonate at a concentration of approximately 10 ppm (0.001% by weight) or less. (iii) Nitrites are white to off-white solids. (iv) Nitrites have positive identification for the cation, as determined according to the relevant methods in the relevant USP. (v) Nitrites have a positive identification test for nitrites, determined according to the relevant methods of the relevant USP. (vi) The nitrite content is optionally determined by relevant USP calorimetry assays, such as ion chromatography combined with a suppressed electrical conductivity detection method, to be approximately 97% or more by weight or 98% or more by weight, and / or 102% or less by weight or 101% or less by weight. (vii) Nitrites, when measured in a 10% solution at 25°C, optionally, when measured according to the relevant USP, and / or using a pH meter, have a pH of about 7 to about 9, or about 8 to about 9. (viii) Nitrites have a drying loss of approximately 0.25% by weight or less or approximately 0.01% by weight. (ix) Nitrites, optionally measured by the Karl Fischer method, have a water content of approximately 0.5% by weight or less. (x) The heavy metal content in nitrites is approximately 10 ppm or less, and is optional. The heavy metal content in nitrites is approximately 10 ppm or less. (xi) The nitrite contains approximately 0.4% by weight or less of nitrate, and optionally, if the nitrite is sodium nitrite, it contains approximately 0.4% by weight or less of sodium nitrate, and if the nitrite is potassium nitrite, it contains approximately 0.4% by weight or less of potassium nitrate. (xii) Nitrites contain approximately 0.005% by weight or less or approximately 0.001% by weight of insoluble substances. (xiii) Nitrites contain approximately 0.005% by weight or less of chloride. (xiv) Nitrites contain approximately 0.01% by weight or less of sulfate. (xv) Nitrites contain iron at a rate of approximately 0.001% by weight or less. (xvi) Nitrite contains approximately 0.01% by weight or less of calcium. (xvii) If the nitrite is not potassium nitrite, the nitrite contains about 0.005% by weight or less or about 0.001% by weight of potassium, and if the nitrite is not sodium nitrite, it contains about 0.005% by weight or less or about 0.001% by weight of sodium. (xviii) Nitrites contain organic volatile compounds in amounts of approximately 0.1% by weight or less, approximately 5000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less. (xix) Nitrite contains ethanol in amounts of approximately 0.1% by weight or less, approximately 5000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less. (xx) Nitrite contains methanol at approximately 3000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less. (xxi) Nitrite contains non-volatile organic carbon in amounts of approximately 50 ppm or less, approximately 25 ppm or less, approximately 20 ppm or less, approximately 10 ppm or less, approximately 7.9 ppm or less, approximately 8 ppm or less, approximately 6 ppm or less, approximately 5.6 ppm or less, or approximately 2.5 ppm or less. (xxii) Nitrite contains mercury at a concentration of approximately 0.05 ppm or less. (xxiii) Nitrite contains approximately 2 ppm or less of aluminum or 0.2 ppm. (xxiv) Nitrites contain approximately 3 ppm or less of arsenic, or 1 ppm. (xxv) Nitrite contains approximately 0.003% by weight or less or 0.001% by weight of selenium. (xxvi) The total aerobic bacterial load in nitrite is approximately 100 CFU / g or less. (xxvii) The total number of yeasts and molds in nitrates is approximately 20 CFU / g or less. (xxviii) Nitrites contain approximately 0.25 EU / mg or less or 0.018 EU / mg of bacterial endotoxin, and (xxix) The nitrite contains less than 0.1 ppm of phosphate such as sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate, and preferably the nitrite does not contain any detectable amount of phosphate.

[0080] In certain embodiments, the nitrite has two or more of the characteristics (i) to (xxix). In further embodiments, the nitrite has five or more of the characteristics (i) to (xxix). In yet further embodiments, the nitrite has ten or more of the characteristics (i) to (xxix). In yet further embodiments, the nitrite has fifteen or more of the characteristics (i) to (xxix). In some embodiments, the nitrite has twenty or more of the characteristics (i) to (xxix). In certain embodiments, the nitrite has all of the characteristics (i) to (xxix). In more specific embodiments, the nitrite is sodium nitrite having all of the characteristics (i) to (xxix).

[0081] In some embodiments, nitrites are optionally determined by the relevant USP calorimetry assay, for example, in combination with an ion-suppressed electrical conductivity detection method. The nitrite content is determined by ion chromatography, such as chromatography, to be in the range of approximately 97% to approximately 101% by weight. In alternative embodiments, the nitrite content is optionally determined by the relevant USP calorimetry assay, such as ion chromatography combined with suppressed conductivity detection, to be in the range of approximately 98% to approximately 102% by weight.

[0082] In certain embodiments, the nitrite has the following characteristics: (i) The nitrite contains approximately 0.02% by weight or less of sodium carbonate. (ii) The nitrite contains a solidification inhibitor of approximately 10 ppm or less. (vi) The nitrites are determined by a USP calorimetry assay to contain 97% or more by weight of nitrites and 101% or less by weight of nitrites. (viii) Nitrites have a drying loss of approximately 0.25% by weight or less. (ix) Nitrites have a water content of approximately 0.5% by weight or less. (x) The heavy metal content in nitrite is approximately 10 ppm or less. (xi) Nitrite contains nitrates of approximately 0.4% by weight or less. (xii) Nitrites contain insoluble substances at a rate of approximately 0.005% by weight or less. (xiii) Nitrites contain approximately 0.005% by weight or less of chloride. (xiv) Nitrites contain approximately 0.01% by weight or less of sulfate. (xv) Nitrites contain iron at a rate of approximately 0.001% by weight or less. (xvi) Nitrite contains approximately 0.01% by weight or less of calcium. (xviii) Nitrites contain organic volatile compounds in amounts of approximately 5000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less. (xxi) Nitrite contains approximately 10 ppm or less of non-volatile organic carbon or approximately 2.5 ppm or less. (xxii) Nitrite contains mercury at a concentration of approximately 0.05 ppm or less. (xxiii) Nitrite contains aluminum at a concentration of approximately 2 ppm or less. (xxiv) Nitrite contains arsenic at a concentration of approximately 3 ppm or less. (xxv) Nitrite contains selenium at a rate of approximately 0.003% by weight or less. (xxvi) The total aerobic bacterial load in nitrite is approximately 100 CFU / g or less. (xxvii) The total number of yeasts and molds in nitrates is approximately 20 CFU / g or less, and (xxviii) Nitrite contains bacterial endodoxins of approximately 0.25 EU / mg or less.

[0083] In these embodiments, the nitrite may be sodium nitrite and may contain about 0.005% by weight or less of potassium. Preferably, 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, as determined according to the relevant methods in the relevant USP. (v) Sodium nitrite has a positive identification test for nitrite, determined according to the relevant methods of the relevant USP. (vii) Sodium nitrite, when measured in a 10% solution at 25°C, optionally, when measured according to the relevant USP, and / or using a pH meter, has a pH of about 7 to about 9, or about 8 to about 9. (xix) Sodium nitrite contains ethanol in amounts of approximately 0.1% by weight or less, approximately 5000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less. (xx) Nitrites containing methanol at approximately 3000 ppm or less, approximately 1000 ppm or less, approximately 500 ppm or less, approximately 100 ppm or less, or approximately 10 ppm or less, and (xxix) The nitrite contains less than 0.1 ppm of phosphate such as sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate, and preferably the nitrite does not contain any detectable amount of phosphate.

[0084] The characteristics of (i) to (xxix) may be determined according to the relevant methods of USP XXXII (2009). Methods for determining the characteristics of (i) to (xxix) are provided in WO2010 / 093746, the disclosure of which is incorporated herein in whole by reference. Methods for preparing sodium nitrite having one or more of the characteristics of (i) to (xxix) are also described in WO2010 / 093746.

[0085] A proton source containing one or more organic carboxylic acids, and components of the proton source. Aspects of this disclosure include a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids. Hereinafter, the term “proton source components” encompasses the proton source itself and any components of a reaction system for producing its precursor containing the proton source, nitric oxide, optionally other oxides of nitrogen, and / or optionally.

[0086] This paragraph provides more detailed examples of organic carboxylic acids.

[0087] In this specification, the term "organic carboxylic acid" refers to any organic acid containing one or more -COOH groups in its molecule. Organic carboxylic acids may be linear or branched. Carboxylic acids may be saturated or unsaturated. Carboxylic acids may be aliphatic or aromatic. Carboxylic acids may be acyclic or cyclic. Carboxylic acids may be vinylogous carboxylic acids.

[0088] Organic carboxylic acids may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids that may be used in this disclosure include α-hydroxycarboxylic acids, β-hydroxycarboxylic acids, and γ-hydroxycarboxylic acids.

[0089] One or more organic carboxylic acids, or each of two or more, should preferably have a pKa1 of less than about 7, more preferably less than 7.0.

[0090] One or more carboxylic acids may be, contain, or consist of one or more reducing carboxylic acids.

[0091] Carboxylic acids can be acid hydrogels containing pendant-COOH groups covalently bonded to polymer molecules forming the three-dimensional polymer matrix of the hydrogel. Examples of such carboxylic acid-containing hydrogels are described, for example, in WO2007 / 007115, WO2008 / 087411, WO2008 / 087408, WO2014 / 188174, and WO2014 / 18175, and the documents referenced therein, all of which are incorporated herein by reference. Such hydrogels typically contain pendant carboxylic acids and sulfonyl groups in acid or salt form covalently bonded to the three-dimensional polymer matrix. For further consideration, see the paragraph under the heading “Other Reservoirs of Components: Hydrogels” below.

[0092] Nevertheless, at least one of one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids is used for polymers or macromolecules, such as hydrogels. It is generally preferable that the polyol(or polyol) is not covalently bonded to the polymer or macromolecule forming a three-dimensional polymer matrix or macromolecular matrix. While we do not wish to be bound by theory, the evidence, for example, the dependence of the effect on stereoisomerism of polyol(or polyol) as discussed below in the paragraph under the heading "Organic Polyols," suggests that the effects of this disclosure on improving the output of a reaction between one or more nitrites and a proton source are achieved, at least in part, by the effect of the organic polyol(or polyol) molecule(or polyol) interacting with the nitrite and proton during the oxidation reaction, implying that the mobility of reactant molecules during the reaction under the influence of polyol molecules may be important. It can be inferred that even in the absence of polyols, as in the eighth aspect of this disclosure, the same mobility between reactants in a reaction between one or more nitrites and a proton source may be important.

[0093] Organic carboxylic acids 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-hydroxypropanoic acid, β-hydroxypropanoic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamo(emboic)ic acid, stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobioic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, their salts, and combinations thereof. In certain embodiments, the organic carboxylic acid is selected from citric acid, its salts, and combinations thereof. In a particular embodiment, the organic carboxylic acid is citric acid or a salt thereof. Carboxylic acids may be polymers or polymerized carboxylic acids, such as polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid, or copolymers of lactic acid and glycolic acid, or may include such polymers or polymers. The term “organic carboxylic acid” as used herein also encompasses partial or complete esters or partial or complete salts thereof of organic carboxylic acids, provided that they can function as proton sources in the uses according to the present invention.

[0094] It is preferable to buffer the pH of the proton source immediately before contact between one or more nitrites and the proton source to control the pH within a known range and limit the rate at which the pH increases as the nitrites are consumed. For further details, see the section under the heading "pH Control, Optional Buffer Systems" below. In particular, it is assumed that at least one organic carboxylic acid of the proton source may preferably be present with its conjugate base. The acid and its conjugate base may preferably form a buffer in an aqueous carrier. Thereafter, the buffer can be selected so that the pH is maintained at a desired level, preferably about 3 to 9, for example, in the range of about 4 to 8, physiological contact, or in the range of about 5 to about 8, which is preferable for contact with living cells and organisms, as the NOx generation reaction proceeds. 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 a mineral acid and / or mineral base.

[0095] The initial pH of an aqueous solution of a proton source containing any desired buffer, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, before (e.g., immediately before) the addition of other components of the NOx-producing reaction mixture that would affect the pH, is preferably in the range of about 3 to 9, e.g., about 4 to 8, e.g., about 5 to 8. As used herein in relation to the proton source, the expression “initial pH” means the pH of an aqueous solution of a proton source containing any desired buffer, before (e.g., immediately before) the addition of other components of the NOx-producing reaction mixture (including some but not all components) that would affect the pH. A dry powder proton source material or other precursor of the aqueous solution of the proton source would be used in an appropriate amount that would produce an aqueous solution having the desired initial pH.

[0096] If it is desirable that the proton source components be stored in a gel or other support system, such as an aqueous gel or solution, the system containing the proton source is preferably buffered to a pH suitable for preventing acidification during storage and preventing the decomposition of the proton source. A pH of about 3 to 6, for example, about 3 to 5, is preferred. If desired, the pH can be increased by adding a base immediately before use of the proton source components.

[0097] For example, some patients have intolerance to citrate. Patients should be tested for potential acid intolerance before treatment, and the acid components should be selected accordingly.

[0098] In one embodiment, the proton source component or portion thereof may be provided for use in the present disclosure in a dry form, optionally in a particulate form such as a powder. Where desired, the proton source component or portion thereof may be encapsulated or microencapsulated for the purpose of controlling or delaying the reaction between one or more nitrites and the proton source, for example. The encapsulated form may be particularly useful if the proton source is normally in a liquid or gel state at room temperature. The dry form and / or encapsulation can assist in the storage of the proton source component, whether alone or in a mixture with other components of the reaction to produce nitric oxide according to the present disclosure. Furthermore, the dry form and / or encapsulation can assist in the incorporation of the proton source component into small objects such as medical devices, whether alone or in a mixture with other components of the reaction to produce nitric oxide according to the present disclosure. Examples of such objects include wound dressings, bandages, vascular stents and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic surgical screws and pins, and other thin medical articles and / or implantable articles. For further details, see the section under the heading "Optional Encapsulation of Components (e.g., Microencapsulation)" below.

[0099] If desired, one or more organic carboxylic acids, optionally encapsulated or microencapsulated, may be present in the proton source component as a dry powder or crystal, or in association with an aqueous carrier, such as a gel or other carrier system, such as an aqueous gel or a solution thereof. Proton source components containing organic carboxylic acids in dry or powder form can be easily dissolved by adding water before use. The molar concentration of the total proton source in such solution (including any organic non-carboxylic acid reducing acids present) before (e.g. immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g. immediately before) the start of the reaction with nitrite, may be in the range of about 0.001 M to about 5 M. In some embodiments, the molar concentration of the total proton source in such solution before (e.g. immediately before) the addition of any other components of the NOx-producing reaction mixture, particularly before (e.g. immediately before) the start of the reaction with nitrite, may be in the range of about 0.01 M to about 2 M. In some embodiments, the molar concentration of the total proton source in such a solution before the start of the reaction with nitrite is in the range of about 0.1 M to about 2 M. In more specific embodiments, the molar concentration of the total proton source in such a solution before the start of the reaction with nitrite is in the range of about 0.2 M to about 1.6 M. In embodiments, the molar concentration of the total proton source in such a solution before the start of the reaction with nitrite may be in the range of 0.8 to 1.2 M. For example, the molar concentration of the total proton source in such a solution before the start 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.

[0100] The terms "molar concentration of total proton source" and "concentration of total proton source" used herein refer to protons (H + ) Donor portion or (if there are two or more) multiple protons ( +) should be understood to mean the concentration of any of the organic carboxylic acids and / or organic non-carboxylic acids used as proton sources in accordance with the present invention at a pH at which at least one of the donor parts is preferentially protonated, i.e., more than 50% on a molar basis is protonated. In other words, before the initiation of the NOx production reaction, the pH is higher If the pH is adjusted, thereby reducing the degree of protonation, the molar concentration or concentration of the total proton source is not considered to have decreased accordingly.

[0101] It should be noted that combining two or more precursor solutions of a NOx-producing reaction mixture will result in 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 two 1 M solutions of equal volumes of solutes A and B will result in a change in the concentration of A to 0.5 M and a change in the concentration of B to 0.5 M. Unless otherwise stated or implied, the concentrations of proton sources described herein are those in the initial solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture added as a liquid, e.g., a solution. The actual concentrations in the NOx-producing reaction mixture can be readily derived by knowing the components of the reaction mixture and how they are prepared.

[0102] Proton source components in dry or powder form can be easily dissolved by adding water before use.

[0103] If desired, one or more organic carboxylic acids may be present in a mixture or solution with one or more polyols or some of such polyols, whether in a dry form or in a carrier liquid.

[0104] It is preferable that the nitrite components are not brought into reactive contact with the proton source until it is desired to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally their precursors. For this reason, the proton source components or a portion thereof are preferably held in a reservoir or container of the kit, apparatus, or device. However, alternatively, it may be possible to hold one or more nitrites, or the dry components of the nitrite components, proton source, and one or more polyols, as a dry composition, e.g., a particulate mixture, and to initiate the reaction by simply adding water or another suitable solvent or liquid carrier.

[0105] Proton source components containing one or more organic non-carboxylic acid reducing acids The above considerations for proton source components containing or consisting of one or more organic carboxylic acids apply similarly to proton source components containing or consisting of one or more organic non-carboxylic acid reducing acids. This paragraph provides more detailed examples of organic non-carboxylic acid reducing acids.

[0106] In this specification, the term "organic non-carboxylic acid reducing acid" refers to any organic reducing acid that does not contain a -COOH group in its molecule. Organic non-carboxylic acid reducing acids may be linear or branched. Non-carboxylic acid reducing acids may be saturated or unsaturated. Non-carboxylic acid reducing acids may be aliphatic or aromatic. Non-carboxylic acid reducing acids may be acyclic or cyclic. Non-carboxylic acid reducing acids may be vinylogases.

[0107] One or more organic non-carboxylic acid reducing acids, or each of two or more, should preferably have a pKa1 of less than about 7, more preferably less than 7.0.

[0108] For the reasons described above, it is generally preferable 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 polymer molecules, such as polymer molecules forming the three-dimensional polymer matrix of a hydrogel.

[0109] Organic non-carboxylic acid reducing acids include, for example, ascorbic acid; ascorbic acid palmitic acid (ascorbyl palmitate); 3-O-ethyl ascorbic acid; and other 3-alkyl ascorbic acid. Ascorbic acid, ascorbate derivatives such as 6-O-octanoylascorbic acid, 6-O-dodecanoylascorbic acid, 6-O-tetradecanoylascorbic acid, 6-O-octadecanoylascorbic acid, and 6-O-dodecanedioylascorbic acid; acidic reductones such as reducing acids; erythorbic acid; oxalic acid; salts thereof; and combinations thereof may be selected. In a particular embodiment, the organic noncarboxylic acid reducing acid is ascorbic acid or a salt thereof.

[0110] Organic non-carboxylic acid reducing acids may support one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic acid reducing acids that may be used in this disclosure include acidic reductones, such as the reducing acid (2,3-dihydroxy-2-cyclopentanone).

[0111] It is preferable to buffer the pH of the proton source and / or reaction mixture after contact between one or more nitrites and the proton source to control the pH within a known range and to control the increase in pH as the nitrites are consumed. For further details, see the paragraph under the heading "pH Control, Optional Buffer Systems" below. In particular, it is assumed that at least one organic non-carboxylic acid reducing acid of the proton source may be present, preferably together with its conjugate base. The acid and its conjugate base may preferably form a buffer in an aqueous carrier. Thereafter, the buffer may be selected so that the pH is maintained at a desired pH, preferably about 3 to 9, for example, in the range of about 4 to 8, physiological contact, or in the range of about 5 to about 8, which is preferable for contact with living cells and organisms, as the NO production reaction proceeds. 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 a mineral acid and / or mineral base.

[0112] The initial pH of the aqueous solution of the proton source containing any desired buffer, before (e.g., immediately before) the addition of other components of the NOx-producing reaction mixture that would affect the pH, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, is preferably in the range of about 3 to 9, for example, about 4 to 8, for example, about 5 to 8. The dry powder proton source material or other precursor of the aqueous solution of the proton source will be used in an appropriate amount that will produce an aqueous solution having the desired initial pH.

[0113] If it is desirable that the proton source components be stored in a gel or other support system, such as an aqueous gel or solution, the system containing the proton source is preferably buffered to a pH suitable for preventing acidification during storage and preventing the decomposition of the proton source. A pH of about 3 to 6, for example, about 3 to 5, is preferred. If desired, the pH can be increased by adding a base immediately before use of the proton source components.

[0114] Some reducing acids, such as oxalic acid, are toxic. The components of an acid should be selected accordingly.

[0115] One or more organic non-carboxylic acid reducing acids may be used in addition to or instead of one or more organic carboxylic acids in the proton source components in the manner described above. For further details, see the paragraph with the heading "Proton Sources Containing One or More Organic Carboxylic Acids, and Proton Source Components."

[0116] Organic polyols and organic polyol components Aspects of this disclosure include one or more organic polyols. Hereinafter, the terms “organic polyol component” or “polyol component” encompass the organic polyol itself and any component of a reaction system for producing its precursor containing the organic polyol, nitric oxide, optionally other oxides of nitrogen, and / or optionally.

[0117] In this specification, the term "organic polyol" refers to an organic molecule that is not a proton source and is not a sugar or polysaccharide (examples of "sugars" and "polysaccharides" include oligosaccharides, glycans, and glycosaminoglycans), and in particular has two or more hydroxyl groups for nitrite reactions. Therefore, organic polyols will have a pKa1 of about 7 or more, for example, 7.0 or more.

[0118] In this specification, the term “organic polyol” preferably excludes reducing agents. Therefore, in all embodiments of one embodiment, the organic polyol excludes reducing agents. Examples of reducing agents that are organic molecules having two or more hydroxyl groups and are not sugars or polysaccharides include thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate, erythorbic acid, and erythorbate. Therefore, thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate, and erythorbate are preferably excluded from the term “organic polyol” because they are reducing agents. In any case, ascorbic acid and erythorbic acid are excluded from the term because they are proton sources, particularly for nitrite reactions. To avoid any doubt, we confirm that reducing agents that are proton sources, such as ascorbic acid and / or erythorbic acid, are not excluded from the proton sources of the present invention, or from the proton source components, combinations, kits, compositions, uses, methods, or any other parts of the present invention, and the means of practical application in which they exist as proton sources.

[0119] Organic polyols may be cyclic or acyclic, or a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, 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 polyols do not support any substituents other than OH groups.

[0120] Preferably, one or more organic polyols are one or more acyclic organic polyols. Preferred one or more acyclic organic polyols are selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferred one or more acyclic organic polyols are selected from algitols, for example, algitols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferably, one or more organic polyols do not contain saponins, sapogenins, steroids, or steroidal glycosides.

[0121] Alternatively, one or more organic polyols may be one or more cyclic organic polyols. In these embodiments, one or more cyclic organic polyols may be cyclic sugar alcohols or cyclic alditols. For example, 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.

[0122] In some embodiments, one or more organic polyols have seven or more hydroxyl groups. In certain embodiments, one or more organic polyols are sugar alcohols or algitols having seven or more hydroxyl groups. In more specific embodiments, one or more organic polyols have nine or more hydroxyl groups. In further embodiments, one or more organic polyols are sugar alcohols or algitols having nine or more hydroxyl groups. In some embodiments, one or more organic polyols have 20 or fewer hydroxyl groups. It has groups. In certain embodiments, one or more organic polyols are sugar alcohols or alditols having 20 or fewer hydroxyl groups. In more specific embodiments, one or more organic polyols have 15 or fewer hydroxyl groups. In further embodiments, one or more organic polyols are sugar alcohols or alditols having 15 or fewer hydroxyl groups. One or more organic polyols may have a number of hydroxyl groups in the range of 7 to 20, more specifically in the range of 9 to 15. In certain embodiments, one or more organic polyols contain 9, 12, 15, or 18 hydroxyl groups.

[0123] Preferably, one or more organic polyols are, for example, sugar alcohol compounds comprising one or more monosaccharide units and one or more acyclic sugar alcohol units. One or more organic polyols may be, for example, sugar alcohol compounds comprising a linear chain of one or more monosaccharide units and one or more acyclic sugar alcohol units, or a branched chain of one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0124] As used herein, a monosaccharide unit refers to a monosaccharide covalently bonded to at least one other unit in a compound (whether another monosaccharide unit or an acyclic sugar alcohol unit). As used herein, an acyclic sugar alcohol unit refers to an acyclic sugar alcohol covalently bonded to at least one other unit in a compound (whether another monosaccharide unit or an acyclic sugar alcohol unit). Units in a compound may be linked via ether bonds. In some embodiments, one or more monosaccharide units are covalently bonded to other units of the compound via glycosidic bonds. In certain embodiments, each monosaccharide unit is covalently bonded to other units of the compound via glycosidic bonds. In certain embodiments, the sugar alcohol compound is a glycoside having a monosaccharide, or an oligosaccharide glycone, and an acyclic sugar alcohol aglycone.

[0125] Preferred acyclic sugar alcohol units are those having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. In certain embodiments, the acyclic sugar alcohol units are selected from the group consisting of erythritol, sreitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, and boremitol units.

[0126] 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 specific embodiments, each monosaccharide unit is a C5 or C6 monosaccharide unit. In certain embodiments, one or more of the sugar alcohol units are C5 or C6 sugar alcohol units. In more specific embodiments, each sugar alcohol unit is a C5 or C6 sugar alcohol unit.

[0127] In certain embodiments, the sugar alcohol compound comprises n monosaccharide units and m acyclic sugar alcohol units, for example, consisting of such units, where n is an integer and at least 1, m is an integer and at least 1, and (n+m) is 10 or less. In certain embodiments, the sugar alcohol compound comprises a chain of n monosaccharide units terminated by one acyclic sugar alcohol unit, for example, consisting of such units, where n is an integer from 1 to 9. In these embodiments, the chain of monosaccharide units may be covalently bonded by glycosidic bonds. In certain embodiments, each monosaccharide unit is covalently bonded to another monosaccharide unit or acyclic sugar alcohol unit by glycosidic bonds. In certain embodiments, the sugar alcohol compound comprises a chain of 1, 2, or 3 monosaccharide units terminated by one acyclic alcohol unit, for example, consisting of such units, where 1, 2, 3, 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); Examples include maltotriitol (n=2) and maltotetraitol (n=3).

[0128] Such sugar alcohol compounds may be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, oligosaccharides refer to sugars consisting of 3 to 10 monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides may 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 may be formed from the dehydration reaction of monosaccharides and sugar alcohols. 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, maltotetraitol.

[0129] Suitable organic polyols may include erythritol, slayitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boremitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and any combination thereof. If used and present, glycerol is preferably associated with one or more other organic polyols, such as erythritol, slayitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, boremitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, or any combination thereof.

[0130] Many organic polyols contain one or more chiral centers and therefore exist in stereoisomeric forms. All stereoisomeric forms, optical isomers, and isomeric mixtures of organic polyols are intended to be included within the scope of this disclosure and patent. In particular, the D and / or L forms of all chiral organic polyols, as well as all mixtures thereof, may be used.

[0131] Interestingly, the effect of using polyols in this disclosure has been found to depend on stereochemistry. Therefore, the selection of one or more optical isomers or mixtures of optical isomers of organic polyols for use in this disclosure may affect the outcome of the reaction between the nitrite and the proton source, at least in terms of the amount of NO produced.

[0132] For example, sorbitol is a stereoisomer of mannitol that differs from each other in the orientation of one hydroxyl group. As shown in Examples 2D and 2E below (Figures 5 and 6), the effects of sorbitol and mannitol on the output of the reaction between the nitrite and the proton source differ in the same reaction system.

[0133] In certain embodiments, the organic polyol is selected from the group consisting of arabitol, xylitol, mannitol, sorbitol, and any combination thereof. Arabitol may be D or L arabitol, or a mixture thereof. Xylitol may be D or L xylitol, or a mixture thereof. Sorbitol may be D or L sorbitol, or a mixture thereof. Mannitol may be D or L mannitol, or a mixture thereof.

[0134] In certain embodiments, one or more polyols are sugar alcohol compounds comprising, for example, one or more monosaccharide units and one or more acyclic sugar alcohol units (including sugar alcohols derived from disaccharides or oligosaccharides) as described herein, and the system described herein When used in methods, combinations, kits, and compositions, they are intended for use in antibacterial methods for treating tuberculosis bacteria or reducing the number of tuberculosis bacteria, or for treatment or methods.

[0135] In one embodiment, the organic polyol component may be provided for use in the Disclosure in a dry form, optionally in a particulate form such as a powder. Where desired, the organic polyol may be encapsulated or microencapsulated for the purpose of controlling or delaying the involvement of the polyol in a reaction between one or more nitrites and a proton source, for example. The encapsulated form may be particularly useful if the organic polyol is normally in a liquid or gel state at room temperature. The dry form and / or encapsulation can aid in the storage of the organic polyol component, whether alone or in a mixture with other components of the reaction to produce nitric oxide according to the Disclosure. Furthermore, the dry form and / or encapsulation can aid in the incorporation of the organic polyol component into small objects such as medical devices, whether alone or in a mixture with other components of the reaction to produce nitric oxide according to the Disclosure. Examples of such objects include wound dressings, bandages, vascular stents and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic surgical screws and pins, and other thin medical articles and / or implantable articles. For further details, see the section under the heading "Optional Encapsulation of Components (e.g., Microencapsulation)" below.

[0136] Alternatively, the organic polyol components may include a support medium, such as an aqueous support liquid or a gel support. If the organic polyol is normally liquid at room temperature, it may be used on its own without any additional support components, or it may be used with one or more support additives, such as a misculation with water.

[0137] If desired, one or more organic polyols, optionally encapsulated or microencapsulated, may be present in the polyol component as a dry powder or crystal, or in association with a gel or other support system, such as an aqueous gel or solution thereof. Polyol components containing organic polyols in dry or powder form can be easily dissolved by adding water before use. The total molar concentration of one or more polyols in such a solution before the start of the reaction with nitrite can be any concentration up to the saturation limit of the polyol, or of each polyol in the solution. For example, the total molar concentration of one or more polyols may be in the range of about 0.001 M to about 5 M. In some embodiments, the total molar concentration of one or more polyols in such a solution before the start of the reaction with nitrite is in the range of about 0.01 M to about 2 M. In some embodiments, the total molar concentration of one or more polyols in such a solution before the start of the reaction with nitrite is in the range of about 0.1 M to about 2 M. In more specific embodiments, the total molar concentration of one or more polyols in such a solution before the start of the reaction with nitrite is in the range of about 0.2 M to about 1.6 M. In embodiments, the total molar concentration of one or more polyols in such a solution before the start of the reaction with nitrite may be in the range of 0.8 to 1.2 M. For example, the total molar concentration of one or more polyols in such a solution before the start 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.

[0138] It should be noted that combining two or more precursor solutions of a NOx production reaction mixture will result in 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 two 1 M solutions of equal volumes of solutes A and B will result in a change in the concentration of A to 0.5 M and a change in the concentration of B to 0.5 M. Unless otherwise stated or implied, the concentrations of organic polyols described herein are before (e.g., immediately before) the addition of any other components of the NOx production reaction mixture added as a liquid, e.g., a solution. This is the concentration in the initial solution. The actual concentration in the NOx-producing reaction mixture can be easily derived by knowing the components of the reaction mixture and how it was prepared.

[0139] Polyol components in dry or powder form can be easily prepared into a solution by adding water before use.

[0140] If desired, the polyol may be present in a mixture or solution with one or more nitrites or proton sources, or with some of such proton sources, whether in a dry form or in a carrier liquid.

[0141] In certain embodiments, the nitrite is retained by separating it from other components of the reaction to produce nitric oxide before use, and the nitrite component may contain one or more polyols. In these embodiments, the organic carboxylic acid component may be substantially free of polyols. In alternative embodiments, the organic carboxylic acid component contains one or more polyols. In these embodiments, the nitrite component may be substantially free of polyols. In further embodiments, the organic carboxylic acid component and the nitrite component may each contain one or more polyols, which may be the same or different between the two components.

[0142] In another embodiment, the organic carboxylic acid components and nitrite components may be substantially free of polyols, and one or more polyols may be contained in separate polyol components.

[0143] Relative concentrations of nitrite, proton source, and any polyol in the reaction mixture The total molar concentration of one or more organic polyols in the polyol components or reaction solution at the start (or before) of the NOx generation reaction is preferably about 0.05 to about 3 times the total molar concentration of nitrite ions, for example, about 0.1 to about 2 times, for example, about 0.25 to about 1.5 times, for example, about 0.3 to about 1.2 times the total molar concentration of nitrite ions in the nitrite components or reaction solution. The same relative molar concentrations between one or more organic polyols and nitrite ions are preferably provided in the components of the combination or kit according to the present invention, or in the composition according to the present invention, before the start of the NOx generation reaction (e.g., immediately before the start).

[0144] The total molar concentration of one or more organic polyols in the polyol components or reaction solution at the start (or before) of the NOx generation reaction is preferably 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 components or reaction solution. The same relative molar concentration between one or more organic polyols and the proton source is preferably provided in the components of the combination or kit according to the present invention, or in the composition according to the present invention, before the start of the NOx generation reaction (e.g., immediately before the start).

[0145] Optional additional components The combinations, kits, or compositions for use in this disclosure may be incorporated into a variety of diluents, carriers, and excipients, and / or may be provided in association with one or more additional components, specific functional components, intended to provide one or more particular benefits to the combinations, kits, or compositions in which they are used. Such diluents, carriers, excipients, and / or additional components would generally be physiologically compatible if in vivo use is desired.

[0146] 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, and croscarmellose sodium. Examples include sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, and calcium chloride.

[0147] Generally, depending on the intended mode of administration, the pharmaceutical formulation will contain about 0.005% to about 95% by weight, preferably about 0.5% to about 50% by weight, of the combination or composition of the present invention, or its components. Practical methods for preparing such dosage forms will be known or obvious to those skilled in the art.

[0148] Excipients may be selected from known excipients depending on the intended use or route of administration, thereby delivering reactants and / or reaction products to the target site for the delivery of nitric oxide, optionally other nitrogen oxides, and / or their precursors. For example, creams, lotions, and ointments may be formulated by incorporating nitrites into excipients such as cream, lotion, and ointment bases, or other thickeners and viscosifiers (e.g., Eudragit L100, Carbopol, carboxymethylcellulose, or hydroxymethylcellulose). Proton sources may be incorporated into excipients selected from Carbopol, carboxymethylcellulose, hydroxymethylcellulose, and methylcellulose, or into aqueous bases. If film formation is desired, film-forming excipients such as propylene glycol, polyvinylpyrrolidone (povidone), gelatin, guar gum, and shellac may be used.

[0149] Optional additional components may be selected from, for example, sweeteners, flavoring agents, thickeners, viscosities, wetting agents, lubricants, binders, film-forming agents, emulsifiers, solubilizers, stabilizers, colorants, odorants, salts, coating agents, antioxidants, pharmaceutically active agents, and preservatives. Such components are well known in the art, and a detailed discussion of them is unnecessary 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, and triethanolamine oleate. Examples of sweeteners or flavoring agents include sugars, saccharin, aspartame, sucralose, neotame, or other compounds that have beneficial effects on taste, aftertaste, unpleasant saltiness, sourness, or bitterness, and reduce the tendency to irritate recipients of oral or inhaled formulations (for example, by causing cough or sore throat, or other undesirable side effects such as reducing the dose delivered or negatively affecting patient compliance with the prescribed treatment regimen). Certain flavoring agents may form complexes with one or more nitrites. Examples of thickeners, viscosities, and film-forming agents are provided above.

[0150] The selection of pharmaceutically active agents and other additional components, such as those functioning as diluents, carriers, and excipients, may be determined by their suitability for the treatment regimen of the relevant disease or medical condition, as well as the desired route of administration of the combination or composition according to this disclosure. Martindale, 39 th Edition(2017), the Merck Index,15 th Edition (2013), Goodman & Gilman's “The Pharmacological Basis of Therapeutics”, 13 thEdition(2017), the British National Formulary on-line(https: / / bnf.nice.org.uk / ), Remington: “The Science & Practice of Pharmacy”, 22 nd Edition(2012), or the Physician’s Desk Reference, 71 st Edition(2017), etc. Standard references may be referred to.

[0151] Examples of administration routes by which the components and compositions according to the present disclosure can be administered to an animal (including a human) subject for therapeutic purposes include topical (e.g., cream, lotion, gel, ointment, paste, emollient, spray), ear canal, nose (e.g., spray), vagina, rectum (e.g., suppository), oral (e.g., mist, spray, mouthwash, aerosol), enteral (e.g., tablet, troche, lozenge, capsule, syrup, elixir), and parenteral (e.g., injection solution), eye, ear, nose, or throat (e.g., drops), or via the respiratory or lung (e.g., mist, aerosol, powder inhalation).

[0152] Examples of pharmaceutically active agents that may be incorporated into or administered concurrently with the components and compositions of this disclosure include antibiotics, steroids, anesthetics (e.g., local anesthetics such as lignocaine (lidocaine), amesocaine (tetracaine), xylocaine, bupivacaine, prilocaine, ropivfacaine, benzocaine, mepivocaine, cocaine, or any combination thereof), analgesics, anti-inflammatory agents (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs)), anti-infective agents, vaccines, immunosuppressants, anticonvulsants, antidementia drugs, prostaglandins, antipyretics, anticycotics, antipsoriasis agents, antivirals, vasodilators or vasoconstrictors, sunscreen preparations (e.g., PABA), antihistamines, hormones such as estrogen, progesterone, or androgens, and antiseborrhea agents. Examples include cardiovascular agents such as alpha or beta blockers, or rogaine, vitamins, emollients, enzymes, mast cell stabilizers, scabies insecticides, lice insecticides, keratolytics, lubricants, narcotics, shampoos, anti-acne preparations, burn treatment preparations, cleansers, deodorants, bleaching agents, diaper (diaper) eczema treatment products, emollients, moisturizers, photosensitizers, poison ivy or American poison ivy or Smack products, sunburn treatment preparations, proteins, peptides, proteoglycans, nucleotides, oligonucleotides (DNA, RNA, etc.), minerals, growth factors, tar-containing preparations, honey-containing preparations (e.g., preparations containing Manuka honey), wart treatment preparations, poultices, wound care products, or any combination thereof.

[0153] Specific examples include analgesics such as ibuprofen, indomethacin, diclofenac, acetylsalicylic acid, paracetamol, propranolol, metoprolol, and oxycodone; thyroid-releasing hormone; sex hormones such as estrogen, progesterone, and testosterone; insulin; verapamil; vasopressin; hydrocortisone; scopolamine; nitroglycerin; and isosorbide dinitrate. Examples include dintirates; antihistamines such as terfenadine; clonidine; nicotine; nonsteroidal immunosuppressants such as cyclosporine, methothotrexate, azathioprine, mycophenolate, cyclophosphamide, TNF-α antagonists, and anti-IL5, -IL4Ra, -IL6, -IL13, -IL17, -IL23 cytokine monoclonal antibodies; anticonvulsants; and drugs for Alzheimer's disease, dementia, and / or Parkinson's disease such as apamorphine, apolamine, and rivastigmine. Where desired, any of the optional additional components may be encapsulated or microencapsulated, for example, to control or delay their release. For further details, see the paragraph under the heading “Optional Encapsulation of Components (e.g., Microencapsulation)” below.

[0154] Selective encapsulation of constituent components (e.g., microencapsulation) At least some of the components of the combinations, kits, and compositions used in this disclosure may be encapsulated, for example, microencapsulated.

[0155] The use of microencapsulated components for NO production provides the long-term production of relatively unstable compounds (such as NO) from precursors that are in a chemically stable form. It is useful. Multiple microencapsulated reactants and / or one or more optional additional components can be readily mixed and stored in contact with each other in a dry environment, and NO generation can be initiated simply by providing 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., the skin, mucous membrane surface, or, according to the present invention, to the nose, mouth, respiratory tract, and / or lungs of the subject, where the physiological environment itself provides enough water to trigger the release of therapeutic amounts of NO. A further advantage is that because the volume occupied by the microencapsulated reactants and / or one or more optional additional components is relatively small, they can be readily incorporated into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, vascular stents and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic surgical screws and pins, and other thin medical articles and / or implantable articles.

[0156] One example of a production method for encapsulating or microencapsulating reactants and / or one or more optional additional components is to spray-dry 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 may also be used, such as pan coating, air suspension coating, centrifugal extrusion, fiber spinning, fiber extrusion, nozzle vibration, ionotropic gelation, coacervate phase separation, interfacial crosslinking, in-situ polymerization, and matrix polymerization. The encapsulated polymer is preferably biocompatible. Examples of such polymers include natural polymers such as ethylcellulose, zein (prolamin seed storage protein found in certain grass species including maize and corn), chitosan, hyaluronic acid, and alginic acid, or biodegradable polyesters, polyanhydrides, poly(orthoesters), polyphosphazenes, or polysaccharides (see Park et al, Molecules 10 (2005), pages 141-161). Compositions in which a single chemical substance is microencapsulated as shown above for the delivery of pharmaceuticals and other drugs are well known. See U.S. Patent No. 4,130,639 by Shalaby and Jamiolkowski and U.S. Patent No. 6,491,748 by Buchholz and Meduski. However, in virtually all such compositions, it is the therapeutic agent that is microencapsulated, and the therapeutic agent is not produced by the reaction of the microencapsulated reagent. However, appropriate modifications to the teachings of the prior art will be within the scope of the art of those skilled in the art. Nitric oxide-releasing polymers for medical articles containing NO adducts / donors have been described. See, for example, U.S. Patent No. 7,829,553 by Arnold (carbon-based diazeniumdiolates attached to hydrophobic polymers) and U.S. Patent No. 7,135,189 by Knapp (a nitrosothiol precursor and a nitric oxide donor).

[0157] pH control, optional buffer systems The composition may have a controlled pH value. In particular, the composition may have a pH value in the range of 3.0 to 8.0, or more specifically, in the range of 4.0 to 8.0. In a more specific embodiment, the composition may have a pH value in the range of 4.0 to 7.4. In an even more specific embodiment, 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.

[0158] The pH of the composition can be controlled in any known manner. In certain embodiments, the pH of the organic carboxylic acid component or the organic reducing acid component is controlled before combination with the nitrite component. In some embodiments, the pH of the organic carboxylic acid component or the organic reducing acid The components include a buffer solution. The buffer solution may be a pharmacologically acceptable buffer solution, such as a phosphate buffer.

[0159] In some embodiments, the buffer is formed by mixing an organic carboxylic acid or an organic non-carboxylic acid reducing acid and their salt counterparts. For example, the organic carboxylic acid component may include an organic carboxylic acid and a salt of the organic carboxylic acid. The organic non-carboxylic acid reducing acid component may include an organic non-carboxylic acid reducing acid and a salt of the organic non-carboxylic acid reducing acid. In certain embodiments, the organic carboxylic acid component includes citric acid and a citrate. In other embodiments, the organic carboxylic acid component or organic reducing acid component includes ascorbic acid and an ascorbate. In some embodiments, the organic carboxylic acid component includes an organic carboxylic acid and a salt of a further organic acid. For example, the organic carboxylic acid component may include citric acid and an ascorbate. In even further embodiments, the organic carboxylic acid component may include an organic carboxylic acid, a salt of the organic carboxylic acid, and a salt of a further organic carboxylic acid. For example, the organic carboxylic acid component may include citric acid, a citrate, and an ascorbate.

[0160] In other embodiments, the buffer is formed by adjusting the pH of an organic carboxylic acid or organic non-carboxylic acid reducing acid so that the acid (protonated form) is miscible with its salt counterpart. This is preferably achieved by adding a strong mineral base and optionally a strong mineral acid to the organic carboxylic acid or organic non-carboxylic acid reducing acid in an amount that generates a buffer system in situ. Suitable examples of strong mineral bases include sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, and cesium hydroxide. Suitable examples of strong mineral acids include hydrochloric acid, sulfuric acid, hydrobromic acid, and nitric acid.

[0161] In particular, when the combination or composition according to this disclosure is brought into contact with cells or the skin, mucous membranes, or other tissues of an animal (including a human), such as in the case of administration to the nose, mouth, respiratory tract, or lungs according to the present invention, the buffer may comprise one or more physiological buffers.Suitable physiologically compatible buffers include Good's buffers that buffer in a pH range of approximately 5 to 9, such as 2-amino-2-methyl-1,3-propanediol, N-2-aminoethanesulfonic acid (ACES), N-(2-acetamide)-iminodiacetic acid (ADA), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and N,N-bis(2-hydroxyethyl) Lysine (BICINE), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS), 1,3-bis[tris(hydroxymethyl)methylamino]-propane (BIS-TRISpropane), 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), 4-(2-hydroxyethyl)-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) dihydrate (POPSO), dibasic phosphate Sodium, monobasic sodium phosphate, dibasic potassium phosphate, monobasic potassium phosphate, [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. One example is 1,3-propanediol (TRIZMA).

[0162] Osmotic pressure of the composition In particular, the solute strength of any solution of nitrites, proton sources, organic polyols, or any combination thereof delivered to the nasal, oral, respiratory, or pulmonary physiological systems of human or animal subjects via routes that would result in contact with the skin or mucous membranes, or in accordance with the present invention, should be controlled to avoid any undesirable dehydration of the organs and tissues of the subject.

[0163] Osmotic pressure (Osm), defined as the number of moles of solute dissolved in one kilogram of solvent, can be expressed as osmotic pressure per kilogram (Osmol / kg). The osmotic pressure of any solution administered to human or animal subjects pursuant to this disclosure should generally be in the range of about 100 to about 5000 mOsmol / kg, for example, about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 to about 2000, 2250, 2750, 300, 3250, 350, 3750, 400, 4250, 450, 4750, or 5000 mOsmol / kg.

[0164] Mixing of components that initiate NOx generation We found that the order in which the components of the NOx generation system are mixed to initiate NOx generation can have an effect on the results obtained using the NOx produced. Evidence of this effect is provided in Example 6 below.

[0165] In this embodiment, it is demonstrated that the effectiveness of the composition according to the present invention for killing the bacterium M. tuberculosis HN878 in THP-1 cells differs depending on whether, on the one hand, the components of nitrite, proton source, and organic polyol are first mixed in a desired proportion at a higher concentration than the desired concentration in the composition used, and then preferably the concentrate is diluted with water to reach the composition used, or on the other hand, the components of nitrite, proton source, and organic polyol are first mixed in a desired proportion at the desired concentration in the composition used.

[0166] Furthermore, it is not predictable which mixing method of the components will yield better results in terms of antibacterial efficacy. Generally, diluting a relatively concentrated pre-mixture to achieve the composition used may result in better antibacterial efficacy against M. tuberculosis HN878 in THP-1 cells, but in some cases, this may not yield as good results as when the components are first mixed at the desired concentration for use.

[0167] Accordingly, in one embodiment of the present invention, a method for preparing a NOx-generating composition includes mixing a nitrite, a proton source, and components of an organic polyol in a desired proportion at a higher concentration than desired in the composition to be used to form a concentrated premixture, and then preferably diluting the concentrated premixture with water to provide the composition to be used.

[0168] Therefore, in another embodiment of the present invention, a method for preparing a NOx-generating composition includes mixing a nitrite, a proton source, and components of an organic polyol in desired proportions at desired concentrations in the form of composition to be used to provide the composition to be used.

[0169] Preferred Embodiment Preferred embodiments of the first to eighth aspects of this disclosure are embodiments having one or more of the following: -One or more nitrites include one or more alkali metal or alkaline earth metal nitrites, such as sodium nitrite, potassium nitrite, or any combination thereof. (For example, including them, or essentially consisting of them, or consisting only of them), - The proton source includes ascorbic acid or ascorbic acid / ascorbic acid buffer, citrate or citrate / citric acid buffer, or any combination of two or more of these (for example, including them, essentially consisting of them, or consisting only of them), - The ascorbic acid or ascorbic acid / ascorbic acid buffer, citric acid or citric acid / citric acid buffer, or any combination of two or more of these molecules are not covalently bonded to a polymer or macromolecule. -One or more organic polyols comprising a linear sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, such as sorbitol, mannitol, arabitol, xylitol, or any combination of two or more thereof (e.g., comprising them, essentially consisting of them, or consisting solely of them), - For example, a sugar alcohol compound comprising one or more organic polyols, each containing a chain of 1, 2, or 3 monosaccharide units terminated by one acyclic alcohol unit, wherein optionally, 1, 2, 3, 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, such as isomalt, maltitol, lactitol, maltotriitol, or maltotetraitol. - The total molar concentration of one or more organic polyols in the polyol components or reaction solution at or before the start of the NOx generation reaction is 0.05 to 3 times the total molar concentration of nitrite ions in the nitrite components or reaction solution. - The total molar concentration of one or more organic polyols in the polyol components or reaction solution at or before the start of the NOx generation reaction is 0.05 to 3 times the total molar concentration of the proton source in the proton source components or reaction solution. - Before the initiation of the NO generation reaction, especially immediately before, the pH of the proton source should be in the range of 3.0 to 9.0 in applications that do not involve contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of cells or animals (including humans). - Before the initiation of the NO generation reaction, especially immediately before, the pH of the proton source should be in the range of 4.0 to 8.0 in applications involving contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of cells or animals (including humans). - Before the initiation of the NO generation reaction, particularly immediately before, the pH of the proton source is in the range of 5.0 to 8.0 in applications involving contact between the reaction mixture and the nose, mouth, respiratory tract, or lungs of an animal (including human) subject, according to the present invention. - The target bacteria are selected from the bacteria listed in the paragraph under the heading "Targets for Antibacterial Use" below, for example, but not limited to their antibiotic-resistant strains, including influenza viruses, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa.

[0170] A preferred embodiment of the ninth aspect of this disclosure is an embodiment in which one or more of the following exist: -One or more nitrites include one or more alkali metal or alkaline earth metal nitrites, e.g., sodium nitrite, potassium nitrite, or any combination thereof (e.g., including them, essentially consisting of them, or consisting solely of them), - The proton source includes ascorbic acid or ascorbic acid / ascorbic acid buffer, citrate or citrate / citric acid buffer, or any combination of two or more of these (for example, including them, essentially consisting of them, or consisting only of them), - The ascorbic acid or ascorbic acid / ascorbic acid buffer, citric acid or citric acid / citric acid buffer, or any combination of two or more of these molecules are not covalently bonded to a polymer or macromolecule. -One or more organic polyols comprising a linear sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, such as sorbitol, mannitol, arabitol, xylitol, or any combination of two or more thereof (e.g., comprising them, essentially consisting of them, or consisting solely of them), - For example, a sugar alcohol compound comprising one or more organic polyols, each containing a chain of 1, 2, or 3 monosaccharide units terminated by one acyclic alcohol unit, wherein optionally, 1, 2, 3, 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, such as isomalt, maltitol, lactitol, maltotriitol, or maltotetraitol. - The total molar concentration of one or more organic polyols in the polyol components or reaction solution at or before the start of the NOx generation reaction is 0.05 to 3 times the total molar concentration of nitrite ions in the nitrite components or reaction solution. - The total molar concentration of one or more organic polyols in the polyol components or reaction solution at or before the start of the NOx generation reaction is 0.05 to 3 times the total molar concentration of the proton source in the proton source components or reaction solution. - Before the initiation of the NO generation reaction, especially immediately before, the pH of the proton source should be in the range of 3.0 to 9.0 in applications that do not involve contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of cells or animals (including humans). - Before the initiation of the NO generation reaction, especially immediately before, the pH of the proton source should be in the range of 4.0 to 8.0 in applications involving contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of cells or animals (including humans). - Before the initiation of the NO generation reaction, particularly immediately before, the pH of the proton source is in the range of 5.0 to 8.0 in applications involving contact between the reaction mixture and the nose, mouth, respiratory tract, or lungs of an animal (including human) subject, according to the present invention. - The target bacteria are selected from the bacteria listed in the paragraph under the heading "Targets for Antibacterial Use" below, for example, but not limited to their antibiotic-resistant strains, including influenza viruses, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, and Pseudomonas aeruginosa.

[0171] Combinations and compositions NOx production reactions can be initiated in several ways. These generally involve contacting one or more nitrites with a proton source under conditions that can initiate NOx production.

[0172] The reaction may be initiated by combining the distinct components of the combination. This combination may be achieved in vitro, and the resulting composition may then be administered to a subject or applied to any surface treated according to this disclosure. Alternatively, the generated gas may be administered to a subject or applied to any surface treated according to this disclosure. Furthermore, the use of both resulting compositions may proceed with a time interval between them, such that some gas generation occurs before the composition is administered to the subject or applied to any surface treated.

[0173] The combination can be stepwise; for example, the dry powder forms of the components are mixed first, and then mixed with water or another liquid carrier medium to initiate the reaction. Alternatively, the dry powder forms of the components can be mixed individually with water or another liquid carrier medium, and then two or more... The reaction may be initiated by mixing the liquids above.

[0174] Alternatively, at least some of the components of the NOx-producing reaction according to this disclosure may be present in a mixture in a single composition, and the NOx-producing reaction may be initiated on the composition. One possible way to initiate the NOx-producing reaction is, for example, by adding a key component or additive to initiate 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.

[0175] A kit according to this disclosure typically comprises one or more components of the combination or composition according to this disclosure, under conditions that prevent the occurrence of a NOx production reaction. The components of the kit are typically held in a container, which may be separated or adapted to facilitate the mixing that would be necessary to initiate a NOx production reaction. A key initiating component for initiating a NOx production reaction, which needs to be introduced by the user of the kit into other necessary components, may be, for example, one of the following: a nitrite component, a proton source component, or a polyol component, or an additional component, typically a commonly available component such as water, which may be supplied by the user.

[0176] The parameters of combinations and compositions defined and described in this patent typically include physical parameters such as pH, concentration, and osmotic pressure. These are, where possible, measured before the initiation of the NOx production reaction. Unless otherwise stated, the pH parameter refers to the pH of the proton source in deionized water at the concentration intended to initiate the NOx production reaction. Unless otherwise stated, the concentration of the solution refers to the concentration before mixing with other components to initiate the NOx production reaction. Typically, when nitrites and organic carboxylic acids or organic reducing acids react upon mixing to produce nitric oxide gas, it is not readily possible to measure such parameters while the NOx production reaction is progressing.

[0177] Furthermore, it should be noted that in the case of a reaction mixture, the concentrations of the components will not necessarily correspond to their concentrations in the parts of the combination before mixing. For example, suppose the composition for initiating the NOx production reaction according to this disclosure is formed from approximately equal volumes of nitrite components and proton source components, which are added together as a pre-prepared solution. In that embodiment, the mixed reaction composition has a nitrite concentration that is half the concentration of the nitrite components and a proton source concentration that is half the concentration of the proton source components.

[0178] The components of the combination and composition may be in any suitable physical form during or after the NOx production reaction, depending on the intended use of the system. For example, since each component of the combination and composition may be in the form of a liquid, gel, or film, the NOx production reaction mixture may also be in the form of a liquid, gel, or film. The liquid may be adapted to be sprayed for inhalation into the respiratory system or lungs. If the NOx production reaction 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 beverage. Alternatively, if the NOx production reaction mixture is intended to be applied to the skin by topical administration, the components of the combination and composition may be in the form of an ointment, lotion, or cream.

[0179] Multi-component systems, kits, and dispensers The multi-component systems described herein are defined in accordance with this disclosure and may optionally include a nitrite component and a proton source component together with a polyol component, as described herein. The components of the multi-component system are brought into contact with each other, and the reaction mixture and / or generated gas is distributed to a suitable container or reservoir for holding the components before use, as well as for mixing the components and distributing the reaction mixture and / or generated gas, and generally the mixture The mixture is adapted to be distributed by means for controlling the mixing and distribution. In one preferred embodiment, the reaction mixture may be distributed in the form of a mist or aerosol of droplets contained in a stream.

[0180] The kits and dispensers of this disclosure generally include a container for holding the components before use, at least one device or other means for mixing the components, distributing the reaction mixture and / or the gases produced, and generally controlling such mixing and distribution, and, if present, its or their components contained in the container(s) of the kit or dispenser before use. Preferably, there may be instructions for use, or instructions from which instructions for use can be found, such as online instructions for use. Such kits and dispensers constitute further embodiments of this disclosure.

[0181] The kits of this disclosure may be relatively simple assemblies of containers and means for mixing components, distributing reaction mixtures and / or gases produced, and generally controlling such mixing and distribution. Preferably, such kits may be provided for research purposes, or when a wide range of modifications in the mixing and distribution operations are expected and permissible.

[0182] Other kits of this disclosure may be more sophisticated assemblies of one or more containers containing consumables, together with one or more dispensers of this disclosure (optionally, together with water or other commonly available components supplied by the user, and any combination and / or composition required by the user to initiate a NOx production reaction).

[0183] The dispensers of this disclosure will generally be adapted for repeated similar acts of dispensing a reaction mixture, a carrier containing the reaction mixture, and / or a generated gas. The dispenser may comprise a pump or propulsion system for dispensing a composition containing the reaction mixture that produces NOx, or a generated gas, from the dispenser and directing it to a target. The propulsion system may preferably consist of a pressurized gas and / or liquefied gas, e.g., pressurized air or pressurized / liquefied butane, which would be pharmaceutically acceptable or biocompatible for medical use. Alternatively, inhalation from the user's lungs may be used to dispensing a composition containing the reaction mixture that produces NO, or a generated gas, from the dispenser and directing it to a target. Dispensers for use in this disclosure may preferably comprise an actuation device, such as a manually operable trigger or button, which allows the user to operate the dispenser. Such dispensers may be adapted for professional use, research use, consumer use, or patient use and may be adapted accordingly to facilitate the intended route for treating the target.

[0184] A wide range of kits and dispenser devices are known in principle and can be used or readily adapted to hold components before use, mix components or facilitate such mixing, distribute compositions containing reaction mixtures and / or generated gases, and generally control or facilitate such mixing and distribution.

[0185] For example: - Syringes, for example, twin-barrel dispensing syringes. - A container system, for example, a pumping container, a squeezing container, or a shaking container, for example, comprising two containers for mixing at least a nitrite component and a proton source component and distributing a composition containing NOx-producing reactants or the gas generated. Such a system is described in US2019 / 0134080, the disclosure of which is incorporated herein by reference. - To hold the components before use in an aqueous solution, mix the components, spray the liquid reaction mixture, distribute it for inhalation into the lungs of a human, and generally to control the mixing and distribution. Apparatus for such use. Examples include soft mist inhalers, jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. Suitable selections of nebulizers, droplet sizes, co-agents, packaging forms, etc., for inhalation of atomized NOx-generating reaction media by acidification of nitrites are described in WO03 / 032928 and WO2009 / 086470, whose disclosures are incorporated herein by reference. - The above apparatus can be adjusted to generally control the mixing and distribution, after filling a nebulizer with a pre-mixed liquid reaction mixture, spraying it, and distributing it for inhalation into a human lung. - Apparatus for holding components in aqueous solution before use, mixing components, aerosolizing liquid reaction mixtures, distributing them for inhalation into human lungs, and generally controlling such mixing and distribution. An example is a constant-dose inhaler. Suitable selections of droplet size, co-agents, packaging forms, etc., for inhalation of sprayed NOx-generating reaction media by acidification of nitrites are described in WO03 / 032928 and WO2009 / 086470, whose disclosures are incorporated herein by reference. - Techniques and apparatus for spraying a nitric oxide-releasing solution into the upper respiratory tract are described in U.S. Patent No. 9,730,956, the disclosure of which is incorporated herein by reference. -A device for holding the components before use in dry powder form and distributing it for inhalation into the lungs of a human. An example is a dry powder inhaler (DPI), which may be formulated as either a reservoir powder or separate blisters of multiple doses, as a single-dose capsule or a multi-dose dry powder inhaler. Selections of suitable powder particle size, co-formulation, packaging form, etc., for inhalation of dry powder combinations to provide a reaction medium into the lungs to generate NO in situ by acidification of nitrite are described in WO2009 / 086470, the disclosure of which is incorporated herein by reference. Dispensers for holding components in solution form before use, aerating them, and dispensing them as a foam for use as a skin disinfectant or for treating skin disorders are described in U.S. Patent Application No. 2013 / 0200109, U.S. Patent No. 7066356, and U.S. Patent Application No. 2019 / 0134080, whose disclosures are incorporated herein by reference. - A transdermal patch assembly for holding its components and distributing them to target skin is described in WO2014 / 188175, the disclosure of which is incorporated herein by reference.

[0186] The combinations and compositions of the present disclosure, or the dosage of the generated gases, may vary within a broad range of limitations depending 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 severity of the treatment required, and the condition, age, and health of the person being treated, or, in the case of non-medical treatment, the nature of the target being treated. In the case of medical treatment, the appropriate dosage to be used will ultimately be determined by a physician. In the case of non-medical treatment, those skilled in the art will be able to study appropriate dosages and treatment methods by reviewing relevant literature through reasonable testing.

[0187] In some embodiments, the composition in which the NOx-generating reaction is taking place, or the gas produced therefrom, can be administered to a target site, such as bacterial cells, living tissue, organs, structures, or objects, within 600 seconds after combining the nitrite component and the proton source component. In this configuration, the target site may be exposed to a large eruption of nitric oxide.

[0188] In some embodiments, the composition in which the NOx-generating reaction is occurring may be formed in situ or near the target site, for example, on, in, or near bacterial cells, living tissue, organs, structures, or objects, including non-living surfaces and spaces. In these embodiments, administration is effective within 0 seconds after combining the nitrite component and the proton source component. In other embodiments, the composition is administered to the target site or near it within a range of more than 0 seconds but less than 600 seconds after combining the nitrite component and the proton source component. In more specific embodiments... In one embodiment, the composition is administered within a range of 0 to 120 seconds. In a further embodiment, the composition is administered within a range of 0 to 60 seconds.

[0189] In other embodiments, the composition in which the NOx-producing reaction is occurring, or the gas produced therefrom, may be administered to a target site or its vicinity, e.g., bacterial cells, living tissue, organs, structures, or subjects, 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 this case, the target site, e.g., bacterial cells, living tissue, organs, structures, or subjects, may not necessarily be exposed to a large eruption of nitric oxide, but may still experience beneficial properties such as antibacterial effects. In these embodiments, the composition in which the NOx-producing reaction is occurring, or the gas produced therefrom, may be administered for up to 48 hours after combining the nitrite component and the proton source component. In certain embodiments, the composition, or the gas generated therefrom, may be administered for 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 about 24 hours, after combining the nitrite component and the proton source component.

[0190] Compositions in which NOx-producing reactions are occurring, or the gases generated therefrom, can be administered for more than 48 hours after combining the nitrite component and the proton source component, provided they are stored properly. For example, the composition can be stored in a sealed container, for example, under vacuum. Storage in a sealed container is typically carried out within 24 hours after combining the nitrite with an organic carboxylic acid or organic reducing acid. The composition can also be stored in a sealed container within 600 seconds after combining the nitrite component and the proton source component. In this method, a certain proportion of nitric oxide gas can be retained. If the NOx-producing composition is stored at low temperatures, for example in the range of about -30°C to about +10°C, for example in the range of about 1°C to about 10°C, the rate of gas generation can be substantially slower, and the storage time of the composition can be very long.

[0191] In certain embodiments, the aerosol dispenser may comprise a plurality of reservoirs, each having a first reservoir containing a nitrite component in liquid form (e.g., aqueous solution) and a second reservoir containing a proton source component in liquid form (e.g., aqueous solution). In this embodiment, each component may preferably be mixed with the propellant before, during, or after the nitrite and proton source components are mixed.

[0192] 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 action or by electric operation of the syringe. For example, the composition may be a liquid or a gel.

[0193] In another specific embodiment, the dispenser may be a multi-barrel syringe having a first barrel containing a nitrite component and a second barrel containing a proton source component. The viscosity of the components may be selected so that they can be dispensed from the syringe by manual action or by electric operation of the syringe. For example, each component may be independently a liquid or a gel.

[0194] Other reservoirs for constituent components: hydrogels In some embodiments of this disclosure, molecular reservoirs, such as hydrogels, may be used. A hydrogel is a highly hydrated, usually crosslinked, three-dimensional polymer network (homopolymer or copolymer) or macromolecule network having the ability to absorb and retain many times the dry weight of water, other aqueous liquids, or other non-aqueous hydrophilic liquids. The absorption of liquid is usually accompanied by swelling of the hydrogel. Covalently bonded polymers or macromolecules By selecting the appropriate constituent chemical groups, acidic hydrogels or hydrogels with other special chemical properties can be prepared.

[0195] Hydrogels capable of functioning as proton source components are known in this disclosure. Examples of such acidic-COOH group-containing hydrogels are described, for example, in WO2007 / 007115, WO2008 / 087411, WO2008 / 087408, WO2014 / 188174, and WO2014 / 18175, and the documents referenced therein, all of which are incorporated herein by reference. The use of such hydrogels in skincare using NOx generation, including transdermal delivery of pharmaceuticals in conjunction with NOx generation, is described in particular in WO2014 / 188174 and WO2014 / 188175. Specific examples of such hydrogels include acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (ATBS, available from Vinati Organics Ltd), and homopolymers and copolymers of their salts. A polymer formed from monomers containing (meth)acrylic acid or thereof may contain a pendant carboxylic acid group for use as a proton source according to the present disclosure.

[0196] Therefore, for example, a multi-component system may include a first acidic hydrogel pad or layer component containing a proton source component and optionally further containing an organic polyol, the other components of which may be nitrite components. The nitrite component may be, for example, a liquid medium containing dissolved nitrite. In this configuration, the NOx generation reaction can be initiated by bringing the surface of the hydrogel pad or layer into contact with the nitrite component. Alternatively, the nitrite component may be a solid carrier, such as a pad or layer, containing nitrite in a form accessible for dissolution in the liquid absorbed by the hydrogel upon contact between the solid carrier and the hydrogel.

[0197] Typically, the solid carrier pad or layer is permeable to the diffusion of nitric oxide (fully permeable or at least semi-permeable). In this configuration, the solid carrier pad or layer and the hydrogel are combined, and when the nitrite component and the proton source component are combined, nitric oxide can diffuse into the treatment area. The solid carrier pad or layer may be, for example, a mesh, a nonwoven bat, a film, a foam, an alginate layer, or a membrane.

[0198] In certain embodiments, the solid carrier layer is a mesh. The mesh may be a number of solid, typically flexible, interconnected strands that form a grid of holes or gaps through which a particular substance passes. The mesh may be woven or nonwoven. In some embodiments, the mesh is nonwoven.

[0199] The solid support layer, such as a mesh, may be made from a polymer material. 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 certain embodiments, the solid support layer is a polypropylene mesh.

[0200] In certain embodiments, the solid carrier is absorbent, and the nitrite component is at least partially absorbed, inhaled, or impregnated within the solid carrier. The absorbed, inhaled, or impregnated nitrite component may be a (dried) solid or may be present in an aqueous solution within the solid carrier.

[0201] In certain embodiments, the solid carrier comprises two or more layers, wherein the nitrite component is absorbed, absorbed, or impregnated into at least one layer, or coated into at least one outer layer. For example, the solid carrier may comprise two, three, four, five, six, seven, eight, nine, or ten or more layers, such as a polypropylene mesh layer, which is coated with one or more nitrites in dry and / or solution form.

[0202] Acidic hydrogels have a natural buffering capacity due to the large supply of protonated pendant acidic groups inside, and H from acidic hydrogels + Ions can migrate through the inhaled aqueous medium when the acidic portion of the pendant on the surface is deprotonated during the NOx generation reaction, thereby maintaining a relatively acidic pH on the surface of the hydrogel structure.

[0203] Non-acidic (e.g., neutral or basic) hydrogels are also known, and nitrite components and / or polyol components may be inhaled and contained for use in the present disclosure. Proton source components can be brought into contact with such hydrogels by providing the proton source in a liquid medium in contact with the hydrogel, and / or by absorbing, inhaling, impregnating, or coating the proton source with a solid support. In such hydrogels, it may be required that none of the nitrite components, proton source components, or polyol components are covalently bonded to the polymer network or macromolecular network of the hydrogel, and considering, for example, that the nitrite components and proton source components should not react together until the initiation of the NOx production reaction is desired, all components required in the present disclosure may be inhaled into the hydrogel and contained in an aqueous medium within the hydrogel mass, but not covalently bonded to the polymer or macromolecules of the hydrogel.

[0204] The thickness of the hydrogel pad or layer can be in the range of 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.

[0205] In general, the characteristics described above regarding proton source components will apply equally to any acidic hydrogel that functions as a proton source component. Therefore, for example, a hydrogel may contain a buffer to maintain the pH of the hydrogel in the range of 4.0–9.0 or 5.0–8.0.

[0206] In some embodiments, the hydrogel may include a barrier layer. The barrier layer is typically a polymer film, such as a polyurethane film, and is located on the outer surface of the hydrogel. In use, the barrier layer is typically located on the surface of the hydrogel opposite the target skin, for example, 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 configuration, the barrier layer may 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 to the target skin, for example, during use.

[0207] In certain embodiments, this disclosure is, a) One or more meshes that have been inhaled, impregnated, or coated with one or more nitrites such as NaNO2, b) A hydrogel comprising a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, Component (a) is separated from component (b), and one or more of components (a) and (b) are A two-component system further comprising one or more organic polyols characterized by one or more of the following: To provide: (a) One or more organic polyols are present in an amount that enhances the reaction output. (b) The proton source is not simply a hydrogel containing pendant carboxylic acid groups covalently bonded to a three-dimensional polymer matrix. (c) One or more organic polyols are not simply glycerols. (d) If one or more viscosity increasing agents are used, one or more organic polyols must not simply be glycerols. (e) If one or more plasticizers are used, one or more organic polyols must not be simply glycerols. (f) One or more organic polyols are not simply polyvinyl alcohols. (g) If one or more viscosity increasing agents are used, one or more organic polyols must not be simply polyvinyl alcohols. (h) The word "simply not" is replaced by "does not include" in one or more of the above (b) to (g). (i) One or more organic polyols are not simply propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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. (j) One or more organic polyols are not propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butanediol, butenediol, butinediol, 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 specified 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.

[0208] To avoid any doubt, we hereby confirm that the embodiments and priorities described above for characterizing features (a) to (h) in relation to the nature of the disclosure are equally applicable to this embodiment.

[0209] Such a system may be used, for example, by combining components (a) and (b) to initiate a NOx production reaction. Such a combination may then be used, for example, by topical application, in the therapy or other treatment of the human or animal body. Uses may be as described in WO2014 / 188174 and WO2014 / 188175, or as described below. The system may also be as described below. It may be used for non-medical purposes. When the system is used for topical medical applications in which it comes into contact with the target skin (including mucous membranes), one or more meshes may form the skin contact layer.

[0210] Use in treatment or surgery Compositions in which a NOx-generating reaction proceeds according to this disclosure, and the gases generated thereby, have many applications in therapy and surgery, including curative and / or preventive treatment, surgical procedures to correct diseases and disorders and conditions, cosmetic surgery, and reconstructive surgery, including human and veterinary medicine and surgical procedures. If a physical malformation or abnormality in response to treatment with a composition or gas generated therefrom causes or exacerbates anxiety, depression, or another mental illness or disorder, then the treatment, prevention, or mitigation of the physical condition may, accordingly, treat, prevent, or mitigate the mental condition, thereby extending the use of this disclosure to the field of mental health.

[0211] Numerous physiological effects of nitric oxide and nitric oxide-producing compositions and medical treatments based thereon have been reported in the literature, resulting in the development of many therapeutic treatments. The following non-limiting list is provided as an example. The listed uses, as well as other uses not listed, are incorporated within this disclosure and patent. Vasodilation caused by nitric oxide increases blood supply and / or lowers blood pressure (see van Faassen et al., Med.Res.Rev.2009 Sept;29(5), pages 683-741). The rapid effects of oral nitric oxide supplementation in lowering blood pressure, improving vascular compliance, and restoring epithelial function in patients with hypertension were reported by Houston et al. in J. Clin. Hypertens. (Greenwich), July 2014, 16(7), pages 524-529. Protection of tissues from damage due to low blood supply (see van Faassen et al., Med.Res.Rev.2009 Sept;29(5), pages 683-741). Action of nitric oxide as a neurotransmitter in nitrergic neurons, for example, nitrergic neurons are active on smooth muscle, for example, in the gastrointestinal tract and erectile tissue (see Toda et al., Pharmacol.Ther., 2005 May; 106(2), pages 233-266). Inhibition by nitric oxide of vascular smooth muscle contraction and growth, platelet aggregation and leukocyte adhesion to the endothelium, assisting vessel homeostasis (see Dessey and Ferron, Current Medical Chemistry - Anti-inflammatory and Anti-allergy Agents in Medicinal Chemistry, 2004;3(3), pages 207-216). Action of nitric oxide to decrease heart contractility and heart rate (See Navin et al., J. Cardiovascular Pharmacology, 2002;39(2), pages 298-309). Critical neonatal care to promote capillary and pulmonary dilation,for example treatment of primary pulmonary hypertens Ion in neonatal patients, and post-meconium aspiration (see Barrington et al., The Cochrane Database of Systematic Reviews, 2017;1,CD000399 (https: / / www.ncbi.nlm.nih.gov / pubmed / 17375630), also Chotigeat et al., J.Med.Assoc.Thai., 2007;90(2), pages 266-271, and Hayward et al., Cardiovascular Research, 1999;43(3), pages 628-638). Prevention of vascular damage,endothelial dysfunction and vascular inflammation,neuropathy and non-healing ulcers,and reducing the consequent danger of requiring lower limb amputation,in diabetes patients(nfb University Studies-“Nitric Oxide Holds Promise for Diabetes”, http: / / www.nfb.org / Images / nfb / Publications / vod / vod212 / vodspr0613.htm), Improvement of hypoxemia in acute lung injury,acute respiratory distress syndrome and severe pulmonary hypertension;treatment of reversible causes of hypoxemic Respiratory distress (see Mark et al., N.Eng.J.Med., Dec.22, 2005;353(25), pages 2683-2695). Administration of nitric oxide as salvage therapy in patients with acute right ventricular failure secondary to pulmonary embolism (See Summerfield et al., 2011; Respir.Care57(3), pages 444-448). Treatment of angina, the effects of paraquat poisoning and other cardiovascular disorders(Abrams, The American Journal of See Cardiology, 1996;77(13), pages 31C-37C. Treatment of bladder contractile dysfunctions (See Moro et al., Eur.J.Pharmacol., January 2012;674(2-3), pages 445-449, and Andersson et al., Br.J.Pharmacol. February 2008;153(7), pages 1438-1444). This is described in Treatment of acute and chronic lung infections and sepsis (see Fang et al., Nature Reviews. Microbiology, October 2004;2(10), pages 820-832, and Goldfarb et al., Critical Care Medicine, January 2007;35(1), pages 290-292), Nitric oxide-containing toxic reactive nitrogen intermediates (RNIs) have been proposed as effector molecules in the anti-mycobacterial effects of activated mouse macrophages against the bacterial toxicity of Mycobacterium tuberculosis (see Chan et al., J. Exp. Med., April 1992; 175, pages 1111-1122). Gaseous nitric oxide may be effective in the treatment of lung infections by antibiotic-resistant bacteria and fungi in patients with cystic fibrosis (see Deppisch et al., 9 February 2016; "Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung infections in patients with cystic fibrosis: a Phase I clinical study", Springer, DOI 10.1007 / s15010-016-0879-x). Nitric oxide has been reported as a potential broad-spectrum topical antibacterial agent for dermatological diseases with low potential for resistance development (see B L Adler and A J Friedman, Future Sci. OA, 2015; 1(1), FSO37). Nitric oxide is a neurotransmitter and is associated with various functions ranging from neuroactivity to sexual organ erection in both males and females in avoidance responses (see Kim et al., J. Nutrition, 2004, 134, page 28735). The use of nitric oxide to treat male erectile dysfunction and impotence has been described by Sullivan et al., Cardiovascular Research, August 1999, 43(3), pages 658-665. The potential use of nitric oxide as a surgical adjuvant for assisting wound healing, reducing ischemia-reperfusion injury, assisting heart and lung recovery from surgery, and assisting recovery from vascular surgery, as well as postoperative recovery from plastic surgery has been reported (see A Krausz and A J Friedman, Future Sci. OA, 2015; 1(1), FSO56). The antibacterial effect and wound healing effect of NO have been described in WO95 / 22335, and Hardwick, et al., 2001, Clin, Sci. 100, pages 395-400. European Patent No. 1411908 (Aberdeen University) reports data suggesting that nitric oxide is effective in treating nail bed infections, including Aspergillus niger. Topical application of NOx generating 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., 1998 April, 38(4), pages 559 - 563), Topical application of NOx generating compositions to the skin for the treatment of viral skin infections (see WO99 / 44622), (Also known as Raynaud's phenomenon) Topical application of NOx products to the skin for the treatment of conditions where vasoconstriction is a underlying problem, such as Raynaud's syndrome (see Tucker, et al. Lancet, 13 November 1999, 354, 9191, pages 1670 - 1675). The use of acidified nitrite as an agent for generating local production of nitric oxide at the skin surface for the treatment of peripheral ischemia, as well as related conditions such as Raynaud's phenomenon, and for the treatment of wounds such as postoperative wounds and burns, is described in WO2000 / 053193. The use of liquid nitric oxide-releasing solution (NORS) for treating wounds in humans is claimed by U.S. Patent No. 9,730,956 (Stenzler, et al.). NORS is also claimed to possess antibacterial, antifungal, and / or antiviral properties, and data is provided that claim antibacterial efficacy against Acetobacter baumanii, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Mannheimia haemolytica. Data is provided that claim antiviral efficacy of NORS against H1N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory polynuclear virus, and bovine parainfluenza-3 virus. Trichophyton rubrum and Trichophyton mentagrophy Data has been provided that suggests the antifungal efficacy of NORS against tes. Chou SH,et al.,The effects of debanding on the lung expression of ET-1,eNOS,and cGMP in rats with left ventricular pressure overload.Exp.Biol.Med.2005,231,pages954-959, Gladwin MT,et al.,Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling,cytoprotection,and vasodilation.Am.J.Physiol.Heart Circ.Physiol.2006,291,pages H2026-H2035, Hunter CJ,et al.,Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator.Nat.Med.2004,10,pages1122-1127、 Ozaki M,et al.,Reduced hypoxic pulmonary vascular remodeling by nitric oxide from the endothelium.Hypertension.2001,37,pages322-327、 Rubin LJ,2006.Pulmonary arterial hypertension.Proc.Am.Thorac.Soc.3,pages111-115、 Yellon D.M.,et al.,2007.Myocardial Reperfusion Injury,N.Engl.J.Med.,357,pages1121-35、 Duranski M.R.,et al.,Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver.J.Clin.Invest.2005,115,pages1232-1240、 Jung K-H.,et al.,Early intravenous infusion of sodium nitrite protects brain against in vivo ischemia-reperfusion injury,Stroke,2006,37,pages2744-2750、 Esme H.,et al.,Beneficial Effects of Supplemental Nitric Oxide Donor Given during Reperfusion Period in Reperfusion-Induced Lung Injury.Thorac.Cardiovasc.Surg.2006,54,pages477-483、 The use of acidified nitrites to release NO as an agent for improving skin quality in humans is described in Chinese Patent Application No. CN101028229. The use of acidified nitrites to release NO as agents for promoting hair growth in humans and preventing or treating alopecia is described in Chinese Patent Application No. CN101062050.

[0212] Other general considerations regarding the physiological effects of nitric oxide can be found, for example, in Lancaster et al., Proc Natl Acad Sci, 1996, 91, pages 8137-8141, and Ignarro et al., Proc Natl Acad Sci, 1987, 84, pages 9265-9269, and are outlined in Brent, J Cell Science, 2003, 116, pages 9-15. This is outlined in Murad, N Engl J Med, 2006, 355, pages 2003-2011.

[0213] The pharmacological forms of NO available for delivery are outlined in Butler and Feelisch, Circulation, 2008, 117, pages 2151-2159.

[0214] Each disclosure of the publications cited above is incorporated herein by reference.

[0215] This disclosure is applicable to all therapeutic and surgical uses of nitric oxide and nitric oxide production systems, including, but not limited to, certain therapeutic and surgical uses published in the above references, as well as all other published therapeutic and surgical uses, and therapeutic and surgical uses based on basic knowledge of the physiological effects of nitric oxide and the products of nitric oxide production reactions.

[0216] Vasodilation The vasodilatory properties of nitric oxide characterize many of the treatments that utilize combinations and compositions and gases generated therefrom.

[0217] Specific examples of diseases, disorders, and conditions that respond to vasodilation include, but are not limited to, conditions associated with ischemia and skin lesions.

[0218] Conditions associated with tissue ischemia include Raynauld's syndrome, severe primary vasospasm, and tissue ischemia caused by surgery, septic shock, radiation, or peripheral vascular disease (e.g., diabetes and other chronic systemic diseases).

[0219] When used to treat or prevent conditions associated with tissue ischemia as a result of surgery, the combinations or compositions of the Disclosure, or nitric oxide generated from NOx-producing reactions using the Disclosure, may be administered to a subject before, during, or after surgery. The combinations, compositions, or generated gases may be administered to or near the surgical site. Examples of surgical procedures in which this treatment or prevention of tissue ischemia may be used include transplantation, tissue or organ transplantation surgery, coronary artery surgery, carotid artery catheter insertion, surgery to provide indwelling arterial or venous catheters for systemic administration of drugs such as chemotherapeutic drugs, cosmetic surgery procedures including, but not limited to, pedicled or revolving flaps, repeat surgery in which incisions are made at the same site as a previous surgical procedure, surgery performed in areas of poor skin and / or poor underlying tissue perfusion (such as in patients with arteriosclerosis or diabetes) or in areas where poor perfusion is expected as a result of an associated disease, surgery in cases of trauma in which blood vessels are damaged or injured, and surgery to remove or excise arteriovenous malformations in or under the skin.

[0220] For example, combinations, compositions, or generated gases may be used to treat or prevent organ ischemia-reperfusion injury by administering the combinations, compositions, or generated gases according to this disclosure to an organ. The organ may 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 / or infarction (stroke)), the lungs (e.g., to treat or prevent pulmonary ischemia-reperfusion injury), the kidneys (e.g., to treat or prevent renal ischemia-reperfusion injury), and the liver (e.g., to treat or prevent hepatic ischemia-reperfusion injury). Surgical intervention may be organ transplantation. Administration of the combinations, compositions, or generated gases may be post-ischemic or prophylactic.

[0221] Use of transdermal drug delivery The properties of nitric oxide that induce transdermal delivery of drugs represent another important utility of the combinations and compositions disclosed herein, as well as the gases generated therefrom.

[0222] WO02 / 17881 and WO2014 / 188175, whose disclosures are incorporated herein by reference, describe combinations and compositions for generating nitric oxide and the use of the gases therefrom for transdermal drug delivery. The same conditions, priorities, and examples described in those publications for such use are also applicable to the combinations and compositions and the gases therefrom of this disclosure.

[0223] Typically, the combinations and compositions of the present disclosure will comprise one or more pharmaceutically active agents delivered transdermally to a subject and will be provided in the form of topical combinations or compositions for application to the skin of the subject. For examples of pharmaceutically active agents(s) that may be used, see the paragraph above under the heading “Optional Additional Components”.

[0224] A suitable topical combination may include a nitrate-containing mesh and a separate proton source-containing hydrogel, the two of which are adapted to be used together on the skin of a subject as described above in the paragraph headed "Other reservoirs for compositions or composition systems; hydrogels". The polyol(s) and pharmaceutically active agent(s) may be provided by 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(s) and pharmaceutically active agent(s) respectively.

[0225] Treatment of wounds, skin lesions, and burns The properties of nitric oxide, which induces vasodilation and transdermal drug delivery and kills bacteria or prevents bacterial growth, give rise to another important utility of the combinations and compositions of the present disclosure, and the gases generated therefrom, in the treatment of wounds, skin lesions, and burns.

[0226] Conditions treatable using the present disclosure include ulcers, skin donor sites, surgical wounds (post-operative), burns (thermal, superficial partial thickness burns, and full thickness burns, etc.), lacerations, and abrasions. The wounds can be chronic or acute. The ulcers can be of various origins such as arterial or venous. Examples of ulcers include leg ulcers, such as chronic leg ulcers or acute leg ulcers, pressure ulcers, such as chronic pressure ulcers or acute pressure ulcers, venous ulcers, and ulcers associated with diabetes such as diabetic foot ulcers.

[0227] WO2014 / 188174, the disclosure of which is incorporated herein by reference, describes combinations and compositions for generating nitric oxide for treating wounds, skin lesions, and burns, and the use of the gases generated therefrom, and the same conditions described in this publication are applicable to the combinations and compositions of the present disclosure, and the gases generated therefrom.

[0228] Typically, the combinations and compositions of the present disclosure will comprise one or more pharmaceutically active agents and will be provided in the form of topical combinations or compositions for application to the skin of interest. For examples of pharmaceutically active agents(s) that may be used, see the paragraph above under the heading “Optional Additional Components”. For the treatment of wounds, skin lesions, and burns, one or more pharmaceutically active agents may preferably be selected from analgesics and / or anesthetics (e.g., topical anesthetics) (e.g., analgesics and / or anesthetics for reducing chronic pain, acute pain, or neuropathic pain), antibacterial agents, disinfectants, anti-inflammatory agents, and anti-scarring agents.

[0229] A suitable topical combination may include a nitrite-containing mesh and a separate proton source-containing hydrogel, both adapted for use together on the skin of interest, as described above in the paragraph under the heading “Other reservoirs for compositions or systems of compositions; hydrogels.” Polyols and pharmaceutically active agents may be provided as one or more separate components of the combination, incorporated into the hydrogel, or any combination of these options may be used for the polyols and pharmaceutically active agents, respectively.

[0230] Topical antibacterial use In antibacterial applications, therapeutically effective NO doses can be low, for example, as low as parts per million (ppm), e.g., 100–600 ppm (see, for example, Ghaffari et al., Nitric Oxide Biology and Chemistry, 2009, 14, pages 21–29, the disclosure of which is incorporated herein by reference), but the effectiveness of nitric oxide depends substantially on how long it is in contact with the skin (see, for example, Ormerod et al., BMC Research Notes, 2011, 4, pages 458–465, the disclosure of which is incorporated herein by reference).

[0231] Proposals for the slow, local release of nitric oxide have been published (see, for example, U.S. Patent No. 6,103,275). However, the resulting local NO dose lasts for less than one hour, providing only poor local antibacterial action. As discussed above in the paragraph under the heading "Multi-component systems, kits, and dispensers" and elsewhere, and as shown in the following examples, the present disclosure enables a much longer duration of NO administration in both local and non-local administration systems, resulting in substantial clinical benefits.

[0232] In particular, the combinations and compositions of the present disclosure have been found to provide a strong output of nitric oxide during the first approximately 200–500 seconds after the NOx generation reaction has commenced ("initial eruption"), followed optionally by the provision of a slower release of nitric oxide ("tail") over a prolonged period, extended over a long time, before gas generation ceases or falls below an effective level. The amount of NO generated by the combinations and compositions of the present disclosure exceeds the minimum effective antibacterial dose disclosed, resulting in the potential effective topical antibacterial use of the combinations and compositions of the present disclosure, and the gas generated therefrom.

[0233] Formulations of NOx-generating combinations and compositions for topical antibacterial applications are well described in the prior art, for example, in U.S. Patent Application No. 2014 / 0056957, the disclosure of which is incorporated herein by reference, and such formulations are also applicable to the combinations and compositions of this disclosure. Another preferred topical combination may comprise a nitrite-containing mesh and a separate proton source-containing hydrogel, both adapted for use together on the target skin, as described above in the paragraphs heading “Other Reservoirs for Compositions or Compositional Systems; Hydrogels”. Polyols and any pharmaceutically active agents may be provided as one or more separate components of the combination, or incorporated into a hydrogel, or any combination of these options may be used for the polyols and pharmaceutically active agents, respectively.

[0234] Other skin treatments or topical treatments Other topical uses of nitric oxide and nitric oxide-producing compositions include stimulating hair growth and treating erectile dysfunction and impotence.

[0235] The combinations and compositions disclosed herein may be formulated for topical use in such therapies.

[0236] Local bandages and bandaging systems, such as wound bandages. In topical treatment, it is often desirable to cover or protect the treatment area of ​​the skin while the treatment is being applied. This helps prevent contamination of the wound, aids in the removal of pus or necrotic tissue fragments from the healing process, prevents or limits the loss of the treatment composition during bathing or showering, through contact with clothing, or as a result of the subject's normal activities, and can buffer bumps or abrasions to the treated area.

[0237] For this purpose, it is common to incorporate treatment into a topical bandage or bandage system, such as a wound bandage or bandage system. At least one component portion of the bandage or bandage system typically includes a backing sheet, which may be waterproof or permeable and optionally comprise a skin-adhesion portion and optionally other layers such as a gauze or pad layer.

[0238] In a further embodiment, the Disclosure provides a topical bandage, e.g., a wound or skin bandage, or bandage system comprising a combination or composition according to a fifth embodiment of the Disclosure, wherein at least one component of the bandage or bandage system comprises a backing sheet and optionally one or more other layers, such as a layer selected from, for example, gauze and a pad layer. The combination or composition according to a fifth embodiment of the Disclosure is preferably positioned on the skin-facing side of the backing sheet, so that when the bandage is applied to the skin and the NOx-generating reaction is initiated, a desired skin area is treated with the NOx-generating reaction mixture or the gas generated therefrom.

[0239] Bandages or bandage systems may preferably be provided in a sealed sterile pack before use.

[0240] Use of nose, mouth, respiratory system, and lungs The properties of nitric oxide to induce vasodilation and transdermal delivery of drugs, and to kill or prevent bacterial growth, give rise to another important utility of the combinations and compositions of the present invention, as well as the gases generated therefrom, in the treatment of the mucous membranes and tissues of the nose, mouth, respiratory tract, and lungs, and / or in the use of the nose, mouth, respiratory tract, and lungs as routes of administration for delivering the combinations and compositions of the present invention to human or animal subjects.

[0241] Conditions treatable using the present invention include, for example, viral infections such as influenza, SARS-CoV, or SARS-CoV-2; pulmonary hypertension; ischemia-reperfusion injury of the heart, brain, and organs involved in transplantation; chronic obstructive pulmonary disease (COPD) (particularly emphysema and chronic bronchitis); severe asthma and asthma and refractory (irreversible) asthma exacerbations caused by viruses and bacteria; intranasal or pulmonary bacterial infections such as pneumonia, tuberculosis, and non-tuberculous Mycobacteria infections; and lung diseases such as other bacterial and viral lung infections, such as secondary bacterial infections following viral infections of the respiratory tract.

[0242] WO2009 / 086470, whose disclosure is incorporated herein by reference, describes sprayable liquid combinations and compositions for generating nitric oxide for treating diseases of the nose, mouth, respiratory tract, and lungs, as well as the use of gases generated therefrom, and / or the use of the nose, mouth, respiratory tract, and lungs as routes of administration for delivering such combinations and compositions to human or animal subjects, and the same conditions, priorities, and examples described in that publication for such uses are also applicable to the combinations and compositions of the present invention, as well as gases generated therefrom.

[0243] Typically, the combinations and compositions of the present invention for delivery to the nose, mouth, respiratory tract, and lungs will comprise one or more pharmaceutically active agents. For examples of pharmaceutically active agents(s) that may be used, see the paragraph under the heading “Optional Additional Components” above. sea ​​bream.

[0244] Two main delivery methods are possible for carrying out the present invention via delivery routes of the nose, mouth, respiratory tract, or lungs(s). The first is that the combination or composition of the present invention is delivered directly to the nose, mouth, respiratory tract, or lungs(s). The second is that the gas generated from the NOx production reaction using the present invention is delivered to the nose, mouth, respiratory tract, or lungs(s)(s) without the combination or composition of the present invention entering the patient's body.

[0245] 1. Direct delivery of combinations or compositions to the nose, mouth, respiratory tract, or lungs (or multiple organs). The combination or composition, or its components, may be delivered directly to the nose, mouth, respiratory tract, or lungs (or more) in a dry solid form, thereby causing the fluids of the mucous membrane to dissolve the solid component material and initiate the NOx production reaction.

[0246] The components of the combination may be administered separately or together. In one preferred embodiment, the proton source or at least one component may be administered before the remaining components so that when the nitrite component comes into situ with the proton source component, a relatively acidic environment is established within the mucosa that facilitates the rapid initiation of the NOx production reaction.

[0247] Direct delivery of any combination of dry components or dry compositions to the nose, mouth, respiratory tract, or lungs(s) may preferably be performed 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 of nitrite components, proton source components, and polyol components) or dry powder compositions to a target, the dry powder inhaler which delivers an aerosol containing particles with a volume mean diameter of less than 6 microns to a target. The dry powder inhaler may be fitted with dry powder in single or multiple doses so as to deliver one or more dry powder components or dry powder compositions in amounts of about 0.1 mg to about 100 mg per inhaled breath, with particles having a volume mean diameter of less than 6 microns to a target.

[0248] In addition or alternatively, the combination or composition, or its components, may be delivered directly to the nose, mouth, respiratory tract, or lungs(s) as a mist or spray of a solution of one or more of the nitrite components, proton source components, and polyol components.

[0249] The embodiments of the present invention described herein are generally applicable to direct delivery to the nose, mouth, respiratory tract, or lungs(s) of a subject. For example, combinations or compositions, or their components, may be directly administered to the nose, mouth, respiratory tract, or lungs(s) of a subject in association with one or more physiologically compatible diluents, carriers, and / or excipients, and / or may be provided in association with one or more additional components, one or more specific functional components intended to provide a particular benefit. 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. If desired, small amounts of non-toxic auxiliary substances such as wetting agents, emulsifiers, lubricants, binders, and solubilizers, for example, sodium phosphate, potassium phosphate, acacia gum, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc., may also be present. Generally, depending on the intended mode of administration, the pharmaceutical formulation will contain about 0.005% to about 95% by weight, preferably about 0.5% to about 50% by weight, of the combination or composition of the present invention, or its components. Practical methods for preparing such dosage forms are known to those skilled in the art. It is either knowledge or obvious. For example, Martindale, 39 th Edition(2017), the Merck Index,15 th Edition (2013), Goodman & Gilman's “The Pharmacological Basis of Therapeutics”, 13 th Edition(2017), the British National Formulary on-line(https: / / bnf.nice.org.uk / ), Remington: “The Science&Practice of Pharmacy”, 22nd Edition (2012), or the Physician's Desk Reference, 71 st Please refer to Edition (2017).

[0250] In one preferred embodiment, the combination or composition for delivery to the nose, mouth, respiratory tract, or lungs (or more) of the target may preferably take the form of a liquid, suspended solid, dry powder, freeze-dried agent, or vial containing the combination or composition, along with components of the NOx-generating reaction such as, for example, a diluent such as lactose, sucrose, or dicalcium phosphate; a lubricant such as magnesium stearate; and a binder such as starch, acacia gum, polyvinylpyrrolidone, gelatin, cellulose, or cellulose derivatives.

[0251] Direct delivery of any droplets or compositions in droplet form containing the components of the combination to the nose, mouth, respiratory tract, or lungs(s) may preferably be performed by inhalation using a nebulizer that delivers a therapeutically effective dose of one or more liquid components (e.g., one or more of nitrite components, proton source components, and polyol components) or compositions in liquid form, the nebulizer which delivers an aerosol containing particles with a volume mean diameter of less than 5 microns. The nebulizer may be fitted with single or multiple doses of the liquid components or compositions of the combination so as to deliver one or more liquid components or compositions in liquid form in doses of about 0.1 mg to about 100 mg per inhaled breath, with the nebulizer being filled with the liquid components or compositions of the combination in droplets with a volume mean diameter of less than 5 microns, preferably having a size in the range of about 2 to about 5 μm.

[0252] In one embodiment, the nebulizer is selected based on its ability to enable the formation of aerosols of compositions in droplet form, or primarily having a mass median aerodynamic diameter (MMAD) of about 2 to about 5 microns, containing the components of the combination.

[0253] In one embodiment, the amount of droplets or droplet-shaped compositions containing the combined components delivered provides a therapeutic effect for pulmonary pathology, respiratory infections, and / or extrapulmonary, systemic distribution, thereby treating extrapulmonary and systemic diseases as well.

[0254] It has been previously shown that two types of nebulizers, jet and ultrasonic, are capable of generating and delivering aerosol particles having a size of 2-4 μm. These particle sizes have been shown to be optimal for intermediate airway deposits and therefore for treating bacterial infections of the lungs caused by Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa. However, unless a specially formulated solution is used, these nebulizers are not effective. Jet nebulizers typically require larger volumes to administer a sufficient amount of drug to achieve a therapeutic effect. Jet nebulizers utilize the air pressure disruption of an aqueous solution to create aerosol droplets. Ultrasonic nebulizers utilize the shearing of an aqueous solution by piezoelectric crystals. However, typically, jet nebulizers are only about 10% efficient under clinical conditions, while ultrasonic nebulizers are only about 5% efficient. Thus, the amount of drug that is deposited in and absorbed in the lungs is only about 10%, despite the large amount of drug placed in the nebulizer. Smaller particle sizes or slower inhalation rates allow for deposition in deeper lungs. Deposition in both the mid-lung fields and alveoli, e.g., mid-airway deposition for antibacterial activity, or mid-lung field and / or alveolar deposition for pulmonary hypertension and systemic delivery, may be desirable in the present invention, depending on the symptoms. Exemplary disclosures of compositions and methods for drug delivery using nebulizers can be found, for example, in US2006 / 0276483, which includes a description of techniques, protocols, and characterization of aerosolized mist delivery using a vibrating mesh nebulizer. The disclosure in US2006 / 0276483 is incorporated herein by reference.

[0255] Therefore, in one embodiment, a vibrating mesh nebulizer is used, in a preferred embodiment, to deliver an aerosol of droplets containing the components of a combination, or of a composition in droplet form. The vibrating mesh nebulizer comprises a liquid storage container in fluid contact with a partition wall, and an inhalation valve and an exhalation valve. In one embodiment, about 1 to about 5 ml of the liquid formulation to be delivered is placed in the storage container, and the aerosol generator is involved in selectively producing atomized aerosols with a volume-average diameter of about 1 to about 5 μm.

[0256] Therefore, for example, in a preferred embodiment, one or both of the nitrite component formulation or proton source component according to the present invention, optionally comprising one or more organic polyols, are placed in a liquid spray inhaler and prepared and delivered in doses of about 7 to about 700 mg, preferably about 17.5 to about 700 mg, more preferably about 17.5 to about 350 mg, more preferably about 0.1 to about 300 mg, more preferably about 0.25 to about 90 mg, in about 1 to about 5 ml of administration solution, while generating a particle size with a volume average diameter of about 1 to about 5 μm.

[0257] As a non-limiting example, a liquid or droplet-form composition containing the components of the combination, which is sprayed, may be administered in a respiratory-appropriate delivery dose as described, preferably in less than 20 minutes, more preferably less than 10 minutes, more preferably less than 7 minutes, more preferably less than 5 minutes, more preferably less than 3 minutes, and most preferably less than 2 minutes.

[0258] As a non-limiting example, in other circumstances, a liquid or droplet-form composition containing the components of the combination, when sprayed, may achieve improved tolerability and / or exhibit improved area under curve (AUC) shape when administered over a longer period of time. Under these conditions, the described respiratory-friendly delivery dose is over about 2 minutes, preferably over 3 minutes, more preferably over 5 minutes, more preferably over 7 minutes, more preferably over 10 minutes, and in some cases, most preferably over 10 to 20 minutes.

[0259] An example of a separate component formulation may include (i) a nitrite in an aqueous solution having a pH greater than about 6, for example in the range of about 6 to about 8, for example about 7, and (ii) a proton source component in an aqueous solution, and at least two separate liquid solution components (i) and (ii) can be mixed to form a NOx-generating composition that can be used to fill a nebulizer for delivery to a human patient or veterinary subject.

[0260] For aqueous and other non-pressurized liquid systems, a variety of nebulizers (including small-volume nebulizers) are available to aerosolize the components of the combination or composition. Compressor Driving nebulizers incorporate jet technology and use compressed air to generate liquid aerosols. Such devices are commercially available from, for example, Healthdyne Technologies, Inc., Invacare, Inc., Mountain Medical Equipment, Inc., Pari Respiratory, Inc. (Midlothian, VA), Mada Medical, Inc., Puritan-Bennet, Schuco, Inc., DeVilbiss Health Care, Inc., and Hospitak, Inc. Ultrasonic nebulizers rely on the mechanical energy of a piezoelectric crystal vibration to generate breathable droplets and are commercially available from, for example, Omron Healthcare, Inc. and DeVilbiss Health Care, Inc. Vibrating mesh nebulizers rely on either piezoelectric or mechanical pulses to generate breathable droplets.Other examples of nebulizers for use with nitrites, nitrite salts, or nitrite-donating compounds or nitric oxide-donating compounds described herein are all incorporated herein by reference in their entirety by U.S. Patents 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, and 5,96 No. 0,792, No. 5,971,951, No. 6,070,575, No. 6,192,876, No. 6,230,706, No. 6,349,71 9, 6,367,470, 6,543,442, 6,584,971, 6,601,581, 4,263,907, It is described in documents No. 5,709,202, No. 5,823,179, No. 6,192,876, No. 6,644,304, No. 5,549,102, No. 6,083,922, No. 6,161,536, No. 6,264,922, No. 6,557,549, and No. 6,612,303.

[0261] Commercially available nebulizers that can be used with droplets or droplet-shaped compositions containing the components of the combinations described herein include: Respirgard II®, Aeroneb®, Aeroneb® Pro, AeroEclipse XL®, and Aeroneb® Go, manufactured by Aerogen (Aerogen, Inc., Galway, Ireland); AERx® and AERx Essence®, manufactured by Aradigm; Porta-Neb®, Freeway Freedom®, SideStream, SideStream Plus, Ventstream, and I-neb, manufactured by Respironics, Inc. (Murrysville, Pennsylvania, USA); and PARI LC-Plus®, PARI, GmbH, manufactured by PARI Respiratory Equipment, Inc. (Pari Respiratory Equipment, Inc., Midlothian, Virginia, USA; PARI GmbH, Starnberg, Germany). Examples include LC-Star®, PARI LC-Sprint®, and e-Flow®. Any of these nebulizers may be used with either a face mask or a mouthpiece, according to the manufacturer's specifications. As a further non-limiting example, U.S. Patent No. 6,196,219 is incorporated herein by reference in its entirety.

[0262] In one embodiment, an aqueous formulation containing soluble or nanoparticle drug particles is provided. In the aqueous aerosol formulation, the drug may be present at a concentration of about 0.67 mg / mL to a maximum of about 700 mg / mL, and in a particular preferred embodiment, the nitrite is present at a concentration of about 0.667 mg of nitrite anions per ml to about 100 mg of nitrite anions per ml. Such formulations provide effective delivery to the appropriate area of ​​the lungs and are more concentrated than aerosol formulations. The formulation has the additional advantage of allowing a large amount of drug substance to be delivered to the lungs in a very short period of time. In one embodiment, the formulation is optimized to provide a well-tolerated formulation. Thus, certain preferred embodiments are formulated to contain a nitrite (such as sodium nitrite, potassium nitrite, or magnesium nitrite), have a good taste, a pH of about 4.7 to about 6.5, an osmotic pressure of about 100 to about 3600 mOsmol / kg, and in certain further embodiments, optionally, an osmotic ion (e.g., chloride, bromide) concentration of about 30 to about 300 mM.

[0263] In one embodiment, the solution or diluent used to prepare the aerosol formulation, in a single vial configuration, 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 admixture), or about 7.0 to about 9.0. This pH range improves tolerability as it incorporates flavoring agents according to certain embodiments as described elsewhere in this specification. If the aerosol is acidic or basic, it can cause bronchospasm and cough. The safe range of pH is relative, and some patients may tolerate mildly acidic aerosols, while others will experience bronchospasm. Any aerosol with a pH below about 4.5 will typically induce bronchospasm. Aerosols with a pH of about 4.5 to about 5.5 will occasionally cause bronchospasm. Any aerosol with a pH above about 8 may have low tolerability, as body tissues are generally unable to buffer alkaline aerosols. Aerosols with a pH controlled between approximately 4.5 and 8.0 typically cause lung irritation accompanied by severe bronchospasm, cough, and inflammatory responses. For these reasons, and to avoid bronchospasm, cough, or inflammation in patients, the optimal pH for aerosol formulations was determined to be approximately pH 5.5 to approximately pH 8.0.

[0264] As a result, in one embodiment, the aerosol formulations for use described herein are adjusted to a pH between about 4.5 and about 7.5, with a pH range of about 4.7 to about 6.5 being most preferred for acidic admixtures and a pH range of about 7.0 to about 8.0 being most preferred for single vial configurations. In non-limiting examples, compositions according to certain embodiments disclosed herein may also include pH buffers or pH adjusters, typically salts prepared from organic acids or bases, and in preferred embodiments, may include acidic excipients described herein (e.g., non-reducing acids such as citrates such as citric acid or sodium citrate), or buffers such as citrates or other buffers as described above and refer to Table 1. Accordingly, these and other representative buffers include organic salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, tromethamine, hydrochloride, or phosphate buffers.

[0265] Many patients exhibit increased sensitivity to various chemical tastes, including bitter, salty, sweet, and metallic tastes. To produce well-tolerated drug products, flavoring can be achieved through the addition of flavoring agents and excipients, adjustment of osmotic pressure, and the use of sweeteners.

[0266] Many patients exhibit increased sensitivity to various chemical agents and have a high incidence of bronchospasm, asthma, or other coughs. The patient's airways are particularly sensitive to hypotonic or hypertonic conditions, acidic or alkaline conditions, and the presence of osmotic ions such as chlorides. Imbalances in these conditions or the presence of chlorides exceeding certain concentrations can lead to bronchospasmodic or inflammatory events and / or coughs, severely impairing treatment with inhalation-suitable formulations. Both of these conditions can hinder the efficient delivery of aerosolized drugs into the bronchial space without the advantageous use of regulated pH, osmotic pressure, and flavoring agents as disclosed in certain embodiments herein.

[0267] In some embodiments (or in separate embodiments of nitrite-donating compounds or nitric oxide-donating compounds), the osmotic pressure of an aqueous solution of the nitrite-donating compound disclosed herein is such that the excipient The delivery is adjusted by providing. In some cases, certain amounts of permeable ions, such as chlorides, bromides, or other anions, may facilitate the successful and effective delivery of aerosolized nitrites. However, it has been found that in the nitrite components disclosed herein, the amount of such permeable ions may typically be less than the amount used for the aerosolized administration of other drug compounds.

[0268] Bronchospasm or cough reflexes are not always improved by the use of a diluent for aerosolization having a given osmotic pressure. However, these reflexes can often be well controlled and / or suppressed if the osmotic pressure of the diluent is within a certain range. Preferred solutions for aerosolization of safe and acceptable therapeutic compounds have a total osmotic pressure of about 100 to about 3600 mOsmol / kg at various chloride concentrations of about 30 mM to about 300 mM, preferably about 50 mM to about 150 mM. This osmotic pressure controls bronchospasm, and the chloride concentration as an osmotic anion controls cough. Since both bromide and iodide anions are osmotic ions, chloride may be substituted with them. In addition, chloride ions may be substituted with bicarbonates.

[0269] Furthermore, the nanoparticle drug dispersion can be freeze-dried to obtain a powder suitable for nasal or pulmonary delivery. Such a powder may contain aggregated nanoparticle drug particles having a surface modifier. Such aggregates may have a size of MMAD within a range suitable for respiration, for example, about 2 to about 5 microns.

[0270] 2. Delivery of gas generated from the NO production reaction to the nose, mouth, respiratory tract, or lungs (or multiple organs). Inhalers for quantitatively delivering nitric oxide to a patient's lungs are well known. Generally, nitric oxide is generated remotely and delivered to a hospital or clinic in a pressurized cylinder connected to a dedicated delivery device for use. As an example, the INOmax therapeutic system can be mentioned (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 for the cylinders of the INOmax therapeutic system and the INOvent delivery device. An evaluation of the INOmax therapeutic system is published, for example, in Kirmse, et al., Chest, June 1998, 113(6), pages 1650–1657. The disclosures of this publication are incorporated herein by reference.

[0271] The method according to the first aspect of the present invention may preferably be carried out in a dedicated NO production facility, and the gaseous product according to the second aspect of the present invention is provided to the user in a pressurized cylinder in a conventional manner. The pressurized gas cylinder is then used in a known manner in conjunction with a dispensing, monitoring, dosing, mixing, and delivery device.

[0272] Targets of antibacterial use As described above, the NOx generation reaction of this disclosure and the gas produced thereby have potentially broad-spectrum biocidal or biostatic effects against microorganisms, leading to numerous antibacterial applications.

[0273] Bacteria can be one or more selected from, for example, bacterial cells, viral particles, and / or fungal cells, or microparasites, and can be individual cells, organisms, or colonies. Bacterial cells, viral particles, and / or fungal cells, or microparasites can be present on or within a host organism, for example, as enterobacteria in humans or other animals, or as bacterial infections in humans or other animals. Bacteria, and / or fungal cells, and / or viral particles, and / or microparasites can be studied in vitro, in It could be Vivo or Exvivo.

[0274] This disclosure may be particularly useful in the treatment or prevention of bacterial infections at sites of skin lesions in subjects. This disclosure may be particularly useful in the treatment of bacterial infection prevention in immunosuppressed subjects.

[0275] In bacterial, fungal, viral, or microparasitic infections in humans or other animals, if bacteria are present, the infection may be in the context of a disease such as the common cold, influenza, tuberculosis, SARS, COVID-19, pneumonia, or measles.

[0276] 1. Bacterial cells Bacteria can be pathogenic bacterial species. Bacterial infections can be caused by pathogenic bacterial species, including Gram-positive and Gram-negative, aerobic and anaerobic, and antibiotic-susceptible and antibiotic-resistant bacteria.

[0277] Examples of bacterial species that can be targeted using the present invention include species of the genera Actinomyces, Bacillus, Bartonella, Bordetalla, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Heliobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia. Any combination thereof can also be targeted by the present invention.

[0278] In certain embodiments, the bacteria may be pathogenic species of Corynebacterium, Mycobacterium, Streptococcus, Staphylococcus, Pseudomonas, or any combination thereof.

[0279] In a more specific embodiment, the targeted bacteria are Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii; Borrelia afzelii; Borrelia recurrentis; Brucella abortus; Brucella canis; Brucella melitensis; Brucella suis; Campylobacter jejuni; Chlamydia pneumoniae; Chlamydia trachomatis; Chlamydophila psittaci; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheria; Ehrlichia canis; Ehrlichia chaffeensis; Enterococcus faecalis; Enterococcus faecium; Escherichia coli including Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli, Enteroinvasive E. coli (EIEC), and Enterohemorrhagic (EHEC) such as E. coli O157:H7; Francisella tularensis; Haemophilus influenza; Helicobacter pylori; Klebsiella pneumoniae; Legionella pneumophila; Leptospira species; Listeria monocytogenes; Mycobacterium leprae; Mycobacterium tuberculosis; Mycobacterium abscessus; Mycobacterium ulcerans; Mycoplasma pneumoniae; Neisseria gonorrhoeae; Neisseria meningitides; Pseudomonas aeruginosa; Nocardia asteroids; Rickettsia rickettsia; Salmonella typhi; Salmonella typhimurium; Shigella sonnei;Shigella dysenteriae;Staphylococcus aureus;Staphylococcus epidermidis;Staphylococcus saprophyticus;Streptococcus agalactiae; Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus viridans; Treponema pallidum subspecies pallidum; Vibrio cholera; Yersinia pestis; and any combination thereof may be selected.

[0280] In particular, the bacteria may be selected from Chlamydia pneumoniae, Bacillus anthracis, Corynebacterium diphtheria, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or any combination thereof.

[0281] The bacteria may be antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species, or antibiotic-resistant or antibiotic-sensitive strains of a bacterial species. The treatment of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) using nitric oxide is described, for example, in WO02 / 20026, the disclosure of which is incorporated herein by reference. Thus, examples of antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species that can be killed or treated using the present invention are methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

[0282] 2.Fungal cells Bacteria can be pathogenic fungal species. Bacterial infections can be infections caused by pathogenic fungal species, including pathogenic yeasts.

[0283] Examples of fungal species that can be targeted using the present invention include Aspergillus, Blastomyces, Candida (e.g., Candida auris), Coccidioides, Cryptococcus (especially Cryptococcus neofromans or Cryptococcus gattii), Hisoplamsa, Murcomycetes, Pneumocystis (e.g., Pneumocystis jirovecii), Sporothrix, Talaromyces, or any combination thereof.

[0284] Examples of fungal infections include aspergillosis (such as allergic bronchopulmonary aspergillosis), tinea pedis (athlete's foot), infections caused by pathogenic Candida species such as vaginal candidiasis, fungal onychomycosis, as well as diaper rash, tinea cruris (ringworm), and tinea corporis (ringworm). It can be listed.

[0285] 3. Virus particles Bacteria can be viral particles. Infection can be caused by pathogenic viruses.

[0286] Examples of viruses that can be targeted using the present invention include influenza viruses, parainfluenza viruses, adenoviruses, noroviruses, rotaviruses, rhinoviruses, coronaviruses, respiratory syncytial virus (RSV), astroviruses, and hepatitis viruses. In particular, the compositions of the present invention can be used to treat or prevent infections caused by one of the groups selected from H1N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory polynuclear virus, bovine parainfluenza-3 virus, SARS-CoV, SARS-CoV-2, and any combination thereof.

[0287] In particular, the present invention may be applied to the treatment of diseases or disorders caused by viral infections. Examples of such diseases that may be targeted by the present invention include respiratory viral diseases, gastrointestinal viral diseases, febrile viral diseases, hepatitis viral diseases, skin viral diseases, hemorrhagic viral diseases, and neuroviral diseases.

[0288] Respiratory viral infections include influenza, rhinovirus (i.e., the common cold virus), respiratory syncytial virus, adenovirus, coronavirus infection, such as COVID-19, and severe acute respiratory syndrome (SARS). Gastrointestinal viral diseases include norovirus infection, rotavirus infection, adenovirus infection, and astrovirus infection. Febrile viral diseases include measles, rubella, chickenpox, herpes zoster, roseola, smallpox, disease V, and chikungunya virus disease. Hepatitis viral diseases include hepatitis A, B, C, D, and E. Skin viral diseases include genital warts, oral herpes, genital herpes, and warts such as molluscum contagiosum. Hemorrhagic viral diseases include Ebola, Lhasa fever, dengue fever, yellow fever, Marbug 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.

[0289] 4. Parasitic microorganisms Bacteria can be parasitic microorganisms (microparasites). Infections can be caused by pathogenic parasitic microorganisms.

[0290] Examples of parasitic microorganisms that can be targeted using the present invention include protozoa.

[0291] In particular, the present invention may target protozoa such as sarcozoans (e.g., amoebas 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.

[0292] Parasitic infections that can be treated using the present invention include malaria, amoebic dysentery, and leishmaniasis (e.g., cutaneous leishmaniasis, mucocutaneous leishmaniasis, or visceral leishmaniasis).

[0293] Human / animal host or subject The subject may be an animal or a human subject. The term “animal” as used herein may generally include humans; however, when the term “animal” appears in phrases such as “animal or human subject,” the context will be understood to mean either a non-human animal in particular, or the reference to “human” is merely specifying the option that an animal could be a human in order to avoid ambiguity.

[0294] In certain embodiments, the subject is a human subject. The human subject may be an infant or an adult.

[0295] In certain embodiments, the subject is a vertebrate subject. Vertebrates may be from the classes Agnatha (jawless fish), Chondrichthyes (chondriac fish), Osteichthyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), or Mammalia (mammals). In certain embodiments, the subject is an animal subject from the classes Mammalia or Aves.

[0296] In certain embodiments, the subject is a domesticated animal. The domesticated animal may be one of the following: - Commensal organisms adapted to human niches (e.g., dogs, cats, guinea pigs) - Game or farm animals obtained or farmed for food (e.g., cattle, sheep, pigs, goats), and - Animals primarily used for towing (e.g., horses, camels, donkeys)

[0297] Examples of livestock include, but are not limited to, alpacas, adaxes, bison, camels, canaries, capybaras, cats, cattle (including Balinese cattle), chickens, collared peccaries, deer (including fallow deer, Japanese deer, white-lipped deer, and white-tailed deer), dogs, donkeys, silver doves, ducks, elands, moose, emus, ferrets, galloes, goats, geese, guinea fowl, guinea pigs, kudus, horses, llamas, minks, moose, mice, mules, musk oxen, ostriches, parrots, pigs, doves, quail, rabbits, rats (including African spiders), reindeer, white oryx, sheep, turkeys, buffalo, yaks, and zebu cattle.

[0298] Animal / human host or subject organs, structures, and internal spaces The organs to which the compositions or multi-component systems of this disclosure are administered are not limited. Examples of organs include the skin, as well as organs of the respiratory system, urinary system, cardiovascular system, digestive system, endocrine system, excretory system, lymphatic system, immune system, cutaneous system, muscular system, nervous system, reproductive system, and skeletal system.

[0299] Examples of cardiovascular organs include the heart, lungs, blood, and blood vessels. Examples of digestive organs include the salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, and anus. Examples of endocrine organs include the hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland, and adrenal glands. Examples of excretory organs include the kidneys, ureters, bladder, and urethra. Examples of lymphatic organs include lymph, lymph nodes, and blood vessels. Examples of immune organs include the tonsils, adenoids, thymus, and spleen. Examples of cutaneous organs include the skin, hair, and nails of mammals, as well as the scales of fish, reptiles, and birds, and the feathers of birds. Examples of nervous organs include the brain, spinal cord, and nerves. Examples of reproductive organs include the ovaries, fallopian tubes, uterus, vulva, vagina, testes, vas deferens, seminal vesicles, prostate gland, and penis. Examples of skeletal organs include bones, cartilage, ligaments, and tendons.

[0300] Human cavities, while not limited to those mentioned above, include the mouth, nose, ears, throat, respiratory tract, lungs, digestive tract, dorsal cavities such as the cranial or spinal cavity, ventral cavities such as the thoracic cavity, abdominal cavity, or pelvic cavity. The administration routes through the nose, mouth, respiratory tract, and lungs (multiple routes are possible) are characteristic features of this invention. .

[0301] In vitro antibacterial treatment of the surface The components and compositions of this disclosure, as well as the gases generated from NOx production reactions according to this disclosure, may be used to apply antibacterial treatment in vitro. "In vitro" means that the surface being treated is not a living organism, even if it may ultimately be intended for medical use.

[0302] Examples of such utility include methods for sterilizing surgical instruments, subcutaneous needles, and other medical devices before use, and for cleaning or treating surfaces in hospitals, clinics, or any other location to reduce or prevent the spread of pathogens.

[0303] Other examples include methods for sterilizing prosthetic organs 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 instruments, intraocular lenses, cochlear implants, implants that reduce pain by electrical stimulation, implantable contraceptive devices, nerve stimulators, artificial heart valves, electrodes, and intravenous infusion and drug delivery devices before placing the device inside the body of the subject.

[0304] Where desired, components or compositions of the present disclosure may be coated onto the surface of an artificial organ or implantable device so that NO generated in a NOx production reaction may perfuse other tissues or organs, or exert other physiological effects in the vicinity of the artificial organ or implantable device.

[0305] Techniques for biocompatible the surface of prostheses or implantable devices, including the incorporation of functional coatings such as coatings containing the components or compositions of the present disclosure, are well known to those skilled in the art. For example, all of their disclosures are incorporated herein by reference by Gultepe et al., Advanced Drug Delivery See Reviews, 8 March 2010, 62(3), pages 305–315, and U.S. Patent Nos. 5,702,754 and 6,270,788, and the publications referenced therein.

[0306] Compositions and methods for more general antibacterial treatment of non-living surfaces are well known in the art and do not require further explanation herein. Antibacterial compositions are used, for example, in the healthcare industry, food service industry, meat processing industry, and the private sector by individual consumers. Antibacterial cleaning compositions typically contain, in an aqueous and / or alcoholic carrier, one or more active antibacterial agents or their components, surfactants, and one or more other components, such as dyes, fragrances, pH adjusters, thickeners, skin conditioners, etc. A wide range of disinfectant or antibacterial compositions are intended to reduce the pathogenic load of various pathogens on a surface. Typically, the composition is a liquid (or is made liquid from a solid premixture before use), and the liquid, after any desired concentration adjustment, is preferably diffused or sprayed onto the surface to be treated, often with the help of a cloth or other wiping device, preferably by adding water, and then left to dry or wiped off. Conventional compositions and methods for surface treatment are, in principle, applicable to use in conjunction with the present invention, thereby the active antibacterial agent being or comprising the NOx-generating composition or its components according to the present invention.

[0307] Further consideration and examples of known antibacterial compositions and methods of use that may be used in connection with the present invention are, for example, incorporated herein by reference in their entirety. This document refers to U.S. Patent Nos. 6,110,908, 5,776,430, 5,635,462, 6,107,261, 6,034,133, 6,136,771, and 8,034,844, European Patent Application No. EP0505935, and PCT Patent Applications WO98 / 01110, WO95 / 32705, WO95 / 09605, and WO98 / 55096.

[0308] Use to improve the well-being of humans and / or animals In addition to the medical uses discussed above, this disclosure may be used for non-therapeutic uses in human or animal subjects. Non-therapeutic uses are distinguished from therapeutic uses in that the subject is healthy or the application does not target the treatment of any diagnosed disease, disorder, or condition the subject has.

[0309] Non-therapeutic uses include treatments aimed at improving the well-being or sense of well-being of an individual, or increasing their metabolic efficiency or immune system activity, so that they can function normally or better resist future infections. Non-therapeutic uses also include treatments that support cognitive function or create a sense of confidence and control in the individual.

[0310] For such non-therapeutic uses, the combinations and compositions disclosed herein may be formulated in a manner similar to or in a non-pharmaceutical manner than pharmaceutical formulations. For further details of formulations similar to pharmaceutical formulations, see the paragraph under the heading “Optional Additional Components” above. Non-pharmaceutical formulations may preferably include food additives, nutritional supplement formulations, foods, beverages, and beverage additives. Formulations adapted for addition to foods and beverages may preferably be in liquid or powder form. Nutritional supplement formulations may preferably be in the form of tablets, capsules, or orally ingestible liquids.

[0311] As noted above in the paragraph with the heading “Use in Therapeutic or Surgical Procedures,” the medical and / or surgical uses of this disclosure may provide secondary benefits to patients in terms of improved well-being or confidence.

[0312] plant use The beneficial effects of nitric oxide on living or dead plants are known. This disclosure includes methods, apparatus, combinations, kits, compositions, uses, and applications of gases therefrom for providing beneficial effects on living or dead plants.

[0313] Known uses of nitric oxide and nitric oxide production systems in plants include: Nitric oxide is used to prevent or delay wilting of cut flowers and plants (see Siegel-Itzkovich, BMJ, 1999; 319(7205), page 274, and Mur et al., 2013; “Nitric oxide in plants: an assessment of the current state of knowledge”, AoB PLANTS doi:10.1093 / aobpla / pls052 (https: / / doi.org / 10.1093%2Faobpla%2Fpls052)). Nitric oxide plays a role in regulating plant-pathogen interactions, promoting hypersensitive plant responses, symbiosis with organisms in nitrogen-fixing nodules, development of lateral roots, adventitious roots and root hairs, and control of stomatal opening (see Mur et al., 2013 cited above); The role of antioxidants and nitric oxide in reactive oxygen species responses in plants (Verma et al., 2013; “Nitric oxide (NO) counteracts cadmium-induced cytotoxicity” in BioMetals). processes mediated by reactive oxygen species(ROS)in Brassica juncea:cross-talk See "between ROS, NO, and antioxidant responses"); 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), pages 1548-1553).

[0314] Each disclosure of the publications cited above is incorporated herein by reference.

[0315] Furthermore, the antibacterial effects of the nitric oxide production systems and gases generated therefrom of this disclosure, as described above in the paragraphs with headings “Use in Therapeutic or Surgical Procedures,” “Topical Antibacterial Use,” “Use in the Nose, Mouth, Respiratory System, and Lungs,” and “Targets for Antibacterial Use,” are equally applicable to targeting bacterial infections of plants, and this disclosure extends to such uses as well.

[0316] Known uses of nitric oxide and nitric oxide production systems in plants, and all other uses thereof, constitute further embodiments of the present disclosure when used in nitric oxide production reactions and / or together with nitric oxide, optionally other oxides of nitrogen produced thereby, and / or optionally its precursors.

[0317] The plants being treated may, in particular, be crops or household plants, i.e., plant species cultivated by humans.

[0318] Examples of crops include, but are not limited to, food crops such as grains, vegetables, and fruits; crops for pharmacokinetically active ingredients such as quinine; fiber crops such as cotton or flax; crops for other materials such as rubber and wood; and flower crops such as roses and tulips.

[0319] Further examples of crops for human food consumption include, but are not limited to, crops for producing rice, wheat, sugarcane, and other sugar crops; corn, soybean oil, potatoes, palm oil, cassava, legumes, sunflower seed oil, rapeseed oil, mustard oil, sorghum, millet, peanuts, beans, sweet potatoes, bananas, soybeans, cottonseed oil, peanuts, peanut oil, yams, tomatoes, grapes, onions, apples, coffee, mangoes, mangosteens, guavas, chilies, bell peppers, tea, cucumbers, oranges, walnuts, almonds, carrots, turnips, coconuts, tangerines, lemons, limes, strawberries, and hazelnuts. [Brief explanation of the drawing]

[0320] [Figure 1] The cumulative plot of nitric oxide (nmol NO per mg of nitrite) generated over time under different reaction conditions in Example 1 is shown. [Figure 2] The results from various tests described in Example 2 are shown below. [Figure 3] The results from various tests described in Example 2 are shown below. [Figure 4] The results from various tests described in Example 2 are shown below. [Figure 5] The results from various tests described in Example 2 are shown below. [Figure 6] The results from various tests described in Example 2 are shown below. [Figure 7] The results from various tests described in Example 2 are shown below. [Figure 8] The results from various tests described in Example 2 are shown below. [Figure 9] The results from various tests described in Example 2 are shown below. [Figure 10] The results from various tests described in Example 2 are shown below. [Figure 11] The results from various tests described in Example 2 are shown below. [Figure 12] The results from various tests described in Example 2 are shown below. [Figure 13] The results from various tests described in Example 2 are shown below. [Figure 14] The results from various tests described in Example 2 are shown below. [Figure 15] The results from various tests described in Example 2 are shown below. [Figure 16] The results from various tests described in Example 2 are shown below. [Figure 17] A schematic diagram of the equipment used for SIFT-MS measurement is shown. [Figure 18] The results from various tests described in Example 3 regarding the antibacterial activity of known antibiotic combinations, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions against M. abscessus are shown. [Figure 19] The results from various tests described in Example 3 regarding the antibacterial activity of known antibiotic combinations, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions against M. abscessus are shown. [Figure 20] The results from various tests described in Example 3 regarding the antibacterial activity of known antibiotic combinations, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions against M. abscessus are shown. [Figure 21] The results from various tests described in Example 3 regarding the antibacterial activity of known antibiotic combinations, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions against M. abscessus are shown. [Figure 22] The results from the tests described in Example 4 regarding the minimum inhibitory concentrations (MICs) of a solution containing citric acid, sodium nitrite, and mannitol against numerous clinical isolate cultures are shown. [Figure 23] The results from the tests described in Example 5 regarding the antibacterial activity of carboxylic acid-nitrite solutions, with and without polyols, against Pseudomonas aeruginosa are shown. [Figure 24]The results from the test described in Example 6 regarding the antibacterial activity against M. tuberculosis HN878 in THP-1 cells are shown. [Figure 25] The results from the test described in Example 6 regarding the antibacterial activity against M. tuberculosis HN878 in THP-1 cells are shown. [Figure 26] The results from the test described in Example 6 regarding the antibacterial activity against M. tuberculosis HN878 in THP-1 cells are shown. [Figure 27] The results from the test described in Example 6 regarding the antibacterial activity against M. tuberculosis HN878 in THP-1 cells are shown. [Figure 28] The results from the tests described in Example 7 regarding cytotoxicity (LDH cytotoxicity assay) and antibacterial activity against H1N1 influenza A virus in MDCK cells are shown below: (a) Cytotoxicity at MOI=0.002 (●) and MOI=0.02 (■) at various dilutions (horizontal axis is nitrite molar concentration) is shown in gray, with the cytotoxicity scale shown on the right (cytotoxicity at measured nitrite concentrations up to 0.015M was <1% of that of the LDH control); (b) Plate photographs are shown comparing MOI=0.002 and nitrite concentrations of 0.15M, 0.015M, and 0.0015M with oseltamivir (1 μM). The order of the plates cited in the preamble is the same as the order of the plates from left to right in the figure (there are two experiments, and the plates for each corresponding experiment are shown above and below). The rightmost pair of plates to the right of the pair of oseltamivir plates is the virus control. Cytotoxicity is shown below each pair of test plates as a percentage of the LDH control (average of three LDH assays at 24 hours post-infection). [Figure 29] The results of tests on the effectiveness of acidified solutions of citric acid and mannitol, buffered to pH 5.8 with sodium nitrite and sodium hydroxide, in killing M. abscessus, compared with amikacin and a negative control, under similar conditions (as described in Example 3), are shown. [Figure 30]An embodiment of the present invention described in Example 10 for use in the treatment of lung infections in humans is schematically shown. [Figure 31] A schematic diagram (right side of Figure 31) shows the point of contact between the liquid NO generating preparation according to the present invention and lung tissue, compared to inhaled gaseous nitric oxide (left side of Figure 31), for use in the treatment of lung infections in human subjects. [Figure 32] The results of the LDH cytotoxicity assay (Experiments 1 & 2) in Example 8 are shown. Data are expressed as the mean + standard deviation (SD) of the two experiments. The 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 bars (2-hour culture) are the left bar of each pair of bars in each case, and the red bars (24-hour culture) are the right 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 MOI 3.0 are shown. In Experiment 1, one virus yield reduction assay was performed using SARS-CoV-2 at four multiple infections (MOIs), and confirmed using back titration of the inoculum virus. In cells inoculated at MOI 3, 2.1 log 10 TCID 50 / ml was found in the virus control well after titration. A reduction in SARS-CoV-2 yield may be observed in some of the conditions tested. After 24 hours of incubation, almost no virus was detected in 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 two titrations. SD is shown as an error bar. The horizontal dotted line level at log10 TCID50 / ml values ​​for chloroquine and cell control represents the limit of detection (LOD) of the assay. [Figure 34(a)]The results of the antiviral test against SARS-CoV-2 in Example 8 (Experiment 2) at MOI 3.0 are shown. The methodology corresponds to the part of Experiment 1 at their respective MOIs, except that the formulation is the Experiment 2 formulation and the incubation was performed for 48 hours instead of 24 hours to increase the level of progeny virus. Data are expressed as the mean + standard deviation (SD) of the two titer measurements. The SD is shown as an error bar. The horizontal dotted line level at the log10 TCID 50 / ml values ​​for chloroquine and cell control is the limit of detection (LOD) of the assay. [Figure 34(b)] The results of the antiviral test against SARS-CoV-2 in Example 8 (Experiment 2) at MOI 0.3 are shown. The methodology corresponds to the part of Experiment 1 at their respective MOIs, except that the formulation is the Experiment 2 formulation and the incubation was performed for 48 hours instead of 24 hours to increase the level of progeny virus. Data are expressed as the mean + standard deviation (SD) of the two titer measurements. The SD is shown as an error bar. The horizontal dotted line level at the log10 TCID 50 / ml values ​​for chloroquine and cell control is the limit of detection (LOD) of the assay. [Figure 35] The results of the antiviral test against SARS-CoV in Example 9 at MOI 3.0 are shown. Two plates were microscopically checked and scored for cytopathic effects (CPE) before cell monolayer staining with crystal violet. In these plates, CPE was found in the form of necrotic tissue fragments on top of the underlying monolayer. The results for the two microscopically checked plates are shown. Data are single titer measurements for each condition. For the remaining plate, CPE could not be scored after crystal violet staining due to the excessively high density of the cell monolayer. The horizontal dotted line level at the log10 TCID 50 / ml value for the cell control is the limit of detection (LOD) of the assay. [Examples]

[0321] The following non-limiting embodiments are provided for further illustration of the present invention.

[0322] Materials, apparatus, and methods used in Examples 1 and 2 solution Stock solutions were prepared by dissolving appropriate masses of 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., Ohio, US) in deionized water. Deionized water (18.2 MΩ) was obtained from the Arium Mini lab water system (Sartorius, Germany).

[0323] Citric acid / citric acid buffer solution was prepared by the following two methods: 1. Titrate stock solutions of 0.1 M citric acid and 0.1 M sodium citrate using the volumes described by Sigma Aldrich, 2018 (https: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html). 2. For the preparation of either 0.1M or 1M, dissolve a known mass of citric acid in a small amount of deionized water, then titrate the 3M sodium hydroxide and deionized water stock solution to achieve the desired buffer solution pH (pH 3 to pH 6.2).

[0324] A similar ascorbic acid / ascorbic acid buffer solution was prepared by using ascorbic acid and sodium ascorbate instead of citric acid and sodium citrate in Method 1.

[0325] Polyol inclusion was achieved by dissolving a known mass of sodium nitrite in a stock solution of the polyol (e.g., either sorbitol or mannitol).

[0326] The order in which the buffer solution and stock components are added is not important, and any mixing order may be used.

[0327] All standard solutions were used within 48 hours of preparation. Phthalate (pH 4) and phosphate (pH 7) tablets dissolved in deionized water (Fisher Scientific UK) Calibration buffer solutions were prepared using (Ltd, Leicestershire, UK).

[0328] Starting up and verifying Selective Ion Flow Tube Mass Spectrometry (SIFT-MS) A Voice 200 selective 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.

[0329] Before 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 confirm that the capillary flow rate was within the operating tolerance. This was followed by benzene, toluene, ethylbenzene, and Manufacturer's calibration gas standard containing xylene (Syft Technologies) An automated verification procedure was performed using (Ltd, New Zealand). Finally, an in-house performance check was performed using a 10 ppm nitrogen dioxide standard (Air Products PLC, Surrey, UK).

[0330] NO generation procedure The SIFT-MS apparatus, reaction chamber, and gas pathway were configured as shown in Figure 17.

[0331] The temperature inside the reaction chamber was continuously monitored using an HT1 Temperature Smart Sensor (SensorPush, New York, US). The reaction chamber and a 670 mL plastic (bisphenol A-free (BPA-free)) clip-lock tube with a silicone seal (Tesco, Welwyn Garden City, UK) were attached to a pump that continuously circulated humid air through the chamber to the SIFT-MS inlet capillary. Vernon, W., and Humidification was achieved by pumping air through two Dreschel bottles containing deionized water, similar to the method described by Whitby, L. (1931) The quantitative humidification of air in laboratory experiments, Trans. Faraday Soc. 27, 248-255. This system was homogenized for 30 minutes before use. Continuous SIFT-MS scanning was initiated for real-time detection and quantification of NO, NO2, and HONO. Once stable baseline readings (consistent concentrations >2 minutes) were observed for these compounds, the samples were placed in the reaction chamber and monitored for 3 hours.

[0332] After SIFT-MS verification, the capillary inlet extension, heated to 120°C, was attached to the reaction chamber outlet via a T-junction, enabling SIFT-MS to sample the gas flowing out of the reaction chamber in real time.

[0333] Samples were prepared by weighing approximately 20 grams (20 gsm) of combed nonwoven polypropylene mesh per square meter (approximately 0.3 cm × 0.3 cm) from RKW-Group, Frankenthal, Germany, using a weighing boat (approximately 3 mg). A 10 μL droplet of the test solution or control solution was added to the center of the mesh, and then reweighed (ensuring the droplet was thoroughly absorbed into the mesh). Finally, the packed mesh in the weighing boat was placed in the reaction chamber, and a final 10 μL droplet of buffer solution was pipetteed to the center of the mesh. The reaction chamber was quickly sealed, and the generation of nitrogen species was observed instantaneously using a SIFT-MS interface.

[0334] Analysis of the generated gas The generated gas was analyzed using the selective ion mode of SIFT-MS, and scans were performed in continuous batches lasting 1000 seconds each. The mass of the following products was repeatedly scanned: nitrite at 30 m / z, nitrite at 48 m / z, nitrogen dioxide at 46 m / z, and nitric oxide at 30 m / z. Precursor cation: hydronium(H3O) + ), nitrosium (NO + ), and dioxygenyl (O2 + All three of the above were used to achieve these measurements. Air was flowed into the chamber at 660 ml / min, and this airflow was sampled at a flow rate of 2.7 ml / min at the SIFT-MS inlet.

[0335] pH measurement of all examples All pH measurements were performed using a Five Easy pH meter (Mettler Toledo, Switzerland) equipped with a glass electrode LE438 probe. A second pH meter, a handheld 205 probe (Testo, Alton, US), was used to measure the pH of this electrode. Accuracy was ensured. Fresh calibration buffer solution was used for daily calibration of the pH meter.

[0336] Example 1 Nitric oxide is produced by contacting 1M / c.pH3 citric acid with a mesh that has been inhaled with 1M sodium nitrite, with or without 1M polyol. The SIFT-MS apparatus, reaction chamber, and gas pathway were configured as shown in Figure 17 above.

[0337] Two test meshes were prepared by drawing 1M sodium nitrite solutions, each containing 1M mannitol and 1M sorbitol, into a mesh as described above.

[0338] A control mesh was prepared by inhaling a 1M sodium nitrite control solution containing polyol into the mesh as described above.

[0339] The samples were prepared by either of the two methods 1 and 2 described above. In each test, a 1 M citrate / citric acid buffer solution with a pH of approximately 3 was added to the test mesh and the control mesh respectively to initiate gas generation as described above.

[0340] The results are shown in Figure 1.

[0341] The data shows that when the mesh also contained 1M mannitol or 1M sorbitol (mannitol has a greater effect than sorbitol), a 1M sodium nitrite inhalation mesh contacted with 1M / c. pH3 citrate produced significantly more nitric oxide than when the polyol was not present.

[0342] Example 2 Investigation of the effects of different carboxylic acids, acid concentrations, pH, and polyols on nitric oxide generation. Samples were prepared as shown above, while varying the organic acid, pH, and polyol as follows. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0343] The SIFT-MS apparatus, reaction chamber, and gas pathway were configured as shown in Figure 17 above.

[0344] The aforementioned test solution was drawn into the aforementioned mesh to create a test mesh.

[0345] When used, a control mesh was prepared by drawing a 1M sodium nitrite control solution containing polyol into the mesh as described above.

[0346] The buffer solution described above, or each buffer solution having the pH described above, was prepared by either of the two methods 1 and 2 described above and added to the test mesh and, if used, the control mesh in each test to initiate gas generation as described above.

[0347] The results are shown in Figures 2-13. In the figures, "normal" refers to the absence of polyols.

[0348] Figure 2 compares the NO generation rates produced by citrate / citric acid buffer or ascorbic acid / ascorbic acid buffer (pH approximately 3) in the absence of polyols. The graph clearly shows that citrate / citric acid buffer produces a higher initial eruption at a higher level than ascorbic acid / ascorbic acid buffer, and the generation lasts longer. Citrate / citric acid buffer peaks at approximately 55,000 ppb, while ascorbic acid / ascorbic acid buffer peaks at approximately 28,000 ppb.

[0349] Figure 3 relates to citrate / citric acid buffer and nitrite systems with and without polyols. The polyol concentration is 1 M. The generation rate, initial eruption, and resulting release over time change in the presence of polyols compared to the absence of polyols. Xylitol and mannitol produce the highest peaks, followed by sorbitol, then without polyols, and then arabitol. In the 500-1000 sec range, xylitol and arabitol have the highest output, followed by mannitol, sorbitol, and then without polyols. Peak eruption: mannitol = xylitol (approx. 64000 ppb) > sorbitol (approx. 53000 ppb) > without polyols (approx. 50000 ppb) > arabitol (approx. 40000 ppb).

[0350] Figure 4 shows the ascorbic acid / ascorbic acid buffer and nitrite systems with and without polyols. The polyol concentration is 1 M. The peak eruptions are mannitol (approx. 40,000 ppb) > arabitol (approx. 35,000 ppb) > xylitol = without polyol (approx. 30,000 ppb) > sorbitol (approx. 23,000 ppb), which is a different order from the citrate / citric acid buffer system in Figure 3.

[0351] Figure 5 shows the citrate / citric acid buffer and nitrite systems with and without polyols (the "no polyol" line, which has almost the same peak eruption as the mannitol line, is omitted for clarity). The polyol concentration is 0.5 M. Peak eruption: Arabitol (approx. 76,000 ppb) >> No polyol = Mannitol (approx. 48,000 ppb) > Xylitol = Sorbitol (approx. 40,000 ppb). This is a different order compared to a similar 1 M polyol citrate / citric acid buffer system (Figure 3), and it can be seen that the polyol effect depends on the polyol concentration.

[0352] Figure 6 shows the results for ascorbic acid / ascorbic acid buffer and nitrite systems with and without polyols (the "no polyol" line, which has almost the same peak eruption as the sorbitol line, is omitted for clarity). The polyol concentration is 0.5 M. Peak eruptions are xylitol (approx. 50,000 ppb) > mannitol (approx. 38,000 ppb) > sorbitol = no polyol (approx. 30,000 ppb) > arabitol (approx. 23,000 ppb). Again, a different order is observed when compared with similar citrate / citric acid buffer (0.5 M polyol) and ascorbic acid / ascorbate (1 M polyol) systems (Figures 5 and 4, respectively). Thus, it is demonstrated that the polyol effect depends on the polyol chemistry / stereochemistry and polyol molar concentration.

[0353] Figures 7 and 8 compare the NO generation rates using citrate / citrate buffer or ascorbic acid / ascorbic acid buffer, and in the presence of polyol (0.5 M). These graphs highlight some of the differences observed in Figures 2-6. In Figure 7, the citrate / citrate buffer peaks at approximately 76,000 ppb, while the ascorbic acid / ascorbic acid buffer peaks at approximately 22,000 ppb. In Figure 8, the citrate / citrate buffer peaks at approximately 48,000 ppb, while the ascorbic acid / ascorbic acid buffer peaks at approximately 38,000 ppb.

[0354] Figure 9 compares the cumulative output of 1M polyol concentrations. The difference at 3000 seconds in ascorbic acid / ascorbic acid buffer is smallest in the order of mannitol > sorbitol = arabitol > xylitol. In citrate / citric acid buffer at 3000 seconds, the order is xylitol > arabitol > mannitol > sorbitol > no polyol. The data shows that the nitric oxide output is, for example, without polyol (accumulated nitric oxide generation of approximately 10,000 nmol per mg of nitrite after 3000 seconds, and still increasing thereafter, curve E) compared to xylitol (accumulated nitric oxide generation of approximately 20,000 nmol per mg of nitrite after the same time). This shows that nitric oxide generation can be obtained, and the rate can be increased by up to approximately 100%, or even more than approximately 100%, compared to curve A), which is still rising.

[0355] Figure 10 compares the cumulative output of 0.5 M polyol concentrations. At 3000 seconds, with citrate / citric acid buffer, the order is arabitol > mannitol = xylitol > sorbitol > no polyol (for clarity, the "no polyol" line for citrate / citric acid buffer below the sorbitol line is omitted). At 3000 seconds, with ascorbic acid / ascorbic acid buffer, the order is xylitol > mannitol > sorbitol > arabitol. Again, this order differs compared to 1 M polyol (Figure 9).

[0356] Figures 11-13 compare cumulative plots of 1M citrate / citric acid buffer and 1M sodium nitrite containing mannitol (0.5M) at different pH levels. The difference decreased as the pH increased, disappearing at pH 6.2. Thus, these experiments suggest that the polyol effect is also pH-dependent.

[0357] Figure 14 shows the cumulative NO (nmol / cm² of mesh area) present in 1M sodium nitrite solution with and without glycerol (1M and 2M), and in citrate / citric acid buffer (1M, pH approximately 2). 2 The output is shown. Over the first 2000 seconds, the NO output of 1M and 2M glycerol is slightly lower than in the absence of polyol. Over longer periods, the glycerol-containing formulations have a greater output, and 2M glycerol has a greater output.

[0358] Figure 15 shows the cumulative NO (nmol / cm² of mesh area) in citrate / citrate buffer (1M, pH approximately 2) and 1M sodium nitrite solution, with or without polyols present in the nitrite solution. 2The output is shown. The plot shows that the presence of glycerol in the mannitol / nitrite solution reduces the output compared to the absence of glycerol. However, surprisingly, unlike with mannitol, the presence of glycerol in the sorbitol / nitrite solution increases the NO output compared to the absence of glycerol.

[0359] When using glycerol, a 1M glycerol solution was first prepared, which was then used to prepare a 1M sorbitol or 1M mannitol solution, and subsequently, a 1M nitrite solution.

[0360] Figure 16 shows the cumulative NO output (mol / mg of nitrite) of citrate / citric acid buffer (1M, pH 5.8) with and without mannitol (0.5M) present in a sodium nitrite (1M) solution. The plot shows that the presence of polyols results in a greater NO output after approximately 2000 seconds of reaction time.

[0361] Figure 16 shows that at physiologically important pH levels above approximately 5, and especially above approximately 5.5, mannitol enhances nitric oxide production compared to the same system without mannitol, providing a cumulative level of 1400 nmol NO per mg of nitrite after 10,000 seconds (167 minutes).

[0362] Example 3 Activity of various organic acid and nitrite solutions, with and without polyols, on M. abscessus cultures material 4.7g Middlebrook 7H9 Bros Base (Sigma-Aldric) h) was reconstituted with 900 ml of distilled water and autoclaved at 121°C for 15 minutes. Middlebrook ADC growth supplement (Sigma-Aldrich) was added to the autoclaved 7H9 solution (50 ml added per 450 ml, total 100 ml).

[0363] 1M Sodium Nitrite (Emsure): Dissolve 6.9g of sodium nitrite powder in 100ml of distilled water in a clean screw-top glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0364] 1M Citric Acid (Sigma-Aldrich): Dissolve 19.2g of citric acid powder in 100ml of distilled water in a clean screw-top glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0365] 1M Ascorbic Acid (Sigma-Aldrich): Add 17.6g of ascorbic acid powder to a sterile glass bottle. Dissolve completely in 100ml of sterile distilled water. Due to its short half-life, it was prepared daily using strict sterilization techniques. Due to its inherent instability, it was not autoclaved, but filtered through a 0.2μ filter before use.

[0366] 1M sodium citrate tribase dihydrate (Sigma-Aldrich): Dissolve 29.4g of sodium citrate powder in 100ml of distilled water in a clean screw-top glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0367] 1M sodium L-ascorbate (Acros Organics): Dissolve 19.8g of sodium ascorbate powder in 100ml of distilled water in a clean screw-top glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0368] D-mannitol (Sigma-Aldrich) was used in experiments involving polyols. The following stock solutions were formed by adding polyols to the sodium nitrite stock solution described above: Stock solution A - 1M sodium nitrite & 0.5M mannitol Stock solution B - 1.5M sodium nitrite and 0.5M mannitol

[0369] A 1.5M citric acid stock solution was also prepared.

[0370] The molar concentrations of each component were adjusted according to the dilution ratio, and the correct final molar concentration of each experimental solution was confirmed.

[0371] Mycobacterium abscessus (MAB) In this embodiment, the laboratory reference strain Mycobacterium abscessus ATCC19977lux was used for all experimental conditions.

[0372] methodology 50 ml Falcon tubes were labeled as Tube T (test suspension), Tube A (acid control), and Tube C (control).

[0373] 8 ml of 7H9+ADC supplement was added to each tube. Then, 100 μl of MAB suspension (previously grown to approximately 3-4 McFarland standards) was added. The baseline relative luminescence (RLU) of the MAB suspension was read. The contents were mixed by vortexing.

[0374] Tube contents when polyol (mannitol) is not present Tube T: 1 ml of sodium nitrite (1M) solution was added to the tube, and immediately afterwards, 1 ml of citric acid solution (1M) or ascorbic acid solution (1M) was added to obtain 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.

[0375] Tube A: 1 ml of citric acid solution (1M) or ascorbic acid solution (1M) was added to the tube, and 1 ml of sterile distilled water was added to produce a final volume of 10 ml, testing a concentration of 0.1 M relative to the acid. The contents were mixed by gentle inversion and incubated at 37°C for 24 hours.

[0376] Tube C: 2 ml of sterile distilled water was added to the tube to create a total volume of 10 ml. This serves as a control to evaluate growth under optimal conditions. The contents were mixed by gentle inversion and incubated at 37°C for 24 hours.

[0377] Contents of tube T when polyol (mannitol) is present When mannitol was present, the contents of tube T were as follows: 1. Tube T: 1 ml of sodium nitrite (1M) & mannitol (0.5M), and 1 ml of citric acid (1M) 2. Tube T: 1 ml of sodium nitrite (1.5 M) & mannitol (0.5 M), and 1 ml of citric acid (1 M) 3. Tube T: 1 ml of sodium nitrite (1M) & mannitol (0.5M), and 1 ml of citric acid (1.5M)

[0378] The activity of T, A, and C solutions was evaluated by measuring the RLU after 30-minute, 60-minute, and 24-hour incubations.

[0379] After a 24-hour incubation, tubes C, A, and T were plated onto Columbia Blood Agar (VWR chemical). The plates were incubated at 37°C for 72 hours. Colony-forming units (CFUs) were read on days 3, 5, and 7 of the incubation. All work was performed in a CL2 biological safety cabinet within the CL2 laboratory.

[0380] The results are shown in Figures 18-21.

[0381] Figure 18 shows that a solution of 0.1 M citric acid and 0.1 M nitrite (tube T) is effective in eliminating and reducing M. abscessus cultures after 7 days at pH values ​​of 5 and 5.5, compared to a solution of 0.1 M citric acid alone (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) is effective in eliminating and reducing M. abscessus cultures after 7 days at pH values ​​of 5.0, 5.5, and 6.0, compared to a solution of ascorbic acid alone (tube A) at pH values ​​of 6.5, 7.0, and 7.4.

[0382] Figure 19a) shows that a solution of 0.1 M citric acid and 0.1 M nitrite is effective in reducing the CFU of M. abscessus cultures after 3 days of incubation, and a solution of 0.1 M citric acid and 0.1 M nitrite containing 0.05 M mannitol is effective in almost completely eliminating M. abscessus cultures after 3 days of incubation. Figure 19b) shows that a solution of 0.1 M citric acid and 0.1 M nitrite without mannitol is effective. The figure shows that a nitrite solution is effective in maintaining the reduced CFU of M. abscessus after 5 days of incubation. The figure also shows that a solution of 0.1 M citrate and 0.1 M nitrite containing 0.05 M mannitol is effective in reducing the CFU of M. abscessus cultures after 5 days of incubation.

[0383] Figure 20a) shows that a solution of 0.15 M citrate and 0.1 M nitrite is effective in reducing the CFU of M. abscessus cultures after 3 days of incubation, and a solution of 0.15 M citrate and 0.1 M nitrite containing 0.05 M mannitol is effective in eliminating M. abscessus cultures after 3 days of incubation. Figure 20b) shows that a solution of 0.15 M citrate and 0.1 M nitrite without mannitol is effective in maintaining the reduced CFU of M. abscessus after 5 days of incubation. The figure also shows that a solution of 0.15 M citrate and 0.1 M nitrite containing 0.05 M mannitol is effective in eliminating M. abscessus cultures after 5 days of incubation.

[0384] Figure 21 shows that a solution of 0.1 M citrate and 0.15 M nitrite is effective in reducing the CFU of M. abscessus cultures after 3 days of incubation and in maintaining the reduction of the CFU after 5 days of incubation. The figure also shows that a solution of 0.1 M citrate and 0.15 M nitrite, containing 0.05 M mannitol, is effective in eliminating M. abscessus cultures after 3 and 5 days of incubation.

[0385] Example 4 Minimum inhibitory concentrations (MICs) of carboxylic acid-nitrite-polyol solutions against Mycobacterium abscessus (Mabs) and Mycobacterium tuberculosis (Mtb) in various clinical isolate cultures. Healthy applicants Peripheral blood samples were collected from healthy volunteers who provided written informed consent (see Ethical Approval REC No. 12 / WA / 0148).

[0386] Mycobacteria strains Both strains of Mycobacterium abscessus (ATCC19977) and Mycobacterium tuberculosis (H37RV) contained a bacterial luciferase (lux) gene cassette (luxCDABE) that enabled the measurement of relative luminescence (RLU) and conventional colony-forming unit (CFU) measurements of bacterial survival.

[0387] General Reagents [Table 2]

[0388] Treatment conditions Treatment 1: 0.15M citrate, 0.1M sodium nitrite, and 0.05M mannitol Treatment 2: 0.1M citrate, 0.15M sodium nitrite, and 0.05M mannitol

[0389] Broth microdilution minimum inhibitory concentration (MIC) The MICs for each treatment against Ma abscessus and Ma tuberculosis were measured according to the guidelines (M07-A9) developed by the Clinical and Laboratory Standards Institute for antibacterial susceptibility testing. Double dilutions of each treatment were prepared for all plates, and the plates were incubated at 37°C. Readings were taken on days 3 and 7 for Mabs and on days 14 and 21 for Mabs. The tests were performed twice.

[0390] All work was performed inside the CL2 biological safety cabinet within the CL2 experimental facility.

[0391] The minimum inhibitory concentration of a solution of 1.5 M citrate, 1 M sodium nitrite, and 0.5 M mannitol against M. abscessus was found to be 4.7 mM. Furthermore, the minimum inhibitory concentration of a solution of 1.5 M citrate, 1 M sodium nitrite, and 0.5 M mannitol against M. tuberculosis was found to be 2.3 mM.

[0392] The minimum inhibitory concentration of a solution of 1 M citrate, 1.5 M sodium nitrite, and 0.5 M mannitol against M. abscessus was found to be 3.1 mM. For M. tuberculosis, the minimum inhibitory concentration of a solution of 1 M citrate, 1.5 M sodium nitrite, Furthermore, it was found that the minimum inhibitory concentration of a 0.5 M mannitol solution was 1.6 mM.

[0393] Additionally, the following isolates from the M. abscessus clinical isolate library at Floto Laboratory, Cambridge University, UK (https: / / www.flotolab.com / ) are available: 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, 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, 1045, 1047, 1049, 1054, Minimum inhibitory concentrations (MICs) were determined by microdilutions of broth using strains 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 twice.

[0394] The test results for the clinical isolate are shown in Figures 22a) and b). The graphs show two MICs of nitric oxide against M. abscessus, using readings obtained at 3, 4, and 5 days after incubation of the isolate. A plate reading was also taken on day 7 of incubation, but no difference was observed compared to day 5. The laboratory strain ATCC19977lux was used as a control in both experiments, and the comparative results against the clinical isolate are shown.

[0395] Figure 22 shows that the citrate-nitrite-mannitol solution is effective across a wide range of clinical isolates. The minimum inhibitory concentration for most clinical isolates was within 0.02 M for the 0.1 M citrate, 0.15 M nitrite, and 0.05 M mannitol solution (Figure 22a), and within 0.04 M for the 0.15 M citrate, 0.1 M nitrite, and 0.05 M mannitol solution (Figure 22b).

[0396] In both figures, the MIC of a particular sample varied from day to day. These are the samples where two or more dots are shown above the identification code of the isolated strain sample. Generally, after several days of incubation, high MICs were observed, rather than low MICs, under those conditions. Overall, the combination of low citrate (0.1M) and high sodium nitrite (0.15M) (Figure 22(a)) is more effective than the combination of high citrate (0.15M) and low sodium nitrite (0.1M) (Figure 22(b)).

[0397] Additional data demonstrating the in vitro killing of M. abscessus by carboxylic acid-nitrite-polyol solutions are shown in Figure 29. This figure demonstrates the effectiveness of aqueous formulations of sodium nitrite, citrate buffered to pH 5.8 using sodium hydroxide solution, and mannitol in killing M. abscessus over a 24-hour period under similar conditions, compared to amikacin and a negative control.

[0398] Example 5 Antibacterial activity of carboxylic acid-nitrite solutions containing and without polyols against Pseudomonas aeruginosa Apparatus and culture medium UKAS calibrated pipette (100-1000 μL range) - Proline® Plus UKAS Calibrated Multichannel Pipettes (P300 and P20) - Gilson®, UK Universal Tube - SLS, UK Calibrated balance - HR-100A Microbial incubator - Heratherm®, ThermoFisher Scientific, UK Tryptone Soy Agar (TSA) - Southern Group Laboratories, UK Tryptone Soy Broth (TSB) - Acumedia®, SLS, UK Malt agar - Acumedia®, Acumedia®, SLS, UK Brain Heart Infusion Broth (BHIB) - Acumedia®, SLS, UK Sabouraud Dextrose Broth (SDB) - Acumedia®, SLS, UK Dey-Engley Neutralizer (DE-N) - Acumedia®, SLS, UK Citric Acid - Sigma, UK Sodium nitrite - Sigma, UK Mannitol - Sigma, UK Sorbitol - Sigma, UK

[0399] Test microorganisms Pseudomonas aeruginosa NCTC13618 - Isolated from a patient with cystic fibrosis.

[0400] formulation [Table 3]

[0401] 1-1M citric acid plus 1M sodium nitrite (with or without 0.5M polyol) 2-0.5M citric acid plus 1M sodium nitrite (with or without 0.5M polyol) 3-0.5M citric acid plus 0.5M sodium nitrite (0.5M polyoxide) (Includes or does not include the letter)

[0402] Dey-Engley Neutralizer Verification A 24-hour culture of Pseudomonas aeruginosa was collected from tryptone soy agar (TSA), and this was used to create 1 × 10⁶ cultures. 8 ±5 × 10 7 CFUmL -1 A suspension was prepared. This was further diluted in Brain Heart Infusion Broth (BHIB) to 1 × 10⁻⁶. 5 ±5 × 10 4 CFUmL -1 A diluted standard suspension was prepared.

[0403] The initial inoculum was identified by serial dilution and smear plating. The neutralizing agent was validated using control (9 mL of phosphate-buffered saline (PBS) and 1 mL of inoculum), toxicity (9 mL of Dey-Engley neutralizing agent (DE-N) and 1 mL of inoculum), and neutralizing agent efficacy (8 mL of neutralizing agent, 1 mL of test agent, and 1 mL of inoculum) samples. Following a 5-minute treatment, 200 μL of suspension was taken from each tube, serially diluted, and 100 μL was plated onto TSA. The agar plates were incubated at 37±2°C for 18–24 hours.

[0404] Antibacterial effectiveness against planktonic organisms A 24-hour culture of P. aeruginosa was collected from TSA and used to obtain 1 × 10⁶ 8 ±5 × 10 7 CFUmL -1 A suspension was prepared. This was further diluted in BHIB to 1 × 10⁻⁶ 6 ±5 × 10 4CFUmL -1 A diluted standard suspension was prepared. A universal tube was filled with 8 mL of bacterial solution.

[0405] 1 ml of citric acid solution and 1 ml of sodium nitrite solution were added to each test reagent to obtain the required concentrations as described above. The solutions were incubated at 37 ± 2 °C for 24 hours. Following the incubation period, 1 mL was taken from each tube and transferred to a tube containing 9 mL of neutralizing agent. Surviving organisms were quantified using serial dilution and plate counting.

[0406] The results are shown in Figure 23.

[0407] The data demonstrates antibacterial efficacy against the following Pseudomonas: - Citric acid (1M) mixed with nitrite (1M) ("Conc. 1"), with or without polyol (0.5M), -Citric acid (0.5M) mixed with nitrite (1M) ("Conc.2"), with and without polyol (0.5M), and - Citric acid (1M) mixed with nitrite (0.5M), with or without polyol (0.5M) ("Conc.3").

[0408] The citric acid solution has a pH of 5.2 (for formulations 1, 3, and 5) and 6.0 (for formulations 2, 4, and 6). Formulations 1 and 2 do not contain polyols, formulations 3 and 4 contain mannitol, and formulations 5 and 6 contain sorbitol.

[0409] All formulations demonstrate good efficacy at pH 5.2. At pH 6, formulations containing mannitol are slightly more effective.

[0410] Example 6 The efficacy of formulations containing nitrite, organic acid, and polyol against M. tuberculosis HN878 in THP-1 cells was evaluated.

[0411] formulation The formulations were prepared as shown in the table below, indicated by the suffix FC in the sample reference. When the preparation method is described as "concentration," it means that the formulation is first prepared as a concentrated premixture 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 respective desired concentrations listed in the table. When the preparation method is described as "dilution," indicated by FD at the end of the sample reference, it means that the formulation is first prepared as a premixture containing all three components at the desired concentrations, namely sodium nitrite (0.15 M), polyol (0.05 M), and acid (0.1 M), and then diluted with distilled water to reach the respective desired concentrations listed in the table.

[0412] For in vitro bacterial inhibition assays against M. tuberculosis HN878, various concentrations of sodium nitrite, namely 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml, were prepared in each formulation by serial dilution. [Table 4]

[0413] An MIC macrophage test was performed using the THP-1 macrophage (1) compound screening assay.

[0414] Macrophage preparation and culture: THP-1 cells were grown for 2 weeks. Then, 5 × 10⁻⁶ cells were cultured. 5 THP-1 cells were suspended in macrophage-containing complete DMEM medium at a concentration of cells / mL. 24-well tissue culture plates, 2mL per well (1×10⁶ cells per well). 6Cells were seeded in ). One 24-well plate of cells was tested three times with seven different drug concentrations plus an untreated control. In addition to the drug assay plate, one additional plate (or at least three additional wells) was seeded on the infection day to determine bacterial uptake. Cells were incubated in a humidified chamber at 37°C and 5% CO2. Complete DMEM medium, free of antibiotics / antifungals, remained unchanged during the 3-day assay.

[0415] Complete DMEM medium for macrophages: The following were added to Dulbecco's modified Eagle medium (Cellgro15-017-cv): Thermoinactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO, F-0500-A) (10%) L929 Prepared Medium (10%) L-Glutamine (Sigma G-7513) (2mM) HEPES buffer (Sigma H-0887) (10mM) Antibiotic / antifungal (Sigma A-9909) (1×) MEM non-essential amino acids (Sigma M-7145) (1×) 2-Mercaptoethanol (Sigma M-6250) (50nM)

[0416] L-929 Prepared Medium: 75cm 2 In a flask, 4.7 × 10⁶ L-929 (CCL-1) cells derived from ATCC were added to 55 mL of DMEM + 10% fetal bovine serum. 5 Cells were seeded. In THP-1 cells, the cells were grown for 3 days. On day 3, the supernatant was collected, filtered through a 0.45 μm filter, aliquoted, and frozen at -20°C. For THP-1 infection, the cell-free filtrate was used in DMEM medium.

[0417] Infection with THP-1 cells: On day 0, the culture medium was removed from the cells and replaced with 0.2 ml of antibiotic / antifungal-free DMEM containing M. tuberculosis HN878 at an MOI ratio of 1 macrophage to 10 bacteria. The tissue culture plate was placed inside a closed Ziploc bag and returned to the incubator. The bag was opened and placed inside the incubator. The cells were incubated with the bacteria for 2 hours. After infection, bacteria adhering to the outside of the cells were removed, each well was washed once with phosphate-buffered saline (PBS), and 2 ml of antibiotic / antifungal-free complete DMEM medium with various drug concentrations was added. To prepare drug concentrations, serial 2-fold dilutions were performed by adding the previous 10 ml suspension to 10 ml of complete medium plus serum in the next tube. The tissue culture plate was returned to a 37°C + 5% CO2 incubator (drugs were left in the wells for 3 days). Each drug concentration was tested in three wells.

[0418] Cell lysates were plated and the viability of THP-1 cells was evaluated 2 hours, 1, 2, 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 redistilled water + 0.05% Tween-80 was added to each well, and the cells were left 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 placed on 7H11 / OADC agar through a 1 / 1,000 dilution step. (To prepare serial dilutions, each 24-well TC plate of cells requires 4 24-well TC plates and 24 agar "quad" plates). Plates were incubated at 32°C for 30 minutes. The cells were incubated for several days, and the colonies were counted to determine the CFU / ml concentration.

[0419] result: In vitro THP-1 HN878 optical density results The minimum inhibitory concentration (MIC) reported as the most diluted composition that inhibits bacteria (i.e., the maximum dilution level of a particular formulation on a scale shown as 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / ml) [Table 5]

[0420] The results are shown in Figures 24-27.

[0421] Figure 24: The efficacy of 30RESP001FC and FD (concentrated and diluted) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of formulations 30RESP001FC (concentrated) (A) and 30RESP001FD (diluted) (B) was evaluated by intracellular death of M. tuberculosis HN878 (square, open) in THP-1 macrophages at 2 hours after infection (day 0), 1, 2, and 5 days after infection, and at 16 μg / ml (▲), 8 μg / ml (inverted triangle, open), 4 μg / ml (diamond, open), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (diamond, filled), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each of the plots in Figure 24, the lines marked with ▲ and (inverted triangle, white) for treatments at 16 μg / ml and 8 μg / ml, respectively, can be distinguished from the lines marked with ▲ and ▼ for treatments at 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments at 16 μg / ml and 8 μg / ml are more effective. In other words, the lines in the plots for treatments at 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than the treatments at 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the line marked with □ for treatment at 1 μg / ml can be easily distinguished from the line in the (square, white) plot for no treatment, because the treatment at 1 μg / ml is more effective. The line in the (square, white) plot for no treatment rises from day 1 onwards, reaching 1 × 10⁻⁶ 4 It has a CFU value that maintains a high level.

[0422] The 30RESP001FC and FD compositions, listed as "16 μg / ml" and referred to in the above MIC table and Figure 24, contain 0.15 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), respectively. The compositions at concentrations of 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml are 50% dilutions of the previous compositions in the order of 16 to 0.125 μg / ml (i.e., the concentration is halved).

[0423] THP-1 macrophages were infected with M. tuberculosis at a 1:10 MOI, and intracellular bacterial counts were determined immediately using bacterial colony counting (CFU) at 2 hours after infection (day 0), and at 1, 2, and 5 days. The values ​​shown are mean ± SD from one independent experiment. In particular, increased efficacy against M. tuberculosis HN878 was observed in treatment with 30RESP001FC and FD (concentrated and diluted) at 16 μg / ml and 8 μg / ml compared to the untreated control (*, p<0.05).

[0424] Figure 25: The efficacy of 30RESP002FC and FD (concentrated and diluted) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP002FC (concentrated) (A) and 30RESP002FD (diluted) (B) formulations was evaluated by intracellular death of M. tuberculosis HN878 (square, open) in THP-1 macrophages at 2 hours, 1, 2, and 5 days after infection, and at concentrations of 16 μg / ml (▲), 8 μg / ml (inverted triangle, open), 4 μg / ml (diamond, open), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (diamond, filled), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each of the plots in Figure 25, the lines marked with ▲ and (inverted triangle, white) for treatments at 16 μg / ml and 8 μg / ml, respectively, can be distinguished from the lines marked with ▲ and ▼ for treatments at 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments at 16 μg / ml and 8 μg / ml are more effective. In other words, the lines in the plots for treatments at 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than the treatments at 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the line marked with □ for treatment at 1 μg / ml can be easily distinguished from the line in the (square, white) plot for no treatment, because the treatment at 1 μg / ml is more effective. The line in the (square, white) plot for no treatment rises from day 1 onwards, reaching 1 × 10⁻⁶ 4 It has a CFU value that maintains a high level.

[0425] The 30RESP002FC and FD compositions, listed as "16 μg / ml" in the MIC table and Figure 25 above, contain 0.15 M sodium nitrite, 0.05 M lactitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), with the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions being 50% dilutions (i.e., halved concentrations) of the previous compositions, in the order of 16 to 0.125 μg / ml, respectively.

[0426] THP-1 macrophages were infected with M. tuberculosis at a 1:10 MOI, and intracellular bacterial counts were immediately determined using bacterial colony counting (CFU) 2 hours, 1, 2, and 5 days after infection. The values ​​shown are mean ± SD from one independent experiment. Increased efficacy against M. tuberculosis HN878 was observed in treatment with 30RESP002FC (concentrated) 16 μg / ml, and 30RESP002FD (diluted) 16 μg / ml and 8 μg / ml compared to the untreated control (*, p<0.05).

[0427] Figure 26: The efficacy of 30RESP003FC and FD (concentrated and diluted) against M. tuberculosis HN878 was evaluated in THP-1 cells. At 2 hours after infection (day 0), 1, 2, and 5 days later, and at 16 μg / ml (▲), 8 μg / ml (inverted triangle, white), 4 μg / ml (diamond, white), 2 μg / ml (○), and 1 μg / ml ( The effectiveness of 30RESP003FC (concentrated) (A) and 30RESP003FD (diluted) (B) for treatment at 16 μg / ml (□), 0.5 μg / ml (diamond, filled), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) was evaluated in terms of intracellular death of M. tuberculosis HN878 (square, open white) in THP-1 macrophages. In each plot in Figure 26, the ▲ and (inverted triangle, open white) plots for treatment at 16 μg / ml and 8 μg / ml, respectively, can be distinguished from the ▲ and ▼ plots for treatment at 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments at 16 μg / ml and 8 μg / ml are more effective. In other words, the lines in the plots for treatments at 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than the treatments at 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the line in the square plot for treatment at 1 μg / ml can be easily distinguished from the line in the no-treatment (square, open) plot because the 1 μg / ml treatment is more effective. The line in the no-treatment (square, open) plot rises from day 1 onwards, reaching 1 × 10⁻⁶ 4It has a CFU value that maintains a high level.

[0428] THP-1 macrophages were infected with M. tuberculosis at a 1:10 MOI, and intracellular bacterial counts were immediately determined using bacterial colony counting (CFU) 2 hours, 1, 2, and 5 days after infection. The values ​​shown are mean ± SD from one independent experiment. Increased efficacy against M. tuberculosis HN878 was observed in treatment with 30RESP003FC (enriched) 16 μg / ml and 8 μg / ml, and 30RESP003FD 16 μg / ml compared to the untreated control (*, p<0.05).

[0429] The 30RESP003FC and FD compositions, listed as "16 μg / ml" in the MIC table and Figure 26 above, contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), with the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions being 50% dilutions (i.e., halved concentrations) of the previous compositions, in the order of 16 to 0.125 μg / ml, respectively.

[0430] Figure 27: The efficacy of 30RESP004FC and FD (concentrated and diluted) against M. tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP004FC (concentrated) (A) and 30RESP004FD (diluted) (B) formulations was evaluated by intracellular death of M. tuberculosis HN878 (square, open) in THP-1 macrophages at 2 hours after infection (day 0), 1, 2, and 5 days after infection, and at concentrations of 16 μg / ml (▲), 8 μg / ml (inverted triangle, open), 4 μg / ml (diamond, open), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (diamond, filled), 0.25 μg / ml (▲), and 0.125 μg / ml (▼). In each of the plots in Figure 27, the lines marked with ▲ and (inverted triangle, white) for treatments at 16 μg / ml and 8 μg / ml, respectively, can be distinguished from the lines marked with ▲ and ▼ for treatments at 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments at 16 μg / ml and 8 μg / ml are more effective. In other words, the lines 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 line marked with □ for treatment at 1 μg / ml can be easily distinguished from the line for no treatment (square, white) because the treatment at 1 μg / ml is more effective. The line for no treatment (square, white) rises from day 1 onwards, reaching 1 × 10⁻⁶ 4 It has a CFU value that maintains a high level.

[0431] The 30RE mentioned in the MIC table and Figure 27 above is listed as "16 μg / ml". The SP004FC and FD compositions each contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M ascorbic acid / ascorbic acid (final molar concentration after dilution), with each of the 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / ml compositions being 50% dilutions of the previous compositions (i.e., halved in concentration) in the order of 16 to 0.125 μg / ml, respectively.

[0432] THP-1 macrophages were used to infect M. tuberculosis at a 1:10 MOI, and intracellular bacterial counts were immediately determined at 1, 2, and 5 days using bacterial colony counting (CFU). The values ​​shown are mean ± SD from one independent experiment. Treatment with 30RESP004FC (enriched) at 16 μg / ml and 8 μg / ml showed increased efficacy against M. tuberculosis HN878 compared to the untreated control (*, p<0.05).

[0433] The formulation is concluded to exhibit in vitro inhibition of M. tuberculosis HN878 at a suitable dosage exceeding the MIC.

[0434] Furthermore, it should be noted that the method of preparing the formulations may affect their in vitro antibacterial effect against M. tuberculosis HN878 in the test of Example 6.

[0435] This is demonstrated by comparing the efficacy of formulation 1 at an 8 μg / ml concentration between its FC and FD versions (Figure 24A vs. 24B). The efficacy of the FC version increases strongly over at least 5 days after incubation, while the efficacy of the FD version does not increase as strongly over the same period. This is in contrast to the 16 μg / ml concentration, which shows very similar good efficacy between the FC and FD versions over the same period.

[0436] Different behavior is observed in formulation 2 (25A vs. 25B). The efficacy of the FD version at a concentration of 16 μg / ml increases more strongly than the FC version for the first two days after incubation, and then remains unchanged. However, the efficacy of the FD version is good and the efficacy of the FC version is very good up to 5 days after incubation. At a concentration of 8 μg / ml, the efficacy of the FD version increases strongly to good efficacy for at least 5 days after incubation, while the efficacy of the FC version does not increase as strongly over the same period.

[0437] Therefore, at least at higher concentrations, it has been shown that the step of adding water to reach the final inhibitory formulation can materially affect the antibacterial activity of the formulation, both in terms of initial antibacterial activity and the degree of bacterial death over 5 days. Generally, although not universal, a better antibacterial activity over a period of 0 to 5 days after incubation is obtained by first preparing a formulation as a concentrated premixture of sodium nitrite, polyol, and acid components at a desired relative molar ratio but at a higher concentration than desired for use (e.g., at least 3 times, e.g., at least 5 times or more, e.g., about 3 to about 80 times or more, e.g., about 3 to about 80 times or more), and then simply diluting the concentrate to obtain a formulation for use.

[0438] Example 7 Cytotoxicity and antiviral activity of carboxylic acid-nitrite-polyol solution against H1N1 influenza A virus The test formulations designated as F1C1, F1C2, and F1C3, corresponding to formulation 30RESP001FC in Example 6, its 10-fold dilution, and its 100-fold dilution, respectively, were used in the Oceanic experiment. When used with lutemivir solution (1 μM) and a viral control, comparative cytotoxicity and H1N1 influenza A virus killing effect were obtained in MDCK cells after 24 hours. Cytotoxicity was assayed using an LDH cytotoxicity assay similar to that in Example 8. Antibacterial activity against H1N1 influenza A virus was measured in MDCK cells diluted in various ranges (horizontal axis is nitrite molar concentration) at MOI = 0.002 (●) and MOI = 0.02 (■). Cytotoxicity is shown in gray, and the cytotoxicity scale is shown to the right (cytotoxicity measured at nitrite concentrations of 0.015 M or less was ≤1% of that of the LDH control). Plate photographs were obtained comparing oseltamivir (1 μM) with lutemivir at MOI = 0.002, and at nitrite concentrations of 0.15 M, 0.015 M, and 0.0015 M. The results are shown in Figure 28. The order of the plates cited second to last is the same as the order of the plates from left to right in the diagram (there are two experiments, and the plates for each corresponding experiment are shown above and below). The rightmost pair of plates, to the right of the pair of oseltamivir plates, is the viral control. Cytotoxicity is shown below each pair of test plates as a percentage of the LDH control (average of three LDH assays at 24 hours post-infection).

[0439] The results show that at suitable doses of the nitrite / citric acid / polyol formulation, the virus is completely eradicated, demonstrably superior to oseltamivir. Similar antiviral activity of the nitrite / citric acid / polyol formulation has been demonstrated against rhinovirus and respiratory syncytial virus (RSV).

[0440] These results demonstrate that therapeutic and prophylactic treatments for respiratory viral infections in humans and animals are provided by the nitrite / acid / polyol formulations according to the present invention.

[0441] Example 8 Cytotoxicity and antiviral activity of carboxylic acid-nitrite-polyol solution against coronavirus SARS-CoV-2 material Test formulation F1 (pH 5.8) Six test concentrations of formulation 1(F1), which consisted of an aqueous solution of sodium nitrite, citric acid at pH 5.8, and mannitol (polyol), were prepared from a stock solution of 1.5 M sodium nitrite, 0.91 M citric acid / citric acid buffer at pH 5.8, and 0.5 M mannitol by the method described below to obtain the following test compositions: [Table 6]

[0442] Comparison used with F1 A control formulation with a pH of 5.8 was prepared from 0.1 M citrate + assay buffer + cells.

[0443] The negative control was assay buffer + cells.

[0444] The positive control was chloroquine-positive cells.

[0445] Test formulation F2 (pH 5.4) Six test concentrations of formulation 2(F2), which consisted of aqueous solutions of sodium nitrite, citric acid at pH 5.4, and mannitol (polyol), were prepared from a stock solution of 1.5 M sodium nitrite, 0.91 M citric acid / citric acid buffer at pH 5.4, and 0.5 M mannitol by the method described below to obtain the following test compositions: [Table 7]

[0446] Control used with F2 A control formulation with a pH of 5.4 was prepared from 0.1 M citrate + assay buffer + cells.

[0447] The negative control was assay buffer + cells.

[0448] The positive control was chloroquine-positive cells.

[0449] Chemical reagents Sodium nitrite: Grade: Sodium Nitrite Extra Pure Ph Eur, USP. Sodium Nitrite CAS No. 7632-00-0, EC No. 231-555-9. Extra Pure Ph Eur, USP, Sigma Aldrich, Product Code 1.065441000.

[0450] Citric acid: Grade: Anhydrous Citric Acid Powder EMPROVE® ESSENTIAL Ph Eur, BP, JP, USP, E330, FCC, Sigma Aldrich, Product Code 1.002425000.

[0451] D-Mannitol: Grade: D-mannitol meeting EP, FCC, and USP test specifications, from Sigma Aldrich, product code M8429-100G.

[0452] Chloroquine phosphate: Grade: Pharmaceutical secondary reference material, Sigma Aldrich, product code PHR1258-1G.

[0453] Preparation of stock solution To prepare the citric acid solution, add 19.2 g of citric acid to 90 ml of distilled water, then 1 Add 0 ml of 3 M sodium hydroxide, then dilute with distilled water to adjust the pH (up to 160 ml for pH 5.4, up to 190 ml for pH 5.8). Alternatively, add 19.2 g of citric acid, followed by 1.2 g of sodium hydroxide, then 20 ml of distilled water, adjust the pH with 10 M sodium hydroxide, and dilute with distilled water to 100 ml. Sterilize the solution by syringe filtration using a 0.22 μm filter.

[0454] To prepare a 1.0 M sodium nitrite solution, 100 mL of distilled water was added ...

Claims

1. One or more agents selected from nitric oxide generating compositions, combinations or combinable aggregates of components of nitric oxide generating compositions, and mixtures thereof, for use as antibacterial agents against Mycobacterium tuberculosis and Mycobacterium tuberculosis, wherein the agent is a nitric oxide generating composition or a component thereof, and the nitric oxide generating composition, or the combination or combinable aggregate of components of the nitric oxide generating composition, comprises one or more nitrites, a proton source containing one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids, and one or more organic polyols. The one or more organic polyols are selected from mannitol, lactitol, or a mixture thereof. A drug wherein the proton source is selected from citric acid, ascorbic acid, or a mixture thereof.

2. The agent according to claim 1, wherein the acid is buffered to a pH higher than that exhibited by an aqueous solution of the same concentration of the acid.

3. The agent according to claim 2, wherein the pH is higher than the above and in the range of approximately 5 to 8.

4. The agent according to claim 2, wherein the pH higher than the above is 5.2 or higher.

5. A drug for use in methods of treating, alleviating, or preventing infections caused by Mycobacterium tuberculosis or the bacterium M. tuberculosis in humans or animals, The agent according to any one of claims 1 to 4, wherein the method comprises administering an antibacterial effective amount of one or more agents to the human or animal subject.

6. The drug according to claim 5, wherein the drug is administered to the lungs of the target.

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