Nitric oxide or nitric oxide releasing compositions for the treatment of sars-cov and sars-cov-2

Abstract

The invention provides one or more agents selected from the group consisting of organic carboxylic acids, organic non-carboxylic reducing acids, nitric oxide (NO), nitric oxide generating compositions, combinations or combination packs of ingredients of nitric oxide generating compositions, and mixtures thereof, for use as an antiviral agent against SARS-CoV or SARS-CoV-2 coronavirus.

Description

Technical Field

[0001] The present invention relates to methods and compositions for treating and combating coronaviruses SARS-CoV and SARS-CoV-2, which are viruses that cause the diseases SARS and COVID-19, respectively. Background Technology

[0002] Respiratory viruses are a major cause of morbidity and mortality. Influenza A, human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (CoV) are single-stranded RNA viruses that can cause pneumonia or exacerbate chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) (Johnston, SL, Proc. Am. Thorac. Soc. 2, 150-156 (2005); Kurai, D et al., Front. Microbiol. 4, 1-12 (2013)). Nitric oxide has been shown in vitro to inhibit the replication of many respiratory viruses, including influenza A and B viruses (Rimmelzwaan, GF et al., J. Virol 73, 8880-8883 (1999); Regev-Shoshani, G et al., Nitric Oxide 31, 48-53 (2013)), human rhinovirus (HRV) (Sanders, SP et al., J. Virol 72, 934-942 (1998)), respiratory syncytial virus (RSV) (Ahmad, A et al., Frontiers in Biosciences 8, a48-53 (2003)), and severe acute respiratory syndrome (SARS) coronavirus (CoV) (Akerstrom, S et al., J. Virology 79, 1966-1969 (2005)). The publications cited above are incorporated herein by reference.

[0003] The respiratory illness COVID-19 is caused by the SARS-CoV-2 coronavirus, which the World Health Organization (WHO) declared a pandemic in March 2020.

[0004] This invention is based on the unexpected discovery that one or more agents selected from organic carboxylic acids, organic noncarboxylic acid reducing acids, nitric oxide (NO), nitric oxide-producing compositions, combinations or combinations of components or ingredients of nitric oxide-producing compositions, and mixtures thereof are effective in vitro antiviral agents against SARS-CoV-2 coronavirus, providing an effective in vivo treatment (treatment and prevention) for COVID-19 caused by SARS-CoV-2 coronavirus in humans and animals. Experimental work investigating the similarity between antiviral activities against SARS-CoV-2 and SARS-CoV, along with prior knowledge of these viruses, has shown or indicated that the same agents are also active against SARS-CoV. This invention also provides effective antiviral treatment of surfaces (including inanimate surfaces as well as hands, arms, and other external surfaces of the human or animal body) and spaces, thereby preventing the spread of SARS-CoV and SARS-CoV-2 coronaviruses and surface contamination. In a preferred embodiment of the invention, nitric oxide can be generated by an NO-generating system that itself includes a proton source containing one or more acids selected from organic carboxylic acids and organic noncarboxylic acids. Such systems can be embodied in NO-generating compositions that can be applied to the lungs of a patient.

[0005] The active agent, such as one or more carboxylic acids, one or more organic noncarboxylic acid reducing acids, NO-producing compositions, nitric oxide-producing compositions, or combinations or mixtures of components or ingredients, may be in any suitable physical form, such as in liquid form or in droplet form entrained in a carrier gas or air, for example, delivered to the patient's lungs as an aerosol or mist.

[0006] According to the invention, it has also been found that acid activators or acids used as proton sources for generating nitric oxide can be effective when buffered to relatively high pH values, such as pH values ​​between about 5 and about 8, for example, at or above about 5.2, for example in the range of 5.2 to 5.8, i.e., pH values ​​that are physiologically tolerated by the tissues of the patient's oral cavity, nasal passages, airways and lungs.

[0007] Compositions that produce nitric oxide and NO possess a range of antimicrobial and other beneficial physiological activities, as discussed herein. Therefore, the antiviral activity against SARS-CoV and SARS-CoV-2 provided by this invention may be accompanied by beneficial activities against other pathogens that may infect patients or are susceptible to infection (including secondary bacterial, viral, parasitic, and fungal infections).

[0008] The composition that produces NO can be prepared by one of the following methods:

[0009] (a) A method for preparing a composition that generates NOx, the method comprising mixing a nitrite, a proton source and an organic polyol component in a desired proportion at a concentration higher than that required in the composition in the form to be used, to form a concentrated premix, and subsequently diluting the concentrated premix appropriately with water to provide a composition to be used;

[0010] (b) A method for preparing a composition that generates NOx, the method comprising mixing a nitrite, a proton source and an organic polyol component in a desired concentration and proportion in a form to be used, thereby providing a composition to be used.

[0011] These alternative methods constitute specific aspects of the present invention.

[0012] Certain antiviral activities of organic carboxylic acids have been previously reported. Koromyslova et al. described the use of citric acid in treating norovirus particles in 2015 (A D Koromyslova et al., Virology 485, 199-204). U.S. Patent No. 8,034,844 (Fox et al.) claims a method for reducing viral populations on a surface, the method comprising contacting the surface with a solid composition comprising a powdered solid matrix and a virally effective amount of an organic acid comprising (i) two or more polycarboxylic acids selected from the group consisting of malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, octanoic acid, azelaic acid, sebacic acid, fumaric acid, maleic acid, tartaric acid, malic acid, maleic acid, citric acid, aconitic acid, and mixtures thereof, and (ii) a water-soluble or water-dispersible polymeric acid selected from the group consisting of polycarboxylic acids, polysulfonic acids, sulfated polymers, polyphosphates, and mixtures thereof. U.S. Patent No. 6,034,133 (Hendley et al.) claims a method for killing rhinovirus and preventing the spread of rhinovirus-induced colds, the method comprising the steps of: identifying a patient suffering from a rhinovirus cold or possibly exposed to rhinovirus; and applying an antiviral composition to the patient's hand in an amount suitable for eradicating rhinovirus, the antiviral composition comprising citric acid, malic acid, and C. 1-6 The alcohol is administered after the patient is identified as having a rhinovirus cold or before the patient is exposed to the rhinovirus. The disclosures of the publications cited above are incorporated herein by reference. However, none of the prior art teaches or suggests activity of organic carboxylic acids against coronaviruses SARS-CoV and SARS-CoV-2.

[0013] Nitric oxide (NO) and its precursors have been extensively studied as potential pharmaceutical agents. Nitric oxide is a potent vasodilator, synthesized and released by vascular endothelial cells, and plays a crucial role in regulating local vascular resistance and blood flow, particularly. In mammalian cells, nitric oxide is primarily produced with L-citrulline via the enzymatic oxidation of L-arginine. Nitric oxide is also released from the skin through a mechanism seemingly independent of NO synthase. Nitric oxide is also involved in the inhibition of platelet and leukocyte aggregation and adhesion, inhibition of cell proliferation, scavenging of superoxide radicals, and regulation of endothelial cell layer permeability. The role of nitric oxide in cancer treatment is discussed in Biochemistry (Moscow), 63(7), 802-809 (1998), the contents of which are incorporated herein by reference. Nitric oxide has been shown to possess antimicrobial properties, as described by FC Fang in J. Clin. Invest. 99(12), 2818-2825 (1997), and as disclosed, for example, in WO 95 / 22335 and WO 02 / 20026 (Aberdeen University), the disclosures of which are incorporated herein by reference. Other known uses and applications of systems for producing nitric oxide, other nitrogen oxides, and their precursors are set forth in the following description of the invention.

[0014] Many questions remain regarding the efficient production and delivery of nitric oxide, other nitrogen oxides, and their precursors to organisms and cells for therapeutic purposes. Systems widely used to produce nitric oxide rely on acidifying nitrite with an inorganic acid, initially producing an equimolar amount of nitrite (HNO2) relative to the starting nitrite. The nitrite then readily decomposes into nitric oxide and nitrate with hydrogen ions and water. The decomposition can be represented by the following equilibrium equation (1):

[0015] 3 HNO2→2 NO+NO3 - +H + +H2O (1)

[0016] Nitrite acidification is typically carried out at pH values ​​less than about 4, which generally favor the formation of nitrite in order to maximize NO production. However, use at pH < 4 is unsuitable for in vivo use where the acid comes into contact with animal tissues. Higher pH values ​​are milder to cells and biological systems, but above pH 4, previous systems have not been able to produce satisfactory yields of NO. To increase the amount of NO produced above pH 4, large amounts of nitrite are required, which is impractical and uneconomical for therapeutic applications. In addition, given the short half-life of nitrite, the conversion rate represented by equation (1) is not easily controlled, making it difficult to control the release of nitric oxide for therapeutic purposes. The reaction between one or more nitrites and a proton source to produce nitric oxide, optionally other nitrogen oxides and / or optionally their precursors is referred to herein as a "NOx-producing reaction" or "NOx-generating reaction" or similar terms, and "NOx" is used to refer to the products of nitrite acidification, particularly nitric oxide, other nitrogen oxides and their precursors, individually and in any combination. It should be understood that each component of the NOx produced may be released as a gas, or may be dissolved in a solution in the reaction mixture, or may be initially dissolved in a solution and subsequently released as a gas, or any combination thereof.

[0017] WO 00 / 53193 (the disclosure of which is incorporated herein by reference) describes a cream or ointment for treating localized skin ischemia and promoting wound healing, wherein the proton source is ascorbic acid. Example 1 describes a KYJelly-based... TM The gel, in Example 7, was tested in both direct skin contact and with the skin protected by a membrane. The use of ascorbic acid reportedly prevented significant skin inflammation (WO 00 / 53193, page 2). However, in practice, the degree of skin inflammation caused by the gel's low pH was unsatisfactory when the gel was in direct contact with the skin, and the skin protective film weakened the gel's effect in the presence of a membrane. As a result, the gel could not be marketed. The composition of WO 00 / 53193 does not contain polyols.

[0018] WO 02 / 20026 (the disclosure of which is incorporated herein by reference) describes a skin preparation for treating drug-resistant skin infections, wherein the proton source is citric acid or salicylic acid. A nitrite-containing composition and an acid-containing composition are dispensed from a dual-cylinder dispenser, the compositions are then mixed to allow the acid to react with the nitrite, and then applied to 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 is used in a pair of creams containing citric acid and nitrite, respectively, mixed on-site to initiate a reaction between the acid and the nitrite (e.g., Formulation 1 of Example 3 in WO 02 / 20026). Glycerol is used together with cetearyl alcohol in a pair of lotions containing citric acid and nitrite, respectively, mixed on-site to initiate a reaction between the acid and the nitrite (e.g., Formulation 3 of Example 3 in WO 02 / 20026). The preferred pH of the reaction mixture is 5 or lower, particularly 4 or lower, as this pH is expected to cause adverse skin inflammation. Nasal sprays are also taught that may use reducing acids, such as ascorbic acid or ascorbyl palmitate, to allow for the use of higher pH values ​​to avoid irritating sensitive nasal mucosa. However, it is generally accepted (WO 02 / 20026, page 16, paragraph 2) that higher pH values ​​will slow down the reaction.

[0019] US 6103275 (published August 15, 2000) (the disclosure of which is incorporated herein by reference) describes the use of a reducing agent, such as ascorbic acid, in conjunction with an organic acid, such as maleic acid, with a pKa between 1 and 4, to acidify nitrite. A viscous (gel) composition is used to slow the release of the reaction products for topical application. The acid and nitrite are kept separate until nitric oxide is generated, and it is stated that the reducing agent is included in at least one of a first gel and a second gel. The pH range used in the method is not specified. However, the fact that the buffering component is called an acid indicates that these compounds are present primarily in a protonated form, and therefore the pH of the composition should be substantially below 4. The presence of an acid with a pKa between 1 and 4 ensures good buffering capacity of the formulation at that pH. While incorporating such an acid is a suitable way to ensure that the pH is maintained at a level that sustains the efficiency of nitrite to nitric oxide conversion, significant adverse skin irritation is expected upon skin contact. The composition of US 6103275 does not contain polyols.

[0020] In WO 2003 / 013489 (the disclosure of which is incorporated herein by reference), 3% polyvinyl alcohol (PA) is proposed as a gelling agent for citric acid and nitrite, respectively, to be mixed together on the spot (WO 2003 / 013489 Example 7). However, test data (WO 2003 / 013489 Tables 11 and 12) show that PA cannot form a stable gel, and the PA compositions are never mixed together or used together. Apart from the above proposal not adhering to the final composition, the compositions of WO 2003 / 013489 do not contain polyols.

[0021] U.S. Patent Application No. 2005 / 0037093 (the disclosure of which is incorporated herein by reference) describes a composition for generating nitric oxide based on a nitrite-acid reaction and mentions optional excipients, including polyvinyl alcohol, propylene glycol, and polyethylene glycol.

[0022] Chinese Patent Application No. CN 101028229 (the disclosure of which is incorporated herein by reference) describes a cosmetic product that produces nitric oxide through the reaction of nitrite with an acid. It teaches the optional use of, in particular, glycerin, propylene glycol, and glyceryl monostearate as additional ingredients. Trihydroxyethylamine is further mentioned as an ingredient in a specific example.

[0023] Chinese Patent Application No. CN 101062050 (the disclosure of which is incorporated herein by reference) describes a product that promotes hair growth by producing nitric oxide through the reaction of nitrite with acid. It teaches the optional use of, in particular, glycerin, propylene glycol, and glyceryl monostearate as additional ingredients. D-panthenol and combinations of panthenol and inositol are mentioned as ingredients in specific examples.

[0024] WO 2008 / 110872 (the disclosure of which is incorporated herein by reference) describes foamable nitric oxide donor compositions that optionally contain a polar solvent, for example, selected from polyols and polyethylene glycol (paragraphs

[0055] and

[0056] ). Specific polyols are indicated as propylene glycol, butanediol, butene glycol, butynediol, pentanediol, hexanediol, octanediol, neopentanediol, 2-methyl-1,3-propanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, glycerol, butane-1,2,3-triol, butane-1,2,4-triol, and hexane-1,2,6-triol. Polyvinyl alcohol, polyethylene glycol 1000 (PEG 1000), PEG 4000, PEG 6000, and PEG 8000 are mentioned in a list of many polymerizing agents as optional other components (paragraph

[0062] ). The paragraphs

[0190] and

[0191] also mention polyols such as glycerol, propylene glycol, hexanediol, diethylene glycol and propylene glycol, as well as ethylene glycol, hexanediol, other diols and polyethylene glycol.

[0025] WO 2009 / 019498 (the disclosure of which is incorporated herein by reference) describes non-thiol reducing agents with pKa not between 1 and 4 as components other than nitrites and proton sources. Examples of non-thiol reducing agents are indicated as iodide anions, butylated hydroquinone, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and β-carotene. Except for butylated hydroquinone, the compositions of WO 2009 / 019498 do not contain polyols.

[0026] WO 2014 / 188174 and WO 2014 / 188175 (the disclosures of which are incorporated herein by reference) describe an application system for skin lesions and a transdermal delivery system, wherein the proton source is a hydrogel containing carboxylic acid and sulfonic acid side groups covalently bonded to a three-dimensional polymer matrix. The first layer in contact with the skin is a polypropylene mesh to which nitrite is absorbed. When the mesh is placed on the skin and the hydrogel is placed over the mesh as a top layer, it has been found that the reaction products of the acid and nitrite are adequately delivered to the skin without unacceptable skin irritation. In WO 2014 / 188175, an alternative primary layer in contact with the skin is disclosed, which is a soluble film formed from, for example, polyvinyl alcohol and containing nitrite. Both references teach that the hydrogel may contain glycerol, without specifying its purpose. However, it is well known that glycerol is added to such hydrogels as a plasticizer (see, for example, WO 00 / 06215, page 14, the disclosure of which is incorporated herein by reference). The references reveal a preference for certain hydroxyl-containing ingredients that are not present, particularly 1-thioglycerol, isoascorbate, ascorbic acid, and butylated hydroquinone.

[0027] U.S. Patent Application No. 2014 / 0335207 (the disclosure of which is incorporated herein by reference) describes a local mixture that produces nitric oxide when a “nitrite medium” is mixed with an “acidifying medium.” Specific embodiments of the “nitrite medium” are described separately in paragraphs

[0050] through

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

[0054] and

[0055] contains a polyol selected from glycerol, glyceryl stearate, caprylyl glycol, ethylhexylglycerol, and hexanediol, while specific embodiments described in other paragraphs contain some of the aforementioned polyols and butanediol. These polyols are also components of embodiments of the “acidifying medium” described in paragraphs

[0056] through

[0062] .

[0028] U.S. Patent Application No. 2015 / 0030702 (the disclosure of which is incorporated herein by reference) describes a skin dressing based on a nitrite-acid reaction. The skin dressing contains a non-thiol reducing agent, such as hydroquinone or butylated hydroquinone. The skin dressing may contain a hydrogel, which, for example, contains a hydrophilic polymer, such as polyvinyl alcohol or polyethylene glycol.

[0029] U.S. Patent Application No. 2017 / 0209485 (the disclosure of which is incorporated herein by reference) describes an apparatus and method for locally applying nitric oxide in a foam or serum carrier. Paragraph

[0070] describes glycerol and (unspecified) "glycerol-like components" as optional additives to increase surface tension and / or reduce vapor pressure.

[0030] U.S. Patent Application No. 2019 / 0134080 (the disclosure of which is incorporated herein by reference) describes a composition and method for topically applying a nitric oxide generating system formed from a foam of a multi-component combination to the skin, said multi-component combination comprising a first solution containing at least one nitrite reactant and a second solution containing at least one acidic reactant. Devices for maintaining, aerating, and dispensing the components of the combination in foam form are also described. Glycerol is mentioned as an optional additive to increase surface tension and / or reduce vapor pressure (paragraph

[0068] ).

[0031] As mentioned above, this invention is based on the unexpected discovery that one or more active agents selected from organic carboxylic acids, organic non-carboxylic acid reducing acids, nitric oxide (NO), compositions that generate nitric oxide, combinations or combinations of components or ingredients of compositions that generate nitric oxide, and mixtures thereof are effective in vitro antiviral agents against SARS-CoV-2 coronavirus, providing an effective in vivo treatment (treatment and prevention) for COVID-19 caused by SARS-CoV-2 coronavirus in humans and animals. Experimental work investigating the similarity between antiviral activities against SARS-CoV-2 and SARS-CoV, together with prior knowledge of the viruses, has shown or indicated that the same agents are also active against SARS-CoV. This invention also provides effective antiviral treatment for surfaces (including inanimate surfaces as well as hands, arms, and other external surfaces of the human or animal body) and spaces, thereby preventing the spread of SARS-CoV and SARS-CoV-2 coronaviruses and surface contamination.

[0032] In a preferred embodiment of the invention, nitric oxide can be generated via an NO generating system, which itself includes a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acids. Such NO generating systems may include nitrite and a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids. Such systems can be embodied in NO-generating compositions that can be applied to the lungs of a patient.

[0033] The active agent, such as one or more organic carboxylic acids, one or more organic noncarboxylic acid reducing acids and / or NO-producing compositions, may be in any suitable physical form, such as in liquid form or in droplet form entrained in a carrier gas or air, for example, delivered to the patient's lungs as an aerosol or mist.

[0034] Examples of one or more organic carboxylic acids and one or more organic noncarboxylic acid reducing acids used in this invention are the same as examples of “proton sources” in the category of one or more organic carboxylic acids and one or more organic noncarboxylic acid reducing acids given below and in the examples. Citric acid is specifically mentioned without departing from most of the disclosure herein. In one embodiment, the acid or proton source is not one of the antiviral mixtures taught in the above-recognized U.S. Patent Nos. 8,034,844 and 6,034,133, namely: (a) an organic acid comprising (i) two or more polycarboxylic acids selected from the group consisting of: malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, octanoic acid, azelaic acid, sebacic acid, fumaric acid, maleic acid, tartaric acid, malic acid, maleic acid, citric acid, aconitic acid, and mixtures thereof; and (ii) a water-soluble or water-dispersible polymeric acid selected from the group consisting of: polycarboxylic acids, polysulfonic acids, sulfated polymers, polyphosphates, and mixtures thereof; or (b) an antiviral composition comprising citric acid, malic acid, and C 1-6 alcohol.

[0035] According to the invention, it has also been found that acid activators or acids used as proton sources for generating nitric oxide can be effective when buffered to relatively high pH values, such as pH values ​​of about 5.2 or higher, for example in the range of 5.2 to 5.8, i.e., pH values ​​that are physiologically tolerable for the tissues of the patient's oral cavity, nasal passages, airways and lungs.

[0036] Compositions that produce nitric oxide and NO possess a range of antimicrobial and other beneficial physiological activities, as discussed herein. Therefore, the antiviral activity against SARS-CoV and SARS-CoV-2 provided by this invention may be accompanied by beneficial activities against other pathogens that may infect patients or are susceptible to infection (including bacterial, secondary viral, parasitic, and fungal infections).

[0037] The composition that produces NO can be prepared by one of the following methods:

[0038] (a) A method for preparing a composition that generates NOx, the method comprising mixing a nitrite, a proton source and an organic polyol component in a desired proportion at a concentration higher than that required in the composition in the form to be used, to form a concentrated premix, and subsequently diluting the concentrated premix appropriately with water to provide a composition to be used;

[0039] (b) A method for preparing a composition that generates NOx, the method comprising mixing a nitrite, a proton source and an organic polyol component in a desired concentration and proportion in a form to be used, thereby providing a composition to be used.

[0040] These alternative methods constitute specific aspects of the present invention.

[0041] Compared to the current situation, using a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids as a nitrite acidifying agent in the presence of one or more organic polyols can more effectively generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors (collectively referred to as NOx), and enhances reaction output. Furthermore, it has been found that the antimicrobially effective reaction products of such reaction systems using organic reducing acids as nitrite acidifying agents can be delivered at physiologically tolerable pH levels, such as between about 5 and about 8, with or without the use of one or more organic polyols. This allows the reaction system to operate at these pH levels, deliver the composition directly, and possess beneficial physiological activities, such as in vivo antimicrobial activity. It has been found that the method for generating nitric oxide, which forms the basis of this invention, optionally generates physiologically effective amounts of nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, after an initial strong burst release of NOx gas over a prolonged period, such as more than about 2 hours, more than about 5 hours, or more than about 10 hours, which could have significant uses in pharmaceuticals and other applications. If an initial strong burst release is not required, the reaction mixture may be administered to the subject some time after the onset of the NOx-producing reaction, such as about 10 minutes, 30 minutes, or an hour or more after the onset of the NOx-producing reaction. Summary of the Invention

[0042] This invention, as defined in and by the appended claims, is a specific embodiment of the more general inventive development disclosed in the following description. As defined in and by the appended claims, this invention relates to the application of a general inventive development to combinations and compositions that carry out a reaction to produce NO, and the gaseous products of that reaction delivered to a human or animal subject via the nose, mouth, respiratory tract, or lungs of a subject. All aspects, embodiments, embodiments, and preferred solutions described herein with respect to this disclosure are equivalent and independently applicable to the invention as defined in and by the appended claims.

[0043] This disclosure provides systems, methods, combinations, kits, and compositions for treating or preventing the spread of SARS-CoV and SARS-CoV-2 using effective amounts of one or more organic carboxylic acids, one or more organic noncarboxylic acid reducing acids, or any mixture thereof. The description herein of carboxylic acids and organic noncarboxylic acid reducing acids suitable as proton sources for generating nitric oxide and optionally other nitrogen oxides and / or optionally their precursors applies accordingly to carboxylic acids and organic noncarboxylic acid reducing acids suitable as antiviral agents against SARS-CoV and SARS-CoV-2.

[0044] In embodiments employing the generation of nitric oxide and optionally other nitrogen oxides and / or optional precursors thereof, the systems, methods, combinations, kits, and compositions comprise one or more nitrites and a proton source as reactants, said proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids. The systems, methods, combinations, kits, and compositions also include one or more organic polyols. The use of reducing acids (i.e., carboxylic acid reducing acids and noncarboxylic acid reducing acids) allows the generation of nitric oxide and optionally other nitrogen oxides and / or optional precursors thereof at a pH slightly above 4, for example, in the range of 5 to 8. This disclosure also provides systems, methods, combinations, kits, and compositions for antimicrobial applications, wherein one or more organic polyols are optional and the reaction is carried out at an initial pH of a proton source in the range of 5 to 8.

[0045] According to a first aspect, this disclosure provides a method for generating nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, the method comprising reacting one or more nitrites with a proton source under reaction conditions suitable for generating nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, said proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids, said reaction being carried out in the presence of one or more organic polyols.

[0046] It is characterized by one or more of the following:

[0047] (a) One or more organic polyols are present to enhance the amount of reaction output;

[0048] (b) The proton source is not merely a hydrogel containing carboxylic acid side groups covalently bonded to a three-dimensional polymer matrix;

[0049] (c) One or more organic polyols, not just glycerol;

[0050] (d) When using one or more tackifiers, one or more organic polyols are not limited to glycerol;

[0051] (e) When using one or more plasticizers, one or more organic polyols are not limited to glycerol;

[0052] (f) One or more organic polyols, not just polyvinyl alcohol;

[0053] (g) When using one or more tackifiers, one or more organic polyols are not limited to polyvinyl alcohol;

[0054] (h) any one or more of (b) to (g), wherein the word “not only” is replaced by “does not include”;

[0055] (i) One or more organic polyols, not limited to propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof or any combination of the above with glycerol and / or polyvinyl alcohol.

[0056] (j) One or more organic polyols that do not contain propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof, or any combination of the above with glycerol and / or polyvinyl alcohol.

[0057] The second aspect of this disclosure is constituted by nitric oxide, optional other nitrogen oxides and / or optional precursors prepared by the method according to the first aspect of this disclosure.

[0058] According to a third aspect, this disclosure provides a method for enhancing the output of a reaction involving one or more nitrites with a proton source to produce nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, said method comprising carrying out the reaction using a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids, and in the presence of one or more organic polyols in an amount that enhances the reaction output. 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.

[0059] According to a fourth aspect, this disclosure provides the use of one or more organic polyols in a reaction mixture to enhance the output of a reaction in which one or more nitrites react with a proton source to produce nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, wherein the proton source comprises one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids. The enhancement of the reaction output is compared to a reaction carried out under the same conditions but without one or more organic polyols.

[0060] According to a fifth aspect, this disclosure provides a combination, kit, or composition for generating nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof through a reaction of one or more nitrites with a proton source, said combination, kit, or composition comprising:

[0061] (i) one or more nitrites;

[0062] (ii) a proton source, said proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids; and

[0063] (iii) One or more organic polyols;

[0064] It is characterized by one or more of the following:

[0065] (a) One or more organic polyols are present to enhance the amount of reaction output;

[0066] (b) The proton source is not merely a hydrogel containing carboxylic acid side groups covalently bonded to a three-dimensional polymer matrix;

[0067] (c) One or more organic polyols, not just glycerol;

[0068] (d) When using one or more tackifiers, one or more organic polyols are not limited to glycerol;

[0069] (e) When using one or more plasticizers, one or more organic polyols are not limited to glycerol;

[0070] (f) One or more organic polyols, not just polyvinyl alcohol;

[0071] (g) When using one or more tackifiers, one or more organic polyols are not limited to polyvinyl alcohol;

[0072] (h) any one or more of (b) to (g), wherein the word “not only” is replaced by “does not include”;

[0073] (i) One or more organic polyols, not limited to propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof or any combination of the above with glycerol and / or polyvinyl alcohol.

[0074] (j) One or more organic polyols that do not contain propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof, or any combination of the above with glycerol and / or polyvinyl alcohol.

[0075] When the proton source comprises a hydrogel containing carboxylic acid side groups covalently bonded to a three-dimensional polymer matrix and the combination or kit contains two or more separate compositions, it is preferred that one or more polyols are not in direct contact with the hydrogel or are present in the separate compositions.

[0076] The chemical composition, kit, or formulation of the fifth aspect of this disclosure may, for example, consist substantially of the aforementioned components (i), (ii), and (iii) and optionally water and / or a pH buffer. The statement "consist substantially of..." may, for example, allow the presence of small amounts of one or more additional components, provided that this does not adversely affect the action of the aforementioned components (i), (ii), and (iii) and optionally water and / or a pH buffer. The total amount of such one or more additional components is preferably less than about 20% by weight or volume of the chemical composition, kit, or formulation, 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.

[0077] The chemical composition of the combination, kit, or composition may, for example, consist of the above-mentioned components (i), (ii), and (iii) and optional water and / or pH buffer, and / or one or more additional components in amounts less than about 20% by weight or volume of the chemical composition of the combination, kit, or 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.

[0078] According to a sixth aspect, this disclosure provides a method for preparing a combination, kit, or composition comprising:

[0079] (i) one or more nitrites;

[0080] (ii) a proton source, said proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids; and

[0081] (iii) One or more organic polyols;

[0082] The method includes bringing components (i), (ii) and (iii) close to each other to form a combination or kit or mixture to form a composition;

[0083] It is characterized by one or more of the following:

[0084] (a) One or more organic polyols are present to enhance the amount of reaction output;

[0085] (b) The proton source is not merely a hydrogel containing carboxylic acid side groups covalently bonded to a three-dimensional polymer matrix;

[0086] (c) One or more organic polyols, not just glycerol;

[0087] (d) When using one or more tackifiers, one or more organic polyols are not limited to glycerol;

[0088] (e) When using one or more plasticizers, one or more organic polyols are not limited to glycerol;

[0089] (f) One or more organic polyols, not just polyvinyl alcohol;

[0090] (g) When using one or more tackifiers, one or more organic polyols are not limited to polyvinyl alcohol;

[0091] (h) any one or more of (b) to (g), wherein the word “not only” is replaced by “does not include”;

[0092] (i) One or more organic polyols, not limited to propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof or any combination of the above with glycerol and / or polyvinyl alcohol.

[0093] (j) One or more organic polyols that do not contain propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof, or any combination of the above with glycerol and / or polyvinyl alcohol.

[0094] As used herein, the term "combination" refers to separate substances or compositions (referred to as "components") that are brought close together and used together. Bringing components close together can be achieved in multiple stages, where some, but not all, of the components are initially brought together into a sub-combination or partial combination, followed by proximity with one or more other components or other sub-combinations or partial combinations. "Proximity" can include close mixtures, solutions, or suspensions, or can refer to close physical proximity that is not equivalent to close mixtures, solutions, or suspensions, such as in separate containers within a kit where the components are provided together for later use. For example, a nitrite component and a proton source component, each containing one or more nitrites (or some of them) and one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids (or some of them), can be stored separately or in separate containers within a kit and brought together by mixing for use in initiating a NOx-generating reaction. One or more organic polyols can be provided in one or both of the nitrite component and the proton source component, or can be provided separately as organic polyol components, also mixed when initiating a NOx-generating reaction. Any or more of the components may be present in multiple portions and multiple containers. The combination can be brought close together in a way that immediately initiates a NOx-producing reaction, for example, because the nitrite and the proton source are in the same solution, thus enabling the reaction to occur. Alternatively, the combination can be brought close together in a way that does not immediately initiate a NOx-producing reaction, but requires one or more other steps or actions before initiation, for example, because the nitrite and the proton source are present as a dry powder mixture or as encapsulated particles, water (e.g., from a mucous membrane in contact with the combination) is required before the NOx-producing reaction can begin.

[0095] In the implementation, the first to sixth aspects of this disclosure may be characterized independently of each other by: only the features mentioned above (a); or only feature (b); or only feature (c); or only feature (d); or only feature (e); or only feature (f); or only feature (g); or feature (h) when only (b) is involved; or feature (h) when only (c) is involved; or feature (h) when only (d) is involved; or feature (h) when only (e) is involved. ; or feature (h) when only (f) is involved; or feature (h) when only (g) is involved; or features (a) and (b) only; or feature (h) when features (a) and (b) are involved; or features (a) and (c) only; or feature (h) when features (a) and (c) are involved; or features (a) and (d) only; or feature (h) when features (a) and (d) are involved; or features (a) and (e) only; or feature (a) only. Features (h) when (e) are involved; or features (a) and (f) only; or features (h) when features (a) and (f) are involved; or features (a) and (g) only; or features (h) when features (a) and (g) are involved; or features (b) and (c) only; or features (h) when features (b) and (c) are involved; or features (b) and (d) only; or features (h) when features (b) and (d) are involved; or features (b) and (e) only. e); or feature (h) when features (b) and (e) are involved; or features (b) and (f) only; or features (h) when features (b) and (f) are involved; or features (a), (b), (c) and (f) only; or features (h) when features (a), (b), (c) and (f) are involved; or all of features (a) to (g); or features (a) and (b) together with features (h) when features (c) to (g) are involved.

[0096] In other embodiments, the first to sixth aspects of the invention may be characterized independently of each other by: only the features (c), (f), and (i) mentioned above; or only features (c), (f), and (j); or feature (i) and feature (h) when referring to features (c) and (f); or feature (j) and feature (h) when referring to features (c) and (f); or only features (d), (g), and (i); or only features (d), (g), and (j); or feature (i) and feature (h) when referring to features (d) and (g); or feature (j) and feature (h) when referring to features (d) and (g); or only features (e), (f), and (i); or only features (e), (f), and (j); or feature (i) and feature (h) when referring to features (e) and (f); or feature (j) and feature (h) when referring to features (e) and (f).

[0097] Preferably, the first to sixth aspects of this disclosure are characterized by: all of features (a) to (g); or features (a) and (b) together with feature (h) when all of features (c) to (g) are involved; or only features (c), (f), and (i); or only features (c), (f), and (j); or feature (i) and feature (h) when features (c) and (f) are involved; or feature (j) and feature (h) when features (c) and (f) are involved; or only features (d), (g) Features (c) and (i); or only features (d), (g) and (j); or features (i) and features (h) when referring to features (d) and (g); or features (j) and features (h) when referring to features (d) and (g); or only features (e), (f) and (i); or only features (e), (f) and (j); or features (i) and features (h) when referring to features (e) and (f); or features (j) and features (h) when referring to features (e) and (f). Note that features (d), (e) and (g) are redundant when features (c) and (f) characterize this disclosure; in that case, features (d), (e) and (g) (or features (h) when referring to features (d), (e) and (g)) may be omitted from the list and are considered as instances of characteristic features (c) and (f) (or features (h) when referring to features (c) and (f)).

[0098] As used herein, the expression “one or more organic polyols that enhance the amount of reaction output” means that such an amount of one or more organic polyols causes an increase in the amount of nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, and / or a longer period of reaction output from which NOx is produced compared to a reaction carried out under the same conditions but without one or more organic polyols. The expression “amount” means the total mass of gaseous nitric oxide released per gram of nitrite that can be used in the initial reaction system. Experimental work that forms the basis of this invention has measured the amount of gaseous nitric oxide released, optionally other gases, and found that these amounts are enhanced. It is therefore believed that this invention enhances the total mass of NOx produced; therefore, the expression “amount” should also be understood to include the total mass of nitric oxide dissolved in the solution in the reaction mixture and the total mass of the NOx reaction products. The expression “output time” specifically means the duration during which at least one of gaseous nitric oxide, optionally other gases, is released in the reaction before the reaction is completed. Similar to the reasoning explained above in the discussion of the phrase "enhancing the amount of one or more organic polyols output from the reaction," it is believed that the phrase "output time period" also includes the duration for which nitric oxide dissolves in the reaction mixture and the duration for which NOx reaction products are generated. It is well known that the reaction with the proton source eventually depletes nitrite, the pH value that has risen during the NOx-producing reaction reaches its maximum, and the reaction ceases. Preferably, the method of the first aspect of the invention enhances the yield of the NOx-producing reaction, particularly but not only the amount of NO produced, such as 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 up to about 150%, for example up to about 125%, for example up to about 100%, for example up to about 75%. Preferably, the method of the first aspect of the invention enhances the duration for which at least one of the reaction, preferably nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, is released in the reaction before the reaction ends, by at least about 5%, for example at least about 10%. Using this invention, the time period for releasing at least one of nitric oxide, optionally other nitrogen oxides and / or optionally their precursors, preferably nitric oxide and most preferably gaseous nitric oxide, and especially for releasing an effective amount, can be enhanced to at least about 2 hours, for example at least about 5 hours, for example up to or more than about 10 hours. The degree of enhancement in the time of nitric oxide release can be expressed as, for example, up to or more than about 150% of the time period for releasing the same amount of nitric oxide without the use of the polyol component, for example up to about 125%, for example up to about 100%, for example up to about 75%.

[0099] The production of nitric oxide, optional other nitrogen oxides, and / or optional precursors can be used to achieve any purpose. Therapeutic and non-therapeutic purposes are illustrated and discussed below.

[0100] According to a seventh aspect, this 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, such as any cell, organ, surface, structure, subject, or internal space therein, the method comprising: (a) administering a combination or composition according to a fifth aspect of this disclosure to the target site or vicinity; or (b) using a method according to a first or third aspect of this disclosure, or performing an application according to a fourth aspect of this disclosure, or using a combination, kit, or composition according to a fifth aspect of this disclosure to generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors, and delivering the resulting nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors to the target site or vicinity; or (c) delivering nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors according to a second aspect of this disclosure to the target site or vicinity.

[0101] The method of the seventh aspect of this disclosure can be, for example, a method for treating a microbial infection in a subject in need. The subject can be, for example, a human subject or other mammalian subject. The microbial infection can be, for example, bacteria, viruses, fungi, microparasites, or any combination thereof.

[0102] The method of the seventh aspect of this disclosure can be, for example, a vasodilatory method performed on a subject. The subject can be, for example, a human subject or other mammalian subject.

[0103] The method of the seventh aspect of this disclosure can be, for example, an antimicrobial method. An antimicrobial method can be to reduce the number of microorganisms, such as bacteria, viruses, fungal cells and / or microparasitic organisms, in the local environment, prevent their reproduction, or limit their reproduction rate. The microorganisms targeted by such methods can be, for example, planktonic cells or particles, or exist as biofilms or other colonies. Any microbial community targeted by this disclosure, whether or not planktonic, can consist of one microbial species or strain, or can contain more than one species or strain.

[0104] According to the eighth aspect, this disclosure provides combinations, kits, or compositions according to the fifth aspect of this disclosure, or nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof according to the second aspect of this disclosure, for use in therapy.

[0105] The combination, kit, or composition used according to the eighth aspect of this disclosure, or nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof, may be used, for example, in a treatment method that delivers nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof to a subject or an internal space therein, the method comprising: (a) administering the combination or composition according to the fifth aspect of this disclosure to the subject or internal space or its vicinity; or (b) using the method according to the first or third aspect of this disclosure, or performing the use according to the fourth aspect of this disclosure, or using the combination, kit, or composition according to the fifth aspect of this disclosure, to generate nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof, and delivering the resulting nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof to the subject or internal space or its vicinity; or (c) delivering nitric oxide, optional other nitrogen oxides, and / or optional precursors thereof according to the second aspect of this disclosure to the subject or internal space or its vicinity.

[0106] According to this disclosure, it was unexpectedly discovered that when the proton source is citric acid (an organic carboxylic acid) or ascorbic acid (an organic noncarboxylic acid reducing acid) with an initial pH in the range of 5 to 8, it also provides good antimicrobial activity in terms of biostability and biocidal action, as demonstrated by the following: killing up to 100% of Mycobacterium abscessus after 3 days, and / or killing Mycobacterium tuberculosis, H1N1 influenza virus, SARS-CoV virus, and SARS-CoV-2 virus. The term "initial pH" as used herein refers to the pH of the aqueous solution initially formed from the proton source before the presence of other components in the reaction mixture that would affect the initial pH, including any desired pH buffer. This antimicrobial activity is independent of the presence of one or more organic polyols, although the presence of one or more organic polyols, such as mannitol or sorbitol, appears to enhance this activity. The discovery that acids (such as citric acid or ascorbic acid) with NOx-generating reaction products exhibiting strong antimicrobial activity at initial pH values ​​in the range of 5 to 8 is particularly surprising. This holds promise for the treatment of respiratory and lung infections, including those that are difficult to treat and / or resistant to antibiotics, including tuberculosis, multidrug-resistant tuberculosis, and nontuberculous mycobacterial infections. Treatment of such infections may be suggested via inhalation of an aqueous composition containing the reaction mixture or its components or precursors at a pH value in the range of 5 to 8. The invention is also capable of treating infections involving a wide range of pathogens, potentially including pathogens from more than one group of bacteria, viruses, fungi, and parasites, referred to as “broad-spectrum” treatment (including therapeutic and / or prophylactic treatment as well as extracorporeal treatment of living and non-living surfaces and spaces to prevent pathogen transmission).

[0107] According to the ninth aspect, this disclosure provides an improvement to the antimicrobial method according to the seventh aspect, the improvement comprising: (a) administering a combination or composition according to the fifth aspect of this disclosure to the microorganism to be targeted or in its vicinity or to a subject infected with the microorganism or the internal space of such a subject; or (b) using a method according to the first or third aspect of this disclosure, or performing the use according to the fourth aspect of this disclosure, or using a combination, kit, or composition according to the fifth aspect of this disclosure to generate nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof, and delivering the resulting nitric oxide, optionally other nitrogen oxides, and / or optionally precursors thereof to the microorganism to be targeted. The target microorganism or its vicinity or the internal space of a subject infected with the microorganism or such a subject; or (c) delivery of nitric oxide, optionally other nitrogen oxides and / or optionally their precursors, according to the second aspect of this disclosure, to the target microorganism or its vicinity or the internal space of a subject infected with the microorganism or such a subject; provided that the initial pH of the aqueous solution of any desired buffer solution, including the reaction mixture, is in the range of 5 to 8 before the presence of other components that would affect the pH of the reaction mixture that generate NOx, or at the start of the reaction with one or more nitrites, and one or more polyols are optional and may be omitted.

[0108] When performing the method according to the ninth aspect of this disclosure, the combination, kit, or composition according to the fifth or eighth aspect of this disclosure may be used to generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors; provided that the initial pH of the aqueous solution of any desired buffer solution, including the reaction mixture containing other components that will affect the pH of the reaction mixture that generate NOx, is in the range of 5 to 8 at the start of the reaction with one or more nitrites, and one or more polyols are optional and may be omitted.

[0109] The method of the ninth aspect of this disclosure can be, for example, a method of treating a subject with a microbial infection. The subject can be, for example, a human subject or other mammalian subject. The microbial infection can be, for example, bacteria, viruses, fungi, microparasites, or any combination thereof. The microbial infection can be on the subject's skin, including mucous membranes. The microbial infection can be in the subject's internal spaces, for example, according to the invention, in the inner lining of the subject's nose, mouth, respiratory tract, lungs, or pleura.

[0110] The components and mixtures used in all aspects of this disclosure for application to humans or animals, as well as any carriers and excipients used for application to humans or animals, are preferably biocompatible and / or pharmaceutically acceptable to minimize tissue irritation and inflammation upon application.

[0111] The combinations, kits, and compositions according to this disclosure can be stored and used in a variety of suitable devices and apparatuses, which will be described in more detail below. Methods according to this disclosure are best performed using such devices and apparatuses, as will be described in more detail below.

[0112] It will be understood that all embodiments, examples, and preferred embodiments specifically described with respect to any one or more aspects of this disclosure are applicable to any one or more other aspects of this disclosure. Additionally, if necessary, any method or use according to one aspect of this disclosure may be performed using combinations, kits, or compositions according to any other aspect. Detailed Implementation

[0113] The aspects of this disclosure will now be described in detail with reference to specific implementation methods. The specific implementation methods described below can be applied to any aspect of this disclosure unless clearly contradictory to such aspects. The specific implementation methods can also be combined with each other specific implementation method unless doing so would create a contradiction.

[0114] Nitrite and nitrite components

[0115] This disclosure relates to 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 containing one or more nitrites for generating nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors.

[0116] There are no particular limitations on the choice of nitrite. 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 mixtures thereof.

[0117] In a specific implementation, the nitrite is either NaNO2 or KNO2. In one implementation, the nitrite is NaNO2.

[0118] In one embodiment, the nitrite component may be provided in a dry form, optionally in particulate form such as powder, for use in this disclosure. Where necessary, the nitrite component may be encapsulated or microencapsulated, for example, to control or delay the reaction between one or more nitrites and a proton source. Dry form and / or encapsulation can facilitate the storage of the nitrite component, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Furthermore, dry form and / or encapsulation can facilitate the incorporation of the nitrite component into small objects such as medical devices, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Such objects include, for example, wound dressings, bandages, blood vessels and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles and inhalers (handheld and nebulizers). Further details can be found in the section below entitled “Optional Encapsulation of Components (e.g., Microencapsulation)”.

[0119] If necessary, the optionally encapsulated or microencapsulated nitrite component may be present as a dry powder or crystals, or combined with a gel or other carrier system, such as an aqueous carrier, for example, as a hydrogel or solution thereof. The nitrite component in dry or powder form is preferably prepared into a solution by adding water before use. The molar concentration of nitrite ions in such nitrite solutions may be in the range of about 0.001 M to about 5 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before acidification (e.g., just before acidification). In some embodiments, the molar concentration of nitrite ions in the nitrite solution is in the range of about 0.01 M to about 2 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before acidification (e.g., just before acidification). In some embodiments, the molar concentration of nitrite ions in the nitrite solution is in the range of about 0.1 M to about 2 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before acidification (e.g., just before acidification). In a more specific embodiment, the molar concentration of nitrite ions in the nitrite solution is in the range of about 0.2 M to about 1.6 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before acidification (e.g., just before acidification). In another embodiment, the molar concentration of nitrite ions in the nitrite solution may be in the range of 0.8 to 1.2 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before combination with the organic carboxylic acid component (e.g., just before combination). For example, the molar concentration of nitrite ions in the nitrite solution 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 before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before combination with the organic carboxylic acid component (e.g., just before combination).

[0120] It should be noted that combining two or more precursor solutions of a NOx-generating reaction mixture will dilute the concentration of each solute or solute combination in each solution, as is well known to those skilled in the art. For example, mixing two 1M solutions of solutes A and B in equal volumes will result in the concentration of A becoming 0.5M and the concentration of B becoming 0.5M. Unless otherwise stated or implied, the concentration of nitrite described herein is its concentration in the initial solution before (e.g., immediately before) the addition of any other components of the NOx-generating reaction mixture in liquid, such as solution form. Given the composition and preparation method of the reaction mixture, the actual concentration in the NOx-generating reaction mixture can be readily derived.

[0121] When necessary, whether in dry form or as a carrier liquid, the nitrite component may include one or more polyols or some of these polyols.

[0122] If it is desired that the nitrite component be stored in a gel or other carrier system, such as an aqueous carrier, for example, in the form of an hydrogel or solution, then preferably, the system containing the nitrite is buffered to a suitable pH value to prevent the nitrite from degrading during storage. A pH value of about 6-9, for example, about 7, is preferred.

[0123] Preferably, the nitrite component is not contacted with the proton source until it is desired to generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors. For this purpose, the nitrite component is preferably stored in a reservoir or container of the kit, device, or apparatus. Alternatively, however, the nitrite component, the proton source, and a dried composition of one or more polyols may be stored as a dry composition, such as a particulate mixture, and the reaction can be initiated by simply adding water or another suitable solvent or liquid carrier.

[0124] Nitrites can be pharmaceutically acceptable grades of nitrites. In some implementations, nitrites are pharmacopoeia grade. In other words, nitrites can comply with one or more current pharmacopoeia thesaurus papers on nitrites. For example, nitrites can comply with one or more of the nitrite thesaurus papers in the United States Pharmacopeia (USP), the European Pharmacopoeia, or the Japanese Pharmacopoeia.

[0125] In specific implementation schemes, the nitrites used have one or more of the following limitations:

[0126] (i) The nitrite contains up to about 0.02% by weight, about 0.01% by weight or about 0.001% by weight of sodium carbonate;

[0127] (ii) The nitrite contains up to about 10 ppm (0.001% by weight) of an anti-caking agent, such as sodium alkyl-naphthalenesulfonate;

[0128] (iii) Nitrites are white to grayish-white solids;

[0129] (iv) Nitrite has a positive identification for cations as determined by the relevant method in the relevant USP;

[0130] (v) Nitrite has a positive identification test for nitrite ions as determined by the relevant method in the relevant USP;

[0131] (vi) Optionally, as determined by relevant USP calorimetry, such as by ion chromatography, such as by ion chromatography combined with suppressed conductivity detection, the nitrite contains not less than about 97% or not less than 98% by weight of nitrite and / or up to 102% or up to 101% by weight of nitrite.

[0132] (vii) Optionally, the pH of nitrite is between about 7 and about 9 or between about 8 and about 9 when measured in a 10% solution at 25°C, according to the relevant USP and / or using a pH meter.

[0133] (viii) Nitrites have a drying loss of up to about 0.25% by weight or about 0.01% by weight;

[0134] (ix) Optionally, as determined by the Karl Fischer method, the nitrite has a water content of up to about 0.5% by weight;

[0135] (x) The heavy metal content in nitrite is at most about 10 ppm of heavy metals, and optionally, the heavy metal content in nitrite is at most about 10 ppm;

[0136] (xi) The nitrite contains up to about 0.4% by weight of nitrate, optionally up to about 0.4% by weight of sodium nitrite when the nitrite is sodium nitrite, and up to about 0.4% by weight of potassium nitrite when the nitrite is potassium nitrite;

[0137] (xiii) Nitrites contain up to about 0.005% by weight or about 0.001% by weight of insoluble matter;

[0138] (xiii) Nitrites contain up to about 0.005% by weight of chloride;

[0139] (xiv) Nitrites contain up to about 0.01% by weight of sulfate;

[0140] (xv) Nitrites contain up to about 0.001% by weight of iron;

[0141] (xvi) Nitrites contain up to about 0.01% by weight of calcium;

[0142] (xvii) When the nitrite is not potassium nitrite, the nitrite contains up to about 0.005% by weight or about 0.001% by weight of potassium, or when the nitrite is not sodium nitrite, the nitrite contains up to about 0.005% by weight or about 0.001% by weight of sodium;

[0143] (xviii) Nitrites contain up to about 0.1% by weight, up to about 5,000 ppm, up to about 1,000 ppm, up to about 500 ppm, up to about 100 ppm or up to about 10 ppm of volatile organic compounds;

[0144] (xix) Nitrites contain up to about 0.1% by weight, up to about 5000 ppm, up to about 1000 ppm, up to about 500 ppm, up to about 100 ppm or up to about 10 ppm of ethanol;

[0145] (xx) Nitrite contains up to about 3000 ppm, up to about 1000 ppm, up to about 500 ppm, up to about 100 ppm or up to about 10 ppm of methanol;

[0146] (xxi) Nitrites contain up to about 50 ppm, up to about 25 ppm, up to about 20 ppm, up to about 10 ppm, up to about 7.9 ppm, up to about 8 ppm, up to about 6 ppm, up to about 5.6 ppm or up to about 2.5 ppm of non-volatile organic carbon;

[0147] (xxii) Nitrites contain up to about 0.05 ppm of mercury;

[0148] (xxiii) Nitrites contain up to about 2 ppm or 0.2 ppm of aluminum;

[0149] (xxiv) Nitrites contain up to about 3 ppm or 1 ppm of arsenic;

[0150] (xxv) Nitrites contain up to about 0.003% by weight or about 0.001% by weight of selenium;

[0151] (xxvi) The total aerobic count of the microbial load in nitrite is at most about 100 CFU / g;

[0152] (xxvii) The total yeast and mold count in nitrates is at most about 20 CFU / g;

[0153] (xxviii) Nitrites contain up to about 0.25 EU / mg or 0.018 EU / mg of bacterial endotoxin; and

[0154] (xxix) Nitrite contains less than about 0.1 ppm of phosphate, such as sodium phosphate, disodium hydrogen phosphate or trisodium phosphate, and preferably, nitrite does not contain detectable amounts of phosphate.

[0155] In some embodiments, the nitrite has two or more of the characteristics (i) to (xxix). In other embodiments, the nitrite has five or more of the characteristics (i) to (xxix). In other embodiments, the nitrite has ten or more of the characteristics (i) to (xxix). In other 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 one specific embodiment, the nitrite has all of the characteristics (i) to (xxix). In a more specific embodiment, the nitrite is sodium nitrite having all of the characteristics (i) to (xxix).

[0156] In some embodiments, nitrite is optionally present in the range of about 97% by weight to about 101% by weight, as determined by a relevant USP calorimetric method, such as ion chromatography, or ion chromatography combined with suppressed conductivity detection. In alternative embodiments, nitrite is optionally present in the range of about 98% by weight to about 102% by weight, as determined by a relevant USP calorimetric method, such as ion chromatography, or ion chromatography combined with suppressed conductivity detection.

[0157] In specific implementation schemes, nitrites have the following characteristics:

[0158] (i) The nitrite contains up to about 0.02% by weight of sodium carbonate;

[0159] (ii) Nitrites contain up to about 10 ppm of anti-caking agent;

[0160] (vi) As determined by USP calorimetry, the nitrite contains not less than 97% by weight and not more than 101% by weight of nitrite.

[0161] (viii) Nitrites have a drying loss of up to about 0.25% by weight;

[0162] (ix) Nitrites have a water content of up to about 0.5% by weight;

[0163] (x) The heavy metal content in nitrite is at most about 10 ppm;

[0164] (xi) Nitrites contain up to about 0.4% by weight of nitrates;

[0165] (xii) Nitrites contain up to about 0.005% by weight of insoluble matter;

[0166] (xiii) Nitrites contain up to about 0.005% by weight of chloride;

[0167] (xiv) Nitrites contain up to about 0.01% by weight of sulfate;

[0168] (xv) Nitrites contain up to about 0.001% by weight of iron;

[0169] (xvi) Nitrites contain up to about 0.01% by weight of calcium;

[0170] (xviii) Nitrites contain up to about 5,000 ppm, up to about 1,000 ppm, up to about 500 ppm, up to about 100 ppm or up to about 10 ppm of volatile organic compounds;

[0171] (xxi) Nitrites contain up to about 10 ppm or up to about 2.5 ppm of non-volatile organic carbon;

[0172] (xxii) Nitrites contain up to about 0.05 ppm of mercury;

[0173] (xxiii) Nitrites contain up to about 2 ppm of aluminum;

[0174] (xxiv) Nitrites contain up to about 3 ppm of arsenic;

[0175] (xxv) Nitrites contain up to about 0.003% by weight of selenium;

[0176] (xxvi) The total aerobic count of the microbial load in nitrite is at most about 100 CFU / g;

[0177] (xxvii) The total yeast and mold count in nitrates is at most about 20 CFU / g; and

[0178] (xxviii) Nitrites contain up to about 0.25 EU / mg of bacterial endotoxin.

[0179] In these embodiments, the nitrite may be sodium nitrite and contains up to about 0.005% by weight of potassium. Preferably, the sodium nitrite also has one or more of the following limitations:

[0180] (iii) Sodium nitrite is a white to grayish-white solid;

[0181] (iv) Sodium nitrite has a positive identification for sodium as determined by the relevant method in the relevant USP;

[0182] (v) Sodium nitrite has a positive identification test for nitrite ions as determined by the relevant method in the relevant USP;

[0183] (vii) Optionally, the pH of sodium nitrite, when measured in a 10% solution at 25°C, is between about 7 and about 9 or between about 8 and about 9, according to the relevant USP and / or using a pH meter.

[0184] (xix) Sodium nitrite contains up to about 0.1% by weight, up to about 5000 ppm, up to about 1000 ppm, up to about 500 ppm, up to about 100 ppm or up to about 10 ppm of ethanol;

[0185] (xx) Nitrites contain up to about 3000 ppm, up to about 1000 ppm, up to about 500 ppm, up to about 100 ppm, or up to about 10 ppm of methanol; and

[0186] (xxix) Nitrite contains less than about 0.1 ppm of phosphate, such as sodium phosphate, disodium hydrogen phosphate or trisodium phosphate, and preferably, nitrite does not contain detectable amounts of phosphate.

[0187] Characteristic (i) to (xxix) can be determined according to the relevant methods in USP XXXII (2009). Methods for determining characteristics (i) to (xxix) are provided in WO 2010 / 093746 (the disclosure of which is incorporated herein by reference in its entirety). WO 2010 / 093746 also describes methods for preparing sodium nitrite having one or more of characteristics (i) to (xxix).

[0188] Proton source and proton source components containing one or more organic carboxylic acids

[0189] This disclosure relates to a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids. Hereinafter, the term "proton source component" encompasses the proton source itself and any component of the reaction system containing the proton source used to generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors.

[0190] In this section, organic carboxylic acids will be illustrated with more detailed examples.

[0191] In this document, "organic carboxylic acid" refers to any organic acid whose molecule contains one or more -COOH groups. Organic carboxylic acids can be straight-chain or branched. They can be saturated or unsaturated. They can be aliphatic or aromatic. They can be acyclic or cyclic. They can be vinyl carboxylic acids.

[0192] Organic carboxylic acids may carry one or more substituents, such as one or more hydroxyl groups. Examples of organic carboxylic acids that can be used for hydroxyl substitution in this disclosure include α-hydroxy-carboxylic acids, β-hydroxy-carboxylic acids, and γ-hydroxy-carboxylic acids.

[0193] One or more organic carboxylic acids, or if there are more than one, each of them should preferably have a pKa of less than about 7, more preferably less than 7.0.

[0194] One or more carboxylic acids may be, contain one or more reduced carboxylic acids, or consist of them.

[0195] Carboxylic acids can be acid hydrogels containing -COOH side groups covalently linked to polymer molecules of the three-dimensional polymer matrix forming the hydrogel. Examples of such carboxylic acid-containing hydrogels are described, for example, in WO2007 / 007115, WO 2008 / 087411, WO 2008 / 087408, WO 2014 / 188174 and WO2014 / 188175 and the references therein, the disclosures of all of which are incorporated herein by reference. Such hydrogels typically contain carboxylic acids and sulfonyl side groups in acid or salt form covalently bonded to the three-dimensional polymer matrix. For further discussion, see the section below entitled “Other Reservoirs of Components: Hydrogels”.

[0196] However, generally preferably, at least one of the acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids is not covalently bonded to the polymer or macromolecule, such as the three-dimensional polymer forming a hydrogel or the polymer or macromolecule forming the macromolecular matrix. Not wishing to be bound by theory, evidence, such as the evidence of the dependence on the stereoisomerism of polyols discussed in the section entitled “Organic Polyols” below, indicates that the enhancing effect of this disclosure on the reaction output of one or more nitrites with a proton source is achieved at least in part through the action of organic polyol molecules interacting with the nitrite and proton during the acidification reaction. This suggests that the mobility of reactant molecules in orientation and repositioning under the influence of polyol molecules during the reaction can be important. Even if the polyol is not necessarily present, for example, as in the eighth aspect of this disclosure, it can be presumed that the same mobility between reactants in the reaction of one or more nitrites with a proton source can be important.

[0197] Organic carboxylic acids may be selected, for example, from salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropionic acid, β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid, stearic acid, malonic acid, succinic acid, fumaric acid, glucoheponic acid, glucuronic acid, lactobionic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, their salts, and combinations thereof. In a specific embodiment, the organic carboxylic acid is selected from citric acid, its salts, and combinations thereof. In one specific embodiment, the organic carboxylic acid is citric acid or a salt thereof. Carboxylic acids may be or comprise polymers or polymeric 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. The term “organic carboxylic acid” as used herein also covers partial or complete esters of organic carboxylic acids or their partial or complete salts, provided that those esters or salts can be used as a proton source according to the invention.

[0198] Preferably, the pH of the proton source is buffered just before contact between one or more nitrites and the proton source to control the pH within a known range and limit the rate of pH increase as the nitrites are consumed. See below the section entitled “pH Control; Optional Buffering Systems” for more details. Specifically, it is contemplated that at least one organic carboxylic acid of the proton source is suitable to be present with its conjugate base. The acid and its conjugate base are suitable to form a buffer solution in an aqueous carrier. The buffer solution can be selected such that the desired pH is maintained during the NOx-generating reaction, preferably in the range of about 3 to 9, for example, about 4 to 8, and preferably in the range of about 5 to about 8 for physiological contact or contact with living cells and organisms. The conjugate base can be added separately in the presence, or it can be generated on-site from the proton source by adjusting the pH using an acid and / or base, preferably an inorganic acid and / or inorganic base.

[0199] The initial pH of the aqueous proton source, including any desired buffer solution, is suitable for the reaction mixture at the start of a reaction with one or more nitrites, prior to the addition of other components that will affect the pH of the NOx-generating reaction mixture (e.g., immediately before addition). The term "initial pH" as used herein in conjunction with "proton source" means the pH of the aqueous proton source, including any desired buffer solution, prior to the addition of other components (including some, but not all, of the NOx-generating reaction mixture) that will affect the pH (e.g., immediately before addition). Dry powder proton source material or other precursors to the aqueous proton source will be used in an amount suitable for producing an aqueous solution with the desired initial pH.

[0200] If it is desired that the proton source component be stored in the form of a gel or other carrier system, such as an aqueous carrier, for example, in the form of a hydrogel or solution, then preferably, the system containing the proton source is buffered to a suitable pH value to maintain the acidity of the proton source and prevent its degradation during storage. A pH value of about 3-6, for example, about 3-5, is preferred. If necessary, the pH value can be increased by adding alkali when the proton source component is about to be used.

[0201] Some patients, for example, are intolerant to citric acid. Patients should be tested for potential acid intolerances before treatment, and the appropriate acid component should be selected accordingly.

[0202] In one embodiment, the proton source component or a portion thereof may be provided in a dry form, optionally in particulate form such as powder, for use in this disclosure. Where necessary, the proton source component or a portion thereof may be encapsulated or microencapsulated, for example, to control or delay the reaction between one or more nitrites and the proton source. Encapsulation is particularly useful when the proton source is typically in a liquid or gel state at room temperature. Dry form and / or encapsulation can facilitate the storage of the proton source, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Furthermore, dry form and / or encapsulation can facilitate the incorporation of the proton source component into small objects such as medical devices, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Such objects include, for example, wound dressings, bandages, blood vessels and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. Further details can be found in the section below entitled “Optional Encapsulation of Components (e.g., Microencapsulation)”.

[0203] If necessary, one or more organic carboxylic acids, optionally encapsulated or microencapsulated, may exist as dry powder or crystals in the proton source component, or be combined with a gel or other carrier system, such as an aqueous carrier, for example, as an hydrogel or solution thereof. The proton source component containing organic carboxylic acids in dry or powder form is preferably prepared into a solution by adding water before use. The molar concentration of the total proton source (including any organic non-carboxylic acid reducing acid present) in such a solution may be in the range of about 0.001 M to about 5 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before the initiation of the reaction with nitrite (e.g., just before initiation). In some embodiments, the molar concentration of the total proton source in such a solution is in the range of about 0.01 M to about 2 M before the addition of any other component of the NOx-generating reaction mixture (e.g., just before its addition) and especially before the initiation of the reaction with nitrite (e.g., just before initiation). In some embodiments, the molar concentration of the total proton source in such a solution is in the range of about 0.1 M to about 2 M before the initiation of the reaction with nitrite. In a more specific embodiment, the molar concentration of the total proton source in such a solution is in the range of about 0.2 M to about 1.6 M prior to initiating the reaction with nitrite. In another embodiment, the molar concentration of the total proton source in such a solution may be in the range of 0.8 to 1.2 M prior to initiating the reaction with nitrite. For example, the molar concentration of the total proton source in such a solution prior to initiating 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.

[0204] The terms "molar concentration of total proton source" and "concentration of total proton source" used in this article should be understood as referring to the concentration of protons (H2O) at a given molar concentration. + Donor portion or proton (H) + At least one of the donor portions (in cases of more than one) is substantially protonated, i.e., more than 50% protonated in molar at a pH value, which is used as the concentration of any one of the organic carboxylic acids and / or organic non-carboxylic acids as the proton source according to the invention. In other words, if the pH is adjusted to a higher pH value before initiating a reaction that produces NOx, thereby reducing the degree of protonation, then the molar concentration or concentration of the total proton source is not considered to decrease accordingly.

[0205] It should be noted that combining two or more precursor solutions of a NOx-producing reaction mixture will dilute the concentration of each solute or solute combination in each solution, as is well known to those skilled in the art. For example, mixing two 1M solutions of solutes A and B in equal volumes will result in the concentration of A becoming 0.5M and the concentration of B becoming 0.5M. Unless otherwise stated or implied, the concentration of the proton source described herein is its concentration in the initial solution before (e.g., immediately before) the addition of any other components of the NOx-producing reaction mixture in liquid, such as solution form. Given the composition and preparation method of the reaction mixture, the actual concentration in the NOx-producing reaction mixture can be readily derived.

[0206] Proton source components in dry or powder form should be prepared into a solution by adding water before use.

[0207] When necessary, one or more organic carboxylic acids can exist or form a solution mixed with one or more polyols or some of these polyols, whether in dry form or as a carrier liquid.

[0208] Preferably, the nitrite component is not brought into contact with the proton source until it is desired to generate nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors. For this purpose, the proton source component, or a portion thereof, is preferably stored in a reservoir or container of the kit, device, or apparatus. Alternatively, however, a dried composition of one or more nitrites or nitrite components, the proton source, and one or more polyols may be stored as a dry composition, such as a particulate mixture, and the reaction can be initiated by simply adding water or another suitable solvent or liquid carrier.

[0209] Proton source components containing one or more organic noncarboxylic acid reducing acids

[0210] The above discussion of proton source components comprising or composed of one or more organic carboxylic acids is similarly applicable to proton source components comprising or composed of one or more organic noncarboxylic acid reducing acids. Organic noncarboxylic acid reducing acids will be illustrated in more detail in this section.

[0211] The term "organic non-carboxylic acid reducing acid" in this article refers to any organic reducing acid whose molecule does not contain a -COOH group. Organic non-carboxylic acid reducing acids can be straight-chain or branched. They can be saturated or unsaturated. They can be aliphatic or aromatic. They can be acyclic or cyclic. They can be vinyl.

[0212] One or more organic noncarboxylic acid reducing acids, or if more than one, each of which should preferably have a pKa of less than about 7, more preferably less than 7.0.

[0213] For the reasons explained above, it is generally preferred that at least one of 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 a three-dimensional polymer matrix of a hydrogel.

[0214] Organic noncarboxylic acid reducing acids may be selected, for example, from ascorbic acid; ascorbyl palmitic acid (ascorbyl palmitate); ascorbic acid derivatives, such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid, and 6-O-dodecanoic acid; acidic reducing ketones, such as reducing acids; isoascorbic acid; oxalic acid; their salts; and combinations thereof. In one specific embodiment, the organic noncarboxylic acid reducing acid is ascorbic acid or a salt thereof.

[0215] Organic noncarboxylic acid reducing acids may carry one or more substituents, such as one or more hydroxyl groups. Examples of organic noncarboxylic acid reducing acids that can be used for hydroxyl substitution in this disclosure include acidic reducing ketones, such as reducing acid (2,3-dihydroxy-2-cyclopentanone).

[0216] Preferably, the pH of the proton source and / or reaction mixture is buffered after contact between one or more nitrites and the proton source to control the pH within a known range and to control the increase of pH as the nitrite is consumed. See below the section entitled “pH Control; Optional Buffering Systems” for further details. Specifically, it is contemplated that at least one organic noncarboxylic acid reducing acid of the proton source is suitable to be present with its conjugate base. The acid and its conjugate base are suitable to form a buffer solution in an aqueous carrier. The buffer solution can be selected such that the desired pH is maintained during the NO-generating reaction, preferably in the range of about 3 to 9, for example, about 4 to 8, and preferably in the range of about 5 to about 8 for physiological contact or contact with living cells and organisms. The conjugate base can be added separately in the presence, or the conjugate base can be generated on-site from the proton source by adjusting the pH using an acid and / or base, preferably an inorganic acid and / or inorganic base.

[0217] Before adding other components that will affect the pH of the reaction mixture that generate NOx (e.g., just before addition), the initial pH of the aqueous solution of the proton source, including any desired buffer, or the pH of the reaction mixture at the start of the reaction with one or more nitrites, should be in the range of about 3 to 9, for example, about 4 to 8, for example, about 5 to 8. Dry powder proton source material or other precursors of the aqueous solution of the proton source will be used in an amount suitable for producing an aqueous solution with the desired initial pH.

[0218] If it is desired that the proton source component be stored in the form of a gel or other carrier system, such as an aqueous carrier, for example, in the form of a hydrogel or solution, then preferably, the system containing the proton source is buffered to a suitable pH value to maintain the acidity of the proton source and prevent its degradation during storage. A pH value of about 3-6, for example, about 3-5, is preferred. If necessary, the pH value can be increased by adding alkali when the proton source component is about to be used.

[0219] Some reducing acids, such as oxalic acid, are toxic. Therefore, the acid component should be selected accordingly.

[0220] In addition to or in place of one or more organic carboxylic acids as described above, one or more organic non-carboxylic acid reducing acids may also be used in the proton source component. For further details, please refer to the section entitled "Proton Sources and Proton Source Components Containing One or More Organic Carboxylic Acids".

[0221] Organic polyols and organic polyol components

[0222] This disclosure relates to 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 containing an organic polyol for generating nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors.

[0223] The term "organic polyol" as used herein refers to an organic molecule having two or more hydroxyl groups that is not a proton source, especially for nitrite reactions, and is neither a sugar nor a polysaccharide (the terms "sugar" and "polysaccharide" include oligosaccharides, polysaccharides, and aminopolysaccharides). Therefore, an organic polyol will have a pKa of approximately 7 or greater, for example, 7.0 or greater.

[0224] The term "organic polyol" in this document preferably excludes reducing agents. In one embodiment of the invention, reducing agents are therefore excluded in all aspects of organic polyols. Examples of reducing agents that are organic molecules having two or more hydroxyl groups but are not sugars or polysaccharides are thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate, isoascorbic acid, and isoascorbate. Therefore, thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate, and isoascorbate are preferably excluded from the term "organic polyol" because they are reducing agents. Ascorbic acid and isoascorbic acid are excluded from this term in any case because they are proton sources, especially for nitrite reactions. To avoid any doubt, it is confirmed that reducing agents such as ascorbic acid and / or isoascorbic acid, which are proton sources, are not excluded from the proton sources of the present invention or from the components, combinations, kits, compositions, uses, methods, or any other parts of the proton source of the present invention, as well as from the means of putting them into practice as proton sources.

[0225] Organic polyols can be cyclic or acyclic, or they can be a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, one or more organic polyols can be selected from one or more alkanes substituted with two or more OH groups, one or more cycloalkanes substituted with two or more OH groups, one or more cycloalkylalkanes substituted with two or more OH groups, and any combination thereof. Most preferably, the organic polyol does not carry any substituents other than OH.

[0226] Preferably, the one or more organic polyols are one or more acyclic organic polyols. Preferredly, the one or more acyclic organic polyols are selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferredly, the one or more acyclic organic polyols are selected from aldehyde alcohols, such as aldehyde alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferably, the one or more organic polyols do not include saponins, saponin aglycones, steroids, or steroidal glycosides.

[0227] Alternatively, one or more organic polyols may be one or more cyclic organic polyols. In these embodiments, the one or more cyclic organic polyols may be cyclic sugar alcohols or cyclic aldehydes. 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 aldehydes having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. A specific example of a cyclic polyol is inositol.

[0228] In some embodiments, one or more organic polyols have seven or more hydroxyl groups. In a specific embodiment, one or more organic polyols are sugar alcohols or aldehydes having seven or more hydroxyl groups. In a more specific embodiment, one or more organic polyols have nine or more hydroxyl groups. In other embodiments, one or more organic polyols are sugar alcohols or aldehydes having nine or more hydroxyl groups. In some embodiments, one or more organic polyols have 20 or fewer hydroxyl groups. In a specific embodiment, one or more organic polyols are sugar alcohols or aldehydes having 20 or fewer hydroxyl groups. In a more specific embodiment, one or more organic polyols have 15 or fewer hydroxyl groups. In other embodiments, one or more organic polyols are sugar alcohols or aldehydes having 15 or fewer hydroxyl groups. One or more organic polyols may have multiple hydroxyl groups in the range of 7 to 20, more specifically in the range of 9 to 15. In some embodiments, one or more organic polyols include 9, 12, 15, or 18 hydroxyl groups.

[0229] Preferably, one or more organic polyols are sugar alcohol compounds, said sugar alcohol compounds comprising one or more monosaccharide units and one or more acyclic sugar alcohol units, for example, composed of these units. One or more organic polyols may be sugar alcohol compounds comprising a straight 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, for example, composed of said straight chain or branched chain.

[0230] As used herein, a monosaccharide unit refers to a monosaccharide covalently linked to at least one other unit in the compound (whether another monosaccharide unit or a noncyclic sugar alcohol unit). As used herein, a noncyclic sugar alcohol unit refers to a noncyclic sugar alcohol covalently linked to at least one other unit in the compound (whether a monosaccharide unit or another noncyclic sugar alcohol unit). Units in the compound may be linked by ether bonds. In some embodiments, one or more monosaccharide units are covalently linked to other units in the compound via glycosidic bonds. In specific embodiments, each monosaccharide unit is covalently linked to other units in the compound via a glycosidic bond. In some embodiments, the sugar alcohol compound is a glycoside having a monosaccharide or oligosaccharide glycosyl group and a noncyclic sugar alcohol glycosidic aglycone.

[0231] Preferred acyclic sugar alcohol units are sugar alcohol units having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. In specific embodiments, the acyclic sugar alcohol units are selected from the group consisting of units of erythritol, threitol, araitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idoteol, and heptadecyl alcohol.

[0232] In a specific embodiment, one or more monosaccharide units are C5 or C6 monosaccharide units. In other words, one or more monosaccharide units are pentose or hexose units. In a more specific embodiment, each monosaccharide unit is a C5 or C6 monosaccharide unit. In a specific embodiment, one or more sugar alcohol units are C5 or C6 sugar alcohol units. In a more specific embodiment, each sugar alcohol unit is a C5 or C6 sugar alcohol unit.

[0233] In some embodiments, the sugar alcohol compound comprises n monosaccharide units and m acyclic sugar alcohol units, for example, consisting of these units, where n is an integer, at least 1, m is an integer, at least 1, and (n+m) is at most 10. In some embodiments, the sugar alcohol compound comprises a chain of n monosaccharide units terminated with a noncyclic sugar alcohol unit, for example, consisting of such a chain, where n is an integer between 1 and 9. In these embodiments, the chain of monosaccharide units can be covalently linked by glycosidic bonds. In specific embodiments, each monosaccharide unit is covalently linked to another monosaccharide unit or noncyclic sugar alcohol unit by a glycosidic bond. In some embodiments, the sugar alcohol compound comprises a chain of one, two, or three monosaccharide units terminated with a noncyclic alcohol unit, for example, consisting of such a chain. One, two, three, or each monosaccharide unit can be a C5 or C6 monosaccharide unit. The noncyclic alcohol unit can be a C5 or C6 sugar alcohol unit. Examples of sugar alcohol compounds include, but are not limited to: isomaltitol, maltitol and lactitol (n=1); maltitol (n=2); and maltetitol (n=3).

[0234] These sugar alcohol compounds can be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, an oligosaccharide is a sugar composed of three to ten monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides can be synthesized (e.g., by hydrogenation) from disaccharides, oligosaccharides, or polysaccharides (e.g., by hydrolysis and hydrogenation), but are not limited to compounds synthesized from disaccharides, oligosaccharides, or polysaccharides. For example, sugar alcohols derived from disaccharides can be formed by the dehydration reaction of a monosaccharide and a sugar alcohol. One or more organic polyols can be sugar alcohols derived from disaccharides, trisaccharides, or tetrasaccharides. Examples of sugar alcohols derived from disaccharides include, but are not limited to, isomaltitol, maltitol, and lactitol. One example of sugar alcohols derived from trisaccharides includes, but is not limited to, maltotriol. One example of sugar alcohols derived from tetrasaccharides includes, but is not limited to, maltotetratitol.

[0235] Suitable organic polyols may be selected from any of the following: erythritol, threitol, araitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idoteol, inositol, heptenetroitol, isomaltitol, maltitol, lactitol, maltotriol, maltotetratitol, polyols, and any combination thereof. Glycerol may be used, and when present, is preferably combined with one or more other organic polyols, such as erythritol, threitol, araitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idoteol, inositol, heptenetroitol, isomaltitol, maltitol, lactitol, maltotriol, maltotetratitol, polyols, or any combination thereof.

[0236] Many organic polyols contain one or more chiral centers and therefore exist in stereoisomeric forms. All stereoisomers, optical isomers, and mixtures of isomers of organic polyols are included within the scope of this disclosure and patent. Specifically, the D and / or L forms of all chiral organic polyols, and all mixtures thereof, can be used.

[0237] Interestingly, it has been found that the role of polyols in this disclosure is stereochemically dependent. Therefore, the choice of the optical isomer or mixture of optical isomers of one or more organic polyols used in this disclosure may affect the reaction outcome between nitrite and the proton source, at least in terms of the amount of NO produced.

[0238] For example, sorbitol is a stereoisomer of mannitol, differing from it in the orientation of one of its hydroxyl groups. This is illustrated in Examples 2D and 2E below. Figure 5 and 6 The effects of sorbitol and mannitol on the reaction output between nitrite and proton source differ in reaction systems that are otherwise identical.

[0239] In a specific embodiment, the organic polyol is selected from the group consisting of aritol, xylitol, mannitol, sorbitol, and any combination thereof. Aritol may be D or L aritol 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.

[0240] In a particular implementation scheme, When used in the systems, methods, combinations, kits, and compositions described herein, in the treatment of tuberculosis infection, or in antimicrobial methods for treating tuberculosis infection or for reducing the number of tuberculosis bacteria, one or more polyols are, as described herein, sugar alcohol compounds comprising one or more monosaccharide units and one or more noncyclic sugar alcohol units (including sugar alcohols derived from disaccharides or oligosaccharides), such as sugar alcohol compounds composed of these units.

[0241] In one embodiment, the organic polyol component may be provided in a dry form, optionally in particulate form such as powder, for use in this disclosure. Where necessary, the organic polyol may be encapsulated or microencapsulated, for example, to control or delay the participation of the polyol in the reaction between one or more nitrites and a proton source. Encapsulation is particularly useful when the organic polyol is typically in a liquid or gel state at room temperature. Dry form and / or encapsulation can facilitate the storage of the organic polyol component, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Furthermore, dry form and / or encapsulation can facilitate the incorporation of the organic polyol component into small objects such as medical devices, whether alone or mixed with other components that generate nitric oxide according to this disclosure. Such objects include, for example, wound dressings, bandages, blood vessels and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. Further details can be found in the section below entitled “Optional Encapsulation of Components (e.g., Microencapsulation)”.

[0242] Alternatively, the organic polyol component may include a carrier medium, such as an aqueous carrier liquid or a gel carrier. If the organic polyol is typically a liquid at room temperature, it can be used as a carrier medium without any additional carrier components, or it can be mixed with one or more carrier additives such as water.

[0243] If necessary, one or more organic polyols, optionally encapsulated or microencapsulated, may exist in the polyol component as a dry powder or crystals, or in combination with a gel or other carrier system, such as an aqueous carrier, for example, as a hydrogel or solution thereof. The polyol component containing organic polyols in dry or powder form is preferably prepared into a solution by adding water before use. The total molar concentration of one or more polyols in such a solution can be any concentration up to the saturation limit of the polyols in the solution or of each polyol before initiating the reaction with nitrite. For example, the total molar concentration of one or more organic 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 is in the range of about 0.01 M to about 2 M before initiating the reaction with nitrite. In some embodiments, the total molar concentration of one or more polyols in such a solution is in the range of about 0.1 M to about 2 M before initiating the reaction with nitrite. In a more specific embodiment, the total molar concentration of one or more polyols in such a solution is in the range of about 0.2 M to about 1.6 M before initiating the reaction with nitrite. In the implementation scheme, the total molar concentration of one or more polyols in such solutions prior to initiating the reaction with nitrite can be in the range of 0.8 to 1.2 M. For example, the total molar concentration of one or more polyols in such solutions prior to initiating the reaction with nitrite can be about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.5 M, or about 1.7 M.

[0244] It should be noted that combining two or more precursor solutions of a NOx-generating reaction mixture will dilute the concentration of each solute or solute combination in each solution, as is well known to those skilled in the art. For example, mixing two 1M solutions of solutes A and B in equal volumes will result in the concentration of A becoming 0.5M and the concentration of B becoming 0.5M. Unless otherwise stated or implied, the concentrations of organic polyols described herein are their concentrations in the initial solution prior to (e.g., immediately before) the addition of any other components of the NOx-generating reaction mixture in liquid, such as solution form. Given the composition and preparation method of the reaction mixture, the actual concentrations in the NOx-generating reaction mixture can be readily derived.

[0245] Polyol components in dry or powder form should be prepared into a solution by adding water before use.

[0246] When necessary, polyols can exist or form solutions mixed with one or more nitrites or proton sources, or some of these proton sources, whether in dry form or as a carrier liquid.

[0247] In specific embodiments where the nitrite is kept separate from other components involved in the reaction that generate nitric oxide before use, the nitrite component may include one or more polyols. In these embodiments, the organic carboxylic acid component may be substantially polyol-free. In alternative embodiments, the organic carboxylic acid component includes one or more polyols. In these embodiments, the nitrite component may be substantially polyol-free. In other embodiments, each of the organic carboxylic acid component and the nitrite component may include one or more polyols, and the polyols may be the same or different between the two components.

[0248] In another embodiment, the organic carboxylic acid component and the nitrite component may be substantially free of polyols, and one or more polyols may be included in a separate polyol component.

[0249] The relative concentrations of nitrite, proton source, and any polyol in the reaction mixture.

[0250] At the start of (or before) the NOx-generating reaction, the total molar concentration of any one or more organic polyols in the polyol component or the reaction solution is preferably between about 0.05 and about 3 times the total molar concentration of nitrite ions in the nitrite component or the reaction solution, for example, between about 0.1 and about 2 times the total molar concentration of nitrite ions, for example, between about 0.25 and about 1.5 times, for example, between about 0.3 and about 1.2 times. Before (or just before) initiating the NOx-generating reaction, the same relative molar concentration of one or more organic polyols to nitrite ions is preferably provided in the components of the combination or kit according to the invention or in the composition according to the invention.

[0251] At the start of (or before) the NOx-generating reaction, the total molar concentration of any one or more organic polyols in the polyol component or the reaction solution is preferably between about 0.05 and about 3 times the total molar concentration of the proton source in the proton source component or the reaction solution, for example, between about 0.1 and about 2 times the total molar concentration of the proton source. Prior to (or just before) initiating the NOx-generating reaction, the same relative molar concentrations of one or more organic polyols and the proton source are preferably provided in the components of the combination or kit according to the invention or in the composition according to the invention.

[0252] Optional additional components

[0253] Combinations, kits, or compositions used in this disclosure may be provided incorporating a range of diluents, carriers, and excipients and / or in combination with one or more additional components, the specific functional components of which are intended to provide one or more specific benefits for the combination, kit, or composition in which they are used. Such diluents, carriers, excipients, and / or additional components are generally physiologically compatible when intended for use in vivo.

[0254] Examples of suitable physiologically compatible diluents, carriers, and / or excipients include, but are not limited to, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talc, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, etc.

[0255] Generally, depending on the intended administration method, the pharmaceutical formulation will contain about 0.005% by weight to about 95% by weight, preferably about 0.5% by weight 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 or readily apparent to those skilled in the art.

[0256] Excipients may be selected from known excipients, depending on the intended use or the route of administration for delivering reactants and / or reaction products to a target site to deliver nitric oxide, optionally other nitrogen oxides, and / or optionally their precursors. For example, creams, lotions, and ointments can be formulated by incorporating nitrites into excipients such as cream, lotion, and ointment bases, or other thickeners and viscous agents (e.g., Eudragit L100, carbopol, carboxymethyl cellulose, or hydroxymethyl cellulose). Proton sources may be incorporated into excipients selected from carbopol, carboxymethyl cellulose, hydroxymethyl cellulose, methyl cellulose, or aqueous bases. If film formation is desired, film-forming excipients such as propylene glycol, polyvinylpyrrolidone (polyvinyl ketone), gelatin, guar gum, and shellac can be used.

[0257] Optional additional components may be selected, for example, from sweeteners, masking agents, thickeners, viscous agents, wetting agents, lubricants, binders, film-forming agents, emulsifiers, solubilizers, stabilizers, colorants, flavor enhancers, salts, coating agents, antioxidants, pharmaceutical actives, and preservatives. These components are well known in the art and need not be described in detail by a skilled reader. Examples of auxiliary substances for wetting agents, emulsifiers, lubricants, binders, and solubilizers include, for example, sodium phosphate, potassium phosphate, gum arabic, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc. Sweeteners or masking agents may include, for example, sugars, saccharin, aspartame, sucralose, neotame, or other compounds that advantageously affect taste, aftertaste, unpleasant saltiness, sourness, or bitterness, or reduce the tendency of oral or inhaled formulations to irritate the recipient (e.g., by causing cough or sore throat or other undesirable side effects, such as potentially reducing the delivered dose or adversely affecting patient compliance with prescribed treatment). Some masking agents may form complexes with one or more nitrites. Examples of thickeners, viscous agents, and film-forming agents have been given above.

[0258] The selection of pharmaceutical active agents and other additional components, such as those used as diluents, carriers, and excipients, can be determined by their suitability for the treatment of the relevant disease or medical condition and the desired route of administration of the combination or composition according to this disclosure. References may be made to standard references such as Martindale, 39th edition (2017); the Merck Index, 15th edition (2013); Goodman & Gilman's "The Pharmacological Basis of Therapeutics", 13th edition (2017); the British National Formulary online (https: / / bnf.nice.org.uk / ); Remington: "The Science & Practice of Pharmacy", 22nd edition (2012); or the Physician's Desk Reference, 71st edition (2017).

[0259] Examples of routes of administration for administering the components and compositions according to this disclosure to animal (including human) subjects to achieve a therapeutic purpose include topically (e.g., creams, lotions, gels, ointments, pastes, emollients, sprays), through the ear, nose (e.g., sprays), vaginally, rectal (e.g., suppositories), orally (e.g., nebulizers, sprays, mouthwashes, aerosols), enterically (e.g., tablets, soft lozenges, lozenges, capsules, licks, elixirs), and parenterally (e.g., injectable liquids), through the eyes, ears, nose, or throat (e.g., drops), or via the respiratory tract or lungs (e.g., nebulizers, aerosols, powder inhalation).

[0260] Examples of pharmaceutically active agents that may be incorporated into or co-administered with the components and compositions according to the present disclosure include antibiotics, steroids, anesthetics (e.g., local anesthetics such as lignocaine, 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, and anticonvulsants. Antidementia drugs, prostaglandins, antipyretics, antifungal agents, antiskin agents, anti-psoriasis agents, antiviral agents, vasodilators or vasoconstrictors, sunscreens (e.g., PABA), antihistamines, hormones (e.g., estrogens, progesters, or androgens), anti-seborrheic agents, cardiovascular treatments (e.g., alpha or beta blockers or rogaine), vitamins, skin softeners, enzymes, mast cell stabilizers, scabies mites, lice killers, keratolytic agents, lubricants, anesthetics, shampoos, anti-acne agents, burn treatments, cleansers, deodorants, bleaching agents, diaper rash treatments, emollients, moisturizers, photosensitizers, poison ivy or poison oak or sumac products, sunburn treatments, proteins, peptides, proteoglycans, nucleotides, oligonucleotides (e.g., DNA, RNA, etc.), minerals, growth factors, tar-containing preparations, honey-containing preparations (e.g., Manuka honey). Preparations of honey), wart treatment preparations, wet dressings, wound care products, or any combination thereof.

[0261] Specific examples include analgesics such as ibuprofen, indomethacin, diclofenac, acetylsalicylic acid, paracetamol, propranolol, metoprolol, and oxycodone; thyroxine-releasing hormone; sex hormones such as estrogen, progesterone, and testosterone; insulin; verapamil; vasopressin; hydrocortisone; scopolamine; nitroglycerin; isosorbide dinitrate; antihistamines such as terfenadine; clonidine; and nicotine. Nonsteroidal immunosuppressive drugs, such as cyclosporine, methotrexate, azathioprine, mycophenylate, cyclophosphamide, TNF-α antagonists, and monoclonal antibodies against cytokines anti-IL5, anti-IL4Ra, anti-IL6, anti-IL13, anti-IL17, and anti-IL23; anticonvulsants; and drugs used for Alzheimer's disease, dementia, and / or Parkinson's disease, such as apomorphine and rivastigmine. If necessary, any of the optional additional components may be encapsulated or microencapsulated, for example, to control or delay its release. See the section below entitled "Optional Encapsulation of Components (e.g., Microencapsulation)" for more details.

[0262] Optional encapsulation of components (e.g., microencapsulation)

[0263] At least some components used in the combinations, kits and compositions of this disclosure may be encapsulated, for example, by microencapsulation.

[0264] Using microencapsulated components to generate NO is suitable because it delays the generation of relatively unstable compounds (e.g., NO) from precursors in their chemically stable form. Multiple microencapsulated reactants and / or one or more optional additional components are readily mixable and in contact with each other when stored in a dry environment, and NO generation can be initiated simply by providing a small amount of water to the precursor mixture. Alternatively, such mixtures of microencapsulated reactants and / or one or more optional additional components can be applied directly to a subject, such as to the skin, mucous membranes, or, according to the invention, to the nose, mouth, respiratory tract, and / or lungs, where the physiological environment itself provides sufficient water to induce a therapeutic amount of NO release. Another advantage is that the microencapsulated reactants and / or one or more optional additional components occupy a relatively small volume, thus facilitating their incorporation into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, blood vessels and other stents, catheters, pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles.

[0265] An example of a method for producing reactants and / or one or more optional additional components for encapsulation or microencapsulation is the spray drying of a melt or polymer solution of the reactants and / or one or more optional additional components to produce an extremely fine powder of individual particles containing 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, ionogelation, condensed phase separation, interfacial crosslinking, in-situ polymerization, and template polymerization. The encapsulating polymer is preferably biocompatible. Such polymers include ethyl cellulose, natural polymers (e.g., corn protein) (a glucogluten seed storage protein found in certain grass species, including corn and maize), chitosan, hyaluronic acid and alginate, or biodegradable polyesters, polyanhydrides, poly(orthoesters), polyphosphazenes, or polysaccharides (see Park et al., Molecules 10 (2005), pp. 141-161). It is well known that compositions of chemical substances microencapsulated as indicated above are used for the delivery of drugs and other agents. See Shalaby and Jamiolkowski, U.S. Patent No. 4,130,639; Buchholz and Meduski, U.S. Patent No. 6,491,748. However, in practice, in all such compositions, the therapeutic agent is microencapsulated, and the therapeutic agent is not generated by the reaction of the microencapsulated reagent. However, suitable modifications to the teachings of the prior art are within the skill of those skilled in the art. Polymers that release nitric oxide have been described for medical articles involving NO adducts / donors. See, for example, Arnold, U.S. Patent No. 7,829,553 (Carbon-based diol diazoxide ionon linked to a hydrophobic polymer); Knapp, U.S. Patent No. 7,135,189 (Nitrothiool precursor and nitric oxide donor).

[0266] pH control; optional buffer system

[0267] The composition may have a controlled pH value. Specifically, the composition may have a pH value in the range of 3.0 to 8.0, or more particularly in the range of 4.0 to 8.0. In a more specific embodiment, the composition has a pH value in the range of 4.0 to 7.4. In a more specific embodiment, the composition may have a pH value in the range of 4.0 to 6.0. In these embodiments, the composition may have a pH value in the range of 4.5 to 6.0.

[0268] The pH of the composition can be controlled by any known means. In a specific embodiment, the pH of the organic carboxylic acid component or the organic reducing acid component is controlled prior to contact with the nitrite component. In some embodiments, the organic carboxylic acid component or the organic reducing acid component comprises a buffer solution. The buffer solution can be a pharmacologically acceptable buffer solution, such as a phosphate buffer.

[0269] In some embodiments, a buffer solution is formed by mixing an organic carboxylic acid or an organic non-carboxylic acid reducing acid with its salt counterpart. For example, the organic carboxylic acid component may comprise an organic carboxylic acid and a salt of an organic carboxylic acid. The organic non-carboxylic acid reducing acid component may comprise an organic non-carboxylic acid reducing acid and a salt of an organic non-carboxylic acid reducing acid. In a specific embodiment, the organic carboxylic acid component comprises citric acid and citrate. In other embodiments, the organic carboxylic acid component or organic reducing acid component comprises ascorbic acid and ascorbate. In some embodiments, the organic carboxylic acid component comprises an organic carboxylic acid and a salt of another organic acid. For example, the organic carboxylic acid component may comprise citric acid and ascorbate. In other embodiments, the organic carboxylic acid component may comprise an organic carboxylic acid, a salt of an organic carboxylic acid, and a salt of another organic carboxylic acid. For example, the organic carboxylic acid component may comprise citric acid, citrate, and ascorbate.

[0270] In other embodiments, a buffer solution is formed by adjusting the pH of the reducing acid of an organic carboxylic acid or an organic non-carboxylic acid to allow the acid (protonated form) to coexist with its salt counterpart. This is preferably achieved by adding a strong inorganic base and optionally a strong inorganic acid to the reducing acid of the organic carboxylic acid or organic non-carboxylic acid in amounts sufficient to generate a buffer system on the spot. Examples of suitable strong inorganic bases include sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, and cesium hydroxide. Examples of suitable strong inorganic acids include hydrochloric acid, sulfuric acid, hydrobromic acid, and nitric acid.

[0271] Buffer solutions may include one or more physiological buffer solutions, particularly when combinations or compositions according to this disclosure come into contact with cells or the skin, mucous membranes, or other tissues of an animal (including a human), such as when applied to the nose, mouth, respiratory tract, or lungs according to the invention. Examples of suitable physiologically compatible buffer solutions include Good's buffer, which buffers in a pH range of about 5 to about 9, such as 2-amino-2-methyl-1,3-propanediol, N-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)-iminodiacetic acid (ADA), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis(2-hydroxyethyl)-2-amino 2-Bis(2-hydroxyethyl)glycine (BICINE), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS), 1,3-bis[tris(hydroxymethyl)methylamino]propane (BIS-TRIS propane), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(N,N-bis[2-hydroxyethyl]amino)-2-aminopropanesulfonic acid (DIPSO), 4-(2-hydroxyethyl) 1-Piperazine propanesulfonic acid (EPPS), diglycine, N-(2-hydroxyethyl)piperazine-N'-(4-butyric acid) (HEPBS), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic 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) dehydrate (POPSO) Disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen 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-[tris(hydroxymethyl)-methyl]glycine (Tricine) or 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA).

[0272] molar osmotic pressure concentration of the composition

[0273] The solute strength of any solution of nitrite, proton source, organic polyol, or any combination thereof delivered to the physiological system, especially through contact with the skin, mucous membranes, or, according to the invention, with the nose, mouth, respiratory tract, or lungs of a human or animal subject, should be controlled to avoid any undesirable dehydration of the subject's organs and tissues.

[0274] Osm is defined as the number of moles of solute dissolved in one kilogram of solvent, and can be expressed as osmol / kg. The osmotic pressure of any solution administered to human or animal subjects according to this disclosure is generally in the range of about 100 to about 5000 mOsmol / kg, for example, from about 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 to about 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750 or 5000 mOsmol / kg.

[0275] The mixing of components can trigger the generation of NOx.

[0276] It has been found that the order in which the components of a NOx-generating system are mixed to initiate NOx production may affect the results of using the resulting NOx. Evidence of this effect is provided in Example 6 below.

[0277] In this embodiment, we demonstrated that the efficacy of the composition according to the invention in killing Mycobacterium tuberculosis HN878 in THP-1 cells varies depending on whether: on the one hand, the nitrite, proton source, and organic polyol components are first mixed at a desired concentration higher than the required concentration in the composition to be used, and then the concentrate is appropriately diluted with water to provide the composition to be used; or on the other hand, the nitrite, proton source, and organic polyol components are first mixed at a desired concentration in the composition to be used, and then the required concentration is provided.

[0278] Furthermore, it is impossible to predict which method of mixing the components will produce better results in terms of antimicrobial activity. While it generally seems that diluting a relatively concentrated premix to provide the composition to be used may produce better antimicrobial activity against Mycobacterium tuberculosis HN878 in THP-1 cells, in some cases, the results are not as good as those obtained by mixing the components first at the concentration required for the intended use.

[0279] In one embodiment of the invention, the method of preparing a NOx-generating composition comprises mixing a nitrite, a proton source, and an organic polyol component in a desired proportion at a concentration higher than that required in the composition in its usable form to form a concentrated premix, and then appropriately diluting the concentrated premix with water to provide a composition for use.

[0280] In another embodiment of the invention, the method of preparing a NOx-generating composition thus includes mixing nitrite, a proton source, and an organic polyol component in a desired concentration and proportion to provide a composition to be used.

[0281] Preferred implementation scheme

[0282] Preferred embodiments of the first to eighth aspects of this disclosure are embodiments in which one or more of the following are present:

[0283] - One or more nitrites comprise the following (e.g., including the following or consisting essentially of the following or consisting only of the following): one or more alkali metal or alkaline earth metal nitrites, such as sodium nitrite, potassium nitrite or any combination thereof;

[0284] - The proton source contains the following (e.g., including the following or consisting essentially of the following or consisting only of the following): ascorbic acid or ascorbic acid / ascorbate buffer, citrate or citrate / citrate buffer or any combination of two or more of these;

[0285] - The molecules of ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citric acid buffer, or any combination thereof, are not covalently bonded to polymers or macromolecules;

[0286] - One or more organic polyols comprise the following (e.g., including the following or consisting essentially of the following or consisting only of the following): straight-chain sugar alcohols or aldehydes having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, such as sorbitol, mannitol, aritol, xylitol, or any combination of two or more of these;

[0287] - One or more organic polyols are chains comprising one, two, or three monosaccharide units terminated with a noncyclic alcohol unit, such as sugar alcohol compounds composed of such chains, optionally wherein one, two, three, or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the noncyclic alcohol unit is a C5 or C6 sugar alcohol unit; for example, isomaltitol, maltitol, lactitol, maltotriol, maltotetratitol;

[0288] - At the start of a reaction that generates NOx, or before the start of a reaction, the total molar concentration of one or more organic polyols in the polyol component or the reaction solution is between 0.05 and 3 times the total molar concentration of nitrite ions in the nitrite component or the reaction solution.

[0289] - At the start of a NOx-generating reaction or before the start of a reaction, the total molar concentration of one or more organic polyols in the polyol component or the reaction solution is between 0.05 and 3 times the total molar concentration of the proton source component or the proton source in the reaction solution;

[0290] - For 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), the pH of the proton source should be in the range of 3.0 to 9.0 before, and especially just before, the initiation of a reaction that produces NO.

[0291] - For 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), the pH of the proton source should be in the range of 4.0 to 8.0 before, and especially just before, the initiation of the NO-producing reaction.

[0292] - For applications that do not involve contact between the reaction mixture and the nose, mouth, respiratory tract or lungs of animal (including human) subjects according to the invention, the pH of the proton source is in the range of 5.0 to 8.0 before, and especially just before, the initiation of the reaction that produces NO.

[0293] - The targeted microorganisms are selected from the microorganisms listed below in the section entitled "Targets for Antimicrobial Use", including, but not limited to, influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, including their antibiotic-resistant strains.

[0294] A preferred embodiment of the ninth aspect of this disclosure is an embodiment in which one or more of the following are present:

[0295] - One or more nitrites comprise the following (e.g., including the following or consisting essentially of the following or consisting only of the following): one or more alkali metal or alkaline earth metal nitrites, such as sodium nitrite, potassium nitrite or any combination thereof;

[0296] - The proton source contains the following (e.g., including the following or consisting essentially of the following or consisting only of the following): ascorbic acid or ascorbic acid / ascorbate buffer, citrate or citrate / citrate buffer or any combination of two or more of these;

[0297] - The molecules of ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citric acid buffer, or any combination thereof, are not covalently bonded to polymers or macromolecules;

[0298] - One or more organic polyols comprise the following (e.g., including the following or consisting essentially of the following or consisting only of the following): straight-chain sugar alcohols or aldehydes having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, such as sorbitol, mannitol, aritol, xylitol, or any combination of two or more of these;

[0299] - One or more organic polyols are chains comprising one, two, or three monosaccharide units terminated with a noncyclic alcohol unit, such as sugar alcohol compounds composed of such chains, optionally wherein one, two, three, or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the noncyclic alcohol unit is a C5 or C6 sugar alcohol unit; for example, isomaltitol, maltitol, lactitol, maltotriol, maltotetratitol;

[0300] - At the start of a reaction that generates NOx, or before the start of a reaction, the total molar concentration of one or more organic polyols in the polyol component or the reaction solution is between 0.05 and 3 times the total molar concentration of nitrite ions in the nitrite component or the reaction solution.

[0301] - At the start of a NOx-generating reaction or before the start of a reaction, the total molar concentration of one or more organic polyols in the polyol component or the reaction solution is between 0.05 and 3 times the total molar concentration of the proton source component or the proton source in the reaction solution;

[0302] - For 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), the pH of the proton source should be in the range of 3.0 to 9.0 before, and especially just before, the initiation of a reaction that produces NO.

[0303] - For 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), the pH of the proton source should be in the range of 4.0 to 8.0 before, and especially just before, the initiation of the NO-producing reaction.

[0304] - For applications that do not involve contact between the reaction mixture and the nose, mouth, respiratory tract or lungs of animal (including human) subjects according to the invention, the pH of the proton source is in the range of 5.0 to 8.0 before, and especially just before, the initiation of the reaction that produces NO.

[0305] - The targeted microorganisms are selected from the microorganisms listed below in the section entitled "Targets for Antimicrobial Use", such as, but not limited to, influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, including their antibiotic-resistant strains.

[0306] Combinations and compositions

[0307] Reactions that produce NOx can be initiated in a variety of ways. Generally, these methods are characterized by contacting one or more nitrites with a proton source under conditions that allow the reaction to begin producing NOx.

[0308] The reaction can be initiated by combining the individual components of the composition. Combination can be achieved in vitro, and the resulting composition can then be applied to a subject or to any surface to be treated according to this disclosure. Alternatively, the released gas can be applied to a subject or to any surface to be treated according to this disclosure. Furthermore, the two applications of the resulting composition can be performed at time intervals, such that the composition is applied to a subject or to any surface to be treated after a certain amount of gas has been released.

[0309] Combination can be carried out stepwise, for example, initially mixing the components in dry powder form and then mixing them with water or another liquid carrier medium to initiate the reaction. Alternatively, the components in dry powder form can be initially mixed with water or another liquid carrier medium separately, followed by mixing two or more liquids to initiate the reaction.

[0310] Alternatively, at least some components of the NOx-generating reaction according to this disclosure may be mixed in a single composition and initiate the NOx-generating reaction on the composition. One possible way to initiate the NOx-generating reaction is, for example, by adding a key component or additive that initiates the reaction, such as water if the components of the composition are in a dry or packaged form; or by adding a proton source if the components of the composition lack a proton source.

[0311] In order to prevent the occurrence of NOx-producing reactions, the kits according to this disclosure typically contain one or more components of combinations or compositions according to this disclosure. A portion of the kit is typically stored in a container; these components may be separate or modified to facilitate the mixing required to initiate a NOx-producing reaction. The key initiating component for initiating the NOx-producing reaction, which needs to be introduced into the kit by the user, may be, for example, one of a nitrite component, a proton source component, or a polyol component, or may be another component, typically a commonly used component, such as water, which may be supplied by the user.

[0312] The parameters of the combinations and compositions defined and described in this patent typically include physical parameters such as pH, concentration, and osmotic pressure. These parameters will be measured whenever possible before initiating the reaction that produces NOx. Unless otherwise stated, the pH parameter refers to the pH value of the proton source in deionized water at the concentration intended to initiate the reaction that produces NOx. Unless otherwise stated, the concentration of the solution refers to the concentration before mixing with other components to initiate the reaction that produces NOx. Typically, these parameters are not readily measurable while the reaction that produces NOx is underway, once nitrites and organic carboxylic acids or organic reducing acids have been mixed to react and produce nitric oxide gas.

[0313] Furthermore, it should be noted that the concentration of a component in the reaction mixture does not necessarily correspond to its concentration in a portion of the composition prior to mixing. For example, suppose the composition used to initiate a NOx-generating reaction according to this disclosure is formed from approximately equal volumes of a nitrite component and a proton source component, both added together as a pre-prepared solution. In this embodiment, the nitrite concentration of the mixed reaction composition is half the concentration of the nitrite component, and the proton source concentration is half the concentration of the proton source component.

[0314] The combination and a portion thereof may be in any suitable physical form, depending on the intended use of the system during or after the NOx-generating reaction. For example, the combination and a portion thereof may each be in the form of a liquid, gel, or membrane, such that the NOx-generating reaction mixture is similarly in the form of a liquid, gel, or membrane. Liquids may be modified to be nebulized and inhaled into the respiratory tract or lungs. If the NOx-generating mixture is intended to be applied to the mouth or throat, then a portion thereof may be in the form of a mouthwash or beverage. Alternatively, if the NOx-generating reaction mixture in topical application is intended to be applied to the skin, then a portion thereof may be in the form of an ointment, lotion, or cream.

[0315] Multicomponent systems, reagent kits and dispensers

[0316] The multi-component system described herein may include a nitrite component and a proton source component, optionally containing a polyol component, as defined and described herein. The components in the multi-component system are suitably in contact with each other, and this contact is achieved by means of suitable containers or reservoirs for preserving the components before use and by means of components for mixing the components, distributing the reaction mixture and / or releasing gases, and overall control of the mixing and distribution of the reaction mixture and / or the released gases. In a preferred embodiment, the reaction mixture may be distributed in the form of a mist or aerosol of droplets entrained in a gas stream.

[0317] The kits and dispensers disclosed herein generally include: at least some containers for preserving components before use; at least one device or other component for mixing components, dispensing reaction mixtures and / or releasing gases, and overall controlling said mixing and dispensing; and the component or those components, if present, contained in the container of the kit or dispenser before use. Instructions for use are preferably available, or a guide to using instructions for use can be found, such as online instructions for use. Such kits and dispensers constitute another aspect of this disclosure.

[0318] The kits disclosed herein can be a relatively simple collection of containers and devices for mixing components, dispensing reaction mixtures and / or releasing gases, and overall control of said mixing and dispensing. Such kits are suitable for research purposes or for situations where a wide range of variations in mixing and dispensing operations are expected and permissible.

[0319] Other kits disclosed herein may be a more complex collection of one or more containers containing consumables (combinations and / or compositions required by the user to initiate a reaction that produces NOx, optionally, water or other generally available ingredients supplied by the user) together with one or more dispensers disclosed herein.

[0320] The dispensers disclosed herein are generally adapted to perform repetitive similar actions of dispensing a reaction mixture, a carrier containing the reaction mixture, and / or an expelled gas. The dispenser may include a pump or propellant system to carry and direct a composition containing a NOx-producing reaction mixture or a gas expelled from the dispenser to a target. The propellant system may use pressurized and / or liquefied gases, preferably pharmaceutically acceptable or biocompatible for medical use, such as pressurized air or pressurized / liquefied butane. Alternatively, aspiration from the user's lungs may be used to carry and direct a composition containing a NO-producing reaction mixture or a gas expelled from the dispenser to a target. Dispensers used in this disclosure are adapted to include an actuation device, such as a manually operated trigger or button, by which a user can activate the dispenser. Such dispensers can be adapted for professional, research, consumer, or patient use, and accordingly modified to facilitate the treatment of a target via a predetermined pathway.

[0321] In principle, a variety of reagent kits and dispenser devices are known that can be used or easily modified to be suitable for preserving components before use, mixing components or facilitating said mixing, dispensing compositions containing reaction mixtures and / or releasing gases and overall controlling said mixing and dispensing or facilitating said control.

[0322] For example:

[0323] - Syringe, such as a double-barrel dispensing syringe.

[0324] - Container systems, such as pump-driven, pressure-driven, or shaking containers, comprising, for example, two containers, for mixing at least a nitrite component and a proton source component and dispensing a composition or emitted gas comprising a reaction that generates NOx. Such systems are described in US 2019 / 0134080, the disclosure of which is incorporated herein by reference.

[0325] - Devices for preserving components, mixing components, nebulizing liquid reaction mixtures, and dispensing the reaction mixtures for inhalation into the lungs of a person before use in aqueous solutions, and for overall control of said mixing and dispensing. Examples include soft fog inhalers, jet nebulizers, ultrasonic nebulizers, and vibrating screen nebulizers. Selection of nebulizers, droplet sizes, adjuvants, packaging forms, etc., suitable for inhalation nebulization of reaction media that generate NOx through nitrite acidification are described in WO 03 / 032928 and WO 2009 / 086470, the disclosure of which is incorporated herein by reference.

[0326] - The above devices can be arranged to atomize and dispense the premixed liquid reaction mixture after it has been loaded into the nebulizer for inhalation into the lungs, and to control the mixing and dispensing of the mixture.

[0327] - A device for preserving components before use in aqueous solutions, mixing components, dispersing liquid reaction mixtures into a vapor form, distributing the reaction mixture for inhalation into the lungs, and overall controlling the mixing and distribution. Examples include metered-dose inhalers. Selection of droplet size, adjuvants, packaging forms, etc., of reaction media suitable for NOx generation by nitrite acidification via inhalation nebulization is described in WO03 / 032928 and WO 2009 / 086470, the disclosures of which are incorporated herein by reference.

[0328] - Techniques and apparatus for spraying a solution that releases nitric oxide into the upper respiratory tract are described in U.S. Patent No. 9,730,956, the disclosure of which is incorporated herein by reference.

[0329] - Devices for preserving and dispensing components in dry powder form for inhalation into the lungs of a person prior to use. Examples include dry powder inhalers (DPIs) that can be formulated as single-dose capsules or multi-dose dry powder inhalers, in the form of stored powder or multiple individual blister packs. The selection of powder particle size, excipients, packaging forms, etc., suitable for inhaling dry powder combinations to provide a reaction medium in the lungs for the in situ generation of NO via nitrite acidification is described in WO 2009 / 086470, the disclosure of which is incorporated herein by reference.

[0330] - A dispenser for preserving, aerating, and dispensing as foam components before use in solution form for skin disinfection or treatment of skin conditions is described in U.S. Patent Application No. 2013 / 0200109, U.S. Patent No. 7066356, and U.S. Patent Application No. 2019 / 0134080, the disclosures of which are incorporated herein by reference.

[0331] - A transdermal patch assembly for storing components and distributing them to the skin of a subject is described in WO 2014 / 188175, the disclosure of which is incorporated herein by reference.

[0332] The dosage of the combinations and compositions or emitted gases disclosed herein can vary within a wide range, depending on the disease, condition, or ailment to be treated (in the case of medical treatment) or the desired effect (in the case of non-medical treatment), the severity of the required treatment, and the condition, age, and health of the subject to be treated, or, in the case of non-medical treatment, the nature of the target to be treated. In the case of medical treatment, the physician will ultimately determine the appropriate dosage. In the case of non-medical treatment, a person skilled in the art will be able to determine the appropriate dosage and method of treatment by reviewing relevant literature or through reasonable experimentation.

[0333] In some implementations, within 600 seconds of the combination of the nitrite component and the proton source component, the composition that undergoes a NOx-producing reaction, or the gas released therefrom, can be applied to a target site, such as microbial cells, living tissue, organs, structures, or a subject. In this way, the target site can be exposed to a large amount of nitric oxide.

[0334] In some embodiments, the composition that causes the NOx-generating reaction can be formed in situ at or near the target site, such as on, inside, or near microbial cells, living tissue, organs, structures, or subjects (including inanimate surfaces and spaces). In these embodiments, the composition is effectively applied within 0 seconds after the combination of the nitrite component and the proton source component. In other embodiments, the composition is applied to the target site or its vicinity within a range of more than 0 seconds and less than 600 seconds after the combination of the nitrite component and the proton source component. In a more specific embodiment, the composition is applied within a range of 0 to 120 seconds. In other embodiments, the composition is applied within a range of 0 to 60 seconds.

[0335] In other embodiments, the composition or gas emitted therefrom that undergoes a NOx-producing reaction can be applied to a target location or its vicinity, such as microbial cells, living tissue, organs, structures, or subjects, more than 600 seconds, for example, more than 2000 seconds, more than 4000 seconds, or more than 8000 seconds, after the combination of the nitrite component and the proton source component. In that case, the target location, such as microbial cells, living tissue, organs, structures, or subjects, is not necessarily exposed to a large amount of nitric oxide, but may still experience beneficial properties, such as antimicrobial effects. In these embodiments, the composition or gas emitted therefrom that undergoes a NOx-producing reaction can be applied up to 48 hours after the combination of the nitrite component and the proton source component. In specific embodiments, the composition or gas emitted therefrom can be applied up to several weeks or months, such as up to about 6 months, or up to about 2 months, or up to about 1 month, or up to about 3 months, 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 the combination of the nitrite component and the proton source component.

[0336] Under proper storage conditions, the composition that produces NOx or the gas released therefrom can be applied more than 48 hours after the combination of the nitrite component and the proton source component. For example, the composition can be stored in a sealed container, such as under vacuum. Storage in a sealed container typically takes place no more than 24 hours after the combination of the nitrite and the organic carboxylic acid or organic reducing acid. The composition can be stored in a sealed container no more than 600 seconds after the combination of the nitrite component and the proton source component. In this way, a certain proportion of nitric oxide gas can be retained. If the NOx-producing composition is stored at low temperatures, such as in the range of about -30°C to about +10°C, for example, at a temperature of about 1°C to about 10°C, the rate of gas release can be greatly slowed down, allowing the composition to be stored for a very long time.

[0337] In one specific embodiment, the aerosol dispenser may include multiple reservoirs, wherein a first reservoir contains a nitrite component in liquid form (e.g., an aqueous solution) and a second reservoir contains a proton source component in liquid form (e.g., an aqueous solution). In this embodiment, each component is suitable for mixing with the propellant before, during, or after mixing the nitrite and proton source components.

[0338] In another specific embodiment, the dispenser may be a single-barrel syringe containing the composition of this disclosure. The viscosity of the composition is selected to enable dispensing from the syringe by manual action or powered operation. For example, the composition may be a liquid or a gel.

[0339] In another specific embodiment, the dispenser may be a multi-cylinder syringe having a first cylinder containing a nitrite component and a second cylinder containing a proton source component. The viscosity of the components is selected to enable dispensing from the syringe by manual action or powered operation. For example, each component may be independently a liquid or a gel.

[0340] Other reservoirs for components: hydrogel

[0341] In some embodiments of this disclosure, molecular reservoirs, such as hydrogels, may be used. Hydrogels are highly hydrated, typically cross-linked, three-dimensional polymer (homopolymer or copolymer) or macromolecular networks capable of absorbing and retaining many times their dry weight of water, other aqueous liquids, or other non-aqueous hydrophilic liquids. Liquid absorption is typically accompanied by swelling of the hydrogel. Acidic hydrogels or hydrogels with other special chemical properties can be prepared by appropriately selecting the component chemical groups covalently bonded to the polymer or macromolecule.

[0342] Hydrogels known to be used as proton source components in this disclosure. Examples of such hydrogels containing acidic -COOH groups are described, for example, in WO 2007 / 007115, WO 2008 / 087411, WO 2008 / 087408, WO 2014 / 188174 and WO 2014 / 188175 and referenced herein, the disclosures of all of which are incorporated herein by reference. Uses of such hydrogels in the production of NOx for skin care, including the production of transdermal drug delivery from NOx, are particularly described in WO 2014 / 188174 and WO 2014 / 188175. Specific examples of such hydrogels include homopolymers and copolymers of acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid (ATBS, available from Vinati Organics Ltd.), and their salts. Polymers formed from monomers including (meth)acrylic acid or composed of (meth)acrylic acid will include carboxylic acid side groups to serve as a proton source according to this disclosure.

[0343] Therefore, for example, a multi-component system may include a first acidic hydrogel pad or layer component comprising a proton source component, optionally also containing an organic polyol, and another component may be a nitrite component. The nitrite component may, for example, be a liquid medium containing dissolved nitrite. In this way, the surface of the hydrogel pad or layer can contact the nitrite component, initiating a reaction that generates NOx. Alternatively, the nitrite component may be a solid support, such as a pad or layer, containing nitrite in a form that dissolves in the absorbent liquid of the hydrogel when in contact between the solid support and the hydrogel.

[0344] Typically, the solid carrier pad or layer is nitric oxide diffusion-permeable (fully permeable or at least semi-permeable). In this way, nitric oxide can diffuse into the treatment area when the solid carrier pad or layer is combined with a hydrogel to combine the nitrite component and the proton source component. The solid carrier pad or layer can be, for example, a screen, a nonwoven sheet, a film, a foam, an alginate layer, or a membrane.

[0345] In specific implementations, the solid carrier layer is a screen. The screen can be a network of interconnected solid threads, typically flexible, forming a mesh with holes or openings that allow certain substances to pass through. The screen can be made of fabric or nonwoven fabric. In some implementations, the screen is nonwoven fabric.

[0346] The solid support layer, such as a screen, can be made of a polymeric material. Examples of suitable polymeric materials include, but are not limited to, cellulose adhesives, polyamides, polyesters, polypropylene, or blends thereof. The polymeric material can be treated to, for example, increase its hydrophilicity. In a specific embodiment, the solid support layer is a polypropylene mesh.

[0347] In a specific implementation, the solid carrier has absorption capacity, and the nitrite component is at least partially absorbed, inhaled, or impregnated in the solid carrier. The absorbed, inhaled, or impregnated nitrite component can be a solid (dry) or in an aqueous solution within the solid carrier.

[0348] In specific embodiments, the solid carrier comprises more than one layer, with the nitrite component absorbed, inhaled, or impregnated in at least one layer or coated on at least one outer layer. For example, the solid carrier may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more layers, such as a polypropylene mesh layer, absorbing, inhaling, impregnating, or coating one or more nitrites in dry and / or solution form.

[0349] Acidic hydrogels possess natural buffering capacity due to their abundant internal supply of protonated acidic side groups, H... + Ions can migrate from the absorbed aqueous medium to maintain the relative acidity of the surface pH during the deprotonation of the lateral acidic portion of the hydrogel structure surface during the NOx-generating reaction.

[0350] Non-acidic (e.g., neutral or basic) hydrogels are also known, in which nitrite components and / or polyol components can be absorbed and contained for use in this disclosure. Proton source components can contact such hydrogels by means of a proton source provided in a liquid medium in contact with the hydrogel, and / or by means of absorption, adsorption, impregnation, or coating onto a solid support. In such hydrogels, the condition is that none of the nitrite component, proton source component, or polyol component is covalently bonded to the polymer or macromolecular network of the hydrogel; for example, considering that the nitrite component and proton source component can only react together until a reaction to produce NOx is required, all components required by this disclosure can be adsorbed into the hydrogel and contained in an aqueous medium within the hydrogel block, but not covalently bonded to the polymer or macromolecules of the hydrogel.

[0351] The thickness of the hydrogel pad or layer can range from 0.5 to 2 mm. In some embodiments, the thickness of the hydrogel pad or layer ranges from 1 to 2 mm. In a specific embodiment, the thickness of the hydrogel pad or layer ranges from 1.0 to 1.6 mm.

[0352] The characteristics described above regarding the proton source component will generally apply equally to any acidic hydrogel used as a proton source component. Therefore, for example, the hydrogel may contain a buffer solution to maintain the pH of the hydrogel in the range of 4.0 to 9.0 or 5.0 to 8.0.

[0353] 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 opposite surface of the hydrogel, such as the subject's skin, to provide a barrier between the combined multi-component system and the atmosphere. The surface of the barrier film adjacent to the hydrogel typically has a larger surface area than the adjacent hydrogel surface. In this way, the barrier layer may extend beyond the periphery of the hydrogel. In these embodiments, the barrier layer may have an adhesive around its periphery to bond the hydrogel to, for example, the subject's skin in use.

[0354] In one specific embodiment, this disclosure provides a two-component system comprising:

[0355] a) One or more screens that absorb, impregnate, or coat with one or more nitrites, such as NaNO2; and

[0356] b) A hydrogel containing a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acid reducing acids.

[0357] Component (a) and component (b) are separate, and one or more of components (a) and (b) further contain one or more organic polyols;

[0358] It is characterized by one or more of the following:

[0359] (a) One or more organic polyols are present to enhance the amount of reaction output;

[0360] (b) The proton source is not merely a hydrogel containing carboxylic acid side groups covalently bonded to a three-dimensional polymer matrix;

[0361] (c) One or more organic polyols, not just glycerol;

[0362] (d) When using one or more tackifiers, one or more organic polyols are not limited to glycerol;

[0363] (e) When using one or more plasticizers, one or more organic polyols are not limited to glycerol;

[0364] (f) One or more organic polyols, not just polyvinyl alcohol;

[0365] (g) When using one or more tackifiers, one or more organic polyols are not limited to polyvinyl alcohol;

[0366] (h) any one or more of (b) to (g), wherein the word “not only” is replaced by “does not include”;

[0367] (i) One or more organic polyols, not limited to propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof or any combination of the above with glycerol and / or polyvinyl alcohol.

[0368] (j) One or more organic polyols that do not contain propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butene glycol, butynediol, pentylene glycol, hexanediol, octylene glycol, neopentylene glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butanediol, butanediol, butanediol, hexanediol, octyl glycol, diols other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerol, any combination thereof, or any combination of the above with glycerol and / or polyvinyl alcohol.

[0369] To avoid any doubt, it is hereby confirmed that the embodiments and preferred embodiments of the characteristic features (a) to (h) described above with respect to aspects of this disclosure are equally applicable to the embodiments.

[0370] Such systems can be used, for example, by combining components (a) and (b) to induce a reaction that produces NOx. These combinations can then be used, for example, by topical application in therapies or other treatments in humans or animals. Uses can be as described in WO 2014 / 188174 and WO 2014 / 188175, or as described below. The system can also be used for non-medical purposes, as described below. When used for topical medical applications where the system comes into contact with the skin (including mucous membranes) of a subject, a screen or mesh may be in contact with the skin layer.

[0371] Uses in therapy or surgery

[0372] The compositions and gases emitted therefrom that produce NOx according to this disclosure have numerous applications in therapies and surgeries, including curative and / or preventative therapies, surgeries to correct diseases, conditions and disorders, cosmetic surgery, reconstructive surgery, including human medicine and veterinary medicine. In cases where bodily deformities or abnormalities in response to treatment with the compositions or gases emitted therefrom cause or exacerbate anxiety, depression, or another mental illness or condition, the treatment, prevention, or relief of the physical ailment can correspondingly treat, prevent, or alleviate the mental illness; therefore, the uses of this disclosure also extend to the field of mental health.

[0373] Numerous physiological effects of nitric oxide and compositions that produce nitric oxide, as well as medical treatments based on them, have been reported in the literature, and thus many therapeutic treatments have been developed. The following non-exhaustive list is provided as an example. Uses listed, and other uses not listed, are covered within this disclosure and the patent.

[0374] Nitric oxide dilates blood vessels, increases blood supply and / or lowers blood pressure (see van Faassen et al., Med. Res. Rev. 2009 Sep; 29(5), pp. 683-741);

[0375] The acute effects of oral nitric oxide supplementation in lowering blood pressure, improving vascular compliance and restoring epithelial function in hypertensive patients are described in Houston et al., J. Clin. Hypertens. (Greenwich), July 2014, 16(7), pp. 524-529;

[0376] Nitric oxide protects tissues from damage caused by poor blood supply (see van Faassen et al., Med. Res. Rev. 2009 Sep; 29(5), pp. 683-741);

[0377] Nitric oxide is used as a neurotransmitter in nitric oxide neurons, such as those active in smooth muscle in the gastrointestinal tract and erectile tissues (see Toda et al., Pharmacol. Ther., May 2005; 106(2), pp. 233-266).

[0378] Nitric oxide inhibits vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte attachment to the endothelium, helping blood vessels achieve dynamic homeostasis (see Dessey and Ferron, Current Medical Chemistry - Anti-inflammatory and Anti-allergy Agents in Medicinal Chemistry, 2004; 3(3), pp. 207-216).

[0379] Nitric oxide is used to reduce cardiac contractility and heart rate (see Navin et al., J. Cardiovascular Pharmacology, 2002; 39(2), pp. 298-309);

[0380] Neonatal intensive care aims to promote capillary and lung expansion, for example, in the treatment of primary pulmonary hypertension and meconium aspiration in newborns (see Barrington et al., The Cochrane Database of Systematic Reviews, 2017; 1, CD000399). https: / / www.ncbi.nlm.mh.gov / pubmed / 17375630See also Chotigeat et al., J. Med. Assoc. Thai., 2007; 90(2), pp. 266-271; see also Hayward et al., Cardiovascular Research, 1999; 43(3), pp. 628-638);

[0381] Preventing vascular injury, endothelial dysfunction and vasculitis, neuropathy and non-healing ulcers in diabetic patients, and reducing the risk of subsequent lower limb amputation (see NFB University Studies - “NitricOxide Holds Promise for Diabetes”, http: / / www.nfb.org / Images / nfb / Publications / vod / vod212 / vodspr0613.htm);

[0382] Improve hypoxemia in acute lung injury, acute respiratory distress syndrome and severe pulmonary hypertension; treat reversible causes of hypoxic respiratory distress (see Mark et al., N. Eng. J. Med., Dec 22, 2005; 353(25), pp. 2683-2695);

[0383] Nitric oxide was used as a salvage therapy in patients with pulmonary embolism-induced acute right heart failure (see Summerfield et al., 2011; Respir.Care 57(3), pp. 444-448);

[0384] The effects of treating angina pectoris, paraquat poisoning and other cardiovascular diseases (see Abrams, The American Journal of Cardiology, 1996; 77(13), pp. 31C-37C);

[0385] Treatment of bladder contraction dysfunction (see Moro et al., Eur. J. Pharmacol., January 2012; 674(2-3), pp. 445-449; see also Andersson et al., Br. J. Pharmacol., February 2008; 153(7), pp. 1438-1444);

[0386] Treatment of acute and chronic lung infections and sepsis (see Fang et al., Nature Reviews Microbiology, October 2004; 2(10), pp. 820-832; see also Goldfarb et al., Critical Care Medicine, January 2007; 35(1), pp. 290-292);

[0387] Reactive nitrogen intermediates (RNIs), including nitric oxide, have been proposed as effector molecules in the anti-mycobacterial activity of activated mouse macrophages against Mycobacterium tuberculosis (see Chan et al., J. Exp. Med., April 1992; 175, pp. 1111-1122).

[0388] Gaseous nitric oxide can be effective in treating antibiotic-resistant bacterial and fungal lung infections in patients with cystic fibrosis (see Deppisch et al., February 9, 2016; "Gaseous nitric oxide to treat antibiotic-resistant bacterial and fungal lung infections in patients with cystic fibrosis: a Phase I clinical study", Springer, DOI 10.1007 / s15010-016-0879-x);

[0389] Nitric oxide has been reported as a potential topical broad-spectrum antimicrobial agent for skin diseases with little likelihood of developing resistance (see BL Adler and AJ Friedman, Future Sci. OA, 2015; 1(1), FSO37);

[0390] Nitric oxide is a neurotransmitter and is associated with neuronal activity and avoidance learning of multiple functions within the range of genital erection in both men and women (see Kim et al., J. Nutrition, 2004, 134, 287-35).

[0391] The use of nitric oxide in the treatment of male impotence and erectile dysfunction is described in Sullivan et al., Cardiovascular Research, August 1999, 43(3), pp. 658-665.

[0392] Nitric oxide has been reported as a surgical adjuvant to help wound healing, reduce local ischemia-reperfusion injury, help the heart and lungs recover from surgery and help recover from vascular surgery, and help recover from postoperative plastic surgery (see A Krausz and AJ Friedman, Future Sci. OA, 2015; 1(1), FSO56);

[0393] The antimicrobial and wound-healing effects of NO are described in WO 95 / 22335 and Hardwick et al., 2001, Clin, Sci. 100, pp. 395-400.

[0394] European Patent No. 1411908 (Aberdeen University) reports data that supposedly shows nitric oxide is effective in treating subungual infections, including those caused by Aspergillus niger.

[0395] NOx-producing compositions are applied topically to the skin for the treatment of fungal skin infections, such as athlete's foot (see Weller et al. J. Am. Acad. Dermatol., April 1998, 38(4), pp. 559-563);

[0396] NOx-producing compositions are applied topically to the skin for the treatment of viral skin infections (see WO99 / 44622);

[0397] NOx-producing compositions are applied topically to the skin to treat conditions where vasoconstriction is the underlying problem, such as Raynaud syndrome (also known as Raynaud's phenomenon) (see Tucker et al., Lancet, November 13, 1999, 354, 9191, pp. 1670-1675).

[0398] WO 2000 / 053193 describes the use of acidified nitrite as an agent that locally generates nitric oxide on the skin surface for the treatment of peripheral local ischemia and related conditions, such as Raynaud's phenomenon and wounds (e.g., postoperative wounds) and burns.

[0399] U.S. Patent No. 9,730,956 (Stenzler et al.) claims the use of liquid nitric oxide-releasing solution (NORS) for treating human wounds. It also claims that NORS has antibacterial, antifungal, and / or antiviral properties, and provides data supposedly demonstrating antibacterial efficacy against *Acetobacter baumanii*, methicillin-resistant *Staphylococcus aureus*, *Escherichia coli*, and *Mannheimia haemolytica*. It provides data supposedly demonstrating antiviral efficacy of NORS against H1N1 influenza virus, bovine infectious rhinotracheitis virus, bovine respiratory syncytial virus, and bovine parainfluenza-3 virus. It provides data supposedly demonstrating antifungal efficacy of NORS against *Trichophyton rubrum* and *Trichophyton mentagrophytes*.

[0400] 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, pages 954-959;

[0401] 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, pp. H2026-H2035;

[0402] Hunter CJ et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat. Med. 2004, 10, pp. 1122-1127;

[0403] Ozaki M, et al., Reduced hypoxic pulmonary vascular remodeling by nitricoxide from the endothelium. Hypertension. 2001, 37, pp. 322-327;

[0404] Rubin LJ, 2006. Pulmonary arterial hypertension. Proc. Am. Thorac. Soc. 3, pp. 111-115;

[0405] Yellon DM et al., 2007. Myocardial Reperfusion Injury, N. Engl. J. Med., 357, pp. 1121-35;

[0406] Duranski MR et al., Cytoprotective effects of nitrite during in vivoischemia-reperfusion of the heart and liver.J.Clin.Invest.2005,115, pages 1232-1240;

[0407] Jung KH. et al., Early intravenous infusion of sodium nitrite protectsbrain against in vivo ischemia-reperfusion injury, Stroke, 2006, 37, pp. 2744-2750;

[0408] Esme H. et al. Beneficial Effects of Supplemental Nitric Oxide DonorGiven during Reperfusion Period in Reperfusion-Induced Lung Injury.Thorac.Cardiovasc.Surg.2006, 54, pages 477-483;

[0409] Chinese patent application No. CN 101028229 describes the use of acidified nitrite for releasing NO as an agent to improve human skin quality;

[0410] Chinese patent application No. CN 101062050 describes the use of acidified nitrite for releasing NO as an agent to promote hair growth and prevent or treat human hair loss.

[0411] Other comprehensive discussions on the physiological role of nitric oxide can be found in, for example, Lancaster et al., Proc Natl Acad Sci, 1996, 91, pp. 8137-8141; Ignarro et al., Proc Natl Acad Sci, 1987, 84, pp. 9265-9269; Brent, J Cell Science, 2003, 116, pp. 9-15; and Murad, N Engl J Med, 2006, 355, pp. 2003-2011.

[0412] The pharmacological forms of NO that have been published are reviewed in Butler and Feelisch, Circulation, 2008, 117, pp. 2151-2159.

[0413] The public content of each of the publications cited above is incorporated herein by reference.

[0414] This disclosure applies to all therapeutic and surgical uses of nitric oxide and systems that produce nitric oxide, including but not limited to the specific therapies and surgical uses disclosed in the foregoing references and all other disclosed therapies and surgical uses, as well as therapies and surgical uses based on the understanding of the physiological effects of nitric oxide and the reaction products that produce nitric oxide.

[0415] Vasodilator

[0416] The vasodilatory properties of nitric oxide characterize many treatments using the combinations and compositions of this disclosure and the gases emitted therefrom.

[0417] Specific examples of diseases, conditions, and disorders that respond to vasodilation include (but are not limited to) conditions associated with local ischemia and skin damage.

[0418] Disorders associated with local tissue ischemia include Raynaud's syndrome, severe primary vasospasm, and local tissue ischemia, such as that caused by surgery, septic shock, radiation, or peripheral vascular disease (e.g., diabetes and other chronic systemic diseases).

[0419] When used to treat or prevent conditions associated with surgically induced tissue ischemia, the combinations or compositions of this disclosure, or the nitric oxide released from the NOx-generating reaction of this disclosure, may be administered to the subject before, during, or after the procedure. The combinations, compositions, or released gases may be applied to or near the surgical site. Examples of surgical procedures that may utilize this treatment or prevention of tissue ischemia include transplantation procedures, tissue or organ transplantation procedures, coronary artery procedures, carotid artery catheterization, procedures to provide arterial or venous catheters for the administration of systemic agents (such as chemotherapy drugs), cosmetic surgery including but not limited to pedicled or rotational flaps, repeat surgeries performed at the same site as previous surgeries, surgeries performed in areas of poor skin and / or poor underlying tissue perfusion or in areas where poor perfusion is expected as a result of comorbidities (e.g., in patients with arteriosclerosis or diabetes), surgeries in cases of wounds with vascular injury or damage, and surgeries to remove or correct skin or subcutaneous arteriovenous malformations.

[0420] For example, by administering a combination, composition, or emitted gas according to this disclosure to an organ, the combination, composition, or emitted gas can be used to treat or prevent ischemia-reperfusion injury of the organ. The organ can be selected from one or more of the following: heart (e.g., for prevention or treatment of myocardial ischemia), brain (e.g., for treatment or prevention of cerebral ischemia and / or infarction (stroke)), lung (e.g., for treatment or prevention of pulmonary ischemia-reperfusion injury), kidney (e.g., for treatment or prevention of renal ischemia-reperfusion injury), and liver (e.g., for treatment or prevention of hepatic ischemia-reperfusion injury). The surgery can be organ transplantation. The administration of the combination, composition, or emitted gas can be subsequent or preventative.

[0421] Transdermal drug delivery

[0422] The nitric oxide-induced transdermal drug delivery property represents another important utility of the combinations and compositions disclosed herein, as well as the gases emitted therefrom.

[0423] The disclosures described herein by reference in WO 02 / 17881 and WO 2014 / 188175 describe the use of combinations and compositions for generating nitric oxide and the gas emitted therefrom for transdermal drug delivery, and the preferred embodiments and examples described in those publications for such uses also apply to the combinations and compositions of this disclosure and the gas emitted therefrom.

[0424] Typically, the combinations and compositions disclosed herein will comprise one or more pharmaceutically active agents that are delivered perdermally to a subject and are provided in the form of a topical combination or composition for application to the subject's skin. For examples of pharmaceutically active agents that may be used, please see the section above entitled "Optional Additional Components".

[0425] Suitable topical combinations may comprise a nitrite-containing sieve and a separate proton-source-containing hydrogel, both suitable for application together to the subject's skin, as described above in the section entitled "Other Reservoirs for Compositions or Composition Systems; Hydrogels". Polyols and pharmaceutical active agents may be provided in one or more individual components of the combination or incorporated into the hydrogel, or polyols and pharmaceutical active agents may be used separately in any combination of these options.

[0426] Treatment of wounds, skin lesions and burns

[0427] The properties of nitric oxide inducing vasodilation and transdermal drug delivery, as well as killing or preventing microbial proliferation, result in another important utility of the combinations and compositions disclosed herein, and the gases emitted therefrom, in the treatment of wounds, skin lesions, and burns.

[0428] Conditions that can be treated with this disclosure include ulcers, donor skin sites, surgical wounds (postoperative), burns (e.g., scalds, superficial burns, partial-thickness burns, and full-thickness burns), lacerations, and abrasions. Wounds can be chronic or acute. Ulcers can originate from various sources, such as arterial or venous sources. Examples of ulcers include leg ulcers, such as chronic or acute leg ulcers; pressure ulcers, such as chronic or acute pressure ulcers; varicose ulcers; and diabetes-related ulcers, such as diabetic foot ulcers.

[0429] The disclosure incorporated herein by reference in WO 2014 / 188174 describes combinations and compositions for generating nitric oxide and the use of the gas emitted therefrom in the treatment of wounds, skin lesions and burns, and the same conditions described in that disclosure also apply to the combinations and compositions and the gas emitted therefrom.

[0430] Typically, the combinations and compositions disclosed herein will comprise one or more pharmaceutically active agents and be provided in the form of a topical combination or composition for application to the skin of a subject. For examples of pharmaceutically active agents that may be used, please refer to the section above entitled “Optional Additional Components.” For the treatment of wounds, skin lesions, and burns, one or more pharmaceutically active agents are suitably selected from analgesics and / or anesthetics (e.g., local anesthetics) (e.g., analgesics and / or anesthetics for reducing chronic pain, acute pain, or neuropathic pain), antimicrobial agents, disinfectants, anti-inflammatory agents, and anti-scarring agents.

[0431] Suitable topical combinations may comprise a nitrite-containing sieve and a separate proton-source-containing hydrogel, both suitable for application together to the subject's skin, as described above in the section entitled "Other Reservoirs for Compositions or Composition Systems; Hydrogels". Polyols and pharmaceutical active agents may be provided in one or more individual components of the combination or incorporated into the hydrogel, or polyols and pharmaceutical active agents may be used separately in any combination of these options.

[0432] Topical antimicrobial use

[0433] In antimicrobial applications, the therapeutically effective dose of NO can be very small, for example as low as several hundred parts per million (ppm), such as 100 to 600 ppm (see, for example, Ghaffari et al., Nitric Oxide Biology and Chemistry, 2009, 14, pp. 21–29, the disclosure of which is incorporated herein by reference). However, the effectiveness of nitric oxide depends largely on how long the skin contact is maintained (Ormerod et al., BMC Research Notes, 2011, 4, pp. 458–465, the disclosure of which is incorporated herein by reference).

[0434] Proposals for slow, localized 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 poor local antimicrobial activity. As discussed above in the section entitled “Multicomponent Systems, Kits, and Dispensers” and elsewhere, and as illustrated in the following examples, this disclosure enables significantly longer NO administration periods in both local and non-local application systems, resulting in substantial clinical advantages.

[0435] Specifically, it has been found that the combinations and compositions of this disclosure can initiate a strong release of nitric oxide (“initial burst release”) for approximately 200-500 seconds after the start of a NOx-generating reaction, optionally followed by a long, slower release period of nitric oxide lasting for many hours (“tail”), before ceasing gas emission or falling below effective levels. The NO dose emitted by the combinations and compositions of this disclosure exceeds the disclosed minimum effective antimicrobial dose, giving rise to potentially effective local antimicrobial uses of the combinations and compositions of this disclosure and the gas emitted therefrom.

[0436] Formulations of NOx-generating combinations and compositions for topical antimicrobial applications are well described in the prior art, such as 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 disclosed herein. Another suitable topical combination may comprise a nitrite-containing sieve and a separate proton-source-containing hydrogel, both suitable for application together to the skin of a subject, as described above in the section entitled "Other Reservoirs of Compositions or Composition Systems; Hydrogels". Polyols and any pharmaceutically active agents may be provided in one or more individual components of the combination or incorporated into the hydrogel, or polyols and pharmaceutically active agents may be employed separately in any combination of these options.

[0437] Other skin or topical treatments

[0438] Other topical applications of nitric oxide and compositions that produce nitric oxide include stimulating hair growth and treating impotence and erectile dysfunction.

[0439] The combinations and compositions disclosed herein can be formulated for topical application of such treatments.

[0440] Topical medications and medication systems, such as wound dressings

[0441] In topical treatments, it is often desirable to cover or protect the treated skin area while applying the treatment. This helps prevent wound contamination, aids in the removal of pus or debris from the healing process, prevents or limits the loss of the treatment composition during bathing or showering, through contact with clothing, or due to the subject's normal activities, and protects the treated area from impacts or friction.

[0442] Therefore, treatment is often incorporated into a topical dressing or dressing system, such as a wound dressing or dressing system. At least one component of the dressing or dressing system typically includes a base plate, which may be impermeable or permeable, optionally having a skin-adhesive portion and optionally other layers, such as gauze or a padding layer.

[0443] In another aspect, this disclosure provides a topical dressing, such as a wound or skin dressing, or a dressing system comprising a combination or composition according to the fifth aspect of this disclosure, wherein at least one component of the dressing or dressing system comprises a base plate and optionally one or more other layers, such as layers selected from gauze and padding layers. The combination or composition according to the fifth aspect of this disclosure is suitably disposed on the skin-oriented side of the base plate and arranged such that when the dressing is applied to the skin and triggers a NOx-generating reaction, the desired epidermal area receives the NOx-generating reaction mixture or the gas released therefrom for treatment.

[0444] Before use, the medication or medication system should be provided in a sealed, sterile package.

[0445] Uses of the nose, mouth, respiratory tract and lungs

[0446] The properties of nitric oxide inducing vasodilation and percutaneous drug delivery, as well as killing or preventing microbial proliferation, result in another important utility of the combinations and compositions disclosed herein, and the gases emitted therefrom, in the treatment of the mucous membranes and tissues of the nose, mouth, respiratory tract, and lungs, and / or the nose, mouth, respiratory tract, and lungs as a route of administration for delivering the combinations and compositions of the invention to human or animal subjects.

[0447] Diseases that can be treated with this invention include lung diseases such as viral infections (e.g., 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) (especially emphysema and chronic bronchitis), asthma (including severe asthma, and viral and bacterial-induced asthma exacerbations, and refractory (irreversible) asthma), intranasal or pulmonary bacterial infections such as pneumonia, tuberculosis, nontuberculous mycobacterial infections, and other bacterial and viral lung infections, such as secondary bacterial infections following respiratory viral infections.

[0448] The disclosure incorporated herein by reference in WO 2009 / 086470 describes the use of atomized liquid combinations and compositions for generating nitric oxide and the gases emitted therefrom for the treatment of diseases of the nose, mouth, respiratory tract and lungs, and / or the nose, mouth, respiratory tract and lungs as a route of administration for delivering such combinations and compositions to human or animal subjects, and the preferred embodiments and examples described in that disclosure for such uses also apply to the combinations and compositions of this disclosure and the gases emitted therefrom.

[0449] Typically, the combinations and compositions of the present invention delivered to the nose, mouth, respiratory tract, and lungs will contain one or more pharmaceutically active agents. For examples of pharmaceutically active agents that may be used, please refer to the section above entitled “Optional Additional Components.”

[0450] Two main delivery methods may be used to carry out this invention, via the nose, mouth, respiratory tract, or lungs. The first is the direct delivery of the combination or composition of this invention to the nose, mouth, respiratory tract, or lungs. The second is the delivery of gases released from the NOx-producing reaction using the invention to the nose, mouth, respiratory tract, or lungs, without the combination or composition of this invention entering the patient's body.

[0451] 1. Combinations or formulations delivered directly to the nose, mouth, respiratory tract, or lungs.

[0452] Combinations or compositions, or their components, can be delivered directly to the nose, mouth, respiratory tract, or lungs in dry solid form, where mucosal fluids dissolve the solid component material and initiate a reaction that produces NOx.

[0453] The components of the combination can be applied separately or together. In a preferred embodiment, the proton source or at least one of its components may be applied before the remaining components to establish a relatively acidic environment in the mucosa, which facilitates the rapid initiation of a NOx-generating reaction when the nitrite component contacts the proton source component in situ.

[0454] It is suitable to deliver to a subject 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 by inhaling dry powder through a dry powder inhaler, directly to the nose, mouth, respiratory tract, or lungs, wherein the dry powder inhaler delivers to the subject an aerosol containing particles with a volume average diameter of less than 6 micrometers. The dry powder inhaler may be adapted for single or multiple-load dry powder administration, such that with each inhalation, the dry powder inhaler delivers to the subject one or more dry powder components or dry powder compositions containing particles with a volume average diameter of less than 6 micrometers, ranging from about 0.1 mg to about 100 mg.

[0455] Alternatively or alternatively, the combination or composition, or its components, may be delivered directly to the nose, mouth, respiratory tract, or lungs as a mist or spray of one or more of the nitrite component, proton source component, and polyol component.

[0456] The embodiments of the invention described herein are generally applicable to direct delivery to the nose, mouth, respiratory tract, or lungs of a subject. Without limitation, for example, combinations or compositions or components thereof may be administered directly to the nose, mouth, respiratory tract, or lungs of a subject in combination with one or more physiologically compatible diluents, carriers, and / or excipients, and / or in combination with one or more additional components, or specific functional components intended to provide one or more particular benefits. Examples of suitable physiologically compatible diluents, carriers, and / or excipients include, but are not limited to, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talc, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, etc. Where necessary, small amounts of non-toxic excipients may also be present, such as wetting agents, emulsifiers, lubricants, binders, and solubilizers, such as sodium phosphate, potassium phosphate, gum arabic, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc. Generally, depending on the intended administration method, the pharmaceutical formulation will contain about 0.005% by weight to about 95% by weight, preferably about 0.5% by weight 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 or obvious to those skilled in the art. See, for example, Martindale, 39th edition (2017); the Merck Index, 15th edition (2013); Goodman & Gilman's “The Pharmacological Basis of Therapeutics”, 13th edition (2017); the British National Formulary online (https: / / bnf.nice.org.uk / ); Remington: “The Science & Practice of Pharmacy”, 22nd edition (2012); or the Physician's Desk Reference, 71st edition (2017).

[0457] In a preferred embodiment, the combination or composition for delivery to the nose, mouth, respiratory tract, or lungs of a subject will be in the form of a unit dosage form, such as a vial containing a liquid, a solid to be suspended, a dry powder, a lyophilized substance, or other composition, which is suitable to contain the following components that react with NOx generation: diluents, such as lactose, sucrose, dicalcium phosphate, etc.; lubricants, such as magnesium stearate, etc.; binders, such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives, etc.

[0458] Suitable for delivering a therapeutically effective dose of one or more liquid components (e.g., one or more of a nitrite component, a proton source component, and a polyol component) or a composition in liquid form to a subject via inhalation using a nebulizer, delivering any droplet or droplet-form composition containing the combined components directly to the nose, mouth, respiratory tract, or lungs, wherein the nebulizer delivers an aerosol containing particles with a volume average diameter of less than 5 micrometers to the subject. The nebulizer may be adapted for single or multiple loading of the combined liquid components or liquid composition for administration, such that with each inhalation, the nebulizer delivers to the subject one or more liquid components or a composition in liquid form in droplets with a volume average diameter of less than 5 micrometers, preferably in droplets ranging in size from about 2 to about 5 μm, in a dose ranging from about 0.1 mg to about 100 mg.

[0459] In one embodiment, the atomizer is selected based on the fact that it allows the formation of an aerosol containing droplets or droplet-form compositions of combined components with a mass median aerodynamic diameter (MMAD) primarily between about 2 and about 5 micrometers.

[0460] In one embodiment, the delivered amount of a composition comprising the combined components in droplet or droplet form provides therapeutic effects against lung lesions, respiratory infections, and / or extrapulmonary, systemic distribution, thereby also treating extrapulmonary and systemic diseases.

[0461] Previously, both jet and ultrasonic nebulizers have been shown to generate and deliver aerosol particles with sizes between 2 and 4 μm. These particle sizes have been shown to be optimal for deposition in the intermediate airways, thus making them ideal for treating bacterial lung infections caused by Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Staphylococcus aureus, and multidrug-resistant Pseudomonas aeruginosa. However, unless specially formulated solutions are used, these nebulizers typically require larger volumes to deliver a sufficient amount of drug to achieve a therapeutic effect. Jet nebulizers utilize air pressure to ablate aqueous solutions into aerosol droplets. Ultrasonic nebulizers utilize piezoelectric crystals to shear aqueous solutions. However, typically, jet nebulizers are only about 10% effective in clinical conditions, while ultrasonic nebulizers are only about 5% effective. Therefore, the amount of drug deposited and absorbed in the lungs is only a small fraction of 10%, despite a large amount of drug being placed in the nebulizer. Smaller particle size or slower inhalation rates allow for deep lung deposition. The present invention may favor mid-lung and alveolar deposition, depending on the indication, such as for antimicrobial activity, mid-airway deposition, or for pulmonary hypertension and systemic delivery, mid- and / or alveolar deposition. Exemplary disclosures of compositions and methods for delivering formulations using nebulizers are available, for example, in US 2006 / 0276483, which includes a description of techniques, schemes, and characterization for delivering aerosol mists using a vibrating screen nebulizer. The contents of US 2006 / 0276483 are incorporated herein by reference.

[0462] Therefore, in one embodiment, and in a preferred embodiment, a vibrating screen nebulizer is used to deliver an aerosol comprising droplets or a composition in droplet form containing combined components. The vibrating screen nebulizer includes a liquid reservoir in fluid contact with a diaphragm and inhalation and exhalation valves. In one embodiment, about 1 ml to about 5 ml of the liquid formulation to be delivered is contained in the reservoir, and an aerosol generator is engaged to produce an aerosol with a particle size selectively between about 1 μm and about 5 μm in volume average diameter.

[0463] Therefore, for example, in a preferred embodiment, one or both of these components, including the nitrite component formulation or the proton source component, optionally comprising one or more organic polyols according to the invention, are placed in a liquid nebulizer and prepared in a dose to deliver about 7 mg to about 700 mg from about 1 ml to about 5 ml of dosing solution, preferably about 17.5 mg to about 700 mg from about 1 ml to about 5 ml, more preferably about 17.5 mg to about 350 mg from about 1 ml to about 5 ml, preferably about 0.1 mg to about 300 mg from about 1 ml to about 5 ml, more preferably about 0.25 mg to about 90 mg from about 1 ml to about 5 ml, producing a volume average diameter particle size between about 1 μm and about 5 μm.

[0464] By way of non-limiting examples, compositions comprising the combined components in the form of atomized liquids or droplets can be administered in the said inhalable delivery dose in less than about 20 minutes, preferably less than about 10 minutes, more preferably less than about 7 minutes, more preferably less than about 5 minutes, more preferably less than about 3 minutes, and in some cases most preferably less than about 2 minutes.

[0465] By way of non-limiting examples, in other cases, compositions comprising the combined components in the form of atomized liquids or droplets can achieve improved tolerability and / or exhibit enhanced area under the curve (AUC) shape characteristics when administered over a longer period of time. Under these conditions, the inhalable delivery dose is delivered within a period of about 2 minutes, preferably more than about 3 minutes, more preferably more than about 5 minutes, more preferably more than about 7 minutes, more preferably more than about 10 minutes, and in some cases most preferably about 10 minutes to about 20 minutes.

[0466] An example of a separate component formulation may comprise (i) a nitrite in an aqueous solution with a pH value 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, wherein at least two separate liquid solution components (i) and (ii) are capable of being mixed to form a composition that generates NOx, which may be used to load a nebulizer for delivery to a human patient or veterinary subject.

[0467] For aqueous and other unpressurized liquid systems, various nebulizers (including small-volume nebulizers) can be used to disperse combined components or compositions in a vapor-like manner. Compression-driven nebulizers incorporate jet technology and use compressed air to generate a liquid aerosol. These devices are available from, for example, Healthdyne Technologies; Invacare; Mountain Medical Equipment; Pari Respiratory (Midlothian, VA); MadaMedical; Puritan-Bennet; Schuco; DeVilbiss Health Care; and Hospitaltak. Ultrasonic nebulizers rely on mechanical energy in the form of piezoelectric crystal vibrations to generate inhalable droplets and are available from, for example, Omron Heathcare and DeVilbiss Health Care. Vibrating screen nebulizers rely on piezoelectric or mechanical pulses to generate inhalable droplets. Other examples of atomizers used with nitrite, nitritesalt, or compounds providing nitrite or nitric oxide as described herein are described in U.S. Patent Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,7 92; 5,971,951; 6,070,575; 6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 are all incorporated herein by reference in their entirety.

[0468] Commercial examples of atomizers that can be used with the compositions comprising the combined components described herein, either in droplet or droplet form, include the Respirgard manufactured by Aerogen (Aerogen Corporation, Galway, Ireland). Pro, AeroEclipse and Go; produced by Aradigm and AERxEssence TM Respironics (Murrysville, Pennsylvania, USA) manufactures... Freeway Freedom TM SideStream, SideStream Plus, Ventstream, and I-neb; and PARI products manufactured by PARI Ltd. (PARI Respiratory Equipment, Midlothian, Virginia, USA; PARI Ltd., Starnberg, Germany). PARI PARI and e-Flow TM According to the manufacturer's instructions, any of these atomizers can be used with a mask or inhaler. By way of other non-limiting examples, U.S. Patent No. 6,196,219 is incorporated herein by reference in its entirety.

[0469] In one embodiment, an aqueous formulation containing soluble or nanoparticle drug particles is provided. For aqueous aerosol formulations, the drug may be present at a concentration from 0.67 mg / mL to a maximum of 700 mg / mL; in some preferred embodiments, the nitrite is present at a concentration from about 0.667 mg nitrite anion per milliliter to about 100 mg nitrite anion per milliliter. Such formulations are effectively delivered to appropriate areas of the lungs, and more concentrated aerosol formulations have the additional advantage of being able to deliver large amounts of the active pharmaceutical ingredient to the lungs in a very short time. In one embodiment, the formulation is optimized to provide a well-tolerated formulation. Thus, some preferred embodiments contain a nitrite (e.g., sodium nitrite, potassium nitrite, or magnesium nitrite) and are formulated to have a pleasant taste, a pH of about 4.7 to about 6.5, and a molar osmolality of about 100 to about 3600 mOsmol / kg; optionally, in some other embodiments, the concentration of osmotic ions (e.g., chloride ions, bromide ions) is about 30 to about 300 mM.

[0470] In one embodiment, the solution or diluent used to prepare the aerosol formulation has a pH value in the range of about 4.5 to about 9.0, preferably about 4.7 to about 6.5 (e.g., in an acidic mixture), or, when configured as a single vial, about 7.0 to about 9.0. This pH range improves tolerability, as masking agents are included in some embodiments, as described elsewhere herein. When the aerosol is acidic or alkaline, it may cause bronchospasm and coughing. While safe pH ranges are relative, and some patients tolerate slightly acidic aerosols, others will experience bronchospasm. Any aerosol with a pH value less than about 4.5 typically induces bronchospasm. Aerosols with a pH value of about 4.5 to about 5.5 sometimes cause bronchospasm. Any aerosol with a pH value greater than about 8 may have low tolerability because body tissues generally cannot buffer alkaline aerosols. Aerosols with pH values ​​below approximately 4.5 and above approximately 8.0 typically cause lung irritation, accompanied by severe bronchospasm, cough, and inflammatory response. For these reasons, and to avoid bronchospasm, cough, or inflammation in patients, the optimal pH value for aerosol formulations is determined to be between approximately pH 5.5 and approximately pH 8.0.

[0471] Therefore, in one embodiment, the aerosol formulation used as described herein is adjusted to a pH value between about 4.5 and about 7.5, wherein the most preferred pH range for the acidic mixture is about 4.7 to about 6.5, and the most preferred pH range for a single vial configuration is about 7.0 to about 8.0. By way of non-limiting examples, according to certain embodiments disclosed herein, the composition may also include a pH buffer or pH adjuster, typically a salt prepared from an organic acid or organic base, and in a preferred embodiment, an acidic excipient (e.g., a non-reducing acid, such as citric acid or citrate, such as sodium citrate) or a buffer, such as citrate or other buffers described above, and refer to Table 1. Thus, these and other representative buffers may include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, thiazoline, hydrochloride or phosphate buffers.

[0472] Many patients experience increased sensitivity to a variety of chemical tastes, including bitter, salty, sweet, and metallic. To create well-tolerated medications, taste masking can be achieved by adding masking agents and excipients, adjusting osmotic pressure, and using sweeteners.

[0473] Many patients exhibit increased sensitivity to multiple chemotherapeutic agents and a high incidence of bronchospasm, asthma, or other cough events. Their airways are particularly sensitive to hypotonic or hypertonic and acidic or alkaline conditions, and to the presence of any osmotic ions such as chloride ions. Any imbalance in these conditions or the presence of chloride ions exceeding a certain concentration value will cause bronchospasm or inflammatory events and / or cough, which significantly diminishes the therapeutic effect of inhaled formulations. Both of these conditions can prevent the effective delivery of aerosolized drugs into the bronchial space, and the advantageous use of pH, osmolarity, and masking agents according to certain embodiments disclosed herein is lacking.

[0474] In some embodiments, the osmotic pressure of the aqueous solution of the nitrite compound disclosed herein (or in a unique embodiment of nitrite or a compound providing nitric oxide) is adjusted by providing an excipient. In some cases, a certain amount of osmotic ions, such as chloride ions, bromide ions, or another anion, can promote successful and efficient delivery of aerosolized nitrite. However, it has been found that the amount of such osmotic ions may be lower for the nitrite components disclosed herein than is typically used for the aerosolized application of other pharmaceutical compounds.

[0475] For aerosolization with a given osmotic pressure, it may not be possible to improve bronchospasm or cough reflex in all cases by using a diluent. However, when the osmotic pressure of the diluent is within a certain range, these reflexes can often be adequately controlled and / or suppressed. Preferred solutions for the safe and tolerable aerosolization of therapeutic compounds have a total osmotic pressure of about 100 to about 3600 mOsmol / kg, and a chloride ion concentration in the range of about 30 mM to about 300 mM, and preferably about 50 mM to about 150 mM. This osmotic pressure induces bronchospasm, while the chloride ion concentration, as an osmotic anion, controls cough. Because bromide or iodide anions are both osmotic ions, they can substitute for chloride ions. Additionally, bicarbonate ions can substitute for chloride ions.

[0476] Nanoparticle drug dispersions can also be freeze-dried to obtain powders suitable for nasal or transpulmonary delivery. These powders may contain aggregated nanoparticle drug particles with surface modifiers. These aggregates may have sizes within the inhalable range, such as approximately 2 to approximately 5 micrometers of MMAD.

[0477] 2. The gas released from the reaction that produces NO is delivered to the nose, mouth, respiratory tract, or lungs.

[0478] Inhalers used to deliver a dose of nitric oxide to a patient's lungs are well known. Generally, nitric oxide is generated outside the site and delivered to a hospital or clinic for use in a pressurized cylinder connected to a specialized delivery device. The INOmax Therapy system can be cited as an example (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 generally used for the cylinder of the INOmax Therapy system, and INOvent for the delivery device. Evaluations of the INOmax Therapy system have been published, for example, in Kirmse et al. Chest, June 1998, 113(6), pp. 1650-1657. The contents of that publication are incorporated herein by reference.

[0479] The method according to the first aspect of the invention is suitable for carrying out in a dedicated NO manufacturing facility, and the gaseous product according to the second aspect of the invention is provided to the user in a pressurized cylinder in a normal manner. The pressurized cylinder is then used in conjunction with distribution, monitoring, dosing, mixing, and delivery equipment in a known manner.

[0480] Target of antimicrobial use

[0481] As previously described, the NOx-generating reactions and the gases released therefrom of this disclosure have biocidal or biostabilizing effects on a wide range of microorganisms, leading to numerous antimicrobial applications.

[0482] Microorganisms can be, for example, any or more of 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 in a host organism, such as as the gut microbiome of humans or other animals, or in bacterial infections of humans or other animals. Bacterial and / or fungal cells and / or viral particles and / or microparasites can be in vitro, in vivo, or ex vivo.

[0483] This disclosure is particularly useful for the treatment or prevention of microbial infections at sites of skin lesions in subjects. This disclosure is particularly useful for the treatment or prevention of microbial infections in immunosuppressed subjects.

[0484] When microorganisms are present in bacterial, fungal, viral, or microparasitic infections in humans or other animals, the infection may occur in the context of diseases such as the common cold, influenza, tuberculosis, SARS, COVID-19, pneumonia, or measles.

[0485] 1. Bacterial cells

[0486] Bacteria can be pathogenic bacterial species. Microbial infections can be caused by pathogenic bacterial species, which include Gram-positive and Gram-negative, aerobic and anaerobic, antibiotic-sensitive and antibiotic-resistant bacteria.

[0487] Examples of bacterial species that can be targeted by this invention include the following bacterial species: *Actinomyces*, *Bacillus*, *Bartonella*, *Bordetalla*, *Borrelia*, *Brucella*, *Campylobacter*, *Chlamydia*, *Chlamydophila*, *Clostridium*, *Corynebacterium*, *Enterococcus*, *Escherichia*, *Francisella*, and *Haemophilus*. The present invention can target any combination of these bacteria, including *Helicobacter*, *Legionella*, *Leptospira*, *Listeria*, *Mycobacterium*, *Mycoplasma*, *Neisseria*, *Pseudomonas*, *Rickettsia*, *Salmonella*, *Shigella*, *Staphylococcus*, *Streptococcus*, *Treponema*, *Ureaplasma*, *Vibrio*, and *Yersinia*.

[0488] In specific implementation schemes, the microorganisms may be the following pathogenic species: Corynebacterium, Mycobacterium, Streptococcus, Staphylococcus, Pseudomonas, or any combination thereof.

[0489] In a more specific implementation scheme, the targeted microorganism may be selected from *Actinomycesisraelii*, *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*, and *Chlamydophila psittaci*. Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheriae; Ehrlichia canis; Ehrlichiachaffeensis; Enterococcus faecalis; Enterococcus faecium; Escherichia coli, such as enterotoxigenic E. coli (ETEC), enteropathogenic E. coli, enteroinvasive E. coli (EIEC), and enterohemorrhagic E. coli (EHEC), including E. coli O157.H7; *Francisella tularensis*; *Haemophilus influenzae*; *Helicobacter pylori*; *Klebsiella pneumoniae*; *Legionellapneumophila*; *Leptospira* species; *Listeria monocytogenes*; *Mycobacterium leprae*; *Mycobacterium tuberculosis*; *Mycobacterium abscessumensis*; *Mycoplasma pneumoniae*; *Neisseria gonorrhoeae*; *Neisseria meningitides*; *Pseudomonas aeruginosa*; *Nocardia asteroides*; *Rickettsia*. *Salmonella 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 cholerae*; *Yersinia* (pestis); and any combination thereof.

[0490] Specifically, the microorganism may be selected from Chlamydia pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or any combination thereof.

[0491] The microorganism can be an antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species, or an antibiotic-resistant or antibiotic-sensitive strain of a bacterial species. The use of nitric oxide to treat methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) is described, for example, in WO02 / 20026, the disclosure of which is incorporated herein by reference. Therefore, 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).

[0492] 2. Fungal cells

[0493] Microorganisms can be pathogenic fungal species. Microbial infections can be caused by pathogenic fungal species, including pathogenic yeasts.

[0494] Examples of fungal species that can be targeted by this invention include the following fungal species: Aspergillus, Blastomyces, Candida (e.g., Candida auris), Coccidioides, Cryptococcus (specifically, Cryptococcus neofromans or Cryptococcus gattii), Hisoplamsa, Murcomycetes, Pneumocystis (e.g., Pneumocystis jirovecii), Sporothrix, Talaromyces, or any combination thereof.

[0495] Examples of fungal infections include aspergillosis (e.g., allergic bronchopulmonary aspergillosis), tinea pedis (athlete's foot), infections caused by Candida species such as vaginal yeast infections, fungal toenail infections and diaper rash, tinea cruris (jock itch), and tinea corporis (ringworm).

[0496] 3. Virus particles

[0497] Microorganisms can be viral particles. Infections can be caused by pathogenic viruses.

[0498] Examples of viruses that can be targeted by this invention include influenza virus, parainfluenza virus, adenovirus, norovirus, rotavirus, rhinovirus, coronavirus, respiratory syncytial virus (RSV), astrovirus, and hepatitis virus. Specifically, the compositions of this invention can be used to treat or prevent infections caused by one of the following groups: H1N1 influenza virus, bovine infectious rhinotracheitis virus, bovine respiratory syncytial virus, bovine parainfluenza-3 virus, SARS-CoV, SARS-CoV-2, and any combination thereof.

[0499] Specifically, this invention can be applied to the treatment of diseases or conditions caused by viral infections. Examples of diseases to which this invention can target include respiratory viral diseases, gastrointestinal viral diseases, herpetic viral diseases, hepatic viral diseases, skin viral diseases, hemorrhagic viral diseases, and neuroviral diseases.

[0500] Respiratory viral infections include influenza, rhinovirus (i.e., cold viruses), respiratory syncytial virus, adenovirus, coronavirus infections (such as COVID-19), and severe acute respiratory syndrome (SARS). Gastrointestinal viral diseases include norovirus infection, rotavirus infection, adenovirus infection, and astrovirus infection. Exanthematous viral diseases include measles, rubella, chickenpox, herpes zoster, roseola, smallpox, hepatitis V, and Chekungunya virus disease. Hepatitis viral diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E. Skin viral diseases include warts, such as genital warts, oral herpes, genital herpes, and molluscum contagiosum. Hemorrhagic viral diseases include Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, and Crimean-Congo hemorrhagic fever. Neuroviral diseases that can be targeted by this invention include poliomyelitis, viral meningitis, viral encephalitis, and rabies.

[0501] 4. Parasitic microorganisms

[0502] Microorganisms can be parasitic microorganisms (microparasites). Infections can be caused by pathogenic parasitic microorganisms.

[0503] Examples of parasitic microorganisms that can be targeted by this invention include protozoa.

[0504] Specifically, the present invention can target the following protozoan groups: Sarcodina (e.g., amoebas, such as Entamoeba, such as Entamoeba histolytica or Entamoeba dispar)), Mastigophora (e.g., flagellates, such as Giardia and Leishmania)), Ciliophora (e.g., ciliate protozoa, such as Balantidium)), Sporozoa (e.g., Plasmodium and Cryptosporidium) and any combination thereof.

[0505] Parasitic infections that can be treated with this invention include malaria, amoebic dysentery, and leishmaniasis (e.g., cutaneous leishmaniasis, mucocutaneous leishmaniasis, or visceral leishmaniasis).

[0506] Human / animal host or subject

[0507] Subjects may be animal or human subjects. The term “animal” in this text may generally include humans; however, where the term “animal” appears in phrases such as “animal or human subjects”, it should be understood from the context to refer specifically to non-human animals, or the mention of “human” is merely to list the option that an animal may be a human to avoid ambiguity.

[0508] In the specific implementation plan, the subjects are human subjects. Human subjects can be infants or adult subjects.

[0509] In the specific implementation plan, the subjects are vertebrate subjects. Vertebrates can belong to the classes Agnathea (jawless fish), Cartilaginous fish (cartilaginous fish), Bony fish (boneless fish), Amphibia (amphibians), Reptiles (reptiles), Aves (birds), or Mammals (mammals). In the specific implementation plan, the subjects are animal subjects belonging to the classes Mammalia or Aves.

[0510] In the specific implementation plan, the subjects are livestock species. Livestock species can be one of the following:

[0511] - Symbiotic animals adapted to human habitats (e.g., dogs, cats, guinea pigs)

[0512] - Animals or livestock (e.g., cattle, sheep, pigs, goats) that are hunted or raised for food; and

[0513] - Animals primarily used for pulling (e.g., horses, camels, donkeys)

[0514] Examples of livestock include (but are not limited to): alpacas, antelopes, bison, camels, canaries, capybaras, cats, cattle (including Bali cattle), chickens, peccaries, deer (including sika deer, white-lipped deer, and white-tailed deer), dogs, donkeys, wild pigeons, ducks, eland, elk, emus, ferrets, bighorn cattle, goats, geese, guinea fowl, guinea pigs, oryx, horses, llamas, mink, moose, mice, mules, musk oxen, ostriches, parrots, pigs, pigeons, quails, rabbits, rats (including canary rats), reindeer, canary rats, sheep, turkeys, buffalo, yaks, and zebu cattle.

[0515] Organs, structures, and internal spaces of animal / human hosts or subjects

[0516] The organs to which the compositions or multi-component systems of this disclosure are applied are not limited. Examples of organs include the skin, and organs of the respiratory, reproductive and urinary, cardiovascular, digestive, endocrine, excretory, lymphatic, immune, autonomic, muscular, nervous, reproductive, and skeletal systems.

[0517] Examples of organs in the cardiovascular system include the heart, lungs, blood, and blood vessels. Examples of organs in the digestive system include the salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rectum, and anus. Examples of organs in the endocrine system include the hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid glands, and adrenal glands. Examples of organs in the excretory system include the kidneys, ureters, bladder, and urethra. Examples of organs in the lymphatic system include lymph nodes and blood vessels. Examples of organs in the immune system include the tonsils, adenoids, thymus, and spleen.

[0518] Examples of organs in the body system 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 organs in the nervous system include the brain, spinal cord, and nerves. Examples of organs in the reproductive system include sex organs such as the ovaries, fallopian tubes, uterus, vulva, vagina, testes, vas deferens, seminal vesicles, prostate, and penis. Examples of organs in the skeletal system include bones, cartilage, ligaments, and tendons.

[0519] Cavities of human subjects include, but are not limited to, the oral cavity, nose, ear, pharynx, respiratory tract, lungs, gastrointestinal tract, dorsal body cavity (e.g., cranial cavity or vertebral cavity), or ventral body cavity (e.g., thoracic cavity, abdominal cavity, or pelvic cavity). Administration via the nose, oral cavity, respiratory tract, and lungs is a characteristic feature of this invention.

[0520] In vitro antimicrobial treatment of the surface

[0521] The components and compositions disclosed herein, as well as the gases emitted from the reaction that produces NOx according to this disclosure, can be used for applying antimicrobial treatments in vitro. "In vitro" means that the surface being treated is not a living organism, even if it may ultimately be intended for medical applications.

[0522] Examples of such uses include methods for sterilizing surgical instruments, hypodermic needles and other medical devices before use, and for cleaning or treating surfaces in hospitals, clinics or anywhere else to reduce or prevent the spread of pathogens.

[0523] Other examples include methods for sterilizing the following before placing a device in a subject's body: prostheses and implantable devices such as stents (e.g., coronary stents), surgical screws, rods, plates and splints, orthopedic implants, pacemakers, insulin infusion devices, catheters, ostomy instruments, intraocular lenses, cochlear implants, electro-analgesic implants, implantable contraceptives, neurostimulators, artificial heart valves, electrodes, intravenous drip and drug delivery devices, etc.

[0524] If necessary, the components or compositions disclosed herein may be coated onto the surface of a prosthesis or implantable device, whereby the NO released in the NOx-generating reaction may be perfused into other tissues or organs or exert other physiological effects in the vicinity of the prosthesis or implantable device.

[0525] Techniques for making the surface of prostheses or implantable devices biocompatible are well known to those skilled in the art, including the incorporation of functional coatings, such as coatings containing components or compositions of the present disclosure. See, for example, Gultepe et al., Advanced Drug Delivery Reviews, March 8, 2010, 62(3), pp. 305-315; and U.S. Patent Nos. 5,702,754 and 6,270,788, and the publications mentioned therein, all of which are disclosed herein by reference.

[0526] Compositions and methods for more general antimicrobial treatment of inanimate surfaces are well known in the art and do not need to be described extensively herein. Antibacterial compositions are used, for example, in the medical industry, food industry, meat processing industry, and the private sector for individual users. Antibacterial cleaning compositions typically contain one or more active antimicrobial agents or components thereof, surfactants, and one or more other ingredients, such as dyes, fragrances, pH adjusters, thickeners, skin conditioners, etc., in an aqueous and / or alcohol-based carrier. Broad-spectrum bactericides or antimicrobial compositions are designed to reduce the pathogen load on a range of pathogens on a surface. Typically, the composition is a liquid (or prepared from a solid premix before use), which, after being appropriately adjusted to the desired concentration by adding water, is often applied or sprayed onto the surface to be treated by means of a cloth or other wiping device, and then air-dried or wiped off. Conventional compositions and methods for treating surfaces are applicable in principle to the present invention, whereby the active antimicrobial agent is or contains a NOx-generating composition or component thereof according to the present invention.

[0527] Further discussion and examples of known antimicrobial compositions and methods of use that can be used in conjunction with the present invention are referenced, for example, U.S. Patent Nos. 6,110,908; 5,776,430; 5,635,462; 6,107,261; 6,034,133; 6,136,771; 8,034,844; European Patent Application No. EP 0505935; and PCT Patent Application Nos. WO 98 / 01110; WO95 / 32705; WO 95 / 09605; and WO 98 / 55096; the contents of which are incorporated herein by reference in their entirety.

[0528] Uses to improve the well-being of people and / or animals

[0529] In addition to the medical uses discussed above, this disclosure can be used for non-therapeutic applications in human or animal subjects. The difference between non-therapeutic and therapeutic applications is that the subject is healthy, or the purpose of this application is not to treat any diagnosed disease, ailment, or disorder suffered by the subject.

[0530] Non-therapeutic applications may include treatments designed to enhance a subject's well-being or sense of well-being, or to improve their metabolic efficiency or immune system activity, enabling them to function better or fight future infections. Non-therapeutic applications also include treatments that contribute to a subject's cognitive function or induce feelings of confidence and control.

[0531] For use in these non-therapeutic applications, the combinations and compositions of this disclosure may be formulated similarly to pharmaceutical preparations or in a non-pharmaceutical manner. Further details regarding pharmaceutical-like formulations can be found in the section above entitled "Optional Additional Components." Non-pharmaceutical formulations preferably include food additives, nutritional pharmaceutical preparations, and food, beverage, and beverage additives. Formulations suitable for addition to food and beverages are preferably in liquid or powder form. Nutritional pharmaceutical preparations are preferably in tablet, capsule, or oral edible liquid form.

[0532] As mentioned above in the section entitled "Use in Therapeutic or Surgical Procedures," the medical and / or surgical uses disclosed herein may provide indirect benefits to patients in terms of increased well-being or confidence.

[0533] Plant Uses

[0534] 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 the gases emitted therefrom to provide beneficial effects on living or dead plants.

[0535] Examples of known uses of nitric oxide and systems that produce nitric oxide in plants include the following:

[0536] Nitric oxide prevents or delays wilting in cut flowers and plants (see Siegel-Itzkovich, BMJ, 1999; 319(7205), p. 274; see also 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 ));

[0537] Nitric oxide regulates plant-pathogen interactions, promotes plant hypersensitive responses, commensalism with organisms in nitrogen-fixing root nodules, development of lateral and adventitious roots and root hairs, and control of stomatal opening (see Mur et al., 2013; cited above).

[0538] The role of nitric oxide in antioxidant and reactive oxygen species responses in plants (see Verma et al., 2013; "Nitric oxide (NO) counteracts cadmium-induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses"; BioMetals).

[0539] The role of nitric oxide in the signal transduction pathways of auxin, cytokinin and other plant hormones (see Liu et al., Proceedings of the National Academy of Sciences, 2013; 110(4), pp. 1548-1553).

[0540] The public content of each of the publications cited above is incorporated herein by reference.

[0541] Furthermore, the nitric oxide generating system and the antimicrobial effect of the gas emitted therefrom, as specifically described but not limited to the above sections entitled “Use in Therapeutic or Surgical Applications,” “Topical Antimicrobial Uses,” “Use in the Nose, Oral, Respiratory Tract, and Lungs,” and “Target of Antimicrobial Uses,” are equally applicable to microbial infections of targeted plants, and this disclosure extends to such uses.

[0542] The above-mentioned known uses and all other uses of nitric oxide and systems for producing nitric oxide in plants constitute other aspects of this disclosure when used in conjunction with reactions that produce nitric oxide using this disclosure and / or the resulting nitric oxide, optional other nitrogen oxides and / or optional precursors thereof.

[0543] Specifically, the plants being treated can be crops or houseplants, i.e., plant species cultivated by humans.

[0544] Crops include (but are not limited to) food crops (such as cereals, vegetables and fruits), pharmaceutical active ingredient crops (such as quinine), fiber crops (such as cotton or flax), other material crops (such as rubber and wood), and floriculture crops (such as roses and tulips).

[0545] Other examples of crops consumed for human food include (but are not limited to) crops that produce the following: rice, wheat, sugarcane and other sugar crops, corn, soybean oil, potatoes, palm oil, cassava, legume seeds, sunflower 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, red peppers, black peppers, tea, cucumbers, oranges, walnuts, almonds, carrots, radishes, coconuts, citrus fruits, lemons, limes, strawberries, and hazelnuts. Attached Figure Description

[0546] Figure 1 The cumulative curves of nitric oxide (nmol NO / mg nitrite) released under different reaction conditions in Example 1 over time are shown.

[0547] Figures 2 to 16 The results of several tests described in Example 2 are shown.

[0548] Figure 17 A schematic diagram of the equipment used for measuring SIFT-MS is shown.

[0549] Figures 18 to 21 Results of multiple tests on the antimicrobial activity against Mycobacterium abscessus described in Example 3 for combinations of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions are shown.

[0550] Figure 22 The results of tests conducted in Example 4 regarding the minimum inhibition concentration (MIC) of solutions containing citric acid, sodium nitrite, and mannitol against a variety of clinical isolates are shown.

[0551] Figure 23 The results of the tests on the antimicrobial activity of carboxylic acid-nitrite solutions with and without polyols against Pseudomonas aeruginosa, as described in Example 5, are shown.

[0552] Figures 24 to 27 The results of the test for antimicrobial activity against Mycobacterium tuberculosis HN 878 in THP-1 cells, as described in Example 6, are shown.

[0553] Figure 28Results of the tests for cytotoxicity (LDH cytotoxicity assay) and antimicrobial activity against H1N1 influenza A virus in MDCK cells as described in Example 7 are shown: (a) at a series of dilutions (horizontal axis is nitrite molar concentration) at MOI = 0.002 (●) and MOI = 0.02 (■), where cytotoxicity is shown in gray and the cytotoxicity scale is on the right (cytotoxicity at measured nitrite concentrations of up to and including 0.015 M ≤ 1% of LDH control); and (b) plate photographs at MOI = 0.002 and nitrite concentrations of 0.15 M, 0.015 M, and 0.0015 M, compared with oseltamivir (1 μM). The order of the plates described in the previous sentence 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 one on top of the other). The rightmost plate pair, immediately to the right of the oseltamivir plate pair, is the viral control. Cytotoxicity is shown below each test plate pair as the % LDH control (average of three LDH measurements at 24 hours post-infection).

[0554] Figure 29 The results show the effectiveness of sodium nitrite, acidified solution of citric acid and mannitol buffered to pH 5.8, in killing Mycobacterium abscesses compared to amikacin and a negative control (described in Example 3) under similar conditions.

[0555] Figure 30 and 31 In the form of a diagram ( Figure 30 The following illustrates an embodiment of the invention for treating lung infection in human subjects, as described in Example 10, and ( Figure 31 ) and inhaled nitric oxide ( Figure 31 Compared to the left side, the contact point between the liquid formulation that generates NO according to the present invention and the lung tissue ( Figure 31 (The view on the right side).

[0556] Figure 32 Results of the LDH cytotoxicity assay in Example 8 (Procedures 1 and 2) are shown. Data can be expressed as the mean plus standard deviation (SD) of the two experiments. SD is shown as gray error bars. Maximum LDH activity (cells + lysis buffer) was set at 100%, and all example results are relative to this value. The LDH positive control is the positive control from the kit. The black bar (incubated for 2 hours) is the left bar of each pair of bars in each case, and the red bar (incubated for 24 hours) is the right bar of each pair of bars in each case.

[0557] Figure 33Results of the antiviral assay against SARS-CoV-2 in Example 8 (Operation 1) at MOI 3.0 are shown. In Operation 1, a viral yield reduction assay was performed using SARS-CoV-2 at four multiples of infection (MOIs), confirmed by reverse titration of the inoculated virus. For cells inoculated at MOI 3, 2.1 log10 TCID50 / ml was found in the virus control wells after titration. A reduction in SARS-CoV-2 yield was observed for some of the conditions tested. After 24 hours of incubation, almost no virus was detected at the three lowest MOIs (i.e., 0.3, 0.03, and 0.003). It is possible that 24 hours of replication on VeroE6 cells was insufficient to obtain high levels of progeny virus. Data are presented as mean + standard deviation (SD) of two titrations. SD is shown as error bars. The horizontal dotted line level with the log 10 TCID50 / ml values ​​for chloroquine and cell controls represents the limit of detection (LOD) of the assay.

[0558] Figure 34 shows the results of the antiviral test against SARS-CoV-2 (Operation 2) of Example 8 at (a) MOI 3.0 and (b) MOI 0.3. This method corresponds to a portion of Operation 1 at those MOIs, with the exception that the formulation is the Operation 2 formulation, incubated for 48 hours instead of 24 hours to increase progeny virus levels. Data are presented as the mean + standard deviation (SD) of two titrations. SD is shown as error bars. The dotted line level with the log 10 TCID50 / ml value of chloroquine and cell controls represents the limit of detection (LOD).

[0559] Figure 35 Results of the antiviral test against SARS-CoV in Example 9 at MOI 3.0 are shown. Two plates were examined under a microscope and scored for cytopathic effect (CPE) before crystal violet staining of the cell monolayer. CPE was found in these plates as cell debris on top of the basal monolayer. Results of the two plates examined under a microscope are shown. Data are single titrations for each condition. For the remaining plates, CPE could not be scored after crystal violet staining due to overly dense cell monolayers. The horizontal dotted line level with the log10 TCID50 / ml value of the cell control represents the limit of detection (LOD).

[0560] Example

[0561] The following non-limiting embodiments are provided to further illustrate the invention.

[0562] Materials, apparatus and methods used in Examples 1 and 2

[0563] solution

[0564] Stock solutions of 0.1M and 1M citric acid (Health Supplies Limited, Thornton Heath, UK), 0.1M sodium citrate (Fisher Scientific, Loughborough, UK), 1M sodium nitrite (Sigma Aldrich, Dorset, UK), 0.5M and 1M sorbitol (Special Ingredients, Chesterfield, UK), 0.5M and 1M D-mannitol (Sigma Aldrich, Dorset, UK), 3M sodium hydroxide (Fisher Scientific, Loughborough, UK), and 0.1M and 1M L-ascorbic acid (ICN Biomedicals, Ohio, US) were prepared by dissolving appropriate masses of these substances in deionized water. Deionized water (18.2 MΩ) was obtained from the Arium Mini laboratory water system (Sartorius, Germany).

[0565] Citric acid / citrate buffer solution was prepared by two methods:

[0566] 1. Titrate the stock solutions of 0.1 M citric acid and 0.1 M sodium citrate using the volumetric titration method described in Sigma Aldrich 2018 (https: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html);

[0567] 2. Dissolve a known mass of citric acid for the 0.1M or 1M formulation in a small volume of deionized water, and then titrate a stock solution of 3M sodium hydroxide and deionized water to achieve the desired buffer solution pH (pH 3 to pH 6.2).

[0568] For Method 1, ascorbic acid and sodium ascorbate are used instead of citric acid and sodium citrate in Method 1 to prepare an ascorbic acid / ascorbate buffer solution in a similar manner.

[0569] The polyol is included by dissolving a known mass of sodium nitrite in a stock solution of the polyol (e.g., sorbitol or mannitol).

[0570] The order in which the components of the buffer solution and stock solution are added is not critical; any mixing order can be used.

[0571] All standard solutions were used within 48 hours of preparation. Calibration buffer solutions were prepared using phthalate (pH 4) and phosphate (pH 7) tablets dissolved in deionized water (Fisher Scientific UK Ltd, Leicestershire, UK).

[0572] Selected ion flow tube mass spectrometry (SIFT) MS) Startup and Verification

[0573] All gas analyses described in this report were performed using a Voice200 Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) (Syft Technologies Ltd, New Zealand). This instrument uses helium (BOC, Surrey, UK) as the carrier gas.

[0574] Before analysis, the SIFT-MS is prepared for use using a simple startup procedure. The instrument is then switched out of standby mode, and a series of pressure checks are performed to ensure that capillary flow is within acceptable operating ranges. An automated validation procedure is then performed using calibration gas standards from the manufacturer (Syft Technologies Ltd., New Zealand) containing benzene, toluene, ethylbenzene, and xylene. Finally, internal performance checks are performed using a 10 ppm nitrogen dioxide standard (Air Products PLC, Surrey, UK).

[0575] NO generation procedure

[0576] SIFT-MS equipment, reaction chamber, and gas pathways, such as Figure 17 As shown in the diagram, it was established.

[0577] The temperature in the reaction chamber was continuously monitored using an HT1 smart temperature sensor (SensorPush, New York, US). The reaction chamber was a 670 mL plastic (BPA-free) clamp-on container with a silicone seal (Tesco, Welwyn Garden City, UK), connected to a pump that continuously circulated humidified air through the reaction chamber and via the SIFT-MS inlet capillary. Humidification was performed by pumping air through two Dreschel bottles containing deionized water, similar to the method described below: Vernon, W. and Whitby, L. (1931) The quantitative humidification of air in laboratory experiments, Trans. Faraday Soc. 27, 248-255. The system was equilibrated for 30 minutes before use. Continuous SIFT-MS scans were initiated to detect and quantify NO, NO2, and HONO in real time. Once stable baseline readings (consistent concentrations >2 min) were observed for these compounds, the sample was placed in the reaction chamber and monitored for three hours.

[0578] After SIFT-MS validation, the extended capillary inlet, heated to 120°C, was connected to the outlet of the reaction chamber via a T-connector, allowing SIFT-MS to sample the gas flowing out of the reaction chamber in real time.

[0579] Samples were prepared by weighing approximately 0.3 cm × 0.3 cm carded nonwoven 20 g / m² (20 g sm) polypropylene sieve from RKW-Group (Frankenthal, Germany) into a weighing boat (approximately 3 mg). A 10 μL droplet of test or control solution was added to the center of the sieve (ensuring the droplet saturates the sieve). Finally, the sieve loaded in the weighing boat was placed in the reaction chamber, and a final 10 μL droplet of buffer solution was aspirated onto the center of the sieve. The reaction chamber was rapidly sealed, and the generation of nitrogen-containing species was instantaneously observed on the SIFT-MS interface.

[0580] Analysis of the generated gases

[0581] The generated gases were analyzed using SIFT-MS in selected ion mode, with each batch scanned sequentially for 1000 seconds. The following product masses were repeated: nitrite 30 m / z, nitrite 48 m / z, nitrogen dioxide 46 m / z, and nitric oxide 30 m / z. These measurements were performed using all three positive precursor ions: hydrated hydrogen ions (H₃O₂). + ), nitrosamines (NO) + ) and dioxygen ions (O2) +Air flowed through the reaction chamber at 660 ml / min, and the airflow was sampled at the SIFT-MS inlet at a flow rate of 2.7 ml / min.

[0582] pH measurement in all embodiments

[0583] All pH measurements were taken using a Five Easy pH meter (Mettler Toledo, Switzerland) with a glass electrode LE438 probe. The accuracy of this electrode was ensured using a second pH meter: a portable 205 probe (Testo, Alton, US). Fresh calibration buffer was used to calibrate the pH meter daily.

[0584] Example 1

[0585] Using 1M / c.pH3 citric acid, contact was made with a sieve containing inhaled 1M sodium nitrite in both the presence and absence of 1M polyol. Nitric oxide is produced

[0586] The SIFT-MS equipment, reaction chamber, and gas passage are as described above. Figure 17 As shown in the diagram, it was established.

[0587] Two test solutions, each containing 1M mannitol and 1M sorbitol respectively, were drawn into the sieves as described above to create two test sieves.

[0588] A control solution of 1M sodium nitrite without polyols was aspirated into the sieve as described above to prepare a control sieve.

[0589] In each test, a 1M citrate / citric acid buffer solution prepared by either of the two methods 1 and 2 described above, with a pH of approximately 3, is added to each of the test and control sieves to induce gas generation as described above.

[0590] Figure 1 The results are shown in the figure.

[0591] Data show that, compared to the absence of polyols, when the sieve also contains 1M mannitol or 1M sorbitol (mannitol has a greater effect than sorbitol), the sieve that inhales 1M sodium nitrite in contact with 1M / c.pH 3 citric acid produces significantly more nitric oxide.

[0592] Example 2

[0593] This study investigated the effects of different carboxylic acids, acid concentrations, pH values, and polyols on nitric oxide production.

[0594] The changes in organic acid, pH value, and polyol in the samples prepared as described above are as follows:

[0595]

[0596]

[0597]

[0598] The SIFT-MS equipment, reaction chamber, and gas passage are as described above. Figure 17 As shown in the diagram, it was established.

[0599] The test solution, as described above, is drawn into the sieve to create the test sieve.

[0600] When in use, a control solution of 1M sodium nitrite without polyols is drawn into the sieve as described above to create a control sieve.

[0601] In each test, a buffer solution prepared by either of the two methods 1 and 2 described above and having the pH value described above, or each buffer solution, is added to the test and control sieves (if used) to induce gas generation as described above.

[0602] Figures 2 to 13 The results are shown in the figure. "Normal" in the figure means that polyols are not present.

[0603] Figure 2 The NO release rates were compared between citrate / citrate buffer and ascorbic acid / ascorbate buffer (pH approximately 3) in the absence of polyols. The graph clearly shows that citrate / citrate buffer exhibits a higher initial burst release and a longer duration and higher level of gas release compared to ascorbic acid / ascorbate buffer. The citrate / citrate buffer trace peaks at approximately 55,000 ppb, while the ascorbic acid / ascorbate buffer trace peaks at approximately 28,000 ppb.

[0604] Figure 3 This study involved citrate / citrate buffer and nitrite systems with and without polyols. The polyol concentration was 1 M. The gas release rates, initial burst release, and post-release varied over time in the presence of polyols compared to systems without polyols. Xylitol and mannitol produced the highest peak values, followed by sorbitol, then polyol-free systems, and finally araitol. In the 500–1000 s region, xylitol and araitol had the highest output, followed by mannitol, sorbitol, and then polyol-free systems. Peak burst release of mannitol = xylitol (approx. 64,000 ppb) > sorbitol (approx. 53,000 ppb) > polyol-free systems (approx. 50,000 ppb) > araitol (approx. 40,000 ppb).

[0605] Figure 4This involves ascorbic acid / ascorbate buffer and nitrite systems with and without polyols. The polyol concentration is 1M. The peak burst release order is: mannitol (approx. 40,000 ppb) > araitol (approx. 35,000 ppb) > xylitol = no polyol (approx. 30,000 ppb) > sorbitol (approx. 23,000 ppb), i.e., with... Figure 3 Different sequences of citric acid / citrate buffer systems.

[0606] Figure 5 This involves citrate / citrate buffer and nitrite systems with and without polyols (the "no polyol" line, with a peak burst nearly identical to the mannitol line, has been omitted for clarity). The polyol concentration is 0.5M. Peak burst release of aritol (approx. 76000 ppb) >> no polyol = mannitol (approx. 48000 ppb) > xylitol = sorbitol (approx. 40000 ppb). It will be seen that this is similar to a 1M polyol citrate / citrate buffer system (…). Figure 3 The different orders indicate that the effect of polyols depends on the concentration of polyols.

[0607] Figure 6 The study involved ascorbic acid / ascorbate buffer and nitrite systems with and without polyols (the "no polyol" line, with a peak burst nearly identical to the sorbitol line, has been omitted for clarity). The polyol concentration was 0.5 M. Peak burst releases were: xylitol (approx. 50,000 ppb) > mannitol (approx. 38,000 ppb) > sorbitol = no polyol (approx. 30,000 ppb) > araitol (approx. 23,000 ppb). Again, similar results were observed with citrate / citrate buffer (0.5 M polyol) and ascorbic acid / ascorbate (1 M polyol) systems (which were respectively...). Figure 5 and 4 The order of these factors differs. Therefore, it is confirmed that the effect of polyols depends on the polyol's chemical / stereochemistry and its molar concentration.

[0608] Figure 7 and 8 Compare the NO release rates in citrate / citrate buffer or ascorbic acid / ascorbate buffer and in the presence of polyols (0.5 M). These figures highlight the NO release rates in... Figures 2 to 6 Some differences were observed. Figure 7 The citrate / citrate buffer trace peaked at approximately 76,000 ppb, while the ascorbic acid / ascorbate buffer trace peaked at approximately 22,000 ppb. Figure 8 The citrate / citrate buffer trace peaked at approximately 48,000 ppb, while the ascorbic acid / ascorbate buffer trace peaked at approximately 38,000 ppb.

[0609] Figure 9 Compare the cumulative output of 1M polyol concentrations. For ascorbic acid / ascorbate buffer, the difference is small at, for example, 3000 s, with the order being mannitol > sorbitol = araitol > xylitol. For citrate / citrate buffer, at 3000 s, the order is xylitol > araitol > mannitol > sorbitol > no polyol. Data show that, for example, between no polyol (curve E, after 3000 s, a cumulative nitric oxide release of approximately 10,000 nmol per mg nitrite is obtained, and it is still increasing at this point) and xylitol (curve A, a cumulative nitric oxide release of approximately 20,000 nmol per mg nitrite is obtained, and it is still increasing), nitric oxide output can increase by up to approximately 100%, or even more.

[0610] Figure 10 Compare the cumulative output of 0.5M polyol concentrations. For citrate / citrate buffer, at 3000s, the order is araitol > mannitol = xylitol > sorbitol > no polyol (the "no polyol" line for citrate / citrate buffer is below the sorbitol line and has been omitted for clarity). For ascorbic acid / ascorbate buffer, at 3000s, the order is xylitol > mannitol > sorbitol > araitol. Again, this order is different compared to 1M polyols. Figure 9 ).

[0611] Figures 11 to 13 The cumulative curves of polyols with and without mannitol (0.5M) and with citric acid / citrate buffer 1M and sodium nitrite (1M) at different pH values ​​were compared. The differences became smaller as pH increased, disappearing at pH 6.2. Therefore, these experiments show that the effect of polyols also depends on pH.

[0612] Figure 14 The cumulative NO (nmol / cm³) in citrate / citrate buffer (1M, pH approximately 2) with and without glycerol (1M and 2M) in 1M sodium nitrite solution is shown. 2 (Sieve area) output. Within the first 2000 seconds, the NO output of 1M and 2M glycerol was slightly lower than when no polyol was present. Over a longer period, formulations containing glycerol showed a greater output, with 2M glycerol showing a significantly higher output.

[0613] Figure 15 The cumulative NO (nmol / cm³) of citric acid / citrate buffer (1M, pH approximately 2) and 1M sodium nitrite solution with or without polyols is shown. 2(Screen area) output. The graph shows that the presence of glycerol in the mannitol / nitrite solution reduces the output compared to the absence of glycerol. However, unexpectedly, unlike the case with mannitol, the output compared to the absence of glycerol... Sorbitol Nitrite solution contains glycerol Enhance No output.

[0614] When using glycerol, first prepare a 1M glycerol solution and use it to prepare a 1M sorbitol or 1M mannitol solution, and then use it to prepare a 1M nitrite solution.

[0615] Figure 16 The cumulative NO output (mol / mg nitrite) in citrate / citrate buffer (1M, pH 5.8) with and without mannitol (0.5M) in sodium nitrite (1M) solution is shown. The graphs indicate that the inclusion of polyols after approximately 2000 s of reaction time results in a greater NO output.

[0616] Figure 16 The results showed that at physiologically important pH levels above approximately 5, and especially above approximately 5.5, mannitol enhanced nitric oxide production compared to the same system without mannitol, providing a cumulative level of 1400 nmol NO per milligram of nitrite after 10,000 s (167 min).

[0617] Example 3

[0618] The effects on Mycobacterium abscessus cultures in the presence and absence of polyols under various organic acid and nitrite solutions. active

[0619] Material

[0620] Reconstitute 4.7g of Middlebrook 7H9 broth base (Sigma-Aldrich) with 900ml of distilled water and autoclave at 121°C for 15 minutes. Add Middlebrook ADC growth supplement (Sigma-Aldrich) to the autoclaved 7H9 solution (50ml per 450ml, for a total of 100ml).

[0621] 1M Sodium Nitrite (Emsure): Dissolve 6.9g of sodium nitrite powder in 100ml of distilled water in a clean screw-cap glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0622] 1M Citric Acid (Sigma-Aldrich): Dissolve 19.2g of citric acid powder in 100ml of distilled water in a clean screw-cap glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0623] 1M Ascorbic Acid (Sigma-Aldrich): Add 17.6g of ascorbic acid powder to a sterile glass vial. Dissolve completely in 100ml of sterile distilled water. Due to its short half-life, it is prepared daily using strict aseptic techniques. Due to its inherent instability, it is not autoclaved but filtered through a 0.2μ filter before use.

[0624] 1M Trisodium Citrate Dihydrate (Sigma-Aldrich): Dissolve 29.4 g of sodium citrate powder in 100 ml of distilled water in a clean screw-cap glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0625] 1M L-Sodium Ascorbate (Acros Organics): Dissolve 19.8g of sodium ascorbate powder in 100ml of distilled water in a clean screw-cap glass bottle. Autoclave the mixture at 121°C for 15 minutes.

[0626] For experiments utilizing polyols, D-mannitol (Sigma-Aldrich) was used. The polyol was added to the above sodium nitrite stock solution to form the following stock solution:

[0627] Stock solution A - 1M sodium nitrite and 0.5M mannitol

[0628] Stock solution B - 1.5M sodium nitrite and 0.5M mannitol

[0629] A stock solution of 1.5M citric acid was also prepared.

[0630] Adjust the molar concentration of each component according to the dilution factor to ensure the correct final molar concentration for each experimental solution.

[0631] Mycobacterium abscessus (MAB)

[0632] The laboratory reference strain Mycobacterium abscessus ATCC 19977 lux was used for all experimental conditions in this example.

[0633] method

[0634] Label the 50ml Falcon tubes as tube T (test suspension), tube A (acid control), and tube C (control).

[0635] Add 8 ml of 7H9+ADC supplement to each tube. Then add 100 μl of MAB suspension (previously grown to approximately 3–4 times the McFarland standard). Read the baseline relative optical units (RLU) of the MAB suspension. Mix the inclusions by vortexing.

[0636] The contents of the tube when polyols (mannitol) are absent

[0637] Pipe T Add 1 ml of sodium nitrite (1M) solution to the tube, followed by 1 ml of citric acid solution (1M) or ascorbic acid solution (1M) to obtain a final concentration of 0.1M in 10 ml. Mix the contents by slowly inverting the tube and incubate at 37°C for 24 hours.

[0638] Pipe A Add 1 ml of citric acid solution (1M) or ascorbic acid solution (1M) to the tube, and add 1 ml of sterile distilled water to produce a final volume of 10 ml. Test the 0.1M concentration acid. Mix the contents by slowly inverting the tube and incubate at 37°C for 24 hours.

[0639] Pipe C Add 2 ml of sterile distilled water to the tube to make a total volume of 10 ml. This is a control to evaluate growth under optimal conditions. Mix the contents by slowly inverting the tube and incubate at 37°C for 24 hours.

[0640] The contents of tube T when polyols (mannitol) are present

[0641] The contents of tube T in the presence of mannitol are as follows:

[0642] 1. Tube T: 1 ml sodium nitrite (1M), mannitol (0.5M), and 1 ml citric acid (1M)

[0643] 2. Tube T: 1 ml sodium nitrite (1.5 M), mannitol (0.5 M), and 1 ml citric acid (1 M)

[0644] 3. Tube T: 1 ml sodium nitrite (1M) and mannitol (0.5M) and 1 ml citric acid (1.5M)

[0645] RLU was measured at 30 minutes, 60 minutes, and 24 hours of incubation to assess the activity of T, A, and C solutions.

[0646] After 24 hours of incubation, tubes C, A, and T were plated onto Columbia blood agar (VWR Chemicals). The plates were incubated at 37°C for 72 hours. Colony forming units (CFU) were read on days 3, 5, and 7 of incubation. All work was performed in a CL2 biosafety cabinet within the CL2 laboratory facility.

[0647] Figures 18 to 21 The results are shown in the figure.

[0648] Figure 18The results showed that after 7 days, solutions of 0.1 M citric acid and 0.1 M nitrite (tube T) at pH 5 and 5.5 effectively eliminated Mycobacterium abscessus cultures and reduced Mycobacterium abscessus cultures compared to solutions of only 0.1 M citric acid at pH 6.0, 6.5, 7.0, and 7.4 (tube A). Figure 18 The study also showed that after 7 days, solutions of 0.1 M ascorbic acid and 0.1 M nitrite (tube T) at pH 5.0, 5.5, and 6.0 effectively eliminated Mycobacterium abscessus cultures and reduced Mycobacterium abscessus cultures compared to solutions of ascorbic acid alone at pH 6.5, 7.0, and 7.4 (tube A).

[0649] Figure 19 a) It was shown that after three days of incubation, a solution of 0.1 M citric acid and 0.1 M nitrite effectively reduced the CFU of Mycobacterium abscessus cultures, and after three days of incubation, a solution of 0.1 M citric acid and 0.1 M nitrite with 0.05 M mannitol effectively almost completely eliminated Mycobacterium abscessus cultures. Figure 19 b) shows that after five days of incubation, a solution containing 0.1 M citric acid and 0.1 M nitrite without mannitol effectively maintained the reduced CFU of Mycobacterium abscessus. The figure also shows that after five days of incubation, a solution containing 0.05 M mannitol, 0.1 M citric acid, and 0.1 M nitrite effectively reduced the CFU of Mycobacterium abscessus cultures.

[0650] Figure 20 a) It was shown that after three days of incubation, a solution of 0.15 M citric acid and 0.1 M nitrite effectively reduced the CFU of Mycobacterium abscessus cultures, and after three days of incubation, a solution of 0.15 M citric acid and 0.1 M nitrite with 0.05 M mannitol effectively eliminated Mycobacterium abscessus cultures. Figure 20 b) shows that after five days of incubation, a solution containing 0.15 M citric acid and 0.1 M nitrite without mannitol effectively maintained the reduced CFU of Mycobacterium abscessus. The figure also shows that after five days of incubation, a solution containing 0.05 M mannitol, 0.15 M citric acid, and 0.1 M nitrite effectively eliminated Mycobacterium abscessus cultures.

[0651] Figure 21 The results showed that a solution of 0.1M citric acid and 0.15M nitrite effectively reduced the CFU of Mycobacterium abscessus cultures after three days of incubation, and maintained this reduction after five days of incubation. The figure also showed that a solution of 0.1M citric acid and 0.15M nitrite containing 0.05M mannitol effectively eliminated Mycobacterium abscessus cultures after three and five days of incubation.

[0652] Example 4

[0653] A series of clinical isolates and cultures were tested using a carboxylic acid-nitrite-polyol solution against Mycobacterium abscessus (Mab). Minimum inhibitory concentration (MIC) for Mycobacterium tuberculosis (Mtb)

[0654] health volunteers

[0655] Peripheral blood samples were obtained from healthy volunteers, who provided written informed consent (ethical approval reference REC No. 12 / WA / 0148).

[0656] Mycobacterium strains

[0657] Both Mycobacterium abscessus (ATCC 19977) and Mycobacterium tuberculosis (H37RV) strains contain a bacterial luciferase (lux) gene cassette (luxCDABE), which can measure relative light units (RLU) and routine colony-forming units (CFU) for bacterial viability.

[0658] General reagents

[0659]

[0660]

[0661] Processing conditions

[0662] Treatment 1: Citric acid 0.15M, sodium nitrite 0.1M, and mannitol 0.05M

[0663] Treatment 2: Citric acid 0.1M, sodium nitrite 0.15M, and mannitol 0.05M

[0664] Minimum inhibitory concentration (MIC) of broth after micro-dilution

[0665] The MICs for each treatment against Mycobacterium abscessus and Mycobacterium tuberculosis were determined according to the Clinical and Laboratory Standards Institute (CSIS) guidelines for antimicrobial susceptibility testing (M07-A9). Each treatment was diluted twofold on plates and incubated at 37°C. Readings for Mab were performed on days 3 and 7, and for Mtb on days 14 and 21. Tests were performed in duplicate.

[0666] All work is carried out in CL2 biosafety cabinets within the CL2 laboratory facility.

[0667] The minimum inhibitory concentration (MIC) of a solution of 1.5M citric acid, 1M sodium nitrite, and 0.5M mannitol against Mycobacterium abscessus was found to be 4.7 mM. The MIC of the same solution against Mycobacterium tuberculosis was also found to be 2.3 mM.

[0668] The minimum inhibitory concentration (MIC) of a solution of 1M citric acid, 1.5M sodium nitrite, and 0.5M mannitol against Mycobacterium abscessus was found to be 3.1 mM. The MIC of the same solution against Mycobacterium tuberculosis was also found to be 1.6 mM.

[0669] The samples were also microdiluted with broth and analyzed using samples numbered 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, and 9... 32, 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 The minimum inhibitory concentration (MIC) was determined for isolates 1049, 1054, 1063, 1066, 1067, 1070, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1082, 1086, 1094, 1096, 1101, 1103, 1104, and 1106. Each individual isolate was evaluated in duplicate.

[0670] Figure 22 Results of tests on clinical isolates are shown in a) and b). The figures show the MICs of nitric oxide against Mycobacterium abscessus reads in duplicate after three, four, and five days of incubation. Plates were also read on day 7 of incubation, but no difference was observed compared to day 5. The laboratory strain ATCC 19977lux was used as a control in both experiments, and results are shown compared to the clinical isolates.

[0671] Figure 22The results showed that citric acid-nitrite-mannitol solutions had an effect on a wide range of clinical isolates. The minimum inhibitory concentrations (MICs) of 0.1 M citric acid, 0.15 M nitrite, and 0.05 M mannitol solutions were within 0.02 M for most clinical isolates. Figure 22 a) and the minimum inhibitory concentrations (MICs) of 0.15 M citric acid, 0.1 M nitrite, and 0.05 M mannitol solutions against most clinical isolates were within 0.04 M. Figure 22 b).

[0672] In both figures, the MIC differed for some samples at different incubation days. Those samples are those that show more than one dot above the isolate sample identification code. Generally, in that case, a higher MIC was observed at later incubation days compared to a lower MIC. Overall, the combination of lower citric acid (0.1M) and higher sodium nitrite (0.15M)... Figure 22 (a) Compared to the combination of higher citric acid (0.15M) and lower sodium nitrite (0.1M) Figure 22 (b)) More effective.

[0673] Figure 29 Further data show that a carboxylic acid-nitrite-polyol solution kills Mycobacterium abscesses in vitro. In this figure, the effectiveness of an aqueous formulation of sodium nitrite, citric acid buffered to pH 5.8 with sodium hydroxide solution, and mannitol in killing Mycobacterium abscesses was confirmed compared to amikacin and a negative control over a 24-hour period under similar conditions.

[0674] Example 5

[0675] Antimicrobial activity against Pseudomonas aeruginosa with and without carboxylic acid-nitrite solutions containing polyols

[0676] Equipment and media

[0677] UKAS calibrated pipettes (100-1000μL range) - Plus

[0678] UKAS calibrated multi-channel pipettes (P300 and P20) - UK

[0679] Universal Pipe - SLS, UK

[0680] Calibration Balance - HR-100A

[0681] Heratherm Microbial Incubator TM ThermoFisher Scientific, UK

[0682] Tryptone Soy Agar (TSA) - Southern Group Laboratories, UK

[0683] Tryptone Soy Broth (TSB) SLS, UK

[0684] Malt agar- SLS, UK

[0685] Brain and Heart Infusion Broth (BHIB) SLS, UK

[0686] Sabouraud Dextrose Broth (SDB) SLS, UK Dey-Engley Neutralizer (DE-N) SLS, UK

[0687] Citric acid - Sigma, UK

[0688] Sodium nitrite - Sigma, UK; Mannitol - Sigma, UK

[0689] Sorbitol - Sigma, UK

[0690] Test microorganisms

[0691] Pseudomonas aeruginosa NCTC 13618 - isolated from patients with cystic fibrosis

[0692] preparation

[0693]

[0694]

[0695] 1-1M citric acid plus 1M sodium nitrite (with or without 0.5M polyol)

[0696] 2-0.5M citric acid plus 1M sodium nitrite (with or without 0.5M polyol)

[0697] 3-0.5M citric acid plus 0.5M sodium nitrite (with or without 0.5M polyol)

[0698] Dey-Engley Neutralizer Validation

[0699] A 24-hour culture of Pseudomonas aeruginosa was harvested from tryptone soybean agar (TSA) and used to prepare 1×10⁸±5×10⁷ CFU / mL. -1 The suspension was further diluted in brain heart infusion broth (BHIB) to prepare 1×10⁵±5×10⁴ CFU / mL.-1 Working suspension.

[0700] The starting inoculum was validated through serial dilution and plating. Neutralizer validation was performed using control (9 mL phosphate-buffered saline (PBS) and 1 mL inoculum), toxicity (9 mL Dey-Engley neutralizer (DE-N) and 1 mL inoculum), and neutralizer efficacy (8 mL neutralizer, 1 mL test agent, and 1 mL inoculum) samples. After 5 minutes of treatment, 200 μL of suspension was removed from each tube, serially diluted, and 100 μL was plated onto a TSA. The agar plates were incubated at 37 ± 2 °C for 18–24 hours.

[0701] Antimicrobial efficacy against plankton

[0702] A 24-hour culture of Pseudomonas aeruginosa was harvested from the TSA and used to prepare 1×10 8 ±5×10⁷ CFU / mL -1 The suspension was further diluted in BHIB to prepare 1×10⁻⁶ ppm. 6 ±5×10 4 CFUmL -1 Working suspension. A general-purpose tube was filled with 8 mL of bacterial solution.

[0703] Add 1 mL of citric acid solution and 1 mL of sodium nitrite solution to each test reagent to obtain the desired concentration as described above. Incubate the solution at 37 ± 2 °C for 24 hours. After the incubation period, remove 1 mL from each tube and transfer it to a tube containing 9 mL of neutralizing agent. Quantify the number of viable organisms using serial dilutions and plate counts.

[0704] Figure 23 The results are shown in the figure.

[0705] The data shows the following antimicrobial efficacy against Pseudomonas spp.:

[0706] - Citric acid (1M) mixed with nitrite (1M), with and without polyol (0.5M) ("Concentration 1");

[0707] - Citric acid (0.5M) mixed with nitrite (1M), with and without polyol (0.5M) (“Concentration 2”); and

[0708] - Citric acid (1M) mixed with nitrite (0.5M), with and without polyol (0.5M) ("Concentration 3").

[0709] Citric acid solutions were prepared at pH 5.2 (formulations 1, 3, and 5) and 6.0 (formulations 2, 4, and 6). Formulations 1 and 2 do not contain polyols; formulations 3 and 4 include mannitol; and formulations 5 and 6 contain sorbitol.

[0710] All formulations showed good efficacy at pH 5.2. At pH 6, the formulation containing mannitol was slightly more effective.

[0711] Example 6

[0712] The efficacy of a formulation containing nitrite organic acids and polyols against Mycobacterium tuberculosis HN878 in THP-1 cells was evaluated.

[0713] preparation

[0714] The formulations are prepared as described in the table below. When the preparation method is described as “concentrated” and indicated by the suffix FC in the sample reference, this means that the formulation was initially prepared as a concentrated premix containing all three components: sodium nitrite (0.75M), polyol (0.25M), and acid (0.5M), and then diluted with distilled water to achieve the desired concentrations of each as described in the table. When the preparation method is described as “diluted” and indicated by the suffix FD in the sample reference, this means that the formulation was initially prepared as a premix containing all three components at the initially desired concentrations, namely sodium nitrite (0.15M), polyol (0.05M), and acid (0.1M), and then diluted with distilled water to achieve the desired concentrations of each as described in the table.

[0715] Within each formulation, a series of concentrations of sodium nitrite were prepared by sequential dilution, namely 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml and 0.125 μg / ml, for in vitro antibacterial assays against Mycobacterium tuberculosis HN878.

[0716]

[0717]

[0718] MIC macrophage assay was performed using the THP-1 macrophage (1) compound screening assay.

[0719] Macrophage preparation and culture: THP-1 cells were expanded for 2 weeks. Afterward, THP-1 cells were suspended in complete DMEM medium for macrophages at a concentration of 5 × 10⁻⁶. 5 Cells / mL. Seed cells into 24-well tissue culture plates at 2 mL per well (1 × 10⁶ cells / mL). 6A 24-well cell plate allows for triplicate testing of a series of seven drug concentrations plus an untreated control. In addition to the drug assay plate, an extra plate (or at least three additional wells) is inoculated to measure bacterial uptake on the day of infection. Cells are incubated in a humidified chamber at 37°C with 5% CO2. The DMEM antibiotic-free / antifungal complete medium remained unchanged during the 3-day assay period.

[0720] Complete DMEM medium for macrophages:

[0721] Dalberke modified Eagle medium (Cellgro 15-017-cv), supplemented with: heat-inactivated fetal bovine serum (AtlasBiologicals, Fort Collins, CO, F-0500-A) (10%).

[0722] L929 conditioned medium (10%)

[0723] L-Glutamine (Sigma G-7513) (2mM)

[0724] HEPES buffer (Sigma H-0887) (10mM) Antibiotic / Antifungal agent (Sigma A-9909) (1X)

[0725] MEM non-essential amino acids (Sigma M-7145) (1X)

[0726] 2-Mercaptoethanol (Sigma M-6250) (50 nM)

[0727] L-929 conditional culture medium:

[0728] L-929 (CCL-1) cells from ATCC were in 55 mL of DMEM + 10% fetal bovine serum at a concentration of 4.7 × 10⁻⁶ cells / mL. 5 Cells were seeded at 75 cm 2 Flasks. For THP-1 cells, allow cells to grow for 3 days. On day 3, collect the supernatant and filter through a 0.45 μm filter, aliquot, and freeze at -20°C. The cell-free filtrate is used for THP-1 infection in DMEM medium.

[0729] THP-1 cell infection:

[0730] On day 0, the culture medium was removed from the cells and replaced with 0.2 ml of antibiotic-free / antifungal DMEM containing Mycobacterium tuberculosis HN878, with an MOI of 1 macrophage to 10 bacteria. The tissue culture plates were placed in sealed Ziploc bags for transport back to the incubator. Once in the incubator, the bags were unzipped. The cells were incubated with the bacteria for 2 hours. After infection, bacteria attached to the cells were removed, and each well was washed once with phosphate-buffered saline (PBS) and 2 ml of antibiotic-free / antifungal complete DMEM medium with multiple drug concentrations was added. To prepare drug concentrations, a 2-fold serial dilution was performed in the secondary tube by adding 10 ml of the previous suspension to 10 ml of complete medium plus serum. The tissue culture plates were returned to the incubator and kept at 37°C + 5% CO2 (drugs held in the wells for 3 days). Each drug concentration was tested in triplicate in the wells.

[0731] Cell lysates were plated and THP-1 cell viability was assessed at 2 hours, 1 day, 2 days, and 5 days post-infection. Tissue culture medium was removed from all wells, and cells were washed twice with 1 ml PBS. Then, 1 ml sterile redistilled water + 0.05% Tween-80 was added to each well; cells were incubated at room temperature for 5–10 minutes. Cell lysates were serially diluted 1:10 in sterile saline in 24-well tissue culture plates. The diluted cell lysates were plated onto 7H11 / OADC agar using a 1 / 1,000 dilution step. (Four 24-well TC plates and 24 agar 'quadrilateral' plates were required for each 24-well TC plate of cells). Plates were incubated at 32°C for 30 days, and colonies were counted to determine CFU / ml.

[0732] result:

[0733] In vitro optical density results of THP-1HN878

[0734] The minimum inhibitory concentration (MIC) is reported as the dilute composition that inhibits bacteria (i.e., the maximum dilution level of a specific formulation on a scale expressed as 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml).

[0735]

[0736]

[0737] Figures 24 to 27 The results are shown in the figure.

[0738] Figure 24Efficacy of 30RESP001FC and FD (concentrated and diluted) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of formulations 30RESP001FC (concentrated) (A) and 30RESP001FD (diluted) (B) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 macrophages at 16 μg / ml (▲) and 8 μg / ml. Treatment with 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (◆), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) resulted in the intracellular killing of Mycobacterium tuberculosis HN878 2 hours (day 0), day 1, day 2, and day 5. Its effects. In Figure 24 In each curve graph, ▲ and ▲ were treated with 16 μg / ml and 8 μg / ml, respectively. The curves may differ from the ▲ and ▼ curves treated with 0.25 μg / ml and 0.125 μg / ml, respectively, because treatment with 16 μg / ml and 8 μg / ml is more effective. In other words, the curves treated with 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than those treated with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the □ curve treated with 1 μg / ml is easily different from the untreated curve. The curve is more effective because treatment at 1 μg / ml is more efficient. No treatment. The CFU value on the curve is rising and remains above 1×10 after day 1. 4 .

[0739] In the MIC table above and Figure 24 The 30RESP001FC and FD compositions described as "16 μg / ml" contain 0.15 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), wherein each of the 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml compositions is diluted 50% (i.e., the concentration is halved) from the previous composition in the order described above, from 16 μg / ml to 0.125 μg / ml.

[0740] THP-1 macrophages were infected with Mycobacterium tuberculosis at an MOI of 1:10, and intracellular bacterial counts were determined by colony counting (CFU) immediately after infection at 2 hours (day 0), day 1, day 2, and day 5. Values ​​shown are mean ± SD from a single independent experiment. Specifically, treatment with 16 μg / ml and 8 μg / ml 30RESP001FC and FD (concentration and dilution) showed increased efficacy against Mycobacterium tuberculosis HN878 relative to the untreated control (*, p < 0.05).

[0741] Figure 25 Efficacy of 30RESP002FC and FD (concentrated and diluted) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of formulations 30RESP002FC (concentrated) (A) and 30RESP002FD (diluted) (B) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 macrophages at 16 μg / ml (▲) and 8 μg / ml. Treatment with 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (◆), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) resulted in the intracellular killing of Mycobacterium tuberculosis HN878 at 2 hours, day 1, day 2, and day 5. Its effects. In Figure 25 In each curve graph, ▲ and ▲ were treated with 16 μg / ml and 8 μg / ml, respectively. The curves may differ from the ▲ and ▼ curves treated with 0.25 μg / ml and 0.125 μg / ml, respectively, because treatment with 16 μg / ml and 8 μg / ml is more effective. In other words, the curves treated with 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than those treated with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the □ curve treated with 1 μg / ml is easily different from the untreated curve. The curve is more effective because treatment at 1 μg / ml is more efficient. No treatment. The CFU value on the curve is rising and remains above 1×10 after day 1. 4 .

[0742] In the MIC table above and Figure 25The 30RESP002FC and FD compositions described as "16 μg / ml" contain 0.15 M sodium nitrite, 0.05 M lactitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), wherein each of the 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml compositions is diluted 50% (i.e., the concentration is halved) from the previous composition in the order described above, from 16 μg / ml to 0.125 μg / ml.

[0743] THP-1 macrophages were infected with Mycobacterium tuberculosis at an MOI of 1:10, and intracellular bacterial counts were determined by colony-forming units (CFU) immediately after infection at 2 hours, day 1, day 2, and day 5. Values ​​shown are mean ± SD from one independent experiment. Treatment with 16 μg / ml 30RESP002FC (concentrated) and 16 μg / ml and 8 μg / ml 30RESP002FD (diluted) showed increased efficacy against Mycobacterium tuberculosis HN878 relative to the untreated control (*, p < 0.05).

[0744] Figure 26 Efficacy of 30RESP003FC and FD (concentrated and diluted) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of 30RESP003FC (concentrated) (A) and 30RESP003FD (diluted) (B) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 macrophages at 16 μg / ml (▲) and 8 μg / ml. Treatment with 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (◆), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) resulted in the intracellular killing of Mycobacterium tuberculosis HN878 2 hours (day 0), day 1, day 2, and day 5. Its effects. In Figure 26 In each curve graph, ▲ and ▲ were treated with 16 μg / ml and 8 μg / ml, respectively. The curves may differ from the ▲ and ▼ curves treated with 0.25 μg / ml and 0.125 μg / ml, respectively, because treatment with 16 μg / ml and 8 μg / ml is more effective. In other words, the curves treated with 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than those treated with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the □ curve treated with 1 μg / ml is easily different from the untreated curve. The curve is more effective because treatment at 1 μg / ml is more efficient. No treatment. The CFU value on the curve is rising and remains above 1×10 after day 1.4 .

[0745] In the MIC table above and Figure 26 The 30RESP003FC and FD compositions described as "16 μg / ml" contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), wherein each of the 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml compositions is diluted 50% (i.e., the concentration is halved) from 16 μg / ml to 0.125 μg / ml in the aforementioned order.

[0746] THP-1 macrophages were infected with Mycobacterium tuberculosis at an MOI of 1:10, and intracellular bacterial counts were determined by colony counting (CFU) immediately after infection at 2 hours, day 1, day 2, and day 5. Values ​​shown are mean ± SD from one independent experiment. Treatment with 16 μg / ml and 8 μg / ml 30RESP003FC (concentrated) and 16 μg / ml 30RESP003FD (diluted) showed increased efficacy against Mycobacterium tuberculosis HN878 relative to the untreated control (*, p < 0.05).

[0747] Figure 27 Efficacy of 30RESP004FC and FD (concentrated and diluted) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 cells. The efficacy of formulations 30RESP004FC (concentrated) (A) and 30RESP004FD (diluted) (B) against Mycobacterium tuberculosis HN878 was evaluated in THP-1 macrophages at 16 μg / ml (▲) and 8 μg / ml. Treatment with 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (◆), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) resulted in the intracellular killing of Mycobacterium tuberculosis HN878 2 hours (day 0), day 1, day 2, and day 5. Its effects. In Figure 27 In each curve graph, ▲ and ▲ were treated with 16 μg / ml and 8 μg / ml, respectively. The curves may differ from the ▲ and ▼ curves treated with 0.25 μg / ml and 0.125 μg / ml, respectively, because treatment with 16 μg / ml and 8 μg / ml is more effective. In other words, the curves treated with 16 μg / ml and 8 μg / ml show significantly lower CFU values ​​than those treated with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the □ curve treated with 1 μg / ml is easily different from the untreated curve. The curve is more effective because treatment at 1 μg / ml is more efficient. No treatment. The CFU value on the curve is rising and remains above 1×10 after day 1. 4 .

[0748] In the MIC table above and Figure 27 The 30RESP004FC and FD compositions described as "16 μg / ml" contain 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M ascorbic acid / ascorbate (final molar concentration after dilution), wherein each of the 8 μg / ml, 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, and 0.125 μg / ml compositions is diluted 50% (i.e., the concentration is halved) from the previous composition in the order described above, from 16 μg / ml to 0.125 μg / ml.

[0749] THP-1 macrophages were infected with Mycobacterium tuberculosis at an MOI of 1:10, and intracellular bacterial counts were determined immediately after infection on days 1, 2, and 5. Values ​​shown are mean ± SD from one independent experiment. Treatment with 16 μg / ml and 8 μg / ml 30RESP004FC (concentrated) showed increased efficacy against Mycobacterium tuberculosis HN878 relative to the untreated control (*, p < 0.05).

[0750] The formulation is inferred to inhibit Mycobacterium tuberculosis HN878 in vitro at appropriate doses exceeding the MIC.

[0751] In the tests of Example 6, it was also noted that the manner in which the formulations were manufactured affected their in vitro antibacterial efficacy against Mycobacterium tuberculosis HN878.

[0752] This was demonstrated by comparing the efficacy of formulation 1 at a concentration of 8 μg / ml between FC and FD formulations. Figure 24 To illustrate this, let's compare A with B (24). The efficacy of the FC formulation increased strongly after at least 5 days of incubation, while the efficacy of the FD formulation increased less strongly over the same period. This contrasts with a concentration of 16 μg / ml, at which the FC and FD formulations showed very similar and good efficacy over the same time period.

[0753] Different behaviors were observed under formulation 2. Figure 25(Comparison A with 25B). During the first two days after incubation, the efficacy of the FD formulation at a concentration of 16 μg / ml increased more strongly than that of the FC formulation, then remained unchanged. However, by day 5 after incubation, the efficacy of the FD formulation was good, while that of the FC formulation was very good. At a concentration of 8 μg / ml, the efficacy of the FD formulation increased strongly to good efficacy after at least 5 days of incubation, while the efficacy of the FC formulation increased less strongly during the same period.

[0754] This indicates that, at least at higher concentrations, the stage of adding water to achieve final inhibition of the formulation substantially affects the antibacterial activity of the formulation in terms of both initial antibacterial action and the degree of bactericidal activity within 5 days. Generally speaking, although not universally applicable, initially preparing a concentrated premix of sodium nitrite, polyol, and acid components in the desired relative molar ratios but at a concentration higher than the concentration to be used (e.g., at least 3 times, for example, at least 5 times, or for example, about 3 times to about 80 times the concentration to be used), and then only diluting the concentrate to obtain the formulation to be used, will provide better antibacterial activity during the 0 to 5 day period following incubation.

[0755] Example 7

[0756] Carboxylic acid-nitrite-polyol solution exhibits cytotoxic and antiviral activity against H1N1 influenza A virus.

[0757] Test formulations, designated F1C1, F1C2, and F1C3, corresponding to formulation 30RESP001FC, its 10-fold dilution, and its 100-fold dilution respectively in Example 6, were used with oseltamivir solution (1 μM) and a viral control to obtain comparative cytotoxicity and H1N1 influenza A virus killing activity in MDCK cells after 24 hours. Similar to Example 8, cytotoxicity was determined by an LDH cytotoxicity assay. Antimicrobial activity against H1N1 influenza A virus in MDCK cells was measured at a series of dilutions (horizontal axis being nitrite molar concentration) at MOI = 0.002 (●) and MOI = 0.02 (■), where cytotoxicity is shown in gray, and the cytotoxicity scale is on the right (cytotoxicity at measured nitrite concentrations up to and including 0.015 M ≤ 1% of the LDH control). Plate images were obtained at MOI = 0.002 and nitrite concentrations of 0.15 M, 0.015 M, and 0.0015 M, and compared with oseltamivir (1 μM). Figure 28 The results are shown in the figure. The order of the plates described in the penultimate sentence 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 one on top of the other). The rightmost pair of plates, immediately to the right of the oseltamivir pair, is the virus control. Cytotoxicity is shown below each pair of test plates as LDH control % (the average of three LDH measurements at 24 hours post-infection).

[0758] The results showed that, at appropriate doses of the nitrite / citric acid / polyol formulation, the virus could be completely eradicated, significantly outperforming oseltamivir. The nitrite / citric acid / polyol formulation has also shown similar antiviral activity against rhinovirus and respiratory syncytial virus (RSV).

[0759] These results indicate that the nitrite / acid / polyol formulations according to the present invention provide therapeutic and preventative treatment for respiratory viral infections in human and animal subjects.

[0760] Example 8

[0761] Carboxylic acid-nitrite-polyol solution exhibits cytotoxic and antiviral activity against SARS-CoV-2 coronavirus.

[0762] Material

[0763] Test formulation F1 (pH 5.8)

[0764] Six test concentrations of formulation 1 (F1) were prepared from a stock solution of 1.5 M sodium nitrite, 0.91 M citric acid / citrate buffer at pH 5.8, and 0.5 M mannitol solution (as an aqueous solution of sodium nitrite, citric acid at pH 5.8, and mannitol (a polyol)) using the method described below, resulting in the following test compositions:

[0765] Formulation 1 (F1)

[0766]

[0767] Control used with F1

[0768] A pH 5.8 control preparation was prepared by adding 0.1M citric acid, assay buffer, and cells.

[0769] The negative control is assay buffer + cells.

[0770] The positive control was chloroquine + cells.

[0771] Test formulation F2 (pH 5.4)

[0772] Six test concentrations of formulation 2 (F2) were prepared from a stock solution of 1.5M sodium nitrite, 0.91M citric acid / citrate buffer at pH 5.4, and 0.5M mannitol solution (as an aqueous solution of sodium nitrite, citric acid at pH 5.4, and mannitol (a polyol)) using the method described below, resulting in the following test compositions:

[0773] Formulation 2 (F2)

[0774]

[0775] Control used with F2

[0776] A pH 5.4 control preparation was prepared by adding 0.1M citric acid, assay buffer, and cells.

[0777] The negative control is assay buffer + cells.

[0778] The positive control was chloroquine + cells.

[0779] Chemical reagents

[0780] Sodium nitrite:

[0781] Grade: Sodium nitrite, Ph Eur, USP. Sodium nitrite CAS No. 7632-00-0, EC No. 231-555-9, Ph Eur, USP, from Sigma Aldrich, product code 1.065441000.

[0782] Citric acid:

[0783] Grade: Anhydrous Citric Acid Powder ESSENTIAL Ph Eur, BP, JP, USP, E 330, FCC, from Sigma Aldrich, product code 1.002425000.

[0784] D-Mannitol:

[0785] Grade: D-mannitol that meets EP, FCC and USP testing standards, from Sigma Aldrich, product code M8429-100G.

[0786] Chloroquine phosphate:

[0787] Grade: Level II drug standard, from Sigma Aldrich, product code PHR1258-1G.

[0788] Preparation of stock solutions

[0789] To prepare a citric acid solution, add 90 ml of distilled water to 19.2 g of citric acid, followed by 10 ml of 3M sodium hydroxide, and then dilute with distilled water to adjust the pH (to 160 ml for pH 5.4, or to 190 ml for pH 5.8). In an alternative method, add 20 ml of distilled water to 19.2 g of citric acid, followed by 1.2 g of solid sodium hydroxide, and then adjust the pH to 100 ml with 10M sodium hydroxide and distilled water. Sterilize the solution by syringe filtration using a 0.22 μm filter.

[0790] To prepare a 1.0 M sodium nitrite solution, add 100 mL of distilled water to 6.9 g of sodium nitrite. To prepare a 1.5 M sodium nitrite solution, add 100 mL of distilled water to 10.35 g of sodium nitrite.

[0791] When specified, 9.1 g of mannitol was added to obtain a concentration of 0.5 M. The solution was sterilized by syringe filtration using a 0.22 μm filter.

[0792] Preparation of formulation

[0793] The pH of the buffered citric acid solution is controlled to the desired value before mixing with the nitrite and mannitol solutions. The pH value described for the formulation is the pH value of the buffered citric acid solution prepared before mixing with the nitrite and mannitol solutions.

[0794] A suitable preparation method is as follows: Add sodium nitrite (1.5 M) containing 0.5 M mannitol to a mixing vessel, and randomly add a pH-controlled citric acid solution to the 1:1 mixture (nitrite + polyol: citric acid). Mix the solution by gently inverting. Once mixed, store the mixture in a sealed container (e.g., a 50 ml Falcon tube) at ambient temperature for 5 minutes. Then, dilute the resulting solution containing 0.75 M nitrite, 0.25 M mannitol, and citric acid 5-fold (1.2-fold concentration) in assay buffer to obtain the final test concentrations of 0.15 M nitrite, 0.05 M mannitol, and, for example, 0.1 M citric acid. Continuously dilute the 1:1 mixture (e.g., a mixture starting with 0.75 M nitrite, 0.25 M mannitol, and 0.5 M citric acid) with distilled water and / or assay buffer. All formulation concentrations can be stored at ambient temperature. Prepare fresh solutions for each operation.

[0795] Other control materials

[0796] S-nitroso-N-acetylpenicillamine (SNAP) was used as an additional control at a range of concentrations known to be suitable for achieving its purpose and denoted as SNAP50, SNAP100, SNAP200, SNAP300, and SNAP400. SNAP is a known NO donor and was used as a positive control in these assays to provide NO and confirm that NO is not cytotoxic in vitro. To control for any potential influence of the N-acetylpenicillamine (...

Claims

1. Use of one or more agents selected from nitric oxide-generating compositions, combinations or complexes of components of nitric oxide-generating compositions, and mixtures thereof in the preparation of a medicament for treating or alleviating or preventing infection caused by SARS-CoV coronavirus and disease SARS or SARS-CoV-2 coronavirus and disease COVID-19, wherein said nitric oxide-generating composition or combination or complexes of components of said nitric oxide-generating composition comprises one or more nitrites, a proton source comprising one or more acids selected from organic carboxylic acids and organic noncarboxylic acid reducing acids, and one or more organic polyols. Furthermore, the proton source comprises citric acid; the one or more organic polyols comprise mannitol; and the molar concentrations of nitrite ions, the proton source, and all of the one or more organic polyols in the solution before the reaction are each independently from 0.001 M to 5 M.

2. Use of the agent according to claim 1, wherein the agent is used to treat or alleviate or prevent the disease SARS.

3. Use of the agent according to claim 1, wherein the agent is used to treat or alleviate or prevent the disease COVID-19.

4. Use of the agent according to claim 1, wherein the one or more organic polyols comprise sugar alcohols, and the sugar alcohols further comprise one or more monosaccharide units and one or more acyclic sugar alcohol units.

5. Use of the agent according to claim 1, wherein the organic polyol further comprises lactitol.

6. Use of the agent according to claim 1, wherein the proton source further comprises ascorbic acid.

7. Use of the agent according to claim 1, wherein the one or more organic carboxylic acids or one or more organic non-carboxylic acid reducing acids are buffered to a pH value higher than that exhibited by an aqueous solution of the acid at the same concentration.

8. Use of the agent according to claim 7, wherein the higher pH value is in the range of 5 to 8.

9. Use of the agent according to claim 7, wherein the higher pH value is greater than or equal to 5.

2.

10. Use of the agent according to claim 9, wherein the higher pH value is in the range of 5.2 to 6.

11. Use of the agent according to any one of claims 1-10, wherein the treatment or relief or prevention comprises administering an antivirally effective amount of the agent to a human or animal subject.

12. Use of the agent according to claim 11, wherein the treatment or relief or prevention comprises administering the drug to the lungs of the subject.

Images