Methods and compositions for treating and combating tuberculosis

Abstract

The present invention provides one or more agents selected from the group consisting of nitric oxide (NO), a nitric oxide generating composition, a combination of components of a nitric oxide generating composition or a combination therapy and mixtures thereof for use as an antibacterial agent against tuberculosis and Mycobacterium tuberculosis.

Description

Technical Field

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

[0002] The respiratory disease tuberculosis is caused by the bacterium Mycobacterium tuberculosis. To date, no fully satisfactory treatment has been found for this disease, which is a serious killer in many countries. Tuberculosis often presents with other pathogenic infections, including viral infections. It would be a great advantage if an agent effective against tuberculosis had a broad spectrum of activity, including activity against viruses and other pathogens commonly present in tuberculosis patients.

[0003] The present invention is based on the following unexpected discovery: one or more agents selected from nitric oxide (NO), the composition that produces nitric oxide, the component of the composition that produces nitric oxide or the combination of ingredients or the combinational combination and their mixture are effective antibacterial agents in vitro for mycobacterium tuberculosis, provide effective in vivo treatment (treatment and prevention) of tuberculosis in humans and animals. The present invention also provides effective antibacterial treatment to surface (comprising inanimate surface and other outer surfaces of hand, arm and human or animal body) and space, thereby prevent the spread of mycobacterium tuberculosis and the contamination of surface. In a preferred embodiment of the present invention, nitric oxide can be produced by NO generation system, and this system comprises nitrite and proton source, and described proton source comprises one or more acids selected from organic carboxylic acid and organic non-carboxylic acid.Organic non-carboxylic acid can be organic non-carboxylic acid reducing acid.This type of system can be embodied as the composition that produces NO, and it can be applied to patient's lung.

[0004] The NO generating system may comprise one or more organic polyols. The one or more organic polyols, when present, suitably comprise a sugar alcohol comprising one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0005] The active agent, such as the NO-generating composition, can be delivered to the patient's lungs in any suitable physical form, such as in liquid form or in the form of droplets entrained in a carrier gas or air, such as in the form of an aerosol or mist.

[0006] According to the present invention, it has also been discovered that acids used as a proton source for generating nitric oxide can be effective when buffered to relatively high pH values, such as a pH between about 5 and about 8, such as at or above about 5.2, such as within 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] Nitric oxide and NO generating compositions have a range of antimicrobial and other beneficial physiological activities as discussed herein, and thus the antibacterial effect against M. tuberculosis provided by the present application can be accompanied by simultaneous beneficial activity against other pathogens that can infect or predispose a patient to infection, including secondary bacterial, viral, parasitic and fungal infections.

[0008] As reported herein, the antibacterial effect against M. tuberculosis is enhanced if the NO generating composition is prepared in a particular way, namely by one of the following methods:

[0009] (a) A method of preparing a NOx generating composition, the method comprising mixing nitrite, a proton source and an organic polyol component in the required proportions in concentrations higher than required in the composition in the form in which it is to be used, to form a concentrated premix, and subsequently diluting the concentrated premix with water as appropriate to provide the composition in the form in which it is to be used;

[0010] (b) A method of preparing a NOx generating composition, the method comprising mixing nitrite, a proton source and an organic polyol component in the required proportions in concentrations required in the composition in the form in which it is to be used, to provide the composition in the form in which it is to be used.

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

[0012] Nitric oxide (NO) and nitric oxide precursors have been extensively studied as potential pharmaceutical agents. Nitric oxide is a potent vasodilator, which is synthesised and released by vascular endothelial cells and plays an important role in the regulation of, inter alia, local vascular resistance and blood flow. In mammalian cells, nitric oxide is produced primarily with L-citrulline by the enzymatic oxidation of L-arginine. Nitric oxide is also released from the skin by a mechanism that appears to be 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 therapy is discussed in Biochemistry (Moscow), 63(7), 802-809 (1998), the disclosure of which is incorporated herein by reference. Nitric oxide has been shown to have antimicrobial properties, as reviewed by F C Fang in J. Clin. Invest. 99(12), 2818-2825 (1997), and as described, 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 generating nitric oxide, other nitrogen oxides and precursors thereof are given in the following description of the present application.

[0013] There remain considerable problems associated with the effective production and delivery of nitric oxide, other oxides of nitrogen and precursors thereof to organisms and cells for therapy. Systems widely used to produce nitric oxide rely on the use of a mineral acid to acidify nitrite salt, initially producing equimolar amounts of nitrous acid (HNO2) compared to the starting nitrite salt, which then readily decomposes to nitric oxide and nitrate with hydrogen ion and water. The decomposition can be represented by the following equilibrium equation (1):

[0014] 3 HNO2→ 2 NO + NO3 - + H + + H2O (1)

[0015] The acidification of nitrite salt is typically carried out at a pH of less than about 4, at which pH the formation of nitrous acid is generally facilitated in an attempt to maximize the production of NO. However, the use of a pH < 4 is not suitable for in vivo use where the acid comes into contact with animal tissue. The higher the pH, the more benign to the cellular and biological systems, but at pH values above 4, the prior systems are unable to produce satisfactory yields of NO. In an attempt to increase the amount of NO produced at pH values above 4, large amounts of nitrite salt are required, which is impractical and uneconomical in therapeutic applications. Additionally, given the short half-life of nitrous acid, the conversion represented by equation (1) is not easily controlled, and thus the release of nitric oxide for therapeutic use is difficult to control. The reaction to produce nitric oxide, optionally other oxides of nitrogen and / or optionally precursors thereof, between one or more nitrite salts and a source of protons is referred to herein as a "NOx-producing reaction" or "NOx production reaction" or similar language, and "NOx" is used to refer to the products of nitrite acidification, particularly nitric oxide, other oxides of nitrogen and precursors thereof, individually and in any combination. It will be appreciated that each component of the NOx produced can be released as a gas, or can dissolve in solution in the reaction mixture, or can initially dissolve in solution and subsequently be released as a gas, or any combination thereof.

[0016] WO 00 / 53193 (the disclosure of which is incorporated herein by reference) describes a cream or ointment for treating skin ischemia and promoting wound healing, in which the source of protons is ascorbic acid. Example 1 describes a gel based on KY Jelly® TM , which in Example 7 was tested in direct contact with the skin and with a membrane protecting the skin. The use of ascorbic acid was said to avoid significant skin inflammation (WO 00 / 53193 page 2). In practice, however, the degree of skin inflammation caused by the low pH of the gel was unsatisfactory when the gel was in direct contact with the skin, and when the membrane was present, the skin-protecting membrane attenuated the effect of the gel. As a result the gel was not marketable. The composition of WO 00 / 53193 does not contain a polyol.

[0017] WO 02 / 20026 (the disclosure of which is incorporated herein by reference) describes a skin formulation for treating drug-resistant infections of the skin in which the proton source is citric acid or salicylic acid. A composition containing a nitrite and a composition containing an acid are dispensed from a dual barrel dispenser and then mixed to react the acid with the nitrite and then applied to the skin. Propylene glycol and polyethylene glycol are taught as optional preservatives for the nitrite composition. Glycerol (glycerin) is taught as an optional thixotropic agent for the nitrite composition. Propylene glycol is used in a pair of creams for citric acid and nitrite respectively, which are mixed on site to initiate the reaction between the acid and the nitrite (e.g. WO 02 / 20026 Example 3, Preparation 1). Glycerin is used with cetostearyl alcohol in a pair of lotions for citric acid and nitrite respectively, which are mixed on site to initiate the reaction between the acid and the nitrite (e.g. WO 02 / 20026 Example 3, Preparation 3). The preferred pH of the reaction mixture is 5 or lower, particularly 4 or lower, which is expected to cause undesirable skin inflammation. Nasal sprays are also taught which can use a reducing acid such as ascorbic acid or ascorbyl palmitate so that a higher pH can be used to avoid irritating the sensitive nasal mucosa. However, it is acknowledged (WO 02 / 20026, page 16, second paragraph) that a higher pH will slow the reaction.

[0018] US 6103275 (published August 15, 2000) (the disclosure of which is incorporated herein by reference) describes a reducing agent such as ascorbic acid with an organic acid having a pKa between 1 and 4 such as maleic acid for acidifying a nitrite. A viscous (gel) composition is used to slow the release of the reaction product for topical use. The acid and nitrite are kept separate until the production of nitric oxide is initiated and it is stated that the reducing agent is included in at least one of the first and second gels. The pH range used by the method is not specified. However, the fact that the buffer components are referred to as acids can indicate that these compounds are present primarily in protonated form so the pH of the composition should be substantially below 4. The presence of an acid having a pKa between 1 and 4 ensures good buffering capacity of the formulation at this pH. While the incorporation of such an acid is one suitable way of ensuring that the pH is maintained at a level at which the efficiency of the conversion of the nitrite to nitric oxide is sustained, a low pH is expected to cause significant undesirable skin irritation on contact with the skin. The composition of US 6103275 does not contain a polyol.

[0019] In WO 2003 / 013489 (the disclosure of which is incorporated herein by reference), 3% polyvinyl alcohol (PA) is proposed as a gelling base for citric acid and nitrite salt, respectively, mixed together on the spot (WO 2003 / 013489 Example 7). However, the test data (WO 2003 / 013489 Tables 11 and 12) show that PA is unable to form a stable gel, and the PA compositions are never mixed together or used together. In addition to the above proposal which does not hold to the final composition, the compositions of WO 2003 / 013489 do not contain a polyol.

[0020] U.S. Patent Application No. 2005 / 0037093 (the disclosure of which is incorporated herein by reference) describes nitrite-acid reaction based nitric oxide generating compositions and mentions optional excipients including polyvinyl alcohol, propylene glycol and polyethylene glycol.

[0021] Chinese Patent Application No. CN 101028229 (the disclosure of which is incorporated herein by reference) describes a cosmetic product which generates nitric oxide by reaction of a nitrite salt with an acid. The use of, inter alia, glycerol, propylene glycol and glycerol monostearate as additional ingredients is taught. Trihydroxyethylamine is further mentioned as an ingredient in specific examples.

[0022] Chinese Patent Application No. CN 101062050 (the disclosure of which is incorporated herein by reference) describes a product for promoting hair growth which generates nitric oxide by reaction of a nitrite salt with an acid. The use of, inter alia, glycerol, propylene glycol and glycerol monostearate as additional ingredients is taught. D-pantothenol and a combination of panthenol and inositol are mentioned as ingredients in specific examples.

[0023] WO 2008 / 110872 (the disclosure of which is incorporated herein by reference) describes foamable nitric oxide donor compositions which optionally contain polar solvents such as selected from polyols and polyethylene glycols (paragraphs

[0055] and

[0056] ). It is stated that specific polyols are propylene glycol, butylene glycol, butene diol, butyne diol, pentane diol, hexane diol, octane diol, neopentyl glycol, 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 as optional further ingredients in a list of many polymeric agents (paragraph

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

[0190] and

[0191] .

[0024] 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 nitrite and proton source. Examples of non-thiol reducing agents are iodide anion, butylated hydroquinone, tocopherol, butylated hydroxyanisole, butylated hydroxytoluene and beta-carotene. The compositions of WO 2009 / 019498 contain no polyols except for butylated hydroquinone.

[0025] WO 2014 / 188174 and WO 2014 / 188175 (the disclosures of which are incorporated herein by reference) describe a dressing system for skin lesions and a transdermal delivery system in which the proton source is a hydrogel comprising pendant carboxylic and sulfonic acid groups covalently bonded to a three-dimensional polymeric matrix. The first layer in contact with the skin is a polypropylene mesh onto which the nitrite is absorbed. When the mesh is placed on the skin and the hydrogel is applied as a top layer over the mesh, the reaction products of the acid with the nitrite are found to be delivered sufficiently 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 dissolvable film formed of, for example, polyvinyl alcohol and containing the nitrite. In both references it is taught that the hydrogel can comprise glycerol, without stating the purpose thereof. 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 disclose a preference for certain hydroxyl-containing ingredients in the absence of others, in particular 1-thioglycerol, erythorbate, ascorbic acid and butylated hydroquinone.

[0026] U.S. Patent Application No. 2014 / 0335207 (the disclosure of which is incorporated herein by reference) describes a topical mixture which produces nitric oxide when a "nitrite medium" is mixed with an "acidifying medium". Specific embodiments of the "nitrite medium" are described in paragraphs

[0050] to

[0055] alone, in which the nitrite is present 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, ethylhexylglycerin and hexylene glycol, and the specific embodiments described in the other paragraphs contain some of the above polyols and butylene glycol. These polyols are also components of the embodiments of the "acidifying medium" described in paragraphs

[0056] to

[0062] .

[0027] U.S. Patent Application No. 2015 / 0030702 (the disclosure of which is incorporated herein by reference) describes a skin patch based on the nitrite-acid reaction. The skin patch comprises a non-thiol reducing agent, such as hydroquinone or butylated hydroquinone. The skin patch can comprise a hydrogel, which for example comprises a hydrophilic polymer, such as polyvinyl alcohol or polyethylene glycol.

[0028] U.S. Patent Application No. 2017 / 0209485 (the disclosure of which is incorporated herein by reference) describes a device and method for topical application of nitric oxide in a foam or serum vehicle. Glycerol and (unspecified) "glycerol-like components" are described in paragraph

[0070] as optional additives to increase surface tension and / or reduce vapor pressure.

[0029] U.S. Patent Application No. 2019 / 0134080 (the disclosure of which is incorporated herein by reference) describes a composition and method for topical application to the skin of a nitric oxide generation system formed from a multi-part combination comprising a first solution comprising at least one nitrite reactant and a second solution comprising at least one acidic reactant. Apparatus for holding, 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] ).

[0030] As mentioned above, the present invention is based on the unexpected finding that one or more active agents selected from nitric oxide (NO), a nitric oxide generating composition, a combination or combination of components or ingredients of a nitric oxide generating composition, and mixtures thereof are effective in vitro antibacterial agents against Mycobacterium tuberculosis, providing effective in vivo treatment (both therapeutic and prophylactic) of tuberculosis in humans and animals. The present invention also provides effective antibacterial treatment of surfaces (including inanimate surfaces as well as external surfaces of hands, arms and other external surfaces of the human or animal body) and spaces, thereby preventing the spread and contamination of surfaces with Mycobacterium tuberculosis.

[0031] In a preferred embodiment of the present invention, nitric oxide can be generated by a NO generation system comprising a nitrite and a proton source, the proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic acids. The organic non-carboxylic acid can be an organic non-carboxylic acid reducing acid. Such a system can be embodied as a NO generating composition, which can be administered to the lungs of a patient.

[0032] The NO generation system can comprise one or more organic polyols. The one or more organic polyols, when present, suitably comprise a sugar alcohol comprising one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0033] The active agent, e.g. the combination or combination of components or ingredients of the NO generating composition or nitric oxide generating composition, can be in any suitable physical form, e.g. in liquid form or in the form of droplets entrained in a carrier gas or air, e.g. delivered into the lungs of a patient as an aerosol or mist.

[0034] It has also been found in accordance with the present application that acids used as sources of protons for the generation of nitric oxide can be effective when buffered to a relatively high pH, e.g. a pH of between about 5 and about 8, e.g. at or above about 5.2, e.g. in the range 5.2 to 5.8, i.e. a pH which is physiologically tolerable by the tissues of the oral cavity, nasal passages, airways and lungs of a patient.

[0035] The nitric oxide and NO generating compositions have a range of antimicrobial and other beneficial physiological activities as discussed herein, and thus the antibacterial effect against Mycobacterium tuberculosis provided by the present application can be accompanied by simultaneous beneficial activity against other pathogens which can infect or predispose a patient to infection, including secondary bacterial, viral, parasitic and fungal infections.

[0036] As reported herein, it has been found in vitro that the antibacterial effect against Mycobacterium tuberculosis can be enhanced if the NO generating composition is prepared in a particular manner, i.e. by one of the following methods:

[0037] (a) a method of preparing a NOx generating composition, said method comprising mixing the nitrite salt, the source of protons and the organic polyol component in the required proportions in concentrations higher than required in the composition in the form in which it is to be used, to form a concentrated premix, and subsequently diluting the concentrated premix with water as appropriate to provide the composition in the form in which it is to be used;

[0038] (b) a method of preparing a NOx generating composition, said method comprising mixing the nitrite salt, the source of protons and the organic polyol component in the required proportions in concentrations required in the composition in the form in which it is to be used, to provide the composition in the form in which it is to be used.

[0039] These alternative methods constitute specific aspects of the present application.

[0040] In comparison to the current state of the art, the production of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof (collectively, NOx), and the enhancement of reaction output, can be more effectively achieved using a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reductive acids as nitrite acidifying agents in the presence of one or more organic polyols. In addition, the antimicrobially effective reaction products of such reaction systems using organic reductive acids as nitrite acidifying agents can be delivered at physiologically tolerable pH values, e.g., between about 5 and about 8, with or without the use of one or more organic polyols, which allows the reaction system to be operated at such pH values, delivered directly as a composition, and have beneficial physiological activities, e.g., antimicrobial activity in vivo. The methods of producing nitric oxide that form the basis of the present invention have been found to optionally produce physiologically effective amounts of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, over a significant period of time, e.g., over about 2 hours, e.g., over about 5 hours, e.g., over about 10 hours, following an initial intense burst of NOx gas production, which makes significant use possible in pharmaceutical and other applications. If the initial intense burst is not desired, the reaction mixture can be administered to a subject some time after the initiation of the NOx-producing reaction, e.g., about 10 minutes, 30 minutes, or one hour or more after the initiation of the NOx-producing reaction. SUMMARY

[0041] The present invention is defined in the appended claims and is particular embodiments of the presently more generally inventive progress disclosed in the following description. The present invention as in and defined by the appended claims relates to the application of the general inventive progress to combinations and compositions that conduct the NO-producing reaction and the gaseous products of that reaction delivered to a human or animal subject via the nose, mouth, respiratory tract, or lungs of the subject. All aspects, embodiments, implementations, and preferences described herein with respect to the present disclosure apply equally and independently to the present invention as in and defined by the appended claims.

[0042] The present disclosure provides systems, methods, combinations, kits, and compositions for the production of nitric oxide and optionally other oxides of nitrogen and / or optionally precursors thereof. The systems, methods, combinations, kits, and compositions include one or more nitrite salts and a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reductic acids as reactants. The systems, methods, combinations, kits, and compositions further include one or more organic polyols. The use of reductic acids (i.e., carboxylic reductic acids and non-carboxylic reductic acids) allows for the production of nitric oxide and optionally other oxides of nitrogen and / or optionally precursors thereof at a pH value slightly above 4, e.g., in the range of 5 to 8. The present disclosure also provides systems, methods, combinations, kits, and compositions for antimicrobial use, wherein one or more organic polyols are optional and the reaction is carried out at a starting pH value of the proton source in the range of 5 to 8.

[0043] According to a first aspect, the present disclosure provides a method for the production of nitric oxide, optionally other oxides of nitrogen and / or optionally precursors thereof, the method comprising reacting one or more nitrite salts with a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reductic acids under reaction conditions suitable for the production of nitric oxide, optionally other oxides of nitrogen and / or optionally precursors thereof, wherein the reaction is carried out in the presence of one or more organic polyols;

[0044] characterized by one or more of the following:

[0045] (a) the one or more organic polyols are present in an amount to enhance the reaction output;

[0046] (b) the proton source is not merely a hydrogel comprising carboxylic acid pendant groups covalently bonded to a three-dimensional polymeric matrix;

[0047] (c) the one or more organic polyols are not merely glycerol;

[0048] (d) the one or more organic polyols are not merely glycerol when one or more tackifiers are used;

[0049] (e) the one or more organic polyols are not merely glycerol when one or more plasticizers are used;

[0050] (f) the one or more organic polyols are not merely polyvinyl alcohol;

[0051] (g) the one or more organic polyols are not merely polyvinyl alcohol when one or more tackifiers are used;

[0052] (h) any one or more of (b) to (g) above, wherein the word "not merely" is replaced with "does not comprise";

[0053] (i) the one or more organic polyols are not only propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the diols listed here, hydroquinone, butylated hydroquinone, 1 -thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol;

[0054] (j) the one or more organic polyols do not comprise propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the diols listed here, hydroquinone, butylated hydroquinone, 1 -thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol.

[0055] Nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof made by the method according to the first aspect of the disclosure constitute a second aspect of the disclosure.

[0056] According to a third aspect, the disclosure provides a method of enhancing the output of a reaction of one or more nitrite salts with a proton source to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, the method comprising conducting the reaction using a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids and in the presence of one or more organic polyols in an amount that enhances the output of the reaction. The enhancement of the output of the reaction is compared to a reaction conducted under the same conditions but without the one or more organic polyols.

[0057] According to a fourth aspect, the disclosure provides the use of one or more organic polyols in a reaction mixture to enhance the output of a reaction of one or more nitrite salts with a proton source to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof in the reaction mixture, wherein the proton source comprises one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids. The enhancement of the output of the reaction is compared to a reaction conducted under the same conditions but without the one or more organic polyols.

[0058] According to a fifth aspect, the disclosure provides a combination, kit or composition for use in the production of nitric oxide, optionally other oxides of nitrogen and / or optionally precursors thereof, by one or more reactions of a nitrite salt with a proton source, the combination, kit or composition comprising:

[0059] (i) one or more nitrite salts;

[0060] (ii) a proton source, the proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids; and

[0061] (iii) one or more organic polyols;

[0062] characterized by one or more of:

[0063] (a) the one or more organic polyols are present in an amount to enhance the output of the reaction;

[0064] (b) the proton source is not merely a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix;

[0065] (c) the one or more organic polyols are not merely glycerol;

[0066] (d) the one or more organic polyols are not merely glycerol when one or more tackifiers are used;

[0067] (e) the one or more organic polyols are not merely glycerol when one or more plasticizers are used;

[0068] (f) the one or more organic polyols are not merely polyvinyl alcohol;

[0069] (g) the one or more organic polyols are not merely polyvinyl alcohol when one or more tackifiers are used;

[0070] (h) any one or more of (b) to (g) above, wherein the words "not merely" are replaced by "comprising";

[0071] (i) the one or more organic polyols are not only propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the ones listed here, hydroquinone, butylated hydroquinone, 1 -thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol;

[0072] (j) the one or more organic polyols do not comprise propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the ones listed here, hydroquinone, butylated hydroquinone, 1 -thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol.

[0073] When the proton source comprises a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix and the combination or kit comprises two or more separate compositions, preferably the one or more polyols are not in direct contact with the hydrogel or are present in the separate compositions.

[0074] The chemical substance of the combination, kit or composition of the fifth aspect of the present disclosure may, for example, essentially consist of the above-mentioned components (i), (ii) and (iii) and optionally water and / or a pH buffer. The expression "essentially consist of" may, for example, allow the presence of small amounts of one or more further components, provided that the action of the above-mentioned components (i), (ii) and (iii) and optionally water and / or a pH buffer is not adversely affected. The total amount of such one or more further components is desirably less than about 20% by weight or volume, 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, of the chemical ingredients of the combination, kit or of the composition.

[0075] The chemical(s) of the combination, kit or composition may, for example, consist of components (i), (ii) and (iii) described above and optionally water and / or a pH buffer, and / or one or more additional components in an amount less than about 20% by weight or volume of the chemical constituents of the combination, kit or composition, 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.

[0076] According to a sixth aspect, the disclosure provides a method of making a combination, kit or composition comprising:

[0077] (i) one or more nitrite salt(s);

[0078] (ii) a proton source comprising one or more acid(s) selected from organic carboxylic acids and organic non-carboxylic reducing acids; and

[0079] (iii) one or more organic polyol(s);

[0080] The method comprises bringing components (i), (ii) and (iii) into proximity with one another, forming a combination or kit or mixing to form a composition;

[0081] characterized by one or more of:

[0082] (a) the one or more organic polyol(s) is / are present in an amount to enhance the output of the reaction;

[0083] (b) the proton source is not merely a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix;

[0084] (c) the one or more organic polyol(s) is / are not merely glycerol;

[0085] (d) the one or more organic polyol(s) is / are not merely glycerol when one or more tackifier(s) is / are used;

[0086] (e) the one or more organic polyol(s) is / are not merely glycerol when one or more plasticizer(s) is / are used;

[0087] (f) the one or more organic polyol(s) is / are not merely polyvinyl alcohol;

[0088] (g) the one or more organic polyol(s) is / are not merely polyvinyl alcohol when one or more tackifier(s) is / are used;

[0089] (h) any one or more of (b) to (g) above, wherein the words "not merely" are replaced by "comprising";

[0090] (i) the one or more organic polyols are not only propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the diols listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol;

[0091] (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantoic acid, panthenol, panthenol combined with inositol, butanediol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the diols listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination thereof, or any of the above in combination with glycerol and / or polyvinyl alcohol.

[0092] As used herein, the expression "combination" refers to separate substances or compositions (referred to as "components") that are brought into proximity and used together. Bringing the components into proximity can be accomplished in multiple stages, where initially some, but not all, of the components are brought into association as a subcombination or partial combination, followed by proximity with one or more other components or other subcombinations or partial combinations. "Proximity" can include intimate mixture, solution or suspension, or can mean close physical proximity that is not equivalent to intimate mixture, solution or suspension, such as in separate containers in 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 nitrite salts (or some of them) and one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids (or some of them), respectively, can be stored separately or in separate containers of a kit, and brought together by mixing for use in initiating a reaction to produce NOx. 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 an organic polyol component, again mixed at the time of initiating the reaction to produce NOx. Any one or more of the components can itself exist in multiple parts and multiple containers. The combination can be brought into proximity in a manner that immediately initiates the reaction to produce NOx, such as because the nitrite and proton source are in the same solution, thus enabling the reaction. Alternatively, the combination can be brought into proximity in a manner that does not immediately initiate the reaction to produce NOx, but instead requires one or more additional steps or actions to be performed prior to initiation, such as because the nitrite and proton source are present as a dry powder mixture or as encapsulated particles, requiring water (such as from a mucous membrane in contact with the combination) prior to initiation of the reaction to produce NOx.

[0093] In embodiments, the first to sixth aspects of the present disclosure can be characterized independently of each other by only the above-mentioned feature (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 referring to (b) only; or feature (h) when referring to (c) only; or feature (h) when referring to (d) only; or feature (h) when referring to (e) only; or feature (h) when referring to (f) only; or feature (h) when referring to (g) only; or only features (a) and (b); or feature (h) when referring to features (a) and (b); or only features (a) and (c); or feature (h) when referring to features (a) and (c); or only features (a) and (d); or feature (h) when referring to features (a) and (d); or only features (a) and (e); or feature (h) when referring to features (a) and (e); or only features (a) and (f); or feature (h) when referring to features (a) and (f); or only features (a) and (g); or feature (h) when referring to features (a) and (g); or only features (b) and (c); or feature (h) when referring to features (b) and (c); or only features (b) and (d); or feature (h) when referring to features (b) and (d); or only features (b) and (e); or feature (h) when referring to features (b) and (e); or only features (b) and (f); or feature (h) when referring to features (b) and (f); or only features (a), (b), (c) and (f); or feature (h) when referring to features (a), (b), (c) and (f); or all of features (a) to (g); or features (a) and (b) together with feature (h) when referring to all of features (c) to (g).

[0094] In other embodiments, the first to sixth aspects of the present disclosure can be characterized independently of each other by only the above-mentioned features (c), (f) and (i); 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).

[0095] Preferably the first to sixth aspects of the present disclosure are characterised 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 just features (c), (f) and (i); or features just (c), (f) and (j); or features (i) and (h) when features (c) and (f) are involved; or features (j) and (h) when features (c) and (f) are involved; or just features (d), (g) and (i); or just features (d), (g) and (j); or features (i) and (h) when features (d) and (g) are involved; or features (j) and (h) when features (d) and (g) are involved; or just features (e), (f) and (i); or just features (e), (f) and (j); or features (i) and (h) when features (e) and (f) are involved; or features (j) and (h) when features (e) and (f) are involved. Note that when features (c) and (f) characterise the present disclosure, features (d), (e) and (g) are redundant; in that case, features (d), (e) and (g) (or (h) when features (d), (e) and (g) are involved) can be omitted from the list and are considered as instances of features (c) and (f) (or (h) when features (c) and (f) are involved).

[0096] The expression "one or more organic polyols that enhance the amount of reaction output" as used herein means that such amount of one or more organic polyols causes the amount of at least one of nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof and / or the time period from which the NOx is produced to be higher than a reaction conducted under the same conditions but without the one or more organic polyols. The expression "amount" means the total mass of gaseous nitric oxide that is evolved per gram of nitrite available for reaction in the starting reaction system. The experimental work underlying the present invention has measured the amount of gaseous nitric oxide, optionally also other gases, evolved and found that these amounts are enhanced. It is thus believed that the present invention enhances the total mass of NOx produced and it is also to be understood that the expression "amount" includes the total mass of nitric oxide dissolved in solution in the reaction mixture as well as the total mass of NOx reaction products. The expression "output time period" means inter alia the length of time during which at least one of gaseous nitric oxide, optionally also other gases, is evolved in the reaction before the reaction ends. For the same reasons as explained above in the discussion of the phrase "one or more organic polyols that enhance the amount of reaction output", it is believed that the phrase "output time period" also includes the length of time during which nitric oxide is dissolved in solution in the reaction mixture as well as the length of time during which NOx reaction products are produced. It is well known that the reaction with the proton source eventually depletes the nitrite, the elevated pH value during the NOx producing reaction reaches its maximum and the reaction stops. Preferably, the method of the first aspect of the present invention enhances the yield of the NOx producing reaction, inter alia but not exclusively the amount of NO produced, for example the amount of gaseous NO, by at least about 5%, for example by at least about 10%, for example by at least about 25%, for example by an enhancement of up to about 150%, for example by an enhancement of up to about 125%, for example by an enhancement of up to about 100%, for example by an enhancement of up to about 75%. Preferably, the method of the first aspect of the present invention enhances the length of time during which at least one of nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, preferably nitric oxide, is evolved in the reaction before the reaction ends, by at least about 5%, for example by at least about 10%. Using the present invention, the time period during which at least one of nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, preferably nitric oxide and most preferably gaseous nitric oxide, is evolved, in particular an effective amount, can be enhanced by at least about 2 hours, for example by at least about 5 hours, for example by up to or more than about 10 hours. The enhancement in the length of time during which nitric oxide is evolved can be expressed for example by an enhancement of up to or more than about 150% of the time period during which the same amount of nitric oxide is evolved without the use of the polyol component, for example by an enhancement of up to about 125%, for example by an enhancement of up to about 100%, for example by an enhancement of up to about 75%.

[0097] The production of nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof can be used for any purpose. Therapeutic and non-therapeutic purposes are exemplified and discussed below.

[0098] According to a seventh aspect, the present disclosure provides a therapeutic or non- therapeutic method of delivering nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof to a target location, e.g., any cell, organ, surface, structure, subject, or internal space therein, the method comprising: (a) administering to the target location or vicinity thereof a combination or composition according to the fifth aspect of the present disclosure; or (b) using a method according to the first or third aspects of the present disclosure, or performing a use according to the fourth aspect of the present disclosure, or using a combination, kit, or composition according to the fifth aspect of the present disclosure, to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, and delivering the nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof thus produced to the target location or vicinity thereof; or (c) delivering nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof according to the second aspect of the present disclosure to the target location or vicinity thereof.

[0099] The method of the seventh aspect of the present disclosure can be, for example, a method of treating a microbial infection in a subject in need thereof. The subject can be, for example, a human subject or other mammalian subject. The microbial infection can be, for example, a bacterial, viral, fungal, micro-parasitic, or any combination thereof.

[0100] The method of the seventh aspect of the present disclosure can be, for example, a method of vasodilation performed on a subject. The subject can be, for example, a human subject or other mammalian subject.

[0101] The method of the seventh aspect of the present disclosure can be, for example, an antimicrobial method. The antimicrobial method can be one that reduces the number of microorganisms, e.g., bacterial, viral, fungal cells, and / or micro-parasitic organisms, at a locus, prevents their multiplication, or limits the rate of their multiplication. The microorganisms targeted by such a method can be, for example, planktonic cells or particles or present in a biofilm or other colony. Any population of microorganisms targeted by the present disclosure, whether planktonic or not, can consist of one species or strain of microorganism, or can comprise more than one species or strain.

[0102] According to an eighth aspect, the present disclosure provides a combination, kit, or composition according to the fifth aspect of the present disclosure, or nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof according to the second aspect of the present disclosure, for use in therapy.

[0103] The combination, kit or composition or nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof for use according to the eighth aspect of the disclosure can be, for example, for use in a method of treatment which delivers nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof to a subject or an internal space therein, the method comprising: (a) administering to the subject or internal space or vicinity thereof a combination or composition according to the fifth aspect of the disclosure; or (b) generating nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof using a method according to the first or third aspect of the disclosure, or carrying out a use according to the fourth aspect of the disclosure, or using a combination, kit or composition according to the fifth aspect of the disclosure, and delivering the nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof thus generated to the subject or internal space or vicinity thereof; or (c) delivering nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof according to the second aspect of the disclosure to the subject or internal space or vicinity thereof.

[0104] According to the present disclosure, it was unexpectedly found that when the proton source is citric acid (an organic carboxylic acid) or ascorbic acid (an organic non-carboxylic reducing acid) with an initial pH in the range of 5 to 8, good antimicrobial activity in terms of biostatic and biocidal action is also provided, evidenced by up to 100% killing of M. abscessus after 3 days, and / or killing of M. tuberculosis, H1N1 influenza virus, SARS-CoV virus and SARS-CoV-2 virus. The expression "initial pH" herein refers to the pH of an aqueous solution in which the proton source is initially formed, including any required pH buffer, prior to the presence of other components of the reaction mixture which will affect the initial pH. This antimicrobial action 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 the action. This finding that the reaction products producing NOx with an initial pH in the range of 5 to 8 of an acid, such as citric acid or ascorbic acid, produces a strong antimicrobial action is particularly unexpected and holds promise for application in the treatment of respiratory and lung infections, including difficult to treat and / or antibiotic resistant infections, including tuberculosis, multi-drug resistant tuberculosis and non-tuberculous mycobacterial infections. Treatment of such infections via inhalation of an aqueous composition containing a reaction mixture or components or precursors thereof with a pH in the range of 5 to 8 can be suggested. The present invention is also capable of treating infections comprising a plurality of pathogens, possibly including pathogens from more than one group of bacteria, viruses, fungi and parasites, referred to as "broad spectrum" treatment (including therapeutic treatment and / or prophylactic treatment as well as in vitro treatment of animate and inanimate surfaces and spaces to prevent the spread of pathogens).

[0105] According to a ninth aspect, the present disclosure provides a modification of the antimicrobial method according to the seventh aspect, the modification comprising: (a) administering to the microorganism to be targeted or in the vicinity thereof or to a subject infected with the microorganism or to an internal space of such subject the combination or composition according to the fifth aspect of the present disclosure; or (b) using the method according to the first or third aspect of the present disclosure, or performing the use according to the fourth aspect of the present disclosure, or using the combination, kit or composition according to the fifth aspect of the present disclosure, producing nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof, and delivering the nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof thus produced to the microorganism to be targeted or in the vicinity thereof or to a subject infected with the microorganism or to an internal space of such subject; or (c) delivering the nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof according to the second aspect of the present disclosure to the microorganism to be targeted or in the vicinity thereof or to a subject infected with the microorganism or to an internal space of such subject; provided that the initial pH value of the aqueous solution comprising the proton source of any required buffer or the pH value of the reaction mixture at the start of the reaction with the nitrite(s) is in the range of 5 to 8 in the presence of the other components of the reaction mixture that will affect the pH value and that the one or more polyols are optional and can be omitted.

[0106] In performing the method according to the ninth aspect of the present disclosure, the combination, kit or composition according to the fifth or eighth aspect of the present disclosure can be used to produce nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof; provided that the initial pH value of the aqueous solution comprising the proton source of any required buffer or the pH value of the reaction mixture at the start of the reaction with the nitrite(s) is in the range of 5 to 8 in the presence of the other components of the reaction mixture that will affect the pH value and that the one or more polyols are optional and can be omitted.

[0107] The method of the ninth aspect of the present disclosure can be, for example, a method of treating a microbial infection in a subject in need thereof. The subject can be, for example, a human subject or other mammalian subject. The microbial infection can be, for example, a bacterial, viral, fungal, micro-parasitic infection or any combination thereof. The microbial infection can be on the skin of the subject, including mucous membranes. The microbial infection can be in an internal space of the subject, for example in the lining of the nose, mouth, respiratory tract, lungs or pleura of the lungs of the subject according to the present disclosure.

[0108] The components and mixtures used in all aspects of the present disclosure for administration to the human or animal body and any carriers and excipients for administration to the human or animal body used in all aspects of the present disclosure are preferably biocompatible and / or pharmaceutically acceptable to minimize irritation and inflammation to the tissue upon administration.

[0109] Combinations, kits, and compositions according to the present 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 the present disclosure are preferably carried out using such devices and apparatuses, as will be described in more detail below.

[0110] It will be understood that all embodiments, examples, and preferences specifically described in relation to any one or more aspects of the present disclosure are applicable to any one or more other aspects of the present disclosure. Additionally, any method or use according to one aspect of the present disclosure can be carried out using a combination, kit, or composition according to any other aspect, if necessary. DETAILED DESCRIPTION

[0111] Aspects of the present disclosure are now described in detail with reference to specific embodiments. The specific embodiments described below can be applicable to any aspect of the present disclosure, unless specifically noted otherwise. The specific embodiments can also be combined with each other, unless doing so would create a contradiction.

[0112] Nitrite and nitrite component

[0113] Aspects of the present disclosure relate to the use of one or more nitrite salts. In the following, the term "nitrite component" encompasses one or more nitrite salts as such, and any component containing one or more nitrite salts of a reaction system for the production of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof.

[0114] The choice of nitrite salt is not particularly limited. Particular examples of nitrite salts that can be used in the compositions of the present disclosure include alkali metal nitrite salts or alkaline earth metal nitrite salts. In some embodiments, the one or more nitrite salts are selected from the group consisting of LiN02, NaN02, KN02, RbN02, CsN02, FrN02, AgN02, Be(N02)2, Mg(N02)2, Ca(N02)2, Sr(N02)2, Mn(N02)2, Ba(N02)2, Ra(N02)2, and any mixture thereof.

[0115] In particular embodiments, the nitrite salt is NaN02or KN02. In one embodiment, the nitrite salt is NaN02.

[0116] In one embodiment, the nitrite component can be provided in dry form, optionally in particulate form such as a powder, for use in the present disclosure. If desired, the nitrite component can be encapsulated or microencapsulated, for example to control or delay the reaction between the one or more nitrites and the proton source. Dry form and / or encapsulation can facilitate storage of the nitrite component, whether alone or in admixture with other components of a reaction to generate nitric oxide according to the present disclosure. Still further, dry form and / or encapsulation can facilitate incorporation of the nitrite component into small objects such as medical devices, whether alone or in admixture with other components of a reaction to generate nitric oxide according to the present disclosure. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, heart assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles and inhalers (hand-held and nebulizers). See the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components" for further details.

[0117] The nitrite component, optionally encapsulated or microencapsulated as necessary, can be present as a dry powder or crystals, or in combination with a gel or other carrier system, e.g., an aqueous carrier, e.g., as an aqueous gel or solution thereof. The nitrite component in dry or powder form is preferably made into a solution by the addition of water prior to use. The molarity of nitrite ions in such a nitrite solution can range from about 0.001 M to about 5 M, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the acidification (e.g., immediately prior to the acidification). In some embodiments, the molarity of nitrite ions in the nitrite solution ranges from about 0.01 M to about 2 M, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the acidification (e.g., immediately prior to the acidification). In some embodiments, the molarity of nitrite ions in the nitrite solution ranges from about 0.1 M to about 2 M, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the acidification (e.g., immediately prior to the acidification). In more particular embodiments, the molarity of nitrite ions in the nitrite solution ranges from about 0.2 M to about 1.6 M, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the acidification (e.g., immediately prior to the acidification). In embodiments, the molarity of nitrite ions in the nitrite solution can range from 0.8 to 1.2 M, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the acidification (e.g., immediately prior to the acidification). For example, the molarity of nitrite ions in the nitrite solution 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, prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the combination with the organic carboxylic acid component (e.g., immediately prior to the combination).

[0118] It should be noted that the act of combining two or more precursor solutions of the NOx-generating reaction mixture will dilute the concentration of each solute or combination of solutes in each solution, as is well known to those skilled in the art. For example, the act of mixing two 1 M solutions of solutes A and B in equal volumes results in a concentration of 0.5 M for A and 0.5 M for B. Unless otherwise stated or implied, the concentrations of nitrite described herein are the concentrations in the initial solution prior to the addition of any other components of the NOx-generating reaction mixture added as a liquid, e.g., solution (e.g., immediately prior to the addition). The actual concentrations in the NOx-generating reaction mixture are readily derived given the known reaction mixture components and mode of preparation.

[0119] If desired, the nitrite component, whether in dry form or in a carrier liquid, can include one or more polyols or some of such polyols.

[0120] If it is desired that the nitrite component be stored as a gel or other carrier system, such as an aqueous carrier, e.g., as an aqueous gel or solution, then preferably the nitrite-containing system is buffered to an appropriate pH to prevent degradation of the nitrite during storage. A pH of about 6-9, e.g., about 7, is preferred.

[0121] Preferably, the nitrite component is not contacted with the proton source until it is desired to generate nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof. To this end, the nitrite component is preferably stored in a reservoir or container of a kit, device, or apparatus. Alternatively, however, the dry components of the nitrite component, the proton source, and one or more polyols can be stored as a dry composition, e.g., a particulate mixture, and the reaction initiated by simply adding water or another suitable solvent or liquid carrier.

[0122] The nitrite can be a pharmaceutically acceptable grade of nitrite. In some embodiments, the nitrite is a pharmacopoeial grade. In other words, the nitrite can follow one or more current pharmacopoeial monographs for nitrites. For example, the nitrite can follow the monograph for nitrites in one or more of the United States Pharmacopeia (USP), the European Pharmacopoeia, or the Japanese Pharmacopoeia.

[0123] In particular embodiments, the nitrite used is characterized by one or more of the following limitations:

[0124] (i) the nitrite contains at most about 0.02 wt%, about 0.01 wt%, or about 0.001 wt% sodium carbonate;

[0125] (ii) the nitrite contains at most about 10 ppm (0.001 wt%) of an anti-caking agent, such as sodium alkyl-naphthalene sulfonate;

[0126] (iii) the nitrite is a white to off-white solid;

[0127] (iv) the nitrite has a positive identification for cations as determined according to the relevant method in the relevant USP;

[0128] (v) the nitrite has a positive identification test for nitrite as determined according to the relevant method in the relevant USP;

[0129] (vi) optionally contains no less than about 97% or no less than 98% by weight of nitrite and / or no more than 102% or no more than 101% by weight of nitrite, as determined, for example, by ion chromatography, e.g., ion chromatography coupled with suppressed conductivity detection, such as by the relevant USP calorimetric assay;

[0130] (vii) optionally has a pH value of between about 7 and about 9 or between about 8 and about 9 when measured in a 10% solution at 25°C, as measured according to the relevant USP and / or using a pH meter;

[0131] (viii) has a loss on drying of no more than about 0.25% or about 0.01% by weight;

[0132] (ix) optionally has a water content of no more than about 0.5% by weight, as determined, for example, by the Karl Fischer method;

[0133] (x) has a heavy metal content of no more than about 10 ppm of heavy metals in the nitrite, optionally, no more than about 10 ppm of heavy metals in the nitrite;

[0134] (xi) contains no more than about 0.4% by weight of nitrates, optionally no more than about 0.4% by weight of sodium nitrate when the nitrite is sodium nitrite and no more than about 0.4% by weight of potassium nitrate when the nitrite is potassium nitrite;

[0135] (xiii) contains no more than about 0.005% or about 0.001% by weight of insoluble material;

[0136] (xiii) contains no more than about 0.005% by weight of chlorides;

[0137] (xiv) contains no more than about 0.01% by weight of sulfates;

[0138] (xv) contains no more than about 0.001% by weight of iron;

[0139] (xvi) contains no more than about 0.01% by weight of calcium;

[0140] (xvii) contains no more than about 0.005% or about 0.001% by weight of potassium when the nitrite is not potassium nitrite or no more than about 0.005% or about 0.001% by weight of sodium when the nitrite is not sodium nitrite;

[0141] (xviii) the nitrite salt contains at most about 0.1 wt%, at most about 5000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of organic volatile compounds;

[0142] (xix) the nitrite salt contains at most about 0.1 wt%, at most about 5000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of ethanol;

[0143] (xx) the nitrite salt contains at most about 3000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of methanol;

[0144] (xxi) the nitrite salt contains at most about 50 ppm, at most about 25 ppm, at most about 20 ppm, at most about 10 ppm, at most about 7.9 ppm, at most about 8 ppm, at most about 6 ppm, at most about 5.6 ppm, or at most about 2.5 ppm of non-volatile organic carbon;

[0145] (xxii) the nitrite salt contains at most about 0.05 ppm of mercury;

[0146] (xxiii) the nitrite salt contains at most about 2 ppm or 0.2 ppm of aluminum;

[0147] (xxiv) the nitrite salt contains at most about 3 ppm or 1 ppm of arsenic;

[0148] (xxv) the nitrite salt contains at most about 0.003 wt% or about 0.001 wt% of selenium;

[0149] (xxvi) the total aerobic count of the microbial load in the nitrite salt is at most about 100 CFU / g;

[0150] (xxvii) the total yeast and mold count in the nitrite salt is at most about 20 CFU / g;

[0151] (xxviii) the nitrite salt contains at most about 0.25 EU / mg or 0.018 EU / mg of bacterial endotoxins; and

[0152] (xxix) the nitrite salt contains less than about 0.1 ppm of phosphate salts, such as sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate, and preferably, the nitrite salt does not contain a detectable amount of phosphate salts.

[0153] In certain embodiments, the nitrite salt has two or more of properties (i) through (xxix). In other embodiments, the nitrite salt has five or more of properties (i) through (xxix). In other embodiments, the nitrite salt has ten or more of properties (i) through (xxix). In other embodiments, the nitrite salt has fifteen or more of properties (i) through (xxix). In some embodiments, the nitrite salt has twenty or more of properties (i) through (xxix). In one specific embodiment, the nitrite salt has all of properties (i) through (xxix). In one more specific embodiment, the nitrite salt is sodium nitrite having all of properties (i) through (xxix).

[0154] In some embodiments, the nitrite salt contains, optionally as determined by the relevant USP calorimetric assay, e.g., as determined by ion chromatography, e.g., ion chromatography coupled with suppressed conductivity detection, nitrite salt in the range of about 97% to about 101% by weight. In alternative embodiments, the nitrite salt contains, optionally as determined by the relevant USP calorimetric assay, e.g., as determined by ion chromatography, e.g., ion chromatography coupled with suppressed conductivity detection, nitrite salt in the range of about 98% to about 102% by weight.

[0155] In specific embodiments, the nitrite salt has the following properties:

[0156] (i) the nitrite salt contains at most about 0.02% by weight sodium carbonate;

[0157] (ii) the nitrite salt contains at most about 10 ppm anti-caking agent;

[0158] (vi) the nitrite salt contains, as determined by the USP calorimetric assay, not less than 97% by weight nitrite salt and at most 101% by weight nitrite salt;

[0159] (viii) the nitrite salt has a loss on drying of at most about 0.25% by weight;

[0160] (ix) the nitrite salt has a water content of at most about 0.5% by weight;

[0161] (x) the heavy metal content in the nitrite salt is at most about 10 ppm;

[0162] (xi) the nitrite salt contains at most about 0.4% by weight nitrate salt;

[0163] (xii) the nitrite salt contains at most about 0.005% by weight insoluble material;

[0164] (xiii) the nitrite salt contains at most about 0.005% by weight of chloride;

[0165] (xiv) the nitrite salt contains at most about 0.01% by weight of sulfate;

[0166] (xv) the nitrite salt contains at most about 0.001% by weight of iron;

[0167] (xvi) the nitrite salt contains at most about 0.01% by weight of calcium;

[0168] (xviii) the nitrite salt contains at most about 5000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of organic volatile compounds;

[0169] (xxi) the nitrite salt contains at most about 10 ppm or at most about 2.5 ppm of non-volatile organic carbon;

[0170] (xxii) the nitrite salt contains at most about 0.05 ppm of mercury;

[0171] (xxiii) the nitrite salt contains at most about 2 ppm of aluminum;

[0172] (xxiv) the nitrite salt contains at most about 3 ppm of arsenic;

[0173] (xxv) the nitrite salt contains at most about 0.003% by weight of selenium;

[0174] (xxvi) the total aerobic count of the microbial load in the nitrite salt is at most about 100 CFU / g;

[0175] (xxvii) the total yeast and mold count in the nitrite salt is at most about 20 CFU / g; and

[0176] (xxviii) the nitrite salt contains at most about 0.25 EU / mg of bacterial endotoxins.

[0177] In these embodiments, the nitrite salt can be sodium nitrite and contains at most about 0.005% by weight of potassium. Preferably, the sodium nitrite also has one or more of the following limitations:

[0178] (iii) the sodium nitrite is a white to off-white solid;

[0179] (iv) the sodium nitrite has a positive identification for sodium as determined according to the relevant method in the relevant USP;

[0180] (v) the sodium nitrite has a positive identification test for nitrite as determined according to the relevant method in the relevant USP;

[0181] (vii) optionally, the pH of the sodium nitrite is between about 7 and about 9 or between about 8 and about 9 when measured in a 10% solution at 25°C, as measured according to the relevant USP and / or using a pH meter;

[0182] (xix) the sodium nitrite contains at most about 0.1 wt%, at most about 5000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of ethanol;

[0183] (xx) the nitrite salt contains at most about 3000 ppm, at most about 1000 ppm, at most about 500 ppm, at most about 100 ppm, or at most about 10 ppm of methanol; and

[0184] (xxix) the nitrite salt contains less than about 0.1 ppm of a phosphate salt, such as sodium phosphate, disodium hydrogen phosphate, or trisodium phosphate, and preferably, the nitrite salt does not contain a detectable amount of a phosphate salt.

[0185] Properties (i) to (xxix) can be determined according to the relevant methods in USP XXXII (2009). Methods for determining properties (i) to (xxix) are provided in WO 2010 / 093746 (the disclosure of which is incorporated by reference herein in its entirety). Methods of making sodium nitrite having one or more of properties (i) to (xxix) are also described in WO 2010 / 093746.

[0186] Proton source and proton source component comprising one or more organic carboxylic acids

[0187] Aspects of the present disclosure relate to a proton source comprising one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids. Hereinafter, the term "proton source component" encompasses the proton source itself, and any component of a reaction system for the production of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, that contains the proton source.

[0188] In this section, organic carboxylic acids will be exemplified in more detail.

[0189] The expression "organic carboxylic acid" herein refers to any organic acid that contains one or more -COOH groups in the molecule. The organic carboxylic acid can be straight-chain or branched. The carboxylic acid can be saturated or unsaturated. The carboxylic acid can be aliphatic or aromatic. The carboxylic acid can be acyclic or cyclic. The carboxylic acid can be a vinylic carboxylic acid.

[0190] The organic carboxylic acid can carry one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids that can be used in the present disclosure include a-hydroxy-carboxylic acids, β-hydroxy-carboxylic acids, and γ-hydroxy-carboxylic acids.

[0191] The one or more organic carboxylic acids, or each of them if there is more than one, should preferably have a pKa1 of less than about 7, more preferably less than 7.0.

[0192] The one or more carboxylic acids can be, comprise or consist of one or more reduced carboxylic acids.

[0193] The carboxylic acid can be an acid hydrogel containing a -COOH side group covalently attached to the polymer molecules of the three-dimensional polymer matrix forming the hydrogel. Examples of such hydrogels containing carboxylic acids are described in, for example, WO 2007 / 007115, WO 2008 / 087411, WO 2008 / 087408, WO 2014 / 188174, and WO 2014 / 188175, and the literature mentioned therein, the disclosures of all of which are incorporated herein by reference. Such hydrogels typically comprise 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 entitled "Other Reservoirs of Components: Hydrogels" below.

[0194] However, generally preferably, at least one of the acid of one or more selected from organic carboxylic acids and organic non-carboxylic acid reducing acids is not covalently bonded to polymer or macromolecule, such as the three-dimensional polymer of forming hydrogel or the polymer or macromolecule of macromolecular matrix. Without wishing to be bound by theory, evidence, such as the evidence of the dependence of the stereoisomerism of the effect of polyol discussed in the chapters and sections entitled " organic polyol " hereinafter, shows that the effect of the output of the reaction of one or more nitrites enhancing the present disclosure and proton source is realized at least in part by the effect of the organic polyol molecule that interacts with nitrite and proton during acidification reaction, which implies that the mobility of reactant molecule orientation and relocation under the influence of polyol molecule during reaction can be important. Even if polyol does not necessarily exist, such as in the eighth aspect of the present disclosure, it can also be inferred that the same mobility between reactants in the reaction of one or more nitrites and proton source can be important.

[0195] The organic carboxylic acid can for example be selected 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, a-hydroxypropionic acid, β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic acid (embonic acid), stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobionic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, salts thereof and combinations thereof. In a particular embodiment, the organic carboxylic acid is selected from citric acid, salts thereof and combinations thereof. In a particular embodiment, the organic carboxylic acid is citric acid or a salt thereof. The carboxylic acid can be or comprise a polymer or polymeric carboxylic acid, for example polyacrylic acid, polymethacrylic acid, a copolymer of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid or a copolymer of lactic acid and glycolic acid. The term "organic carboxylic acid" as used herein also encompasses partial or complete esters of organic carboxylic acids or partial or complete salts thereof, provided that those esters or salts can be used as proton source for use according to the present application.

[0196] Preferably, the pH of the proton source is buffered at the point of contact between the one or more nitrite salts and the proton source to control the pH within a known range and limit the rate of increase of the pH as the nitrite salts are consumed. Further details are provided in the section below entitled "pH control; optional buffering system". In particular, it is envisaged that at least one organic carboxylic acid of the proton source is adapted to be present with its conjugate base. The acid and its conjugate base are adapted to form a buffer in the aqueous carrier. The buffer can be selected such that it maintains the desired pH as the NOx generating reaction proceeds, preferably the pH is in the range of about 3 to 9, for example about 4 to 8, 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 if present, or 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.

[0197] The initial pH of the aqueous solution of the proton source including any required buffer prior to the addition of other components of the NOx generating reaction mixture which will affect the pH (e.g. immediately prior to addition) or the pH of the reaction mixture at the start of the reaction with the one or more nitrite salts is suitably in the range of about 3 to 9, for example about 4 to 8, for example about 5 to 8. The expression "initial pH" as used herein in connection with the proton source means the pH of the aqueous solution of the proton source including any required buffer prior to the addition of other components of the NOx generating reaction mixture which will affect the pH (e.g. immediately prior to addition). Dry powdered proton source material or other precursors of the aqueous solution of the proton source will be used in amounts appropriate to produce an aqueous solution having the desired initial pH.

[0198] If it is desired that the proton source component be stored in a gel or other carrier system, such as an aqueous carrier, e.g., in the form of an aqueous gel or solution, then preferably the proton source-containing system is buffered to an appropriate pH to maintain the acidity of the proton source during storage and to prevent degradation of the proton source. A pH of about 3-6, e.g., about 3-5, is preferred. If necessary, the pH can be raised by the addition of a base just prior to use of the proton source component.

[0199] Some patients are intolerant of citric acid, for example. Patients should be tested for possible intolerance to the acid prior to treatment, and the acid component should be selected accordingly.

[0200] In one embodiment, the proton source component or portions thereof can be provided in dry form, optionally in particulate form such as a powder, for use in the present disclosure. If desired, the proton source component or portions thereof can be encapsulated or microencapsulated, e.g., to control or delay the reaction between the nitrite(s) and the proton source. Encapsulated forms can be used especially when the proton source typically has a liquid or gel state at room temperature. Dry form and / or encapsulation can facilitate storage of the proton source, whether alone or in admixture with other components of a reaction for producing nitric oxide according to the present disclosure. Still further, dry form and / or encapsulation can facilitate incorporation of the proton source component into small objects such as medical devices, whether alone or in admixture with other components of a reaction for producing nitric oxide according to the present disclosure. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, heart-assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. See the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components" for further details.

[0201] The one or more organic carboxylic acids, optionally encapsulated or microencapsulated, can be present in the proton source component as a dry powder or crystals, or in combination with a gel or other carrier system, e.g., an aqueous carrier, e.g., as an aqueous gel or solution thereof, if desired. Proton source components containing organic carboxylic acids in dry or powder form are preferably made into solution by the addition of water prior to use. The molar concentration of total proton source (including any organic non-carboxylic acid reducing acids present) in such a solution can range from about 0.001 M to about 5 M prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the initiation of the reaction with nitrite (e.g., immediately prior to the initiation). In some embodiments, the molar concentration of total proton source in such a solution ranges from about 0.01 M to about 2 M prior to the addition of any other components of the NOx-generating reaction mixture (e.g., immediately prior to the addition) and especially prior to the initiation of the reaction with nitrite (e.g., immediately prior to the initiation). In some embodiments, the molar concentration of total proton source in such a solution ranges from about 0.1 M to about 2 M prior to the initiation of the reaction with nitrite. In more particular embodiments, the molar concentration of total proton source in such a solution ranges from about 0.2 M to about 1.6 M prior to the initiation of the reaction with nitrite. In embodiments, the molar concentration of total proton source in such a solution can range from 0.8 to 1.2 M prior to the initiation of the reaction with nitrite. For example, the molar concentration of total proton source in such a solution 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 prior to the initiation of the reaction with nitrite.

[0202] The expressions "molar concentration of total proton source," "concentration of total proton source," and the like, as used herein, are understood to refer to the concentration of either of the organic carboxylic acid and / or the organic non-carboxylic acid used as the proton source according to the present application at a pH value at which the proton (H+) donor moiety or at least one of the proton (H+) donor moieties (in the case of more than one) is mostly protonated, i.e., more than 50% protonated on a molar basis. In other words, if the pH value is adjusted to a higher pH value prior to the initiation of the NOx-generating reaction, whereby the degree of protonation is reduced, then the molar concentration or concentration of total proton source is not considered to be correspondingly reduced.

[0203] It should be noted that the act of combining two or more precursor solutions of a NOx-generating reaction mixture will dilute the concentration of each solute or combination of solutes in each solution, as is well known to those skilled in the art. For example, the act of mixing two 1 M solutions of solutes A and B in equal volumes results in a concentration of A of 0.5 M and a concentration of B of 0.5 M. Unless otherwise stated or implied, the concentration of a proton source described herein is the concentration it has in the initial solution prior to the addition of any other components of the NOx-generating reaction mixture in liquid, e.g., solution, form (e.g., at the point of addition). The actual concentration in the NOx-generating reaction mixture is readily derived, given the reaction mixture components and mode of preparation.

[0204] Proton source components in dry or powder form are preferably made into solutions by the addition of water prior to use.

[0205] If desired, one or more organic carboxylic acids, whether in dry form or in a carrier liquid, can be mixed with or form a solution with one or more polyols or some of such polyols

[0206] Preferably, the nitrite component is not contacted with the proton source until it is desired to generate nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof. To this end, the proton source component or a portion thereof is preferably held in a reservoir or container of a kit, device, or apparatus. Alternatively, however, one or more nitrite or nitrite components, the proton source, and one or more polyols can be held in dry combination, e.g., as a particulate mixture, and the reaction initiated by the simple addition of water or another suitable solvent or liquid carrier.

[0207] Proton source component comprising one or more organic non-carboxylic acid reductive acids

[0208] The above discussion of proton source components comprising or consisting of one or more organic carboxylic acids applies similarly to proton source components comprising or consisting of one or more organic non-carboxylic reducing acids. Organic non-carboxylic reducing acids will be exemplified in more detail in this section.

[0209] The expression "organic non-carboxylic reducing acid" herein refers to any organic reducing acid that does not contain a -COOH group in the molecule. The organic non-carboxylic reducing acid can be straight-chained or branched. The non-carboxylic reducing acid can be saturated or unsaturated. The non-carboxylic reducing acid can be aliphatic or aromatic. The non-carboxylic reducing acid can be acyclic or cyclic. The non-carboxylic reducing acid can be vinylic.

[0210] The one or more organic non-carboxylic reducing acids, or each of them if there is more than one, should preferably have a pKai of less than about 7, more preferably less than 7.0.

[0211] For the above-explained reasons, it is generally preferred that at least one of the one or more acids selected from organic carboxylic acids and organic non-carboxylic reducing acids is not covalently bound to the polymer molecules, e.g. to the polymer molecules forming the three-dimensional polymer matrix of the hydrogel.

[0212] The organic non-carboxylic reducing acid can for example be selected from ascorbic acid; ascorbyl palmitate (ascorbyl palmitate); ascorbic acid derivatives such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid and 6-O-dodecanedioyl ascorbic acid; acidic reducing ketones such as reductic acid; erythorbic acid; oxalic acid; salts thereof; and combinations thereof. In a particular embodiment, the organic non-carboxylic reducing acid is ascorbic acid or a salt thereof.

[0213] The organic non-carboxylic reducing acid can carry one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic reducing acids that can be used in the present disclosure include acidic reducing ketones such as reductic acid (2.3-dihydroxy-2-cyclopentanone).

[0214] Preferably, the pH of the proton source and / or the reaction mixture is buffered after contact between the one or more nitrite salts and the proton source to control the pH within a known range and to control the increase in pH as the nitrite salt is consumed. See the section below entitled "pH control; optional buffering system" for further details. In particular, it is envisaged that at least one organic non-carboxylic 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 in the aqueous carrier. The buffer can be selected such that the desired pH is thereby maintained as the NO-generating reaction proceeds, preferably the pH is in the range of about 3 to 9, such as about 4 to 8, 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 if present, or can be generated on site from the proton source by adjusting the pH using an acid and / or a base, preferably an inorganic acid and / or an inorganic base.

[0215] The initial pH of the aqueous solution of the proton source including any required buffer, prior to the addition of other components of the NOx-generating reaction mixture that will affect the pH (e.g. immediately prior to addition) or the pH of the reaction mixture at the start of the reaction with the one or more nitrite salts is suitably in the range of about 3 to 9, such as about 4 to 8, such as about 5 to 8. Dry powder proton source material or other precursors of the aqueous solution of the proton source will be used in amounts suitable to produce an aqueous solution having the desired initial pH.

[0216] If it is desired that the proton source component be stored as a gel or other carrier system, such as an aqueous carrier, e.g., as an aqueous gel or solution, then preferably the system containing the proton source is buffered to an appropriate pH to maintain the acidity of the proton source during storage and to prevent degradation of the proton source. A pH of about 3 to 6, e.g., about 3 to 5, is preferred. If necessary, the pH can be increased by the addition of a base just prior to use of the proton source component.

[0217] Some reducing acids, such as oxalic acid, are toxic. The acid component is accordingly selected with this in mind.

[0218] In addition to or instead of one or more organic carboxylic acids in the manner described above, one or more organic non-carboxylic reducing acids can be used in the proton source component. Further details are found in the section entitled "Proton sources and proton source components comprising one or more organic carboxylic acids".

[0219] Organic polyols and organic polyol component

[0220] Aspects of the disclosure relate to one or more organic polyols. In the following, the term "organic polyol component" or "polyol component" encompasses the organic polyol per se, and any component containing the organic polyol of a reaction system for the production of nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof.

[0221] The expression "organic polyol" herein refers to an organic molecule having two or more hydroxyl groups, which is not a proton source, and in particular for the nitrite reaction, which is also not a sugar or polysaccharide (the terms "sugar" and "polysaccharide" include oligosaccharides, polysaccharides and aminopolysaccharides). Thus, the organic polyol will have a pKai of about 7 or greater, e.g., 7.0 or greater.

[0222] The expression "organic polyol" herein preferably excludes reducing agents. In one embodiment of the invention, the organic polyol excludes reducing agents in all its aspects. Examples of reducing agents which are organic molecules having two or more hydroxyl groups but which are not sugars or polysaccharides are thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate, erythorbic acid and erythorbate. Thus, thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate and erythorbate are preferably excluded from the expression "organic polyol" because they are reducing agents. Ascorbic acid and erythorbic acid are in any case excluded from the expression because they are proton sources, in particular for the nitrite reaction. To avoid doubt, it is confirmed that reducing agents which are proton sources, such as ascorbic acid and / or erythorbic acid, are excluded from the proton source of the invention or from the proton source component, combination, kit, composition, use, method or any other part of the invention and from the means of putting into practice in which they are present as proton sources.

[0223] The organic polyols can be cyclic or acyclic, or can be a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, the 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 polyols do not carry any substituents other than OH groups.

[0224] Preferably, the one or more organic polyols is one or more acyclic organic polyols. Preferred one or more acyclic organic polyols are selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferred one or more acyclic organic polyols are selected from alditols, for example alditols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Preferably, the one or more organic polyols do not include saponins, sapogenins, steroids, or steroid glycosides.

[0225] Alternatively, the one or more organic polyols can be one or more cyclic organic polyols. In these embodiments, the one or more cyclic organic polyols can be cyclic sugar alcohols or cyclic alditols. For example, the one or more cyclic polyols can be cyclic sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms or cyclic alditols having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. A particular example of a cyclic polyol is inositol.

[0226] In some embodiments, the one or more organic polyols have 7 or more hydroxyl groups. In specific embodiments, the one or more organic polyols are sugar alcohols or alditols having 7 or more hydroxyl groups. In more specific embodiments, the one or more organic polyols have 9 or more hydroxyl groups. In other embodiments, the one or more organic polyols are sugar alcohols or alditols having 9 or more hydroxyl groups. In some embodiments, the one or more organic polyols have 20 or fewer hydroxyl groups. In specific embodiments, the one or more organic polyols are sugar alcohols or alditols having 20 or fewer hydroxyl groups. In more specific embodiments, the one or more organic polyols have 15 or fewer hydroxyl groups. In other embodiments, the one or more organic polyols are sugar alcohols or alditols having 15 or fewer hydroxyl groups. The one or more organic polyols can have a number of hydroxyl groups in the range of 7 to 20, more specifically in the range of 9 to 15. In certain embodiments, the one or more organic polyols include 9, 12, 15, or 18 hydroxyl groups.

[0227] Preferably, the one or more organic polyols are sugar alcohol compounds comprising, for example consisting of, one or more monosaccharide units and one or more acyclic sugar alcohol units. The one or more organic polyols can be sugar alcohol compounds comprising, for example consisting of, 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.

[0228] A monosaccharide unit, as used herein, refers to a monosaccharide covalently linked to at least one other unit in the compound, whether another monosaccharide unit or an acyclic sugar alcohol unit. An acyclic sugar alcohol unit, as used herein, refers to an acyclic sugar alcohol covalently linked to at least one other unit in the compound, whether a monosaccharide unit or another acyclic sugar alcohol unit. The units in the compound can be linked by ether bonds. In some embodiments, the one or more monosaccharide units are covalently linked to other units of the compound by glycosidic bonds. In specific embodiments, each monosaccharide unit is covalently linked to other units of the compound by glycosidic bonds. In certain embodiments, the sugar alcohol compound is a glycoside having a monosaccharide or oligosaccharide glycone and an acyclic sugar alcohol aglycone.

[0229] A preferred acyclic sugar alcohol unit is a sugar alcohol unit having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. In specific embodiments, the acyclic sugar alcohol unit is selected from the group consisting of units of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, and volemitol.

[0230] In specific embodiments, the one or more monosaccharide units are C5 or C6 monosaccharide units. In other words, the one or more monosaccharide units are pentose or hexose units. In more specific embodiments, each monosaccharide unit is a C5 or C6 monosaccharide unit. In specific embodiments, the one or more sugar alcohol units are C5 or C6 sugar alcohol units. In more specific embodiments, each sugar alcohol unit is a C5 or C6 sugar alcohol unit.

[0231] In certain embodiments, the sugar alcohol compound comprises, e.g., consists of, n monosaccharide units and m acyclic sugar alcohol units, where n is an integer, at least 1, m is an integer, at least 1, and (n+m) is at most 10. In certain embodiments, the sugar alcohol compound comprises, e.g., consists of, a chain of n monosaccharide units capped with one acyclic sugar alcohol unit, 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 particular embodiments, each monosaccharide unit is covalently linked to another monosaccharide unit or acyclic sugar alcohol unit by a glycosidic bond. In certain embodiments, the sugar alcohol compound comprises, e.g., consists of, a chain of 1, 2, or 3 monosaccharide units capped with one acyclic alcohol unit. The 1, 2, 3, or each monosaccharide unit can be a C5 or C6 monosaccharide unit. The acyclic alcohol unit can be a C5 or C6 sugar alcohol unit. Examples of sugar alcohol compounds include, but are not limited to: isomalt, maltitol, and lactitol (n = 1); maltotriitol (n = 2); and maltotetraitol (n = 3).

[0232] Such sugar alcohol compounds can be described as sugar alcohols derived from a disaccharide or oligosaccharide. As used herein, oligosaccharide refers to a sugar consisting of three to ten monosaccharide units. Sugar alcohols derived from a disaccharide or oligosaccharide can be synthesized from a disaccharide, oligosaccharide, or polysaccharide (e.g., from hydrolysis and hydrogenation), but are not limited to compounds synthesized from a disaccharide, oligosaccharide, or polysaccharide. For example, a sugar alcohol derived from a disaccharide can be formed from a dehydration reaction of a monosaccharide and a sugar alcohol. One or more organic polyols can be a sugar alcohol derived from a disaccharide, a trisaccharide, or a tetrasaccharide. Examples of sugar alcohols derived from a disaccharide include, but are not limited to, isomalt, maltitol, and lactitol. One example of a sugar alcohol derived from a trisaccharide includes, but is not limited to, maltotriitol. One example of a sugar alcohol derived from a tetrasaccharide includes, but is not limited to, maltotetraitol.

[0233] As suitable organic polyols, any one selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and any combination thereof can be mentioned. Glycerol can be used, and when present, is preferably combined with one or more other organic polyols, such as erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, or any combination thereof.

[0234] Many organic polyols contain one or more chiral centers and thus exist in stereoisomeric forms. All stereoisomeric forms and optical isomers and isomer mixtures of the intended organic polyols are included within the scope of the present disclosure and patent. In particular, the D and / or L forms of all chiral organic polyols and all mixtures thereof can be used.

[0235] Interestingly, it has been found that the effect of using polyols in the present disclosure is stereochemically dependent. Thus, the selection of the optical isomeric form or optical isomer mixture of the one or more organic polyols used in the present disclosure can affect the outcome of the reaction between the nitrite and the proton source, at least in terms of the amount of NO produced.

[0236] For example, sorbitol is a stereoisomer of mannitol, differing from one another by the orientation of one hydroxyl group. As shown in Examples 2D and 2E below, Figure 5 and 6 ), sorbitol and mannitol differ in their effect on the reaction output between the nitrite and the proton source in otherwise identical reaction systems.

[0237] In particular embodiments, the organic polyol is selected from the group of arabitol, xylitol, mannitol, sorbitol, and any combination thereof. The arabitol can be D or L arabitol or mixtures thereof. The xylitol can be D or L xylitol or mixtures thereof. The sorbitol can be D or L sorbitol or mixtures thereof. The mannitol can be D or L mannitol or mixtures thereof.

[0238] In particular embodiments, 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, the one or more polyols are saccharol compounds comprising one or more monosaccharide units and one or more acyclic sugar alcohol units, including sugar alcohols derived from a disaccharide or oligosaccharide, for example consisting of these units, as described herein.

[0239] In one embodiment, the organic polyol component can be provided in dry form, optionally in particulate form such as a powder, for use in the present disclosure. If desired, the organic polyol can be encapsulated or microencapsulated, for example to control or delay the participation of the polyol in the reaction between the nitrite salt(s) and the proton source. Encapsulated form can be used especially when the organic polyol normally has a liquid or gel state at room temperature. Dry form and / or encapsulation can facilitate storage of the organic polyol component, whether alone or in admixture with other components of a reaction for producing nitric oxide according to the present disclosure. Still further, dry form and / or encapsulation can facilitate incorporation of the organic polyol component into small objects such as medical devices, whether alone or in admixture with other components of a reaction for producing nitric oxide according to the present disclosure. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, heart-assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles. See the section below entitled "Optional Encapsulation (e.g., Microencapsulation) of Components" for further details.

[0240] Alternatively, the organic polyol component can include a carrier medium, such as an aqueous carrier liquid or a gel carrier. If the organic polyol is normally a liquid at room temperature, it can be used as the carrier medium, without any additional carrier components, or it can be used in admixture with one or more carrier additives such as water.

[0241] The one or more organic polyols, optionally encapsulated or microencapsulated, can be present in the polyol component as a dry powder or crystals, or in combination with a gel or other carrier system, e.g., an aqueous carrier, e.g., as an aqueous gel or solution thereof, if desired. Polyol components containing organic polyols in dry or powder form are preferably made into solution by the addition of water prior to use. The molar concentration of total polyol or polyols in such a solution prior to initiation of the reaction with nitrite can be any concentration up to the saturation limit of the polyol or each polyol in solution. For example, the molar concentration of total organic polyol or polyols can range from about 0.001 M to about 5 M. In some embodiments, the molar concentration of total polyol or polyols in such a solution prior to initiation of the reaction with nitrite ranges from about 0.01 M to about 2 M. In some embodiments, the molar concentration of total polyol or polyols in such a solution prior to initiation of the reaction with nitrite ranges from about 0.1 M to about 2 M. In more specific embodiments, the molar concentration of total polyol or polyols in such a solution prior to initiation of the reaction with nitrite ranges from about 0.2 M to about 1.6 M. In embodiments, the molar concentration of total polyol or polyols in such a solution prior to initiation of the reaction with nitrite can range from 0.8 to 1.2 M. For example, the molar concentration of total polyol or polyols in such a solution prior to initiation of 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.

[0242] It should be noted that the act of combining two or more precursor solutions of the NOx-generating reaction mixture will dilute the concentration of each solute or combination of solutes in each solution, as is well known to those skilled in the art. For example, the act of mixing two 1 M solutions of solutes A and B in equal volumes results in a concentration of 0.5 M for A and 0.5 M for B. Unless otherwise stated or implied, the concentrations of organic polyols described herein are the concentrations in the initial solution prior to the addition of any other components added in liquid, e.g., solution, form, e.g., at the time of addition. The actual concentrations in the NOx-generating reaction mixture are readily derived given the known reaction mixture components and mode of preparation.

[0243] The polyol component in dry or powder form is preferably made into solution by the addition of water prior to use.

[0244] If desired, the polyol, whether in dry form or in carrier liquid, can be mixed with or formed into solution with one or more nitrite salts or proton sources or some of such proton sources.

[0245] In specific embodiments in which the nitrite is kept separate from the other components of the nitric oxide generating reaction until just prior to use, the nitrite component can include one or more polyols. In these embodiments, the organic carboxylic acid component can be substantially free of polyols. In alternative embodiments, the organic carboxylic acid component includes one or more polyols. In these embodiments, the nitrite component can be substantially free of polyols. In other embodiments, each of the organic carboxylic acid component and the nitrite component can include one or more polyols, which can be the same or different between the two components.

[0246] In another embodiment, the organic carboxylic acid component and the nitrite component can be substantially free of polyols and one or more polyols can be included in a separate polyol component.

[0247] Relative concentrations of nitrite, proton source and any polyol in the reaction mixture

[0248] The total molarity of any one or more organic polyols in the polyol component or in the reaction solution at the start of (or prior to initiation of) the NOx generating reaction is suitably between about 0.05 times and about 3 times the total molarity of nitrite ions in the nitrite component or in the reaction solution, for example between about 0.1 times and about 2 times the total molarity of nitrite ions, for example between about 0.25 times and about 1.5 times, for example between about 0.3 times and about 1.2 times the total molarity of nitrite ions. The same relative molarity between the one or more organic polyols and the nitrite ions is suitably provided in the components of the combination or kit according to the application or in the composition according to the application prior to (or immediately prior to) initiation of the NOx generating reaction.

[0249] The total molarity of any one or more organic polyols in the polyol component or in the reaction solution at the start of (or prior to initiation of) the NOx generating reaction is suitably between about 0.05 times and about 3 times the total molarity of the proton source in the proton source component or in the reaction solution, for example between about 0.1 times and about 2 times the total molarity of the proton source. The same relative molarity between the one or more organic polyols and the proton source is suitably provided in the components of the combination or kit according to the application or in the composition according to the application prior to (or immediately prior to) initiation of the NOx generating reaction.

[0250] Optional additional components

[0251] The combinations, kits or compositions for use in the present disclosure can be incorporated into a wide variety of diluents, carriers and excipients and / or provided in association with one or more additional components, the particular functional component intended to provide one or more particular benefits to the combination, kit or composition in which it is used. When in vivo use is intended, such diluents, carriers, excipients and / or additional components will generally be physiologically compatible.

[0252] Examples of suitable physiologically compatible diluents, carriers and / or excipients include, without limitation, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talcum, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride and the like.

[0253] Generally, depending on the intended mode of administration, a pharmaceutical formulation will contain from about 0.005% to about 95%, preferably from about 0.5% to about 50% by weight of the combination or composition of the present application or a component thereof. Actual methods of preparing such dosage forms are known, or will be apparent in light of this disclosure, to those skilled in this art.

[0254] The excipient can be selected from known excipients, depending on the intended use or the route of administration of the reactants and / or reaction products to deliver nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof to the target site. For example, a cream, lotion and ointment can be formulated by incorporating a nitrite salt into an excipient, such as a cream, lotion and ointment base, or other thickening and viscosity agents (e.g. Eudragit L100, carbopol, carboxymethyl cellulose or hydroxymethyl cellulose). The proton source can be incorporated into an excipient selected from carbopol, carboxymethyl cellulose, hydroxymethyl cellulose, methyl cellulose or an aqueous base. If a film is intended to be formed, a film-forming excipient can be used, such as propylene glycol, polyvinylpyrrolidone (povidone), gelatin, guar gum and shellac.

[0255] The optional further components can for example be selected from sweeteners, taste-masking agents, thickening agents, viscosity-increasing agents, humectants, lubricants, binders, film-forming agents, emulsifiers, solubilizers, stabilizers, colorants, flavor enhancers, salts, coating agents, antioxidants, pharmaceutically active agents, and preservatives. Such components are well known in the art and do not need to be discussed in detail for the skilled reader. Examples of auxiliary substances such as humectants, emulsifiers, lubricants, binders, and solubilizers include for example sodium phosphate, potassium phosphate, gum arabic, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like. Sweeteners or taste-masking agents can for example include sugars, saccharin, aspartame, sucralose, neotame, or other compounds that advantageously influence the taste, aftertaste, sensation of unpleas- ant saltiness, sourness, or bitterness, reduce the tendency of oral or inhaled formulations to irritate the recipient, for example by causing coughing or sore throat or other undesired side effects, for example possibly reducing the delivered dose or adversely affecting the patient's compliance with a prescribed treatment regimen. Certain taste-masking agents can form complexes with one or more nitrite salts. Examples of thickening agents, viscosity-increasing agents, and film-forming agents have been given above.

[0256] The choice of pharmaceutically active agents and other further components, for example components used as diluents, carriers, and excipients, can be determined by its suitability for the treatment regimen of the relevant disease or medical condition and the desired route of administration of the combination or composition according to the present disclosure. Reference can be made to standard reference works, for example Martindale, 39thEdition (2017), the Merck Index, 15thEdition (2013); Goodman & Gilman’s “The Pharmacological Basis of Therapeutics”, 13thEdition (2017); the British National Formulary online (https: / / bnf.nice.org.uk / ); Remington: “The Science & Practice of Pharmacy”, 22ndEdition (2012); or the Physician’s Desk Reference, 71stEdition (2017).

[0257] Examples of routes of administration for which components and compositions according to the present disclosure can be administered to animal (including human) subjects for therapeutic purposes include topical (e.g. creams, lotions, gels, ointments, pastes, emollients, sprays), otic, nasal (e.g. sprays), vaginal, rectal (e.g. suppositories), oral (e.g. mists, sprays, mouthwash, aerosols), enteric (e.g. tablets, soft lozenges, troches, capsules, pastilles, elixirs) and parenteral (e.g. injectable liquids), ocular, aural, nasal or laryngeal (e.g. drops) or via the respiratory tract or lungs (e.g. mists, aerosols, powder inhalation).

[0258] Examples of pharmaceutically active agents that can be incorporated into or co-administered with components and compositions according to the present disclosure include antibiotics, steroids, anaesthetics (e.g. local anaesthetics such as lignocaine (lidocaine), amethocaine (tetracaine), xylocaine, bupivacaine, prilocaine, ropivfacaine, benzocaine, mepivocaine, cocaine or any combination thereof), analgesics, anti-inflammatory agents (e.g. non-steroidal anti-inflammatory drugs (NSAIDs)), anti-infective agents, vaccines, immunosuppressants, anticonvulsants, anti-dementia drugs, prostaglandins, antipyretics, antifungals, antipsoriatics, antivirals, vasodilators or vasoconstrictors, sunscreen agents (e.g. PABA), antihistamines, hormones (e.g. oestrogens, progestogens or androgens), anti-seborrhoeics, cardiovascular therapeutics (e.g. alpha or beta blockers or Rogaine), vitamins, skin softening agents, enzymes, mast cell stabilisers, scabicides, pediculicides, keratolytic agents, emollients, anaesthetics, shampoos, anti-acne agents, burn treatment agents, cleansers, deodorants, depigmenting agents, nappy rash treatment products, emollients, moisturisers, photosensitisers, poison ivy or oak or sumac products, sunburn treatment agents, proteins, peptides, proteoglycans, nucleotides, oligonucleotides (e.g. DNA, RNA etc.), minerals, growth factors, tar-containing agents, honey-containing agents (e.g. agents containing Manuka honey), wart treatment agents, wet dressings, wound care products or any combination thereof.

[0259] Specific examples include analgesics such as ibuprofen, indomethacin, diclofenac, acetylsalicylic acid, paracetamol, propranolol, metoprolol and oxycodone; thyroid releasing hormone; sex hormones such as oestrogens, progesterones and testosterone; insulin; verapamil; vasopressin; hydrocortisone; scopolamine; nitroglycerin; isosorbide dinitrate; antihistamines such as terfenadine; clonidine; nicotine; non-steroidal immunosuppressants such as cyclosporine, methotrexate, azathioprine, mycophenylate, cyclophosphamide, TNF-a antagonists and anti-IL5, anti-IL4Ra, anti-IL6, anti-IL13, anti-IL17, anti-IL23 cytokine monoclonal antibodies; anticonvulsants; and drugs for Alzheimer's disease, dementia and / or Parkinson's disease such as apamorphine and rivastigmine. If desired, any of the optional additional components can be encapsulated or microencapsulated, for example to control or delay its release. See the section below entitled "Optional encapsulation (e.g. microencapsulation) of components" for further details.

[0260] Optional encapsulation of components (e.g. microencapsulation)

[0261] At least some components of the combinations, kits and compositions of the disclosure can be encapsulated, for example microencapsulated.

[0262] The use of microencapsulated components to generate NO is advantageous because it delays the generation of a relatively unstable compound (e.g., NO) from precursors that are in a chemically stable form. The various microencapsulated reactants and / or one or more optional additional components are easily mixed and brought into contact with one another in a dry environment, and the generation of NO is initiated simply by providing a small amount of water to the precursor mixture. Alternatively, such a mixture of microencapsulated reactants and / or one or more optional additional components can be applied directly to a subject, e.g., the skin, mucosal surfaces, or into the nose, mouth, respiratory tract, and / or lungs of a subject in accordance with the present application, where the physiological environment itself provides sufficient water to cause the release of a therapeutic amount of NO. Another advantage is that the microencapsulated reactants and / or one or more optional additional components occupy relatively little volume, and thus they are easily incorporated into small objects such as medical devices. Such objects include, for example, wound dressings, bandages, vascular and other stents, catheters, pacemakers, defibrillators, heart-assist devices, artificial valves, electrodes, orthopedic screws and pins, and other thin medical and / or implantable articles.

[0263] One example of a method for producing encapsulates or microencapsulates of the reactants and / or one or more optional additional components is spray drying of a melt or polymer solution of the reactants and / or one or more optional additional components to produce a very fine powder of individual particles comprising the material dispersed within a polymer matrix. Other encapsulation or microencapsulation methods can also be used, such as pan coating, air suspension coating, centrifugal extrusion, fiber spinning, fiber extrusion, nozzle vibration, ionotropic gelation, coacervation phase separation, interfacial crosslinking, in situ polymerization, and template polymerization. The encapsulating polymer is preferably biocompatible. Such polymers include ethyl cellulose, natural polymers (e.g., zein) (alcohol-soluble prolamine seed storage proteins found in certain grass species, including maize and corn), chitosan, hyaluronic acid, and alginic acid or biodegradable polyesters, poly-anhydrides, poly(ortho esters), polyphosphazenes, or polysaccharides (see Park et al., Molecules 10 (2005), pp. 141-161). Compositions of a chemical substance microencapsulated as indicated above are known for delivering 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 virtually all such compositions, the therapeutic agent is microencapsulated, and the therapeutic agent is not produced by a reaction of the microencapsulating agent. However, suitable modifications of the teachings of the prior art are within the skill of the ordinarily skilled artisan. Nitric oxide-releasing polymers have been described for medical articles involving NO adducts / donors. See, e.g., Arnold, U.S. Patent No. 7,829,553 (carbon-based diazeniumdiones based on diols linked to hydrophobic polymers); Knapp, U.S. Patent No. 7,135,189 (nitrosothiol precursors and nitric oxide donors).

[0264] pH control; optional buffer system

[0265] The composition can have a controlled pH. In particular, the composition can have a pH in the range of 3.0 to 8.0 or more particularly in the range of 4.0 to 8.0. In more particular embodiments, the composition has a pH in the range of 4.0 to 7.4. In more particular embodiments, the composition can have a pH in the range of 4.0 to 6.0. In these embodiments, the composition can have a pH in the range of 4.5 to 6.0.

[0266] The pH of the composition can be controlled by any known means. In particular embodiments, the pH of the organic carboxylic acid component or the organic reductive acid component is controlled prior to the nitrite component. In some embodiments, the organic carboxylic acid component or the organic reductive acid component includes a buffer. The buffer can be a pharmacologically acceptable buffer, such as a phosphate buffer.

[0267] In some embodiments, the buffer is formed by mixing an organic carboxylic acid or an organic non-carboxylic reductive acid and its salt counterpart. For example, the organic carboxylic acid component can include an organic carboxylic acid and a salt of the organic carboxylic acid. The organic non-carboxylic reductive acid component can include an organic non-carboxylic reductive acid and a salt of the organic non-carboxylic reductive acid. In particular embodiments, the organic carboxylic acid component includes citric acid and a citrate salt. In other embodiments, the organic carboxylic acid component or the organic reductive acid component includes ascorbic acid and an ascorbate salt. In some embodiments, the organic carboxylic acid component includes an organic carboxylic acid and a salt of another organic acid. For example, the organic carboxylic acid component can include citric acid and an ascorbate salt. In other embodiments, the organic carboxylic acid component can include an organic carboxylic acid, a salt of the organic carboxylic acid, and a salt of another organic carboxylic acid. For example, the organic carboxylic acid component can include citric acid, a citrate salt, and an ascorbate salt.

[0268] In other embodiments, the buffer is formed by adjusting the pH of the organic carboxylic acid or the organic non-carboxylic reductive acid such that the acid (protonated form) and its salt counterpart coexist in mixture. This is conveniently accomplished by adding a strong inorganic base and optionally a strong inorganic acid to the organic carboxylic acid or the organic non-carboxylic reductive acid in such amounts that the buffer system is generated 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.

[0269] The buffer can include one or more physiological buffers, particularly when the combination or composition according to the disclosure is in contact with the skin, mucosa or other tissues of a cell or animal (including a human), for example in the case of administration to the nose, oral cavity, respiratory tract or lungs according to the application. Examples of suitable physiologically compatible buffers include Good's buffers, which buffer in the pH range of about 5 to about 9, for example 2-amino-2-methyl-1.3-propanediol, N-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)-iminodiacetic acid (ADA), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (BICINE), 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (BIS-TRIS), 1,3-bis[tris(hydroxymethyl)methylamino]-propane (BIS-TRIS propane), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-(N,N-bis[2-hydroxyethyl]amino)-2-aminopropanesulfonic acid (DIPSO), 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), bisglycine, N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) 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).

[0270] Osmolality of the composition

[0271] The tonicity of any solution of nitrite, proton source, organic polyol or any combination thereof delivered to the physiological system, particularly through a route of contact with the skin, mucosa of a human or animal subject or according to the application with the nose, oral cavity, respiratory tract or lungs, should be controlled to avoid any unwanted dehydration of the subject's organs and tissues.

[0272] Osmotic pressure (Osm) is defined as the number of moles of solute dissolved in one kilogram of solvent and can be expressed as Osmoles per kilogram (Osmol / kg). The osmotic pressure of any solution administered to a human or animal subject according to the present disclosure is generally in the range of about 100 to about 5000 mOsmol / kg, for example, about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 to about 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 mOsmol / kg.

[0273] Mixing of components to initiate production of NOx

[0274] It was discovered that the order in which the components of the NOx-producing system were mixed to initiate production of NOx can have an impact on the results using the NOx produced thereby. Evidence of this impact is provided in Example 6 below.

[0275] In this example, we demonstrate that the efficacy of a composition according to the present invention to kill Mycobacterium tuberculosis HN878 in THP-1 cells is different depending on whether: on the one hand, the nitrite salt, the proton source and the organic polyol component are first mixed in the required proportions at concentrations higher than those required in the composition in the form in which it is to be used, and this concentrate is then diluted with water as appropriate to provide the composition to be used; or on the other hand, the nitrite salt, the proton source and the organic polyol component are first mixed in the required proportions at the concentrations required in the composition in the form in which it is to be used.

[0276] Furthermore, there is no way to predict which way of mixing the components will produce better results in terms of antimicrobial action. While it would generally be expected that diluting a relatively concentrated pre-mix to provide the composition to be used can produce better antimicrobial action against Mycobacterium tuberculosis HN878 in THP-1 cells, in some cases the results produced are not as good as with the method of mixing the components at the concentrations required for that use.

[0277] In one embodiment of the present invention, therefore, the method of preparing a NOx-producing composition comprises mixing the nitrite salt, the proton source and the organic polyol component in the required proportions at concentrations higher than those required in the composition in the form in which it is to be used to form a concentrated pre-mix, and subsequently diluting this concentrated pre-mix with water as appropriate to provide the composition to be used.

[0278] In another embodiment of the present invention, therefore, the method of preparing a NOx-producing composition comprises mixing the nitrite salt, the proton source and the organic polyol component in the required proportions at the concentrations required in the composition in the form in which it is to be used to provide the composition to be used.

[0279] Preferred embodiments

[0280] Preferred embodiments of the first to eighth aspects of the present disclosure are embodiments in which one or more of the following are present:

[0281] - the one or more nitrite salts comprise (e.g. consist essentially of or consist of) one or more alkali metal or alkaline earth metal nitrite salts, for example sodium nitrite, potassium nitrite, or any combination thereof;

[0282] - the proton source comprises (e.g. consists essentially of or consists of) ascorbic acid or an ascorbic acid / ascorbate buffer, citric acid or a citric acid / citrate buffer, or any combination of two or more thereof;

[0283] - the molecules of the ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citrate buffer, or any combination of two or more thereof are not covalently bound to a polymer or macromolecule;

[0284] - the one or more organic polyols comprise (e.g. consist essentially of or consist of) a linear sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, for example sorbitol, mannitol, arabitol, xylitol, or any combination of two or more thereof;

[0285] - the one or more organic polyols are sugar alcohol compounds comprising (e.g. consisting of) a chain of 1, 2 or 3 monosaccharide units capped with one non-cyclic alcohol unit, optionally wherein the 1, 2, 3 or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the non-cyclic alcohol unit is a C5 or C6 sugar alcohol unit; for example isomaltitol, maltitol, lactitol, maltotriitol, maltotetraitol;

[0286] - the total molarity of the one or more organic polyols in the polyol component or in the reaction solution at the start of or prior to the start of the NOx-producing reaction is between 0.05 and 3 times the total molarity of nitrite ions in the nitrite component or in the reaction solution;

[0287] - the total molarity of the one or more organic polyols in the polyol component or in the reaction solution at the start of or prior to the start of the NOx-producing reaction is between 0.05 and 3 times the total molarity of the proton source in the proton source component or in the reaction solution;

[0288] - for applications not involving contact between the reaction mixture and the skin (including mucous membranes), organs or other tissues of a cell or an animal (including a human), the pH of the proton source prior to, especially immediately prior to, initiation of the NO-generating reaction is in the range of 3.0 to 9.0;

[0289] - for applications not involving contact between the reaction mixture and the skin (including mucous membranes), organs or other tissues of a cell or an animal (including a human), the pH of the proton source prior to, especially immediately prior to, initiation of the NO-generating reaction is in the range of 4.0 to 8.0;

[0290] - for applications not involving contact between the reaction mixture and the nose, oral cavity, respiratory tract or lungs of an animal (including a human) subject according to the application, the pH of the proton source prior to, especially immediately prior to, initiation of the NO-generating reaction is in the range of 5.0 to 8.0;

[0291] - the targeted microorganism is selected from the microorganisms listed below in the section entitled “Targets for antimicrobial use”, such as, without limitation, influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, including antibiotic-resistant strains thereof.

[0292] Preferred embodiments of the ninth aspect of the present disclosure are embodiments in which one or more of the following are present:

[0293] - the one or more nitrite salts comprise (e.g., consist essentially of or consist of only) one or more alkali metal or alkaline earth metal nitrite salts, such as sodium nitrite, potassium nitrite, or any combination thereof;

[0294] - the proton source comprises (e.g., consists essentially of or consists of only) ascorbic acid or an ascorbic acid / ascorbate buffer, citric acid or a citric acid / citrate buffer, or any combination of two or more thereof;

[0295] - the molecules of the ascorbic acid or ascorbic acid / ascorbate buffer, citric acid or citric acid / citrate buffer, or any combination of two or more thereof are not covalently bonded to a polymer or macromolecule;

[0296] - the one or more organic polyols comprise (e.g., consist of or consist essentially of or consist only of) a linear sugar alcohol or alditol having 4 to 12 carbon atoms and 4 to 12 OH groups per molecule, for example sorbitol, mannitol, arabitol, xylitol, or any combination of two or more thereof;

[0297] - the one or more organic polyols are a sugar alcohol compound comprising (e.g., consisting of) a chain of 1, 2, or 3 monosaccharide units capped with one non-cyclic alcohol unit, optionally wherein the 1, 2, 3, or each monosaccharide unit is a C5 or C6 monosaccharide unit and / or the non-cyclic alcohol unit is a C5 or C6 sugar alcohol unit; for example isomaltitol, maltitol, lactitol, maltotriitol, maltotetraitol;

[0298] - the total molarity of the one or more organic polyols in the polyol component or in the reaction solution at the start of or prior to the initiation of the NOx-producing reaction is between 0.05 and 3 times the total molarity of nitrite ions in the nitrite component or in the reaction solution;

[0299] - the total molarity of the one or more organic polyols in the polyol component or in the reaction solution at the start of or prior to the initiation of the NOx-producing reaction is between 0.05 and 3 times the total molarity of the proton source in the proton source component or in the reaction solution;

[0300] - for applications that do not involve contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of a cell or animal (including a human), the pH of the proton source prior to, especially immediately prior to, the initiation of the NO-producing reaction is in the range of 3.0 to 9.0;

[0301] - for applications that do not involve contact between the reaction mixture and the skin (including mucous membranes), organs, or other tissues of a cell or animal (including a human), the pH of the proton source prior to, especially immediately prior to, the initiation of the NO-producing reaction is in the range of 4.0 to 8.0;

[0302] - for applications that do not involve contact between the reaction mixture and the nose, oral cavity, respiratory tract, or lungs of an animal (including a human) subject according to the invention, the pH of the proton source prior to, especially immediately prior to, the initiation of the NO-producing reaction is in the range of 5.0 to 8.0;

[0303] - the targeted microorganism is selected from the microorganisms listed below in the section entitled "Targets for Antimicrobial Uses", for example without limitation influenza virus, SARS-CoV, SARS-CoV-2, Mycobacterium tuberculosis, Mycobacterium abscessus, Pseudomonas aeruginosa, including their antibiotic-resistant strains.

[0304] Combinations and compositions

[0305] The reaction to produce NOx can be initiated in a number of ways. Typically these ways are characterized by contacting one or more nitrite salts with a proton source under conditions that can initiate the reaction to produce NOx.

[0306] The reaction can be initiated by combining the individual components of the combination. The combination can be effected in vitro, and the resulting composition can then be applied to a subject or to any surface to be treated according to the disclosure. Alternatively, the evolved gas can be applied to a subject or to any surface to be treated according to the disclosure. Still further, the two uses of the resulting composition can be separated in time, such that the composition is applied to a subject or to any surface to be treated after a certain gas has evolved.

[0307] The combination can be effected stepwise, for example by initially mixing the components in dry powder form, and then mixing with water or another liquid carrier medium to initiate the reaction. Alternatively, the components in dry powder form can be initially mixed separately with water or another liquid carrier medium, and then the two or more liquids are subsequently mixed to initiate the reaction.

[0308] Alternatively, at least some of the components of the reaction to produce NOx according to the disclosure can be mixed to exist in a single composition, and the reaction to produce NOx is initiated on the composition. One way to initiate the reaction to produce NOx can be, for example, the addition of a key component or additive to initiate the reaction, for example, the addition of water if the components of the composition are in dry or encapsulated form; or the addition of a proton source if the components of the composition lack a proton source.

[0309] In the case of preventing the production of NOx, the kit according to the disclosure typically contains one or more components of a combination according to the disclosure or a composition according to the disclosure. Part of the kit is typically kept in a container, which can be separate, or adapted to facilitate the mixing required to initiate the reaction to produce NOx. The key initiation component of the reaction to produce NOx that needs to be introduced by the user of the kit into the other necessary components can be, for example, one of the nitrite component, the proton source component, or the polyol component, or can be another ingredient, typically a commonly utilized component, such as water, which can be supplied by the user.

[0310] The parameters of the combinations and compositions defined and described in this patent typically include physical parameters such as pH, concentration, and osmolarity. These parameters will be measured, if possible, prior to initiating the NOx-producing reaction. Unless otherwise specified, the pH parameter refers to the pH of the proton source in deionized water at the concentration intended to initiate the NOx-producing reaction. Unless otherwise specified, the concentration of a solution refers to the concentration prior to mixing with other components to initiate the NOx-producing reaction. Typically, such parameters are less easily measured when the NOx-producing reaction is in progress, as the nitrite and organic carboxylic or organic reductive acid react to produce nitric oxide gas upon mixing.

[0311] Further, it is noted that the concentrations of the ingredients in the reaction mixture do not necessarily correspond to their concentrations in the part of the combination prior to mixing. For example, assume that a combination for initiating a NOx-producing reaction according to the present disclosure is formed from approximately equal volumes of a nitrite component and a proton source component, both added together as pre-made solutions. In this embodiment, the nitrite concentration of the mixed reaction combination is half the nitrite component concentration, and the proton source concentration is half the proton source component concentration.

[0312] The combinations and parts of the compositions can be in any suitable physical form, depending on the intended use of the system during or after the NOx-producing reaction. For example, the combinations and parts of the compositions can each be in the form of a liquid, a gel, or a film, so that the NOx-producing reaction mixture is similarly in the form of a liquid, a gel, or a film. The liquid can be adapted to be capable of being aerosolized for inhalation into the respiratory tract or lungs. If the NOx-producing reaction mixture is intended to be applied to the oral cavity or throat, the combinations and parts of the compositions can be in the form of a mouthwash or beverage. Alternatively, if the NOx-producing reaction mixture is intended to be applied to the skin in a topical application, the combinations and parts of the compositions can be in the form of an ointment, lotion, or cream.

[0313] Multi-component systems, kits and dispensers

[0314] The multi-component systems described herein can include a nitrite component and a proton source component, optionally with a polyol component, as defined according to the present disclosure and as described herein. The components in the multi-component systems are adapted to be in contact with each other and to be in contact with the reaction mixture and / or the evolved gas that is distributed by means of suitable containers or reservoirs for preserving the components prior to use and means for mixing the components, dispensing the reaction mixture and / or the evolved gas, and overall control of said mixing and dispensing. In a preferred embodiment, the reaction mixture can be dispensed in the form of a mist or aerosol of droplets entrained in a gas stream.

[0315] Kits and dispensers of the disclosure generally comprise at least some containers for holding components prior to use; at least one device or other means for mixing the components, dispensing the reaction mixture and / or expelling the gas and overall controlling said mixing and dispensing; and the component or those components, if any, contained in the containers of the kit or dispenser prior to use. Instructions for use are desirably present, or directions to find instructions for use are available, for example online. Such kits and dispensers constitute a further aspect of the disclosure.

[0316] Kits of the disclosure can be relatively simple assemblies of containers and devices for mixing the components, dispensing the reaction mixture and / or expelling the gas and overall controlling said mixing and dispensing. Such kits are suitable for research purposes, or for situations where it is anticipated and permissible that mixing and dispensing operations will vary widely.

[0317] Other kits of the disclosure can be more complex assemblies comprising one or more containers of consumables (combinations and / or compositions required by the user to initiate the NOx-generating reaction, optionally water or other generally available ingredients supplied by the user) together with one or more dispensers of the disclosure.

[0318] Dispensers of the disclosure are generally adapted to perform repetitive similar actions of dispensing the reaction mixture, a carrier containing the reaction mixture and / or expelling the gas. The dispensers can contain a pump or propellant system to carry the composition containing the NOx-generating reaction mixture or the gas being expelled from the dispenser and direct it to the target. The propellant system can use a pressurized gas and / or a liquefied gas, suitable for medical use being pharmaceutically acceptable or biocompatible, for example pressurized air or pressurized / liquefied butane. Alternatively, suction from the user's lungs can be used to carry the composition containing the NO-generating reaction mixture or the gas being expelled from the dispenser and direct it to the target. Dispensers for use in the disclosure desirably contain an actuation device, for example a manually operated trigger or button, by which the user can activate the dispenser. Such dispensers can be adapted for professional, research, consumer or patient use and correspondingly adapted to facilitate a predetermined route through which the target passes.

[0319] A wide variety of kit and dispenser devices are known in principle which can be used or readily adapted to hold components prior to use, mix the components or facilitate said mixing, dispense the composition containing the reaction mixture and / or expel the gas and overall control said mixing and dispensing or facilitate said control.

[0320] For example:

[0321] - a syringe, for example a dual barrel dispensing syringe.

[0322] - container systems, e.g., pump-action containers, press-action containers, or shake-action containers, e.g., containing two containers to mix at least the nitrite component and the proton source component and to dispense a composition containing a reaction to generate NOx or an evolved gas. Such systems are described in US 2019 / 0134080, the disclosure of which is incorporated herein by reference.

[0323] - devices for storing components before use in aqueous solution, mixing components, nebulizing the liquid reaction mixture, and dispensing the reaction mixture for inhalation into the lungs of a human, and overall control of the mixing and dispensing. Examples include soft mist inhalers, jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. The selection of nebulizers, droplet size, adjuvants, packaging form, etc. suitable for inhalation nebulization of a reaction medium to generate NOx by nitrite acidification is described in WO 03 / 032928 and WO 2009 / 086470, the disclosures of which are incorporated herein by reference.

[0324] - the above devices can be arranged to nebulize the pre-mixed liquid reaction mixture after it has been loaded into the nebulizer and to dispense the reaction mixture for inhalation into the lungs of a human, and overall control of the mixing and dispensing.

[0325] - devices for storing components before use in aqueous solution, mixing components, atomizing the liquid reaction mixture, and dispensing the reaction mixture for inhalation into the lungs of a human, and overall control of the mixing and dispensing. Examples include metered dose inhalers. The selection of droplet size, adjuvants, packaging form, etc. suitable for inhalation nebulization of a reaction medium to generate NOx by nitrite acidification is described in WO 03 / 032928 and WO 2009 / 086470, the disclosures of which are incorporated herein by reference.

[0326] - techniques and devices for spraying a nitric oxide-releasing solution into the upper respiratory tract are described in U.S. Patent No. 9730956, the disclosure of which is incorporated herein by reference.

[0327] - devices for storing components in dry powder form before use and dispensing them for inhalation into the lungs of a human. Examples include dry powder inhalers (DPIs), which can be formulated as single-dose capsules, or multi-dose dry powder inhalers, as reservoir powders or multi-dose individual blisters. The selection of powder particle size, adjuvants, packaging form, etc. suitable for inhalation of dry powder combinations for providing a reaction medium in the lungs to generate NO in situ by nitrite acidification is described in WO 2009 / 086470, the disclosure of which is incorporated herein by reference.

[0328] - a dispenser for holding the components before use in solution, aerating them and dispensing them as a foam for skin disinfecting use or treating skin conditions is described in U.S. Patent Application No. 2013 / 0200109, U.S. Patent 7066356 and U.S. Patent Application No. 2019 / 0134080, the disclosures of which are incorporated herein by reference;

[0329] - a transdermal patch assembly for holding the components and dispensing them to the skin of a subject is described in WO 2014 / 188175, the disclosure of which is incorporated herein by reference.

[0330] The dose of the combination and composition or gas evolved of the present disclosure can vary within wide limits, depending on the disease, disorder or condition to be treated (in the case of medical treatment) or the effect desired (in the case of non-medical treatment), the severity of the treatment required 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 attending physician will eventually decide on the appropriate dose to be used. In the case of non-medical treatment, the skilled person will be able to investigate the appropriate dose and method of treatment by consulting the relevant literature or by reasonable trial and error.

[0331] In some embodiments, the combination that produces the NOx-producing reaction or gas evolved therefrom can be applied to the target location, e.g., a microbial cell, living tissue, organ, structure or subject, within 600 seconds after the combination of the nitrite component and the proton source component. In this manner, the target location can be exposed to a burst of nitric oxide.

[0332] In some embodiments, the combination that produces the NOx-producing reaction can be formed in situ at or near the target location, e.g., on, in or near a microbial cell, living tissue, organ, structure or subject, including inanimate surfaces and spaces. In these embodiments, application is effective at 0 seconds after the combination of the nitrite component and the proton source component. In other embodiments, the combination is applied to the target location 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 more specific embodiments, the combination is applied within a range of 0 and 120 seconds. In other embodiments, the combination is applied within a range of 0 and 60 seconds.

[0333] In other embodiments, the composition that generates the reaction producing NOx or gas evolved therefrom can be applied to or near a target site, e.g., a microbial cell, living tissue, organ, structure, or subject, more than 600 seconds, e.g., more than 2000 seconds, e.g., more than 4000 seconds, e.g., more than 8000 seconds, after the combination of the nitrite component and the proton source component. In that case, the target site, e.g., a microbial cell, living tissue, organ, structure, or subject, is not necessarily exposed to a bolus of nitric oxide, but can still experience a beneficial property, e.g., antimicrobial action. In these embodiments, the composition that generates the reaction producing NOx or gas evolved therefrom can be applied up to 48 hours after the combination of the nitrite component and the proton source component. In particular embodiments, the composition or gas evolved therefrom can be applied up to several weeks or months, e.g., up to about 6 months, or up to about 2 months, or up to about 1 month, 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.

[0334] In the case of proper storage, the composition that generates the reaction producing NOx or gas evolved 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 closed container, e.g., under vacuum. Storage in a closed container is typically carried out up to 24 hours after the combination of the nitrite and the organic carboxylic acid or organic reductic acid. The composition can be stored in a closed container up to 600 seconds after the combination of the nitrite component and the proton source component. In this way, a proportion of the nitric oxide gas can be retained. If the NOx-generating composition is stored at a low temperature, e.g., in the range of about -30 °C to about +10 °C, e.g., about 1 °C to about 10 °C, the rate of evolution of the gas can be greatly slowed, allowing for very long storage times of the composition.

[0335] In one particular embodiment, the aerosol dispenser can include a plurality of reservoirs, wherein a first reservoir contains the nitrite component in liquid form (e.g., aqueous solution) and a second reservoir contains the proton source component in liquid form (e.g., aqueous solution). In this embodiment, each component is adapted to be mixed with a propellant prior to, during, or after mixing of the nitrite and proton source components.

[0336] In another particular embodiment, the dispenser can be a single barrel syringe containing the composition of the disclosure. The viscosity of the composition is selected to enable dispensing from the syringe by manual action of the syringe or by power operation. For example, the composition can be a liquid or a gel.

[0337] In another specific embodiment, the dispenser can be a multiple barrel syringe having a first barrel containing the nitrite component and a second barrel containing the proton source component. The viscosities of the components are selected to enable dispensing from the syringe by manual action of the syringe or by power operation. For example, each component can independently be a liquid or a gel.

[0338] Further reservoirs of components: hydrogels

[0339] In some embodiments of the present disclosure, a molecular reservoir, such as a hydrogel, can be used. A hydrogel is a highly hydrated, usually cross-linked, three-dimensional polymeric (homopolymer or copolymer) or macromolecular network that is capable of absorbing and retaining many times its dry weight of water, other aqueous liquids, or other non-aqueous, hydrophilic liquids. The absorption of liquid is usually accompanied by swelling of the hydrogel. By appropriate selection of the chemical groups of the components covalently bonded to the polymer or macromolecule, acidic hydrogels or hydrogels with other specific chemical properties can be prepared.

[0340] Hydrogels that can be used as the proton source component in the present disclosure are known. 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 the documents mentioned therein, the disclosures of all of which are incorporated herein by reference. The use of such hydrogels for generating NOx for the care of skin, including the transdermal delivery of drugs in conjunction with NOx, is specifically 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 salts thereof. Polymers formed from monomers comprising (meth)acrylic acid or consisting of (meth)acrylic acid will include carboxylic acid side groups for use as the proton source according to the present disclosure.

[0341] Thus, for example, a multi-component system can include a first acidic hydrogel pad or layer component comprising a proton source component, optionally also containing an organic polyol, and another component can be a nitrite component. The nitrite component may, for example, be a liquid medium containing dissolved nitrite salt. In this way, the surface of the hydrogel pad or layer can be contacted with the nitrite component, initiating the reaction to generate NOx. Alternatively, the nitrite component can be a solid support, such as a pad or layer, containing a nitrite salt in a form that is capable of dissolving in the absorption liquid of the hydrogel upon contact between the solid support and the hydrogel.

[0342] Typically, the solid support mat or layer is nitric oxide diffusion permeable (fully permeable or at least semi-permeable). In this way, nitric oxide can diffuse to the treatment area when the solid support mat or layer and the hydrogel are combined to combine the nitrite component and the proton source component. The solid support mat or layer can be, for example, a screen, a nonwoven cloth sheet, a film, a foam, an alginate layer or a membrane.

[0343] In particular embodiments, the solid support layer is a screen. The screen can be a number of connected solid threads, typically flexible, forming a mesh of holes or gaps through which certain substances can pass. The screen can be a woven or nonwoven cloth. In some embodiments, the screen is a nonwoven cloth.

[0344] The solid support layer, for example a screen, can be made of a polymeric material. Examples of suitable polymeric materials include, but are not limited to, fibreglass, polyamide, polyester, polypropylene or blends thereof. The polymeric material can be treated, for example to increase its hydrophilicity. In particular embodiments, the solid support layer is a polypropylene mesh.

[0345] In particular embodiments, the solid support has absorbent capacity and the nitrite component is at least partially absorbed, imbibed or impregnated in the solid support. The absorbed, imbibed or impregnated nitrite component can be solid (dry) or can be in aqueous solution within the solid support.

[0346] In particular embodiments, the solid support comprises more than one layer and the nitrite component is absorbed, imbibed or impregnated in at least one of the layers or coated on at least one outer layer. For example, the solid support can comprise 2 layers, 3 layers, 4 layers, 5 layers, 6 layers, 7 layers, 8 layers, 9 layers, 10 layers or more, for example polypropylene screen layers, absorbed, imbibed, impregnated or coated with one or more nitrites in dry and / or solution form.

[0347] The acidic hydrogel has a natural buffering capacity as there is a large internal supply of protonated acidic side groups from which H+ions can migrate via the absorbed aqueous medium to maintain the relative acidic pH at the surface of the hydrogel structure as the pendant acidic moieties on the surface remove protons during the NOx-generating reaction.

[0348] Non-acidic (e.g., neutral or basic) hydrogels are also known in which a nitrite component and / or a polyol component can be absorbed and contained for use in the present disclosure. A proton source component can be contacted with such hydrogels, either by being provided in a liquid medium in contact with the hydrogel, and / or by being absorbed, imbibed, impregnated, or coated onto a solid support. In such hydrogels, provided that none of the nitrite component, the proton source component, or the polyol component is covalently bonded to the polymer or macromolecular network of the hydrogel; all components required by the present disclosure can be imbibed into the hydrogel and contained in the aqueous medium within the hydrogel mass, but not covalently bonded to the polymer or macromolecule of the hydrogel, for example, with the understanding that the nitrite component and the proton source component do not react together until the reaction to generate NOx is desired to be initiated.

[0349] The thickness of the hydrogel pad or layer can be in the range of 0.5 to 2 mm. In some embodiments, the thickness of the hydrogel pad or layer is in the range of 1 to 2 mm. In particular embodiments, the thickness of the hydrogel pad or layer is in the range of 1.0 to 1.6 mm.

[0350] The features described above in relation to the proton source component will generally apply equally to any acidic hydrogel used as a proton source component. Thus, for example, the hydrogel can contain a buffer to maintain the pH of the hydrogel in the range of 4.0 to 9.0 or 5.0 to 8.0.

[0351] In some embodiments, the hydrogel can include a barrier layer. The barrier layer is typically a polymeric 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, for example, to the skin of a subject, to provide a barrier between the combined multi-component system and the atmosphere. The surface of the barrier film that is contiguous with the hydrogel typically has a greater surface area than the adjacent surface of the hydrogel. In this way, the barrier layer can extend beyond the perimeter of the hydrogel. In these embodiments, the barrier layer can have an adhesive around its periphery to adhere the hydrogel to, for example, the skin of a subject in use.

[0352] In one particular embodiment, the present disclosure provides a two-component system comprising:

[0353] a) one or more screens imbibed, impregnated, or coated with one or more nitrite salts, such as NaNO2; and

[0354] b) a hydrogel comprising a proton source, the proton source comprising one or more acids selected from the group consisting of organic carboxylic acids and organic non-carboxylic reducing acids,

[0355] wherein component (a) is separate from component (b), and wherein one or more of components (a) and (b) further comprises one or more organic polyols;

[0356] characterized by one or more of the following:

[0357] (a) one or more organic polyols are present in an amount to enhance the reaction output;

[0358] (b) the proton source is not only a hydrogel comprising pendant carboxylic acid groups covalently bonded to a three-dimensional polymeric matrix;

[0359] (c) the one or more organic polyols are not only glycerol;

[0360] (d) the one or more organic polyols are not only glycerol when one or more tackifiers are used;

[0361] (e) the one or more organic polyols are not only glycerol when one or more plasticizers are used;

[0362] (f) the one or more organic polyols are not only polyvinyl alcohol;

[0363] (g) the one or more organic polyols are not only polyvinyl alcohol when one or more tackifiers are used;

[0364] (h) any one or more of (b) through (g) above, wherein the words "not only" are replaced with "not comprising";

[0365] (i) the one or more organic polyols are not only propylene glycol, polyethylene glycol, glycerol monostearate (glyceryl stearate), trihydroxyethylamine, D-pantothenyl alcohol, panthenol, panthenol in combination with inositol, butylene glycol, butenediol, butynediol, pentanediol, hexanediol, octanediol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexanediol, caprylyl glycol, a diol other than the diols listed here, hydroquinone, butylated hydroquinone, 1-thioglycerol, erythorbate, ethylhexylglycerin, any combination of them, or any of the above in combination with glycerol and / or polyvinyl alcohol;

[0366] (j) the one or more organic polyols do not include propylene glycol, polyethylene glycol, glyceryl monostearate (glyceryl stearate), trihydroxyethylamine, D-panthenol, panthenol, panthenol combined with inositol, butylene glycol, butylenediol, butynediol, pentylene glycol, hexylene glycol, caprylyl glycol, neopentyl glycol, 2-methyl-1,3-propanediol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, dibutylene glycol, butane-1,2,3-triol, butane-1,2,4-triol, hexane-1,2,6-triol, hexylene glycol, caprylyl glycol, a glycol other than those listed herein, hydroquinone, butylated hydroquinone, 1-thioglycerol, isoascorbate, ethylhexylglycerin, any combination thereof, or any combination of any of the foregoing with glycerol and / or polyvinyl alcohol.

[0367] For the avoidance of doubt, it is hereby acknowledged that the embodiments and preferences of the characteristic features (a) to (h) described above in relation to aspects of the present disclosure apply equally to this embodiment.

[0368] Such systems can be used, for example, by combining components (a) and (b) to induce a reaction that produces NOx. Such combinations can then be used, for example, in the therapy or other treatment of humans or animals by topical application. Uses can be as described in WO 2014 / 188174 and WO 2014 / 188175, or can be as described below. The system can also be used for non-medical purposes, as described below. When used for topical medical applications in which the system contacts the subject's skin (including mucous membranes), one or the screen can be the contact skin layer.

[0369] Use in therapy or surgery

[0370] Compositions that undergo NOx-generating reactions according to the present disclosure and the gases evolved therefrom have numerous applications in therapy and surgery, including curative and / or preventative therapy, surgery to correct diseases, conditions, and disorders, cosmetic surgery, reconstructive surgery, and both human and veterinary medicine, as well as surgery. Where a physical deformity or abnormality responsive to treatment with the composition or the gases evolved therefrom causes or exacerbates anxiety, depression, or another mental illness or condition, the treatment, prevention, or alleviation of the physical condition may correspondingly treat, prevent, or alleviate the mental condition, and thus the use of the present disclosure also extends to the field of mental health.

[0371] Many physiological effects of nitric oxide and nitric oxide-generating compositions and medical treatments based thereon have been reported in the literature, and therefore many therapeutic treatments have been developed. The following non-exhaustive list is provided as an example. The listed uses, as well as other uses not listed, are encompassed within the present disclosure and patents.

[0372] 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), p. 683-741);

[0373] The acute effects of oral nitric oxide supplementation in hypertensive patients to lower blood pressure, increase vascular compliance and restore epithelial function are described in Houston et al., J. Clin. Hypertens. (Greenwich), 2014 Jul; 16(7), p. 524-529;

[0374] Nitric oxide protects tissues from damage caused by low blood supply (see van Faassen et al., Med. Res. Rev. 2009 Sep; 29(5), p. 683-741);

[0375] Nitric oxide acts as a neurotransmitter in nitric oxide-ergic neurons, for example in smooth muscle with activity in the gastrointestinal tract and erectile tissue (see Toda et al., Pharmacol. Ther., 2005 May; 106(2), p. 233-266);

[0376] Nitric oxide inhibits vascular smooth muscle contraction and growth, platelet aggregation and leukocyte attachment to the endothelium, helping blood vessels to achieve homeostasis (see Dessey and Ferron, Current Medical Chemistry - Anti-inflammatory and Anti-allergy Agents in Medicinal Chemistry, 2004; 3(3), p. 207-216);

[0377] Nitric oxide is used to reduce cardiac contractility and heart rate (see Navin et al., J. Cardiovascular Pharmacology, 2002; 39(2), p. 298-309);

[0378] Neonatal intensive care to promote capillary and lung dilation, for example to treat primary pulmonary hypertension and after meconium aspiration in neonatal patients (see Barrington et al., The Cochrane Database of Systematic Reviews, 2017; 1, CD000399 https: / / www.ncbi.nlm.mh.gov / pubmed / 17375630); see also Chotigeat et al., J. Med. Assoc. Thai., 2007; 90(2), pages 266-271 ; see also Hayward et al., Cardiovascular Research, 1999; 43(3), pages 628-638);

[0379] Prevention of vascular damage, endothelial dysfunction and vascular inflammation, neuropathy and non-healing ulcers in diabetic patients and reduction of the consequent risk of amputation of the lower limbs (see nfb University Studies - "Nitric Oxide Holds Promise for Diabetes", http: / / www.nfb.org / Images / nfb / Publications / vod / vod212 / vodspr0613.htm);

[0380] Improvement of hypoxemia in acute lung injury, acute respiratory distress syndrome and severe pulmonary arterial hypertension; treatment of reversible causes of hypoxemic respiratory distress (see Mark et al., N. Eng. J. Med., December 22, 2005; 353(25), pages 2683-2695);

[0381] Nitric oxide as rescue therapy in patients with pulmonary embolism secondary acute right heart failure (see Summerfield et al., 201 1 ; Respir. Care 57(3), pages 444-448);

[0382] Treatment of angina pectoris, effects of paraquat poisoning and other cardiovascular disorders (see Abrams, The American Journal of Cardiology, 1996; 77(13), pages 31 C-37C;

[0383] Treatment of bladder contractile dysfunction (see Moro et al., Eur. J. Pharmacol., January 2012; 674(2-3), pages 445-449; see also Andersson et al., Br. J. Pharmacol. February 2008; 153(7), pages 1438-1444);

[0384] Treatment of acute and chronic lung infections and sepsis (see Fang et al., Nature Reviews. Microbiology, October 2004; 2(10), pages 820-832; see also Goldfarb et al., Critical Care Medicine, January 2007; 35(1), pages 290-292);

[0385] Toxic reactive nitrogen intermediates (RNIs) including nitric oxide have been proposed as effector molecules in the anti-mycobacterial action of activated murine macrophages against Mycobacterium tuberculosis (see Chan et al., J. Exp. Med., April 1992, pp. 1111-1122);

[0386] Gaseous nitric oxide can be effective in treating antibiotic-resistant bacterial and fungal lung infections in patients with cystic fibrosis (see Deppisch et al., 9th February 2016;“Gaseous nitric oxide to treat antibiotic resistant bacterial and fungal lung infections in patients with cystic fibrosis: a Phase I clinical study”, Springer, DOI 10.1007 / s15010-016-0879-x);

[0387] Nitric oxide has been reported as a potential topical broad-spectrum antimicrobial agent for dermatological conditions with little potential for developing resistance (see B L Adler and AJ Friedman, Future Sci. OA, 2015; 1 (1), FSO37);

[0388] Nitric oxide is a neurotransmitter and is associated with a variety of functions in the range of genital erectile in males and females to neuronal activity and avoidance learning (see Kim et al., J. Nutrition, 2004, 134, pp. 28735);

[0389] The use of nitric oxide for the treatment of male impotence and erectile dysfunction is described in Sullivan et al., Cardiovascular Research, August 1999, 43 (3), pp. 658-665;

[0390] Nitric oxide has been reported as a potential use as a surgical aid to help wound healing, reduce ischaemia-reperfusion injury, help the heart and lungs recover from surgery and help recover from vascular surgery and help recover from plastic surgery after surgery (see A Krausz and AJ Friedman, Future Sci. OA, 2015; 1 (1), FSO56);

[0391] 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;

[0392] European Patent No. 1411908 (Aberdeen University) reports data which is said to show that nitric oxide is effective in treating subungual infections, including Aspergillus niger;

[0393] NOx-generating compositions are topically applied to the skin for the treatment of fungal skin infections, such as Athlete's Foot (Hong Kong Foot) (see Weller et al. J. Am. Acad. Dermatol., April 1998, 38(4), pp. 559-563);

[0394] NOx-generating compositions are topically applied to the skin for the treatment of viral skin infections (see WO 99 / 44622);

[0395] NOx-generating compositions are topically applied to the skin for the treatment of conditions in which vasoconstriction is a fundamental problem, such as Raynaud syndrome (also known as Raynaud's phenomenon) (see Tucker et al. Lancet, 13 November 1999, 354, 9191, pp. 1670-1675);

[0396] The use of acidified nitrite salts as agents for the topical generation of nitric oxide at the surface of the skin for the treatment of peripheral local ischemia and related conditions, such as Raynaud's phenomenon and wounds (e.g. post-surgical wounds) and burns, is described in WO 2000 / 053193;

[0397] U.S. Patent No. 9,730,956 (Stenzler et al.) claims the use of a liquid nitric oxide releasing solution (NORS) to treat wounds in humans. NORS is also claimed to be antibacterial, antifungal, and / or antiviral, and data is provided purporting to demonstrate antibacterial efficacy against Acetobacter baumanii, methicillin-resistant Staphylococcus aureus, Escherichia coli, and Mannheimia haemolytica. Data is provided purporting to demonstrate antiviral efficacy of NORS against H1N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, and bovine parainfluenza-3 virus. Data is provided purporting to demonstrate antifungal efficacy of NORS against Trichophyton rubrum and Trichophyton mentagrophytes;

[0398] Chou S-H 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, pp. 954-959;

[0399] 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;

[0400] Hunter CJ et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat. Med. 2004, 10, pp. 1122-1127;

[0401] Ozaki M, et al., Reduced hypoxic pulmonary vascular remodeling by nitric oxide from the endothelium. Hypertension. 2001, 37, pp. 322-327;

[0402] Rubin LJ, 2006. Pulmonary arterial hypertension. Proc. Am. Thorac. Soc. 3, pp. 111-115;

[0403] Yellon D.M. et al., 2007. Myocardial Reperfusion Injury, N. Engl. J. Med., 357, pp. 1121-35;

[0404] Duranski M.R. et al., Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J. Clin. Invest. 2005, 115, pp. 1232-1240;

[0405] Jung K-H. et al., Early intravenous infusion of sodium nitrite protects brain against in vivo ischemia-reperfusion injury, Stroke, 2006, 37, pp. 2744-2750;

[0406] Esme H. et al. Beneficial Effects of Supplemental Nitric Oxide Donor Given during Reperfusion Period in Reperfusion-Induced Lung Injury. Thorac. Cardiovasc. Surg. 2006, 54, pp. 477-483;

[0407] The use of acidified nitrite for releasing NO as an agent for improving the quality of human skin is described in Chinese patent application No. CN 101028229;

[0408] The use of acidified nitrite salts for the release of NO as an agent to promote hair growth and prevent or treat hair loss in humans is described in Chinese patent application No. CN 101062050.

[0409] Further comprehensive discussions of the physiological roles 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; Murad, N Engl J Med, 2006, 355, pp. 2003-2011.

[0410] Pharmacological forms for the delivery of NO have been published in Butler and Feelisch, Circulation, 2008, 117, pp. 2151-2159.

[0411] The disclosures of each of the above-cited publications are incorporated herein by reference.

[0412] The present disclosure is applicable to all therapeutic and surgical uses of nitric oxide and nitric oxide-generating systems, including without limitation the specific therapies and surgical uses published in the above references and all other published therapies and surgical uses, as well as therapies and surgical uses based on the fundamental understanding of the physiological roles of nitric oxide and the reaction products that generate nitric oxide.

[0413] Vasodilation

[0414] The property of nitric oxide to induce vasodilation characterizes many of the therapies in which the combinations and compositions of the present disclosure and the gases emitted therefrom are used.

[0415] Specific examples of diseases, disorders, and conditions that respond to vasodilation include, but are not limited to, conditions associated with ischemia and skin damage.

[0416] Conditions associated with tissue ischemia include Raynaud's syndrome, severe primary vasospasm, and tissue ischemia, for example, resulting from surgery, septic shock, radiation, or peripheral vascular disease (e.g., diabetes and other chronic systemic diseases).

[0417] When used to treat or prevent a condition associated with surgical-induced tissue ischemia, the combination or composition of the disclosure or the nitric oxide evolved from the reaction producing NOx of the disclosure can be administered to the subject before, during or after surgery. The combination, composition or evolved gas can be administered to the surgical site or near the surgical site. Examples of surgical procedures for which the treatment or prevention of tissue ischemia can be used include transplant surgery, tissue or organ transplant surgery, coronary artery surgery, carotid catheterization, surgery to provide an intra-arterial or intravenous cannula for administration of a systemic agent such as a chemotherapy drug, cosmetic surgery including but not limited to a pedicle flap or a rotation flap, repeat surgery in the same site incision as a previous surgical procedure, surgery performed in an area of poor skin and / or underlying tissue perfusion or an area in which poor perfusion is expected to occur as a result of a concomitant disease (for example in a patient with arteriosclerosis or diabetes), surgery in the case of a wound with vascular damage or compromise and surgery to remove or correct a cutaneous or subcutaneous arteriovenous malformation.

[0418] By way of example, the combination, composition or evolved gas of the disclosure can be used to treat or prevent ischemic reperfusion injury of an organ by administering the combination, composition or evolved gas to the organ. The organ can be one or more selected from the group consisting of the heart (for example to prevent or treat myocardial ischemia), the brain (for example to treat or prevent cerebral ischemia and or infarction (stroke)), the lung (for example to treat or prevent ischemic reperfusion injury of the lung), the kidney (for example to treat or prevent ischemic reperfusion injury of the kidney) and the liver (for example to treat or prevent ischemic reperfusion injury of the liver). The surgery can be an organ transplant. The administration of the combination, composition or evolved gas can be post ischemic event or can be prophylactic.

[0419] Transdermal drug delivery uses

[0420] The property of nitric oxide to induce transdermal delivery of a drug represents another important utility of the combinations and compositions of the disclosure and the gas evolved therefrom.

[0421] WO 02 / 17881 and WO 2014 / 188175, the disclosures of which are incorporated herein by reference, describe the use of combinations and compositions for the production of nitric oxide and the gas evolved therefrom for transdermal drug delivery, and the preferred options and examples described in those publications for such use are also applicable to the combinations and compositions of the disclosure and the gas evolved therefrom.

[0422] Typically, the combinations and compositions of the disclosure will comprise one or more pharmaceutically active agents to be delivered transdermally to a subject and will be provided in a topical combination or composition for application to the skin of a subject. For examples of pharmaceutically active agents that can be used, see the section above entitled "Optional Additional Components".

[0423] A suitable topical combination can comprise a nitrite-containing mesh and a separate proton source-containing hydrogel, both suitable for use together on the skin of a subject, as described above in the section entitled "Other reservoirs of a composition or combination system; hydrogels". A polyol and a pharmaceutically active agent can be provided in one or more separate components of the combination or incorporated into the hydrogel, or a polyol and a pharmaceutically active agent can each take any combination of these options.

[0424] Treatment of wounds, skin lesions and burns

[0425] The properties of nitric oxide to induce vasodilation and drug transdermal delivery and to kill or prevent proliferation of microorganisms gives rise to another important utility of the combinations and compositions of the disclosure and gases emanating therefrom in the treatment of wounds, skin lesions and burns.

[0426] Conditions treatable using the disclosure include ulcers, skin donor sites, surgical wounds (post-surgery) burns (e.g. scalds, superficial burns, partial thickness burns and full thickness skin burns), lacerations and abrasions. The wounds can be chronic or acute. The ulcers can be of various origins, e.g. arterial or venous origin. Examples of ulcers include leg ulcers, e.g. chronic leg ulcers or acute leg ulcers; pressure ulcers, e.g. chronic pressure ulcers or acute pressure ulcers; venous ulcers and ulcers associated with diabetes, e.g. diabetic foot ulcers.

[0427] WO 2014 / 188174, which is incorporated herein by reference, describes combinations and compositions for the production of nitric oxide and the use of gases emanating therefrom to treat wounds, skin lesions and burns, and the same conditions described in that publication are also applicable to the combinations and compositions of the disclosure and gases emanating therefrom.

[0428] Typically, the combinations and compositions of the disclosure will comprise one or more pharmaceutically active agents and be provided in a topical combination or composition for application to the skin of a subject. For examples of pharmaceutically active agents that can be used, see the section above entitled "Optional additional components". For the treatment of wounds, skin lesions and burns, the one or more pharmaceutically active agents are suitably selected from analgesics and / or anaesthetics (e.g. local anaesthetics) (e.g. analgesics and / or anaesthetics that reduce chronic pain, acute pain or neuropathic pain), antimicrobial agents, antiseptics, anti-inflammatory agents and anti-scarring agents.

[0429] A suitable topical combination can comprise a nitrite-containing mesh 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." The polyol and pharmaceutically active agent can be provided in one or more separate components of the combination or incorporated into the hydrogel, or the polyol and pharmaceutically active agent can each employ any combination of these options.

[0430] Topical antimicrobial uses

[0431] In antimicrobial applications, therapeutically effective NO doses can be small, e.g., as low as a few hundred parts per million (ppm), e.g., 100 to 600 ppm (see, e.g., Ghaffari et al., Nitric Oxide Biology and Chemistry, 2009, 14, pp. 21-29, the disclosure of which is incorporated herein by reference), but the effectiveness of nitric oxide depends fundamentally on how long skin contact is maintained (Ormerod et al., BMC Research Notes, 2011, 4, pp. 458-465, the disclosure of which is incorporated herein by reference).

[0432] Proposals for slow local release of nitric oxide have been published (see, e.g., U.S. Patent No. 6,103,275). However, the resulting local NO dose lasts less than one hour, which provides poor local antimicrobial effect. As discussed above in the section entitled "Multicomponent Systems, Kits, and Dispensers," and elsewhere, and as shown in the following examples, the present disclosure enables much longer periods of NO administration in both local and non-local administration systems, resulting in significant clinical advantages.

[0433] Specifically, it has been found that the combinations and compositions of the present disclosure are capable of an initial intense output of nitric oxide ("initial burst") approximately 200-500 seconds after the start of the reaction that produces NOx, optionally followed by a long, slower release period of nitric oxide that extends for many hours ("tail"), after which gas evolution ceases or falls below effective levels. The dose of NO evolved by the combinations and compositions of the present disclosure exceeds the published minimum effective antimicrobial dose, leading to potentially effective topical antimicrobial uses for the combinations and compositions of the present disclosure and the gases evolved therefrom.

[0434] Formulations of NOx-generating combinations and compositions for topical antimicrobial applications are well described in the art, for example, U.S. Patent Application No. 2014 / 0056957, the disclosure of which is incorporated herein by reference, and such formulations are suitable for use with the combinations and compositions of the present disclosure. Another suitable topical combination can comprise a nitrite-containing mesh and a separate proton source-containing hydrogel, both suitable for use together on 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 can be provided in one or more of the separate components of the combination or incorporated into the hydrogel, or the polyols and pharmaceutically active agents can each take any combination of these options.

[0435] Other skin or topical treatments

[0436] Other topical applications of nitric oxide and nitric oxide-generating compositions include stimulating hair growth and treating impotence and erectile dysfunction.

[0437] The combinations and compositions of the present disclosure can be formulated for topical application of such treatments.

[0438] Topical dressings and dressing systems, e.g., wound dressings

[0439] In topical treatments, it is often desirable to cover or protect the area of skin being treated while the treatment is being applied. This can help prevent the wound from becoming contaminated, help remove pus or debris from the healing process, prevent or limit the treatment composition from being lost during bathing or showering or through contact with clothing or from the subject's normal activities, and protect the treated area from buffeting or rubbing.

[0440] To this end, the treatment is often incorporated into a topical dressing or dressing system, e.g., a wound dressing or dressing system. At least one component of the dressing or dressing system typically comprises a backing, which can be water-impermeable or water-permeable, optionally with a skin-adhesive portion and optionally other layers, e.g., gauze or a pad.

[0441] In another aspect, the present disclosure provides a topical dressing, e.g., a wound or skin dressing, or a dressing system, comprising a combination or composition according to the fifth aspect of the present disclosure, at least one component of the dressing or the dressing system comprising a backing and optionally one or more other layers, e.g., a layer selected from gauze and a pad. The combination or composition according to the fifth aspect of the present disclosure is suitably disposed on the skin-facing side of the backing and arranged to provide the desired area of epidermis with the NOx-generating reaction mixture or gas evolved therefrom when the dressing is applied to the skin and the NOx-generating reaction is initiated.

[0442] The dressing or dressing system is suitably provided in a sealed, sterile package prior to use.

[0443] Nasal, oral, respiratory, and lung uses

[0444] The properties of nitric oxide to induce vasodilation and transdermal delivery of drugs and to kill microorganisms or prevent microbial proliferation create another important utility for the combinations and compositions of the present disclosure and the gases evolved therefrom in treating mucous membranes and tissues of the nose, mouth, respiratory tract and lungs, and / or the use of the nose, mouth, respiratory tract and lungs as routes of administration for delivering the combinations and compositions of the present invention to human or animal subjects.

[0445] Conditions that can be treated using the present 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) (in particular, emphysema, 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.

[0446] WO 2009 / 086470, the disclosure of which is incorporated herein by reference, describes the use of aerosolized liquid combinations and compositions for generating nitric oxide and gases emitted therefrom for treating diseases of the nose, mouth, respiratory tract and lungs, and / or the use of the nose, mouth, respiratory tract and lungs as routes of administration for delivering such combinations and compositions to human or animal subjects, and the preferred embodiments and embodiments described in that publication for such uses also apply to the combinations and compositions of the present disclosure and gases emitted therefrom.

[0447] Typically, combinations and compositions of the invention for delivery to the nose, oral cavity, respiratory tract and lungs will contain one or more pharmaceutically active agents. See the section above entitled "Optional Additional Components" for examples of pharmaceutically active agents that may be used.

[0448] Via the delivery route of nose, oral cavity, respiratory tract or lung, two kinds of main delivery methods may be used to carry out the present invention.The first is that combination of the present invention or composition are directly delivered to nose, oral cavity, respiratory tract or lung.The second is to use the present invention to be delivered to nose, oral cavity, respiratory tract or lung from the gas emitted by the reaction that produces NOx, without combination of the present invention or composition entering patient's health.

[0449] 1. Direct delivery of the combination or composition to the nose, mouth, respiratory tract or lungs

[0450] The combination or compositions or their components can be delivered in dry solid form directly to the nose, mouth, respiratory tract or lungs, where mucosal fluids dissolve the solid component materials and initiate the NOx-generating reaction.

[0451] The components of the combination can be administered separately or together. In a preferred embodiment, the proton source or at least one component thereof can be administered prior to the remaining components in order to establish a relatively acidic environment in the mucosa which facilitates rapid initiation of the NOx-generating reaction upon in situ contact of the nitrite component with the proton source component.

[0452] A therapeutically effective dose of one or more dry powder components (e.g., one or more of the nitrite component, the proton source component, and the polyol component) or dry powder compositions of the combination can be delivered to a subject by inhalation of the dry powder using a dry powder inhaler that delivers an aerosol containing particles having a volume mean diameter of less than 6 microns to the subject. The dry powder inhaler can be adapted for single or multiple loading of the dry powder for administration such that the dry powder inhaler delivers between about 0.1 mg and about 100 mg of the one or more dry powder components or dry powder compositions in particles having a volume mean diameter of less than 6 microns to the subject per inhalation breath.

[0453] Additionally or alternatively, the combination or composition or components thereof can be delivered directly to the nose, mouth, respiratory tract, or lungs as a mist or spray of droplets of a solution of one or more of the nitrite component, the proton source component, and the polyol component.

[0454] The embodiments of the application described herein are generally suitable for direct delivery to the nose, mouth, respiratory tract, or lungs of a subject. Without limitation, for example, the combination or composition, or components thereof, can be directly administered 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, specific functional components intended to provide one or more particular benefits. Examples of suitable physiologically compatible diluents, carriers, and / or excipients include, without limitation, lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talcum, cellulose, cellulose derivatives, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, and the like. If desired, minor amounts of nontoxic auxiliary substances, such as wetting agents, emulsifying agents, lubricants, binders, and solubilizers, such as sodium phosphate, potassium phosphate, gum acacia, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like, can also be present. Generally, the pharmaceutical formulations will contain from about 0.005% to about 95% by weight, preferably from about 0.5% to about 50% by weight, of the combination or composition of the application, or components thereof, depending on the intended mode of administration. Actual methods of preparing such dosage forms are known, or will be apparent in light of this disclosure, to those skilled in this art. See, e.g., Martindale, 39thEd. (2017), the Merck Index, 15thEd. (2013); Goodman & Gilman’s “The Pharmacological Basis of Therapeutics”, 13thEd. (2017); the British National Formulary online (https: / / bnf.nice.org.uk / ); Remington: “The Science & Practice of Pharmacy”, 22ndEd. (2012); or the Physician’s Desk Reference, 71stEd. (2017).

[0455] In a preferred embodiment, the combination or composition for delivery to the nose, mouth, respiratory tract, or lungs of a subject will take the form of unit dosage forms, such as vials containing liquids, solids to be suspended, dry powders, lyophilizates, or other compositions, suitably containing the following components together with the NOx-generating reaction: diluents, such as lactose, sucrose, dicalcium phosphate, and the like; lubricants, such as magnesium stearate and the like; binders, such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives, and the like.

[0456] 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 liquid form of a composition is suitably delivered to a subject by inhalation using a nebulizer that delivers to the subject an aerosol containing particles having a volume mean diameter of less than 5 microns, with any droplets or droplet form of a composition comprising the combined components being delivered directly to the nose, mouth, respiratory tract, or lungs. The nebulizer can be adapted for single or multiple loading of the administration of the liquid components or liquid composition of the combination, such that the nebulizer delivers to the subject between about 0.1 mg and about 100 mg of the one or more liquid components or liquid form of a composition in droplets having a volume mean diameter of less than 5 microns, preferably in droplets having a size in the range of about 2 to about 5 pm, per inhalation breath.

[0457] In one embodiment, the nebulizer is selected according to an aerosol that allows formation of droplets or droplet form of a composition comprising the combined components having a mass median aerodynamic diameter (MMAD) primarily between about 2 to about 5 microns.

[0458] In one embodiment, the amount of droplets or droplet form of a composition comprising the combined components delivered provides a therapeutic effect against pulmonary pathologies, respiratory tract infections, and / or extra-pulmonary, systemic distribution, thereby also treating extra-pulmonary and systemic diseases.

[0459] Previously, both jet and ultrasonic nebulizers have been shown to be capable of generating and delivering aerosol particles sized between 2 and 4 μιη. These particle sizes have been shown to be optimal for deposition in the intermediate airways, and thus optimal for treating bacterial infections of the lung caused by gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, P. aeruginosa, Serratia marcescens, Haemophilus influenzae, Burkholderia cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, Staphylococcus aureus, and multi-drug resistant P. aeruginosa. However, unless specially formulated solutions are used, these nebulizers typically require larger volumes of drug to be administered in order to achieve a therapeutic effect. Jet nebulizers utilize air pressure to break up aqueous solutions into aerosol droplets. Ultrasonic nebulizers utilize a piezoelectric crystal to shear aqueous solutions. However, typically, jet nebulizers are only about 10% efficient under clinical conditions, while ultrasonic nebulizers are only about 5% efficient. Thus, the amount of drug that is deposited and absorbed in the lung is a small fraction of 10%, despite the large amount of drug that is placed into the nebulizer. Smaller particle size or slow inhalation rates allow for deep lung deposition. The present invention can desire middle lung and alveolar deposition, depending on the indication, such as for antimicrobial activity, where intermediate airway deposition is desired, or for pulmonary hypertension and systemic delivery, where intermediate and / or alveolar deposition is desired. Exemplary disclosures of compositions and methods for delivering formulations using a vibrating mesh nebulizer can be found, for example, in US 2006 / 0276483, including a description of techniques, protocols, and characterization for delivering aerosolized mist using a vibrating mesh nebulizer. The disclosure of US 2006 / 0276483 is incorporated herein by reference.

[0460] Thus, in one embodiment, in a preferred embodiment a vibrating mesh nebulizer is used to deliver an aerosol of a composition comprising a combination component or combination in the form of droplets. The vibrating mesh nebulizer comprises a liquid reservoir in fluid contact with a membrane and inhalation and exhalation valves. In one embodiment, about 1 ml to about 5 ml of liquid formulation to be delivered is in the reservoir, and engages an aerosol generator, generating a nebulized aerosol with particle size selectively between about 1 μιη and about 5 μιη volume mean diameter.

[0461] Thus, for example, in preferred embodiments, the nitrite component formulation or one or both of these components optionally including one or more organic polyols according to the present application are placed in a liquid nebulizer and dosed to deliver from about 7 mg to about 700 mg, preferably from about 17.5 mg to about 700 mg in about 1 ml to about 5 ml, more preferably from about 17.5 mg to about 350 mg in about 1 ml to about 5 ml, preferably from about 0.1 mg to about 300 mg in about 1 ml to about 5 ml, more preferably from about 0.25 mg to about 90 mg in about 1 ml to about 5 ml, resulting in a volume mean diameter particle size of between about 1 μιη to about 5 μιη.

[0462] By way of non-limiting example, the composition comprising the combination components in nebulized liquid or droplet form can be administered in the inhalable delivery dose described in less than about 20 minutes, preferably in less than about 10 minutes, more preferably in less than about 7 minutes, more preferably in less than about 5 minutes, more preferably in less than about 3 minutes, and in some cases most preferably in less than about 2 minutes.

[0463] By way of non-limiting example, in other cases, the composition comprising the combination components in nebulized liquid or droplet form can achieve improved tolerability and / or exhibit an area under the curve (AUC) shape enhancement feature when administered over a longer period of time. In these conditions, the inhalable delivery dose described is in excess of about 2 minutes, preferably in excess of about 3 minutes, more preferably in excess of about 5 minutes, more preferably in excess of about 7 minutes, more preferably in excess of about 10 minutes, and in some cases most preferably from about 10 minutes to about 20 minutes.

[0464] One example of separate component formulations can comprise (i) a nitrite in aqueous solution having a pH in excess of about 6, for example in the range of about 6 to about 8, for example about 7; and (ii) a proton source component in aqueous solution, the at least two separate liquid solution components (i) and (ii) being capable of mixing to form a NOx-generating composition that can be used to load a nebulizer for delivery to a human patient or a veterinary subject.

[0465] For aqueous and other unpressurized liquid systems, a variety of nebulizers, including small volume nebulizers, can be used to atomize the combined components or compositions into a mist. Compressor-driven nebulizers incorporate jet technology and use compressed air to generate a liquid aerosol. Such devices are available from, for example, Healthdyne Technologies, Inc.; Invacare, Inc.; Mountain Medical Equipment, Inc.; Pari Respiratory, Inc. (Midlothian, VA); Mada Medical, Inc.; Puritan-Bennet; Schuco, Inc.; DeVilbiss Health Care; and Hospitak, Inc. 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. Vibration mesh nebulizers rely on piezoelectric or mechanical pulses to generate inhalable droplets. Other examples of nebulizers for use with the nitrite, nitrite salt, or compound providing a nitrite or nitric oxide 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; 5,960,792; 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, all of which are incorporated herein by reference in their entireties.

[0466] Commercial examples of nebulizers that can be used for the droplets or compositions in droplet form described herein comprising the combined components include Respirgard II® nebulizers (Aerogen, Inc., Galway, Ireland) and and Aeroneb Solo® nebulizers (Aerogen, Inc., Galway, Ireland) and Aerx Essence® nebulizers (Aradigm, Inc., Hayward, CA) and TM; Produced by Respironics (Murrysville, Pennsylvania, USA) Freeway Freedom TM , SideStream, SideStream Plus, Ventstream, and I-neb; and PARI manufactured by PARI Respiratory Equipment, Inc., Midlothian, Virginia, USA; and PARI Ltd., Starnberg, Germany. and e-Flow TM Any of these nebulizers can be used with a face mask or mouthpiece according to the manufacturer's instructions. By way of further non-limiting example, U.S. Patent No. 6,196,219 is incorporated herein by reference in its entirety.

[0467] In one embodiment, an aqueous formulation containing soluble or nanoparticulate drug particles is provided. For aqueous aerosol formulations, the drug can be present in a concentration of 0.67 mg / mL to a maximum of 700 mg / mL; in certain preferred embodiments, nitrite is present at a concentration of 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 drug substance to the lungs in a very short period of time. In one embodiment, the formulation is optimized to provide a well-tolerated formulation. Therefore, certain preferred embodiments contain nitrite (e.g., sodium nitrite, potassium nitrite, or magnesium nitrite) and are formulated to have a good taste, a pH of about 4.7 to about 6.5, an osmotic pressure of about 100 to about 3600 mOsmol / kg, and optionally in certain other embodiments, a concentration of penetrant ions (e.g., chloride ions, bromide ions) of about 30 to about 300 mM.

[0468] 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 about 7.0 to about 9.0 as a single vial configuration. This pH value range improves tolerability, as described elsewhere herein, including taste-masking agents according to certain embodiments. When the aerosol is acidic or basic, it can cause bronchospasm and coughing. While the safe range of pH values is relative, and some patients can tolerate slightly acidic aerosols, other patients 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 above about 8 can have low tolerability, as the body tissues generally cannot buffer basic aerosols. Controlling aerosols with a pH value below about 4.5 and above about 8.0 typically causes lung irritation, with severe bronchospasm coughing and inflammatory reactions. For these reasons, and to avoid bronchospasm, coughing, or inflammation in patients, the optimal pH value of the aerosol formulation is determined to be between about pH 5.5 to about pH 8.0.

[0469] Thus, in one embodiment, the aerosol formulation used as described herein is adjusted to a pH value of between about 4.5 and about 7.5, with the most preferred pH range of the acidic mixture being about 4.7 to about 6.5, and the most preferred pH range of the single vial configuration being about 7.0 to about 8.0. By way of non-limiting example, the composition can also include a pH buffer or pH adjusting agent, typically a salt prepared from an organic acid or organic base, and in preferred embodiments, an acidic excipient (e.g., a non-reducing acid, such as citric acid or citrate, e.g., sodium citrate) or buffer as described herein, e.g., citrate or other buffers described above, and with reference to Table 1. Thus, these and other representative buffers can include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, edetate, hydrochloride, or phosphate buffers.

[0470] Many patients have increased sensitivity to a variety of chemical tastes, including bitter, salty, sweet, metallic. To create a well-tolerated drug product, taste-masking can be achieved by adding taste-masking agents and excipients, adjusting osmolality, and sweeteners.

[0471] Many patients have increased sensitivity to a variety of chemical agents and have a high incidence of bronchospasm, asthma or other coughing episodes. Their airways are particularly sensitive to hypotonic or hypertonic and acidic or basic conditions, and to the presence of any permeant ion such as chloride. Any imbalance in these conditions or the presence of chloride above a certain concentration value will cause bronchospasm or an inflammatory episode and / or coughing, which greatly diminishes the therapy of inhalable formulations. Both conditions can prevent the effective delivery of aerosolized drug to the intrabronchial space, lacking the advantageous use of pH adjustment, tonicity and taste masking agents according to certain embodiments disclosed herein.

[0472] In some embodiments, the tonicity of the aqueous solution of the nitrite compound disclosed herein (or in unique embodiments of nitrite or compounds that provide nitric oxide) is adjusted by the provision of excipients. In some cases, a certain amount of permeant ions, such as chloride, bromide or another anion, can facilitate the successful and effective delivery of aerosolized nitrite. However, it has been found that for the nitrite components disclosed herein, the amount of such permeant ions can be lower than that typically used for the aerosolized administration of other pharmaceutical compounds.

[0473] For aerosolization with a given tonicity, it can not be possible to improve bronchospasm or cough reflex in all cases by using diluents. However, when the tonicity of the diluents is within a certain range, it is often possible to adequately control and / or suppress these reflexes. A preferred solution for the aerosolization of therapeutic compounds that is safe and tolerable has a total tonicity of about 100 to about 3600 mOsmol / kg, a chloride concentration in the range of about 30 mM to about 300 mM, and preferably about 50 mM to about 150 mM. This tonicity controls bronchospasm, and the chloride concentration as a permeant anion controls cough. Because bromide or iodide anions are also permeant ions, they can be substituted for chloride. Additionally, bicarbonate can be substituted for chloride.

[0474] The nanoparticulate drug dispersions can also be freeze-dried to obtain a powder suitable for nasal or pulmonary delivery. Such powders can contain aggregated nanoparticulate drug particles with a surface modifier. Such aggregates can have a size in the respirable range, for example, about 2 to about 5 microns MMAD.

[0475] 2. Delivery of the gas evolved from the NO generating reaction to the nose, mouth, respiratory tract or lungs

[0476] Inhalers for delivering metered quantities of nitric oxide to the lungs of patients are well known. In general, nitric oxide is generated on site and delivered in pressurised cylinders connected to a dedicated delivery device for use in a hospital or clinic. The INOmax Therapy system can be mentioned 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 acronym INOmax (inhaled nitric oxide) is generally used for the cylinders of the INOmax Therapy system, and INOvent for the delivery device. An evaluation of the INOmax Therapy system has been published, for example Kirmse et al. Chest, June 1998, 113(6), pages 1650-1657. The published disclosure is incorporated herein by reference.

[0477] The method according to the first aspect of the application is suitably carried out in a dedicated NO manufacturing facility, and the gaseous product according to the second aspect of the application is provided to the user in a pressurised cylinder in the normal manner. The pressurised cylinder is then used in known manner in conjunction with distribution, monitoring, dosing, mixing and delivery equipment.

[0478] Target for antimicrobial use

[0479] As described previously, the reactions of the present disclosure to generate NOx and the gases evolved therefrom have biocidal or biostatic effects on a potentially wide range of microorganisms, leading to many antimicrobial applications.

[0480] The microorganism can for example be any one or more selected from bacterial cells, viral particles and / or fungal cells or micro-parasites, and can be individual cells, organisms or colonies. The bacterial cells, viral particles and / or fungal cells or micro-parasites can be present on or in a host organism, for example as part of the gut microbiome of a human or other animal, or in a bacterial infection of a human or other animal. The bacterial and / or fungal cells and / or viral particles and / or micro-parasites can be in vitro, in vivo or ex vivo.

[0481] The present disclosure is particularly useful for treating or preventing microbial infection of a site of skin damage in a subject. The present disclosure is particularly useful for treating or preventing microbial infection in an immunosuppressed subject.

[0482] When the microorganism is present in a bacterial infection, fungal infection, viral or micro-parasite infection of a human or other animal, the infection can for example be in the context of a disease such as a cold, influenza, tuberculosis, SARS, COVID-19, pneumonia or measles.

[0483] 1. a bacterial cell

[0484] The bacteria can be a pathogenic bacterial species. The microbial infection can be an infection caused by a pathogenic bacterial species, including gram-positive and gram-negative, aerobic and anaerobic, antibiotic-sensitive and antibiotic-resistant bacteria.

[0485] Examples of bacterial species that can be targeted using the present application include species of the following bacteria: Actinomyces, Bacillus, Bartonella, Bordetalla, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Heliobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia. The present application can also target any combination thereof.

[0486] In particular embodiments, the microbe can be a pathogenic species of Corynebacterium, Mycobacterium, Streptococcus, Staphylococcus, Pseudomonas, or any combination thereof.

[0487] In more specific embodiments, the targeted microorganism can be selected from the group consisting of Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii; Borrelia afzelii; Borrelia recurrentis; Brucella abortus; Brucella canis; Brucella melitensis; Brucella suis; Campylobacter jejuni; Chlamydia pneumoniae; Chlamydia trachomatis; Chlamydophila psittaci; Clostridium botulinum; Clostridium difficile; Clostridium perfringens; Clostridium tetani; Corynebacterium diphtheria; Ehrlichia canis; Ehrlichia chaffeensis; Enterococcus faecalis; Enterococcus faecium; Escherichia coli, such as Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli, Enteroinvasive E. coli (EIEC), and Enterohemorrhagic E. coli (EHEC), including E. coli 0157.H7; Francisella tularensis; Haemophilus influenza; Helicobacter pylori; Klebsiella pneumoniae; Legionella pneumophila; Leptospira species; Listeria monocytogenes; Mycobacterium leprae; Mycobacterium tuberculosis; Mycobacterium abscessus; Mycobacterium ulcerans; Mycoplasma pneumoniae; Neisseria gonorrhoeae; Neisseria meningitides; Pseudomonas aeruginosa; Nocardia asteroids; Rickettsia rickettsia; Salmonella typhi; Salmonella typhimurium; Shigella sonnei; Shigella dysenteriae; Staphylococcus aureus; Staphylococcus epidermidis; Staphylococcus saprophyticus; Streptococcus agalactiae; Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus viridans; Treponema pallidum subspecies pallidum; Vibrio cholera; Yersinia pestis; and any combination thereof.

[0488] In particular, the microorganism can be selected from Chlamydia pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Haemophilus influenza, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or any combination thereof.

[0489] The microorganism can be an antibiotic resistant or antibiotic susceptible pathogenic bacterial species, or an antibiotic resistant or antibiotic susceptible strain of a bacterial species. The use of nitric oxide therapy for methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) is described in, for example, WO 02 / 20026, the disclosure of which is incorporated herein by reference. Thus, an example of an antibiotic resistant or antibiotic susceptible pathogenic bacterial species that can be killed or treated using the present application is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-susceptible Staphylococcus aureus (MSSA).

[0490] 2. Fungal cell

[0491] The microorganism can be a pathogenic fungal species. The microorganism infection can be an infection caused by a pathogenic fungal species including pathogenic yeasts.

[0492] Examples of fungal species that can be targeted using the present application include species of the following fungi: Aspergillus, Blastomyces, Candida (e.g. Candida auris), Coccidioides, Cryptococcus (in particular, Cryptococcus neofromans or Cryptococcus gattii), Hisoplamsa, Murcomycetes, Pneumocystis (e.g. Pneumocystis jirovecii), Sporothrix, Talaromyces, or any combination thereof.

[0493] Examples of fungal infections include aspergillosis (e.g. allergic bronchopulmonary aspergillosis), athlete's foot (Hong Kong foot), infections caused by Candida pathogenic species, such as vaginal yeast infections, fungal toenail infections, and diaper rash, tinea cruris (jock itch), and tinea corporis (ringworm).

[0494] 3. Viral particle

[0495] The microorganism can be a viral particle. The infection can be caused by a pathogenic virus.

[0496] Examples of viruses that can be targeted using the present application include influenza virus, parainfluenza virus, adenovirus, norovirus, rotavirus, rhinovirus, coronavirus, respiratory syncytial virus (RSV), astrovirus, and hepatitis virus. In particular, the compositions of the present application can be used to treat or prevent an infection caused by one selected from the group consisting of H1N1 influenza virus, bovine infectious rhinotracheitis virus, bovine respiratory syncytial virus, bovine parainfluenza-3 virus, SARS-CoV, SARS-CoV-2, and any combination thereof.

[0497] In particular, the present application can be applied to the treatment of a disease or condition caused by a viral infection. Examples of such diseases that can be targeted by the present application include respiratory viral diseases, gastrointestinal viral diseases, exanthematous viral diseases, hepatic viral diseases, cutaneous viral diseases, hemorrhagic viral diseases, and neurological viral diseases.

[0498] Respiratory viral infections include influenza, rhinovirus (i.e., the common cold virus), respiratory syncytial virus, adenovirus, coronavirus infection (e.g., 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, shingles, roseola, smallpox, fifth disease, and chikungunya virus disease. Hepatic viral diseases include hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E. Cutaneous viral diseases include warts, such as genital warts, oral herpes, genital herpes, and molluscum contagiosum. Hemorrhagic viral diseases include Ebola, Lassa fever, denghue fever, yellow fever, Marbug hemorrhagic fever, and Crimean-Congo hemorrhagic fever. Neurological viral diseases that can be targeted using the present application include polio, viral meningitis, viral encephalitis, and rabies.

[0499] 4. Parasitic microorganisms

[0500] The microorganism can be a parasitic microorganism (micro-parasite). The infection can be caused by a pathogenic parasitic microorganism.

[0501] Examples of parasitic microorganisms that can be targeted using the present application include protozoa.

[0502] In particular, the present application can target the following groups of protozoa: Sarcodina (e.g. Amoebae, e.g. Entamoeba, e.g. Entamoeba histolytica or Entamoeba dispar), Mastigophora (e.g. flagellates, e.g. Giardia and Leishmania), Ciliophora (e.g. ciliates, e.g. Balantidium), Sporozoa (e.g. Plasmodia and Cryptosporidium), and any combination thereof.

[0503] Parasitic infections that can be treated using the present application include malaria, amoebic dysentery, and leishmaniasis (e.g. cutaneous leishmaniasis, mucocutaneous leishmaniasis, or visceral leishmaniasis).

[0504] Human / animal host or subject

[0505] The subject can be an animal or a human subject. The term "animal" herein can generally include humans; however, where the term "animal" appears in the phrase "animal or human subject" and the like, it will be understood from the context that it refers especially to a non-human animal, or that reference to "human" is merely an option that the animal can be human to avoid doubt.

[0506] In particular embodiments, the subject is a human subject. The human subject can be an infant or an adult subject.

[0507] In particular embodiments, the subject is a vertebrate subject. The vertebrate can belong to the class Agnatha (jawless fish), the class Chondrichthyes (cartilaginous fish), the class Osteichthyes (bony fish), the class Amphibia (amphibians), the class Reptilia (reptiles), the class Aves (birds), or the class Mammalia (mammals). In particular embodiments, the subject is an animal subject that belongs to the class Mammalia or the class Aves.

[0508] In particular embodiments, the subject is a domesticated species. The domesticated species can be one of the following:

[0509] - commensal animals adapted to the human niche (e.g. dogs, cats, guinea pigs)

[0510] - prey animals or farm animals sought or kept for food (e.g. cattle, sheep, pigs, goats); and

[0511] - animals kept primarily for draught purposes (e.g. horses, camels, donkeys)

[0512] Examples of livestock include, but are not limited to, alpaca, nilgai, bison, camel, canary, capybara, cat, cattle (including Bali cattle), chicken, coati, deer (including fallow deer, sika deer, chital, and white-tailed deer), dog, donkey, feral pigeon, duck, giraffe, elk, emu, ferret, gaur, goat, goose, guinea fowl, guinea pig, greater kudu, horse, llama, mink, mouse, mule, musk ox, ostrich, parrot, pig, pigeon, quail, rabbit, rat (including agouti), reindeer, agouti, sheep, turkey, water buffalo, yak, and zebu.

[0513] Organs, structures, and internal spaces of an animal / human host or subject

[0514] The organs to which the compositions or multi-component systems of the present disclosure are administered are not limited. Examples of organs include skin, and organs of the respiratory system, the genitourinary system, the cardiovascular system, the digestive system, the endocrine system, the excretory system, the lymphatic system, the immune system, the integumentary system, the muscular system, the nervous system, the reproductive system, and the skeletal system.

[0515] Examples of organs of the cardiovascular system include the heart, the lungs, the blood, and the blood vessels. Examples of organs of the digestive system include the salivary glands, the esophagus, the stomach, the liver, the gall bladder, the pancreas, the intestines, the colon, the rectum, and the anus. Examples of organs of the endocrine system include the hypothalamus, the pituitary gland, the pineal body or pineal gland, the thyroid gland, the parathyroid glands, and the adrenal glands, i.e., the adrenal glands. Examples of organs of the excretory system include the kidneys, the ureters, the urinary bladder, and the urethra. Examples of organs of the lymphatic system include the lymph and the lymph nodes and vessels. Examples of organs of the immune system include the tonsils, the adenoids, the thymus, and the spleen.

[0516] Examples of organs of the integumentary system include the skin, hair, and nails of mammals, scales of fish, reptiles, and birds, and feathers of birds. Examples of organs of the nervous system include the brain, the spinal cord, and the nerves. Examples of organs of the reproductive system include the sex organs, such as the ovaries, the fallopian tubes, the uterus, the vulva, the vagina, the testes, the vas deferens, the seminal vesicles, the prostate, and the penis. Examples of organs of the skeletal system include the bones, the cartilage, the ligaments, and the tendons.

[0517] Cavities of a human subject include, but are not limited to, the oral cavity, the nose, the ear, the throat, the respiratory tract, the lungs, the gastrointestinal tract, the dorsal body cavity (e.g., the cranial cavity or the vertebral cavity), or the ventral body cavity (e.g., the thoracic cavity, the abdominal cavity, or the pelvic cavity). The nasal, oral, respiratory, and pulmonary routes of administration are characteristic features of the present invention.

[0518] In vitro antimicrobial treatment of surfaces

[0519] The components and compositions of the present disclosure, and the gases evolved from the NOx-generating reactions according to the present disclosure, can be used to apply antimicrobial treatment in vitro. By "in vitro" is meant that the surface treated is not a living organism, even though it can ultimately be intended for medical use.

[0520] Examples of such utilities include methods for disinfecting surgical instruments, hypodermic syringe needles, and other medical devices prior to use, and for cleaning or treating surfaces to reduce or prevent the spread of pathogens, whether in a hospital or clinic or anywhere else.

[0521] Other examples include methods for disinfecting prosthetic and implantable devices, such as stents (e.g., coronary artery stents), surgical screws, rods, plates and splints, orthopedic implants, cardiac pacemakers, insulin infusion devices, catheters, ostomy appliances, intraocular lenses, cochlear implants, electroanalgesic implants, implantable contraceptive devices, neurostimulators, artificial heart valves, electrodes, intravenous drips and drug delivery devices, and the like, prior to their placement in a subject.

[0522] If desired, the components or compositions of the present disclosure can be coated onto the surface of a prosthetic or implantable device, whereby the NO evolved in the NOx-generating reaction can perfuse other tissues or organs or exert other physiological effects in the vicinity of the prosthetic or implanted device.

[0523] Techniques for rendering the surface of a prosthetic or implantable device biocompatible are well known to those of skill in the art, including incorporation of functional coatings, such as coatings comprising the components or compositions of the present disclosure. See, e.g., Gultepe et al., Advanced Drug Delivery Reviews, March 8, 2010, 62(3), pp. 305-315; and U.S. Patent Nos. 5702754 and 6270788, and the publications referenced therein, the disclosures of all of which are incorporated herein by reference.

[0524] More general antimicrobial treatment compositions and methods for inanimate surfaces are well known in the art and need not be broadly described here. Antimicrobial compositions are used in, for example, the medical industry, the food industry, the meat processing industry, and the private sector for individual users. Antimicrobial cleaning compositions typically contain in an aqueous and / or alcoholic carrier one or more active antimicrobial agents or components thereof, surfactants, and one or more further ingredients such as dyes, fragrances, pH regulators, thickeners, skin conditioners, etc. Broad-spectrum bactericides or antimicrobial compositions aim at reducing the pathogen load of a range of pathogens on a surface. Typically, the compositions are liquids (or made from a solid pre-mix into a liquid before use), which after the desired adjustment of the concentration, suitably by the addition of water, are applied by means of a cloth or other wiping means or sprayed onto the surface to be treated and then allowed to dry or wiped off. Conventional compositions and methods of treating surfaces are in principle applicable to the present application, whereby the active antimicrobial agent is or comprises a NOx-generating composition according to the present application or components thereof.

[0525] For further discussion and examples of known antimicrobial compositions and methods of use that can be used in conjunction with the present application, reference is made to, 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; WO 95 / 32705; WO 95 / 09605; and WO 98 / 55096; the contents of which are incorporated herein by reference in their entirety.

[0526] Use to enhance human and / or animal well-being

[0527] In addition to the medical uses discussed above, the present disclosure can be used for non-therapeutic applications in a human or animal subject. Non-therapeutic applications differ from therapeutic applications in that the subject is healthy, or the present application is not targeted at treating any diagnosed disease, disorder, or condition that the subject is suffering from.

[0528] Non-therapeutic applications can include treatments aimed at enhancing the well-being or happiness of the subject, or improving the metabolic efficiency or immune system activity of the subject, such that the subject is able to function better normally or fight off future infections. Non-therapeutic applications also include treatments that help the cognitive function of the subject or cause a feeling of confidence and control.

[0529] For use in such non-therapeutic applications, the combinations and compositions of the disclosure can be formulated similarly to pharmaceutical formulations or in a non-pharmaceutical manner. For further details on formulations similar to pharmaceutical formulations, see the section above entitled "Optional Additional Components". Non-pharmaceutical formulations suitably include food additives, nutraceutical formulations, foods, beverages and beverage additives. Formulations suitable for addition to foods and beverages suitably are in the form of a liquid or a powder. Nutraceutical formulations suitably are in the form of a tablet, capsule or orally consumable liquid.

[0530] As mentioned above in the section entitled "Use in therapy or surgery", the medical and / or surgical use of the disclosure can provide an indirect benefit to the patient in terms of enhanced well-being or confidence.

[0531] Plant use

[0532] Nitric oxide is known to have beneficial effects on living or dead plants. The disclosure includes methods, devices, combinations, kits, compositions, uses and applications of gases emanating therefrom that provide beneficial effects on living or dead plants.

[0533] Examples of known uses of nitric oxide and nitric oxide-generating systems on plants include the following:

[0534] Nitric oxide prevents or delays wilting of cut flowers and plants (see Siegel-Itzkovich, BMJ, 1999; 319(7205), p274; 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 ));

[0535] Nitric oxide modulates plant-pathogen interactions, promotes plant hypersensitive responses, symbiosis with organisms in nitrogen-fixing nodules, development of lateral and adventitious roots and root hairs, and control of stomatal opening (see Mur et al., 2013; cited above);

[0536] 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);

[0537] The role of nitric oxide in the signalling pathways of plant growth hormones, cytokinins and other plant hormones (see Liu et al., Proceedings of the National Academy of Sciences, 2013; 110(4), pp. 1548-1553).

[0538] The disclosures of each of the above-cited publications are incorporated herein by reference.

[0539] Furthermore, the antimicrobial effects of the nitric oxide-generating systems of the present disclosure and the gases emitted therefrom, which are specifically described but not exclusively described above in the sections entitled "Use in therapy or surgery", "Topical antimicrobial use", "Nasal, oral, respiratory and pulmonary use" and "Target of antimicrobial use", equally apply to targeting microbial infections of plants, and the present disclosure extends to such uses as well.

[0540] The above known uses of nitric oxide and nitric oxide-generating systems on plants and all other uses thereof constitute further aspects of the present disclosure when used in conjunction with the reactions for generating nitric oxide using the present disclosure and / or the nitric oxide, optionally other nitrogen oxides and / or optionally precursors thereof produced thereby.

[0541] In particular, the plants treated can be agricultural crops or household plants, i.e. plant species cultivated by humans.

[0542] Agricultural crops include, but are not limited to, food crops (such as cereals, vegetables and fruits), medicinal active ingredient crops (such as quinine), fibre crops (such as cotton or flax), other material crops (such as rubber and wood) and flower crops (such as roses and tulips).

[0543] Other examples of crops for human food consumption include, but are not limited to, crops that produce rice, wheat, sugar cane and other sugar crops, maize (corn), soybean oil, potato, palm oil, cassava, legume seeds, sunflower oil, rapeseed oil, mustard oil, sorghum, millet, groundnuts, sweet potato, banana, soybean, cottonseed oil, peanuts, peanut oil, yams, tomatoes, grapes, onions, apples, coffee, mangoes, mangosteen, guava, peppers, pepper, tea, cucumbers, oranges, walnuts, almonds, carrots, radishes, coconuts, citrus, lemons, limes, strawberries, and hazelnuts, among others. BRIEF DESCRIPTION OF DRAWINGS

[0544] IN THE DRAWINGS:

[0545] Figure 1 A plot showing the cumulative release of nitric oxide (nmol NO / mg nitrite) over time in different reaction conditions of Example 1.

[0546] Figures 2 to 16 A plot showing the results of the tests described in Example 2.

[0547] Figure 17 A schematic showing the apparatus used to measure SIFT-MS.

[0548] Figures 18 to 21 A plot showing the results of the tests described in Example 3 on the antimicrobial activity of known antibiotics, carboxylic acid solutions, carboxylic acid-nitrite solutions, and carboxylic acid-nitrite-polyol solutions against M. abscessus.

[0549] Figure 22 A plot showing the results of the tests described in Example 4 on the minimum inhibition concentration (MIC) of solutions containing citric acid, sodium nitrite, and mannitol against a number of clinical isolates.

[0550] Figure 23 A plot showing the results of the tests described in Example 5 on the antimicrobial activity of carboxylic acid-nitrite solutions with and without polyols against P. aeruginosa.

[0551] Figures 24 to 27 A plot showing the results of the tests described in Example 6 on the antimicrobial activity against M. tuberculosis HN 878 in THP-1 cells.

[0552] Figure 28The results of the tests described in Example 7 for cytotoxicity (LDH cytotoxicity assay) and antimicrobial activity against H1N1 influenza A virus in MDCK cells are shown: (a) at MOI = 0.002 (●) and MOI = 0.02 (■), at a dilution series (nitrite molarity on the horizontal axis), with cytotoxicity shown in gray and a cytotoxicity scale on the right (cytotoxicity ≤ 1% of the LDH control at measured nitrite concentrations up to and including 0.015 M); and (b) photographs of plates 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 were two experiments, and the plates for each corresponding experiment are shown one above the other). The rightmost pair of plates, immediately to the right of the oseltamivir pair of plates, are the virus controls. Cytotoxicity is shown below each pair of test plates as % of the LDH control (average of three LDH assays at 24 hours post infection).

[0553] Figure 29 Shown are results of testing the effectiveness of an acidified solution of sodium nitrite, citric acid buffered to pH 5.8 with sodium hydroxide, and mannitol in killing M. abscessus compared to amikacin and a negative control (described in Example 3) under similar conditions.

[0554] Figure 30 and 31 In schematic form ( Figure 30 ) shows an embodiment of the invention described in Example 10 for treating a lung infection in a human subject, and ( Figure 31 ) and inhaled gas nitric oxide ( Figure 31 Compared with the left side of the figure, the contact point between the liquid preparation generating NO according to the present invention and the lung tissue ( Figure 31 view of the right side of the .

[0555] Figure 32 The results of the LDH cytotoxicity assay of Example 8 are shown (runs 1 and 2). The data can be expressed as the mean + standard deviation (SD) of two experiments. The SD is shown as a gray error bar. The maximum LDH activity (cells + lysis buffer) is set to 100%, and all example results are relative to this value. The LDH positive control is the positive control from the kit. The black bars (incubation for 2 hours) are the left bars of each pair of bars in each case, and the red bars (incubation for 24 hours) are the right bars of each pair of bars in each case.

[0556] Figure 33Results of the antiviral testing of Example 8 against SARS-CoV-2 at MOI 3.0 (Run 1) are shown. In Run 1, using SARS-CoV-2, a one virus yield reduction assay was performed at four multiplicities of infection (MOI) using reverse titration of virus inoculum confirmation. For cells inoculated at MOI 3, 2.1 logio TCIDso / ml was found in the virus control well after titration. SARS-CoV-2 yield reduction was observed for some of the conditions tested. After 24 hours of incubation, little virus was detected in the lowest three MOIs (i.e., 0.3, 0.03, and 0.003). It is possible that 24 hours of replication on VeroE6 cells was not sufficient to obtain high levels of progeny virus. Data are presented as the mean of two titrations + standard deviation (SD). SD is shown as error bars. The horizontal dotted line at the level of the chloroquine and cell control logio TCIDso / ml values is the limit of detection (LOD) of the assay.

[0557] Figure 34 shows results of the antiviral testing of Example 8 against SARS-CoV-2 at (a) MOI 3.0 and (b) MOI 0.3 (Run 2). This method corresponds to part of Run 1 at those MOIs, except that the formulation was Run 2 formulation and the incubation was 48 hours instead of 24 hours to increase the level of progeny virus. Data are presented as the mean of two titrations + standard deviation (SD). SD is shown as error bars. The horizontal dotted line at the level of the chloroquine and cell control logio TCIDso / ml values is the limit of detection (LOD) of the assay.

[0558] Figure 35 Results of the antiviral testing of Example 9 against SARS-CoV at MOI 3.0 are shown. Prior to staining of the cell monolayers with crystal violet, 2 plates were examined microscopically and scored for cytopathic effect (CPE). CPE was found to be present in these plates, in the form of cell debris on top of the underlying monolayer. Results of the two plates examined microscopically are shown. Data are a single titration for each condition. For the remaining plates, scoring for CPE was not possible after crystal violet staining due to the density of the cell monolayers. The horizontal dotted line at the level of the cell control logio TCIDso / ml values is the limit of detection (LOD) of the assay.

[0559] Examples

[0560] The following non-limiting examples are provided to further illustrate the application.

[0561] Materials, apparatus and methods used for Examples 1 and 2

[0562] Solutions

[0563] Stock solutions of 0.1 M and 1 M citric acid (Health Supplies Limited, Thornton Heath, UK), 0.1 M sodium citrate (Fisher Scientific, Loughborough, UK), 1 M sodium nitrite (Sigma Aldrich, Dorset, UK), 0.5 M and 1 M sorbitol (Special Ingredients, Chesterfield, UK), 0.5 M and 1 M D-mannitol (Sigma Aldrich, Dorset, UK), 3 M sodium hydroxide (Fisher Scientific, Loughborough, UK) and 0.1 M and 1 M L-ascorbic acid (ICN Biomedicals Inc., Ohio, US) were prepared by dissolving the appropriate mass in deionised water. Deionised water (18.2 MΩ) was obtained from an Arium Mini laboratory water system (Sartorius, Germany).

[0564] Citric acid / citrate buffer solutions were prepared by two methods:

[0565] 1. Volumetric titration of 0.1 M citric acid and 0.1 M sodium citrate stock solutions as described by Sigma Aldrich 2018 (https: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html);

[0566] 2. Dissolution of a known mass of citric acid for 0.1 M or 1 M formulations in a small volume of deionised water followed by titration of 3 M sodium hydroxide and deionised water stock solutions to achieve the required buffer solution pH (pH 3 to pH 6.2).

[0567] For method 1, ascorbic acid and sodium ascorbate were used instead of citric acid and sodium citrate for method 1 to prepare ascorbic acid / ascorbate buffer solutions similarly.

[0568] Polyols were included by dissolving a known mass of sodium nitrite with a stock solution of a polyol (e.g. sorbitol or mannitol).

[0569] The order of addition of the components of the buffer solutions and stock solutions is not critical and any mixing order can be used.

[0570] All standard solutions were used within 48 hours of preparation. Calibration buffer solutions were prepared using o-phthalate (pH 4) and phosphate (pH 7) tablets dissolved in deionised water (Fisher Scientific UK Limited, Leicestershire, UK).

[0571] Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) initiation and validation NO generation procedure

[0572] All gas analysis described in this report were performed using a Voice200 Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) (Syft Technologies Limited, New Zealand). This instrument uses helium (BOC, Surrey, UK) as the carrier gas.

[0573] Prior to analysis, the SIFT-MS was prepared for use using a simple start-up procedure. The instrument was exited from standby mode and a series of gas pressure checks were performed to ensure that capillary flow was within acceptable limits for operation. An automated validation procedure was then performed using the manufacturer's calibration gas standards containing benzene, toluene, ethylbenzene and xylene (Syft Technologies Limited, New Zealand). Finally, an internal performance check was performed using a 10 ppm nitric dioxide standard (Air Products PLC, Surrey, UK).

[0574] Figure 17

[0575] The SIFT-MS instrument, reaction cell and gas pathway were set up as shown in Analysis of the gas generated

[0576] ​The temperature in the reaction chamber was constantly monitored with an HT1 smart temperature sensor (SensorPush, New York, US). The reaction chamber was a 670 mL plastic (bisphenol A (BPA)-free) interlocking bucket with a silicone seal (Tesco, Welwyn Garden City, UK) that was connected to a pump that constantly circulated humidified air through the reaction chamber via the SIFT-MS inlet capillary. The air was humidified by pumping it through two Dreschel bottles containing deionized water in a similar manner to that described by: Vernon, W. and Whitby, L. (1931) The quantitative humidification of air in laboratory experiments, Trans. Faraday Soc. 27, 248-255. The system was allowed to equilibrate for 30 minutes before use. Continuous SIFT-MS scans were started, detecting and quantifying NO, NO2 and HONO in real time. Once stable baseline readings (consistent concentrations > 2 minutes) were observed for these compounds, the sample was placed in the reaction chamber and monitored for three hours.

[0577] Following SIFT-MS validation, the capillary inlet extension heated to 120 °C was connected via a T-junction to the outlet of the reaction chamber, allowing the SIFT-MS to sample in real time the gases flowing from the reaction chamber.

[0578] Samples were prepared by weighing approximately 0.3 cm x 0.3 cm carded nonwoven 20 gsm polypropylene screens from RKW-Group (Frankenthal, Germany) in a weighing boat (approximately 3 mg). After adding a 10 μΐ^drop of test or control solution onto the center of the screen (ensuring the drop soaked into the screen), it was weighed again. Finally, the loaded screen in the weighing boat was placed in the reaction chamber and a final 10 μΐ^drop of buffer solution was drawn onto the center of the screen. The reaction chamber was quickly sealed and the production of nitrogen-containing species was observed instantaneously at the SIFT-MS interface.

[0579] pH measurements for all examples

[0580] The resulting gases were analysed using a selective ion mode analysis of the SIFT-MS and each batch was scanned sequentially for 1000 seconds. The following product masses were scanned repeatedly: nitrous acid 30 m / z, nitrous acid 48 m / z, nitrogen dioxide 46 m / z, and nitric oxide 30 m / z. These measurements were achieved using all three positive precursor ions: hydronium (H3O+), nitrosyl (NO+) and dioxy (O2+). Air was flowed through the reaction chamber at 660 ml / min and the SIFT-MS inlet sampled this gas stream at a flow rate of 2.7 ml / min.

[0581] Example 1

[0582] All pH measurements were made using a Five Easy pH meter (Mettler Toledo, Switzerland) with a glass electrode LE438 probe. A second pH meter was used to ensure the accuracy of the electrode: a hand-held 205 probe (Testo, Alton, US). Freshly calibrated buffer solutions were used to calibrate the pH meters each day.

[0583] Contacting a mesh containing inhaled 1M sodium nitrite with and without 1M polyol using 1M / c. pH 3 citric acid

[0584] Nitric oxide generation Figure 17

[0585] The SIFT-MS apparatus, reaction chamber and gas pathways were set up as described above and in Figure 1 .

[0586] Two test solutions of 1 M sodium nitrite containing 1 M mannitol and 1 M sorbitol respectively were aspirated into the mesh as described above to make two test meshes.

[0587] A control solution of 1 M sodium nitrite without polyol was aspirated into the mesh as described above to make a control mesh.

[0588] A buffer solution of 1 M citric acid / citrate buffer prepared by either of the above methods 1 and 2 and having a pH of about 3 was added to each of the test and control meshes in each test to initiate gas production as described above.

[0589] Example 2 The results are shown in Table 1.

[0590] The data show that the mesh of inhaled 1 M sodium nitrite in contact with 1 M / c. pH 3 citric acid produced a significantly greater amount of nitric oxide when the mesh also contained 1 M mannitol or 1 M sorbitol (mannitol was more effective than sorbitol) than when no polyol was present.

[0591] Investigation of the effect of different carboxylic acids, acid concentrations, pH and polyols on nitric oxide generation

[0592] Figure 17

[0593] Samples were prepared as above, with the organic acid, pH value and polyol varied as follows:

[0594]

[0595]

[0596]

[0597]

[0598]

[0599] The SIFT-MS apparatus, reaction chamber and gas pathways were set up as described above and in Figures 2 to 13 .

[0600] Test solutions as described above were pipetted into the test mesh as described above to make the test mesh.

[0601] A control solution of 1 M sodium nitrite without polyol was pipetted into the control mesh as described above to make the control mesh, if used.

[0602] A buffer solution or each buffer solution as described above, prepared by either of methods 1 and 2 above and having the pH values described above, was added to the test and control meshes (if used) in each test to initiate gas production as described above.

[0603] Figure 2 Results are shown in the graphs. "Normal" in the graphs means that no polyol was present.

[0604] Figure 3 The rate of NO release produced by citric acid / citrate buffer or ascorbic acid / ascorbate buffer (pH about 3) in the absence of polyol was compared. The graphs clearly show that the citric acid / citrate buffer has a higher initial burst and a longer duration of gas release at a higher level than the ascorbic acid / ascorbate buffer. The citric acid / citrate buffer trace peaks at about 55000 ppb, while the ascorbic acid / ascorbate buffer trace peaks at about 28000 ppb.

[0605] Figure 4Citric acid / citrate buffer and nitrite systems with and without polyols are involved. The polyol concentration is 1 M. In the presence of polyols, the gas evolution rate, initial burst and after release change over time compared to the absence of polyols. Xylitol and mannitol produce the highest peaks, followed by sorbitol, then no polyols, and then arabitol. In the 500-1000s region, xylitol and arabitol have the highest output, followed by mannitol, sorbitol, and then no polyols. Peak burst mannitol = xylitol (about 64000 ppb) > sorbitol (about 53000 ppb) > no polyols (about 50000 ppb) > arabitol (about 40000 ppb).

[0606] Figure 3 Ascorbic acid / ascorbate buffer and nitrite system with and without polyols. Polyol concentration is 1 M. Peak burst release mannitol (about 40,000 ppb) > arabitol (about 35,000 ppb) > xylitol = no polyol (about 30,000 ppb) > sorbitol (about 23,000 ppb), i.e., with Figure 5 The citric acid / citrate buffer system has a different order.

[0607] Figure 3 Citric acid / citrate buffer and nitrite systems with and without polyols are involved (the "no polyol" line, with a peak burst nearly identical to the mannitol line, has been omitted for clarity). The polyol concentration is 0.5 M. Peak burst arabitol (about 76,000 ppb) >> no polyol = mannitol (about 48,000 ppb) > xylitol = sorbitol (about 40,000 ppb). It will be seen that this is comparable to the similar 1 M polyol citric acid / citrate buffer system ( Figure 6 ) in a different order, indicating that the effect of polyols depends on the concentration of polyols.

[0608] Figure 5 Ascorbic acid / ascorbate buffer and nitrite systems with and without polyols are involved (the "no polyol" line, with a peak burst nearly identical to the sorbitol line, has been omitted for clarity). The polyol concentration is 0.5 M. Peak burst xylitol (approximately 50,000 ppb) > mannitol (approximately 38,000 ppb) > sorbitol = no polyol (approximately 30,000 ppb) > arabitol (approximately 23,000 ppb). Again, similar peak bursts are observed with the citric acid / citrate buffer (0.5 M polyol) and ascorbic acid / ascorbate (1 M polyol) systems (respectively). Figure 7 and 4 ) is in a different order than the others. Thus, it was demonstrated that the effect of the polyols depends on the chemical / stereochemistry of the polyols and the molar concentration of the polyols.

[0609] Figures 2 to 6 and 8 The NO release rates were compared in citrate / citrate buffer or ascorbate / ascorbate buffer and in the presence of polyols (0.5 M). These graphs highlight some of the differences observed in Figure 7 Figure 8 The citrate / citrate buffer trace in reaches a peak at about 76,000 ppb, while the ascorbate / ascorbate buffer trace reaches a peak at about 22,000 ppb. Figure 9 The citrate / citrate buffer trace in reaches a peak at about 48,000 ppb, while the ascorbate / ascorbate buffer trace reaches a peak at about 38,000 ppb.

[0610] Figure 10 The cumulative output was compared for 1 M polyol concentrations. The differences in ascorbate / ascorbate buffer at, for example, 3000 s were small, with the order being mannitol > sorbitol = arabitol > xylitol. For citrate / citrate buffer, at 3000 s, the order was xylitol > arabitol > mannitol > sorbitol > no polyol. The data show that the output of nitric oxide can increase by as much as about 100%, and even more, between, for example, no polyol (curve E, after 3000 s, about 10,000 nmol of cumulative nitric oxide release per mg of nitrite is obtained, at which time it is still rising) and xylitol (curve A, about 20,000 nmol of cumulative nitric oxide release per mg of nitrite is obtained, which is still rising).

[0611] Figure 9 The cumulative output was compared for 0.5 M polyol concentrations. For citrate / citrate buffer, at 3000 s, the order was arabitol > mannitol = xylitol > sorbitol > no polyol (the "no polyol" line for citrate / citrate buffer, which is below the sorbitol line, has been omitted for clarity). For ascorbate / ascorbate buffer, at 3000 s, the order was xylitol > mannitol > sorbitol > arabitol. Again, this order is different than for 1 M polyols ( Figures 11 to 13 ).

[0612] Figure 14 Cumulative plots were compared for citrate / citrate buffer 1 M and sodium nitrite (1 M) with and without mannitol (0.5 M) and at different pH values. The differences became smaller as the pH value increased, and at pH 6.2, the differences disappeared. Thus, from these experiments, it is seen that the effect of polyols also depends on the pH value.

[0613] Figure 15 ​Cumulative NO (nmol / cm2 of screen area) output of citrate / citrate buffer (1 M, pH ~ 2) in the presence and absence of glycerol (1 M and 2 M) in 1 M sodium nitrite solution is shown. The NO output of 1 M and 2 M glycerol is slightly lower than in the absence of polyol for the first 2000 s. At longer times, the formulations with glycerol have greater output, with 2 M glycerol having greater output.

[0614] Figure 16 Cumulative NO (nmol / cm2 of screen area) output of citrate / citrate buffer (1 M, pH ~ 2) in the presence and absence of polyol in 1 M sodium nitrite solution is shown. The plot shows that inclusion of glycerol in the mannitol / nitrite solution reduces output compared to the absence of glycerol. However, unexpectedly, unlike the case of mannitol, inclusion of glycerol in the sorbitol / nitrite solution enhances NO output compared to the output in the absence of glycerol.

[0615] When glycerol is used, a 1 M glycerol solution is first made and used to make either a 1 M sorbitol or 1 M mannitol solution, which is in turn used to make a 1 M nitrite solution.

[0616] Figure 16 Cumulative NO output (mol / mg of nitrite) of citrate / citrate buffer (1 M, pH 5.8) in the presence and absence of mannitol (0.5 M) in 1 M sodium nitrite solution is shown. The plot shows that inclusion of polyol results in greater NO output after about 2000 s of reaction time.

[0617] Example 3 It is shown that mannitol enhances nitric oxide production at physiologically important pH levels above about 5, especially above about 5.5, providing a cumulative level of 1400 nmol of NO per mg of nitrite after 10000 s (167 minutes) compared to the same system without mannitol.

[0618] Activity against M. abscessus cultures with and without polyol in a range of organic acid and nitrite solutions

[0619] Tube T Tube A

[0620] MATERIALS

[0621] 4.7 g of Middlebrook 7H9 Broth Base (Sigma-Aldrich) was reconstituted with 900 ml of distilled water and autoclaved at 121 °C for 15 minutes. Middlebrook ADC Growth Supplement (Sigma-Aldrich) was added to the autoclaved 7H9 solution (50 ml per 450 ml, 100 ml added in total).

[0622] 1 M Sodium Nitrite (Emsure): 6.9 g of sodium nitrite powder was dissolved in 100 ml of distilled water in a clean screw-capped glass bottle. The mixture was autoclaved at 121 °C for 15 minutes.

[0623] 1 M Citric Acid (Sigma-Aldrich): 19.2 g of citric acid powder was dissolved in 100 ml of distilled water in a clean screw-capped glass bottle. The mixture was autoclaved at 121 °C for 15 minutes.

[0624] 1 M Ascorbic Acid (Sigma-Aldrich): 17.6 g of ascorbic acid powder was added to a sterile glass bottle. It was thoroughly dissolved in 100 ml of sterile distilled water. Due to the short half-life, it was prepared daily using strict aseptic techniques. Due to its inherent instability, it was not autoclaved but filtered through a 0.2 μ filter prior to use.

[0625] 1 M Trisodium Citrate Dihydrate (Sigma-Aldrich): 29.4 g of sodium citrate powder was dissolved in 100 ml of distilled water in a clean screw-capped glass bottle. The mixture was autoclaved at 121 °C for 15 minutes.

[0626] 1 M L-Ascorbic Acid Sodium Salt (Acros Organics): 19.8 g of sodium ascorbate powder was dissolved in 100 ml of distilled water in a clean screw-capped glass bottle. The mixture was autoclaved at 121 °C for 15 minutes.

[0627] For experiments utilising a polyol, D-mannitol (Sigma-Aldrich) was used. The polyol was added to the above sodium nitrite stock solution to form the following stock solutions:

[0628] Stock Solution A - 1 M Sodium Nitrite and 0.5 M Mannitol

[0629] Stock Solution B - 1.5 M Sodium Nitrite and 0.5 M Mannitol

[0630] A stock solution of 1.5 M citric acid was also prepared.

[0631] The molarity of each component was adjusted for the dilution factor to ensure the correct final molarity of each experimental solution.

[0632] Mycobacterium abscessus (MAB)

[0633] Laboratory reference strain Mycobacterium abscessus ATCC 19977 lux was used for all experimental conditions in this example.

[0634] Method

[0635] Label 50 ml falcon tubes as tube T (test suspension), tube A (acid control) and tube C (control).

[0636] Add 8 ml of 7H9 + ADC supplement to each tube. Then add 100 μΐ of MAB suspension (grown previously to approximately 3-4 McFarland standard). Read the baseline relative light units (RLU) of the MAB suspension. Mix the contents by vortexing.

[0637] Tube contents when polyol (mannitol) is not present

[0638] Tube C : Add 1 ml of sodium nitrite (1 M) solution to the tube, followed by 1 ml of citric acid solution (1 M) or ascorbic acid solution (1 M) to give a final concentration of 0.1 M in 10 ml. Mix the contents by gently inverting and incubate at 37°C for 24 hours.

[0639] Figures 18 to 21 : Add 1 ml of citric acid solution (1 M) or ascorbic acid solution (1 M) to the tube and add 1 ml of sterile distilled water to make a 10 ml final volume, testing 0.1 M concentration of acid. Mix the contents by gently inverting and incubate at 37°C for 24 hours.

[0640] Figure 18 : Add 2 ml of sterile distilled water to the tube to make a 10 ml total volume. This is the control to assess growth under optimal conditions. Mix the contents by gently inverting and incubate at 37°C for 24 hours.

[0641] Tube T contents when polyol (mannitol) is present

[0642] Tube T contents when mannitol is present are as follows:

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

[0644] 2. Tube T: 1 ml of sodium nitrite (1.5 M) and mannitol (0.5 M) and 1 ml of citric acid (1 M)

[0645] 3. Tube T: 1 ml sodium nitrite (1 M) and mannitol (0.5 M) and 1 ml citric acid (1.5 M)

[0646] RLU was measured at 30 minutes, 60 minutes and 24 hours of incubation to assess the activity of the T, A and C solutions.

[0647] After 24 hours of incubation, tube C, tube A and tube 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 day 3, day 5 and day 7 of incubation. All work was performed in a CL2 biological safety cabinet within a CL2 laboratory facility.

[0648] Figure 18 Results are shown in the following table.

[0649] Figure 19 It is shown that the solution of 0.1 M citric acid and 0.1 M nitrite (tube T) was effective in eliminating the M. abscessus culture after 7 days at pH values of 5 and 5.5 and reduced the M. abscessus culture compared to the solution of only 0.1 M citric acid (tube A) at pH values of 6.0, 6.5, 7.0 and 7.4. Figure 19 It is also shown that the solution of 0.1 M ascorbic acid and 0.1 M nitrite (tube T) was effective in eliminating the M. abscessus culture after 7 days at pH values of 5.0, 5.5 and 6.0 and reduced the M. abscessus culture compared to the solution of only ascorbic acid (tube A) at pH values of 6.5, 7.0 and 7.4.

[0650] Figure 20 a) shows that the solution of 0.1 M citric acid and 0.1 M nitrite was effective in reducing the CFU of the M. abscessus culture after three days of incubation and that the solution of 0.1 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in almost completely eliminating the M. abscessus culture after three days of incubation. Figure 20 b) shows that the solution of 0.1 M citric acid and 0.1 M nitrite without mannitol was effective in maintaining the reduced M. abscessus CFU after five days of incubation. The graph also shows that the solution of 0.1 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in reducing the CFU of the M. abscessus culture after five days of incubation.

[0651] Figure 21 a) shows that the solution of 0.15 M citric acid and 0.1 M nitrite was effective in reducing the CFU of the M. abscessus culture after three days of incubation and that the solution of 0.15 M citric acid and 0.1 M nitrite with 0.05 M mannitol was effective in eliminating the M. abscessus culture after three days of incubation. Example 4b) shows that a solution of 0.15M citric acid and 0.1M nitrite, without mannitol, was effective in maintaining a reduced number of M. abscessus CFU after five days of incubation. The graph also shows that a solution of 0.15M citric acid and 0.1M nitrite with 0.05M mannitol was effective in eliminating M. abscessus cultures after five days of incubation.

[0652] Minimum inhibitory concentrations (MIC) of carboxylic acid-nitrite-polyol solutions against M. abscessus (Mab) and M. tuberculosis (Mtb) in a range of clinical isolates shows that a solution of 0.1M citric acid and 0.15M nitrite was effective in reducing the number of M. abscessus CFU after three days of incubation and in maintaining the reduction in the number of M. abscessus CFU after five days of incubation. The graph also shows that a solution of 0.1M citric acid and 0.15M nitrite with 0.05M mannitol was effective in eliminating M. abscessus cultures after three and five days of incubation.

[0653] Figure 22

[0654] Figure 22 Figure 22

[0655] Healthy volunteers

[0656] Peripheral blood samples were obtained from healthy volunteers who provided written informed consent (Ethical approval reference REC No. 12 / WA / 0148).

[0657] Mycobacterial strains

[0658] Both M. abscessus (ATCC 19977) and M. tuberculosis (H37RV) strains contained a bacterial luciferase (lux) gene cassette (luxCDABE) which enabled measurement of relative light units (RLU) as well as routine colony forming unit (CFU) measurements for bacterial survival.

[0659] General reagents

[0660]

[0661]

[0662] Treatment conditions

[0663] Treatment 1 : citric acid 0.15M, sodium nitrite 0.1M and mannitol 0.05M

[0664] Treatment 2: citric acid 0.1M, sodium nitrite 0.15M and mannitol 0.05M

[0665] Broth microdilution minimum inhibitory concentrations (MICs)

[0666] The MIC of each treatment against M. abscessus and M. tuberculosis was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for Antimicrobial Susceptibility Testing (M07-A9). Each treatment was performed in a two-fold dilution on plates and the plates were incubated at 37°C and read for Mab on day 3 and day 7 and for Mtb on day 14 and day 21. The tests were performed in duplicate.

[0667] All work was performed in a CL2 biosafety cabinet within a CL2 laboratory facility.

[0668] It was found that a 1.5 M citric acid, 1 M sodium nitrite, and 0.5 M mannitol solution had a minimum inhibitory concentration of 4.7 mM against M. abscessus. It was also found that a 1.5 M citric acid, 1 M sodium nitrite, and 0.5 M mannitol solution had a minimum inhibitory concentration of 2.3 mM against M. tuberculosis.

[0669] It was found that a 1 M citric acid, 1.5 M sodium nitrite, and 0.5 M mannitol solution had a minimum inhibitory concentration of 3.1 mM against M. abscessus. It was also found that a 1 M citric acid, 1.5 M sodium nitrite, and 0.5 M mannitol solution had a minimum inhibitory concentration of 1.6 mM against M. tuberculosis.

[0670] Minimum inhibitory concentrations (MICs) were also determined by broth microdilution using isolates from the Floto Laboratory (Cambridge University, UK) (https: / / www.flotolab.com / ) library of M. abscessus clinical isolates numbers 570, 571, 573, 575, 578, 579, 580, 581, 582, 583, 584, 585, 589, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 616, 617, 619, 812, 825, 829, 839, 845, 848, 853, 857, 858, 873, 894, 898, 909, 919, 928, 932, 942, 944, 955, 956, 959, 963, 964, 965, 968, 975, 980, 982, 985, 993, 995, 1000, 1001, 1007, 1011, 1017, 1023, 1024, 1026, 1027, 1042, 1043, 1045, 1047, 1049, 1054, 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 assessed in duplicate.

[0671] Figure 22 Results shown in a) and b) on clinical isolates tested. Graphs show the MIC of nitric oxide against M. abscessus read in duplicate after three, four, and five days of incubation of the isolates. Plates were also read at day 7 of incubation, but no difference was seen compared to day 5. Laboratory strain ATCC 19977lux was used as a control in both experiments and shows the comparative results with the clinical isolates.

[0672] Figure 22 The citrate-nitrite-mannitol solution was shown to have an impact in a wide range of clinical isolates. The minimum inhibitory concentration of 0.1 M citrate, 0.15 M nitrite, and 0.05 M mannitol solution was within 0.02 M of the MIC of the majority of clinical isolates Figure 22 a), and the minimum inhibitory concentration of 0.15 M citrate, 0.1 M nitrite, and 0.05 M mannitol solution was within 0.04 M of the MIC of the majority of clinical isolates Figure 29 b).

[0673] In both graphs, the MICs on certain samples varied on different days. Those samples are the ones that show more than one point above the identification code of the isolate sample. Generally, in that case, a higher MIC was observed at a later incubation day compared to a lower MIC. Overall, the combination of lower citric acid (0.1 M) and higher sodium nitrite (0.15 M) ( Example 5 (a)) was more effective than the combination of higher citric acid (0.15 M) and lower sodium nitrite (0.1 M) ( Antimicrobial activity of carboxylic acid-nitrite solutions with and without polyol against P. aeruginosa (b)).

[0674] Figure 23 Additional data showing the in vitro killing of M. abscessus by carboxylate-nitrite-polyol solutions are shown in Figure 6. In this figure, the effectiveness of an aqueous formulation of sodium nitrite, citric acid buffered to pH 5.8 using a sodium hydroxide solution, and mannitol in killing M. abscessus compared to amikacin and a negative control over a 24 hour period under similar conditions was confirmed.

[0675] Example 6

[0676] Formulations

[0677] Equipment and media

[0678] UKAS calibrated pipettes (100-1000 μL range)

[0679] UKAS calibrated multichannel pipettes (P300 and P20)

[0680] Universal tubes - SLS, UK

[0681] Calibrated balance - HR-100A

[0682] Microbiological incubator - Heratherm TM , ThermoFisher Scientific, UK

[0683] Tryptone soya agar (TSA) - Southern Group Laboratories, UK

[0684] Tryptone soya broth (TSB) SLS, UK

[0685] Malt agar SLS, UK

[0686] Brain heart infusion broth (BHIB) SLS, UK

[0687] Sabouraud Dextrose Broth (SDB) SLS, UK Dey-Engley Neutraliser (DE-N) SLS, UK

[0688] Citric Acid - Sigma, UK

[0689] Sodium Nitrite - Sigma, UK Mannitol - Sigma, UK

[0690] Sorbitol - Sigma, UK

[0691] Test Microorganism

[0692] Pseudomonas aeruginosa NCTC 13618 - isolated from a patient with cystic fibrosis

[0693] Formulation

[0694]

[0695] Concentration 1 - 1 M Citric Acid plus 1 M Sodium Nitrite (with or without 0.5 M polyol)

[0696] Concentration 2 - 0.5 M Citric Acid plus 1 M Sodium Nitrite (with or without 0.5 M polyol)

[0697] Concentration 3 - 0.5 M Citric Acid plus 0.5 M Sodium Nitrite (with or without 0.5 M polyol)

[0698] Dey-Engley Neutraliser Validation

[0699] Twenty-four hour cultures of Pseudomonas aeruginosa were harvested from Tryptone Soya Agar (TSA) and used to prepare a 1 x 108±5 x 107CFU mL"1suspension. This was further diluted in Brain Heart Infusion Broth (BHIB) to prepare a 1 x 105±5 x 104CFU mL"1working suspension.

[0700] The starting inoculum was confirmed by serial dilution and plating. Neutraliser validation was performed using control (9 mL Phosphate Buffered Saline (PBS) and 1 mL inoculum), toxicity (9 mL Dey-Engley Neutraliser (DE-N) and 1 mL inoculum) and neutraliser efficacy (8 mL neutraliser, 1 mL test agent and 1 mL inoculum) samples. After 5 minutes treatment, 200 μL of suspension was removed from each tube, serially diluted and 100 μL plated onto TSA. Agar plates were incubated at 37 ± 2°C for 18-24 hours.

[0701] Antimicrobial efficacy against planktonic organisms

[0702] Twenty-four hour cultures of P. aeruginosa were harvested from TSA and used to prepare 1 x 108±5 x 107CFU mL"1suspensions. These were further diluted in BHIB to prepare 1 x 106±5 x 104CFU mL"1working suspensions. Universal tubes were filled with 8 mL of bacterial solution.

[0703] One milliliter of citric acid solution and 1 mL of sodium nitrite solution were added to each of the agents tested, to give the required concentrations as described above. The solutions were incubated at 37 ± 2°C for 24 hours. After the incubation period, 1 mL was removed from each tube and transferred to a tube containing 9 mL of neutralising agent. The viable organisms were quantified using serial dilution and plating.

[0704] Complete DMEM medium for macrophages: Results are shown in Table 2.

[0705] The data show the following antimicrobial effectiveness against Pseudomonas spp:

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

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

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

[0709] The citric acid solutions were at pH 5.2 (formulations 1, 3 and 5) and 6.0 (formulations 2, 4 and 6). Formulations 1 and 2 contained no polyol; formulations 3 and 4 included mannitol; and formulations 5 and 6 contained sorbitol.

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

[0711] L-929 conditioned medium:

[0712] Formulations including nitrite, organic acid and polyol were evaluated for efficacy against M. tuberculosis HN878 in THP-1 cells.

[0713] Infection of THP-1 cells:

[0714] Formulations were prepared as set out in the table below. Where the preparation method is stated to be "concentrated" and the sample reference is denoted by the suffix FC, this means that the formulation was initially made as a concentrated pre-mix containing all three components, sodium nitrite (0.75M), polyol (0.25M) and acid (0.5M), and then diluted with distilled water to achieve the required concentration for each as stated in the table. Where the preparation method is stated to be "diluted" and the sample reference is denoted by the suffix FD, this means that the formulation was initially made as a pre-mix containing all three components at the concentrations required initially, i.e. sodium nitrite (0.15M), polyol (0.05M) and acid (0.1M), and then diluted with distilled water to achieve the required concentration for each as stated in the table.

[0715] Within each formulation, a series of concentrations of sodium nitrite were prepared by serial dilution, i.e. 16μg / ml, 8μg / ml, 4μg / ml, 2μg / ml, 1 μg / ml, 0.5μg / ml, 0.25μg / ml and 0.125μg / ml, for the in vitro bacteriostatic assay against M. tuberculosis HN878.

[0716]

[0717] MIC macrophage testing was performed using the THP-1 macrophage (1) compound screening assay.

[0718] Macrophage preparation and culture: THP-1 cells were expanded for 2 weeks. Thereafter, THP-1 cells were suspended in complete DMEM medium for macrophages at a concentration of 5 x 105cells / ml. Cells were seeded into 24-well tissue culture plates at 2 ml per well (1 x 106per well). One 24-well cell plate allowed testing of a series of 7 drug concentrations plus an untreated control in triplicate. In addition to the drug assay plates, one additional plate (or at least 3 additional wells) was seeded for assay of bacterial uptake on the day of infection. Cells were incubated at 37°C in a humidified chamber under 5% C02. DMEM complete medium without antibiotics / antimycotics was not changed during the 3-day assay period.

[0719] Results:

[0720] Dulbecco's Modified Eagle Medium (Cellgro 15-017-cv), supplemented with: heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO, F-0500-A) (10%)

[0721] L929 conditioned medium (10%)

[0722] L-glutamine (Sigma G-7513) (2mM)

[0723] HEPES buffer (Sigma H-0887) (10 mM) Antibiotic / antimycotic (Sigma A-9909) (1X)

[0724] MEM non-essential amino acids (Sigma M-7145) (1X)

[0725] 2-mercaptoethanol (Sigma M-6250) (50 nM)

[0726] In vitro THP-1 HN878 optical density results

[0727] L-929 (CCL-1) cells from ATCC were seeded at 4.7 x 105cells in 55 mL DMEM + 10% fetal bovine serum in 75 cm2flasks. For THP-1 cells, cells were grown for 3 days. On day 3, supernatant was collected and filtered through a 0.45 μιη filter, aliquoted, and frozen at -20 °C. Cell-free filtrate was used for THP-1 infection in DMEM media.

[0728] Figures 24 to 27

[0729] On day 0, media was removed from cells and replaced with 0.2 ml of antibiotic / antimycotic-free DMEM containing M. tuberculosis HN878 at a MOI of 1 macrophage to 10 bacteria ratio. The tissue culture plates were placed in a closed Ziploc bag for transport back to the incubator. Once in the incubator, the bag was unzipped. The cells were incubated with the bacteria for 2 hours. After infection, bacteria that were attached outside the cells were removed, each well was washed once with phosphate buffered saline (PBS), and 2 mL of antibiotic / antimycotic-free complete DMEM media with various drug concentrations was added. To prepare the drug concentrations, a 2-fold serial dilution was performed in a separate tube by adding 10 ml of the previous suspension to 10 ml of complete media plus serum. The tissue culture plates were returned to the incubator at 37 °C + 5% C02(drug remained in the wells for 3 days). Each drug concentration was tested in triplicate in the wells.

[0730] Cell lysates were plated and THP-1 cell viability was assessed at 2 hours, 1 day, 2 days and 5 days post-infection. The tissue culture medium was removed from all wells and the cells were washed twice with 1 ml PBS. Then, 1 ml sterile double distilled water + 0.05% Tween-80 was added to each well; the cells were left at room temperature for 5-10 minutes. Cell lysates were serially diluted 1 : 10 in sterile saline in 24 well tissue culture plates. The diluted cell lysates were plated onto 7H11 / OADC agar by a 1 / 1,000 dilution step. (Four 24 well TC plates were required for each 24 well TC plate of cells to perform serial dilutions, and 24 agar 'quadrilaterals' plates). The plates were incubated at 32°C for 30 days and colonies were counted to determine CFU / ml.

[0731] Figure 24

[0732] Figure 24

[0733] Minimum inhibitory concentration (MIC), reported as the most dilute composition that inhibits bacteria (i.e. the maximum dilution level of the specific formulation at scales 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)

[0734]

[0735]

[0736] Figure 24 Results are shown in Figure 6.

[0737] Figure 25 Efficacy of 30 RESP001 FC and FD (concentrated and diluted) against M. tuberculosis HN878 was assessed in THP-1 cells. Formulation 30 RESP001 FC (concentrated) (A) and 30 RESP001 FD (diluted) (B) were assessed for efficacy in killing M. tuberculosis HN878 intracellularly in THP-1 macrophages after infection and treatment with 16 μg / ml (A), 8 μg / ml 4 μg / ml (O), 2 μg / ml (·), 1 μg / ml (□), 0.5 μg / ml (♦), 0.25 μg / ml (A) and 0.125 μg / ml (T) at 2 hours (day 0), day 1, day 2 and day 5 post-infection. In each graph in Figure 6, the Efficacy of 30 RESP001 FC and FD (concentrated and diluted) against M. tuberculosis HN878 was assessed in THP-1 cells. Formulation 30 RESP001 FC (concentrated) (A) and 30 RESP001 FD (diluted) (B) were assessed for efficacy in killing M. tuberculosis HN878 intracellularly in THP-1 macrophages after infection and treatment with 16 μg / ml (A), 8 μg / ml Figure 25 4 μg / ml (O), 2 μg / ml (·), 1 μg / ml (□), 0.5 μg / ml (♦), 0.25 μg / ml (A) and 0.125 μg / ml (T) at 2 hours (day 0), day 1, day 2 and day 5 post-infection. In each graph in Figure 6, the The curves can differ from the curves for the 0.25 μg / ml and 0.125 μg / ml treatments, respectively, because the treatments with 16 μg / ml and 8 μg / ml were more effective. In other words, the curves for the 16 μg / ml and 8 μg / ml treatments show significantly lower CFU values than the 0.25 μg / ml and 0.125 μg / ml treatments, especially at day 5. Similarly, the curve for the 1 μg / ml treatment,, can readily differ from the no-treatment curve, because the treatment at 1 μg / ml was more effective. The CFU values for the no-treatment curve rose, remaining above 1 x 104after day 1.

[0738] The 30RESP001 FC and FD compositions described in the above MIC table and Figure 25 as "16 μg / ml" comprise 0.15 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentrations 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 preceding composition in the order stated, respectively.

[0739] THP-1 macrophages were infected with M. tuberculosis at an MOI of 1 : 10, and the number of intracellular bacteria was determined using bacterial colony counts (CFU) at 2 hours post-infection (day 0), day 1, day 2, and immediately after day 5. The values shown are the mean ± SD from one independent experiment. Specifically, efficacy against M. tuberculosis HN878 increased in the 30RESP001 FC and FD (concentrated and diluted) treatments with 16 μg / ml and 8 μg / ml relative to the no-treatment control (*, p < 0.05).

[0740] Figure 26 : Evaluation of 30RESP002 FC and FD (concentrated and diluted) efficacy against M. tuberculosis HN878 in THP-1 cells. Evaluation of formulations 30RESP002 FC (concentrated) (A) and 30RESP002 FD (diluted) (B) efficacy in killing M. tuberculosis HN878 in cells at 2 hours post-infection and day 1, day 2, and immediately after day 5 in THP-1 macrophages treated with 16 μg / ml ( ), 8 μg / ml ( ), 4 μg / ml ( ), 2 μg / ml ( ), 1 μg / ml ( ), 0.5 μg / ml ( ), 0.25 μg / ml ( ), and 0.125 μg / ml ( ). The values shown are the mean ± SD from one independent experiment. : Evaluation of 30RESP002 FC and FD (concentrated and diluted) efficacy against M. tuberculosis HN878 in THP-1 cells. Evaluation of formulations 30RESP002 FC (concentrated) (A) and 30RESP002 FD (diluted) (B) efficacy in killing M. tuberculosis HN878 Figure 26In each of the graphs, the curves for the treatments with 16 μg / ml and 8 μg / ml are The curves can differ from the curves for the treatments with 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments with 16 μg / ml and 8 μg / ml are more effective. In other words, the curves for the treatments with 16 μg / ml and 8 μg / ml show significantly lower CFU values than the treatments with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the curve for the treatment with 1 μg / ml, □, can easily differ from the curve for the untreated The curves can differ from the curves for the treatments with 0.25 μg / ml and 0.125 μg / ml, respectively, because the treatments with 16 μg / ml and 8 μg / ml are more effective. In other words, the curves for the treatments with 16 μg / ml and 8 μg / ml show significantly lower CFU values than the treatments with 0.25 μg / ml and 0.125 μg / ml, especially on day 5. Similarly, the curve for the treatment with 1 μg / ml, □, can easily differ from the curve for the untreated The CFU values for the untreated

[0741] In the above MIC table and Figure 26 The 30RESP002FC and FD compositions described as "16 μg / ml" in the above MIC table and in the text comprise 0.15 M sodium nitrite, 0.05 M lactitol and 0.1 M citric acid / citrate (final molar concentrations 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 preceding composition in the order mentioned, respectively.

[0742] THP-1 macrophages were infected with M. tuberculosis at MOI 1 : 10 and the number of intracellular bacteria was determined using the bacterial colony count method (CFU) at 2 hours post-infection, immediately after day 1, day 2 and day 5. The values shown are the mean ± SD from one independent experiment. Efficacy against M. tuberculosis HN878 increased relative to the untreated control in the treatments with 16 μg / ml 30RESP002FC (concentrated) and 16 μg / ml and 8 μg / ml 30RESP002FD (diluted) (*, p < 0.05).

[0743] Figure 27 Efficacy of 30RESP003FC and FD (concentrated and diluted) against M. tuberculosis HN878 was evaluated in THP-1 cells. Efficacy of 30RESP003FC (concentrated) (A) and 30RESP003FD (diluted) (B) was evaluated in THP-1 macrophages infected with M. tuberculosis HN878 at MOI 1 : 10 and treated with 16 μg / ml (▲), 8 μg / ml Intracellular killing of Mycobacterium tuberculosis HN878 after 2 hours (day 0), 1 day, 2 days, and 5 days after treatment with 4 μg / ml (◇), 2 μg / ml (○), 1 μg / ml (□), 0.5 μg / ml (◆), 0.25 μg / ml (▲), and 0.125 μg / ml (▼) The effect. Figure 27 In each curve, ▲ and 8 μg / ml were treated with The curves can be distinguished from the ▲ and ▼ curves treated with 0.25 μg / ml and 0.125 μg / ml, respectively, because the 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 distinguished from the curve without treatment. curve, because the treatment at 1 μg / ml was more effective. The CFU value of the curve is increasing and remains above 1×10 4 after day 1.

[0744] THP-1 macrophages were infected with Mycobacterium tuberculosis at an MOI of 1:10, and the number of intracellular bacteria was determined using the bacterial colony count method (CFU) 2 hours, 1 day, 2 days, and 5 days after infection. The values ​​shown are the mean ± SD from an independent experiment. In the treatment with 16 μg / ml and 8 μg / ml 30RESP003FC (concentrated) and 16 μg / ml 30RESP003FD (diluted), the efficacy against Mycobacterium tuberculosis HN878 increased relative to the untreated control (*, p < 0.05).

[0745] In the above MIC table and Figure 27 The 30RESP003FC and FD compositions described as "16 μg / ml" referred to in the present invention comprise 0.1 M sodium nitrite, 0.05 M mannitol, and 0.1 M citric acid / citrate (final molar concentration after dilution), wherein 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 are each diluted by 50% (i.e., the concentration is halved) of the previous composition in the order of 16 μg / ml to 0.125 μg / ml.

[0746] Figure 24Efficacy of 30RESP004FC and FD (concentrated and diluted) against M. tuberculosis HN878 was assessed in THP-1 cells. Efficacy of formulation 30RESP004FC (concentrated) (A) and 30RESP004FD (diluted) (B) in killing M. tuberculosis HN878 intracellularly after infection and treatment with 16 μg / ml (A), 8 μg / ml 4 μg / ml (O), 1 μg / ml (□), 0.5 μg / ml (♦), 0.25 μg / ml (▲) and 0.125 μg / ml (▼) was assessed in THP-1 macrophages at 2 hours (day 0), day 1, day 2 and day 5 after infection and treatment. In each of the graphs in Figure 25 The curves for treatment with 16 μg / ml and 8 μg / ml can differ from the curves for treatment 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 for treatment with 16 μg / ml and 8 μg / ml show significantly lower CFU values than the curves for treatment with 0.25 μg / ml and 0.125 μg / ml, especially at day 5. Similarly, the curve for treatment with 1 μg / ml can easily differ from the curve for no treatment, because treatment at 1 μg / ml is more effective. The CFU values for the curve for no treatment are rising, remaining above 1 x 104after day 1. The curves for treatment with 16 μg / ml and 8 μg / ml can differ from the curves for treatment 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 for treatment with 16 μg / ml and 8 μg / ml show significantly lower CFU values than the curves for treatment with 0.25 μg / ml and 0.125 μg / ml, especially at day 5. Similarly, the curve for treatment with 1 μg / ml can easily differ from the curve for no treatment, because treatment at 1 μg / ml is more effective. The CFU values for the curve for no treatment are rising, remaining above 1 x 104after day 1.

[0747] The 30RESP004FC and FD compositions mentioned in the above MIC table and Example 7 described as "16 μg / ml" comprise 0.1 M sodium nitrite, 0.05 M mannitol and 0.1 M ascorbic acid / ascorbate (final molar concentrations 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. halving of the concentration) from the preceding composition in the order mentioned, respectively.

[0748] THP-1 macrophages were infected with M. tuberculosis at MOI 1:10 and the number of intracellular bacteria was determined immediately after infection at day 1, day 2 and day 5 using the bacterial colony count method (CFU). The values shown are the mean values ± SD from one independent experiment. In the treatment with 16 μg / ml and 8 μg / ml 30RESP004FC (concentrated), the efficacy against M. tuberculosis HN878 was increased relative to the untreated control (*, p < 0.05). ​​​

[0749] It was concluded that the formulation showed inhibition of M. tuberculosis HN878 in vitro at appropriate doses exceeding the MIC.

[0750] It was also noted in the tests of Example 6 that the way the formulations were manufactured had an impact on their in vitro antibacterial efficacy against M. tuberculosis HN878.

[0751] This was demonstrated by comparing the efficacy of Formulation 1 at a concentration of 8 μg / ml between FC and FD versions ( ​ A vs. 24B). The efficacy of the FC format increased strongly after at least 5 days of incubation, while the efficacy of the FD format increased less strongly over the same time period. This contrasts with the 16 μg / ml concentration, where the FC and FD formats showed very similar and good efficacy over the same time period.

[0752] Different behavior was observed under Formulation 2 ( ​ A vs. 25B). The efficacy of the FD form at a concentration of 16 μg / ml increased more strongly than the FC form during the first two days after incubation, but remained stable thereafter. However, by day 5 after incubation, efficacy was good in the FD form and very good in the FC form. At a concentration of 8 μg / ml, efficacy of the FD form increased significantly to good efficacy after at least 5 days of incubation, while efficacy of the FC form increased less strongly over the same period.

[0753] Thus, it is shown that, at least at higher concentrations, the stage of adding water to achieve final inhibition of the formulation substantially affects the antibacterial effect of the formulation in terms of initial antibacterial effect and the extent of bactericidal activity within 5 days. Generally speaking, although not universal, initially preparing the formulation as a concentrated premix of sodium nitrite, polyol and acid components in their desired relative molar proportions but at a concentration higher than the desired concentration for use (e.g., at least 3 times, such as at least 5 times, such as about 3 times to about 80 times more concentrated than the desired concentration for use), and then only diluting the concentrate to obtain the formulation for use, will provide better antibacterial activity over a period of 0 to 5 days after incubation.

[0754] ​

[0755] Cytotoxicity and antiviral activity of carboxylate-nitrite-polyol solutions against H1N1 influenza A virus

[0756] Test formulations called F1C1, F1C2 and F1C3, corresponding to Formulation 30 RESP001FC, a 10-fold dilution thereof and a 100-fold dilution thereof in Example 6, respectively, were used with oseltamivir solution (1 μΜ) and virus control to obtain comparative cytotoxicity and H1N1 influenza A virus killing after 24 hours in MDCK cells. Cytotoxicity was determined by LDH cytotoxicity assay, similar to Example 8. Antimicrobial activity against H1N1 influenza A virus in MDCK cells was measured at a range of dilutions (nitrite molarity on the horizontal axis) at MOI = 0.002 (·) and MOI = 0.02 (■), with cytotoxicity shown in gray on the right (cytotoxicity < 1% of the LDH control at nitrite concentrations measured up to and including 0.015 M). Plate photographs were obtained at MOI = 0.002 and nitrite concentrations 0.15 M, 0.015 M and 0.0015 M, in comparison with oseltamivir (1 μΜ). Figure 28 Results are shown in the figure. The order of the plates recited in the last but one sentence is the same as the order of the plates from left to right in the figure (there are two experiments, with the plates of each respective experiment shown one on top of the other). The plate pair on the far right, immediately to the right of the oseltamivir plate pair, is the virus control. Cytotoxicity is shown below each pair of test plates as % of the LDH control (average of 3 LDH determinations at 24 hours post-infection).

[0757] The results show that at a suitable dose of nitrite / citrate / polyol formulation, the virus can be completely eradicated, significantly outperforming oseltamivir. Nitrite / citrate / polyol formulations have shown similar antiviral activity against rhinovirus and respiratory syncytial virus (RSV).

[0758] These results show that nitrite / acid / polyol formulations according to the application provide therapeutic and prophylactic treatment of respiratory viral infections in human and animal subjects.

[0759] Example 8

[0760] Cytotoxicity and antiviral activity of carboxylate-nitrite-polyol solutions against coronavirus SARS-CoV-2

[0761] Materials

[0762] Test formulation F1 (pH 5.8)

[0763] Formulation 1 (F1) was prepared from stock solutions of 1.5M sodium nitrite, 0.91M citric acid / citrate buffer pH 5.8 and 0.5M mannitol solution in water as an aqueous solution of sodium nitrite, citric acid pH 5.8 and mannitol (a polyol) at six test concentrations by the method described below, to give the following test compositions:

[0764] Formulation 1 (F1)

[0765]

[0766] Controls used with F1

[0767] pH 5.8 control formulation was prepared from 0.1M citric acid + assay buffer + cells.

[0768] Negative control was assay buffer + cells.

[0769] Positive control was chloroquine + cells.

[0770] Test formulation F2 (pH 5.4)

[0771] Formulation 2 (F2) was prepared from stock solutions of 1.5M sodium nitrite, 0.91M citric acid / citrate buffer pH 5.4 and 0.5M mannitol solution in water as an aqueous solution of sodium nitrite, citric acid pH 5.4 and mannitol (a polyol) at six test concentrations by the method described below, to give the following test compositions:

[0772] Formulation 2 (F2)

[0773]

[0774] Controls for use with F2

[0775] pH 5.4 control formulation was prepared from 0.1M citric acid + assay buffer + cells.

[0776] Negative control was assay buffer + cells.

[0777] Positive control was chloroquine + cells.

[0778] Chemical reagents

[0779] Sodium nitrite:

[0780] Grade: Sodium nitrite extra pure Ph Eur, USP. Sodium nitrite CAS No. 7632-00-0, EC No. 231-555-9., extra pure Ph Eur, USP, from Sigma Aldrich, product code 1.06544 100.

[0781] Citric acid:

[0782] Grade: Citric acid anhydrous powder ESSENTIAL Ph Eur, BP, JP, USP, E 330, FCC from Sigma Aldrich, product code 1.00242 5000.

[0783] D-mannitol:

[0784] Grade: D-mannitol meeting the test specifications of EP, FCC, USP from Sigma Aldrich, product code M8429-100G.

[0785] Chloroquine phosphate:

[0786] Grade: Pharmaceutical secondary standard from Sigma Aldrich, product code PHR1258-1G.

[0787] Preparation of stock solutions

[0788] To prepare the citric acid solution, 90 ml of distilled water is added to 19.2 g of citric acid, followed by 10 ml of 3M sodium hydroxide, then diluted with distilled water to adjust the pH (pH 5.4 to 160 ml, or pH 5.8 to 190 ml). In an alternative method, 20 ml of distilled water is added to 19.2 g of citric acid, followed by 1.2 g of solid sodium hydroxide, then pH is adjusted to 100 ml with 10M sodium hydroxide and distilled water. The solution is sterilized by syringe filtration using a 0.22 pm filter.

[0789] To prepare a 1.0 M sodium nitrite solution, 100 mL of distilled water is added to 6.9 g of sodium nitrite. To prepare a 1.5 M sodium nitrite solution, 100 mL of distilled water is added to 10.35 g of sodium nitrite.

[0790] When specified, 9.1 g of mannitol is added, resulting in a concentration of 0.5 M. The solution is sterilized by syringe filtration using a 0.22 pm filter.

[0791] Preparation of formulations

[0792] The pH of the buffered citric acid solution is controlled to the desired value prior to mixing with the nitrite and mannitol solutions. The pH values stated for the formulations are for the buffered citric acid solution made prior to mixing with the nitrite and mannitol solutions.

[0793] A suitable way of making the formulation is as follows: sodium nitrite (1.5 M) containing 0.5 M mannitol is added to a mixing vessel, pH controlled citric acid solution is added randomly as a 1 : 1 mixture (nitrite + polyol: citric acid). The solution is mixed by gently inverting. Once mixed, the mixture is stored in a sealed container (e.g. 50 ml falcon tube) at ambient temperature for 5 minutes. The resulting solution containing 0.75 M nitrite, 0.25 M mannitol and citric acid is then diluted 5-fold in assay buffer (1.2-fold concentration) to give a final test concentration of 0.15 M nitrite, 0.05 M mannitol and e.g. 0.1 M citric acid in the assay. Serial dilutions are made with distilled water and / or assay buffer medium from a 1 : 1 mixture (e.g. a mixture starting with 0.75 M nitrite, 0.25 M mannitol, 0.5 M citric acid). All formulation concentrations can be stored at ambient temperature. Fresh solutions are prepared for each manipulation.

[0794] Additional controls

[0795] S-nitroso-N-acetylpenicillamine (SNAP) is 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 used as a positive control to provide NO in these tests to demonstrate that NO is not cytotoxic in vitro. To control for any potential effects of the N-acetylpenicillamine (NAP) moiety of the SNAP molecule on the assay, NAP at the corresponding concentrations is used as a NO blank control and denoted as NAP50, NAP100, NAP200, NAP300 and NAP400.

[0796] Viruses

[0797] SARS-CoV-2 clinical isolates.

[0798] Cell lines

[0799] Vero...

Claims

1. Use of one or more agents selected from the group consisting of a nitric oxide generating composition, a combination or a combined association of components of a nitric oxide generating composition and mixtures thereof in the manufacture of a medicament for the treatment or alleviation or prevention of tuberculosis or infection caused by Mycobacterium tuberculosis, wherein the agent is or comprises a nitric oxide generating composition or a component thereof and wherein the nitric oxide generating composition or the combination or the combined association of components of a nitric oxide generating composition comprises one or more nitrite salts, a proton source comprising one or more acids selected from the group consisting of organic carboxylic acids and organic non-carboxylic reducing acids and one or more organic polyols; wherein the one or more organic polyols are selected from the group consisting of mannitol, lactitol or mixtures thereof; and wherein the proton source is selected from the group consisting of citric acid, ascorbic acid or mixtures thereof.

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

3. Use of the agent according to claim 2, wherein the higher pH value is in the range of 5 to 8.

4. Use of the agent according to claim 2, wherein the higher pH value is greater than or equal to 5.

2.

5. Use of the agent according to claim 4, wherein the higher pH value is in the range of 5.2 to 6.

6. Use of the agent according to any one of claims 1 to 5, wherein the nitric oxide generating composition is or can be prepared by a method comprising mixing the nitrite salt, the proton source and the organic polyol components in the required proportions in concentrations higher than required in the composition in the form to be used to form a concentrated pre-mix, followed by suitably diluting the concentrated pre-mix with water to provide the composition to be used.

7. Use of the agent according to any one of claims 1 to 5, wherein the nitric oxide generating composition is or can be prepared by a method comprising mixing the nitrite salt, the proton source and the organic polyol components in the required proportions in the concentrations required in the composition in the form to be used to provide the composition to be used.

8. Use of the agent according to any one of claims 1 to 5, comprising administering a medicament to a human or animal subject.

9. Use of the agent according to any one of claims 1 to 5, comprising administering a medicament to the lungs of a subject.

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