Compositions comprising nitrite salts, reducing agents and organic carboxylic acids to generate nitric oxide and to treat microbial infection

Abstract

The present invention relates to compositions and kits for generating nitric oxide. The compositions and kits include a nitrite salt, a reducing agent and an organic carboxylic acid. The compositions and kits of the present invention may be used in a variety of applications. In particular, the present invention relates to compositions, kits and methods using such compositions and kits for treating or preventing microbial infections.

Description

COMPOSITIONS AND KITS FOR GENERATING NITRIC OXIDETECHNICAL FIELDThe present invention relates to compositions and kits for generating nitric oxide. The compositions and kits of the present invention may be used in a variety of applications. In particular, the present invention relates to compositions, kits and methods using such compositions and kit for treating or preventing microbial infections.BACKGROUNDNitric oxide (NO) and nitric oxide precursors have been extensively studied as potential pharmaceutical agents. Nitric oxide is a signalling molecule involved in several physiological and pathological processes. In particular, nitric oxide is known to be a vasodilator and to inhibit bacterial growth. For example, WO 2007 / 116102 A describes the use of liquid formulations containing dissolved nitric oxide in the preparation of a medicament for the prevention and treatment of oral, gastric and digestive infections.As a therapeutic agent, nitric oxide is known to have different modes of action. For example, nitric oxide could affect bacteria in multiple mechanisms of action in the intra and extracellular space, including: oxidative DNA damage, inhibition of DNA, lipid peroxidation, protein deamination and tyrosine nitration (Biol. Chem., 385, 2004, 1- 10). Two broad classes of reactions are generally involved in cells: S-nitrosation of thiols and nitrosylation of some metalloenzymes. S-nitrosation involves the conversion of thiol groups (e.g., cysteine residues in proteins) to form S-nitrosothiols in protein cysteine residues, acting as an important post-translational modification and involved in almost every class of cell signalling. Nitrosylation of specific metalloenzymes leads to the formation of metal nitrosyl complexes which may disable normal enzymatic activity. This is particularly the case for metalloenzymes having iron or copper d-transition metal complexes.A variety of methods have been described to deploy nitric oxide in different therapeutic applications (Xie et al. Adv. Sci. 2021 , 8, 2003895, US 005648101A). Notably, acidification of nitrite salts using an acid to produce initially nitrous acid (HNO2) has been widely adopted for the generation of nitric oxide, whereby nitrous acid readily decomposes to nitric oxide and nitrate ions with hydrogen ions and water.The nitrous acid decomposition can be simplistically represented by the following balanced equation (1):3 HNO22 NO + NOT + H++ H2O (1)The acid and nitrite salt are typically provided as separate entities and combined at the point of need to prevent the release of nitric oxide before required.Varying factors, such as the initial nitrite concentration and the acidity (pH) of the reaction conditions, affect the nitric oxide generation profile. For example, low pH values (higher concentrations of hydronium ions) drive the equilibrium involved in the acidification of nitrite salts towards the generation of greater amounts of nitric oxide. However, low pH compositions can cause damage to physiological tissues and cells, and so optimal pH values for nitric oxide generation are often unusable in therapeutic applications. The type of acid used in the acidification of nitrite to generate nitric oxide has not been particularly limited, with organic carboxylic acids generally used in the literature.For example, WO 2022 / 013614 describes compositions for treating conditions in subjects responsive to nitric oxide treatment. The compositions include a combination of a nitric oxide releasing compound, an acidifying agent and a carrier to be topically administered.Nitric oxide may also be generated ex vivo and delivered to a patient as a gas. For example, WO 2008 / 076136 describes a device for controlled intermittent or continuous delivery of gaseous nitric oxide to the lung of a mammal.Alternatively, solutions containing the reactants for generating nitric oxide in situ may be delivered to a patient such that the nitric oxide is generated on or within the patient at the desired target. For example, WO 2020 / 245574 describes compositions for treating respiratory disorders with an acidified nitrite solution which reacts to release nitric oxide in situ. Compared to gaseous delivery, this method provides a far more targeted delivery of NO, and thereby requires much smaller amounts to be administered to achieve clinical effects. As a result, there is much lower alveolar exposure, and hence considerably reduced associated risks of toxicity. By focussing activity at the site of infection, in situ delivery mimics the physiological NO response to microbial attack and minimises environmental exposure to NO or its metabolites.In some therapeutic applications, the nitric oxide may be delivered to the patient through a gas permeable gel. For example, WO 2014 / 188174 describes a dressing system comprising: (i) a layer containing a nitrite; and (ii) a hydrogel that contains hydrogen ions. Nitric oxide is produced in situ in the dressing which is applied to the target area. Encapsulation has also been used to attempt to provide a long-lasting amount of nitric oxide. For example, US 2014 / 0056963 describes methods and compositions for generating and applying therapeutic nitric oxide gas from the reaction of at least one microencapsulated nitrite salt and an activating volume of an aqueous acidified gel.An alternative approach to generating nitric oxide from nitrite salts is to combine the nitrite salt directly with a reducing agent. Ascorbic acid is an example of an acid that is also known to act as a reducing agent, to reduce nitrite to nitric oxide. Licht, et al., Carcinogenesis (1988), 365-372 describes that ascorbic acid and ascorbate ion (denoted together as AA) inhibit nitrosation by competing for the nitrosating agents formed from nitrite. AA is oxidized irreversibly by this reaction and the nitrite equivalents are reduced to nitric oxide. More recently Buettner et al., Advances in Redox Research 9 (2023) 1000079 reports the production of nitric oxide from nitrite, as facilitated by ascorbate, over the pH range of 2.4-7.4. Additionally, WO 2013 / 085784 describes mixing an aqueous solution of sodium nitrite with an aqueous solution of an equimolar amount of citric acid, in the presence of an excess of ascorbic acid to mainly reduce the nitrogen dioxide formed. Furthermore, WO 2009 / 0866470 outlines formulations that incorporate nitrite, citric acid and ascorbic acid in aerosolized form. In these formulations, an excess of sodium nitrite is employed compared to citric acid.All these above examples illustrate that the delivery profile of nitric oxide to the patient is an important factor controlling the efficacy of the therapeutic application.The various effects of nitric oxide in the body, from physiological mechanisms to pathogen dispersal and killing are known to be sensitive to the particular dose range, as summarised for example by Mendhi et al., Applied Materials Today 2020. They suggest that this has diminished progress in translating nitric oxide therapies into the medical or clinical fields. Poh et al., Molecules, 2022 also highlight different biological roles of nitric oxide depending on its dosage level. Friedman [Virulence, 2012] demonstrates the different susceptibility of various pathogens to nitric oxide.The time release profile of nitric oxide formulations is thus important to therapeutic effect, and the discoveries herein are a significant advance in this regard. There is a need to develop a novel approach to influencing the nitric oxide generation profile in ways other than simply the nitrite concentration and / or the pH of the solution such that nitric oxide generation can be tailored from different delivery forms and different therapeutic applications.SUMMARY OF INVENTIONThe inventors have discovered that compositions and kits with a novel combination of components can tailor the nitric oxide generation profile.At its most general, the compositions and kits of the present invention include one or more nitrite salts, one or more reducing agents and one or more organic carboxylic acids, where the molar ratio of the total reducing agent to the nitrite salt is at least about 0.25%, the molar ratio of any one reducing agent to total nitrite salts is at most about 75%, and the molar ratio of total organic carboxylic acid to total nitrite of at least about 0.5%. These compositions include at least one reducing agent and at least one organic carboxylic acid. In embodiments where the reducing agent is an organic carboxylic acid (such as oxalic acid), the composition and kits include at least one organic carboxylic acid that is different to the reducing agent that is an organic carboxylic acid.The present inventors have found that the use of one or more reducing agents in combination with an organic carboxylic acid in the ratios specified above allows the nitric oxide generation profile to be adapted to suit the specific delivery form and / or therapeutic application. The adaptation of the nitric oxide profile may be, for example, increasing or decreasing the production rate in the initial burst of nitric oxide generation, prolonging or shortening the burst of generation of nitric oxide over a specific time period, increasing or decreasing the steady state nitric oxide production level, and / or increasing or reducing the cumulative amount of nitric oxide produced. These features may be described, for example, by the peak NO concentration generated by the composition, the time to peak NO generated, the full width at half maximum (FWHM) from a plot of NO generated over time and the area under the curve from a plot of NO generated over time.Without wishing to be bound by theory, the present inventors believe that the one or more reducing agent acts as an accelerant in the compositions for generating nitric oxide from the acidification of a nitrite salt. The remainder of the nitrite acts as a reservoir to produce nitric oxide. By selecting the molar ratios of reducing agent to the amount of nitrite, the nitric oxide generation profile can be adapted by design to suit a specific application.Further, the inventors have found that by causing some of the reagents to be released on a delayed basis (e.g. by encapsulation, or gels, or by introducing in secondary layers), or alternatively by adding some reagents at subsequent times, the nitric generation profile can be further controlled, in particular by extending the time over which the generation occurs, and by in some formulations, creating bursts of nitric oxide at various times.In a first aspect, the present invention provides a composition for generating nitric oxide, the composition comprising:(i) one or more nitrite salts;(ii) one or more reducing agents;(iii) one or more organic carboxylic acids; and the molar ratio of the total reducing agent or agents in the composition to the total nitrite salt or salts in composition is at least about 0.25%, the molar ratio of any one of the reducing agents present in the composition to the total nitrite salt or salts in the composition is at most about 75%, and the molar ratio of total organic carboxylic acid in the composition to total nitrite salt or salts in the composition is at least about 0.5%.In a second aspect, the present invention provides a kit for providing a composition for generating nitric oxide, the kit including:(i) a nitrite component including one or more nitrite salts; and(ii) an acidic component including one or more organic carboxylic acids; wherein the kit further includes one or more reducing agents, and the molar ratio of the total reducing agent or agents in the kit to the total nitrite salt or salts in kit is at least about 0.25%, the molar ratio of any one of the reducing agents present in the kit to the total nitrite salt or salts in the kit is at most about 75%, and the molar ratio of total organic carboxylic acid in the kit to total nitrite salt or salts in the kit is at least about 0.5%.In embodiments of the second aspect where the nitrite component and / or the acidic component includes a polar solvent (such as being present in an aqueous carrier, e.g., in an aqueous solution or a hydrogel), the nitrite component and the acidic component are typically in separate components. In embodiments of the second aspect where the nitrite component includes a polar solvent (e.g., is present in an aqueous carrier), the one or more reducing agents are typically separate from the nitrite component. The nitrite salt will typically react to generate nitric oxide if in contact with an acid, a reducing agent or both if these components include a polar solvent (e.g., are present in an aqueous carrier). The kits of the present invention are typically in a form where the nitrite salt will not significantly react before required. In this way, the user of the kit may initiate reaction of the nitrite salt to form nitric oxide at the point of use. For completeness, the kits of the second aspect may include nitrite component, acid component and the reducing agent or agents together in the absence of a polar solvent (e.g., in the absence of an aqueous carrier, such as when all components are in a dry solid form).For the avoidance of doubt, the compositions of the first aspect and the kits of the second aspect contain both a reducing agent and an organic carboxylic acid. Where the reducing agent is an acid (such as oxalic acid), the organic carboxylic acid component (iii) is a different organic carboxylic acid to the organic carboxylic acid that is a reducing agent in component (ii).In a third aspect, the present invention provides use of the composition according to the first aspect or the kit according to the second aspect for generating nitric oxide.In a fourth aspect, the present invention provides the composition according to the first aspect or the kit according to the second aspect for use in treating or preventing a microbial infection.In a fifth aspect, the present invention provides a method of treating or preventing a microbial infection, which comprises administering to a subject an antimicrobial amount of the composition according to the first aspect or the kit according to the second aspect.In a sixth aspect, the present invention provides use of the composition according to the first aspect or the kit according to the third aspect for the manufacture of a medicament for treating or preventing a microbial infection.In a seventh aspect, the present invention provides a method of treating a surface or space to reduce the number of viable microbes on the surface or in the space, which comprises applying to the surface or in the space or to a vicinity thereof, an antimicrobial amount of the composition according to the first aspect or the kit according to the second aspect.In an eighth aspect, the present invention provides a method of generating nitric oxide, wherein the method includes the steps of:(i) initiating an initial NO-generating reaction by combining one or more nitrite salts, a polar solvent and one or both of one or more organic carboxylic acids and one or more reducing agents to form an initial NO-generating reaction composition; and(ii) adding or exposing subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents to components of the initial NO-generating reaction composition at one or more subsequent times to form one or more subsequent NO-generating reaction compositions; provided that one or more organic carboxylic acids are present in the initial and / or one or more subsequent reaction compositions and one or more reducing agents are present in the initial and / or one or more subsequent reaction compositions. In other words, both organic carboxylic acid or acids and reducing agent or agents are introduced to the NO-generating reaction composition (initial and / or one or more subsequent reaction compositions) during the method and their introduction may be concomitantly or sequentially (or a combination thereof).The addition or exposure of the subsequent quantities in step (ii) may include the addition of the subsequent quantities from a source external to the initial NO- generating reaction composition or by exposing the subsequent quantities to the components of the initial reaction composition from a source within or adjacent to the initial NO-generating reaction composition where of the subsequent quantities are available or exposed to the NO-generating reaction composition only after a period of time after step i), such as a delayed release mechanism.In a ninth aspect, the present invention provides a method of generating nitric oxide, wherein the method includes combining one or more nitrite salts, a polar solvent, one or more organic carboxylic acids and one or more reducing agents to form a NO-generating reaction composition; wherein at least one of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents is added into the NO-generating reaction composition over a longer time period than at least one of the other of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents.The skilled person will appreciate that, except where mutually exclusive, a feature described in relation to any one of the above aspects may be applied mutatis mutandis to any other aspect. Furthermore, except where mutually exclusive, any feature described herein may be applied to any aspect and / or combined with any other feature described herein.DESCRIPTION OF INVENTIONThe present invention will be described in detail with reference to the Examples and accompanying drawings.Figure 1 shows a plot of the concentration of nitric oxide produced over time for the formulations containing ascorbic acid (AA) of Table 1 in Example 1. The concentration of nitric oxide generated is shown on a logarithmic scale on the y-axis.Figure 2 shows a plot of the cumulative nitric oxide concentration generated in the first 13.5 minutes relative to the control against the molar ratio of [ascorbic acid] to [nitrite] (in %) for the formulations in Example 2.Figure 3 shows a plot of the cumulative nitric oxide profile over time for the formulations in Example 2.Figure 4 shows a plot of the concentration of nitric oxide produced over time for the formulations containing sodium thiosulphate (STS) in Example 3. The concentration of nitric oxide generated is shown on a logarithmic scale on the y-axis.Figure 5 shows a plot of the cumulative nitric oxide concentration over the first 60 minutes against molar ratio of [thiosulphate] to [nitrite] (in %) for the formulations containing sodium thiosulphate in Example 3.Figure 6 shows a plot of the cumulative nitric oxide concentration over time for the formulations containing sodium thiosulphate in Example 3.Figure 7a shows the nitric oxide profile relative to the control over time for formulations containing various concentrations of ascorbic acid without mannitol.Figure 7b shows the nitric oxide profile relative to the control for formulations containing various concentrations of ascorbic acid with mannitol.Figure 8 shows the NO generated after successive additions of ascorbic acid to a formulation containing nitrite salt, citric acid / citrate buffer and an initial amount of ascorbic acid.Figure 9 shows the results from different formulations with respect to antimicrobial activity. Colony forming units / mL (CFU / mL) of Pseudomonas aeruginosa are plotted against different formulations containing citric acid / citrate either with or without additional ascorbic acid. The ascorbic acid is added in two concentrations to show a dose response.Figure 10 shows the results from different formulations with respect to antimicrobial activity. CFU / mL of Pseudomonas aeruginosa are plotted against different formulations containing citric acid / citrate without mannitol, and with additional ascorbic acid.Figure 11 shows a schematic of an NO generation profile over time showing some of the parameters that may be tailored by using the compositions described herein.Figure 12 shows the nitric oxide profile relative to the control over time for formulations containing different ratios of reducing agents, ascorbic acid / sodium thiosulphate.Figure 13 shows the nitric oxide profile relative to the control over time for formulations with different incremental addition of ascorbic acid.Figure 14 shows the nitric oxide profile relative to the control over time for formulations with different sequential addition of ascorbic acid.Figure 15 shows the nitric oxide profile relative to the control over time for a formulation with different incremental addition of iron (II) sulphate heptahydrate.Figure 16 shows the nitric oxide profile relative to the control over time for a formulation to which DL-cysteine is subsequently added.Figure 17 shows the nitric oxide profile relative to the control over time for a formulation to which glutathione is subsequently added.Figure 18 shows the nitric oxide profile relative to the control over time for a formulation to which penicillamine is subsequently added.Figure 19 shows the nitric oxide profile relative to the control over time for a formulation to which dithiothreitol is subsequently added.Figure 20 shows the results from different silica-supported thiol reducing agents with respect to antimicrobial activity. CFU / mL of Pseudomonas aeruginosa are plotted against different formulations containing citric acid / citrate with nitrite / mannitol, with additional silica-supported cysteine or mercaptopropyl-functionalised silica.Figure 21 A shows the results from experiments investigating solid disks of nitrite / mannitol and citric acid / citrate infused into separate gel formulations with respect to antimicrobial activity. CFU / mL of Pseudomonas aeruginosa is plotted against the formulation at different timepoints.Figure 21 B shows the results from experiments investigating solid disks of nitrite / mannitol and citric acid / citrate infused into separate gel formulations, to which glutathione as reducing agent are added, with respect to antimicrobial activity. CFU / mL of Pseudomonas aeruginosa is plotted against the formulation at different timepoints.Figure 22 shows the results from different formulations with respect to antimicrobial activity. CFU / mL of Streptococcus mutans is shown as a function of time for different formulations.The term “about” is used herein to denote that the numerical value is not strictly limiting, and the skilled person will understand that the value may extend above or below (as appropriate) the exact value in line with the skilled person’s understanding of the value. The term “about” may signify a value that is up to ±10% of the value.The term “molarity” (also known as molar concentration) is a measure of the concentration of a chemical species in terms of amount of substance per unit volume of solution. In the context of the present invention, the molarity is provided as the number of moles per liter and has the unit symbol mol / L or “M”. The expression “mM” is also used as an abbreviation for the unit “millimolar”, mmol / L. The term “millimolar” has its usual meaning in the art, i.e. , a concentration of one thousandth of a mole per litre.The term “salts thereof” is used herein to refer to basic addition salt of the mentioned compound(s) formed through reaction with a suitable base, such as the hydroxide, carbonate, bicarbonate, sulphate, of a metal cation (such as a pharmaceutically acceptable metal cation), with ammonia, or with organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminium salts and the like. Illustrative examples of bases include sodium hydroxide, potassium hydroxide, lithium hydroxide, choline hydroxide, sodium carbonate, N+(CI-4 alkyl)4) and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. In certain applications (e.g., medical use), the salts referred to herein may be pharmaceutically acceptable salts.The term “derivatives thereof” is used herein to refer to compounds that are produced from or related to the mentioned compounds.The term “at least” is used to refer to the lower limit of a numerical value and includes such value unless specified otherwise. For example, the term “at least X” is considered to be equivalent to “X or more” (where X is a numerical value). The term “at most” is used to refer to the upper limit of a numerical value and includes such value unless specified otherwise. For example, the term “at most X” is considered to be equivalent to “X or less” (where X is a numerical value).Compositions for generating nitric oxideThe compositions of the present invention include one or more nitrite salt, one or more reducing agent, and one or more organic carboxylic acid. The compositions are further defined by the molar ratio of the reducing agent component and the nitrite component.The compositions of the present invention include both an organic carboxylic acid (or more than one organic carboxylic acid) and a reducing agent (or more than one reducing agent). In embodiments where the reducing agent is an organic carboxylic acid (such as oxalic acid), the compositions include an organic carboxylic acid as component (iii) that is different from the reducing agent that is an organic carboxylic acid.Nitrite salt (component (i))The compositions of the present invention include one or more nitrite salt. The choice of nitrite salt is not particularly limited. The nitrite salt may be selected from one or more alkali metal nitrite salts or alkaline metal nitrite salts. For example, the one or more nitrite salt may be selected from UNO2, NaNCh, KNO2, RbNCh, CsNCh, FrNCh, AgNO2, Be(NO2)2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2, Ra(NO2)2and any mixture thereof.In particular, the one or more nitrite salt may be selected from UNO2, NaNCh, KNO2, RbNO2, CSNO2, AgNO2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2and any mixture thereof. The nitrite salt may be NaNC>2, KNO2, or a mixture thereof. The nitrite salt may be NaNCh.The nitrite salt may be a pharmaceutically acceptable grade of nitrite salt. In other words, the nitrite salt may adhere to one or more active pharmacopoeia monographs for the nitrite salt. For example, the nitrite salt may adhere to the monograph of the nitrite salt of one or more of the United States Pharmacopoeia (USP), European Pharmacopoeia or Japanese Pharmacopoeia.In particular, the nitrite salt used may have one or more of the characteristics as provided in paragraphs

[0032] to

[0060] and / or Table 1 in paragraph

[0204] of WO2010 / 093746, the disclosure of which is incorporated herein by reference in its entirety.The molarity of the one or more nitrite salt in the composition may be in the range 0.001 M to 2.0 M. In some embodiments, the molarity of the one or more nitrite salt in the composition is 0.01 M to 1.5 M, 0.05 M to 1 .0 M or 0.075 M to 0.75 M. In particular embodiments, the molarity of the one or more nitrite salt in the composition is at least 0.075 M, at least 0.080 M, at least 0.085 M, at least 0.090 M, at least 0.095 M, or at least 0.100 M. In particular embodiments, the molarity of the one or more nitrite saltin the composition is at most 0.75 M, at most 0.70 M, at most 0.65 M, at most 0.60 M, at most 0.55 M, or at most 0.50 M.The compositions of the present invention include one or more reducing agents. The expression “one or more reducing agents” herein refers to a compound or substance capable of reducing any nitrogen species with formal oxidation states of nitrogen of III and IV.The reducing agent or agents (when used in combination with an organic carboxylic acid) may provide a mechanism for tailoring the NO generation profile of the compositions described herein.The reducing agent or agents may be an organic acid reducing agent or salt thereof, a reducing sugar or salt thereof, or an inorganic phosphorus-containing or sulphur-containing salt selected from sulfites, dithionates, thiosulphates, phosphites and hypophosphites.Examples of organic acid reducing agents include but are not limited to ascorbic acid, ascorbic acid derivatives, reductic acid (2,3-dihydroxy-2-cyclopentanone), erythorbic acid, oxalic acid, formic acid, glutathione. Examples of reducing sugar reducing agents include but are not limited to glucose, galactose, fructose, ribose, glyceraldehyde, xylose, cellobiose, lactose and erythrose.In certain embodiments, the reducing agent or reducing agents may be a reducing agent containing a sulphur atom (or a sulphur-containing reducing agent). Examples of reducing agent containing a sulphur atom include, but are not limited to, thiols (such as glutathione, cysteine, DL-cysteine, N-acetyl cysteine, penicillamine, dithiobutylamine, dithiothreitol) and salts thereof, sulphates (such as iron (II) sulphate heptahydrate), sulfites, dithionates and thiosulphates. Without wishing to bound by theory, it is believed that sulphur-containing reducing agents may be capable of generating hydrogen sulphide (H2S) in addition to acting as a reducing agent. H2S is an endogenous gasotransmitter (as is nitric oxide) and is known to regulate different cellular biological systems. In this way, sulphur-containing reducing agents may enhance the effect of the compositions.The reducing agent or reducing agents may be selected from ascorbic acid, reductic acid (2,3-dihydroxy-2-cyclopentanone), erythorbic acid, oxalic acid, formic acid, dithionous acid, thiols (such as glutathione, cysteine, DL-cysteine, N-acetyl cysteine, penicillamine, dithiothreitol), reducing sugars (such as glucose, galactose, fructose, ribose, glyceraldehyde, xylose, cellobiose, lactose or erythrose), salts thereof, derivatives thereof, sulphates (such as iron (II) sulphate heptahydrate), sulfites, dithionates, thiosulphates, phosphites and hypophosphites and combinations thereof.In some embodiments, the one or more reducing agent is provided on a solid support. In some embodiments, the solid support comprises a silica support, a glass bead or a polystyrene support.In some embodiments, the one or more reducing agent comprises or is a solid supported material selected from, but not limited to, solid supported ascorbic acid, solid supported reductic acid (such as 2,3-dihydroxy-2-cyclopentanone), solid supported erythorbic acid, solid supported oxalic acid, solid supported formic acid, solid supported dithionous acid, solid supported thiols (such as glutathione, cysteine, N-acetyl cysteine, DL-cysteine, penicillamine, dithiothreitol), solid supported reducing sugars (such as glucose, galactose, fructose, ribose, glyceraldehyde, xylose, cellobiose, lactose or erythrose), solid supported salts thereof, solid supported derivatives thereof, solid supported sulphates (such as iron (II) sulphate heptahydrate), solid supported thiosulphates, solid supported phosphites and silica supported hypophosphites and combinations thereof.Thus, in some embodiments, the one or more reducing agent comprises or is a silica supported material. In some embodiments the silica supported material is selected from, but not limited to, silica supported ascorbic acid, silica supported reductic acid (such as 2,3-dihydroxy-2-cyclopentanone), silica supported erythorbic acid, silica supported oxalic acid, silica supported formic acid, silica supported dithionous acid, silica supported thiols (such as glutathione, cysteine, N-acetyl cysteine, DL-cysteine, penicillamine, dithiothreitol), silica supported reducing sugars (such as glucose, galactose, fructose, ribose, glyceraldehyde, xylose, cellobiose, lactose or erythrose), silica supported salts thereof, silica supported derivatives thereof, silica supported sulphates (such as iron (II) sulphate heptahydrate), silica supported thiosulphates, silica supported phosphites and silica supported hypophosphites and combinations thereof.In some embodiments the silica supported material comprises or is silica supported cysteine.In some embodiments the silica supported material comprises or is 3-mercaptopropyl- functionalized silica gel.In some embodiments, the one or more reducing agent is selected from ascorbic acid, ascorbic acid salts, ascorbic acid derivatives and salts thereof, thiosulphate salts (e.g., sodium thiosulphate and potassium thiosulphate) and combinations thereof.In some embodiments, the one or more reducing agent is selected from ascorbic acid, salts thereof, and combinations thereof. In some embodiments, the one or more reducing agent is ascorbic acid.In some embodiments, the one or more reducing agent is an ascorbic acid derivative selected from ascorbate palmitic acid (ascorbyl palmitate), 3-0-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, 6-0- dodecanedioyl ascorbic acid, L-Ascorbic acid 2-phosphate, 2-O-alpha-D- Glucopyranosyl-L-ascorbic acid and combinations thereof.In some embodiments, the one or more reducing agent comprises or is sodium thiosulphate.In some embodiments, the one or more reducing agent comprises or is iron (II) sulphate heptahydrate.In some embodiments, the composition includes one reducing agent as the sole reducing agent. For example, the composition may include ascorbic acid, an ascorbic acid derivative or a thiosulphate salt as the sole reducing agent. In other embodiments, the compositions include two or more reducing agents. For example, the composition may include ascorbic acid and a thiosulphate salt as reducing agents or include an ascorbic acid derivative and a thiosulphate salt as reducing agents. In particular embodiments, the composition includes sodium thiosulphate and ascorbic acid as reducing agents.When the composition includes two or more reducing agents, the ratio between the reducing agents is not particularly limited. As each reducing agent may affect the NO-generation profile in a different way, the NO-generation profile may be adapted depending on the ratio of the reducing agents. In some embodiments, the composition includes two reducing agents and the molar ratio between the two reducing agents is in the range of about 10:90 to about 90:10. For example, the molar ratio between the two reducing agents may be about 10:90, about 20:80, about 25:75, about 30:70, about 40:60 or about 50:50.When the reducing agent is a salt, the salt may be a pharmaceutically acceptable salt.The molarity of the reducing agent is not particularly limited beyond the limitation placed on it by the molar ratios as described herein. The molarity of the one or more reducing agent in the composition may be at least 0.001 mM. In some embodiments, the molarity of the one or more reducing agent in the composition is at least 0.01 mM, at least 0.05 mM, at least 0.1 mM, or at least 0.5 mM. In some embodiments, the molarity of any one reducing agent in the composition is at most 1.5 M, at most 1.0 M, at most 750 mM, or at most 500 mM.In particular embodiments, the molarity of the reducing agent in the composition is 0.01 mM to 1.5 M, 0.05 mM to 1 .0 M, 0.1 mM to 750 mM, or 0.5 mM to 500 mM.In some embodiments, the reducing agent component may also be an acid. In specific embodiments, the reducing agent may also be an organic carboxylic acid (such as oxalic acid). In such embodiments the composition and kits include at least one organic carboxylic acid that is different to the reducing agent that is an organic carboxylic acid.Where the reducing agent is an acid (such as ascorbic acid), the composition includes at least one other acid as the acid in component (iii). In other words, the compositions include a reducing agent (which may be an acid) and an acid (which is different from the reducing agent that is an acid).Reducing agent - thiolsThiol-containing molecules such as glutathione are known to be able to act as “NO- donors” in that they can react with nitric oxide to form an S-nitrosothiol compound (forexample S-nitrosoglutathione). Over time S-nitrosothiol compounds could undergo enzymatic or non enzymatic decomposition or homolytic cleavage at physiological pH to release nitric oxide and the oxidised disulfide (for example oxidised glutathione (GSSG)). Thiol-containing molecules therefore represent an attractive option for use as a reducing agent in the compositions and kits described herein for their ability to not only act as a reducing agent analogously to other reducing agents described herein but also to act as an NO-donor. (Massa, et al. Antioxidants 2021 , 10 (7), 1111)Thus, in some embodiments, the composition for generating nitric oxide may include:(i) one or more nitrite salts;(ii) one or more reducing agents, wherein at least one reducing agent is a thiol;(iii) one or more organic carboxylic acids; and wherein the molar ratio of the total reducing agent or agents in the composition to the total nitrite salt or salts in composition is at least about 0.25%, the molar ratio of any one of the thiols present in the composition to the total nitrite salt or salts in the composition is at most about 75%, and the molar ratio of total organic carboxylic acid in the composition to total nitrite salt or salts in the composition is at least about 0.5%.In some embodiments, the thiol may comprise or may be glutathione, cysteine, N- acetyl cysteine, DL-cysteine, penicillamine, dithiothreitol, or a combination thereof.In further embodiments, the thiol may comprise or may be a silica supported material (such as silica supported glutathione, silica supported cysteine or N-acetyl cysteine, silica supported silica supported penicillamine, silica supported dithiothreitol, 3- mercaptopropyl-functionalized silica gel, or combination thereof). 3-mercaptopropyl- functionalized silica gel.Organic carboxylic acid (component (iii))The compositions of the present invention include one or more organic carboxylic acid as component (iii). For the avoidance of doubt, carboxylic acids are acids containing one or more -COOH group in the molecule. The organic carboxylic acid is typically the principal component in the acidification of the nitrite to nitric oxide. The organic carboxylic acid may be any organic carboxylic acid or any combination thereof which is suitable for acidifying the nitrite salt.The organic carboxylic acid may be straight-chain or branched. The organic carboxylic acid may be saturated or unsaturated. The organic carboxylic acid may be aliphatic or aromatic. The organic carboxylic acid may be acyclic or cyclic. The organic carboxylic acid may be a vinylogous carboxylic acid. The one or more organic carboxylic acid may, for example, be selected from salicylic acid, acetyl salicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, a-hydroxypropanoic acid, P-hydroxypropanoic acid, p-hydroxybutyric acid, p-hydroxy-p-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobioic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, carnitine, salts thereof, and combinations thereof.The one or more organic carboxylic acid may be selected from citric acid, hyaluronic acid, alginic acid, maleic acid, salts thereof, and combinations thereof. In particular, the one or more organic carboxylic acid may be citric acid or salts thereof.In certain embodiments, the composition includes a mixture of organic carboxylic acids.The molarity of the one or more organic carboxylic acid in component (iii) of the composition may be at least 0.001 M. In some embodiments, the molarity of the one or more organic carboxylic acid in the composition is at least 0.0025 M, at least 0.005 M, at least 0.0075 M or at least 0.01 M. In some embodiments, the molarity of the one or more organic carboxylic acid in the composition is at most 5 M, at most 3M, at most 2 M, or at most 1.5 M. In particular embodiments, the molarity of the one or more organic carboxylic acid in the composition is 0.0025 M to 2 M, 0.005 M to 1.5 M, 0.0075 M to 1 M, or 0.01 M to 0.75 M.In certain embodiments, the organic carboxylic acid may be a polymeric organic carboxylic acid. The one or more polymeric organic carboxylic acids may be, for example, a polymer capable of forming a hydrogel. The one or more polymeric organic carboxylic acids may be, for example, selected from polylactic acid, hyaluronic acid, chitosan, alginic acid and acidified polyacrylamides.Where the composition includes a polymeric organic carboxylic acid, the molarity of the organic carboxylic acid is calculated based on the molarity of the monomeric unit containing the carboxylic acid. In this way, the overall moles of carboxylic acidcontaining units are considered rather than the overall moles of the polymer.The compositions and kits may additionally include other acids, such as an inorganic or organic acid (in addition to the organic carboxylic acid or acids). In this way, the compositions and kits may include a mixture of acids, including at least one organic carboxylic acid. Suitable inorganic acids include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulphuric and carbonic acid.Molar ratiosReducing agent to nitrite molar ratios (“RAN molar ratios")The compositions of the present invention are defined by the molar ratio of reducing agent or agents in the composition to nitrite salt or salts in the composition (sometimes referred herein as the “RAN” or “reducing agent to nitrite” molar ratios).The compositions described herein include a molar ratio of total reducing agents in the composition to total nitrite salt in the composition of at least about 0.25%. In this way, the compositions may have sufficient total amount of reducing agent to show an effect. This ratio may be referred to as the “total RAN molar ratio” as it involves the total reducing agent in the composition.The molar ratio of the total reducing agent in the composition to the total nitrite salt in the composition (the total RAN molar ratio) is calculated by dividing the sum of the molarity of each reducing agent in the composition by the sum of the molarity of each nitrite salt component in the composition, i.e. ,Sum of [molarity of each reducing agent] / sum of [molarity of each nitrite salt].The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%).The upper limit of the molar ratio of total reducing agent in the composition to the total nitrite salt in the composition is not particularly limited. In some embodiments, the molar ratio of the total reducing agent in the composition to the total nitrite salt in the composition is at most 500%. In further embodiments, the molar ratio of the totalreducing agent in the composition to the total nitrite salt in the composition is at most 400%, at most 300%, at most 200%, at most 150%, at most 125% or at most 100%.Where the composition includes a single reducing agent as the sole reducing agent, then the total RAN molar ratio of the composition is based only on the sole reducing agent. Where the composition includes more than one reducing agent, the total RAN molar ratio is based on the sum of the molarity of each reducing agent present in the composition. In this way, the absolute amount of each reducing agent in a composition including more than one reducing agent can be lower than compositions including a sole reducing agent.In some embodiments, the molar ratio of the total reducing agent in the composition to the total nitrite salt in the composition is at least about 0.3%, at least about 0.5%, at least about 0.75% or at least about 1%.The molar ratio of any individual reducing agent in the composition to total nitrite salt in the composition is at most about 75%. In other words, the composition has a limit on the amount of any single reducing agent. This ratio may be referred to as the “individual RAN molar ratio” as it involves calculating the ratio based on the molarity of each individual reducing agent in the composition. In this way, the composition can include more than one reducing agent as long as the amount of each reducing agent does not exceed a molar ratio of any one reducing agent to total nitrite salt of about 75%.The molar ratio of the any individual reducing agent in the composition to the total nitrite salt in the composition (the individual RAN molar ratio) is calculated by dividing the molarity of any one reducing agent in the composition by the sum of the molarity of each nitrite salt component in the composition, i.e. ,[molarity of any one reducing agent] / sum of [molarity of each nitrite salt].The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%).In this way, compositions including more than one reducing agent may have a total RAN molar ratio of above 75% as long as each individual RAN molar ratio is about 75% or less.In some embodiments, the molar ratio of any individual reducing agent to the total nitrite salt is about 50% or less, about 42% or less, about 30% or less, or about 25%.For compositions that include a single reducing agent as the sole reducing agent the total RAN molar ratio and the individual RAN molar ratio will be the same. In these embodiments, the composition includes a molar ratio of the total reducing agent in the composition to the total nitrite salt in the composition in the range of about 0.25% to about 75%. In particular embodiments, the composition includes a single reducing agent as the sole reducing agent and the molar ratio of total reducing agent to the total nitrite salt is from about 0.5% to about 42%, from about 0.75% to about 30%, or from about 1% to about 25%.For compositions that include more than one reducing agent, the compositions include a total RAN molar ratio from about 0.25% and an individual molar ratio of up to about 75%.Organic carboxylic acid to nitrite molar ratio (“OCAN molar ratio")The compositions and kits herein require a minimum molar ratio of the organic carboxylic acid to nitrite ratio (the “OCAN molar ratio”). The organic carboxylic acid is an important element in the acidification of the nitrite salt to generate nitric oxide. As such, the compositions and kits described herein have a molar ratio of total organic carboxylic acid in the composition or kit to total nitrite in the composition or kit of at least about 0.5%.The molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition (the OCAN molar ratio) is calculated by dividing the sum of the molarity of each organic carboxylic acid in the composition by the sum of the molarity of each nitrite salt component in the composition, i.e. ,Sum of [molarity of each organic carboxylic acid] / sum of [molarity of each nitrite salt].The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%). As is understood by the skilled person, ascorbic acid is not an organic carboxylic acid. As such, any ascorbic acid present in the composition or kit does not contribute to the OCAN molar ratio.In some embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is at least about 0.75%. In particular embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is at least about 1.0%. In further embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 4% or at least about 5%.The maximum molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is not particularly limited. In some embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is at most about 500%. In further embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is at most about 400%, at most about 300%, at most about 200%, at most about 150%, at most about 125% or at most about 100%.In some embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is in the range of about 0.5% to about 500%. In further embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is in the range of about 0.75% to about 400%. In particular embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is in the range of about 1 .0% to about 300%. In yet further embodiments, the molar ratio of the total organic carboxylic acid in the composition to the total nitrite salt in the composition is in the range of about 1.5% to about 200%, about 2% to about 200%, about 2.5% to about 150%, about 3% to about 150%, about 4% to about 125% or about 5% to about 100%.In some embodiments, the reducing agent may also be an organic carboxylic acid (such as oxalic acid). In these embodiments, the organic carboxylic acid reducing agent (e.g., oxalic acid) contributes to the OCAN molar ratio.Reducing agent to acid molar ratio (“RAA molar ratio")In some embodiments, the molar ratio of the total reducing agent in the composition to total acid in the composition is from about 0.01 %. This ratio may be referred to as the “total RAA molar ratio” as it involves the total reducing agent in the composition.The molar ratio of the total reducing agent to the total acid (total RAA molar ratio) is calculated by dividing sum of the molarity of the each reducing agent in the composition by the sum of the molarity of each acid in the composition, i.e.,Sum of [molarity of each reducing agent] / sum of [molarity of each acid]).The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%).In some embodiments, the molar ratio of the total reducing agent in the composition to total acid in the composition is at least about 0.01 %, at least about 0.1 %, or at least about 0.5%, or at least about 1%, or at least about 2%.The upper limit of the RAA molar ratio is not particularly limited. In compositions where all of the reducing agents present are also acids, the molar ratio of the total reducing agent in the composition to total acid in the composition will be less than 100% because the compositions must include at least one organic carboxylic acid in addition to the reducing agent or agents present and the reducing agents that are acids will be counted in the total acid molarity. In these embodiments, the molar ratio of the total reducing agent in the composition to total acid in the composition may be at most about 75%, at most 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 24%, at most about 22%, or at most about 20%.In particular embodiments, the reducing agent or all of the reducing agents in the composition are acids and the molar ratio of the total reducing agent in the composition to total acid in the composition is in the range of about 0.01% to about 75%, about 0.1% to about 50 %, about 0.5% to about 40%, about 1% to about 30%, or about 2% to about 25%.Total acid to nitrite molar ratio (“TAN molar ratio")In some embodiments, the molar ratio of the total acid in the composition to total nitrite in the composition is from about 1% (or about 1.0%). This ratio may be referred to as the “TAN molar ratio” as it involves the total acid in the composition.The molar ratio of the total acid to the total nitrite (TAN molar ratio) is calculated by dividing sum of the molarity of each acid in the composition by the sum of the molarity of each nitrite salt in the composition, i.e.,Sum of ([molarity of each acid] / sum of [molarity of each nitrite salt]).The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%).In some embodiments, the molar ratio of the total acid in the composition to total nitrite in the composition is at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%.The upper limit of the TAN molar ratio is not particularly limited. In some embodiments, the molar ratio of the total acid in the composition to total nitrite in the composition is at most 100%. It may be possible to extend the time of release of NO from the composition by including an excess of nitrite to acid to provide a nitrite reservoir. In some embodiments, the molar ratio of the total acid in the composition to total nitrite in the composition is at most 95%, at most 90%, at most 85%, at most 80%, or at most 75%.In certain embodiments, the molar ratio of the total acid in the composition to total nitrite in the composition is in the range of about 1 % to about 100%, about 5% to about 95%, about 10% to about 90%, about 20% to about 85%, about 30% to about 80%, or about 40% to about 75%.Total acid and reducing agent to nitrite molar ratio (“TARAN molar ratio")In some embodiments, the molar ratio of the total acid and total reducing agent in the composition to total nitrite in the composition is from about 1 % (or about 1.0%). This ratio may be referred to as the “TARAN molar ratio” as it involves the total acid and the total reducing agent in the composition.The molar ratio of the total acid and total reducing agent to the total nitrite (TARAN molar ratio) is calculated by dividing sum of the molarity of each acid and the molarityof each reducing agent (which is not an acid) in the composition by the sum of the molarity of each nitrite salt in the composition, i.e.,Sum of ([molarity of each acid] + [molarity of each reducing agent that is not an acid]) / sum of [molarity of each nitrite salt]).The molar ratio is provided as a percentage (i.e., the figure is multiplied by 100%). The molarity of reducing agents that are acids is counted only once to avoid double counting. In this way, the TAN and TARAN are the same where all reducing agents in the compositions are acids.In some embodiments, the molar ratio of the total acid and the total reducing agent in the composition to total nitrite in the composition is at least about 5%, at least about 10%, at least about 20%, at least about 30%, or at least about 40%.The upper limit of the TARAN molar ratio is not particularly limited. In some embodiments, the molar ratio of the total acid and the total reducing agent in the composition to total nitrite in the composition is at most 100%. It may be possible to extend the time of release of NO from the composition by including an excess of nitrite to acid and reducing agent to provide a nitrite reservoir. In some embodiments, the molar ratio of the total acid and the total reducing agent in the composition to total nitrite in the composition is at most 95%, at most 90%, at most 85%, at most 80%, or at most 75%.In certain embodiments, the molar ratio of the total acid and the total reducing agent in the composition to total nitrite in the composition is in the range of about 1 % to about 100%, about 5% to about 95%, about 10% to about 90%, about 20% to about 85%, about 30% to about 80%, or about 40% to about 75%. pH and buffered solutionsThe pH of the composition is typically no greater than 8 to allow generation of nitric oxide from the acidification of the nitrite salt component. The specific pH of the composition may depend on the specific application. For example, lower pH values may be acceptable for applications not involving direct contact with physiological surfaces (such as the treatment of inanimate surfaces and physiological applications where the composition is not in direct contact with the physiological environment).Higher pH values are typically used when direct contact of the compositions with the physiological environment is expected.The compositions of the present invention may have a pH in the range of about 2 to about 8. In some embodiments, the composition has a pH in the range of about 3 to about 7.5, for example, about 3.5 to about 7 or about 4.0 to about 6.8.The pH of the composition may depend on the specific use of the composition. In some embodiments, the compositions are for use in the lungs for treatment or prevention of respiratory diseases or disorders and the pH of the compositions may be in the range of about 4 to about 7.5. In other embodiments, the compositions are for use in the treatment or prevention of diseases or disorders associated with the ear or nose of a patient and the pH of the compositions may be in the range of about 2 to about 6. In other embodiments, the compositions are for use in the treatment or prevention of diseases or disorders associated with the oral cavity and / or teeth of a patient and the pH of the compositions may be in the range of about 2 to about 8. In yet further embodiments, the compositions are for use in the treatment or prevention of diseases or disorders associated with the eye of a patient and the pH of the compositions may be in the range of about 4 to about 7.The pH of the composition or an acidic component of the kit may be buffered to control the pH within a known range. The acidic component of the kit or composition may comprise (for example, include or consist essentially of, or consist only of) one or more acids (e.g., one or more organic carboxylic acids) and its conjugate base. The one or more acids and corresponding conjugate base may suitably form a buffer. For example, the one or more acids and corresponding conjugate base may suitably form a buffer in an aqueous carrier (such as an aqueous gel or solution). The conjugate base, where present, may be added separately, or may be generated in situ from the one or more acids by the adjustment of the pH using an acid and / or base, for example, a mineral acid and / or mineral base.In particular embodiments, the compositions and kits may include a buffer formed from one or more organic carboxylic acid and its conjugate base. The compositions and / or kits may comprise a citric acid I citrate buffer. In some embodiments, the one or more organic carboxylic acid is a citric acid / citrate buffer.In some embodiments, the organic carboxylic acid component is a buffer system comprising at least one organic carboxylic acid and at least one conjugate base and water, and wherein the buffer capacity p of the buffer system is at least 0.06 as calculated by the equation (1):(1), where:Kwis the water dissociation equilibrium constant at 25 °C;[H+] is the concentration of hydrogen ions, based on the pH of buffer system;Cbuf is the buffer concentration in the composition; and Ka is the dissociation constant of the acid at 25 °C.In some embodiments, the buffer system above has a pH in the range of 3 to 7.5.In particular embodiments, the buffer capacity of the buffer system is in the range of 0.065 to 0.95, 0.070 to 0.90, 0.075 to 0.85, 0.080 to 0.80, 0.085 to 0.75, 0.090 to 0.70, 0.095 to 0.65 or 0.100 to 0.60 as defined by equation (1). In certain embodiments, the buffer capacity p of the buffer system is in the range of 0.060 to 0.20 as defined by equation (1).The useful range of buffer capacities can be varied depending on the pH of the buffer system to account for a decreasing efficacy of compositions at or above a pH of 7.5. A lower buffer capacity may be used when the pH of the buffer is significantly lower than a pH of 7.5. Meanwhile, a higher buffer capacity may be used when the pH of the buffer is closer to a pH of 7.5. The relevant useful buffer capacity (RUP) may be used to measure the buffer capacity in the context of the present disclosure, where the Rlip is the integral of p with respect to pH between the pH of the buffer system (pHJ) and a pH of 7.50 and p is the buffer capacity of the buffer system as calculated by the equation (1). The Rlip may be expressed as equation (2).In some embodiments, the organic carboxylic acid component is a buffer system comprising at least one organic carboxylic acid, at least one conjugate base and water, wherein the buffer system has a pH in the range of 4 to 7.5, and wherein the relevant useful buffer capacity Rlip of the buffer system is at least 0.04, where the Ru is defined as in equation (2)As the integral of p with respect to pH between the pH of the buffer system (pHJ) and a pH of 7.5 using the trapezoidal method at pH intervals of 0.01 and p is the buffer capacity of the buffer system as calculated by the equation (1):(D. where:Kwis the water dissociation equilibrium constant at 25 °C;[H+] is the concentration of hydrogen ions, based on the pH of the buffer system; Cbuf is the buffer concentration in the composition; and Ka is the dissociation constant of acid at 25 °C.The trapezoidal method of calculating integrals is well known. As an example, the integral from 1 to 2 (on the x axis) at 0.5 intervals would be calculated as follows. The value on the y axis at 1 and the value on the y axis at 1.5 (interval of 0.5) is added together and halved to get an average y axis value. This average value is then multiplied by the interval, 0.5 to give a first area value. This calculation is repeated for the values on the y axis at 1 .5 and 2 to give a second area value. The first and second area values are then added together to give the integral between 1 and 2 using the trapezoidal method at an interval of 0.5. In the present calculation of the Rup, this method is used with the pH values on the x-axis and at intervals of pH of 0.01 and the buffer capacity p on the y-axis.In particular embodiments, the relevant useful buffer capacity Rup of the buffer system is in the range of 0.040 to 0.75 where the Rup is the integral of p with respect to pH between the pH of the buffer system (pHJ) and a pH of 7.5 using the trapezoidal method at intervals of pH of 0.01 and p is the buffer capacity of the buffer system as calculated by the equation (1). In other embodiments, the relevant useful buffercapacity Rup of the buffer system is in the range of 0.0425 to 0.70, 0.045 to 0.65, 0.050 to 0.60, 0.050 to 0.55, or 0.050 to 0.50 where the Rup is the integral of p with respect to pH between the pH of the buffer system (pHJ) and a pH of 7.5 using the trapezoidal method at intervals of pH of 0.01 and p is the buffer capacity of the buffer system as calculated by the equation (1).Further features of the buffer capacity and relevant buffer capacity can be found in PCT / GB2024 / 050390 and PCT / GB2024 / 050391 , the contents of both documents are incorporated herein in their entirety.Improved nitric oxide generating capabilitiesThe present inventors surprisingly found that compositions of the present invention having the components and molar ratio as defined above can be used to tailor nitric oxide generating capabilities.In general, the output of nitric oxide involves an initial rise in NO concentration to a peak. After the peak, the NO concentration typically reduces. However, the extent to which the NO concentration reduces may vary. For example, the NO concentration may stay close to the peak output or reduce only a relatively small amount from the peak output and exist in a steady state (relatively stable NO concentration) for a period of time. Alternatively, the NO concentration can fall significantly from the peak before reaching a relatively low steady state for a period. In some cases, there may be a mixture of a first (relatively high NO output) steady state followed by a drop in the NO output to a second (relatively low NO output) steady state. Without wishing to be bound by theory, it is thought that the one or more organic non-carboxylic acid acts as an accelerator in the compositions for generating nitric oxide from the acidification of nitrite salt.There are several parameters that can define the output of nitric oxide from the acidification of a nitrite salt. Some of these parameters are shown in the schematic of Figure 11. The parameters include:Time taken to reach the peak NO output from the initial reaction;NO concentration at peak output (optionally relative to a control) - see for example, parameter (i) of Figure 11 ;Cumulative NO production within a set time period (e.g., 60 minutes) (optionally relative to a control); this may be calculated, for example, as area under the curve (AUG) of a plot of NO concentration versus time;Time it takes to return to 50% peak concentration after the peak subtracted from the time it takes to initially reach 50% of peak concentration, e.g., full width at half maximum (FWHM) of peak - see, for example, parameter (ii) of Figure 11 ;Time taken to reach a subsequent lower plateau (‘steady state’);NO concentration at the steady state phase - see, for example, parameter (iii) of Figure 11 ;Time to return to the level of a control or baseline - see, for example, parameter (iv) of Figure 11 .The compositions of the present invention having a molar ratio as described above may be used to tailor one or more of these parameters. For example, the combination of the reducing agent and one or more organic carboxylic acid with the molar ratio described herein may have one or more of the following effects in comparison to a corresponding composition without a reducing agent:Increase NO concentration at the peak;Increase time of peak at full width at half maximum (FWHM);Increase the area under the curve for a given time period based on the plot of NO concentration over time.Increase or decrease the steady state NO concentrationIncrease or decrease the duration of the steady state NO concentration.Furthermore, varying relative amounts of one or more of the reducing agent, organic carboxylic acid and nitrite, the concentrations of one or more of the reducing agent, organic carboxylic acid and nitrite, the pH and the type of reducing agent may have an influence on one or more of the parameters. For example, different reducing agents may produce different NO release characteristics. As such, by combination it may be possible to control the duration and strength of the production in the burst and steady state regimes. In this way, it is possible to control the release of nitric oxide to the specific needs of the user.The compositions of the present invention may release nitric oxide for at least 10 minutes, at least 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or more.The compositions of the present invention may further include one or more organic polyol. The expression “organic polyol” herein refers to an organic molecule with twoor more hydroxy groups that is not an acid, particularly for a nitrite salt reaction, and is not a saccharide or polysaccharide (the terms “saccharide” and “polysaccharide” include oligosaccharide, glycan and glycosaminoglycan). The organic polyol will thus have a pKa1 of about 7 or greater.The expression “organic polyol” herein preferably excludes reductants. Examples of reductants which are organic molecules with two or more hydroxy groups and not a saccharide or polysaccharide are thioglycerol (for example, 1 -thioglycerol), hydroquinone, butylated hydroquinone, erythorbic acid and erythorbate. Thioglycerol, (for example, 1 -thioglycerol), hydroquinone, butylated hydroquinone, erythorbic acid and erythorbate are thus excluded from the expression “organic polyol” because they are reductants.The organic polyol may be cyclic or acyclic or may be a mixture of one or more cyclic organic polyol and one or more acyclic organic polyol. For example, the one or more organic polyol may be selected from one or more alkane substituted by two or more OH groups, one or more cycloalkane substituted by two or more OH groups, one or more cycloalkylalkane substituted by two or more OH groups, and any combination thereof. The organic polyol may not carry any substituents other than OH.The one or more organic polyol may be one or more acyclic organic polyol. The one or more acyclic organic polyol may be selected from the sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more acyclic organic polyol may be selected from the alditols, for example the alditols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more organic polyol may not include a saponin, sapogenin, steroid or steroidal glycoside.Alternatively, the one or more organic polyol may be one or more cyclic organic polyol. The one or more cyclic organic polyol may be a cyclic sugar alcohol or a cyclic alditol. For example, the one or more cyclic polyol may be a cyclic sugar alcohol having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms or a cyclic alditol having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. A specific example of a cyclic polyol is inositol.The one or more organic polyol may have 7 or more hydroxy groups. The one or more organic polyol may be a sugar alcohol or alditol having 7 or more hydroxy groups. The one or more organic polyol may have 9 or more hydroxy groups. The one or more organic polyol may be a sugar alcohol or alditol having 9 or more hydroxy groups. Theone or more organic polyol may have 20 or fewer hydroxy groups. The one or more organic polyol may be a sugar alcohol or alditol having 20 or fewer hydroxy groups. The one or more organic polyol may have 15 or fewer hydroxy groups. The one or more organic polyol may be a sugar alcohol or alditol having 15 or fewer hydroxy groups. The one or more organic polyol may have a number of hydroxy groups in the range of 7 to 20, for example, in the range of 9 to 15. The one or more organic polyol may include 9, 12, 15 or 18 hydroxy groups.The one or more organic polyol may be a sugar alcohol compound comprising, for example consisting of one or more monosaccharide units and one or more acyclic sugar alcohol units. The one or more organic polyol may be a sugar alcohol compound 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.A “monosaccharide unit” as used herein refers to a monosaccharide covalently linked to at least one other unit (whether another monosaccharide unit or an acyclic sugar alcohol unit) in the compound. An “acyclic sugar alcohol unit” as used herein refers to an acyclic sugar alcohol linked covalently to least one other unit (whether a monosaccharide unit or another acyclic sugar alcohol unit) in the compound. The units in the compound may be linked through ether linkages. One or more of the monosaccharide units may be covalently linked to other units of the compound through a glycosidic bond. Each of the monosaccharide units may be covalently linked to other units of the compound through a glycosidic bond. The sugar alcohol compound may be a glycoside with a monosaccharide or oligosaccharide glycone and an acyclic sugar alcohol aglycone.Acyclic sugar alcohol units may be sugar alcohol units having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The acyclic sugar alcohol unit may be selected from the group consisting of units of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol and volemitol.One or more of the monosaccharide units may be a Cs or Cs monosaccharide unit, i.e., a pentose or hexose unit. Each monosaccharide unit may be a Cs or Cs monosaccharide unit. One or more of the sugar alcohol units may be a Cs or Cs sugar alcohol unit. Each sugar alcohol unit may be a Cs or Cs sugar alcohol unit.The sugar alcohol compound may comprise, for example may consist of, n monosaccharide units and m acyclic sugar alcohol units, where n is a whole number and at least one, m is a whole number and at least one and (n + m) is no more than 10. The sugar alcohol compound may comprise of a chain of n monosaccharide units terminated with one acyclic sugar alcohol unit, where n is a whole number between one and nine. The chain of monosaccharide units may be covalently linked by glycosidic bonds. Each monosaccharide unit may be covalently linked to another monosaccharide unit or the acyclic sugar alcohol unit by a glycosidic bond. The sugar alcohol compound may comprise, for example may consist of, a chain of 1 , 2 or 3 monosaccharide units terminated with one acyclic alcohol unit. 1 , 2, 3 or each monosaccharide unit may be a Cs or Cs monosaccharide unit. The acyclic alcohol unit may be a Cs or Cs sugar alcohol unit. Examples of the sugar alcohol compound include but are not limited to: isomalt, maltitol and lactitol (n = 1); maltotriitol (n = 2); and maltotetraitol (n = 3).Such sugar alcohol compounds may be described as sugar alcohols derived from a disaccharide or an oligosaccharide. “Oligosaccharide”, as used herein, refers to a saccharide consisting of three to ten monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides may be synthesised (e.g. by hydrogenation) from disaccharides, oligosaccharides or polysaccharides (e.g. from hydrolysis and hydrogenation), but are not limited to compounds synthesised from disaccharides, oligosaccharides or polysaccharides. For example, sugar alcohols derived from a disaccharide may be formed from the dehydration reaction of a monosaccharide and a sugar alcohol. The one or more organic polyol may be a sugar alcohol derived from a disaccharide, trisaccharide or tetrasaccharide. Examples of sugar alcohols derived from disaccharides include but are not limited to isomalt, maltitol and lactitol. An example of a sugar alcohol derived from a trisaccharide includes but is not limited to maltotriitol. An example of a sugar alcohol derived from a tetrasaccharide includes but is not limited to maltotetraitol.Organic polyols may be selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and any combination thereof. Glycerol can be used, and when present is preferably in association with one or more other organic polyol, for example erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, or any combination thereof.In some embodiments, the one or more organic polyol is selected from arabitol, xylitol, mannitol, sorbitol and combinations thereof. In particular embodiments, the one or more organic polyol is mannitol.Many organic polyols contain one or more chiral centre and thus exist in stereoisomeric forms. All stereoisomeric forms and optical isomers and isomer mixtures of the organic polyols are intended to be included within the scope of this invention. In particular, the D and / or L forms of all chiral organic polyols and all mixtures thereof may be used.When the composition or kit is in solid form and includes one or more organic polyols, it is preferred that the organic polyol is added to the composition after any processing which involves removal of solvent (e.g., after spray drying or lyophilisation steps). In other words, the polyol may be added to a composition including one or more particles containing a nitrite salt and an acid; or added to a composition including one or more particles containing a nitrite salt and / or one or more particles containing an acid (either before or after an agglomeration of these particles is formed) may further include one or more organic polyol.The molarity of the organic polyol in the composition may be in the range of 0.001 M to 3.0 M. In some embodiments, the molarity of the organic polyol in the composition is in the range of 0.002 M to 2.0 M, 0.003 M to 1.0 M, 0.005 to 0.5 M, or 0.01 M to 0.2 M.In particular embodiments, the molarity of the organic polyol in the composition is at least 0.005 M, at least 0.008 M, at least 0.01 M, or at least 0.015 M. In particular embodiments, the molarity of the organic polyol in the composition is at most 1.000 M, at most 0.500 M, at most 0.200 M, or at most 0.150 M.Alternatively, the compositions and kits of the present invention may be substantially free of one or more organic polyol.Form of the compositionThe compositions of the present invention may be in any suitable physical form.The composition of the present invention may include a polar solvent, such as a polar solvent. In particular embodiments, the composition of the present invention is contained in an aqueous carrier. For example, the composition may be contained in an aqueous gel or liquid.Polar and polar protic solvents are known to the skilled person. Polar solvents include but are not limited to water, alcohols (such as methanol, ethanol, propanol, butanol, isopropanol), amines (such as ammonia), dimethyl sulfoxide, dimethylformamide, acetonitrile, dichloromethane and acetone. In particular embodiments, the solvent is water.Alternatively, the composition may be in solid form. For example, the composition may be in particulate form such as a powder. The composition in solid form may conveniently be made up into solution before use by addition of water.Form of components of the compositionOne or more component of the composition of the present invention may be contained in a carrier, such as an aqueous carrier. For example, the nitrite salt component and / or the acid component and / or the reducing agent may be contained in a carrier, which may be an aqueous carrier. The aqueous carrier may be an aqueous gel or an aqueous liquid. In some embodiments, the one or more component of the composition is contained in an aqueous gel carrier.In a particular embodiment, the acid component and the nitrite salt component are both contained in aqueous carrier. In this embodiment, the reducing agent component may be included in the acid component. In these embodiments, the nitrite salt component is typically kept separate from the acid component and the reducing agent component until the point of need to prevent acidification of the nitrite salt before required.Alternatively, or in addition to the above, one or more component of the composition of the present invention may be in solid form. In one such embodiment, the composition may conveniently be made up into solution before use by addition of water. For example, the composition may be accompanied by instructions with the volume of aqueous carrier required to be added to form the composition for generating nitric oxide as described herein. In some embodiments, a separate container with afixed volume of aqueous carrier and instructions to add the fixed volume of aqueous carrier to form the composition is provided.In another embodiment, one or more component of the composition may be dispersed in one or more solid or gel carriers, such as one or more disk, paste or gel. The solid, paste or gel may be an inert, biocompatible material such as a hydrogel. In some embodiments, the solid, paste or gel may be injectable or extrudable.Each carrier may be an inert, porous carrier into which the component(s) are infused or dispersed. In one embodiment, the components of the composition may be infused or dispersed into at least two inert carriers, with a first carrier comprising at least a nitrite salt and a second carrier comprising an organic carboxylic acid. In another embodiment, the components of the composition may be infused into at least three inert carriers, with a first carrier comprising at least a nitrite salt, a second carrier comprising an organic carboxylic acid and a third carrier comprising at least one reducing agent. In another embodiment, the components of the composition may be infused or dispersed into separate inert carriers, with a first carrier comprising at least a nitrite salt, and a second carrier comprising an organic carboxylic acid and at least one reducing agent.The separate solid or gel carrier formulations (e.g., flat disks) may be brought into proximity to one another, directly or within a supporting medium. The supporting medium may be a gel into which the solid forms are embedded, or a solution in which the solid forms are placed. In other examples, the separate extrudable carrier formulations are contained within a dual syringe for co-injection.Over time, the components of the composition diffuse from the solid carriers in a controlled release manner, at which point they can react with each other to form nitric oxide.Kits for generating nitric oxideThe present invention also provides a kit for providing a composition for generating nitric oxide, the kit including:(i) a nitrite component including one or more nitrite salts; and(ii) an acidic component including one or more organic carboxylic acids; andwherein the kit further includes one or more reducing agents, and the molar ratio of the total reducing agent or agents in the kit to the total nitrite salt (total RAN molar ratio) or salts in kit is at least about 0.25%, and the molar ratio of any one of the reducing agents present in the kit to the total nitrite salt or salts (RAN molar ratio) in the kit is at most about 75%.When the components are in solid dry powder form, the components may be kept separate or in the same container. When one or more components include an aqueous carrier, the nitrite component is kept separate from the acidic component and / or reducing agent or agents until the point of use. In certain embodiments, the one or more reducing agents will be included in the acidic component.The composition for generating nitric oxide may be as described herein. For example, the composition for generating nitric oxide may include a nitrite salt, a reducing agent, an organic carboxylic acid, a molar ratio, a polyol, a pH and form as described herein. In other words, all optional and preferred features of the compositions described herein are to be equally considered to be optional and preferred features of the kit described herein (unless clearly incompatible with the kit as described).The kit typically comprises two or more components of the composition whereby the nitric oxide generating reaction is prevented from occurring. The parts of the kit are typically held in containers, which may be separate or adapted to facilitate the mixing that would be required to initiate the nitric oxide generating reaction. The critical initiating component for initiating the nitric oxide generating reaction, which needs to be introduced to the other necessary components by a user of the kit, may for example be one of the nitrite salt component, the reducing agent component, the organic carboxylic acid component, or may be an additional ingredient, such as water, which may be supplied by the user.Method of preparing a compositionThe composition may be prepared by any known method in the art. For example, a method of preparing a composition as disclosed herein includes mixing the nitrite salt, the reducing agent and the acid components to provide the composition.In some embodiments, the reducing agent and the acid components are mixed prior to mixing with the nitrite salt component. In this embodiment, if the addition of the reducing agent component to the acid component causes the pH of the resultingmixture to decrease, a mineral base (such as sodium hydroxide) may be added in an amount sufficient to adjust I increase the pH. For example, the pH may be adjusted I increased to the pH of the acid component prior to addition of the reducing agent component. Readjusting I increasing the pH in this way ensures that any changes in nitric oxide production are due to the presence of reducing agent and not due to any changes in the pH of the solution.In some embodiments, an organic polyol (if present) may be added to the nitrite salt component prior to mixing with the reducing agent and the acid components.Pharmaceutical compositionThe composition of one or more components of the kit disclosed herein may be included in a pharmaceutical composition, optionally with one or more pharmaceutically acceptable carriers, excipients and / or adjuvants. Such carriers, excipients and / or adjuvants may be physiologically compatible when desired for use in vivo.Examples of carriers and / or excipients, for example carriers and or excipients that are physiologically compatible, include without limitation lactose, starch, dicalcium phosphate, magnesium stearate, sodium saccharin, talcum, cellulose, cellulose derivatives, sodium croscarmellose, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulphate, calcium chloride, agar, agarose, polaxamers, pectin, alginic acid, alginate salts, and the like.Depending on the intended mode of administration, the pharmaceutical composition will contain about 0.005% to about 95%, preferably about 0.5% to about 50% by weight of the composition of the present invention or components thereof. Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in the art.Excipients may be selected from known excipients depending on the intended use or administration route whereby the reactants and / or reaction products are to be delivered to the target site for the delivery of the nitric oxide. Optional additional components may, for example, be selected from sweetening agents, taste-masking agents, wetting agents, lubricants, binders, emulsifiers, solubilising agents, stabilising agents, colourants, odourants, salts, coating agents, antioxidants, pharmaceutically active agents and preservatives. Such components are well known in the art and adetailed discussion of them is not necessary for the skilled reader. Examples of auxiliary substances such as wetting agents, emulsifying agents, lubricants, binders, and solubilising agents include, for example, sodium phosphate, potassium phosphate, gum acacia, polyvinylpyrrolidone, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate and the like. A sweetening agent or a taste-masking agent may, for example, include a sugar, saccharin, aspartame, sucralose, neotame or other compound that beneficially affects taste, after-taste, perceived unpleasant saltiness, sourness or bitterness, that reduces the tendency of an oral or inhaled formulation to irritate a recipient (e.g., by causing coughing or sore throat or other undesired side effects, such as reducing the delivered dose or adversely affecting patient compliance with a prescribed therapeutic regimen). Certain taste-masking agents may form complexes with one or more of the nitrite salts.Examples of pharmaceutically active agents that may be incorporated in the components and compositions or co-administered with the components and compositions according to the present invention include antibiotics, steroids, anaesthetics (for example topical anaesthetics such as lignocaine (lidocaine), amethocaine (tetracaine), xylocaine, bupivacaine, prilocaine, ropivafacaine, benzocaine, mepivacaine, cocaine or any combination thereof), analgesics, antiinflammatory agents (for example non-steroidal anti-inflammatory drugs (NSAIDs)), anti-infective agents, vaccines, immunosuppressants, anticonvulsants, anti-dementia drugs, prostaglandins, antipyretics, antipsychotics, anti-psoriasis agents, antiviral agents, vasodilators or vasoconstrictors, sunscreen preparations (e.g. PABA), antihistamines, hormones such as oestrogen, progesterone or androgens, antiseborrheic agents, cardiovascular treatment agents such as alpha or beta blockers or Rogaine, vitamins, or any combination thereof.Particular examples include analgesic agents, such as ibuprofen, indomethacin, diclofenac, acetylsalicylic acid, paracetamol, propranolol, metoprolol, and oxycodone; thyroid release hormone; sex hormones, such as oestrogen, progesterone and testosterone; insulin; verapamil; vasopressin; hydrocortisone; scopolamine; nitroglycerine; isosorbide dinitrate; anti-histamines, such as terfenadine; clonidine; nicotine; non-steroidal immunosuppressant drugs, such as cyclosporine, methotrexate, azathioprine, mycophenolate, cyclophosphamide, TNF-a antagonists and anti-IL5, -IL4Ra, -IL6, -IL13, -IL17, -IL23 cytokine monoclonal antibodies; anti-convulsants; and drugs for Alzheimer’s, dementia and / or Parkinson’s disease, such as apomorphine and rivastigmine.Controlling the nitric oxide release capabilityThe compositions according to the present invention may be used in a process for controlling the release of nitric oxide.The present invention includes a method of generating nitric oxide, wherein the method includes the steps of:(i) initiating an initial NO-generating reaction by combining one or more nitrite salts, a polar solvent and one or both of one or more organic carboxylic acids and one or more reducing agents to form an initial NO-generating reaction composition; and(ii) adding or exposing subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents to components of the initial NO-generating reaction composition at one or more subsequent times to form one or more subsequent NO-generating reaction compositions; provided that one or more organic carboxylic acids are present in the initial and / or one or more subsequent reaction compositions and one or more reducing agents are present in the initial and / or one or more subsequent reaction compositions. In other words, both organic carboxylic acid or acids and reducing agent or agents are introduced to the NO-generating reaction composition (initial and / or one or more subsequent reaction compositions) during the method and their introduction may be concomitantly or sequentially (or a combination thereof).The addition or exposure of the subsequent quantities in step (ii) may include the addition of the subsequent quantities from a source external to the initial NO- generating reaction composition or by exposing the subsequent quantities to the components of the initial reaction composition from a source within or adjacent to the initial NO-generating reaction composition where of the subsequent quantities are available or exposed to the NO-generating reaction composition only after a period of time after step i), such as a delayed release mechanism.The introduction or exposure of subsequent quantities of any of the components (nitrite salt, organic carboxylic acid, reducing agent) to the components of the initial reaction composition may be done in a variety of ways, including but not limited toexternal addition or by delayed release from the initial product, such as via such mechanisms as encapsulated particles, or gels to slow mixing, or secondary layers. In this way, the subsequent quantities may be present in the product at the time of the initial reaction, but the quantities are not available to or exposed to the initial reaction mixture. The subsequent quantities may be introduced to the initial reaction mixture.The method above provides three principal components of the NO-generating reaction (nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents) to the reaction mixture in a way that different amounts of each component may be introduced into the reaction at different times. In this way, the release of NO generated from the components may be controlled.For example, the initial reaction mixture may include nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents and subsequent quantities of reducing agent or agents may be introduced. Alternatively, the initial reaction mixture may include nitrite salt or salts and organic carboxylic acid or acids and reducing agent or agents are added in at least one of the subsequent quantities. In this way, both organic carboxylic acid or acids and reducing agent or agents are introduced during the method, whether concomitantly or sequentially.Using this method, the initial NO-generating reaction composition may act as a reservoir of one of the components to which the other components may be added in order to provide subsequent releases of NO after the initial reaction. For example, the initial NO-generating reaction composition may act as a reservoir of nitrite salt or salts. An initial quantity of organic carboxylic acid or acids and reducing agent or agents may be added to form the initial NO-generating reaction composition. Subsequent quantities of organic carboxylic acid or acids and / or reducing agent or agents may then be added to the initial NO-generating reaction composition. Alternatively, the initial NO-generating reaction composition may act as a reservoir of organic carboxylic acid or acids and / or reducing agent or agents. An initial quantity of nitrite salts may be added to form the initial NO-generating reaction composition. Subsequent quantities of nitrite salts may then be added to the initial NO-generating reaction composition.The nitrite salt, organic carboxylic acid and reducing agent are as described herein. The initial NO-generating reaction composition includes a polar solvent as described herein. In particular embodiments, the polar solvent is water. Before the step ofinitiating the initial NO-generating reaction, the nitrite salt or salts may be separated from the organic carboxylic acid or acids and reducing agent or agents. In alternative embodiments, the nitrite salt or salts may be stored in the same container as the organic carboxylic acid or acids and / or reducing agent or agents before the step of initiating the initial NO-generating reaction, provided that the container is absent of polar solvent. In these embodiments, the nitrite salt or salts and organic carboxylic acid or acids and / or reducing agent or agents may be in a solid form.In some embodiments, the initial reaction composition is a composition as described herein.The step of subsequently adding subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents to the initial reaction composition is a discrete step from the step of initiating the initial reaction. The time between these steps however is not particularly limited and can be selected based on the specific need of the user. In particular embodiments, the step of subsequently adding subsequent quantities of any of the components occurs after a reduction in NO production from the initial reaction composition. In certain embodiments, the step of subsequently adding subsequent quantities of any of the components occurs after NO production from the initial reaction composition reaches a steady state.In some embodiments, the step of subsequently adding subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents is repeated one or more times. In this way, the method may include two or more subsequent additions of one or more of the components (subsequently adding subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents) to the reaction composition. Each repetition may add the same components each time or may be different components for different additions. For example, the method may include adding a reducing agent in a first subsequent addition and then adding an organic carboxylic acid in a second subsequent addition. Alternatively, the method may include the addition of a first reducing agent (e.g., ascorbic acid) in a first subsequent addition and a second reducing agent (e.g., sodium thiosulphate) in a second subsequent addition. Further, the amount of each component may vary between subsequent additions.As will be appreciated, step (ii) may be performed once or more than once. Where step (ii) is performed more than once, the first performance of step (ii) may form a first subsequent NO-generating reaction composition and the second performance of step (ii) involves the formation of a second subsequent NO-generating reaction composition by addition of or exposure of the subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and / or reducing agent or agents.The one or more subsequent additions may add the component directly to the reaction mixture. Alternatively, or in addition, the subsequent additions may arise in a time- delayed release from an ingredient of the initial product. Such time-delayed release mechanisms are known. For example, the component may be encapsulated, in a gel or within a secondary layer that slowly releases the component. The component may be added with one or more other additional components. For example, one or more further reducing agents may be added with additional nitrite salts.In a particular embodiment, the initial NO-generating reaction composition is a composition as described herein and the method includes at least two steps of subsequently adding a reducing agent (such as ascorbic acid or sodium thiosulphate) to the reaction composition. These subsequent addition steps are discrete steps as described above. The amount of reducing agent added in each subsequent addition may vary between additions.The two or more subsequent additions may occur at regular time intervals. For example, the two or more subsequent additions may occur every 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours or 12 hours. Alternatively, the two or more subsequent additions may occur at irregular time intervals depending on need.The present invention also includes a method of generating nitric oxide, wherein the method includes combining one or more nitrite salts, a polar solvent, one or more organic carboxylic acids and one or more reducing agents to form a NO-generating reaction composition; wherein at least one of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents is added into the NO- generating reaction composition over a longer time period than at least one of the other of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents.Using this method, the NO-generating reaction composition may act as a reservoir of one of the components to which the other components may be added in order to vary the time of NO release. The nitrite salt, organic carboxylic acid and reducing agent are as described herein. In particular embodiments, the polar solvent is water. In some embodiments, the NO-generating reaction composition forms a composition as described herein.In certain embodiments, one of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents are added to the NO-generating reaction composition for a time period that is longer than the time taken to add the other two of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents. In certain embodiments, the reducing agent or agents are added to the NO-generating reaction composition over a longer period of time than the nitrite salt or salts and the organic carboxylic acid or acids.Alternatively, two of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents are added to the NO-generating reaction composition for a time period that is longer than the time taken to add the other two of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents. In certain embodiments, the reducing agent or agents and organic carboxylic acid are added to the NO-generating reaction composition over a longer period of time than the nitrite salt or salts.The component or components that are being added to the NO-generating reaction composition over a longer period of time than the other component or components, may be added at a fixed rate, a variable rate, or combinations thereof. The specific rate of addition depends on the exact need.For example, the rate at which the component is added may be fixed over a time interval. Alternatively, the rate at which the component is added may be variable over a time interval. The rate at which the component is added may increase or decrease at a linear rate over a time interval. Alternatively, the rate at which the component is added may increase or decrease in a non-linear rate, such as exponentially, over a time interval.In some embodiments, one or more of the components may be added at a fixed weight or fixed volume per time interval. In other embodiments, one or more of thecomponents may be added at an increasing or decreasing weight or volume over a time interval.The rate of addition may be the same over the total addition of any given component. In other embodiments, the component may be added at different rates over different time intervals. The component may be added over two or more time intervals and the rate of addition of the component is different between at least two of the time intervals. In other words, the component may be added a first rate over a first time interval and at a second rate over a second time interval. The first and second rates may be chosen as described above. For example, the component may be added at a linearly increasing rate for a first time interval, followed by a fixed rate for a second time interval. The addition may continue for further time intervals at other rates of addition. In this way, the addition of the component can be controlled to further control the release of NO.Dilution after applicationThe composition of the present invention may be contained in an aqueous carrier. In these embodiments, the compositions may be diluted, e.g., using an additional aqueous carrier after the application of the composition to the area of interest (e.g., area to be treated in a patient or an ex vivo surface to be treated).In some embodiments, the composition may be diluted by a factor of from about 1 / 2 to about 1 / 50. For example, the composition may be diluted by a factor of about 1 / 2, about 1 / 3, about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20 or about 1 / 50. The inventors surprisingly found that the controllable nature of the nitric oxide generating capabilities of the compositions of the present invention were still observed even after dilution.Applications in therapy or surgeryCompositions for generating nitric oxide according to the present invention, and the evolved gas therefrom, have many applications in therapy and surgery, including curative, cosmetic and / or prophylactic therapy, surgery to correct diseases and disorders and conditions, cosmetic surgery, reconstructive surgery, including human and veterinary medicine and surgery.Disclosed herein are the compositions of the present invention for use in therapy or surgery, for example, use in one or more of the medical applications described below.Also disclosed herein are methods of therapy or surgery using compositions of the present invention, for example, in methods of surgery or therapy in one or more of the medical applications described below.VasodilationThe compositions for generating nitric oxide according to the present invention can be used to induce vasodilation.Particular examples of diseases, disorders and conditions responsive to vasodilation include, but are not limited to conditions associated with ischaemia and skin lesions.Conditions associated with tissue ischaemia include Raynaud syndrome, severe primary vasospasm, and tissue ischaemia, for example tissue ischaemia caused by surgery, septic shock, irradiation or a peripheral vascular disease (for example diabetes and other chronic systemic disease).When used in the treatment or prevention of conditions associated with tissue ischaemia as a result of surgery, a composition of the present invention, or nitric oxide gas evolved therefrom may be administered to a subject before, during or after the surgery. The composition or nitric oxide evolved therefrom may be administered to the site of the surgery or in the vicinity of the site of the surgery. Examples of surgical procedures in which this treatment or prevention of tissue ischaemia may be used include transplantation surgery, tissue or organ grafting surgery, coronary surgery, carotid arterial catheterisation, surgery to provide indwelling arterial or venous catheters for administering systemic agents such as chemotherapy drugs, cosmetic surgery procedures including but not limited to a pedicled or rotation flap, repeat surgery where the incision is made at the same site as a prior surgical procedure, surgical operations performed in areas of poor skin and / or poor underlying tissue perfusion or where poor perfusion might be anticipated as a result of concomitant diseases (such as in patients with arteriosclerosis or diabetes mellitus), surgery in cases of trauma in which the blood vessels are damaged or compromised, and surgery to remove or rectify cutaneous or subcutaneous arteriovenous malformations.For example, the composition or nitric oxide gas evolved therefrom may be used in the treatment or prevention of ischemic reperfusion injury of an organ by administering a composition according to the present invention or nitric oxide gas evolved therefrom to an organ. The organ may be one or more selected from the heart (e.g. to preventor treat myocardial ischemia), the brain (e.g. to treat or prevent cerebral ischemia and or an infarction (stroke)), a lung (e.g. to treat or prevent ischemic reperfusion injury of the lung), a kidney (e.g. to treat or prevent ischemic reperfusion injury of the kidney), and a liver (e.g. to treat or prevent ischemic reperfusion injury of the liver). The surgery may be the transplantation of an organ. Administration of the composition or evolved gas may follow an ischemic episode or may be prophylactic.Treatment or prevention of microbial infectionsThe present invention provides compositions, kits and pharmaceutical composition for use in treating or preventing a microbial infection. The present invention also provides a method for treating or preventing a microbial infection, which comprises administering to a subject an antimicrobial amount of a composition, kit or pharmaceutical composition as disclosed herein. The present invention also provides use of a composition, kit or pharmaceutical composition as disclosed herein for the manufacture of a medicament for treating or preventing a microbial infection.The nitric oxide gas evolved from the composition or pharmaceutical composition may have a biocidal or biostatic effect on a potentially wide range of microorganisms, leading to many anti-microbial treatments. The microbes may, for example, be any one or more selected from bacterial cells, viral particles and / or fungal cells, or microparasites, and may be individual cells, organisms or colonies. For example, the microbes may be bacterial cells and viral particles.When the microbe is present in a bacterial infection, a fungal infection, viral or microparasitic infection of a human or other animal, the infection may, for example, be in the context of a disease such as the common cold, influenza, tuberculosis, SARS, COVID-19, pneumonia or measles.The bacterium may be a pathogenic bacterial species. The microbial infection may be an infection caused by a pathogenic bacterial species, including Gram positive and Gram negative, aerobic and anaerobic, antibiotic-sensitive and antibiotic-resistant bacteria.Examples of bacterial species which may be targeted using the present invention include species of the Actinomyces, Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella,Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, or Yersinia genera. Any combination thereof can also be targeted by the present invention.The microbe may be a pathogenic species of Corynebacterium, Mycobacterium, Streptococcus, Staphylococcus, Pseudomonas or any combination thereof.The microbe to be targeted may be selected from Acinetobacter baumannii, 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 diphtheriae, 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 (EHEC), including E. coli O157:H7, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Mycoplasma pneumoniae, Mycobacterium avium, Mycobacterium Kansasii, Neisseria gonorrhoeae, Neisseria meningitidis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus Mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, Tanerella forsythia, Treponema pallidum subspecies pallidum, Treponema denticola, Vibrio cholerae, Yersinia pestis, and any combination thereof.The microbe may be selected from Acinetobacter baumannii, Chlamydia pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Haemophilus influenzae, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium ulcerans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, or any combination thereof.The microbe may be an antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species or an antibiotic-resistant or antibiotic-sensitive strain of a bacterial species. The use of nitric oxide to treat methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) is described, for example, in WO 2002 / 20026, the disclosure of which is incorporated herein by reference. An example of an antibiotic-resistant or antibiotic-sensitive pathogenic bacterial species which may be killed or treated using the present invention is thus methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).The microbe may be a pathogenic fungal species. The microbial infection may be an infection caused by a pathogenic fungal species, including pathogenic yeasts.Examples of fungal species which may be targeted using the present invention include species of Aspergillus, Blastomyces, Candida (for example Candida auris), Coccidioides, Paracoccidiodes, Cryptococcus (in particular, Cryptococcus neofromans or Cryptococcus gattii), Histoplasma, Mucorales, Pneumocystis (for example Pneumocystis jirovecii or carinii), Sporothrix, Talaromyces or any combination thereof.Examples of fungal infections include aspergillosis (such as allergic bronchopulmonary aspergillosis), histoplasmosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, mucormycosis, cryptococcosis and, infections caused by a pathogenic species of Candida, such as candidiasis.The microbe may be a virus particle. The infection may be caused by a pathogenic virus.Examples of viruses which may be targeted using the present invention include influenza viruses, parainfluenza viruses, adenoviruses, noroviruses, rotaviruses, rhinoviruses, coronaviruses, respiratory syncytial virus (RSV), astroviruses, and hepatic viruses. The compositions of the present invention may be used in the treatment or prevention of an infection caused by one of the group selected from H1 N1 influenza virus, infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine parainfluenza-3 virus, SARS-CoV, SARS-CoV-2, and any combination thereof.The invention may be applied to treat a disease or disorder caused by a viral infection (Zelikin and Chandrawati, Adv. Sci. 2021 , 8, 2003895).Respiratory viral infections include influenza, rhinovirus (i.e. common cold virus), respiratory syncytial virus, adenovirus, coronavirus infections, for example, COVID- 19, and severe acute respiratory syndrome (SARS), cytomegalovirus and HSV. Gastrointestinal viral diseases include norovirus infections, rotavirus infections, adenovirus infections and astrovirus infections. 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.The microbe may be a parasitic microorganism (microparasite). The infection may be caused by a pathogenic parasitic microorganism. Examples of parasitic microorganisms which may be targeted using the present invention include protozoa.In particular, the invention may target the protozoa groups of Sarcodina (e.g. amoeba, for example Entamoeba such as Entamoeba histolytica or Entamoeba dispar), Mastigophora (e.g. flagellates, for example Giardia and Leishmania), Ciliophora (e.g. ciliates, for example Balantidium), Sporozoa (e.g. Plasmodium and Cryptosporidium), and any combination thereof.Parasitic infections that may be treated using the present invention include pulmonary malaria, pulmonary amebiasis, pulmonary babesiosis, pulmonary toxoplasmosis and pulmonary leishmaniasis (e.g. mucocutaneous leishmaniasis).The microbe may be a facultative anaerobic organism. The facultative anaerobic organism may be a gram-positive bacterium. The facultatively anaerobic organism may be one or more of Staphylococcus species including Staphylococcus aureus and Staphylococcus mutans, Escherichia coli, Salmonella spp, Listeria spp, and Yersinia pestis.In some embodiments, the microbial infection is a bacterial, viral, fungal, microparasitic infection or any combination thereof. In particular embodiments, the microbial infection is an infection of rhinovirus, SARS-CoV (SARS), SARS-CoV-2 (COVID-19), Mycobacterium tuberculosis (tuberculosis) or influenza.The present compositions may be for use in treating or preventing a respiratory disease or disorder caused by a microbial infection. For examples, the respiratory diseases or disorders caused a microbial infection include, but are not limited to: lung diseases such as viral infections for example influenza, SARS-CoV or SARS-CoV-2, pulmonary arterial hypertension, pulmonary fibrosis of any cause, bronchiectasis of any cause (including cystic fibrosis and non-cystic fibrosis bronchiectasis), interstitial pneumonia of an unknown cause, chronic obstructive pulmonary disease (COPD) (particularly, emphysema, chronic bronchitis), asthma including severe asthma and viral and bacterial induced exacerbations of asthma and refractory (non-reversible) asthma, intra-nasal or pulmonary bacterial infections such as pneumonia, tuberculosis, non-tuberculosis mycobacterial infections, and other bacterial, protozoal and viral lung infections, for example secondary bacterial infections following virus infections of the respiratory tract.The present compositions may be for use in treating or preventing an ophthalmic disease or disorder caused by a microbial infection. Ophthalmic diseases or disorders caused by a microbial infection include, but are not limited to: conjunctivitis such as viral conjunctivitis, bacterial conjunctivitis, gonococcal conjunctivitis and chlamydial conjunctivitis; keratitis such as herpes keratitis, bacterial keratitis, parasitic keratitis (e.g., caused by Acanthamoeba) and fungal keratitis; endophthalmitis such as exogenous endophthalmitis and endogenous endophthalmitis; cellulitis such as preseptal cellulitis and orbital cellulitis; a stye; blepharitis, such as anterior blepharitis and posterior blepharitis; and uveitis.The present compositions may be for use in treating or preventing an oral disease or disorder caused by a microbial infection. Oral diseases or disorders caused by a microbial infection included but are not limited to: tooth decay and / or dental caries; periodontitis; oral herpes; oral thrush; canker sores; hand, foot and mouth disease; and herpangina. In some embodiments, the oral disease or disorder is caused by Staphylococcus mutans, a facultatively anaerobic bacterium found in the oral cavity.The present compositions may be for use in treating or preventing an infectious disease or disorder associated with the ear or ear canal. Infectious diseases or disorders associated with the ear or ear canal include, but are not limited to: an outer ear infection, such as otitis externa; a middle ear infection caused by bacteria, such as Streptococcus pneumoniae or Haemophilus influenzae’, a middle ear infection caused by a virus, such as those associated with viral respiratory infections (e.g.rhinoviruses, parainfluenza, and seasonal coronaviruses); an inner ear infection, such as labyrinthitis and vestibular neuritis.Treatment of genital diseases and disordersThe properties of nitric oxide to induce vasodilation and to kill or prevent the proliferation of microbes have given rise to another important utility of the compositions according to the present invention or the nitric oxide gas evolved therefrom in the treatment of genital diseases and disorders.The present compositions or kits may be for use in treating or preventing an infectious disease or disorder associated with the genitals. The present compositions may also be used for treating or preventing a non-infectious disease or disorder associated with the genitals.The present compositions or kits may be for use in treating or preventing a genital disease or disorder caused by a microbial infection. Genital diseases or disorders caused by a microbial infection included but are not limited to; genital herpes; genital warts; genital candidiasis; genital ulcers; bacterial cystitis; bacterial vaginosis; chlamydia; gonorrhoea; trichomoniasis; molluscum contagiosum; anal yeast infections; anal gland infections.In some embodiments, the microbial infection is a bacterial, viral, fungal, microparasitic infection or any combination thereof. In particular embodiments, the microbial infection is an infection of herpes simplex virus (e.g. HSV-1 , and HSV-2); human papillomavirus (such as HPV types 6 and 11); molluscum contagiosum virus; Candida albicans’, Treponema pallidum-, Haemophilus ducreyi', Gardnerella vaginalis; Chlamydia trachomatis; Neisseria gonorrhoeae; Trichomonas vaginalis; Staphylococcus aureus; Streptococcus; Escherichia coli; and Proteus species.The present compositions or kits may be for use in treating or preventing a genital disease or disorder related to sexual function. Genital diseases or disorders related to sexual function include but are not limited to; erectile dysfunction; female sexual dysfunction; vulvodynia; pelvic inflammatory disease; penile fibrosis; Peyronie’s disease; genital vascular insufficiency.The present compositions or kits may be for use in treating or preventing a non- infectious genital disease or disorder. Non-infectious genital diseases or disordersinclude but are not limited to interstitial cystitis; non-sexually acquired genital ulceration; chronic prostatitis; endometriosis; genital tissue repair.In certain embodiments, the present compositions or kits for generating nitric oxide are for use in the treatment of genital diseases and / or disorders, and methods of generating nitric oxide for use in the treatment of genital diseases and / or disorders using the present compositions or kits.Known compositions for use in treating genital vascular insufficiency and erection dysfunction include topical nitroglycerine. Nitroglycerine is a prodrug and requires a metabolic process to release NO. Nitroglycerine is mainly metabolised by mitochondrial aldehyde dehydrogenase (ALDH2) to release NO. With repeated nitroglycerine exposure ALDH2 can become inactive by oxidative stress, causing tolerance. The metabolism of nitroglycerine may not be efficient in individuals with ALDH2 polymorphisms, ALDH2 deficiency, and in those that have built a tolerance from previous nitroglycerine uses.The compositions according to the present invention overcome the limitations associated with nitroglycerine. The compositions or kits according to the present invention do not require a metabolic process to be converted into an active form. The compositions or kits of the present invention do not rely on ALDH2-dependent activation and therefore may be used by individuals with ALDH2 polymorphisms, ALDH2 deficiency and in those that have developed a tolerance through previous nitroglycerine uses.In some embodiments, the present compositions and / or kits may be applied to the genitals and / or surrounding areas (e.g. anus, thighs, buttocks) in combination one or more of smart hydrogels; hydrogels; liposomal formulations; targeted nanoparticles; nanofiber composites; any other pharmaceutically acceptable treatment (e.g. berdazimer sodium).Treatment of wounds, skin lesions and burnsThe properties of nitric oxide to induce vasodilation and to kill or prevent the proliferation of microbes have given rise to another important utility of the compositions according to the present invention or the nitric oxide gas evolved therefrom in the treatment of wounds, skin lesions and burns.The conditions treatable using the present invention include ulcers, skin donor sites, surgical wounds (post-operative) burns (such as scalds, superficial, partial thickness and full thickness burns), lacerations and abrasions. Wounds may be chronic or acute. Ulcers may be of various origins, such as of arterial or venous origin. Examples of ulcers include leg ulcers, for example chronic leg ulcers or acute leg ulcers, pressure ulcers, for example chronic pressure ulcers or acute pressure ulcers, venous ulcers and ulcers associated with diabetes, such as diabetic foot ulcers.In topical treatments, it is often desirable to cover or protect the treated area of skin while the treatment is being applied. This may assist in preventing contamination of a wound, assist in removing pus or debris from the healing process, prevent or restrict loss of the treatment composition on bathing or showering or through contact with clothing or as a result of a subject’s normal activity, and cushion the treated area against knocks or rubbing.For this purpose, it is common to incorporate the treatment in a topical cream, gel or dressing or dressing system, for example, a wound dressing or dressing system. The dressing, or at least one component part of the dressing system, typically includes a backing sheet which may be water-impermeable or water permeable and may optionally be provided with skin-adherent portions and optionally other layers such as gauze or pad layers.A wound or skin dressing or dressing system comprising a combination or composition according to the present invention is described. The dressing or at least one component of the dressing system comprises a backing sheet and optionally one or more other layer such as, for example, layers selected from gauze and pad layers. The composition according to the present invention may be suitably disposed on the skin-directed side of the backing sheet (e.g., directly or using a hydrogel containing the composition) and arranged so that the desired skin area is treated with the composition, or the nitric oxide gas evolved therefrom when the dressing is applied to the skin. The dressing or dressing system may suitably be provided in a sealed sterile pack before use.Alternatively, the composition according to the present invention may be applied in combination with an existing dressing.Tissue repair and / or regenerationThe properties of nitric oxide to induce vasodilation has given rise to another important utility of the compositions according to the present invention or the nitric oxide gas evolved therefrom in tissue repair and regeneration.In some embodiments, the tissue is tooth, bone, ocular, skeletal, nervous, skin, cardiovascular tissue or any combination thereof. In embodiments, the present compositions and / or kits may be for use in tissue repair and / or regeneration for, but not limited to, muscle tissue repair and / or regeneration; nerve repair and / or regeneration; oral health healing; oral ulcers; tooth healing; osseous regeneration, reconstruction peri-implantis; gum healing such as treatment of periodontitis; and bonding of dental implants to bone.In embodiments, the present compositions and / or kits may be for use in tooth and / or bone repair and / or regeneration. In particular embodiments, the present compositions and / or kits may be for use in bone and / or tooth repair and regeneration following, but not limited to, fractures, and surgical procedures (e.g. dental surgery, bone grafting or implant placement). In further embodiments, the present compositions and / or kits may be for use in bone repair and / or regeneration in a method of treating osteoporosis.In embodiments, the present compositions and / or kits may be for use in nerve repair and / or regeneration. In particular embodiments, the present compositions and / or kits may be for use in nerve repair and / or regeneration in a method of treating glaucoma. In embodiments, the present compositions and / or kits may be for use in treating or preventing corneal infections (bacterial and fungal). Corneal infections can lead to keratitis, and to corneal ulcers, if untreated, and the present compositions and / or kits may be for use in such treatments.In embodiments, the present compositions and / or kits may be for use in cardiovascular tissue repair and regeneration. In particular embodiments, the present compositions and / or kits may be for use in cardiovascular tissue repair and / or regeneration in a method of treating angina.In certain embodiments, the present compositions and / or kits are for generating nitric oxide for use in tissue repair and / or regeneration, and methods of generating nitric oxide for use in tissue repair and / or regeneration using the present composition or kit.In some embodiments, the present compositions or kits may be applied to the tissue (e.g. bone tissue) in combination one or more of smart hydrogels; hydrogels; liposomal formulations; targeted nanoparticles; nanofiber composites; any other pharmaceutically acceptable treatment.Treatment of hair and / or scalp conditionsThe properties of nitric oxide to induce vasodilation and to kill or prevent the proliferation of microbes have given rise to another important utility of the compositions according to the present invention or the nitric oxide gas evolved therefrom in the treatment of hair and / or scalp conditions.NO enhances scalp microcirculation, aiding hair follicle nourishment, stimulation of hair growth and upregulation of follicle growth markers. NO may work in combination with other treatments for the use in hair and scalp conditions, such as minoxidil and berdazimer sodium.The hair and / or scalp conditions treatable using the present compositions or kits include alopecia (e.g. male and female androgenic alopecia; alopecia areata; senescent alopecia; radiation-induced alopecia); telogen effluvium; scalp psoriasis; seborrheic dermatitis; eosinophilic folliculitis; post-transplant healing; pruritus.The hair and / or scalp conditions treatable using the present compositions or kits may be caused by a microbial infection. Hair and / or scalp conditions caused by a microbial infection include but are not limited to seborrheic dermatitis; tinea capitis; folliculitis; candidiasis; impetigo; shingles; pediculosis capitis (head lice).In some embodiments, the microbial infection is a bacterial, viral, fungal, microparasitic infection or any combination thereof. In particular embodiments, the microbial infection is an infection of Staphylococcus aureus’, Trichophyton tonsurans’, Microsporum canis’, Pseudomonas aeruginosa’, Malassezia’, Candida albicans’, Streptococcus pyogenes’, varicella-zoster virus.In certain embodiments, the present compositions and / or kits are for generating nitric oxide for use in the treatment of hair and / or scalp conditions, and methods of generating nitric oxide for use in the treatment of hair and / or scalp conditions using the present compositions or kits.Existing compositions and kits for use in the treatment of hair and / or scalp conditions include transdermal patches; injections; oral supplements; moisture-triggered, pH- responsive, thermal, and photo-triggered systems are less targeted and less practical for NO delivery to the hair and scalp. The compositions according to the present invention the limitation by providing localised, efficient and more targeted NO delivery to the hair and / or scalp.In some embodiments, the present compositions or kits may be applied to the hair and / or scalp in combination with one or more of smart hydrogels; hydrogels; liposomal formulations; targeted nanoparticles; nanofiber composites; any other pharmaceutically acceptable treatment (e.g. minoxidil, berdazimer sodium).Methods of administrationThe compositions of the present invention are typically administered to a subject shortly after the three constituents (nitrite salt, reducing agent and acid) are combined in the presence of, for example, water. In this way, the subject may be exposed to the reaction products of the acidification of the nitrite shortly after the reaction is initiated. Alternatively, the compositions may be formed in-situ. For example, one or more of the three constituents above may be added to a wound dressing that has already been applied such that the composition is formed in the wound dressing in-situ.The compositions or kits described herein may include the reducing agent, and / or the acid and / or the nitrite salt as separate components, particularly if one or more of the reducing agent, acid and nitrite salt are provided as an aqueous solution. Alternatively, the reducing agent, acid and nitrite salt may be solid powders and maybe admixed in the composition or kit. In these embodiments, the composition or kit includes instructions with the volume of aqueous media (e.g., water) to be added to the admixed solid powder. In some embodiments, the composition or kit includes a separate container with a fixed volume of aqueous media and instructions to add the fixed volume of aqueous media.Prior to administration, the nitrite salt, acid, reducing agent and aqueous media may then be combined to provide the composition of the present invention. The resulting composition may then be administered to the subject.In particular embodiments, the composition is administered to a subject by inhalation of an aerosol of the composition (e.g., by nebulisation).Alternatively, the compositions of the present invention may be administered to a subject in powdered form after the three constituents (nitrite salt, reducing agent and acid) are combined.The subject may be an animal or human subject. The term “animal” herein generally can include human; however, where the term “animal” appears in the phrase “an animal or human subject” or the like, it will be understood from the context to refer particularly to non-human animals or that the reference to “human” merely particularises the option that the animal may be a human to avoid doubt.The subject may be a human subject. The human subject may be an infant or adult subject.The subject may be a vertebrate animal subject. The vertebrate animal may be in the Class Agnatha (jawless fish), Class Chondrichthyes (cartilaginous fish), Class Osteichthyes (bony fish), Class Amphibia (amphibians), Class Reptilia (reptiles), Class Aves (birds), or Class Mammalia (mammals). The subject may be an animal subject in the Class Mammalia or Aves.The subject may be a domestic species of animal. The domestic species of animal may be one of:- commensals, adapted to a human niche (e.g., dogs, cats, guinea pigs) prey or farm animals sought or farmed for food (e.g., cows, sheep, pig, goats); and animals for primarily draft purposes (e.g., horse, camel, donkey)Examples of domestic animals include, but are not limited to: alpaca, addax, bison, camel, canary, capybara, cat, cattle (including Bali cattle), chicken, collared peccary, deer (including fallow deer, sika deer, thorold’s deer, and white-tailed deer), dog, donkey, dove, duck, eland, elk, emu, ferret, gayal, goat, goose, guinea fowl, guinea pig, greater kudu, horse, llama, mink, moose, mouse, mule, muskox, ostrich, parrot, pig, pigeon, quail, rabbit, rat (including the greater cane rat), reindeer, sheep, snakes, turkey, water buffalo, yak and zebu.In vitro antimicrobial treatments of surfacesThe components and compositions of the present disclosure, and the evolved gas from the nitric oxide generating reaction according to the present disclosure, may be used to apply antimicrobial treatments in vitro. By “in vitro" it is meant that the surface being treated is not a living organism.Examples of such utility include methods for sterilising surgical instruments, hypodermic needles and other medical devices before use, as well as cleaning or treatment of surfaces, whether in a hospital or clinic or anywhere else, to reduce or prevent the spread of a pathogen.Other examples include methods for sterilising prostheses and implantable devices such as stents (for example coronary stents), surgical screws, rods, plates and splints, orthopaedic implants, cardiac pacemakers, insulin infusion devices, catheters, ostomy appliances, intraocular lenses, cochlear implants, electrical pain-reducing implants, implantable contraceptive devices, neurostimulators, artificial heart valves, electrodes, intravenous drips and drug delivery devices, and the like before locating the device within a subject’s body. Other examples include methods for sterilising sponges (such as gauze sponges). In this context, the term “surface” covers the surface of the interconnected voids of cells dispersed throughout the sponge.If desired, the components or compositions of the present disclosure may be coated onto the surface of the prosthesis or implantable device. Compositions and methods for more general antimicrobial treatment of inanimate surfaces are well known in the art and do not require extensive description here. Antibacterial compositions are used, for example, in the health care industry, food service industry, meat processing industry and in the private sector by individual consumers. Antibacterial cleansing compositions typically contain one or more active antibacterial agent or components thereof, a surfactant, and one or more other ingredients, for example dyes, fragrances, pH adjusters, thickeners, skin conditioners and the like, in an aqueous and / or alcoholic carrier. Broad spectrum antiseptic or antimicrobial compositions aim to reduce the pathogen load of a range of pathogens on a surface. Typically, the composition is a liquid (or is made up to be a liquid from a solid pre-mix prior to use), the liquid - after any desired adjustment of concentration, suitably by addition of water - being spread or sprayed onto a surface to be treated, often with the aid of a cloth or other wiping device, and may then be left to dry on or wiped off.The conventional compositions and methods of treatment of surfaces are in principle applicable to be used with the present invention, whereby the active antimicrobial agent is or comprises the composition for generating nitric oxide or the components thereof according to the present invention.For further discussion and examples of known antimicrobial compositions and methods of use which may be used in association with the present invention, we refer for example to US Patents 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 Applications 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.The present invention provides a method of treating a surface or space, for example an inanimate surface or space, to reduce the number of viable microbes on the surface or in the space, which comprises applying to the surface or the space or to the vicinity thereof an antimicrobial amount of the composition described herein.Agrochemical applicationsIn one aspect, the present invention provides a composition or kit as described herein for generating nitric oxide to regulate plant or plant product quality, use of a composition or kit as described herein for generating nitric oxide to regulate plant or plant product quality, and methods of generating nitric oxide to regulate plant or plant product quality using a composition or kit as described herein.In certain embodiments, disclosed herein is a composition or kit as described herein for generating nitric oxide to regulate plant-based food quality, use of a composition or kit as described herein for generating nitric oxide to regulate plant-based food quality, and methods of generating nitric oxide to regulate plant-based food quality using a composition or kit as described herein. The plant-based food may be a whole plant-based food or a processed plant-based food.In certain embodiments, disclosed herein is a composition or kit as described herein for generating nitric oxide to regulate postharvest fruit, vegetable, salad or pulses quality, use of a composition or kit as described herein for generating nitric oxide to regulate postharvest fruit, vegetable, salad or pulses quality, and methods ofgenerating nitric oxide to regulate postharvest fruit, vegetable, salad or pulses quality using a composition or kit as described herein.In particular, the compositions or kits as described herein may be included in proximity to fruit, vegetable, salad or pulses post-harvesting. In this way, the compositions or kits may help to extend fruit, vegetable, salad or pulses shelf-life after harvest. As NO has the capability of suppressing the ethylene production, this would result on reducing the oxidation stress of the plant species (Da Veiga et al., Nitric oxide, 2024, 142, 26-37).This invention offers a wide coverage on how the NO could be delivered, especially since different NO dosages are generally required depending on plant species and their application methods.The compositions or kits as described herein may also be included in proximity to fruit, vegetable, salad or pulses during transport, storage, on the shelf and / or in a consumer’s domicile.Uses of compositions or kits for generating nitric oxideThe present invention also provides use of a composition or kit as disclosed herein for generating nitric oxide.The compositions or kits of the present invention having a molar ratio as defined above show improved nitric oxide generating capabilities. For example, the compositions of the present invention provide a sharp initial burst of nitric oxide followed by a slow decrease in nitric oxide concentration, a high steady-state nitric oxide concentration and a high cumulative nitric oxide concentration.Accordingly, the present invention also provides a method of improving the nitric oxide generating capabilities of a composition. The method comprising adding a reducing agent and an acid as disclosed herein in a molar ratio as disclosed herein to a composition comprising a nitrite salt.EXAMPLESThe following non-limiting examples are provided for further illustration of the present invention.General informationStock solutionsStock solutions of citric acid (Fisher, C / 6200 / 60, 2368300), sodium nitrite (Supelco, 1.06549.0500, A1842349317), D-mannitol (ThermoScientific, 125345000, A0452980), sodium hydroxide (2M in wt, J / 7660 / PB15, 2359191), sodium thiosulphate (ThermoScientific, A17629.36, 10249057) and L-ascorbic acid (ThermoScientific, A15613.22, 10241930) were prepared by dissolving the appropriate mass in deionised water (< 18.2 M ) obtained from water system, Elga Veolia.Electrochemistry measurementsThe electrochemistry measurements (to measure NO output) were performed using an Apollo 4000 Free Radical Analyzer (World Precision Instruments (WPI), Inc., Sarasota, FL), with a 2 mm platinum disk nitric oxide electrode (ISO single bond NOP, WPI). The nitric oxide electrode was calibrated daily by the addition of known amounts of NaNO2 to a KI / H2SO4 calibration solution. Calibration curves were obtained by plotting the changes in current (pA) against the changes in Nitric oxide concentration (nM).Slow addition with a Syringe pumpThe slow addition of reactants was performed using a syringe pump (Aladdin AL- 1000, WPI) which has a programmable single channel infusion I withdrawal syringe pump. It is controlled with a microcontroller-based system which drives a step motor, allowing a large range of pumping rates configured to the inside diameter of the loaded syringe. The syringe is driven with a drive-screw and drive-nut mechanism. It can run various programs with up to 41 pumping phases and can be set to change pumping rates and set dispensing volumes.Antimicrobial activityAntimicrobial activity of our formulations was tested, independently, at Imperial College London’s Bioengineering Core facilities by the Deputy Technical Operations Manager via Imperial Consultants.Pseudomonas Aeruginosa and Staphylococcus Aureus from glycerol stocks were grown separately in Mueller-Hinton broth (Merck, 102671723, BCCK2277) (MHB) overnight at 37°C in a 100 mL conical flask in a shaking incubator at 200 rpm. The bacteria were sub-cultured in a 1 :10 dilution, 1 mL of culture in 9 mL of MHB, for 1-2 h until they reached an optical density (OD) value of around 0.5. Next, for the serial decimal dilutions, 100 pL of bacterial culture was added to Mueller-Hinton agar (MHA) plates and spread using a bacterial spreader. The plates were incubated at 37°C in a static incubator until clear colonies were formed to be able to count CFU / mL. Single colonies for all the bacteria strains were picked using a seeding loop and added to 100 mL MHB. The biological culture was then agitated in a 37°C incubator overnight.Formulation preparations and control are as indicated above for 1x concentration.3 mL of the 1x formulations and control were mixed with 3 mL MHB to achieve 0.5x concentrated solutions. 6 mL of ~1.3 x 107CFU / mL of bacterial culture was mixed with 2 mL of either the 1x or 0.5x concentrated solutions for a final concentration of 0.25x and 0.125x, respectively. The negative control used was MHB. Incubation was done at 37°C in an agitating incubator at 180 rpm using round bottom tubes. Aliquots of 1 mL were taken at the determined time points by the experiment (1 h, 2 h and 4 h) Serial dilutions were performed for each time point using MHB and seeded on MHA as detailed earlier. And finally, a visual count of colonies and pictures of the plates was done after 16 hours.Example 1Control FormulationA formulation comprising sodium nitrite, a citric acid / citrate buffer and mannitol was used as a control formulation. In the control formulation, the nitrite was present in a concentration of 0.15 M, the citric acid / citrate buffer was present in a concentration of 0.1 M and the mannitol was present in a concentration of 0.05 M. The pH of the control formulation was 5.5.Addition of Ascorbic AcidThe effect of adding ascorbic acid to the control formulation was examined. The acidity and pH of 10 different concentrations of ascorbic acid in a citric acid / citrate buffer (0.2 M, pH 5.5) were monitored to investigate the pH change.No significant change in pH value was observed upon addition of ascorbic acid in concentrations less than or equal to 5.68 mM. From 11.36 mM of ascorbic acid, the pH of the solution started to decrease more significantly, indicating that ascorbic acid acidifies the formulation. However, before addition of nitrite, these solutions were readjusted by adding the required amount of sodium hydroxide (NaOH) to reach pH 5.5. This was to make sure that any changes in nitric oxide production were due to the presence of the ascorbic acid and not due to any changes in the pH of the solution.Each of the Formulations of Table 1 was then added in equal measure to a solution of nitrite [0.3M] and mannitol [0.1M] so that post-mix the overall concentrations were Citric / Citrate [0.1M], Nitrite [0.15M] and mannitol [0.05M], The formulations were analysed by electrochemistry. The results are shown in Table 1 below as well as Figure 1.Table 1a High signal / noise ratio in the initial increase, made this value hard to determine.Time it takes to reach 50% peak concentration subtracted from the time it takes to return to 50% of peak concentration, full width at half maximum (FWHM).Figure 1 shows a logarithmic plot of NO concentration against time for Formulations 4 to 10 and the control. The figure shows a sharp increase of maximal nitric oxide concentration after ascorbic acid addition for Formulations 4 to 10 compared to the control. A similar maximum nitric oxide concentration of - 1900 times the control was observed for Formulations 8,9 and 10. The figure also shows that for any arbitrary threshold of NO concentration between -10 to -1000 times the control, the time for which the concentration remained above that level showed a strong trend to increase with increasing ascorbic acid concentration (from Formulations 4 to 10).As shown in Table 1 , a positive relationship is observed between ascorbic acid concentration and tpwHM. Furthermore, it can be observed that the peak concentration of nitric oxide increases as ascorbic acid increases from about 0 to 3 mM and that the peak concentration of nitric oxide is approximately the same for concentrations of ascorbic acid above 3 mM.These results demonstrate that addition of ascorbic acid leads to a sharp increase of nitric oxide compared to the control formulation. The results also demonstrate that the concentration of ascorbic acid added is positively correlated with the time and size of the response.Example 2A range of formulations based on the control formulation from Example 1 were prepared by varying the proportions of ascorbic acid to citric acid. The following formulations were prepared: 100% citric acid (which corresponds to the control formulation of Example 1); 0.1 % ascorbic acid; 0.5% ascorbic acid; 1 % ascorbic acid; 2% ascorbic acid; 5% ascorbic acid; 10% ascorbic acid; 20% ascorbic acid; 50% ascorbic acid; 100% ascorbic acid. % ascorbic acid refers to the molar ratio of ascorbic acid to citric acid (RAA molar ratio), which is calculated according to the formula [ascorbic acid] / [citric acid] + [ascorbic acid]. It is noted that only the proportion of ascorbic acid to control was altered. Other variables such as nitrite concentration and pH were kept constant.The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The results are shown in Table 2 below and in Figure 2.Table 2Total acid in these formulations is the sum of [citric acid] + [ascorbic acid].The 0% [ascorbic acid] I [nitrite] formulation provided a control. Figure 2 shows that the 66.7 % [ascorbic acid] I [nitrite] formulation provided a cumulative nitric oxide value of 363x larger than the control. Surprisingly, formulations containing lower ascorbic acid concentrations provided a cumulative nitric oxide value that was even higher than the 66.7 % [ascorbic acid] I [nitrite] formulation. The same surprising effect was seen in the peak NO concentrations. In both peak and cumulative NO measurements, the highest values were observed roughly in the 1.3% to 33% [ascorbic acid] / [nitrite] range.Figure 3 shows the cumulative NO concentration over time for the above formulations, showing the surprising feature that solutions containing 1.3%, 3.3%, 6.7%, 13.3% and 33.3 % [ascorbic acid] / [nitrite] resulted in greater overall NO production than the solution containing 66.7% [ascorbic acid] / [nitrite] .Example 3The effect of adding sodium thiosulphate to the control formulation was examined.A range of formulations based on the control formulation from Example 1 were prepared by varying the proportions of sodium thiosulphate to nitrite salt. The following formulations were prepared: control (which corresponds to the control formulation of Example 1); 2% sodium thiosulphate, 4% sodium thiosulphate, 9% sodium thiosulphate, 19% sodium thiosulphate, 41% sodium thiosulphate and 83% sodium thiosulphate. % sodium thiosulphate refers to the molar ratio of sodium thiosulphate to nitrite salt (RAN molar ratio), which is calculated according to the formula [sodium thiosulphate] / [nitrite salt]. It is noted that only the proportion of sodium thiosulphate to control was altered. Other variables such as nitrite concentration and pH were kept constant.Table 3The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The results are shown in Figures 4 to 6.Figure 4 shows that the nitric oxide concentration generated was higher for all formulations containing sodium thiosulphate compared to the control. Generally, the initial burst as well as the steady state nitric oxide concentration increased as the % thiosulphate was increased. However, after about 2500 seconds, the nitric oxide concentration was higher for the formulation with 19% sodium thiosulphate compared to the formulations with 41% sodium thiosulphate and 83% sodium thiosulphate.Figures 5 and 6 show that the cumulative nitric oxide concentration, over the first 60 minutes, was higher for all formulations containing sodium thiosulphate compared to the control. From 2% to 19% sodium thiosulphate, increasing the % sodium thiosulphate provided higher cumulative nitric oxide concentrations. However, the cumulative nitric oxide concentration in the first 2000 seconds was broadly similar for the formulations with 19% sodium thiosulphate and 41% sodium thiosulphate, albeit lower for the 83% sodium thiosulphate formulation.Example 4The effect of pH on the addition of ascorbic acid to the control formulation was investigated. pH 5.5, 6.6 and 7.3Table 4 shows the effect of adding concentrations of ascorbic acid to a formulation similar to the control formulation of Example 1, at pH 6.6. The nitric oxide generatedby the formulations was measured by the same method employed in Example 1. The results are shown below.Table 4ND - not detected; N / A - not applicableTable 4 compares the addition of concentrations of ascorbic acid to the control formulation of Example 1 at pH 5.5, 6.6 and 7.3. The results show that adding 0.14 mM to 5.68 mM of ascorbic acid at pH 6.6 led to an increase of nitric oxide concentration. Higher concentrations of ascorbic acid also gave a longer lasting response.The testing was also performed at a pH of 7.3 and ascorbic acid concentration of 1.42 mM (all other factors being the same). The peak [NO] relative to control at pH 5.5 is 1 , the tpwHM (s) is 115,000, the AUG first hour (mMs) is 6 and the AUG first 2 hours (mMs) is 13. The results also show that even at pH of 7.3, the addition of ascorbic acid still produces nitric oxide at a similar level as the control (without ascorbic acid) at pH 5.5. The formulations therefore continue to generate effective amounts of nitric oxide at physiological pH’s.The results demonstrate that in the presence of ascorbic acid it is possible to generate nitric oxide at higher pHs, with the nitric oxide concentration at pH 6.6 exceeding the production of the control formulation.Example 5Formulations were prepared and tested to demonstrate the influence of ascorbic acid on the production of NO over time in formulations both with and without mannitol. The formulations were prepared as follows:0.3 mM, 1.4 mM or 2.8 mM of ascorbic acid was added to 10 mL of citrate buffer (0.2 M). The ascorbic acid / citrate buffer solution was then added to a 10 mL solution of sodium nitrite (0.3 M) with or without mannitol (0.1 M), resulting in formulations with ascorbic acid concentrations of 0.15 mM, 0.7 mM or 1.4 mM. The pH of the formulations was 5.5. The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The results are shown in Figures 7a and 7b.The ascorbic acid concentration significantly impacts the production of nitric oxide (NO) in both formulations containing mannitol and those without. As such, the influence of the ascorbic acid on the NO production is demonstrated in both formulations including a polyol (such as mannitol) and without a polyol.Example 6The effect on the generation of nitric oxide of adding successive additions of ascorbic acid to the compositions of the present invention is shown in Figure 8. 20 mL solution of the following formulation was used: nitrite was present in a concentration of 0.15 M, citric acid / citrate buffer was present in a concentration of 0.1 M, initial ascorbic acid was present in a concentration of 0.028 mM and mannitol was present in a concentration of 0.05 M. The pH of the formulation was 5.4. A 0.4mM ascorbic acid stock solution was used for subsequent ascorbic acid additions.The components were mixed to provide the initial generation of nitric oxide. Subsequently, 1 .4 mL of ascorbic acid stock was added to the mixture at around 300 seconds to increase the ascorbic acid concentration by 0.026 mM. A further 1.4 mL of ascorbic acid stock was added to the mixture at around 500 seconds to increase the ascorbic acid concentration by 0.024 mM. A further 0.714 mL of ascorbic acid stock was added to the mixture at around 650 seconds to increase the ascorbic acidconcentration by 0.012 mM. A final 0.36 mL of ascorbic acid stock was added to the mixture at around 800 seconds to increase the ascorbic acid concentration by 0.006 mM. The nitric oxide generated by the formulations was measured by the same method employed in Example 1.Adding successive amounts of reducing agent produces successive bursts of nitric oxide without the need for additional nitrite (as shown in figure 8).Example 7The antimicrobial activity of the compositions on Pseudomonas aeruginosa and Staphylococcus aureus was tested.Formulation F1 contains 0.22M Nitrite, 0.15M Citrate, 0.005 Mannitol at pH 5.0. To show the effect of including a reducing agent, variations of this formulation were used that include ascorbic acid (AA, 11 mM and 28 mM), sodium thiosulphate (STS, 28 mM) and a combination of ascorbic acid and thiosulphate (both at 28 mM).Figure 9 shows that the colony forming unit / mL (CFU / mL) count of Pseudomonas aeruginosa with formulations containing citric acid / citrate with additional ascorbic acid is lower than the formulations without additional ascorbic acid (noting that the CFU / mL is provided on a logarithmic scale). The ascorbic acid is added in two concentrations, 11 mM and 28 mM. The increased concentration of ascorbic acid shows a reduction in the CFU / mL counts for Pseudomonas aeruginosa. As such, there appears to be a dose response. The data are provided in Table 5 below.Table 5Figure 10 shows the colony forming unit / mL (CFU / mL) of Pseudomonas aeruginosa with formulations containing citric acid / citrate without mannitol. Formulation F2 contains 0.22M Nitrite, 0.15M Citrate at pH 5.0. To show the effect of including a reducing agent, a formulation was used including ascorbic acid (AA, 28 mM). Thegraph shows that the addition of AA also reduces the CFU / mL count in formulations without mannitol. The data are provided in Table 6 below.Table 6Table 7 below reports the antimicrobial activity of our formulation with additional ascorbic acid or both additional ascorbic acid and additional sodium thiosulphate (STS)).The formulation containing 28 mM ascorbic acid (AA) and 28 mM sodium thiosulphate (STS) was found to be more potent against Pseudomonas aeruginosa, below the detection threshold. Although STS has been widely accepted as a reducing agent for nitrogen species with oxidation states of III and IV, STS is also viewed either as a source of hydrogen sulfide (H2S) or a signalling molecule for hydrogen sulfide (Xie et al, Nitric oxide, 2024, 149, 67-74). Along with nitric oxide, H2S is an endogenous gasotransmitter and is known to regulate different cellular biological systems (Bairagi et al. Appl. Nano Mater. 2023, 6, 19727-19739). Thus, using STS as a reducing agent could have a dual therapeutic effect with the potential formation of two gasotransmitters, nitric oxide and hydrogen sulfide.Table 7Lower Limit of detection (LLD)Preliminary results from antimicrobial testing of Staphylococcus aureus with formulations containing AA, STS or a combination of AA and STS showed a reduction in the Log 10 (CFU / mL).Example 8A nitrite solution (10 mL of 0.30 M NaNCh + 0.1M mannitol) was added to a citrate solution (10 mL of 0.2M pH 5.4 citrate buffer) (“control”). Then, solutions of AA / STS (adjusted to pH = 5 with NaOH) at various molar ratios (25:75, 50:50 and 75:25 AA:STS) was added to the formulation.The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The resulting NO-generation profile is shown in Figure 12.Example 9A fixed amount of AA (46 mg) was added to acidified nitrite (10 mL of 0.44 M NaNCh + 0.01 M mannitol and 10 mL of citrate buffer 0.3M (pH 5.0)) at three different rates (1.15 mg per hour, 2.3 mg per hour and 4.6 mg per hour).The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The resulting NO-generation profile is shown in Figure 13.At the high rate of 4.6 mg / h, added for 10 hours, the NO concentration reached a plateau of approaching 100x control, and faded after the 10 hours. At the medium rate of 2.3 mg / h for 20 hours, the plateau was in the 20 to 50 range, and faded after the 20 hours. At the low rate of 1.15mg / h for 40 hours, the plateau was around 10 x control, and faded after 40 hours.Example 10Two samples were taken and compared. The first sample started with acidified nitrite (10 mL of 0.44 M NaNCh + 0.01 M mannitol and 10 mL of citrate buffer 0.3M (pH 5.0)) and adding AA (40 mg in 1 mL, over 10 hours. The second sample started with nitrite (10 mL, 0.44 M) alone and adding slowly the AA / Citrate buffer (10 mL of citrate buffer 0.3M (pH 5.0) + 40 mg of AA) at an addition rate of 1 mL / h.The nitric oxide generated by the formulations was measured by the same method employed in Example 1. The resulting NO-generation profile is shown in Figure 14.Example 11The effect on the generation of nitric oxide of adding successive additions of iron (II) sulphate heptahydrate was investigated.A 20 mL solution (containing 0.15 M nitrite + 0.10 M citric acid / citrate buffer + 0.05 M mannitol) was used. The initial pH of the formulation was 5.4. The components were mixed to provide the initial generation of nitric oxide. A 0.18 mM iron (II) sulphate heptahydrate stock solution was used.100 pL of iron (II) sulphate heptahydrate stock was added to the mixture at around 300 seconds to increase the iron (II) sulphate heptahydrate concentration by 0.9 pM. A further 200 pL of iron (II) sulphate heptahydrate stock was added to the mixture at around 530 seconds to increase the iron (II) sulphate heptahydrate concentration by 1.6 pM. A further 400 pL of iron (II) sulphate heptahydrate stock was added to the mixture at around 930 seconds to increase the iron (II) sulphate heptahydrate concentration by 3.6 pM. A further 800 pL of iron (II) sulphate heptahydrate stock was added to the mixture at around 1230 seconds to increase the iron (II) sulphate heptahydrate concentration by 6.6 pM. A final 1.6 mL of iron (II) sulphate heptahydrate stock was added to the mixture at around 1580 seconds to increase the iron (II) sulphate heptahydrate concentration by 12.3 pM.The nitric oxide generated by the formulations was measured by the same method employed in Example 1. Adding successive amounts of iron (II) sulphate heptahydrate produces successive bursts of nitric oxide without the need for additional nitrite (as shown in Figure 15).Example 12The effect on the generation of nitric oxide of adding DL-cysteine was investigated.A 20 mL solution (containing 0.22 M nitrite + 0.15 M citric acid / citrate buffer + 0.005 M mannitol) was used. The pH of the formulation was 5.0. The components were mixed to provide the initial generation of nitric oxide. An 82.6 mM DL-cysteine stock solution was used for DL-cysteine addition.300 pL of DL-cysteine stock was added to the mixture at around 1000 seconds to increase the DL-cysteine concentration by 1.22 mM . The nitric oxide generated by the formulations was measured by the same method employed in Example 1. Adding DL- cysteine produces a burst of nitric oxide without the need for additional nitrite (as shown in Figure 16).Example 13The effect on the generation of nitric oxide of adding glutathione was investigated.A 20 mL solution (containing 0.22 M nitrite + 0.15 M citric acid / citrate buffer + 0.005 M mannitol) was used. The pH of the formulation was 5.0. The components were mixed to provide the initial generation of nitric oxide. The components were mixed to provide the initial generation of nitric oxide. A 65.1 mM glutathione stock solution was used for glutathione addition.470 pL of glutathione stock was added to the mixture at around 200 seconds to increase the glutathione concentration by 1.50 mM. The nitric oxide generated by the formulations was measured by the same method employed in Example 1. Adding glutathione produces a burst of nitric oxide without the need for additional nitrite (as shown in Figure 17).Example 14The effect on the generation of nitric oxide of adding penicillamine to the compositions of were investigated.A 20 mL solution (containing 0.22 M nitrite + 0.15 M citric acid / citrate buffer + 0.005 M mannitol) was used. The pH of the formulation was 5.0. The components were mixed to provide the initial generation of nitric oxide. A 67.0 mM penicillamine stock solution was used for penicillamine addition.300 pL of penicillamine stock was added to the mixture at around 450 seconds to increase the penicillamine concentration by 0.99 mM. The nitric oxide generated by the formulations was measured by the same method employed in Example 1 . Adding penicillamine produces a burst of nitric oxide without the need for additional nitrite (as shown in Figure 18).Example 15The effect on the generation of nitric oxide of adding dithiothreitol was investigated.A 20 mL solution (containing 0.22 M nitrite + 0.15 M citric acid / citrate buffer + 0.005 M mannitol) was used. The pH of the formulation was 5.0. The components were mixed to provide the initial generation of nitric oxide. A 28.4 mM dithiothreitol stock solution was used for dithiothreitol addition.Successive volumes (100 pL, 100 pL, 50 pL and 200 pL) of dithiothreitol stock were added to the mixture to increase the dithiothreitol concentration by up to 0.63 mM. The nitric oxide generated by the formulations was measured by the same method employed in Example 1. Adding increasing amounts of dithiothreitol produces larger nitric oxide concentrations without the need for additional nitrite (as shown in Fig. 19).Example 16The antimicrobial activity of Formulation F1 in the presence of supported silica was investigated.A 1 mL solution of formulation F1 was used (F1 : 0.22 M nitrite + 0.15 M citric acid / citrate buffer + 0.005 M mannitol). The initial pH of the formulation was 5.0.• The supported silica-components (20 mg) were pre-suspended with formulation F1 for 30 minutes at 37°C then diluted with 9 mL of Pseudomonas aeruginosa inoculum at 108colony forming units / mL (CFU / mL). Figure 20 shows that the colony forming units / mL (CFU / mL) count of Pseudomonas aeruginosa with formulations containing silica supported cysteine (Cysteine / SiC>2) or 3-mercaptopropyl-functionalized silica gel (Mercap / SiCh) is lower than the control formulation after 24 hrs of treatment. After 48 hrs, there is a significant reduction with these silica-supported materials. As such, the graph shows that the addition of these functionalized silica materials reduces the CFU / mL count in formulations after a longer exposure time (as shown in Figure 20 and Table 8). With regard to Figure 20, the empty box with an arrow signifies the LCD (Limit of Detection) at this dilution, as no CFU were detected.Table 8Example 17Various gel formulations were prepared using an inert gelling agent. Each formulation contained two gel disks: one infused with 2.2 M nitrite and 0.05M mannitol and the other with citric acid / citrate in a concentration of 0.3 M (Gel Formulation F3). Each disk weighed approximately 80 mg, with a 6 mm diameter. The initial pH of the acidic gel formulation was 5.5.The two disks were stacked and stored in microcentrifuge tubes for different ageing periods — 120 hours, 336 hours and 840 hours. Following each time point, the antimicrobial activity of the gels was evaluated against Pseudomonas aeruginosa microorganisms as shown in Figure 21A. This experimental set-up was repeated by adding 2.5% (%w / w) of Glutathione into the acidic gel (Gel Formulation F4; Figure 21 B).Figure 21A and Table 9 show that the unaged gel gave a larger reduction in CFU / mL than the aged gels. Repeating this experiment by adding Glutathione as a reducing agent shows a significant reduction in CFU / mL and provides a better treatment at different ageing periods — 120 hours, 336 hours and 840 hours (as shown in Figure 21 B and Table 10). With regard to Figure 21 B, the empty box with an arrow signifies the LOD (Limit of Detection) at this dilution, as no CFU were detected.Table 9Table 10Example 18The antimicrobial activity of the present formulations against Streptococcus mutans was studied as shown in Figure 22.The following formulation was used: nitrite was present in a concentration of 0.22 M, citric acid / citrate buffer was present in a concentration of 0.15 M and mannitol was present in a concentration of 0.005 M. The initial pH of the formulation (Formulation F1) was 5.0. Formulation F1 was prepared, added to 1 mL of PBS and left to react for 5 minutes, giving Formulation F2.1mL of this diluted formulation (F2) was added to 1mL of Streptococcus mutans inoculum at 108colony forming units / mL (CFU / mL) resulting in an overall % dilution. In another tube the same process was followed but / V-acetyl-L-cysteine (NAC) was added to formulation F1 to achieve an initial concentration of 612 mM upon exposure to Streptococcus mutans inoculum. Figure 22 shows the change in CFU / mL over a 2- hour period when comparing F2 at % dilution alone to F2 + 612 mM NAC at a % dilution. Adding NAC results in a significant decrease in the Streptococcus mutans CFU / mL count compared to no NAC treatment.List of references cited in this application:EP1984052BWO 2007 / 116102 AWO 2008 / 076136WO 2009 / 0866470WO 2013 / 085784WO 2014 / 188174 A1WO 2020 / 245574 A1WO 2022 / 013614 A1WO 02 / 20026WO 95 / 32705W098 / 0110WO 95 / 09605WO98 / 55096US1997 / 005648101 AUS 2014 / 0056963 A1Licht et al., Carcinogenesis (7988), 365-372Ridnour et al., Biol. Chem., 385, 2004, 1-10Friedman et al., Virulence. 20723(3): 271-279Mendhi et al., Applied Materials Today, 79, 2020, 100562Massa, et al. Antioxidants. 2021 , 10 (7), 1111.Zelikin et al., Adv. Sci. 2027, 8, 2003895Poh et al., Molecules. 2022; 27(3): 674Buettner et al., Advances in Redox Research 9 (2023), 1000079 Bairagi et al. Appl. Nano Mater. 2023, 6, 19727-19739Xie et al, Nitric oxide, 2024, 149, 67-74Da Veiga et al., Nitric oxide, 2024, 142, 26-37Massa, et al. Antioxidants 2021, 10 (7), 1111

Claims

1. 79CLAIMS1. A composition for generating nitric oxide, the composition comprising:(i) one or more nitrite salts;(ii) one or more reducing agents;(iii) one or more organic carboxylic acids; and wherein the molar ratio of the total reducing agent or agents in the composition to the total nitrite salt or salts in composition is at least about 0.25%, the molar ratio of any one of the reducing agents present in the composition to the total nitrite salt or salts in the composition is at most about 75%, and the molar ratio of total organic carboxylic acid in the composition to total nitrite salt or salts in the composition is at least about 0.5%.

2. The composition for generating nitric oxide according to claim 1 , wherein the one or more reducing agents is selected from ascorbic acid, reductic acid (2,3- dihydroxy-2-cyclopentanone), erythorbic acid, oxalic acid, formic acid, dithionous acid, thiols (such as glutathione, N-acetyl cysteine, DL-cysteine, penicillamine or dithiothreitol), reducing sugars (such as glucose, galactose, fructose, ribose, glyceraldehyde, xylose, cellobiose, lactose or erythrose), salts thereof, derivatives thereof, sulfites, dithionates, thiosulphates, phosphites and hypophosphites and combinations thereof.

3. The composition for generating nitric oxide according to claims 1 or 2, wherein the one or more reducing agent is selected from ascorbic acid, ascorbic acid salts, ascorbic acid derivatives, thiosulphate salts and combinations thereof.

4. The composition for generating nitric oxide according to any one of claims 1 to3, wherein the one or more reducing agent is selected from ascorbic acid, salts thereof, and combinations thereof, optionally the one or more reducing agent is ascorbic acid.

5. The composition for generating nitric oxide according to any one of claims 1 to4, wherein the one or more reducing agent is an ascorbic acid derivative selected from ascorbate palmitic acid (ascorbyl palmitate), 3-0-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 ascorbic80 acid, 6-O-dodecanedioyl ascorbic acid, L-Ascorbic acid 2-phosphate, 2-0- alpha-D-Glucopyranosyl-L-ascorbic acid and combinations thereof.

6. The composition for generating nitric oxide according any one of claims 1 to 3, wherein the one or more reducing agent is sodium thiosulphate.

7. The composition for generating nitric oxide according to claims 1 or 2, wherein the one or more reducing agent is selected from glutathione, N-acetyl cysteine, DL-cysteine, penicillamine or dithiothreitol, or a combination thereof.

8. The composition for generating nitric oxide according to any one of claims 1 to 7, wherein the one or more organic carboxylic acid is selected from salicylic acid, acetyl salicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, a-hydroxypropanoic acid, p-hydroxypropanoic acid, p- hydroxy butyric acid, p-hydroxy-p-butyric acid, naphthoic acid, oleic acid, palmitic acid, pamoic (emboic) acid, stearic acid, malonic acid, succinic acid, fumaric acid, glucoheptonic acid, glucuronic acid, lactobioic acid, cinnamic acid, pyruvic acid, orotic acid, glyceric acid, glycyrrhizic acid, sorbic acid, hyaluronic acid, alginic acid, oxalic acid, carnitine, salts thereof, and combinations thereof.

9. The composition for generating nitric oxide according to claim 8, wherein the one or more organic carboxylic acid is selected from citric acid, salts thereof, and combinations thereof.

10. The composition for generating nitric oxide according to any one of claims 1 to9, wherein the one or more organic carboxylic acid is a citric acid I citrate buffer.11 . The composition for generating nitric oxide according to any one of claims 1 to10, wherein the one or more organic carboxylic acid is present in a concentration of at least 0.001 M.

12. The composition for generating nitric oxide according to any one of claims 1 to11 , wherein the molar ratio of the total reducing agent in the composition to the total nitrite salt in the composition is at least about 0.3%, at least about 0.5%, at least about 0.75% or at least about 1% and / or the molar ratio of any individual reducing agent to the total nitrite salt is about 50% or less, about 42% or less, about 30% or less, or about 25%.8113. The composition for generating nitric oxide according to any one of claims 1 to12, wherein the molar ratio of total organic carboxylic acid in the composition to total nitrite salt or salts in the composition is at least about 0.75%, at least about 1.0%, is at least about 1.5%, at least about 2% or at least about 2.5%.

14. The composition for generating nitric oxide according to any one of claims 1 to13, wherein the molar ratio of the total reducing agent in the composition to total acid in the composition is from about 0.01%.

15. The composition for generating nitric oxide according to claim 14, wherein the molar ratio of the total reducing agent in the composition to total acid in the composition is at least about 0.1%, or at least about 0.5%, or at least about 1%, or at least about 2%.

16. The composition for generating nitric oxide according to any one of claims 1 to15, wherein the composition has a pH in the range of about 2 to about 7.5.

17. The composition for generating nitric oxide according to any one of claims 1 to16, wherein the one or more nitrite salt is selected from LiNO2, NaNO2, KNO2, RbNO2, CSNO2, FrNO2, AgNO2, Be(NO2)2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2, Ra(NO2)2and any mixture thereof.

18. The composition for generating nitric oxide according to claim 17, wherein the one or more nitrite salt is NaNO2, KNO2, or a mixture thereof.

19. The composition for generating nitric oxide according to any one of claims 1 to 18, wherein the composition further comprises one or more organic polyol.

20. The composition for generating nitric oxide according to claim 19, wherein the one or more organic polyol is selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.

21. The composition for generating nitric oxide according to claim 20, wherein the one or more organic polyol is selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, glycerol and combinations thereof.8222. The compositions for generating nitric oxide according to claim 22, wherein the one or more organic polyol is selected from arabitol, xylitol, mannitol, sorbitol and combinations thereof.

23. The composition for generating nitric oxide according to any one of claims 1 to 22, wherein one or more component of the composition is contained in a carrier (e.g., an aqueous solution or an aqueous gel) or is in a solid form.

24. A kit for providing a composition for generating nitric oxide, the kit including:(i) a nitrite component including one or more nitrite salts; and(ii) an acidic component including one or more organic carboxylic acids; wherein the kit further includes one or more reducing agents, and the molar ratio of the total reducing agent or agents in the kit to the total nitrite salt or salts in kit is at least about 0.25%, the molar ratio of any one of the reducing agents present in the kit to the total nitrite salt or salts in the kit is at most about 75%, and wherein the molar ratio of total organic carboxylic acid in the kit to total nitrite salt or salts in the kit is at least about 0.5%25. The kit for providing a composition for generating nitric oxide according to claim 24, wherein at least one of the nitrite component, acidic component or the reducing agent or agents are in an aqueous carrier and the nitrite component is in a separate container from acidic component or the reducing agent or agents.

26. Use of a composition according to any one of claims 1 to 23, or a kit according to any one of claims 24or 25, for generating nitric oxide.

27. The composition according to any one of claims 1 to 23, or the kit according to any one of claims 24 or 25, for use in treating or preventing a microbial infection.

28. A method of treating or preventing a microbial infection, which comprises administering to a subject an antimicrobial amount of a composition according to any one of claims 1 to 23, or a kit according to any one of claims 24 to 25.8329. Use of a composition according to any one of claims 1 to 23, or a kit according to any one of claims 24 to 25, for the manufacture of a medicament for treating or preventing a microbial infection.

30. A method of treating a surface or space to reduce the number of viable microbes on the surface or space, which comprises applying to the surface or the space or to a vicinity thereof an antimicrobial amount of a composition according to any one of claims 1 to 23, or a kit according to any one of claims 24 to 25.31 . A method of generating nitric oxide, wherein the method includes the steps of:(i) initiating an initial NO-generating reaction by combining one or more nitrite salts, a polar solvent and one or both of one or more organic carboxylic acids and one or more reducing agents to form an initial NO-generating reaction composition; and(ii) adding or exposing subsequent quantities of one or more of nitrite salt or salts, organic carboxylic acid or acids and reducing agent or agents to components of the initial NO-generating reaction composition at one or more subsequent times to form one or more subsequent NO-generating reaction compositions; provided that one or more organic carboxylic acids are present in the initial and / or one or more subsequent reaction compositions and one or more reducing agents are present in the initial and / or one or more subsequent reaction compositions.

31. A method of generating nitric oxide, wherein the method includes combining one or more nitrite salts, a polar solvent, one or more organic carboxylic acids and one or more reducing agents to form a NO-generating reaction composition; wherein at least one of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents is added into the NO-generating reaction composition over a longer time period than at least one of the other of the nitrite salt or salts, the organic carboxylic acid or acids and the reducing agent or agents.

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