Wound dressing

Abstract

A wound dressing for treating a wound, the wound dressing comprising a nitric oxide generating layer that generates nitric oxide by acidification of nitrite wherein the nitric oxide generating layer comprises a solid powdered nitrite component and a solid proton source component; methods and uses of such wound dressings for treating wounds.

Description

[0001] The present invention relates to a wound dressing for treating wounds, the wound dressing comprising a nitric oxide generating layer that generates nitric oxide by acidification of nitrite, wherein the nitric oxide generating layer comprises a solid powder nitrite component and a solid proton source component.

[0002] Background

[0003] Nitric oxide (NO) and nitric oxide precursors have been widely studied as potential medicaments.

[0004] However, there are still substantial problems in the efficient generation and delivery of nitric oxide, other nitrogen oxides, and their precursors to organisms and cells for treatment. Widely adopted systems for generating nitric oxide rely on acidifying nitrite with a proton source (such as an acid) to initially produce nitrous acid (HNO 2 ), which then readily decomposes into nitric oxide and nitrate, as well as hydrogen ions and water. This decomposition can be represented by the following equilibrium equation (1):

[0005] 3 HNO 2 →2 NO+NO 3 - +H + +H 2 O (1)

[0006] Acids and nitrites are typically provided as separate components in pre-determined amounts. These separate components remain separate until use to minimize the reaction before the moment of need. These two reactants are thus provided in a two-part system that involves a part containing nitrite and a separate part containing acid. In this way, these two separate components in the two separate parts can be combined or mixed when needed to prevent the release of nitric oxide before it is required.

[0007] Properties of nitric oxide, such as the property of killing microorganisms or preventing microbial proliferation, have important utility in the treatment of wounds, skin lesions, and burns. Therefore, means for generating and delivering nitric oxide have also been applied to wound dressings. However, there is still a need to keep the acid and nitrite separate until use, and thus two-part wound dressing systems have been developed.

[0008] Typically, such two-part wound dressings include a first part containing nitrite and a second part containing acid, and these two components remain separate until needed. Thus, the two-part wound dressing is packaged as two separate pieces, and then these two separate pieces need to be combined to initiate the reaction. The use of a two-part system has its drawbacks. For example, combining the two-part system when needed has the possibility of introducing, for example, user error or dosing inaccuracy when combining the two parts.

[0009] In contrast, a one-part (also known as single-part) wound dressing is provided as a single piece before use. One-part wound dressings are simpler to use because they generally require minimal preparation (such as removing a protective film) before application to a subject. Accordingly, it is desirable to provide a source of nitrite and an acidifying agent as a one-part or single-part wound dressing. Additionally, the provision of a single-piece product can reduce manufacturing complexity, cost, and packaging.

[0010] However, difficulties arise in attempting to provide acidification of nitrite as a single-piece system because reactions are initiated and a significant proportion of nitric oxide is lost during manufacture and storage of the system, and there is a risk of not providing sufficient nitric oxide at the point of need.

[0011] WO 2021 / 198461 describes a wound dressing for generating nitric oxide. The described wound dressing requires a nitric oxide releasing agent and an activator (a species for activating and / or facilitating release of nitric oxide from the nitric oxide releasing agent) to be present in the wound dressing as two separate and distinct layers (a nitric oxide source layer and an activator layer). In addition to these separate and distinct layers, a separating layer is described to further prevent contact between the nitric oxide source layer and the activator layer prior to use.

[0012] Accordingly, there is a need for a one-part wound dressing system that provides the species required for generating and delivering nitric oxide, such as nitrite and acid species, without causing premature or unwanted NO release prior to the point of need. Summary of the Invention

[0014] The present inventors have attempted to provide a simple wound dressing that delivers nitric oxide by acidification of nitrite.

[0015] Most generally, the present invention provides a wound dressing having a nitric oxide generating layer, and the nitric oxide generating layer comprises a solid nitrite and a solid proton source. In this way, the nitrite and the proton source are kept in close proximity (or tightly bound) to provide acidification of the nitrite upon contact with an aqueous environment, but do not substantially react until needed, and thus a single-component system can be provided. The solid components comprising both the nitrite and the acid source can avoid including a source of moisture (such as a solution or a water-based gel). In this way, the reactants are less exposed to moisture to minimize the reaction until the reaction is needed.

[0016] In a first aspect, the present invention provides a wound dressing for treating wounds, the wound dressing comprising a nitric oxide generating layer that generates nitric oxide by acidification of nitrite, wherein the nitric oxide generating layer comprises a solid powder nitrite component and a solid proton source component.

[0017] The nitric oxide generating layer may comprise a dry wound dressing substrate. The solid powder nitrite is typically mixed with the dry wound dressing substrate. In certain embodiments, the dry proton source component comprises a solid powder proton source component, and the solid powder proton source component is mixed with the dry wound dressing substrate. In other embodiments, at least a portion of the dry proton source component forms part of the dry wound dressing substrate. For example, the dry wound dressing substrate may comprise proton source fibers. In certain embodiments, the dry proton source component comprises a solid powder proton source component, and another portion of the dry proton source component forms part of the dry wound dressing substrate.

[0018] The dry wound dressing substrate may be composed of woven or non-woven fibers.

[0019] All components of the nitric oxide generating layer may be dry components. Based on the weight of the nitric oxide generating layer, the water content of the nitric oxide generating layer may be 10% or less, 5% or less, 2% or less, or 1% or less.

[0020] The wound dressing may be a one-piece wound dressing. In other words, the wound dressing may be provided as a single piece prior to the time of need.

[0021] The wound dressing may further comprise one or more additional layers in addition to the nitric oxide generating layer. In other words, the wound dressing may be a multi-layer wound dressing. The nitric oxide generating layer may be combined with other layers and / or materials to fabricate the wound dressing.

[0022] The solid powder nitrite component and the solid powder proton source component may be provided by:

[0023] a. A blend of one or more single particles containing nitrite and one or more single particles containing a proton source;

[0024] b. One or more single particles each containing nitrite and a proton source;

[0025] c. Particle aggregates, wherein the particle aggregates comprise one or more single particles containing nitrite, one or more single particles containing a proton source, and an optional binder;

[0026] d. Particle aggregates, wherein the particle aggregates comprise one or more single particles each containing nitrite and a proton source, and an optional binder; or

[0027] e. Combinations thereof.

[0028] Single particles or particle aggregates can be blended with or coated with excipients for influencing the rate of water entry into the particles and / or excipients for influencing the kinetics of nitric oxide formation from the particles.

[0029] Excipients for influencing the rate of water entry into the particles can be polyols or hydrophobic materials such as one or more phospholipids (e.g., dipalmitoyl phosphatidylcholine, DPPC), magnesium stearate, or colloidal silica, and / or excipients for influencing the rate of water entry into the particles can be nitric oxide or nitric oxide precursor chelating materials such as thiols, alcohols, amines, or amides.

[0030] Particles containing nitrite and a proton source can be formed by spray drying a mixture containing a nitrite solution and a proton source solution.

[0031] A blend of one or more single particles containing nitrite and one or more single particles containing a proton source can be formed as follows: (a) micronizing a nitrite solid together with a proton source solid; or (b) combining the two solids as follows:

[0032] (i) spray drying or freeze drying a solution containing nitrite,

[0033] (ii) spray drying or freeze drying a solution containing a proton source, and

[0034] (iii) blending the solids produced in steps (i) and (ii).

[0035] The proton source can comprise an acid precursor such as an ester or a photoacid.

[0036] A wound dressing can include one or more additional dry layers adjacent to the nitric oxide generating layer. Based on the weight of the layer adjacent to the nitric oxide generating layer, the water content of any layer adjacent to the nitric oxide generating layer can be 10% or less, 5% or less, 2% or less, or 1% or less.

[0037] The wound dressing can further include an antimicrobial agent.

[0038] In a second aspect, the present invention provides a packaged wound dressing comprising a wound dressing as described herein within a low moisture permeability package. The low moisture permeability package can include one or more low moisture permeability materials (e.g., aluminum foil) in the walls of the package and / or can be hermetically sealed.

[0039] The packaging atmosphere within the packaged wound dressing can have a low water content at the time of initial packaging, and / or the package includes a packaging insert for sequestering moisture.

[0040] In a third aspect, the present invention provides a method of treating a wound, the method comprising applying a wound dressing as described herein to a wound of a subject. The wound dressing can be a one-piece wound dressing. In other words, the wound dressing can be provided as a single piece prior to the time of need.

[0041] In a fourth aspect, the present invention provides a combination of a solid powder nitrite component and a solid proton source component in a wound dressing as described herein for treating a wound of a subject. The wound dressing can be a one-piece wound dressing. In other words, the wound dressing can be provided as a single piece prior to the time of need.

[0042] Optional or specific features of one aspect of the present invention as described herein apply equally to other aspects of the present invention, provided that the feature is compatible with that aspect. In particular, optional or specific features of the wound dressing apply equally to the packaged wound dressing, the method of treating a wound, and the combination for treating a wound, provided that the features are compatible with these parts.

[0043] Detailed Description

[0044] The present invention will now be described in more detail. The examples and the following drawings provide illustrations of the present invention.

[0045] Figure 1 Showing the deposition patterns of the powders of Examples 1A, 2, 3, and 4 on agarose with Hanks balanced salt solution and a pH indicator (phenol red).

[0046] Figure 2 Showing the cumulative NO generation of Examples 1A, 2, 3, and 4.

[0047] Figure 3 Showing the sprouting intensity of HUVEC spheroids treated with Examples 1B and 6A quantified by an image analysis system to determine the cumulative sprout length (CSL) per spheroid relative to a basal control.

[0048] Figure 4 Showing a schematic diagram of the wound dressing of the present invention.

[0049] Figure 5 Showing the NO release curve of a wound dressing of the present invention over 2000 minutes.

[0050] Figure 6 Showing the NO release curve of another wound dressing of the present invention over 2000 minutes.

[0051] Figure 7 Showing a schematic diagram of the apparatus for measuring and analyzing evolved gaseous nitric oxide by selected ion flow tube mass spectrometry (SIFT-MS).

[0052] The reaction between one or more nitrites and a proton source to produce nitric oxide, optionally other nitrogen oxides and / or optionally their precursors is referred to herein as the "NOx formation reaction" or the "reaction to form NOx" or similar terms, and "NOx" is used to refer to the acidification products of nitrites, particularly nitric oxide, other nitrogen oxides and their precursors, individually and in any combination together. It is to be understood that the individual components of the NOx produced may escape as a gas, or may dissolve in the reaction mixture, or may initially dissolve and then escape as a gas, or any combination thereof.

[0053] The term "about" is used herein to indicate that a numerical value is not strictly limiting, and one of ordinary skill in the art will understand that the value can extend above or below the exact value (as appropriate) according to one's understanding of the value. The term "about" can indicate a value of up to ±10% of the value.

[0054] Unless otherwise stated, the particle size as described herein refers to the volume median diameter (VMD).

[0055] As used herein, the terms "one-part", "single-part", "two-part" and "multi-part" are used to refer to the number of parts of a wound dressing before the time of need (e.g., when applied to a subject). For example, a one-part wound dressing is provided as a single piece before the time of need. A one-part wound dressing is typically applied to a subject as a single piece. In contrast, two-part and multi-part wound dressings are provided as two or more pieces, respectively, before the time of need, and are typically combined into a single-piece wound dressing immediately before application to the subject. It should also be noted that a one-part wound dressing as described herein can be formed by a nitric oxide generating layer and one or more other layers or components (such as a backing layer).

[0056] Wound Dressing

[0057] A "wound dressing" as used herein is a material intended to be applied to the outer surface (e.g., skin or fur) of a subject (e.g., a human or an animal) to cover, protect and / or treat a lesion on the skin of the subject. A wound dressing is suitable for any break or interruption in the skin barrier, which can be caused by, for example, ulcers, surgeries, burns, incisions, lesions, wounds, tears, traumas and / or abrasions.

[0058] Nitric Oxide Generating Layer

[0059] The wound dressing of the present invention comprises a nitric oxide generating layer that generates nitric oxide by acidification of a nitrite, wherein the nitric oxide generating layer comprises a solid powder nitrite component and a solid proton source component.

[0060] In this way, the nitrite and the proton source can be in close proximity to fully react when exposed to an aqueous environment. The nitrite and the proton source are present in a single layer of the wound dressing. In this way, these components do not need to be combined during use (e.g., as part of a two-component system). Additionally, since the nitrite and the proton source are in solid form, nitric oxide generation prior to use is reduced because the water content can be minimized.

[0061] Without wishing to be bound by theory, the inventors of the present invention have found that it is possible to provide the nitrite and the proton source together in a single layer of a wound dressing when the substances are provided as a dry solid powder composition. In this form, the nitrite and the proton source cannot react with each other and thus no NOx is generated. However, when exposed to a moist environment, such as wound exudate, the nitrite and the proton source are able to react and generate NOx. Accordingly, the wound dressing of the present invention enables the nitrite and the proton source to be present in close proximity to each other without the need to be separated in separate layers or by a barrier layer to prevent the generation of NOx prior to use (i.e., enables the nitrite and the proton source to be provided on the same layer in the wound dressing).

[0062] Generally, all components of the nitric oxide generating layer are dry components. In this way, the reaction between the nitrite and the acid component is minimized prior to use. Based on the weight of the nitric oxide generating layer, the water content of the nitric oxide generating layer can be 10% or less, 5% or less, 2% or less, or 1% or less. In this way, the reaction between the nitrite and the proton source reactants is minimized prior to use. The water content can be measured by standard laboratory methods, such as weighing the sample, removing the moisture (e.g., by drying in an oven at over 100 °C), and then weighing the sample again.

[0063] Dry Wound Dressing Substrate

[0064] The nitric oxide generating layer can include a dry wound dressing substrate. The solid powder nitrite is typically mixed with the dry wound dressing substrate. In certain embodiments, the dry proton source component includes a solid powder proton source component, and the solid powder proton source component is mixed with the dry wound dressing substrate. In other embodiments, at least a portion of the dry proton source component forms part of the dry wound dressing substrate. For example, the dry wound dressing substrate can include proton source fibers. In certain embodiments, the dry proton source component includes a solid powder proton source component, and another portion of the dry proton source component forms part of the dry wound dressing substrate.

[0065] Dry wound dressing substrates are known per se. The dry wound dressing can be an adsorbent. The dry wound dressing substrate can be a synthetic or natural polymeric material. The dry wound dressing substrate can consist of woven or non-woven fibers or solid foams. The dry wound dressing substrate can consist of cotton fibers, rayon, polyester (such as PLGA) and / or gelling fibers such as alginates (salts of alginic acid) and carboxymethyl cellulose and its salts. Additionally or alternatively, the dry wound dressing substrate can be a hydrophilic material (such as silicone) and / or a solid foam of alginate.

[0066] Particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be incorporated or encapsulated into the substrate. In this way, the solid powder composition can be retained within the material by the substrate until exposure to moisture or an aqueous environment. The particles of the solid powder composition can be exposed or partially exposed on the surface of the substrate, or can be completely encapsulated within the substrate.

[0067] The material can be a fibrous material comprising the fibers of the substrate, and particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be incorporated or encapsulated into the fibrous material. The particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be exposed or partially exposed on the surface of the substrate fibers, or can be completely encapsulated within the fiber network and fiber cross-section.

[0068] In some instances, the dry wound dressing substrate is porous and at least some of the particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component are in the pores of the substrate. In other words, the substrate can be porous and impregnated with particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component. In some instances, the substrate is made porous by including pores in the surface of the substrate. In other instances, the substrate can be a porous network of substrate elements (such as polymer fibers) and the particles or particle aggregates are in the voids between the substrate elements. As a specific example, the particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be impregnated into the voids of a polymer fiber network.

[0069] The particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be of a particle size suitable for dispersion in the gelling fibers. The particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can have a particle size greater than about 5 μm. For example, the particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can have a particle size greater than about 50 μm, greater than about 100 μm, greater than about 250 μm, greater than about 500 μm, greater than about 750 μm, greater than about 1000 μm.

[0070] To achieve a greater particle size, the particles or particle agglomerates can be granulated. "Granulation" refers to the process of combining particulate materials to form larger particles called granules. Granulation can be carried out, for example, by compressing the particles or agglomerates to provide a tablet, which can then be broken into granules. The particles or agglomerates can be compressed at about 1 to about 10 MT (metric tons), for example, at about 3 to about 7 MT. The particles or agglomerates can be compressed at about 3.8 MT. The particles or agglomerates can be compressed at about 6.5 MT. A sieve, such as a 1 mm sieve, can be used to break the tablet into granules.

[0071] To facilitate compression, a binder can be added to the particles or agglomerates. Suitable binders can include sugars, natural binders, or synthetic or semi-synthetic polymer binders. Sugar materials can include, for example, sucrose or liquid glucose. Natural binders can include, for example, gum arabic, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid, or cellulose. Synthetic or semi-synthetic polymer binders can include, for example, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol, polymethacrylate. The binder can be a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate (copovidone). The binder can be microcrystalline cellulose.

[0072] The binder can be incorporated into the composition at a %w / w of about 5% w / w to about 30% w / w. For example, the binder can be incorporated into the composition at a %w / w of about 10% w / w to about 25% w / w.

[0073] Alternatively, the composition can be substantially binder-free.

[0074] The particle size can be increased in this way to ensure that the particles or agglomerated particles remain trapped (incorporated or encapsulated) between the fibers.

[0075] Particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component can be incorporated into the substrate during the production of the substrate. A method of incorporating or encapsulating particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component into a substrate, the method comprising the steps of: (i) mixing particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component with a non-polar liquid containing the substrate or a substrate precursor to form a liquid-particle mixture, and (ii) curing the liquid-particle mixture to form a material incorporating or encapsulating particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component.

[0076] The liquid-particle mixture can be cured by spinning the mixture into fibers. Techniques known to those skilled in the art for fiber spinning can be used. For example, the liquid-particle mixture can be cured by dry spinning, wet spinning, gel spinning, or electrospinning. The liquid-particle mixture can be cured by electrospinning. "Electrospinning" refers to a fiber production method in which an electrically charged line of a polymer solution or polymer melt is drawn to the fiber diameter using electricity. The liquid-particle mixture can be cured by gel spinning. "Gel spinning" refers to a fiber production method that relies on temperature-induced physical gelation for curing.

[0077] Alternatively, after the substrate is formed, particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be incorporated into the substrate. For example, particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component can be impregnated into a porous substrate, such as a fibrous web substrate. In these examples, the substrate has been formed, and particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component are added thereto. A specific example of a method for impregnating a solid powder composition into a porous substrate includes those described in EP2331309 (and other techniques available from Fibroline France).

[0078] Solid Powder Nitrite Component and Solid Proton Source Component

[0079] The wound dressing of the present invention includes a solid powder nitrite component and a solid proton source component in a single nitric oxide generating layer. In this way, the single nitric oxide generating layer can release nitric oxide by acidification of nitrite when exposed to an aqueous environment or moisture in the atmosphere.

[0080] Solid Powder Nitrite Component

[0081] The solid powder nitrite component includes nitrite. There is no particular limitation on the selection of nitrite. The nitrite can be selected from one or more alkali metal nitrites or alkaline earth metal nitrites. For example, the one or more nitrites can be selected from LiNO 2 , NaNO 2 , KNO 2 , RbNO 2 , CsNO 2 , FrNO 2 , AgNO 2 , Be(NO 2 ) 2 , Mg(NO 2 ) 2 , Ca(NO 2 ) 2, Sr(NO 2 ) 2 , Mn(NO 2 ) 2 , Ba(NO 2 ) 2 , Ra(NO 2 ) 2 and any mixtures thereof. The nitrite can be NaNO 2 or KNO 2 . The nitrite can be NaNO 2 .

[0082] The nitrite can be a pharmaceutically acceptable grade of nitrite. In other words, the nitrite can follow one or more valid pharmacopeial monographs on nitrites. For example, the nitrite can follow the nitrite monographs of one or more of the United States Pharmacopeia (USP), the European Pharmacopeia, or the Japanese Pharmacopeia.

[0083] In particular, the nitrite used can have one or more of the characteristics provided in Table 1 in paragraphs

[0032] to

[0060] and / or paragraph

[0204] of WO 2010 / 093746, the disclosure of which is hereby incorporated by reference in its entirety.

[0084] Solid Proton Source Component

[0085] The solid proton source component includes a proton source. The proton source can be any class of substances capable of acting as a proton source for the acidification of nitrites. There is no particular limitation on the choice of the proton source. The proton source can be, for example, an acid.

[0086] The solid proton source component can be provided as a solid powder proton source component. Additionally or alternatively, the solid proton source component can be provided as part of a dry wound dressing substrate (such as a proton source fiber).

[0087] The acid can be selected from one or more organic carboxylic acids or organic non-carboxylic reducing acids.

[0088] The term "organic carboxylic acid" as used herein refers to any organic acid containing one or more -COOH groups in the molecule. The organic carboxylic acid can be straight-chain or branched-chain. The carboxylic acid can be saturated or unsaturated. The carboxylic acid can be aliphatic or aromatic. The carboxylic acid can be acyclic or cyclic. The carboxylic acid can be an allenic carboxylic acid.

[0089] The organic carboxylic acid can carry one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids useful in the present disclosure include α-hydroxy-carboxylic acids, β-hydroxy-carboxylic acids, and γ-hydroxy-carboxylic acids.

[0090] The expression "organic non-carboxylic reducing acid" as used herein refers to any organic reducing acid that does not contain a -COOH group in its molecule. The organic non-carboxylic reducing acid can be straight-chain or branched-chain. The non-carboxylic reducing acid can be saturated or unsaturated. The non-carboxylic reducing acid can be aliphatic or aromatic. The non-carboxylic reducing acid can be acyclic or cyclic. The non-carboxylic reducing acid can be an enol.

[0091] The organic non-carboxylic reducing acid can carry one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic reducing acids useful in the present disclosure include acidic reducing ketones, such as reductic acid (2,3-dihydroxy-2-cyclopentanone).

[0092] The one or more organic carboxylic acids or non-carboxylic reducing acids can have a pKa of less than about 7 1 .

[0093] The one or more organic carboxylic acids can comprise, consist of, or be one or more reducing carboxylic acids. The organic carboxylic acid can be selected, for example, from salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropionic acid, β-hydroxypropionic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, pamoic acid (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, its salts, and combinations thereof.

[0094] The organic carboxylic acid can be citric acid or its salt.

[0095] The carboxylic acid can be or comprise a polymeric or polymerized carboxylic acid, such as polyacrylic acid, polymethacrylic acid, a copolymer of acrylic acid and methacrylic acid, polylactic acid, polyglycolic acid, or a copolymer of lactic acid and glycolic acid. The term "organic carboxylic acid" as used herein also encompasses partial esters or full esters of organic carboxylic acids or partial salts or full salts thereof, provided that these can act as proton sources for use in accordance with the present invention.

[0096] The organic non-carboxylic reducing acid can be selected, for example, from ascorbic acid; ascorbate palmitic acid (ascorbyl palmitate); ascorbic acid derivatives such as 3-O-ethyl ascorbic acid, other 3-alkyl ascorbic acids, 6-O-octanoyl ascorbic acid, 6-O-dodecanoyl ascorbic acid, 6-O-tetradecanoyl ascorbic acid, 6-O-octadecanoyl ascorbic acid, and 6-O-dodecanedioyl ascorbic acid; acidic reductones such as reductic acid; isoascorbic acid; its salts; and combinations thereof.

[0097] The organic non-carboxylic reducing acid can be ascorbic acid or its salt.

[0098] The one or more organic carboxylic or organic non-carboxylic reducing acids of the proton source can suitably be present together with their conjugate bases. When contacted with or exposed to an aqueous environment, the acid and its conjugate base can suitably form a buffer. The acid and its conjugate base can be provided in a ratio that achieves the desired pH upon exposure to the aqueous environment.

[0099] A buffer system can be selected to achieve the desired pH upon exposure to an aqueous environment and to maintain the desired pH while the NOx generation reaction proceeds. A buffer system can be selected such that the pH of the reaction can be in the range of about 3 to 9, such as about 4 to 8. For physiological contact or contact with living cells and organisms, the pH of the reaction can be in the range of about 5 to about 8. When present, the conjugate base can be added separately or can be generated in situ from the proton source by adjusting the pH using an acid and / or a base, such as an inorganic acid and / or an inorganic base.

[0100] The proton source can be a citric acid / citrate buffer system, such as a citric acid / sodium citrate buffer system.

[0101] The proton source can be or can comprise an acid precursor. An "acid precursor" is a species that can undergo a chemical reaction to provide an acid species. For example, an acid precursor can be a species that can undergo hydrolysis to provide an acid species. In other words, an acid precursor can be a hydrolysable acid precursor for releasing an acid upon hydrolysis. For example, an acid precursor can be an ester. An acid precursor can be a photoacid. In other words, an acid precursor can be a species that becomes more acidic upon absorption of light. For the avoidance of doubt, the term "photoacid" as used herein includes species that undergo reversible proton photodissociation and species that undergo irreversible proton photodissociation.

[0102] Solid proton source components can be provided as part of a dry wound dressing substrate (e.g., as proton source fibers). In some embodiments, the solid proton source assembly includes proton source fibers. In other words, the solid proton source component includes fibers capable of providing protons. Such proton source fibers include, but are not limited to, polyacrylic acid fibers (especially partially neutralized polyacrylic acid fibers) and polyester fibers (especially PLGA fibers).

[0103] In certain embodiments, the solid proton source component can include a combination of a solid powder proton source component and proton source fibers.

[0104] In specific embodiments, the solid proton source component includes a solid powder proton source component.

[0105] Those skilled in the art will understand that the choice of acid component / proton source can be selected according to the desired use. Solid Powder Sub Combination of Nitrate Component and Solid Proton Source Component

[0106] The solid powder nitrite component and the solid proton source component are present in the nitric oxide generating layer. Generally, the solid powder nitrite component and the solid proton source component exist as a mixture of components. In this way, the solid powder nitrite component and the solid proton source component are present in close proximity to react when exposed to an aqueous environment.

[0107] When the nitric oxide generating layer includes the solid proton source component as part of a dry wound dressing substrate, the solid powder nitrite component and the solid powder proton source component (when present) can be combined by the methods described herein for combining solid powder components with a dry wound dressing substrate.

[0108] The mixture of the solid powder nitrite component and the solid powder proton source component can be achieved in various ways. The solid powder nitrite component and the solid powder proton source component can be added to the nitric oxide generating layer independently. In other words, the solid powder nitrite component can be added to the nitric oxide generating layer separately from the solid powder proton source component.

[0109] In specific embodiments, the solid powder nitrite component and the solid powder proton source component are mixed before adding these components to other components of the nitric oxide generating layer or during the formation of the nitric oxide generating layer. There are various ways to mix the solid powder nitrite component and the solid powder proton source component. Specific ways of mixing the solid powder nitrite component and the solid powder proton source component are provided below.

[0110] Combination of Solid Powder Nitrite Component and Solid Powder Proton Source Component

[0111] The solid powder nitrite component and the solid powder proton source component can be provided by one or more particles each containing a nitrite and a proton source. It is to be understood that when a particle contains both a proton source and a nitrite, the particle can contain the nitrite and the proton source within the same particle.

[0112] Additionally or alternatively, the solid powder nitrite component and the solid powder proton source component can be provided by one or more particles containing a nitrite and no proton source and one or more particles containing a proton source and no nitrite. It is to be understood that the particles can contain a nitrite or a proton source, rather than containing a nitrite and a proton source in the same particle. The one or more particles containing a nitrite or a proton source can be blended to provide a substantially uniform particle mixture.

[0113] A “homogeneous mixture” is a mixture that is compositionally uniform such that it always has the same proportion of its components.

[0114] Thus, the one or more particles containing a nitrite or a proton source can be blended to provide a composition having a uniform distribution and proportion of nitrite particles and proton source particles.

[0115] The one or more particles can be present in the wound dressing as single particles or as aggregates of single particles or a combination thereof.

[0116] The expressions “aggregate”, “aggregating” and “aggregated together” herein refer to the aggregation or collection of primary (single) particles that exhibit recognizable collective behaviour.

[0117] In the present invention, an aggregate of single particles can comprise (i) single particles containing a nitrite and single particles containing a proton source, (ii) single particles containing a nitrite and a proton source, or (iii) a combination thereof, and optionally, a binder.

[0118] In the present invention, the recognizable collective behaviour can be resistance to mechanical separation, i.e., the particles adhere to each other.

[0119] The particles or aggregates of the solid powder nitrite component and the solid powder proton source component can be of a particle size suitable for their intended use or application. For example, the particles or aggregates of the solid composition can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less or about 1 μm or less.

[0120] Alternatively, the particles or agglomerates of the solid powder nitrite component and the solid powder proton source component may have a particle size greater than 5 μm. For example, the particles or agglomerates of the solid composition may have a particle size greater than 50 μm, greater than 100 μm, greater than 250 μm, greater than 500 μm, greater than 750 μm, greater than 1000 μm.

[0121] The weight ratio of nitrite to proton source in the mixture of the solid powder nitrite component and the solid powder proton source component may be in the range of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24.

[0122] The mixture of the solid powder nitrite component and the solid powder proton source component may contain additional optional additives, such as a binder (as mentioned above) or an organic polyol.

[0123] Binder

[0124] The mixture of the solid powder nitrite component and the solid powder proton source component may be substantially free of one or more binders. Alternatively, the mixture of the solid powder nitrite component and the solid powder proton source component may further include one or more binders.

[0125] As used herein, "binder" refers to a reagent that promotes particle adhesion, i.e., promotes the formation of particle agglomerates.

[0126] Suitable binders may include sugars, natural binders or synthetic or semi-synthetic polymer binders. Sugar substances may include, for example, sucrose or liquid glucose. Natural binders may include, for example, gum arabic, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid or cellulose. Synthetic or semi-synthetic polymer binders may include, for example, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyvinyl alcohol, polymethacrylate. The binder may be a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate (copovidone). The binder may be microcrystalline cellulose.

[0127] The binder may be incorporated into the mixture of the solid powder nitrite component and the solid powder proton source component at a % w / w of about 5% w / w to about 30% w / w. For example, the binder may be incorporated into the mixture of the solid powder nitrite component and the solid powder proton source component at a % w / w of about 10% w / w to about 25% w / w.

[0128] Organic polyol

[0129] A mixture of a solid powder nitrite component and a solid powder proton source component can be substantially free of one or more organic polyols. Alternatively, a mixture of a solid powder nitrite component and a solid powder proton source component can further include one or more organic polyols. When a mixture of a solid powder nitrite component and a solid powder proton source component includes one or more organic polyols, it is preferred to add the organic polyols to the mixture of the solid powder nitrite component and the solid powder proton source component after any processing involving removal of the solvent (e.g., after a spray drying or freeze drying step). In other words, the polyol can be added to a composition comprising one or more particles containing nitrite and a proton source; or added to a mixture comprising one or more particles containing nitrite and / or one or more particles containing a proton source (before or after formation of aggregates of these particles).

[0130] The expression "organic polyol" herein refers to an organic molecule having two or more hydroxyl groups, which is not a proton source, particularly for nitrite reactions, and is not a sugar or a polysaccharide (the terms "sugar" and "polysaccharide" include oligosaccharides, glycans, and glycosaminoglycans). The organic polyol thus has a pKa of greater than about 7 1 .

[0131] The expression "organic polyol" herein preferably does not include reducing agents. Examples of reducing agents that are organic molecules having two or more hydroxyl groups and are not sugars or polysaccharides are thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate esters, isoascorbic acid, and isoascorbate esters. Thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate esters, and isoascorbate esters are therefore preferably excluded from the expression "organic polyol" because they are reducing agents. In any case, ascorbic acid and isoascorbic acid are excluded from this expression because they are proton sources, particularly for nitrite reactions.

[0132] The organic polyol can be cyclic or acyclic, or can be a mixture of one or more cyclic organic polyols and one or more acyclic organic polyols. For example, the one or more organic polyols can be selected from one or more alkanes substituted with two or more OH groups, one or more cycloalkanes substituted with two or more OH groups, one or more cycloalkylalkanes substituted with two or more OH groups, and any combination thereof. The organic polyol can be unsubstituted with any substituents other than OH.

[0133] The one or more organic polyols may be one or more acyclic organic polyols. The one or more acyclic organic polyols may be selected from sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more acyclic organic polyols may be selected from alditols, such as alditols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The one or more organic polyols may not include saponins, sapogenins, steroids or steroid glycosides.

[0134] Alternatively, the one or more organic polyols may be one or more cyclic organic polyols. The one or more cyclic organic polyols may be cyclic sugar alcohols or cyclic alditols. For example, the one or more cyclic polyols may be cyclic sugar alcohols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms or cyclic alditols having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. A specific example of a cyclic polyol is inositol.

[0135] The one or more organic polyols may have 7 or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 7 or more hydroxyl groups. The one or more organic polyols may have 9 or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 9 or more hydroxyl groups. The one or more organic polyols may have 20 or fewer hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 20 or fewer hydroxyl groups. The one or more organic polyols may have 15 or fewer hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having 15 or fewer hydroxyl groups. The one or more organic polyols may have a number of hydroxyl groups in the range of 7 to 20, such as in the range of 9 to 15. The one or more organic polyols may include 9, 12, 15 or 18 hydroxyl groups.

[0136] The one or more organic polyols may be a sugar alcohol compound comprising one or more monosaccharide units and one or more acyclic sugar alcohol units, such as a sugar alcohol compound composed of one or more monosaccharide units and one or more acyclic sugar alcohol units. The one or more organic polyols may be a straight chain comprising 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, such as a sugar alcohol compound composed thereof.

[0137] As used herein, a "monosaccharide unit" refers to a monosaccharide covalently linked to at least one other unit in a compound, whether another monosaccharide unit or an acyclic polyol unit. As used herein, an "acyclic polyol unit" refers to an acyclic polyol covalently linked to at least one other unit in a compound, whether a monosaccharide unit or another acyclic polyol unit. The units in the compound may be linked via an ether bond. One or more monosaccharide units may be covalently linked to other units in the compound via a glycosidic bond. Each monosaccharide unit may be covalently linked to other units in the compound via a glycosidic bond. A polyol compound may be a glycoside having a monosaccharide or oligosaccharide glycone and an acyclic polyol aglycone.

[0138] The acyclic polyol unit may be a polyol unit having 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. The acyclic polyol unit may be selected from the units of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, and heptitol.

[0139] One or more monosaccharide units may be C 5 or C 6 monosaccharide units, i.e., pentose or hexose units. Each monosaccharide unit may be C 5 or C 6 monosaccharide units. One or more polyol units may be C 5 or C 6 polyol units. Each polyol unit may be C 5 or C 6 polyol units.

[0140] The polyol compound may contain n monosaccharide units and m acyclic polyol units, for example, may be composed of n monosaccharide units and m acyclic polyol units, where n is an integer and at least 1, m is an integer and at least 1, and (n + m) is not greater than 10. The polyol compound may contain a chain of n monosaccharide units capped with one acyclic polyol unit, for example, may be composed thereof, where n is an integer between 1 and 9. The chain of monosaccharide units may be covalently linked via a glycosidic bond. Each monosaccharide unit may be covalently linked via a glycosidic bond to another monosaccharide unit or an acyclic polyol unit. The polyol compound may contain a chain of 1, 2, or 3 monosaccharide units capped with one acyclic polyol unit, for example, may be composed thereof. One, two, three, or each monosaccharide unit may be C 5 or C 6 monosaccharide units. The acyclic polyol unit may be C 5 or C 6 polyol units. Examples of polyol compounds include, but are not limited to: isomaltitol, maltitol, and lactitol (n = 1); maltotriitol (n = 2); and maltotetraitol (n = 3).

[0141] Such sugar alcohol compounds can be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, "oligosaccharide" refers to a sugar composed of 3 to 10 monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides can be synthesized (e.g., by hydrogenation) from disaccharides, oligosaccharides, or polysaccharides (e.g., from hydrolysis and hydrogenation), but are not limited to compounds synthesized from disaccharides, oligosaccharides, or polysaccharides. For example, a sugar alcohol derived from a disaccharide can be formed by a dehydration reaction of a monosaccharide and a sugar alcohol. The one or more organic polyols can be sugar alcohols derived from disaccharides, trisaccharides, or tetrasaccharides. Examples of sugar alcohols derived from disaccharides include, but are not limited to, isomaltitol, maltitol, and lactitol. Examples of sugar alcohols derived from trisaccharides include, but are not limited to, maltotriitol. Examples of sugar alcohols derived from tetrasaccharides include, but are not limited to, maltotetraitol.

[0142] The organic polyol can be selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, heptitol, isomaltitol, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, and any combination thereof. Glycerol can be used, and when present, is preferably combined with one or more other organic polyols, such as erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, heptitol, isomaltitol, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, or any combination thereof.

[0143] Many organic polyols contain one or more chiral centers and thus exist in stereoisomeric forms. All stereoisomeric forms and optical isomers and mixtures of isomers of the organic polyol are intended to be included within the scope of the present invention. In particular, the D and / or L forms of all chiral organic polyols and all mixtures thereof can be used.

[0144] Agglomeration of Particles

[0145] Agglomeration of the particles can be achieved by any suitable means known to those skilled in the art.

[0146] Agglomeration of the particles can be achieved by mechanical means, such as by mechanically pressing the particles together. Agglomeration by mechanical means can be achieved by micronization of nitrite particles and proton source particles. Alternatively, agglomeration by mechanical means can be achieved by providing particles that are substantially free of static electricity.

[0147] Agglomeration of the particles can be achieved by chemical means, such as chemically promoted adhesion or chemical coating. Agglomeration by chemical means can be achieved by an adhesion promoter, such as moisture. Alternatively, agglomeration by chemical means can be achieved by a coating material that binds primary particles of nitrite and primary particles of a proton source together. Suitable binders were previously discussed, and suitable coating materials are discussed in the "Coated Particles" section below.

[0148] Coated Particles

[0149] The one or more particles of the mixture of the solid powder nitrite component and the solid powder proton source component can be coated with an excipient (also referred to herein as coated particles).

[0150] The coated particles can include individual particles containing nitrite and a proton source and coated with an excipient.

[0151] Alternatively, the coated particles can be an agglomerate of particles coated with an excipient, and the particle agglomerate includes (a) particles containing nitrite and a proton source and / or (b) a mixture of one or more nitrite particles containing nitrite and one or more proton source particles containing a proton source.

[0152] In this way, the coated particles include nitrite and a proton source within the same coating.

[0153] The excipient can be hydrophobic. The excipient can be any material capable of coating the particles or agglomerate such that the particles or agglomerate are coated with a hydrophobic layer. The hydrophobic material can be a polymeric material, such as an organic polymeric material, such as a polyol. The hydrophobic material can be an amphiphilic class, such as a surfactant type class, such as a nonionic, anionic, cationic, or zwitterionic surfactant type class. The hydrophobic material can be, for example, an inorganic mineral material and an inorganic mineral material forming a 3D framework. The hydrophobic material can be biocompatible. The hydrophobic material can include one or more of poly(lactic-co-glycolic acid) (PLGA), phospholipids such as dipalmitoylphosphatidylcholine (DPPC), magnesium stearate, and mesoporous silica. The hydrophobic material can contain poly(lactic-co-glycolic acid) (PLGA) that is a polymeric material without acid end groups, or can contain poly(lactic-co-glycolic acid) (PLGA) that is a polymeric material with acid end groups. The excipient can contain polyols, magnesium stearate, colloidal silica.

[0154] As used herein, a "surfactant" refers to a surfactant that can reduce the surface tension of a species in a medium or the interfacial tension between media. Surfactant classes generally have a hydrophilic head and a hydrophobic tail.

[0155] The hydrophobic material can be attached to the particles or agglomerate by chemical bonding or by electrostatic or intermolecular forces.

[0156] When the coated particles or coated aggregates are exposed to an aqueous environment, the coating of the coated particles or coated particle aggregates can affect the reaction kinetics of the acidification of nitrite, such as reaction kinematics.

[0157] The excipient can be a class of substances capable of capturing or sequestering nitric oxide or a nitric oxide precursor. For example, the excipient can include thiols, alcohols, amines, or amides.

[0158] The coated particles or coated particle aggregates of the mixture of the solid powder nitrite component and the solid powder proton source component can be of a particle size suitable for the desired use or application. The coated particles or coated particle aggregates of the mixture of the solid powder nitrite component and the solid powder proton source component can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less. Alternatively, the coated particles or coated aggregates of the mixture of the solid powder nitrite component and the solid powder proton source component can have a particle size greater than about 5 μm. For example, the particles or aggregates of the mixture of the solid powder nitrite component and the solid powder proton source component can have a particle size greater than about 50 μm, greater than about 100 μm, greater than about 250 μm, greater than about 500 μm, greater than about 750 μm, greater than about 1000 μm.

[0159] Forming Particles from a Mixture Containing a Nitrite Solution and a Proton Source Solution

[0160] The mixture of the solid powder nitrite component and the solid powder proton source component can be formed by spray drying or freeze drying a mixture containing a nitrite solution and a proton source solution.

[0161] The particles of the mixture of the solid powder nitrite component and the solid powder proton source component can be formed from a mixture containing a nitrite solution and a proton source solution. The particles formed in this way should be formed by removing the solvent within a short time (such as 30 seconds or less) after mixing the nitrite solution and the proton source solution, and / or by placing the mixture under reaction delay conditions (such as at a temperature below the freezing point of the solvent) after mixing the nitrite solution and the proton source solution in order to remove the solvent. In this way, the solvent is removed from the mixture while minimizing the acidification of nitrite. Thus, an effective amount of nitrite and proton source can be present in the resulting powder composition.

[0162] When the solvent is removed within a short time after mixing the nitrite solution and the proton source solution, the solvent can be removed within 30 seconds or less after mixing the nitrite solution and the proton source solution. In some examples, the solvent is removed within 10 seconds or less, 5 seconds or less, 2 seconds or less, or 1 second or less after mixing the nitrite solution and the proton source solution. In some examples, the solvent is removed within 500 milliseconds or less, 100 milliseconds or less, 50 milliseconds or less, or 10 milliseconds or less after mixing the nitrite solution and the proton source solution.

[0163] In one example, particles can be formed by spray drying a mixture containing a nitrite solution and a proton source solution. Spray drying of the mixture can enable the removal of the solvent within 30 seconds or less after mixing the nitrite solution and the proton source solution. Spray drying of materials is known per se.

[0164] The mixture is typically a mixture of an aqueous nitrite solution and an aqueous proton source solution. When using aqueous solutions, the time between mixing these two aqueous solutions is minimized to inhibit the acidification of the nitrite. The aqueous solution of nitrite and the aqueous solution of acid can be mixed online for about 1 to about 10 milliseconds, such as about 3 to about 5 milliseconds, before spray drying occurs. Spray drying can be carried out immediately after mixing the nitrite and acid solutions. It is to be understood that, as described, mixing and spray drying a mixture containing a nitrite solution and a proton source solution limits the potential reaction time between the proton source and nitrite components.

[0165] Particles formed by spray drying a mixture containing a nitrite solution and an acid solution can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0166] Spray drying a mixture containing a nitrite solution and an acid solution as described can produce a mixture of a solid powder nitrite component and a solid powder proton source component, wherein each particle contains a nitrite and a proton source component.

[0167] Particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in any suitable morphology. For example, particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in crystalline form or amorphous form. Particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in amorphous form.

[0168] Additionally or alternatively, the mixture of the nitrite solution and the proton source solution is placed under reaction delay conditions (e.g., at a temperature below the freezing point of the solvent) immediately before, during, or after mixing the nitrite solution and the proton source solution and used to remove the solvent. In this way, the acidification of nitrite is delayed until the solvent is removed. In particular, the solvent can be an aqueous solvent.

[0169] A specific instance of the reaction delay condition is that the temperature of the mixture is below the freezing point of the solvent. In this way, the reaction rate of the acidification of nitrite can be slowed down while the solvent is removed. When the temperature of the mixture is below the freezing point of the solvent, the nitrite solution and the proton source solution are usually mixed at a temperature above the freezing point of the solvent, and then the temperature of the mixture is lowered to below the freezing point of the solvent. In this way, good mixing of the solution can be achieved.

[0170] In some instances, solvent removal can be carried out under reduced gas pressure. In particular, solvent removal can be carried out under reduced gas pressure and at a temperature below the freezing point of the solvent to be removed.

[0171] A particularly useful technique for removing the solvent under reaction delay conditions is freeze-drying (also known as "lyophilization").

[0172] It should be noted that the terms "removing the solvent" and / or "drying" as used herein are for obtaining a solid powder composition. These terms include but are not limited to completely removing the solvent. In some instances, the solid powder composition may include trace amounts of residual solvent. For example, the powder composition may contain up to about 10% residual solvent, such as up to about 5% residual solvent, up to about 3% residual solvent, or up to about 1% residual solvent. Additional drying techniques, such as vacuum drying, can be employed after the initial solvent removal to provide the solid powder composition.

[0173] Combining Solids to Form Particle Agglomerates

[0174] A mixture of the solid powder nitrite component and the solid powder proton source component can be formed by combining a nitrite-containing solid and a proton source-containing solid to form particle aggregates, where the particle aggregates include one or more nitrite-containing particles and one or more proton source-containing particles.

[0175] Combining the nitrite-containing solid and the proton source-containing solid to form particle aggregates can be achieved, for example, as follows: (a) blending one or more nitrite particles and one or more proton source particles, where the nitrite particles are formed by spray-drying a nitrite solution and the proton source particles are formed by spray-drying a proton source solution; or (b) forming one or more particles by micronizing the nitrite solid and the proton source solid together.

[0176] Blended Spray-Dried Nitrite Particles and Spray-Dried Acid Particles

[0177] A mixture of a solid powder nitrite component and a solid powder proton source component can be formed as follows:

[0178] (i) Spray-dry or freeze-dry a solution containing nitrite,

[0179] (ii) Spray-dry or freeze-dry a solution containing a proton source,

[0180] (iii) Blend the materials from (i) and (ii).

[0181] A mixture of a solid powder nitrite component and a solid powder proton source component can be a blend of nitrite particles and proton source particles, where the nitrite particles are formed by spray-drying a nitrite solution and the proton source particles are formed by spray-drying a proton source solution. The spray-dried nitrite particles and the spray-dried proton source particles can be blended by standard means known to those skilled in the art to provide a blended solid powder composition.

[0182] The spray-dried nitrite particles and the spray-dried proton source particles can be blended at a nitrite / proton source weight ratio in the range of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24.

[0183] The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a time of about 5 to about 60 minutes, such as about 10 to about 40 minutes, or about 15 to about 30 minutes. The spray-dried nitrite particles and the spray-dried proton source particles can be blended for about 20 minutes.

[0184] Particles formed as described by spray-drying a nitrite solution and spray-drying an acid solution and blending these components can have a particle size of about 10 μm or less, such as about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0185] Spray-drying a nitrite solution and spray-drying a proton source solution and blending these components can produce a mixture of a solid powder nitrite component and a solid powder proton source component including particle agglomerates, where the agglomerates include one or more particles containing nitrite and one or more particles containing a proton source.

[0186] Particles formed by spray drying a nitrite solution and a proton source solution and blending these components can be in any suitable form. For example, particles formed by spray drying a nitrite solution and a proton source solution and blending these components can be in crystalline form or amorphous form. Particles formed by spray drying a mixture containing a nitrite solution and a proton source solution can be in amorphous form.

[0187] Particles Formed by Micronizing Nitrite Solid and Acid Solid Together

[0188] Particles can be formed by micronizing a nitrite solid together with a proton source solid.

[0189] As used herein, the expression "micronizing" refers to a method for reducing the average particle size of a solid composition to within the generally micron range. Micronizing can be achieved by standard methods known to those skilled in the art. For example, micronizing can be carried out by grinding or milling particles or by using supercritical fluids.

[0190] In the case where the proton source is a buffered acid system, the proton source solid can be two components - a solid acid component and a solid conjugate base component. The nitrite solid and the proton source solid can be micronized at a ratio in the range of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24, for example a ratio of 1:9 w / w nitrite:proton source.

[0191] Particles formed by micronizing a nitrite solid together with a proton source solid can have a particle size of about 10 μm or less, for example about 5 μm or less, about 4 μm or less, about 3 μm or less, about 2 μm or less, or about 1 μm or less.

[0192] Micronizing the nitrite solution together with the proton source solution as described can produce a solid powder composition containing particles containing nitrite and particles containing the proton source. Micronizing the nitrite solution together with the proton source solution as described can produce a solid powder composition comprising agglomerates, the agglomerates comprising particles containing nitrite and particles containing the proton source.

[0193] Particles formed by micronizing a nitrite solution together with a proton source solution can be in any suitable form. For example, particles formed by micronizing a nitrite solution together with a proton source solution can be in crystalline form or amorphous form. Particles formed by micronizing a nitrite solution together with a proton source solution can be in crystalline form.

[0194] Particles formed by micronization may include one or more optional additives as described above (except for proton sources and nitrites). In particular, particles formed by micronization may include a binder as described above. The binder may be micronized together with nitrite solid and proton source solid.

[0195] Other Features of the Wound Dressing

[0196] The wound dressing may be a one-piece wound dressing. The one-piece wound dressing includes all components required to apply the wound dressing to a subject in a single piece. In this way, the wound dressing does not need to be assembled by a practitioner before being applied to the subject.

[0197] The wound dressing may be a single-layer (nitric oxide generating layer) wound dressing, or may be a multi-layer wound dressing (including a nitric oxide generating layer).

[0198] The wound dressing may particularly include a backing layer. The backing layer is typically located on the outer surface of the wound dressing and on the opposite side of the wound dressing surface suitable for application to the subject. In this way, the backing layer can protect the wound and the active components of the wound dressing from the environment during application. The backing layer may be flexible. The backing layer may be gas permeable or semi-permeable. The backing layer may be made of polyurethane. The backing layer may include an adhesive for adhering the wound dressing to the subject. The backing layer of the wound dressing is known per se.

[0199] The wound dressing may include a removable protective layer on the outer surface of the wound dressing to protect the wound dressing components (such as the nitric oxide generating layer) before applying the wound dressing to the subject. The removable protective layer is typically removed from the wound dressing before applying the wound dressing to the subject to expose the active wound dressing components (such as the nitric oxide generating layer) to the wound. The removable protective layer may be flexible. The removable protective layer may be transparent or translucent.

[0200] The nitric oxide generating layer may be intended to be applied directly to the subject's wound during use. The wound dressing may be configured such that the nitric oxide generating layer is directly applied to the subject's wound during use. For example, the nitric oxide generating layer may form the outer surface of the wound dressing. Alternatively, the nitric oxide generating layer is adjacent to an outer removable protective film or layer that is to be removed before directly applying the nitric oxide generating layer to the subject's wound. In other words, the wound dressing may include a removable protective film that forms the outer surface of the wound dressing, and wherein the nitric oxide generating layer is adjacent to the removable protective film. In this way, the removable protective film can be removed before application, and the nitric oxide generating layer can be directly applied to the subject's wound.

[0201] Alternatively, one or more permeable layers may be adjacent to the nitric oxide generating layer and are intended to be applied to a wound dressing. The wound dressing may include one or more permeable layers adjacent to the nitric oxide generating layer and is configured to directly apply the one or more permeable layers to a subject's wound in use. The wound dressing may further include an outer removable protective film or layer to be removed before directly applying the one or more permeable layers to the subject's wound. In other words, the wound dressing may include a removable protective film forming the outer surface of the wound dressing, wherein the one or more permeable membranes are adjacent to the removable protective film and the one or more permeable layers are adjacent to the nitric oxide generating layer. In this way, the removable protective film may be removed before application, and the one or more permeable layers adjacent to the nitric oxide generating layer may be directly applied to the subject's wound. The one or more permeable layers may be made of any permeable material, typically any gas and / or liquid permeable material. In this way, nitric oxide may enter these layers and / or liquid may pass through these layers into the nitric oxide generating layer.

[0202] The wound dressing may include one or more additional drying layers adjacent to the nitric oxide generating layer. Based on the weight of the layer adjacent to the nitric oxide generating layer, the water content of any layer adjacent to the nitric oxide generating layer may be 10% or less, 5% or less, 2% or less, or 1% or less.

[0203] The material of the additional layer of the wound dressing may be a mesh (woven or non-woven), non-woven bat, film, foam, alginate, amorphous hydrogel, cross-linked hydrogel, or film. The layers of the wound dressing may be formed of natural or synthetic materials. For example, the layers of the wound dressing may be carboxymethyl cellulose fibers and synthetic polymer fabrics. The present invention is not limited to the uses and materials listed above, and other suitable materials and uses of the wound dressing are known to those skilled in the art.

[0204] Additional Antimicrobial Agents

[0205] The acidification of the nitrite component and the proton source component generally has antimicrobial activity. In some instances, the wound dressing includes an additional antimicrobial agent. Antimicrobial agents are known per se. In some instances, the wound dressing includes AgNO 2 both as an antimicrobial agent and as a nitrite.

[0206] Packaged Wound Dressing

[0207] The present invention also provides a packaged wound dressing comprising a wound dressing as described herein within a low moisture permeability package.

[0208] A low moisture permeability package can include one or more low moisture permeability materials (such as aluminum foil) in the walls of the package. In certain embodiments, the low moisture permeability package includes one or more low moisture permeability materials (such as aluminum foil) in the walls of the package and the wound dressing, and is hermetically sealed. The low moisture permeability package can include one or more low moisture permeability materials (such as aluminum foil) in at least a portion of all of the outer walls of the package.

[0209] The package atmosphere within the wound dressing package can have a low water content at the time of initial packaging. The package atmosphere can have a relative humidity of 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. A hygrometer can be used to measure the relative humidity.

[0210] The package atmosphere can include an inert packaging gas such as nitrogen, argon, helium, or CO 2 . The package atmosphere includes 10% or less, 8% or less, 5% or less, 2% or less, 1% or less oxygen. In some embodiments, the package atmosphere is substantially free of oxygen.

[0211] Additionally or alternatively, the package can include one or more moisture-sequestering package inserts. Such a package insert can be a desiccant packet, such as a silica gel packet.

[0212] Methods and Combinations for Treating Wounds

[0213] The present invention provides a method of treating a wound, the method comprising applying a wound dressing as described herein to a wound of a subject. The wound dressing can be a one-piece wound dressing. In other words, the wound dressing can be provided as a single piece prior to the time of need.

[0214] The present invention also provides a combination of a solid powder nitrite component and a solid proton source component in a wound dressing as described herein for treating a wound of a subject. The wound dressing can be a one-piece wound dressing. In other words, the wound dressing can be provided as a single piece prior to the time of need.

[0215] In some embodiments, the method includes applying the packaged wound dressing to a wound of a subject. In other embodiments, the method includes removing a removable outer layer or film of the wound dressing prior to applying it to a wound of a subject. Generally, the method does not include combining two or more parts of the wound dressing prior to applying it to a wound of a subject.

[0216] In some embodiments, the method includes adding water (including aqueous solutions, suspensions, gels, or other forms containing water) to the nitric oxide generating layer before applying the wound dressing to a subject's wound. The addition of water can be direct to the nitric oxide generating layer or can be indirect (e.g., through one or more permeable layers adjacent to the nitric oxide generating layer). The added water can be a sterile aqueous solution. The aqueous environment can be a sterile saline solution.

[0217] Alternatively, the wound dressing is applied to the subject's wound without adding water. In this way, aqueous fluids (e.g., blood and / or exudate) from the subject can be absorbed by the nitric oxide generating layer of the wound dressing and activate the production of nitric oxide.

[0218] The subject can be a human or animal subject. The subject can be a human or a domesticated animal.

[0219] Method for Producing Solid Powder Components

[0220] Method for Producing a Solid Powder Composition by Removing Solvent

[0221] A method of making a mixture of a solid powder nitrite component and a solid powder proton source component can include removing solvent from a mixture of a nitrite solution and a proton source solution to minimize acidification prior to formation of the powder composition.

[0222] In one example, the method includes removing solvent (e.g., by spray drying) within less than 30 seconds after mixing the nitrite solution and the proton source solution to form a solid.

[0223] In another example, the method includes providing reaction delay conditions (e.g., freeze drying) during solvent removal and immediately before, during, and / or after mixing the nitrite solution and the proton source solution.

[0224] In one example, the method can include removing solvent from an aqueous mixture containing a nitrite solution and a proton source solution to form a solid powder.

[0225] The aqueous solution of nitrite can have a concentration in the range of about 0.1M to about 5M. The aqueous solution of nitrite can have a concentration of at least about 0.1M, at least about 0.2M, at least about 0.5M, at least about 0.75M, or at least about 1M. The aqueous solution of nitrite can have a concentration of at most about 5M, at most about 4M, at most about 3M, or at most about 2M. For example, the aqueous solution of nitrite can have a concentration in the range of about 1M to about 2M, such as about 1.5M. The aqueous solution of nitrite can have a pH of about 6.5 to about 9, such as about 7 to about 8.

[0226] The aqueous solution of the proton source can have a concentration in the range of about 0.1 M to about 5 M. The aqueous solution of the proton source can have a concentration of at least about 0.1 M, at least about 0.2 M, at least about 0.5 M, at least about 0.75 M, or at least about 1 M. The aqueous solution of the proton source can have a concentration of at most about 5 M, at most about 4 M, at most about 3 M, or at most about 2 M. For example, the aqueous solution of the proton source can have a concentration in the range of about 0.5 M to about 1.5 M, such as about 1 M. The aqueous solution of citric acid can have a pH of about 4 to 6. The pH of the aqueous solution of the proton source can be adjusted using, for example, an inorganic base such as sodium hydroxide.

[0227] In some instances, the step of removing the solvent takes 20 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, or 1 second or less after mixing the nitrite solution and the proton source solution. In some instances, the solvent is removed within 500 milliseconds or less, 100 milliseconds or less, 50 milliseconds or less, or 10 milliseconds or less after mixing the nitrite solution and the proton source solution.

[0228] Spray Drying

[0229] A mixture of a solid powder nitrite component and a solid powder proton source component can be produced by spray drying a nitrite solution and a proton source solution.

[0230] The aqueous solution of the nitrite and the aqueous solution of the acid can be mixed online for about 1 to about 10 milliseconds, such as about 3 to about 5 milliseconds, before spray drying occurs. Spray drying can be carried out immediately after mixing the nitrite and the proton acid solution. It is to be understood that, as described, mixing and spray drying a mixture containing a nitrite solution and a proton source solution greatly limits the potential reaction time between the proton source and the nitrite component and completely stops the reaction when the moisture is rapidly removed.

[0231] Spray drying can be carried out at an outlet temperature of about 60 to about 80 °C, such as about 65 to about 75 °C or about 68 to about 70 °C. Spray drying can be carried out at an atomization pressure of about 1 to 6 bar. Spray drying can be carried out at a liquid feed rate of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / m, or about 3 g / min.

[0232] Reaction Delay Conditions

[0233] As an alternative, the method can include providing reaction delay conditions (such as freeze drying) during the solvent removal process and immediately before, during, and / or after mixing the nitrite solution and the proton source solution.

[0234] A particular instance of the reaction delay condition is that the temperature of the mixture is below the freezing point of the solvent. In this way, the reaction rate of the acidification of nitrite can be slowed down while removing the solvent. When the temperature of the mixture is below the freezing point of the solvent, the nitrite solution and the proton source solution are usually mixed at a temperature above the freezing point of the solvent, and then the temperature of the mixture is lowered to below the freezing point of the solvent. In this way, good mixing of the solution can be achieved.

[0235] In some instances, solvent removal can be carried out under reduced gas pressure. In particular, solvent removal can be carried out under reduced gas pressure and at a temperature below the freezing point of the solvent to be removed.

[0236] A particularly useful technique for removing the solvent under reaction delay conditions is freeze-drying (also known as "lyophilization").

[0237] The time taken to remove the solvent after mixing the nitrite solution and the proton source solution under delayed reaction conditions can be about 10 minutes or less. Under these conditions, it may not be very important to remove the solvent (such as water) so quickly. However, it is also desirable to remove the solvent within a relatively short time range to further limit the acidification of nitrite. In some instances, the solvent is removed within about 8 minutes or less, such as about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, or about 2 minutes or less after mixing the nitrite solution and the proton source solution. In further instances, the step of removing the solvent takes about 1 minute or less, about 30 seconds or less, about 20 seconds or less, about 15 seconds or less, or about 10 seconds or less after mixing the nitrite solution and the proton source solution.

[0238] It should be noted that the terms "removing the solvent" and / or "drying" as used herein are for obtaining a solid powder composition. These terms include, but are not limited to, completely removing the solvent. In some instances, the solid powder composition may include trace amounts of residual solvent. For example, the powder composition may contain up to about 10% of residual solvent, such as up to about 5% of residual solvent, up to about 3% of residual solvent, or up to about 1% of residual solvent. Additional drying techniques, such as vacuum drying, can be employed after the initial removal of the solvent to provide the solid powder composition.

[0239] Method for Combining Particles to Form Particle Agglomerates

[0240] The formation of particle aggregates including particles containing nitrite and particles containing a proton source can be achieved in a variety of ways.

[0241] In one instance, the method may include the steps:

[0242] (i) Spray drying or freeze drying a nitrite solution to form nitrite particles;

[0243] (ii) Spray drying or freeze drying a proton source solution to form proton source particles; and

[0244] (iii) Blending the nitrite particles and the proton source particles.

[0245] The aqueous solution of nitrite may have a concentration in the range of about 0.1 M to about 5 M. The aqueous solution of nitrite may have a concentration of at least about 0.1 M, at least about 0.2 M, at least about 0.5 M, at least about 0.75 M or at least about 1 M. The aqueous solution of nitrite may have a concentration of at most about 5 M, at most about 4 M, at most about 3 M or at most about 2 M. For example, the aqueous solution of nitrite may have a concentration in the range of about 1 M to about 2 M, such as about 1.5 M. The aqueous solution of nitrite may have a pH of about 6.5 to about 9, such as about 7 to about 8.

[0246] The aqueous solution of the proton source may have a concentration in the range of about 0.1 M to about 5 M. The aqueous solution of the proton source may have a concentration of at least about 0.1 M, at least about 0.2 M, at least about 0.5 M, at least about 0.75 M or at least about 1 M. The aqueous solution of the proton source may have a concentration of at most about 5 M, at most about 4 M, at most about 3 M or at most about 2 M. For example, the aqueous solution of the proton source may have a concentration in the range of about 0.5 M to about 1.5 M, such as about 1 M. The aqueous solution of citric acid may have a pH of about 4 to 6. The pH of the aqueous solution of the proton source can be adjusted using, for example, an inorganic base such as sodium hydroxide.

[0247] Spray drying can be carried out at an outlet temperature of about 60 to about 80 °C, such as about 65 to about 75 °C or about 68 to about 70 °C. Spray drying can be carried out at an atomization pressure of about 1 to 6 bar. Spray drying can be carried out at a liquid feed rate of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / m, or about 3 g / min.

[0248] In some instances, the spray-dried particles are further dried, for example, by vacuum drying.

[0249] The spray-dried or freeze-dried nitrite particles and the spray-dried or freeze-dried proton source particles can be blended by standard means known to those skilled in the art to provide a blended solid powder composition.

[0250] The spray-dried or freeze-dried nitrite particles and the spray-dried or freeze-dried proton source particles can be blended at a nitrite / proton source weight ratio in the range of about 1:1 to about 1:99, such as in the range of about 1:4 to about 1:49 or about 1:7 to about 1:24.

[0251] The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a time period of from about 5 to about 60 minutes, such as from about 10 to about 40 minutes, or from about 15 to about 30 minutes. The spray-dried nitrite particles and the spray-dried proton source particles can be blended for a time period of about 20 minutes.

[0252] Method for Producing a Mixture of Solid Powder Nitrite Component and Solid Powder Proton Source Component by Micronizing

[0253] A method for producing a mixture of a solid powder nitrite component and a solid powder proton source component can include the step of micronizing a nitrite solid together with a proton source solid to produce a solid powder composition. Micronization itself is known. Micronization can be achieved by standard methods known to those skilled in the art. For example, micronization can be carried out by milling or grinding the particles or by using supercritical fluids.

[0254] The nitrite solid can be micronized together with the proton source solid for a time period of from about 5 to about 30 minutes, such as from about 5 to about 20 minutes, or from about 5 to about 15 minutes. The nitrite solid can be micronized together with the proton source solid for a time period of about 10 minutes.

[0255] The nitrite solid can be micronized together with the proton source solid at a Venturi pressure of 8 bar and a grinding pressure of 2 bar.

[0256] The present inventors have found that micronizing a nitrite solid together with a proton source solid (i.e., simultaneously) can produce a solid powder composition that releases nitric oxide better when exposed to an aqueous environment, as compared to a solid powder composition formed by blending separately micronized nitrite powder and separately micronized proton source powder.

[0257] Method for Producing a Solid Powder Composition with Coated Particles

[0258] A mixture of a solid powder nitrite component and a solid powder proton source component can be produced that includes particles coated in a hydrophobic material. The method can include any one of the following steps:

[0259] (i) coating particles containing a nitrite and a proton source with a hydrophobic material; or

[0260] (ii) combining one or more nitrite particles containing a nitrite and one or more proton source particles containing a proton source, and then coating the mixture.

[0261] The hydrophobic material can be the same hydrophobic material as described above.

[0262] The particles or particle aggregates can be coated in any suitable manner known to those skilled in the art.

[0263] Particles or particle aggregates can be coated by dispersing them in a solution containing a hydrophobic material and drying the solution to provide particles or particle aggregates coated with a layer of hydrophobic material. In some instances, the solution includes a non-polar solvent. In certain instances, the solution is free of polar solvents (e.g., methanol). Such polar solvents can dissolve at least a portion of the particles. In particular, the solution can be anhydrous.

[0264] The hydrophobic material can be, for example, PLGA. The particles or particle aggregates can be dried with the hydrophobic material at a 1:1 w / w ratio. The solution in which the particles or particle aggregates are dispersed or suspended can be a solution of DCM and the hydrophobic material.

[0265] In certain embodiments, the suspension of particles in the hydrophobic material solution is dried by spray drying. The solution containing the hydrophobic material in which the particles or particle aggregates are dispersed can be spray dried at an outlet temperature of about 28 to 30 °C. The solution containing the hydrophobic material in which the particles or aggregates are dispersed can be spray dried at an atomization pressure of about 1 bar. The solution containing the hydrophobic material in which the particles or aggregates are dispersed can be spray dried at a liquid feed rate of about 2 g / min.

[0266] The coated particles or coated particle aggregates can have a particle size of less than about 10 μm, such as less than about 9 μm, such as less than about 8 μm, less than about 7 μm, less than about 6 μm, or less than about 5 μm.

[0267] Particles or particle aggregates can be coated by blending them with a hydrophobic material to provide particles or particle aggregates coated with a layer of hydrophobic material. The hydrophobic material can be, for example, DPPC, magnesium stearate, mesoporous silica, or combinations thereof. The particles or aggregates can be blended with the hydrophobic material at a 1:1 w / w ratio. The hydrophobic material can be sieved before blending. Alternatively, the hydrophobic material can not be sieved before blending.

[0268] The particles or particle aggregates can be blended with the hydrophobic material for a time of about 10 to about 40 minutes, or about 15 to about 30 minutes. The spray-dried nitrite particles and the spray-dried proton source particles can be blended for about 20 minutes.

[0269] Aqueous Environment

[0270] The nitric oxide generating layer of the present invention generally releases NOx when in contact with an aqueous environment. There is no particular limitation on the aqueous environment.

[0271] The aqueous environment can be an aqueous biological fluid, such as a body fluid. Such body fluids can include wound exudate or exudates, and / or blood (such as plasma, serum).

[0272] Alternatively, the aqueous environment can be a sterile aqueous solution. The aqueous environment can be a saline solution.

[0273] In some embodiments, the solid powder composition can be sufficiently hygroscopic to absorb moisture from the air, which is sufficient to initiate the release of NOx. Examples

[0274] Preparation of the solid powder composition

[0275] Materials and Analytical Methods

[0276] The following materials were obtained from commercial sources: sodium nitrite from Honeywell, citric acid from Sigma Aldrich, trisodium citrate from Merck, sodium hydroxide from Fisher, PLGA RG502H from Sigma Aldrich, mesoporous silica (Syloid 244FP) from Grace, dipalmitoylphosphatidylcholine (DPPC) from Avanti, Kollidon VA64 Fine from BASF, microcrystalline cellulose from JRS Pharma, and dichloromethane (DCM) from Sigma Aldrich. Deionized (DI) water (18.2 MΩ) was prepared using an ELGA water purification system.

[0277] Unless otherwise noted, the following analytical methods were used.

[0278] Dry powder particle size distribution (PSD) measured by Sympatec

[0279] Laser particle size analysis of spray-dried powders was performed using a Sympatec HELOS particle size analyzer equipped with an R3 lens (0.5 - 175.0 μm range) / R5 lens (0.5 - 875.0 μm range) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The ASPIROS glass tube was filled with powder in a reduced humidity environment (<25% RH) and sealed with Parafilm until measurement. Unless otherwise noted, measurements were performed in triplicate and average data were reported.

[0280] Example 1: Spray drying a mixture of a nitrite solution and a proton source solution to form a solid Powder Group Compound

[0281] A feed solution of 1.5 M sodium nitrite (feed solution 1) was prepared by dissolving the required mass of sodium nitrite in deionized water. A feed solution of 1 M citric acid adjusted to pH 4 (feed solution 2) was prepared by dissolving the required mass of citric acid in deionized water and adjusting the pH to 4 using 10 M aqueous sodium hydroxide solution. The pH of the solution was measured using a Mettler Toledo SevenCompact pH meter.

[0282] The feed solutions 1 and 2 were spray-dried using a Buchi B290 spray dryer equipped with a Buchi two-fluid nozzle. The two feed solutions were pumped simultaneously using separate feed lines (platinum-cured silicone L / S14 tubing), which were connected using a Y-piece fitting and a single Masterflex peristaltic pump that combined the feed solutions just prior to atomization. A standard Buchi cyclone separator and collection vessel were installed for product collection.

[0283] The feed solutions were spray-dried in two batches under the following conditions:

[0284] Examples Outlet Temperature (°C) Atomization Pressure (bar) Liquid Feed Rate (g / min) 1A 68-70 5.5 3.06 1B 68-70 1.5 3.05

[0285] Both of these batches were then vacuum-dried for 24 h using an Edwards Super Modulyo freeze dryer set to 25 °C.

[0286] The two batches were then subjected to particle size distribution measurements using a Sympatec HELOS particle size analyzer equipped with an R3 lens (range 0.5 - 175.0 μm) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The measurements were performed in triplicate.

[0287] The resulting particle size distributions were measured as follows:

[0288]

[0289] VMD = volume median diameter

[0290] Example 2: Separately Spray-Dry Nitrite and Proton Source, then Blend to Produce a Solid Composition

[0291] A 1.5 M sodium nitrite solution was prepared by dissolving the required mass of sodium nitrite in deionized water.

[0292] A 1 M citric acid solution adjusted to pH 5.6 was prepared by dissolving the required mass of citric acid in deionized water and adjusting the pH to 5.6 using 10 M aqueous sodium hydroxide solution. The pH of the solution was measured using a Mettler Toledo Seven Compact pH meter.

[0293] These feed solutions were spray-dried separately using a Buchi B290 spray dryer under the following conditions:

[0294]

[0295] All batches were then vacuum-dried for 24 hours using an Edwards Super Modulyo freeze dryer set to 25 °C.

[0296] The particle size distributions of these three batches were then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (range 0.5 - 175.0 μm) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The measurements were performed in triplicate.

[0297]

[0298] The spray-dried nitrite solid (Component 2A) and the spray-dried citric acid solid at pH 5.6 (Component 2C) were then blended together at a ratio of 9:1 w / w citrate solid:nitrite solid using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 2.

[0299] Example 3: Micronization of nitrite solid with proton source solid to produce a solid powder composition

[0300] Sodium nitrite, citric acid, and trisodium citrate were combined in the following weight proportions: 10.79%, 14.74%, and 74.47% respectively. The mixture was blended using a Turbula T2F mixer at 47 rpm for 10 minutes.

[0301] The blend was micronized using an Atritor M3 fluid energy mill at a Venturi pressure of 8 bar and a grinding pressure of 2 bar. The blend was fed directly into the hopper at a target feed rate of ~2 g / min. The resulting powder (Example 3) was collected in a single collection tank at reduced humidity (20% RH).

[0302] The particle size distribution was then measured using a Sympatec HELOS particle size analyzer equipped with an R3 lens (range 0.5 - 175.0 μm) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The measurements were performed in triplicate.

[0303] The resulting particle size distribution measurements were as follows:

[0304]

[0305] VMD = Volume Median Diameter

[0306] Comparative Example 4: Separately Micronize Nitrite and Proton Source, then Blend to Produce a Solid Composition

[0307] Sodium nitrite was micronized using an Atritor M3 fluid energy mill at a Venturi pressure of 8 bar and a grinding pressure of 2 bar. Sodium nitrite was fed directly into the hopper at a target feed rate of ~2 g / min. The resulting powder (Component 4A) was collected in a single collection tank at reduced humidity (20% RH).

[0308] Citric acid and trisodium citrate were combined in the following weight ratios: 16.51% and 83.49% respectively. The mixture was blended using a Turbula T2F mixer at 47 rpm for 10 minutes.

[0309] The blend was micronized using an Atritor M3 fluid energy mill at a Venturi pressure of 8 bar and a grinding pressure of 2 bar. The blend was fed directly into the hopper at a target feed rate of ~2 g / min. The resulting powder (Component 4B) was collected in a single collection tank at reduced humidity (20% RH).

[0310] The micronized nitrite solid (Component 4A) and the micronized citric acid solid (Component 4B) were then blended at a ratio of 9:1 w / w citrate solid:nitrite solid using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Comparative Example 4.

[0311] NOx Evolution

[0312] Examples 1A, 2, 3 and 4 were loaded into an APTAR Unidose nasal sprayer (https: / / www.aptar.com / products / pharmaceutical / uds / ) supported in a rig 30 cm above a Petri dish (9.8 cm diameter) containing agarose as well as Hanks balanced salt solution and a pH indicator (phenol red). Figure 1 Show the powder deposition pattern due to local pH adjustment caused by the particles where they land.

[0313] The plate was transferred to a sealed chamber immediately after application and nitrogen oxides (NOx) were measured over 15 minutes by Selected Ion Flow Tube Mass Spectrometry (SIFT-MS). All powders released nitric oxide regardless of their method of preparation. However, differences in the total amount of NOx released were observed between the four powders over the 15-minute period.

[0314] It should be noted that the agarose was buffered at a neutral to weakly basic pH, which should inhibit the reaction, but the particles were able to overcome this buffering effect within a short time and counteract this buffering in the local area. The following table and Figure 2 show the cumulative NO generation for Examples 1A, 2, 3, and 4. The cumulative NO / nmols per mg of nitrite was normalized for the experimental results with respect to the percentage of nitrite in the powder.

[0315] Examples Cumulative NO / nmols <![CDATA[Cumulative NO / nmols.mg nitrite -1 > Example 1A 2410 526 Example 3 1571 322 Example 4 562 120 Example 2 2393 552

[0316] Coated solid powder composition

[0317] Example 5: Particles Coated with Hydrophobic Material DPPC or Mesoporous Silica

[0318] Example 1B was blended with mesoporous silica at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5A.

[0319] Example 1B was blended with DPPC at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5B.

[0320] Example 3 was blended with mesoporous silica at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5C.

[0321] Example 3 was blended with DPPC at a ratio of 1:1 w / w using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5D.

[0322] Example 6: Particles Coated with PLGA

[0323] A PLGA RG502H solution was prepared by dissolving 1.5 grams of PLGA in approximately 30 mL of DCM to form a clear and colorless solution. 1.5 grams of Example 1B was added to this solution with stirring to form a feed suspension 6A at a 1:1 w / w ratio as a visually homogeneous white suspension.

[0324] Similarly, a separate PLGA RG 502H solution was prepared by dissolving 1.5 grams of PLGA in approximately 30 mL of DCM to form a clear and colorless solution. 1.5 grams of Example 3 was added to this solution with stirring to form a feed solution 6B at a 1:1 w / w ratio as a visually homogeneous white suspension.

[0325] These feed suspensions were spray dried using a Buchi B290 spray dryer according to the method detailed above. The spray drying parameters are summarized below.

[0326]

[0327] In an environment with reduced humidity (28% RH), the sample vial was placed horizontally in a single weighing dish. The lid was removed and the opening was covered with pierced (punctured with a needle) foil. The sample was transferred to an Edwards Super Modulyo freeze dryer set to 25 °C and vacuum dried for 24 hours (the maximum observed vacuum pressure was ~0.1 mbar). After vacuum drying, the sample was transferred to a low humidity (~24% RH) environment and covered with nitrogen. The vial was then sealed with Parafilm and sealed into a foil bag with desiccant for storage at 2 - 8 °C.

[0328] Particle size distribution measurements were then carried out using a Sympatec HELOS particle size analyzer equipped with an R3 lens (range 0.5 - 175.0 μm) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at a pressure of 3.00 bar and a reduced pressure of 60 mbar. The measurements were carried out in triplicate.

[0329] The resulting particle size distribution measurements are as follows:

[0330]

[0331] VMD = Volume Mean Diameter

[0332] Example 7: NOx Evolution of Coated Particles

[0333] An aliquot (30 mg) of the powder sample was deposited in a 60 mm Petri dish. A cellulose filter paper (50 mm diameter) was placed on top of the sample and a slight pressure was applied. A sodium phosphate solution (10 mM, 250 μl) was dispensed onto the cellulose filter paper. The sample was immediately placed in a 650 ml chamber, which was sealed, and then moist air was aspirated through the chamber at 650 ml / min for 30 minutes. The air stream from the outlet was analyzed by Single Ion Flow Tube Mass Spectrometry (SIFT-MS).

[0334]

[0335]

[0336] Biological evaluation of the solid powder composition

[0337] Example 8: Efficacy of Four Formulations against Pseudomonas aeruginosa Evaluation

[0338] Prepare and solidify Petri dishes containing nutrient agar (NA, available from AcuMedia). Prepare a Pseudomonas aeruginosa (ATCC 9027) inoculum in phosphate-buffered saline (PBS, Sigma-Aldrich) and serially dilute it to a final concentration of 1x10 5 CFU mL -1 . Pipette 100 μL of the inoculum onto the NA plates, spread it, and allow it to dry at room temperature for 15 minutes. Remove the lids from the inoculated agar plates and place the open plates inside an Aptar Unidose nasal sprayer.

[0339] Connect an Aptar delivery device containing Example 1A, Example 3, Comparative Example 4, or Example 2 powder to the Aptar nasal spray device and atomize the powder (approx. 50 mg dose) onto the agar plates. The following table shows the examples used for each formulation.

[0340] Formulation 1 Example 1A Formulation 2 Example 3 Formulation 3 Comparative Example 4 Formulation 4 Example 2

[0341] After 5 seconds, replace the agar plate lids and incubate the agar plates at 37 °C ± 2 °C for 16 hours. After incubation, take pictures of the plates. For all plates, take three biopsy punches from a 2 x 2 cm area in the center of the agar plate. Sterile swabs moistened with PBS are used to remove bacteria from each biopsy, any cells are suspended in 10 mL of PBS, then sonicated for 5 minutes, serially diluted, and plated onto NA.

[0342] Also, test negative control plates not exposed to the atomized powder and positive control plates added with 1 mL of bleach simultaneously. All tests are performed in quintuplicate.

[0343] For each test item, randomly select three replicates and extract DNA from 400 μL of each biopsy using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer's instructions. Elute the samples in AE buffer at a final volume of 100 μL.

[0344] For each extraction, perform qPCR in triplicate using the QuantiNova Pathogen and IC kit (Qiagen) according to the manufacturer's instructions. Each reaction tube contains each primer at a final concentration of 16 μM and a labeled probe at 5 μM.

[0345] The cycling conditions were as follows: 50 °C for 10 min, 95 °C for 2 min, 35 cycles of 95 °C for 5 sec, 55 °C for 30 sec, and 72 °C for 1 min. Each assay run was validated with positive (Pseudomonas aeruginosa) and negative (RNase-free water) controls. Data were analyzed using Q-Rex software (Qiagen) to obtain Cq values from a predefined threshold. For each sample, the mean Cq value was compared to a standard curve with a defined range of 1x10 2 to 1x10 8 CFU mL -1 to calculate the final sample concentration in Log 10 CFUmL -1 .

[0346] Table 1: Mean recovery and reduction of Pseudomonas aeruginosa from three biopsy punches taken at the center of nutrient agar inoculated with 1x 10 5 CFU mL -1 after treatment with Formulations 1, 2, 3, 4, and bleach compared to untreated negative controls (N = 5).

[0347]

[0348] SD = standard deviation, CFU = colony-forming unit, N / A = not applicable, *= p < 0.05, **= p < 0.01, ***= p < 0.001.

[0349] The mean Pseudomonas aeruginosa recovery of 7.44 ± 0.17 Log 10 CFU mL -1 was observed from biopsies taken from the negative control plates. Mean Pseudomonas aeruginosa recoveries of 3.52 ± 3.12 and 1.36 ± 2.13 Log 10 CFU mL -1 were observed from biopsies taken from Formulations 2 and 3. No live Pseudomonas aeruginosa was recovered from biopsies taken from Formulations 1 and 4 or the positive control plates.

[0350] Table 2: Molecular quantification of Pseudomonas aeruginosa from biopsy punches taken from nutrient agar inoculated with 1x 10 5 CFU mL -1 after treatment with Formulations 1, 2, 3, 4, and bleach compared to untreated negative controls.

[0351]

[0352]

[0353] SD = standard deviation, CFU = colony-forming unit. #= Quantification below the detection limit. ～ = The quantification of the positive control sample was carried out to N = 1, so the standard deviation could not be calculated. N / A = Not applicable, ** = p < 0.01, *** = p < 0.001.

[0354] Compared with the untreated negative control, after treatment with Formulation 1 and Formulation 4 powders, a significant decrease in the recovery of viable Pseudomonas aeruginosa was observed from biopsies of nutrient agar plates inoculated with 1x10 5 CFU mL -1 inoculum because no viable Pseudomonas aeruginosa was recovered. Molecular quantification reflects the recovery from colony counts.

[0355] Example 9: Effect of the Powder Composition on the Germination of Human Umbilical Vein Endothelial Cells (HUVEC) in a Spheroid-Based Cell Angiogenesis Assay

[0356] 10x concentrated stock solutions / suspensions of Examples 1B and 6A were prepared in basal medium (without supplements and FCS) by vortexing and pipetting. Subsequently, a semi-logarithmic dilution series was prepared in the same medium.

[0357] # Example 1B [mg / mL] Example 6A [mg / mL] 1 5.000 10.000 2 1.500 3.000 3 0.500 1.000 4 0.150 0.300 5 0.050 0.100 6 0.015 0.030 7 0.005 0.010

[0358] Endothelial Cells Cells: HUVEC, primary human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany), passages 3 to 4.

[0359] Morphology: Adherent, cobblestone-like growth as a monolayer Medium: Endothelial cell growth and basal medium (ECGM / ECBM, PromoCell) Subculture: Split 1:3; every 3 - 5 days, at approximately 1x10 4 cells / cm 2 Seeding density: At 37 °C, 5% CO 2 2

[0360] Doubling time: 24 - 48 hours Storage: Frozen with approximately 1x10 6 cells / vial in 70% medium, 20% FCS, 10% DMSO

[0361] Source: Human umbilical vein, pooled donors

[0362] Test Methods

[0363] Experiments were conducted in a modification of the originally published protocol (Korff and Augustin: J Cell Sci 112:3249-58, 1999). Briefly, spheroids were prepared as described (Korff and Augustin: J Cell Biol 143:1341-52, 1998) by pipetting 400 HUVEC in hanging drops onto plastic dishes to allow spheroid aggregation overnight. Then, 50 HUVEC spheroids were seeded in 0.9 ml of collagen gel and pipetted into individual wells of a 24-well plate to allow polymerization. After 30 minutes, pre-incubated test samples (final assay concentrations are shown in Table 1) were added by pipetting 100 μl of a 10-fold concentrated working solution onto the polymerized gel. The plates were incubated at 37 °C for 24 hours and fixed by adding 4% PFA (Roth, Karlsruhe, Germany).

[0364] Quantification

[0365] The sprouting intensity of HUVEC spheroids treated with test samples was quantified by an image analysis system that measured the cumulative sprout length (CSL) per spheroid. Photographs of individual spheroids were taken using an inverted microscope and digital imaging software NIS-Elements BR 3.0 (Nikon). Subsequently, the spheroid photographs were uploaded to the homepage of Wimasis GmbH for image analysis. The imaging analysis tool WimSprout was used to determine the cumulative sprout length of each spheroid. The mean of the cumulative sprout lengths of 10 randomly selected spheroids was analyzed as a single data point. The mean and SD values of each triplicate experiment were converted to % of the basal control.

[0366] Results

[0367] Figure 3 The CSLs of Examples 1B and 6A relative to the basal control are shown. The effect of Example 1B (spray-dried particles without coating) was less than that of the basal control. In contrast, the PLGA-coated particles of Example 6A showed a significant dose-dependent effect compared to the basal control. This indicates that, despite a basically neutral environment, the coated particles provide a local environment capable of acidifying nitrite.

[0368] Figure 4Schematic showing the wound dressing 100 of the present invention, which has a backing layer 102, a nitric oxide generating layer 106, and a removable protective layer 108. The wound dressing 100 can be in an airtight package before use. When the wound dressing 100 is needed, it can be removed from any package. The removable protective layer 108 can be removed from the wound dressing 100 to expose the nitric oxide generating layer 106. The wound dressing can be applied to a subject's wound by placing the nitric oxide generating layer 106 onto the subject's wound. Optionally, water can be added to the exposed nitric oxide generating layer 106 before applying the wound dressing.

[0369] The adhesive zone 104 can adhere to the subject to attach the wound dressing to the subject. The adhesive zone and the backing layer 102 can help to seal the wound dressing to the subject. Figure 4 Not drawn to scale.

[0370] Example 10: NO Release from a One-Piece Carboxymethylcellulose (CMC) Wound Dressing Containing a Nitrite Source and a Proton Source ( Figure 5 )

[0371] Using a high-intensity alternating electric field, a carboxymethylcellulose-based fabric (120 g / m 2 ) was impregnated with a powder (20 g / m 2 , SFM Ltd, Mercury) containing a nitrite source and a proton source, similar to Example 1A.

[0372] Nitric oxide release was measured by applying the one-piece dressing to a laboratory wound model and analyzing the evolved gaseous nitric oxide by selected ion flow tube mass spectrometry (SIFT-MS). A schematic of the apparatus used is shown in Figure 7 .

[0373] A laboratory wound model consisting of a shallow 5 cm diameter cylindrical cup with an inlet and an outlet was used, placed at a 2° angle of inclination towards the inlet within a heated stainless steel plate (30 °C). Two 4.9 cm diameter cellulose filter papers pre-saturated with sodium chloride solution (0.9% w / v) were placed inside the cup to form a surrogate "wound bed".

[0374] The inlet was connected to a syringe pump, which aspirated sodium chloride solution (0.9% w / v) at a rate of 0.4 ml / hr during the duration of the test. A 5x5 cm square piece of the one-piece dressing was cut from the main sample and weighed.

[0375] The dressing was placed on the "wound bed". To prevent leakage during the test, a 10x10 cm blank carboxymethylcellulose-based fabric (120 g / m 2, the sample is surrounded by a frame of SFM Ltd, Mercury).

[0376] A 10x10 cm stainless steel container with a grid across its surface is placed on top of the dressing, with the grid facing down and touching the dressing to act as a weight.

[0377] A measurement chamber (1100 ml plastic box) with an inlet and an outlet leading to SIFT-MS is placed on the stainless steel plate of the laboratory wound model, which entirely covers the dressing, the surrounding fabric frame, and the stainless steel container. The plastic box is pressed down with a weight of approximately 1 kg.

[0378] A pipe is connected to the outlet of the plastic box, which is connected to a Dreschel bottle containing 2 mM sodium hydroxide solution, then to a Dreschel containing silica beads, and finally to the inlet of SIFT-MS. Air is drawn through the system at approximately 30 ml / min and analyzed by SIFT-MS. The NO release curve over 2000 minutes is shown in Figure 5 .

[0379] Example 11: NO Release from a Multilayer Composite Dressing Containing a One-Piece Nitric Oxide Generating Layer Comprising a Nitrite Source and a Proton Source ( Figure 6 )

[0380] Using a high-intensity alternating electric field, similar to Example 1A, a hydroactive wound pad made of polyester fibers with superabsorbent powder (136 g / m 2 ) is impregnated with a powder containing a nitrite source and a proton source (20 g / m 2 basis weight, Freudenberg, M1520).

[0381] Nitric oxide release is measured by applying the one-piece dressing to the laboratory wound model and analyzing the evolved gaseous nitric oxide by selected ion flow tube mass spectrometry (SIFT-MS).

[0382] A laboratory wound model consisting of a shallow 5 cm diameter cylindrical cup with an inlet and an outlet is placed inside a heated stainless steel plate (30 °C) at a 2° tilt angle towards the inlet. Two 4.9 cm diameter cellulose filter papers pre-saturated with sodium chloride solution (0.9% w / v) are placed inside the cup to form a surrogate "wound bed".

[0383] The inlet is connected to a syringe pump, which draws sodium chloride solution (0.9% w / v) at a rate of 0.4 ml / hr during the duration of this test. A 5x5 cm square piece is cut from the main sample of the one-piece dressing and weighed. An absorbent wicking material (20 g / m2 Polypropylene, a 5x5 cm square sheet from Absorb, Don&Low Ltd).

[0384] Place the laminated dressing on the "wound bed". To prevent leakage during the experiment, surround the sample with a 10x10 cm blank carboxymethylcellulose-based fabric (120 g / m 2 , SFM Ltd, Mercury) with a window of approximately 5x5 cm cut out in the center. Place a 10x10 cm stainless steel container with a grid across its surface on top of the dressing, with the grid facing down and touching the dressing to act as a weight.

[0385] Place a measurement chamber (1100 ml plastic box) with an inlet and an outlet leading to SIFT-MS on the stainless steel plate of the laboratory wound model, which entirely covers the dressing, the surrounding fabric frame, and the stainless steel container.

[0386] Press the plastic box with a weight of approximately 1 kg. Connect a pipe to the outlet of the plastic box, which is connected to a Dreschel bottle containing 2 mM sodium hydroxide solution, then to a Dreschel containing silica beads, and finally to the inlet of SIFT-MS. Draw air through the system at approximately 30 ml / min and analyze it by SIFT-MS. The NO release curve for 2000 minutes is shown in Figure 6 .

Claims

1. A wound dressing for treating wounds, the wound dressing comprising a nitric oxide generating layer that generates nitric oxide through acidification of nitrite, wherein the nitric oxide generating layer comprises a solid powder nitrite component and a solid proton source component, and wherein the solid powder nitrite component and the solid powder proton source component are provided by the following: a. A blend of one or more single particles containing nitrite and one or more single particles containing a proton source; b. One or more single particles each containing nitrite and a proton source; c. Particle aggregates, wherein the particle aggregates comprise one or more single particles containing nitrite, one or more single particles containing a proton source, and an optional binder; d. Particle aggregates, wherein the particle aggregates comprise one or more single particles containing nitrite and a proton source and an optional binder; or e. A combination thereof.

2. The wound dressing according to claim 1, wherein the nitric oxide generating layer comprises a dry wound dressing substrate.

3. The wound dressing according to claim 2, wherein the dry proton source component comprises a solid powder proton source component, and the solid powder proton source component is mixed with the dry wound dressing substrate.

4. The wound dressing according to claim 2 or 3, wherein at least a portion of the dry proton source component forms part of the dry wound dressing substrate.

5. The wound dressing according to any one of claims 2 to 4, wherein the dry proton source component comprises a solid powder proton source component, and another portion of the dry proton source component forms part of the dry wound dressing substrate.

6. The wound dressing according to any one of claims 2 to 5, wherein the dry wound dressing substrate is composed of woven or non-woven fibers.

7. The wound dressing according to any one of claims 1 to 6, wherein all components of the nitric oxide generating layer are dry components.

8. The wound dressing according to any one of claims 1 to 7, wherein, based on the weight of the nitric oxide generating layer, the water content of the nitric oxide generating layer is 10% or less, 5% or less, 2% or less, or 1% or less.

9. The wound dressing according to any one of claims 1 to 8, wherein the wound dressing is a one-piece wound dressing.

10. The wound dressing according to any one of claims 1 to 9, wherein the wound dressing further comprises one or more additional layers in addition to the nitric oxide generating layer.

11. The wound dressing according to claim 1, wherein one or more of the single particles or particle aggregates are blended with or coated with an excipient for influencing the rate of water entry into the particles and / or an excipient for influencing the kinetics of nitric oxide formation from the particles.

12. The wound dressing according to claim 11, wherein the excipient for influencing the rate of water entry into the particles is a polyol or a hydrophobic material such as phospholipid, magnesium stearate, or colloidal silica, and / or the excipient for influencing the rate of water entry into the particles is a nitric oxide or nitric oxide precursor chelating material such as thiol, alcohol, amine, or amide.

13. The wound dressing according to claim 11 or claim 12, wherein the particles containing nitrite and a proton source are formed by spray drying a mixture containing a nitrite solution and a proton source solution.

14. The wound dressing according to claim 11 or claim 12, wherein a blend of one or more single particles containing nitrite and one or more single particles containing a proton source is formed as follows: (a) micronizing a nitrite solid together with a proton source solid; or (b) combining the two solids as follows: (i) spray drying or freeze drying a solution containing nitrite, (ii) spray drying or freeze drying a solution containing a proton source, and (iii) blending the solids produced in steps (i) and (ii).

15. The wound dressing according to any one of claims 1 to 14, wherein the proton source comprises an acid precursor such as an ester or a photoacid.

16. The wound dressing according to any one of claims 1 to 15, wherein the wound dressing comprises one or more additional dry layers adjacent to the nitric oxide generating layer.

17. The wound dressing according to any one of claims 1 to 16, which further comprises one or more additional layers adjacent to the nitric oxide generating layer, provided that the water content of any layer adjacent to the nitric oxide generating layer is 5% or less, 2% or less or 1% or less based on the weight of the layer adjacent to the nitric oxide generating layer.

18. The wound dressing according to any one of claims 1 to 17, wherein the wound dressing comprises an antimicrobial agent.

19. A packaged wound dressing comprising the wound dressing according to any one of claims 1 to 18 within a low moisture permeability package.

20. The packaged wound dressing according to claim 19, wherein the low moisture permeability package comprises one or more low moisture permeability materials (such as aluminum foil) in the walls of the package and / or can be hermetically sealed.

21. The packaged wound dressing according to claim 19 or 20, wherein the packaging atmosphere within the packaged wound dressing has a low moisture content at the time of initial packaging and / or the package comprises a moisture sequestering packaging insert.

22. A method of treating a wound, the method comprising applying the wound dressing according to any one of claims 1 to 18 to a wound of a subject.

23. The method of treating a wound according to claim 22, wherein the wound dressing is a one-piece wound dressing.

24. A combination of a solid powder nitrite component and a solid powder proton source component in the wound dressing according to any one of claims 1 to 18 for treating a wound of a subject.

25. The combination for treating a wound according to claim 24, wherein the wound dressing is a one-piece wound dressing.

Images