Wound dressing material

Abstract

Provided are wound dressings for treating wounds, which include a nitric oxide generating layer for generating nitric oxide by acidification of nitrite, wherein the nitric oxide generating layer includes a solid powder nitrite component and a solid proton source component, as well as methods and uses of such wound dressings for treating wounds.

Description

[Technical Field]

[0001] The present invention relates to a wound dressing for treating a wound, comprising a nitric oxide-generating layer (comprising both a solid powdered nitrite component and a solid proton source component) for generating nitric oxide by acidifying nitrite. [Background technology]

[0002] Nitric oxide (NO) and its precursors have been widely investigated as potential pharmaceutical agents. However, substantial problems remain with regard to the efficient generation and delivery of nitric oxide, other oxides of nitrogen, and their precursors to organs and cells for therapy. A widely adopted system for the generation of nitric oxide is based on the acidification of nitrite with a proton source, such as an acid, to first produce nitrous acid (HNO), which then readily decomposes to nitric oxide and nitrate, along with hydrogen ions and water, according to the following equilibrium equation (1):

number

[0003] The acid and nitrite are typically provided in predetermined amounts as separate components that are kept separate until the time of use to minimize reaction until the time of need. Thus, these two reactants are provided in a two-component system, with one component containing the nitrite and one component containing the acid. In this way, the two separate components in the two separate components can be combined or mixed at the time needed to prevent the release of nitric oxide before they are needed.

[0004] The properties of nitric oxide, such as killing and preventing the growth of microorganisms, make it useful in the treatment of wounds, skin lesions, and burns. Therefore, methods for producing and delivering nitric oxide also find application in wound dressings. However, acid and nitrite salts still need to be stored separately until use, and for this reason, two-component wound dressing systems have been developed.

[0005] Typically, such two-component wound dressings include a first component containing a nitrite and a second component containing an acid, with the two components stored separately until needed. Therefore, two-component wound dressings are packaged as two separate pieces that must be combined to initiate the reaction. The use of two-component systems has drawbacks. For example, combining the two components when needed can lead to application errors or inaccurate dosages.

[0006] In contrast, one-component (also referred to as single-component) wound dressings provide a single piece of wound dressing until the time of use. Single-component wound dressings typically require minimal preparation (e.g., removal of a protective film) before application to a subject, making them easier to use. Therefore, it is desirable to provide the nitrite and acidifying source as a one-component or single-component wound dressing. In addition, providing a single-component product can reduce manufacturing complexity, cost, and packaging.

[0007] However, a problem arises when attempting to provide nitrite acidification in a single component system in that the system loses a significant proportion of its nitric oxide during production and storage as the reaction begins, potentially not providing enough nitric oxide when needed.

[0008] WO 2021 / 198461 describes a nitric oxide-generating wound dressing. The wound dressing described requires that a nitric oxide-releasing agent and an activator (a species that activates and / or promotes the release of nitric oxide from the nitric oxide-releasing agent) are present in the wound dressing as separate, distinct layers (a nitric oxide source layer and an activator layer). In addition to the separate, distinct layers, the document also describes the use of a separation layer to further prevent contact between the nitric oxide source layer and the activator layer prior to use.

[0009] Therefore, there is a need for a single-component wound dressing that provides the species needed to generate and deliver nitric oxide, such as nitrite and acid species, until the time of need without causing premature or unwanted release of NO. Summary of the Invention

[0010] The present inventors sought to provide a simple wound dressing for delivering nitric oxide by acidifying nitrite. In its most general form, the present invention provides a wound dressing having a nitric oxide-generating layer, which includes both a solid nitrite salt and a solid proton source. In this manner, the nitrite salt and proton source can be provided in a single-component system, maintaining close proximity (or intimate association) to provide acidification of the nitrite salt upon contact with an aqueous environment, but not substantially reacting until needed. By including solid components of both the nitrite salt and the acid source, the incorporation of a moisture source (such as a solution or aqueous gel) can be avoided. In this manner, exposure of the reactants to moisture is reduced and reaction is minimized until needed.

[0011] In a first aspect, the present invention provides a wound dressing for treating a wound (comprising a nitric oxide generating layer that generates nitric oxide by acidification of nitrite), wherein the nitric oxide generating layer comprises a component of nitrite in a solid powder form, and a component of a solid proton source.

[0012] The nitric oxide generating layer may include a dry wound dressing substrate. The solid powder nitrite salt 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 may include proton source fibers. In certain embodiments, the dry proton source component includes a solid powder proton source component, and a further portion of the dry proton source component forms part of the dry wound dressing substrate.

[0013] The substrate of the dry wound dressing may be composed of woven or nonwoven fibers. The components of the nitric oxide producing layer may all be dry components. The moisture content of the nitric oxide producing layer may be 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the nitric oxide producing layer.

[0014] The wound dressing may be a one-piece wound dressing, in other words the wound dressing may be provided as a single piece until needed. The wound dressing may comprise one or more further layers in addition to the nitric oxide producing layer, in other words the wound dressing may be a multi-layer wound dressing. The nitric oxide producing layer may be combined with other layers and / or materials to form a wound dressing.

[0015] The components of the solid powder nitrite and the solid powder proton source are: a. a blend of one or more individual particles containing nitrite and one or more individual particles containing a proton source; b. one or more individual particles, each particle containing nitrite and a proton source; c. an agglomerate of particles, wherein the agglomerate comprises one or more individual particles containing nitrite salts and one or more individual particles containing a proton source, and optionally comprises a binder; d. an agglomerate of particles, the agglomerate comprising one or more individual particles, each containing nitrite and a proton source, and optionally a binder; or e. A combination of these may be provided by

[0016] Individual particles or agglomerates of particles may be blended or coated with excipients that affect the rate of water penetration into the particle and / or that affect the rate of nitric oxide formation from the particle. The excipient that affects the rate at which water enters the particle may be a hydrophobic material such as a polyol or one or more phospholipids (e.g., dipalmitoylphosphatidylcholine, DPPC), magnesium stearate or colloidal silica, and / or the excipient that affects the rate at which water enters the particle may be a material that sequesters nitric oxide or a precursor of nitric oxide, such as a thiol, alcohol, amine or amide.

[0017] Particles containing nitrite and a proton source may be formed by spray drying a mixture containing a nitrite solution and a proton source solution. The blend of one or more individual particles containing nitrite and one or more individual particles containing a proton source can be prepared by (a) micronizing the nitrite solids together with the proton source solids; or (b) micronizing the two solids together. (i) spray-drying or freeze-drying a solution containing nitrite; (ii) spray-drying or freeze-drying a solution containing a proton source; (iii) blending the solids produced in steps (i) and (ii) The slits may be formed by either aligning the slits or by fitting the slits together.

[0018] The proton source may include an acid precursor such as an ester or a photoacid. The wound dressing may include one or more additional dry layers adjacent to the nitric oxide producing layer, and the moisture content of any layer adjacent to the nitric oxide producing layer may be 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the layer adjacent to the nitric oxide producing layer.

[0019] The wound dressing may further comprise an antimicrobial agent. In a second aspect, the present invention provides a packaged wound dressing comprising the wound dressing described herein within a low-moisture permeable package, which may comprise one or more low-moisture permeable materials (e.g., aluminum foil) in the walls of the package and / or may be sealed. The packaging atmosphere within the packaged wound dressing may have a low moisture content upon initial packaging and / or the package may include a moisture-sequestering pack insert.

[0020] In a third aspect, the present invention provides a method of treating a wound, the method comprising applying a wound dressing described herein to a wound in a subject. The wound dressing may be a one-piece wound dressing. In other words, the wound dressing may be provided as a single piece until needed.

[0021] In a fourth aspect, the present invention provides a combination of a solid powder nitrite salt component and a solid proton source component in a wound dressing described herein for use in treating a wound in a subject. The wound dressing may be a one-piece wound dressing. In other words, the wound dressing may be provided as a single piece until needed.

[0022] Any or particular features of one aspect of the invention described herein apply equally to other aspects of the invention, provided that the feature is compatible with the aspect. In particular, any or particular features of the wound dressing apply equally to the packaged wound dressing, the method of treating a wound, and the combination used to treat a wound, provided that the features are compatible with those aspects. [Brief explanation of the drawings]

[0023] The present invention will now be described in more detail, the examples and accompanying drawings being given as illustrations of the invention. [Figure 1] 1 shows the deposition patterns of powders of Examples 1A, 2, 3 and 4 on agarose containing Hank's balanced salt solution and a pH indicator (phenol red). [Figure 2] The cumulative NO production for Examples 1A, 2, 3 and 4 is shown. [Figure 3] 1 shows the sprouting intensity of HUVEC spheroids treated with Examples 1B and 6A, quantified with an image analysis system and measured as cumulative sprout length (CSL) per spheroid compared to a reference control. [Figure 4] 1 shows a schematic diagram of a wound dressing of the present invention. [Figure 5] 1 shows the NO release profile over 2000 minutes for a wound dressing of the present invention. [Figure 6] 1 shows the NO release profile over 2000 minutes for another wound dressing of the present invention. [Figure 7] A schematic diagram of an apparatus for measuring and analyzing generated gaseous nitric oxide by Selected-Ion Flow Tube Mass Spectrometry (SIFT-MS) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0024] The reaction between one or more nitrites and a proton source to produce nitric oxide, optionally other oxides of nitrogen, and / or optionally precursors thereof, is referred to herein as the "NOx-forming reaction," or "NOx-forming reaction," etc., where "NOx" is used to refer collectively to the products of the acidification of nitrites, particularly nitric oxide, other oxides of nitrogen, and precursors of both, individually or in any combination. It will be understood that each component of the NOx produced may evaporate as a gas, or may go into solution in the reaction mixture, or may initially go into solution and then evaporate as a gas, or any combination thereof.

[0025] The term "about" is used herein to mean that a numerical value is not strictly limited, and one of ordinary skill in the art will understand that the value may extend above or below the exact value (as appropriate) in accordance with the artisan's understanding of the value. The term "about" may mean a value up to ±10% of the value. Particle sizes referred to herein refer to volume mean diameter (VMD) unless otherwise specified.

[0026] As used herein, the terms "one-component," "single-component," "two-component," and "multi-component" refer to the number of pieces of a wound dressing until it is needed (e.g., application to a subject). For example, a one-component wound dressing is provided as a single piece until it is needed. A one-component wound dressing is typically applied to a subject as a single piece. In contrast, two-component and multi-component wound dressings are each provided in two or more pieces until they are needed, and are typically combined into a single-piece wound dressing just before application to a subject. It should also be noted that the one-component wound dressings described herein can be formed from a nitric oxide-producing layer and other layers or components, such as one or more backing layers.

[0027] wound dressing As used herein, a "wound dressing" is a material that is applied to the exterior of a subject (e.g., a human or animal) and is intended to cover, protect, and / or treat a lesion on the subject's skin. Wound dressings are suitable for use in connection with any breach or disruption of the skin barrier, which may be caused by an ulcer, surgery, burn, cut, lesion, wound, laceration, trauma, and / or abrasion.

[0028] Nitric oxide producing layer The wound dressing of the present invention includes a nitric oxide generating layer for generating nitric oxide by acidifying nitrite, wherein the nitric oxide generating layer includes a solid powder nitrite component and a solid proton source component.

[0029] In this manner, the nitrite and proton source may be in sufficient proximity to react when exposed to an aqueous environment. The nitrite and proton source are present in a single layer of the wound dressing. In this manner, these components do not need to be combined at the point of use (e.g., as part of a two-component system). Additionally, because the nitrite and proton source are in solid form, the water content can be minimized, thereby mitigating pre-use generation of nitric oxide.

[0030] Without being bound by theory, the inventors have found that nitrite and a proton source can be provided together in a single layer of a wound dressing when the species are provided as a dry, solid, powder composition. In this form, the nitrite and the proton source cannot react with each other and therefore do not produce NOx. However, when exposed to a moist environment, such as wound exudate, the nitrite and the proton source can react and produce NOx. Therefore, the wound dressing of the present invention does not need to be separated by a separating layer or a barrier layer to prevent the production of NOx until the time of use, and the nitrite and the proton source can be located adjacent to each other (i.e., the nitrite and the proton source can be provided in the same layer of the wound dressing).

[0031] Typically, all components of the nitric oxide generating layer are dry components. In this way, reaction between the nitrite and acid components is minimized until use. The moisture content of the nitric oxide generating layer can be 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the nitric oxide generating layer. In this way, reaction between the nitrite and the proton source reactants is minimized prior to use. Moisture content can be measured by standard laboratory methods, such as weighing a sample, removing the moisture (e.g., by drying in an oven at 100°C or greater), and then reweighing the sample.

[0032] Dry wound dressing substrate The nitric oxide generating layer may comprise a dry wound dressing substrate. The solid powder nitrite may typically be 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 a further portion of the dry proton source component forms part of the dry wound dressing substrate.

[0033] Dry wound dressing substrates are known per se. The dry wound dressing may be absorbent. The dry wound dressing substrate may be of a synthetic or natural polymer type. The dry wound dressing substrate may consist of woven or nonwoven fibers or solid foams. The dry wound dressing substrate may consist of fibers of cotton, rayon, polyester (such as PLGA) and / or gelling fibers such as alginates (salts of alginic acid) and carboxymethylcellulose and its salts. Additionally or alternatively, the dry wound dressing substrate may be a hydrophilic material (e.g., silicone) and / or a solid alginate foam.

[0034] Particles or particle aggregates of the solid powder nitrite component and / or the solid powder proton source component may be incorporated into or encapsulated in a substrate. In this manner, the solid powder may be held within the material by the substrate until exposed to moisture or an aqueous environment. Particles of the solid powder composition may be exposed or partially exposed on the surface of the substrate, or may be completely encapsulated in the substrate.

[0035] The material may be a fibrous material including substrate fibers, and particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may be incorporated or encapsulated in the fibrous material. The particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may be exposed or partially exposed on the surface of the substrate fibers, or may be fully encapsulated in the fiber network and fiber cross-section.

[0036] In some embodiments, the substrate of the dry wound dressing is porous, and at least some of the particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component are located within the pores of the substrate. In other words, the substrate may be porous and impregnated with particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component. In some embodiments, the substrate is porous by including pores on the surface of the substrate. In other embodiments, the substrate may be a porous mesh of substrate elements, such as polymer fibers, and the particles or particle agglomerates may be located in the void spaces between the substrate elements. In a specific example, the particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may be impregnated into the void spaces of the polymer fibrous mesh.

[0037] The particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may have a particle size suitable for dispersion in gelling fibers. The particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may have a particle size greater than about 5 μm. For example, the particles or particle agglomerates of the solid powder nitrite component and / or the solid powder proton source component may 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, or greater than about 1000 μm.

[0038] To obtain larger particle sizes, particles or particle agglomerates may be granulated. "Granulation" refers to the process of combining fine particle seeds to form larger particles as granules. Granulation can be performed, for example, by compressing the particles or agglomerates to obtain tablets, which are then crushed into granules. The particles or agglomerates may be compressed at about 1 to about 10 MT (metric ton), for example, at about 3 to about 7 MT. The particles or agglomerates may be compressed at about 3.8 MT. The particles or agglomerates may be compressed at about 6.5 MT. Tablets may be crushed into granules using a sieve, for example, a 1 mm sieve.

[0039] Binders may be added to the particles or aggregates to facilitate compression. Suitable binders include sugars, natural binders, or synthetic or semi-synthetic polymer binders. Sugars may include, for example, sucrose or liquid glucose. Natural binders include, for example, acacia, tragacanth, gelatin, starch lake, gelatinized 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, and polymethacrylate. The binder may be a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate (copovidone). The binder may be microcrystalline cellulose.

[0040] The binder may be incorporated into the composition at a % w / w of about 5% w / w to about 30% w / w, for example, the binder may be incorporated into the composition at a % w / w of about 10% w / w to about 25% w / w. Alternatively, the composition may be substantially free of binders. Particle size can be increased by such means in order to keep the particles or agglomerates trapped (embedded or encapsulated) between the fibers.

[0041] Particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component can be incorporated into the substrate during production of the substrate. A method for incorporating or encapsulating particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component in a substrate includes (i) mixing particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component with a non-polar solution containing the substrate or a substrate precursor to form a liquid-particle mixture, and (ii) solidifying the liquid-particle mixture to form a material incorporating or encapsulating particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component.

[0042] The liquid-particle mixture may be solidified by spinning the mixture into fibers. Methods known to those skilled in the art may be used to spin fibers. For example, the liquid-particle mixture may be solidified by dry spinning, wet spinning, gel spinning, or electrospinning. The liquid-particle mixture may be solidified by electrospinning. "Electrospinning" refers to a method of producing fibers in which electrically charged threads of a polymer solution or melt are drawn to the diameter of a fiber using electrical forces. The liquid-particle mixture may be solidified by gel spinning. "Gel spinning" refers to a method of producing fibers that relies on temperature-induced physical gelation for solidification.

[0043] Alternatively, particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component can be incorporated into the substrate after it is formed. For example, particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component may be impregnated into a porous substrate, such as a fibrous mesh substrate. In these examples, the substrate is already formed, and particles or particle agglomerates of the solid powder nitrite component and / or solid powder proton source component are added thereto. Specific examples of methods for impregnating a porous substrate with a solid powder composition include those described in EP 2331309 (and other techniques available from Fibroline France).

[0044] Solid powder nitrite components and solid proton source components The wound dressing of the present invention contains a solid powder nitrite component and a solid proton source component in a single nitric oxide generating layer, which can release nitric oxide through acidification of the nitrite when exposed to an aqueous environment or atmospheric moisture.

[0045] Nitrite solid powder ingredients The solid powder nitrite includes a nitrite. The selection of the nitrite is not particularly limited. The nitrite can be selected from one or more nitrites of alkali metals or alkaline earth metals. For example, the one or more nitrites can be selected from LiNO2, NaNO2, KNO2, RbNO2, CsNO2, FrNO2, AgNO2, Be(NO2)2, Mg(NO2)2, Ca(NO2)2, Sr(NO2)2, Mn(NO2)2, Ba(NO2)2, Ra(NO2)2, and any mixture thereof. The nitrite can be NaNO2 or KNO2. The nitrite can be NaNO2.

[0046] The nitrite may be a pharmaceutically acceptable grade nitrite. In other words, the nitrite may comply with one or more active pharmacopoeia monographs for nitrites. For example, the nitrite may comply with one or more of the United States Pharmacopoeia (USP), the European Pharmacopoeia, or the Japanese Pharmacopoeia monographs for nitrites.

[0047] In particular, the nitrite salt used may have one or more characteristics provided in paragraphs

[0032] to

[0060] and Table 1 in paragraph

[0204] of WO 2010 / 093746 (the entire disclosure of which is incorporated herein by reference).

[0048] Solid proton source components The solid proton source component includes a proton source. The proton source may be any species capable of acting as a source of protons for acidifying nitrite. The selection of the proton source is not particularly limited. The proton source may be, for example, an acid.

[0049] 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 matrix (e.g., as proton source fibers). The acid may be selected from one or more organic carboxylic acids or organic non-carboxylic reducing acids.

[0050] The expression "organic carboxylic acid" as used herein refers to any organic acid containing one or more -COOH groups in the molecule. The organic carboxylic acid may be linear or branched. The carboxylic acid may be saturated or unsaturated. The carboxylic acid may be aliphatic or aromatic. The carboxylic acid may be acyclic or cyclic. The carboxylic acid may be a vinyl carboxylic acid.

[0051] The organic carboxylic acid may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic carboxylic acids that can be used in the present disclosure include α-hydroxycarboxylic acids, β-hydroxycarboxylic acids, and γ-hydroxycarboxylic acids.

[0052] The expression "organic non-carboxylic reducing acid" as used herein refers to any organic reducing acid that does not contain a -COOH group in the molecule. The organic non-carboxylic reducing acid may be linear or branched. The non-carboxylic reducing acid may be saturated or unsaturated. The non-carboxylic reducing acid may be aliphatic or aromatic. The non-carboxylic reducing acid may be acyclic or cyclic. The non-carboxylic reducing acid may be vinylic.

[0053] The organic non-carboxylic reducing acid may have one or more substituents, such as one or more hydroxyl groups. Examples of hydroxyl-substituted organic non-carboxylic reducing acids that can be used in the present disclosure include acidic reductones, such as reductic acid (2,3-dihydroxy-2-cyclopentanone). The one or more organic carboxylic acids or non-carboxylic reducing acids may have a pKa of less than about 7.

[0054] The one or more organic carboxylic acids may comprise, consist of, or be themselves one or more reducing carboxylic acids. The organic carboxylic acid may be selected from, for example, salicylic acid, acetylsalicylic acid, acetic acid, citric acid, glycolic acid, mandelic acid, tartaric acid, lactic acid, maleic acid, malic acid, benzoic acid, formic acid, propionic acid, α-hydroxypropanoic acid, β-hydroxypropanoic acid, β-hydroxybutyric acid, β-hydroxy-β-butyric acid, naphthoic acid, oleic acid, palmitic acid, 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, salts thereof, and combinations thereof.

[0055] The organic carboxylic acid may be citric acid or a salt thereof. The carboxylic acid may be or include a polymeric or polymerized carboxylic acid, such as, for example, polyacrylic acid, polymethacrylic acid, copolymers of acrylic acid and methacrylic acid, polybutyric acid, polyglycolic acid, or copolymers of butyric acid and glycolic acid. As used herein, the term "organic carboxylic acid" also extends to partial or complete esters of organic carboxylic acids or partial or complete salts thereof, provided that they are capable of functioning as a proton source when used in accordance with the present invention.

[0056] The organic non-carboxylic reducing acid may be selected from, for example, ascorbic acid; ascorbate palmitate (ascorbyl palmitate); ascorbate 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; erythorbic acid; salts thereof; and combinations thereof.

[0057] The organic non-carboxylic reducing acid may be ascorbic acid or a salt thereof. The proton source, one or more organic carboxylic acids or organic non-carboxylic reducing acids, may suitably be present together with their conjugate bases. The acids and their conjugate bases may suitably form a buffer when in contact with or exposed to an aqueous environment. The acids and their conjugate bases may be provided in a ratio that achieves a desired pH when exposed to an aqueous environment.

[0058] The buffer system may be selected so that upon exposure to an aqueous environment, a desired pH is achieved and the NOx-producing reaction proceeds and is maintained. The buffer system may be selected so that the pH of the reaction is in the range of about 3 to 9, e.g., about 4 to 8. In physiological contact, or when in contact with living cells and organisms, the pH of the reaction may be in the range of about 5 to about 8. The conjugate base, if present, may be added separately or may be generated in situ from a proton source by adjusting the pH with an acid and / or base, e.g., a mineral acid and / or mineral base. The proton source may be a citric acid / citrate buffer system, for example a citric acid / trisodium citrate buffer system.

[0059] The proton source may be or may include 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 may be a species that can undergo hydrolysis to provide an acid species. In other words, an acid precursor may be hydrolyzable and release an acid upon hydrolysis. For example, an acid precursor may be an ester. An acid precursor may be a photoacid. In other words, an acid precursor may be a species that absorbs light to become more acidic. For the avoidance of doubt, the term "photoacid" as used herein includes species that undergo reversible proton photolysis and species that undergo irreversible proton photolysis.

[0060] The solid proton source component can be provided as a component of the dry wound dressing substrate (e.g., as a proton source fiber). In some embodiments, the solid proton source component includes a proton source fiber. In other words, the term proton source component includes a fiber capable of providing protons. Such proton source fibers include, but are not limited to, polyacrylic acid fibers (particularly, partially neutralized polyacrylic acid fibers) and polyester fibers (particularly, PLGA fibers).

[0061] In certain embodiments, the solid proton source component may include a combination of a solid powder proton source component and proton source fibers. In certain embodiments, the solid proton source component includes a solid powder proton source component. Those skilled in the art will appreciate that the choice of acid component / proton source may be selected depending on the desired application.

[0062] Combining a solid powder nitrite component with a solid proton source component The solid powder nitrite component and the solid proton source component are present in the nitric oxide generating layer. Typically, the solid powder nitrite component and the solid proton source component will be present as a mixture of said components. In this manner, the solid powder nitrite component and the solid proton source component are present in such proximity that they will react when exposed to an aqueous environment.

[0063] When the nitric oxide generating layer includes a solid proton source component as a component of a dry wound dressing substrate, the solid powder nitrite component and, if present, the solid powder proton source component may be combined by the methods described herein for combining solid powder components with a dry wound dressing substrate.

[0064] The mixing of the solid powder nitrite component and the solid powder proton source component can be achieved by a number of methods. The solid powder nitrite component and the solid powder proton source component may be added independently to the nitric oxide generating layer. In other words, the solid powder nitrite component may be added separately from the solid powder proton source component to the nitric oxide generating layer.

[0065] In certain embodiments, the solid powder nitrite component and the solid powder proton source component are mixed either before adding them to the other components of the nitric oxide producing layer or during the formation of the nitric oxide producing layer. There are various methods for mixing the solid powder nitrite component and the solid powder proton source component. A preferred method for mixing the solid powder nitrite component and the solid powder proton source component is described below.

[0066] Combination of a solid powder nitrite component and a solid powder proton source component The nitrite component of the solid powder and the proton source component of the solid powder may be provided by one or more particles each containing a nitrite and a proton source. It should be recognized that when a particle contains both a proton source and a nitrite, the particle may contain the nitrite and the proton source within the same particle.

[0067] Additionally, or alternatively, the nitrite component of the solid powder and the proton source component of the solid powder can be provided by one or more particles containing nitrite but not a proton source, and one or more particles containing a proton source but not a nitrite. It should be recognized that the particles may contain either nitrite or a proton source within the same particle, or may not contain both nitrite and a proton source. One or more particles containing either nitrite or a proton source may be blended to obtain a substantially homogeneous mixture of particles.

[0068] A "homogeneous mixture" is a mixture that is uniform in composition and has its components in the same proportions throughout. Thus, one or more particles containing either nitrite or a proton source may be blended to provide a composition with a uniform distribution and proportion of nitrite particles and proton source particles.

[0069] The particle or particles may be present in the wound dressing as individual particles, or as agglomerates of individual particles, or a combination thereof. As used herein, the terms "agglomerate," "aggregate," and "aggregated together" refer to an aggregation or collection of primary (individual) particles that exhibit a discernible collective behavior.

[0070] In the present invention, the aggregate of individual particles comprises (i) individual particles containing nitrite and individual particles containing a proton source, (ii) individual particles containing nitrite and a proton source, or (iii) a combination thereof, and optionally includes a binder. In the present invention, the distinguishable collective behavior may be resistance to mechanical separation, i.e., particles adhering to each other.

[0071] The particles or agglomerates of the solid powder nitrite component and the solid powder proton source component may have a particle size suitable for their desired use or application. For example, the particles or agglomerates of the solid composition may have a particle size of about 10 μm or less, e.g., 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.

[0072] Alternatively, the particles or agglomerates of the solid powder nitrite salt 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, or greater than 1000 μm.

[0073] The weight ratio of nitrite to proton source in a 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. The mixture of the solid powder nitrite salt component and the solid powder proton source component may contain further optional additives such as a binder (as described above) or an organic polyol.

[0074] Binder The mixture of the solid powder nitrite salt component and the solid powder proton source component may be substantially free of one or more binders, or alternatively, the mixture of the solid powder nitrite salt component and the solid powder proton source component may further comprise one or more binders. As used herein, "binder" refers to an agent that promotes particle adhesion, i.e., promotes the formation of particle aggregates.

[0075] Suitable binders include sugars, natural binders, or synthetic or semi-synthetic polymer binders. Sugars may include, for example, sucrose or liquid glucose. Natural binders may include, for example, acacia, tragacanth, gelatin, starch, 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, and polymethacrylate. The binder may be a copolymer of 1-vinyl-2-pyrrolidone and vinyl acetate (copovidone). The binder may be microcrystalline cellulose.

[0076] The binder may be blended 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 blended 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.

[0077] organic polyol The mixture of the solid powder nitrite salt component and the solid powder proton source component may be substantially free of one or more organic polyols, or alternatively, the mixture of the solid powder nitrite salt component and the solid powder proton source component may further comprise one or more organic polyols. When the mixture of the solid powder nitrite salt component and the solid powder proton source component contains one or more organic polyols, it is preferable to add the organic polyols to the mixture of the solid powder nitrite salt component and the solid powder proton source component after any treatment including solvent removal (e.g., after a spray-drying or freeze-drying step). In other words, the polyol may be added to a composition containing one or more particles containing nitrite salt and a proton source (either before or after the particle aggregates described below are formed); or it may be added to a mixture containing one or more particles containing nitrite salt and / or one or more particles containing a proton source.

[0078] The expression "organic polyol" as used herein refers to an organic molecule having two or more hydroxyl groups that is not a proton source, particularly not a proton source for the reaction of nitrite, and that is not a sugar or polysaccharide (the terms "sugar" and "polysaccharide" include oligosaccharides, glycans, and glycosaminoglycans). Thus, an organic polyol will have a pKa of about 7 or greater.

[0079] The term "organic polyol" as used herein preferably excludes reducing agents. Examples of reducing agents that are organic molecules having two or more hydroxyl groups and that are not sugars or polysaccharides include thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbic acid, ascorbate, erythorbic acid, and erythorbate. Thus, thioglycerol (e.g., 1-thioglycerol), hydroquinone, butylated hydroquinone, ascorbate, and erythorbate are preferably excluded from the term "organic polyol" because they are reducing agents. Ascorbic acid and erythorbic acid are excluded from the term in any case because they are proton sources, particularly for the nitrite reaction.

[0080] The organic polyol may be cyclic or acyclic, or may 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 may be selected from one or more alkanes substituted with two or more OH groups, one or more cycloalkanes substituted with two or more OH groups, one or more cycloalkylalkanes substituted with two or more OH groups, and any combination thereof. The organic polyol may not have any substituents other than OH.

[0081] 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, for example, 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.

[0082] Alternatively, the one or more organic polyols may include 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.

[0083] The one or more organic polyols may have seven or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having seven or more hydroxyl groups. The one or more organic polyols may have nine or more hydroxyl groups. The one or more organic polyols may be sugar alcohols or alditols having nine 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, for example, in the range of 9 to 15. The one or more organic polyols may contain 9, 12, 15, or 18 hydroxyl groups.

[0084] The one or more organic polyols may be sugar alcohol compounds comprising, for example, one or more monosaccharide units and one or more acyclic sugar alcohol units. The one or more organic polyols may be sugar alcohol compounds comprising, for example, linear chains of one or more monosaccharide units and one or more acyclic sugar alcohol units, or branched chains of one or more monosaccharide units and one or more acyclic sugar alcohol units.

[0085] As used herein, a "monosaccharide unit" refers to a monosaccharide covalently linked to at least one other unit (whether another monosaccharide unit or an acyclic sugar alcohol unit) in a compound. As used herein, an "acyclic sugar alcohol unit" refers to an acyclic sugar alcohol covalently linked to at least one other unit (whether a monosaccharide unit or another acyclic sugar alcohol unit) in a compound. The units of a compound may be linked via an ether bond. One or more monosaccharide units may be covalently linked to other units of the compound via glycosidic bonds. Each monosaccharide unit may be covalently linked to other units of the compound via glycosidic bonds. A sugar alcohol compound may be a glycoside having a monosaccharide or oligosaccharide glycone and an acyclic sugar alcohol aglycone.

[0086] The acyclic sugar alcohol unit may be a sugar alcohol having 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms. The acyclic sugar alcohol unit may be selected from the group consisting of erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, and volemitol units.

[0087] The one or more monosaccharide units may be C5 or C6 monosaccharide units, i.e., pentose or hexose units. Each monosaccharide unit may be a C5 or C6 monosaccharide unit. The one or more sugar alcohol units may be a C5 or C6 sugar alcohol unit. Each sugar alcohol unit may be a C5 or C6 sugar alcohol unit.

[0088] A sugar alcohol compound may comprise, e.g., consist of, n monosaccharide units and m acyclic sugar alcohol units, where n is a natural number, m is a natural number, and (n + m) does not exceed 10. A sugar alcohol compound may comprise, e.g., consist of, a chain of n monosaccharide units terminated by one acyclic sugar alcohol unit (where n is an integer from 1 to 9). The chain of monosaccharide units may be covalently linked by glycosidic bonds. Each monosaccharide unit may be covalently linked to another monosaccharide unit or to an acyclic sugar alcohol unit by a glycosidic bond. A sugar alcohol compound may comprise, e.g., consist of, one, two, or three units terminated by one acyclic alcohol unit. One, two, three, or each monosaccharide unit may be a C5 or C6 monosaccharide unit. The acyclic alcohol unit may be a C5 or C6 sugar alcohol unit. Examples of sugar alcohol compounds include, but are not limited to, isomalt, maltitol, and lactitol (n=1); maltotriitol (n=2); and maltotetriitol (n=3).

[0089] Such sugar alcohol compounds may be described as sugar alcohols derived from disaccharides or oligosaccharides. As used herein, "oligosaccharide" refers to a sugar consisting of 3 to 10 monosaccharide units. Sugar alcohols derived from disaccharides or oligosaccharides may be synthesized (e.g., hydrogenated) from disaccharides, oligosaccharides, or polysaccharides (e.g., from hydrolysates and hydrogenated products), but are not limited to compounds synthesized from disaccharides, oligosaccharides, or polysaccharides. For example, sugar alcohols derived from disaccharides may be formed by the dehydration reaction of a monosaccharide with a sugar alcohol. One or more organic polyols may be sugar alcohols derived from disaccharides, trisaccharides, or tetrasaccharides. Examples of sugar alcohols derived from disaccharides include, but are not limited to, isomalt, maltitol, and lactitol. Examples of sugar alcohols derived from trisaccharides include, but are not limited to, maltotriitol. Examples of sugar alcohols derived from tetrasaccharides include, but are not limited to, maltotetriitol.

[0090] The organic polyol may be selected from erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol, polyglycitol, and combinations thereof. When present, glycerol is preferably used in combination with one or more other organic polyols, such as erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetritoitol, polyglycitol, or any combination thereof.

[0091] Many organic polyols contain one or more chiral centers and therefore exist in stereoisomeric forms. All stereoisomeric forms and optical isomers of organic polyols, as well as mixtures of isomers, are intended to be included within the scope of the present invention. In particular, D and / or L forms of any chiral organic polyol, and any mixtures thereof, may be used.

[0092] Particle agglomeration Aggregation of the particles may be achieved by any suitable means known to those skilled in the art. Agglomeration of the particles may be achieved by mechanical means, for example, by mechanically pressing the particles together. Agglomeration by mechanical means may be achieved by micronizing the nitrite particles and the proton source particles. Alternatively, agglomeration by mechanical means may be achieved by providing the particles with a substantially static-free state.

[0093] Particle aggregation may be achieved by chemical means, e.g., chemically facilitating adhesion or by chemical coating. Chemical aggregation may be achieved by an adhesion promoter, e.g., moisture. Alternatively, chemical aggregation may be achieved by a coating material that binds the primary particles of the nitrite salt and the primary particles of the proton source together. Suitable binders have been described above, and suitable coating materials are described in the "Coating Particles" section below.

[0094] Coated particles One or more particles of the mixture of the solid powder nitrite component and the solid powder proton source component may be coated with an excipient (also referred to herein as coated particles). The coated particles may include a single particle containing nitrite and a proton source and coated with an excipient.

[0095] Alternatively, the coated particles may be an agglomerate of particles coated with an excipient, the agglomerate of particles including a mixture of (a) particles containing nitrite and a proton source, and / or (b) one or more nitrite particles containing nitrite and one or more proton source particles containing a proton source. In this way, the coated particles contain nitrite and a proton source within the same coating.

[0096] The excipient may be hydrophobic. The excipient may be any material capable of coating particles or aggregates such that the particles or aggregates are coated with a hydrophobic layer. The hydrophobic material may be a polymeric material, e.g., an organic polymeric material such as a polyol. The hydrophobic material may be an amphiphilic species, e.g., a surface-active species such as a nonionic, anionic, cationic, or amphoteric surface-active species. The hydrophobic material may be an inorganic mineral material, e.g., an inorganic mineral material that forms a 3D framework. The hydrophobic material may be biocompatible. The hydrophobic material may include one or more of poly(lactic-co-glycolic acid) (PLGA), a phospholipid such as dipalmitoylphosphatidylcholine (DPPC), magnesium stearate, and mesoporous silica. The hydrophobic material may include a polymeric material, poly(lactic-co-glycolic acid) (PLGA), that does not have acid end groups, or a polymeric material, poly(lactic-co-glycolic acid) (PLGA), that has acid end groups. Excipients may include polyols, magnesium stearate, colloidal silica.

[0097] As used herein, "surfactant" refers to a surface-active agent that can reduce the surface tension of species in a medium or the interfacial tension between media. Surface-active species generally have a hydrophilic head and a hydrophobic tail. The hydrophobic material may be attached to the particles or aggregates by chemical bonds or by electrostatic or intermolecular forces.

[0098] The coating of the coated particle or coated agglomerate of particles can affect reaction dynamics, for example, the kinetics of nitrite acidification, when the coated particle or coated agglomerate is exposed to an aqueous environment. The excipient may be a species capable of capturing or sequestering nitric oxide or a nitric oxide precursor, for example, the excipient may include a thiol, an alcohol, an amine, or an amide.

[0099] The coated particles or agglomerates of the mixture of the solid powder nitrite salt component and the solid powder proton source component may have a particle size appropriate for the desired use or application. The coated particles or agglomerates of the mixture of the solid powder nitrite salt component and the solid powder proton source component may have a particle size of about 10 μm or less, e.g., 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 agglomerates of the mixture of the solid powder nitrite salt component and the solid powder proton source component may have a particle size greater than about 5 μm. For example, the particles or agglomerates of the mixture of the solid powder nitrite salt component and the solid powder proton source component may 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, or greater than about 1000 μm.

[0100] Particle formation from mixtures containing nitrite and proton source solutions The mixture of the solid powder nitrite salt component and the solid powder proton source component may be formed by spray drying or freeze drying a mixture containing the nitrite salt solution and the proton source solution.

[0101] Particles of a mixture of a solid powder nitrite component and a solid powder proton source component can be formed from a mixture containing a nitrite solution and a proton source solution. The particles thus formed must be formed by removing the solvent within a short time (e.g., within 30 seconds) after mixing the nitrite solution and the proton source solution, and / or by subjecting the mixture to reaction-inhibiting conditions (e.g., below the freezing point of the solvent) to remove the solvent after mixing the nitrite solution and the proton source solution. In this way, the solvent is removed from the mixture while minimizing acidification of the nitrite. Therefore, effective amounts of nitrite and proton source may be present in the resulting powder composition.

[0102] When the solvent is removed quickly after mixing the nitrite solution and the proton source solution, the solvent may be removed within 30 seconds after mixing the nitrite solution and the proton source solution. In some embodiments, the solvent is removed within 10 seconds, within 5 seconds, within 2 seconds, or within 1 second after mixing the nitrite solution and the proton source solution. In some embodiments, the solvent is removed within 500 milliseconds, within 100 milliseconds, within 50 milliseconds, or within 10 milliseconds after mixing the nitrite solution and the proton source solution.

[0103] In one embodiment, the particles can be formed by spray-drying a mixture containing a nitrite solution and a proton source solution. By subjecting the mixture to spray drying, the solvent can be removed within 30 seconds after mixing the nitrite solution and the proton source solution. Spray drying of materials is known per se.

[0104] The mixture is typically a mixture of an aqueous solution of nitrite and an aqueous solution of a proton source. When aqueous solutions are used, the time between mixing the two aqueous solutions is minimized to prevent acidification of the nitrite. The aqueous solution of nitrite and the aqueous solution of acid can be mixed in-line for about 1 to about 10 milliseconds, for example, about 3 to about 5 milliseconds, before spray drying. Spray drying can also be performed immediately after mixing the nitrite solution and the acid solution. As described, it is understood that mixing a mixture containing a nitrite solution and a proton source solution and then spray drying limits the time available for reaction between the proton source and nitrite components.

[0105] The particles formed by spray drying the mixture containing the nitrite solution and the acid solution may have a particle size of about 10 μm or less, e.g., 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. As described, spray drying a mixture containing a nitrite solution and an acid solution results in a mixture of a solid powder nitrite component and a solid powder proton source component, where each particle contains the nitrite salt and the proton source component.

[0106] The particles formed by spray-drying a mixture containing a nitrite solution and a proton source solution may be in any suitable form. For example, the particles formed by spray-drying a mixture containing a nitrite solution and a proton source solution may be in a crystalline form or an amorphous form. The particles formed by spray-drying a mixture containing a nitrite solution and a proton source solution may be in an amorphous form.

[0107] Additionally or alternatively, the mixture of the nitrite solution and the proton source solution may be placed under reaction-suppressing conditions (e.g., a temperature below the freezing point of the solvent) to remove the solvent before, during, or immediately after mixing the nitrite solution and the proton source solution. In this way, acidification of the nitrite salt is suppressed until the solvent is removed. In particular, the solvent may be an aqueous solvent.

[0108] A specific example of a reaction inhibiting condition is to keep the temperature of the mixture below the freezing point of the solvent. In this way, the reaction rate of the acidification of the nitrite can be slowed while the solvent is being removed. When the temperature of the mixture is below the freezing point of the solvent, the nitrite solution and the proton source solution are typically 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 solutions can occur.

[0109] In some embodiments, solvent removal may occur in a low-pressure gas state, particularly in a low-pressure gas state in combination with a temperature below the freezing point of the solvent being removed. A particularly useful technique for removing solvent under reaction-limiting conditions is lyophilization (also called "freeze-drying").

[0110] It should be noted that the terms "solvent removal" and / or "drying" are used herein to obtain a solid powder composition. These terms include, but are not limited to, the complete removal of solvent. In some examples, the solid powder composition may contain trace amounts of residual solvent. For example, the powder composition may contain up to about 10% residual solvent, e.g., 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, may be utilized after the initial removal of solvent to obtain a solid powder composition.

[0111] Combining solids to form particle agglomerates The mixture of the solid powder nitrite component and the solid powder proton source component may be formed by combining a nitrite-containing solid with a proton source-containing solid to form a particle agglomerate, wherein the particle agglomerate includes one or more particles containing nitrite and one or more particles containing the proton source.

[0112] Combining the nitrite salt-containing solid with the proton source-containing solid to form particle agglomerates can be accomplished, for example, by (a) blending particles of one or more nitrite salts with particles of one or more proton sources, where the nitrite salt particles are formed by spray drying a nitrite salt 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 salt solid with the proton source solid.

[0113] Blend of spray-dried nitrite particles and spray-dried acid particles The mixture of the solid powder nitrite component and the solid powder proton source component is (i) subjecting a solution containing nitrite to spray drying or freeze drying; (ii) subjecting a solution containing a proton source to spray drying or freeze drying; (iii) Blending the species (i) and (ii). It may be formed by

[0114] The mixture of the solid powder nitrite salt component and the solid powder proton source component may be a blend of nitrite salt particles and proton source particles, where the nitrite salt particles are formed by spray drying a nitrite salt solution and the proton source particles are formed by spray drying a proton source solution. The spray-dried nitrite salt particles and the spray-dried proton source particles can be blended by standard means known to those skilled in the art to obtain a blended solid powder composition.

[0115] The spray-dried nitrite particles and spray-dried proton source particles may be blended in a weight ratio of nitrite to proton source 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.

[0116] The spray-dried nitrite particles and the spray-dried proton source particles may be blended for a period of about 5 to about 60 minutes, for example, 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 may be blended for a period of about 20 minutes.

[0117] As described above, the particles formed by spray drying the nitrite solution and the acid solution and blending these components may have a particle size of about 10 μm or less, e.g., 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.

[0118] As described above, by subjecting the nitrite solution to spray drying and the proton source solution to spray drying and blending these components, a mixture of a solid powder nitrite component and a solid powder proton source component can be obtained, comprising particle agglomerates, wherein the agglomerates comprise one or more particles containing nitrite and one or more particles containing a proton source.

[0119] The particles formed by subjecting a nitrite solution to spray drying, a proton source solution to spray drying, and blending these components may have any suitable form. For example, the particles formed by subjecting a nitrite solution to spray drying, a proton source solution to spray drying, and blending these components may be crystalline or amorphous. The particles obtained by subjecting a mixture containing a nitrite solution and a proton source solution to spray drying may be amorphous.

[0120] Particles formed by micronizing nitrite solids with acid solids The particles may be formed by micronizing the nitrite solids with the acid solids. As used herein, the term "micronization" refers to a process of reducing the average particle size of a solid composition, typically to within the micrometer scale. Micronization can be achieved by standard processes known to those skilled in the art. For example, micronization can occur by grinding or milling the particles or by utilizing supercritical fluids.

[0121] When the proton source is an acid buffer system, the solid proton source may be two components: a solid acid component and a solid conjugate base component. The solid nitrite and solid proton source may be micronized in a ratio 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, e.g., 1:9 w / w nitrite:proton source. The particles formed by micronizing the nitrite solid with the proton source solid may have a particle size of about 10 μm or less, e.g., 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.

[0122] As described above, a solid powder composition of nitrite-containing particles and proton source-containing particles can be obtained by micronizing a nitrite solution with a proton source solution. As described above, a solid powder composition of agglomerates comprising nitrite-containing particles and proton source-containing particles can be obtained by micronizing a nitrite solution with a proton source solution. The particles formed by micronizing a nitrite solution with a proton source solution may be in any suitable form. For example, the particles formed by micronizing a nitrite solution with a proton source solution may be in a crystalline form or an amorphous form. The particles formed by micronizing a nitrite solution with a proton source solution may be in a crystalline form.

[0123] The particles formed by micronizing may include one or more optional additives (in addition to the proton source and nitrite), as described above. In particular, the particles formed by micronizing may include a binder, as described above. The binder may be micronized together with the nitrite solids and the proton source solids.

[0124] Other characteristics of wound dressings The wound dressing may be a one-piece wound dressing. A one-piece wound dressing contains all of the components needed to apply the wound dressing to a subject in a single piece. In this way, the wound dressing does not require assembly by a skilled person before application to a subject. The wound dressing may be a single layer (nitric oxide producing layer) wound dressing or a multi-layer (including a nitric oxide producing layer) wound dressing.

[0125] The wound dressing may particularly include a backing layer. The backing layer is typically positioned on the outer surface of the wound dressing, opposite the surface of the wound dressing adapted to be applied to a subject. In this way, the backing layer can protect the wound and the active ingredients of the wound dressing from the environment when applied. The backing layer may be stretchable. The backing layer may be permeable or semi-permeable to gas. The backing layer may be made of polyurethane. The backing layer may include an adhesive to attach the wound dressing to a subject. Backing layers for wound dressings are known per se.

[0126] The wound dressing may include a removable protective layer on the outer surface of the wound dressing to protect the components of the wound dressing (e.g., the nitric oxide-producing layer) before applying the wound dressing to a subject. The removable protective layer is typically removed from the wound dressing before applying the wound dressing to a subject, exposing the active components of the wound dressing (e.g., the nitric oxide-producing layer) to the wound site. The removable protective layer may be stretchable. The removable protective layer may be transparent or translucent.

[0127] The nitric oxide-producing layer may be intended to be applied directly to a wound on a subject during use. The wound dressing may be configured so that the nitric oxide-producing layer is applied directly to a wound on a subject during use. For example, the nitric oxide-producing layer may form the outer surface of the wound dressing. Alternatively, the nitric oxide-producing layer is adjacent to a removable outer protective film or layer for removal before the nitric oxide-producing layer is applied directly to a wound on a subject. In other words, the wound dressing may include a removable protective film that forms the outer surface of the wound dressing, where the nitric oxide-producing layer is adjacent to the removable protective film. In this way, the removable protective film can be removed before application, and the nitric oxide-producing layer can be applied directly to a wound on a subject.

[0128] Alternatively, one or more transmission layers may be adjacent to the nitric oxide-producing layer, and the one or more transmission layers may be applied to a wound dressing. The wound dressing may include one or more transmission layers adjacent to the nitric oxide-producing layer, and the one or more transmission layers may be configured to be applied directly to a wound of a subject during use. The wound dressing may further include an outer removable protective film or layer for removal before applying the one or more transmission layers directly to a wound of a subject. In other words, the wound dressing may include a removable film forming the outer surface of the wound dressing, where one or more transmission films are adjacent to the removable protective film and the one or more transmission layers are adjacent to the nitric oxide-producing layer. In this way, the removable protective film can be removed before application, and the one or more transmission layers adjacent to the nitric oxide-producing layer can be applied directly to a wound of a subject. The one or more transmission 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 producing layer.

[0129] The wound dressing may include one or more additional dry layers adjacent to the nitric oxide producing layer, and the moisture content of any layer adjacent to the nitric oxide producing layer may be 10% or less, 5% or less, 2% or less, or 1% or less based on the weight of the layer adjacent to the nitric oxide producing layer.

[0130] The material of the additional layer of the wound dressing may be a mesh (woven or nonwoven), a nonwoven batt, a film, a foam, an alginate, an amorphous hydrogel, a cross-linked hydrogel, or a membrane. The layers of the wound dressing may be formed from natural or synthetic materials; for example, the layers of the wound dressing may be carboxymethylcellulose fibers and synthetic polymer fabrics. The present invention is not limited to the uses and materials described above, and other suitable materials and uses for wound dressings will be known to those skilled in the art.

[0131] Further antimicrobial agents The acidified nitrite component and the proton source component typically have antimicrobial activity. In some embodiments, the wound dressing further comprises an antimicrobial agent. Antimicrobial agents are known per se. In some embodiments, the wound dressing includes AgNO2 as both the antimicrobial agent and the nitrite.

[0132] Packaged wound dressings The present invention also provides a packaged wound dressing comprising the wound dressing described herein within a low moisture permeable package. The low-moisture permeable packaging material may include one or more low-moisture permeable materials (e.g., aluminum foil) on the walls of the packaging material. In certain embodiments, the low-moisture permeable packaging material includes one or more low-moisture permeable materials (e.g., aluminum foil) on the walls of the packaging material and the wound dressing, and is sealed with high airtightness. The low-moisture permeable packaging material may include one or more low-moisture permeable materials (e.g., aluminum foil) on at least a portion of all outer walls of the packaging material.

[0133] The packaging atmosphere inside the packaged wound dressing may have a low moisture content initially in the package. The packaging atmosphere may have a relative humidity of 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. Relative humidity can be measured using a hygrometer. The packaging atmosphere may include an inert packaging gas such as nitrogen, argon, helium, or CO. The packaging atmosphere may include no more than 10%, no more than 8%, no more than 5%, no more than 2%, or no more than 1% oxygen. In some embodiments, the packaging atmosphere is substantially free of oxygen. Additionally or alternatively, the packaging may include one or more pack inserts that sequester moisture. Such pack inserts may be desiccant packs, such as silica gel packs.

[0134] Methods and combinations for treating wounds The present invention provides a method for treating a wound, the method comprising applying a wound dressing described herein to a wound in a subject. The wound dressing may be a one-piece wound dressing. In other words, the wound dressing may be provided as a single piece until needed. The present invention also provides a combination of a solid powder nitrite component and a solid proton source component in a wound dressing described herein for use in treating a wound in a subject. The wound dressing may be a one-piece wound dressing. In other words, the wound dressing may be provided as a single piece until needed.

[0135] In some embodiments, the method includes applying the packaged wound dressing to a wound in a subject. In other embodiments, the method includes removing a removable outer layer or film of the wound dressing before applying it to a wound in a subject. Typically, the method will not include combining two or more components of the wound dressing before applying it to a wound in a subject. In some embodiments, the method includes adding water (including aqueous solutions, suspensions, gels, or other forms containing water) to the nitric oxide-producing layer before applying the wound dressing to the subject's wound. The water may be added directly to the nitric oxide-producing layer or indirectly to the nitric oxide-producing layer (e.g., via one or more permeation layers adjacent to the nitric oxide-producing layer). The added water may be a sterile aqueous solution. The aqueous environment may be a sterile saline solution.

[0136] Alternatively, the wound dressing may be applied to the subject's wound without the addition of water, such that aqueous fluids from the subject (e.g., blood and / or exudate) may be absorbed by the nitric oxide producing layer of the wound dressing and activate nitric oxide production. The subject may be a human or animal subject. The subject may be a human or a domestic animal.

[0137] Manufacturing method of solid powder ingredients Method for producing a solid powder composition by removing the solvent The method of producing the mixture of solid powder nitrite salt component and solid powder proton source component may include removing solvent from the mixture of nitrite salt solution and proton source solution in such a way to minimize acidification before the solid powder composition is formed. In one embodiment, the method includes removing the solvent (by spray drying) in less than 30 seconds after mixing the nitrite solution and the proton source solution to form a solid.

[0138] In another embodiment, the method includes providing reaction retarding conditions (e.g., lyophilization) during solvent removal and before, during, and / or immediately after combining the nitrite solution and the proton source solution. In one embodiment, the method may include removing solvent from an aqueous mixture containing a nitrite solution and a proton source solution to form a solid powder.

[0139] The aqueous solution of nitrite may have a concentration ranging from 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 up to about 5 M, up to about 4 M, up to about 3 M, or up to about 2 M. For example, the aqueous solution of nitrite may have a concentration ranging from about 1 M to about 2 M, such as about 1.5 M. The aqueous solution of nitrite may have a pH of from about 6.5 to about 9, e.g., from about 7 to about 8.

[0140] The aqueous solution of the proton source may have a concentration ranging from about 0.1 M to about 5 M. The aqueous solution of the 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 the nitrite may have a concentration of up to about 5 M, up to about 4 M, up to about 3 M, or up to about 2 M. For example, the aqueous solution of the nitrite may have a concentration ranging from 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 4-6. The pH of the aqueous solution of the proton source may be adjusted using an inorganic base, for example, sodium hydroxide.

[0141] In some embodiments, 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 combining the nitrite solution and the proton source solution, hi some embodiments, the solvent is removed within 500 milliseconds, within 100 milliseconds, within 50 milliseconds, or within 10 milliseconds after combining the nitrite solution and the proton source solution.

[0142] spray drying The mixture of the solid powder nitrite component and the solid powder proton source component can be produced by subjecting a nitrite solution and a proton source solution to spray drying. The aqueous solutions of nitrite and acid may be mixed in-line for about 1 to about 10 milliseconds, e.g., about 3 to about 5 milliseconds, before spray drying. Spray drying may be performed immediately after mixing the nitrite and proton source solutions. It will be appreciated that by subjecting the mixture containing the combined nitrite and proton source solutions to spray drying as described, the time available for reaction between the proton source and nitrite components is significantly limited, and that the reaction may cease entirely if the water is rapidly removed.

[0143] Spray drying may be carried out at an outlet temperature in the range of about 60 to about 80° C., such as about 65 to about 75° C., or about 68 to about 70° C. Spray drying may be carried out at an atomization pressure in the range of about 1 to 6 bar Spray drying may be carried out at a liquid feed rate in the range of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / min, or about 3 g / min.

[0144] Reaction inhibition conditions Alternatively, the method may include providing reaction-quenching conditions (e.g., lyophilization) while removing the solvent, and before, during, and / or immediately after mixing the nitrite solution and the proton source solution. A specific example of a reaction inhibiting condition is to keep the temperature of the mixture below the freezing point of the solvent. In this way, the reaction rate of the acidification of the nitrite can be slowed down while the solvent is being removed. When the temperature of the mixture is below the freezing point of the solvent, the nitrite solution and the proton source solution are typically mixed at a temperature above the freezing point of the solvent, and then the temperature of the mixture is lowered to a temperature below the freezing point of the solvent. In this way, good mixing of the solutions may be achieved.

[0145] In some embodiments, solvent removal may be performed at low gas pressures, particularly low gas pressures in combination with temperatures below the freezing point of the solvent being removed. A particularly useful technique for removing solvent under quenching conditions is lyophilization (also called "freeze-drying").

[0146] The time required to remove the solvent after mixing the nitrite solution and the proton source solution under reaction-quenching conditions is about 10 minutes or less. Under these conditions, such rapid removal of the solvent (e.g., water) may not be critical. However, removal of the solvent within a relatively short time frame is also desirable to further limit acidification of the nitrite. In some embodiments, the solvent is removed within about 8 minutes or less, e.g., 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 under reaction-quenching conditions. In further embodiments, the solvent removal step occurs within 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.

[0147] It should be noted that the terms "solvent removal" and / or "drying" used herein refer to obtaining a solid powder composition. These terms include, but are not limited to, complete removal of the solvent. In some embodiments, the solid powder composition may contain trace amounts of residual solvent. For example, the powder composition may contain up to about 10% residual solvent, e.g., up to about 5% residual solvent, up to about 3% residual solvent, or up to about 1% residual solvent. To obtain a solid powder composition, additional drying techniques, such as vacuum drying, may be utilized after the initial removal of the solvent.

[0148] A method for combining particles to form particle aggregates The formation of particle agglomerates comprising particles containing nitrite and particles containing a proton source can be achieved in a number of ways. In one embodiment, the method comprises: (i) subjecting a nitrite solution to spray drying or freeze drying to form nitrite particles; (ii) subjecting the proton source solution to spray drying or freeze drying to form proton source particles; and (iii) blending the nitrite particles with the proton source particles; may include:

[0149] The aqueous solution of nitrite can have a concentration ranging from about 0.1 M to about 5 M. The aqueous solution of nitrite 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 nitrite can have a concentration of up to about 5 M, up to about 4 M, up to about 3 M, or up to about 2 M. For example, the aqueous solution of nitrite can have a concentration ranging from about 1 M to about 2 M, such as about 1.5 M. The aqueous solution of nitrite can have a pH of from about 6.5 to about 9, e.g., from about 7 to about 8.

[0150] The aqueous solution of the proton source can have a concentration ranging from about 0.1 M to about 5 M. The aqueous solution of the nitrite 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 nitrite can have a concentration of up to about 5 M, up to about 4 M, up to about 3 M, or up to about 2 M. For example, the aqueous solution of the nitrite can have a concentration ranging from 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 4-6. The pH of the aqueous solution of the proton source can be adjusted using an inorganic base, for example, sodium hydroxide.

[0151] Spray drying may be carried out at an outlet temperature in the range of about 60 to about 80° C., such as about 65 to about 75° C. or about 68 to about 70° C. Spray drying may be carried out at an atomization pressure in the range of about 1 to 6 bar. Spray drying may be carried out at a liquid feed rate in the range of about 1 to about 5 g / min, such as about 2 g / min to about 4 g / min or about 3 g / min.

[0152] In some embodiments, the spray-dried particles are further dried, for example, by vacuum drying. The spray-dried or freeze-dried particles of nitrite salt and the spray-dried or freeze-dried particles of proton source may be blended by standard means known to those skilled in the art to obtain a blended solid powder composition. The spray-dried or freeze-dried particles of nitrite and the spray-dried or freeze-dried particles of proton source may be blended in a weight ratio of nitrite to proton source ranging from about 1:1 to about 1:99, such as from about 1:4 to about 1:49 or from about 1:7 to about 1:24.

[0153] The spray-dried nitrite particles and the spray-dried proton source particles may be blended for a period of about 5 to about 60 minutes, for example, 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 may be blended for a period of about 20 minutes.

[0154] Method for producing a mixture of a solid powder nitrite component and a solid powder proton source component by micronization The method for preparing a mixture of a solid powder nitrite salt component and a solid powder proton source component may include micronizing the nitrite salt solid and the proton source solid to prepare a solid powder composition. Micronization is known per se. Micronization can be achieved by standard methods known to those skilled in the art. For example, micronization may be achieved by grinding or milling the particles or by utilizing supercritical fluids.

[0155] The nitrite solids may be micronized with the proton source solids for a period of about 5 to about 30 minutes, e.g., about 5 to about 20 minutes, or about 5 to about 15 minutes. The nitrite solids may be micronized with the proton source solids for a period of about 10 minutes. The nitrite solids may be micronized with the proton source solids at a venturi pressure of 8 bar and a milling pressure of 2 bar.

[0156] The inventors have found that co-micronizing a nitrite salt solid with a proton source solid (i.e., simultaneously) results in a solid powder composition that, when exposed to an aqueous environment, provides a better release of nitric oxide than a solid powder composition formed by blending a separately micronized nitrite salt powder with a separately micronized proton source powder.

[0157] Method for producing a composition of solid powder of coated particles The mixture of the solid powder nitrite component and the solid powder proton source component can be prepared by including particles coated with a hydrophobic material, by any of the following methods: (i) coating particles containing nitrite and a proton source with a hydrophobic material; or (ii) combining one or more nitrite particles containing a nitrite salt and one or more proton source particles containing a proton source, and then coating the mixture. may include:

[0158] The hydrophobic material may be the same hydrophobic material described above. The particles or agglomerates of particles may be coated in any suitable manner known to those skilled in the art.

[0159] Particles or particle aggregates can be coated by dispersing the particles or aggregates in a solution containing a hydrophobic material and drying the solution to obtain particles or particle aggregates coated with a layer of hydrophobic material. In some embodiments, the solution contains a non-polar solvent. In certain embodiments, the solution does not contain a polar solvent (e.g., methanol). Such a polar solvent can dissolve at least a portion of the particles. In particular, the solvent can be anhydrous.

[0160] The hydrophobic material may be, for example, PLGA. The particles or particle aggregates may be dried with the hydrophobic material in a 1:1 w / w ratio. The solution in which the particles or particle aggregates are dispersed or suspended may be a solution of DCM and the hydrophobic material.

[0161] In certain embodiments, a suspension of particles in a solution of a hydrophobic material is dried by spray drying. The solution containing the hydrophobic material in which the particles or particle aggregates are dispersed may be spray dried at an outlet temperature of about 28-30°C. The solution containing the hydrophobic material in which the particles or particle aggregates are dispersed may be spray dried at an atomization pressure of about 1 bar. The solution containing the hydrophobic material in which the particles or particle aggregates are dispersed may be spray dried at a liquid feed rate of about 2 g / min.

[0162] The coated particles or agglomerates of coated particles may have a particle size of less than about 10 μm, such as less than about 9 μm, for example, less than about 8 μm, less than about 7 μm, less than about 6 μm, or less than about 5 μm. The particles or particle aggregates may be coated by blending the particles or particle aggregates with a hydrophobic material to obtain particles or aggregates coated with a layer of the hydrophobic material. The hydrophobic material may be, for example, DPPC, magnesium stearate, mesoporous silica, or a combination thereof. The particles or aggregates may be blended with the hydrophobic material in a 1:1 w / w ratio. The hydrophobic material may be sieved before blending. Alternatively, the hydrophobic material may not be sieved before blending.

[0163] The particles or particle agglomerates may be blended with the hydrophobic material for a period of about 10 to about 40 minutes, or for a period of about 15 to about 30 minutes. The spray-dried nitrite particles and spray-dried proton source particles may be blended for a period of about 20 minutes.

[0164] aqueous environment The nitric oxide generating layer of the present invention typically releases NOx upon contact with an aqueous environment, although the aqueous environment is not particularly limited. The aqueous environment may be an aqueous biological fluid, such as a bodily fluid, which may include wound secretions or exudates and / or blood (plasma, serum, etc.). Alternatively, the aqueous environment may be a sterile aqueous solution. The aqueous environment may be a saline solution.

[0165] In some embodiments, the solid powder composition may be hygroscopic enough to absorb moisture from the air, sufficient to initiate the release of NOx.

[0166] Example Preparation of solid powder compositions Materials and analytical methods The following materials were purchased from commercial sources: sodium nitrite from Honeywell, citric acid from Sigma-Aldrich, trisodium citrate from Merck, sodium hydroxide from Fisher, PLGA RG 502H 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.

[0167] Unless otherwise stated, the following analytical methods were used: Sympatec Dry Powder Particle Size Distribution (PSD) Laser particle size analysis of the spray-dried powders was performed using a Sympatec HELOS particle size analyzer equipped with an R3 lens (range 0.5-175.0 μm) / R5 lens (range 0.5-875 μm) and an ASPIROS dispersion unit. Dispersion was achieved using compressed air at 3.00 bar and a vacuum of 60 mbar. Powders were filled into ASPIROS glass tubes in a low humidity environment (<25% RH) and sealed with parafilm until measurements were taken. Measurements were performed in triplicate unless otherwise noted, and average data were reported.

[0168] Example 1: A mixture containing a solution of a nitrite salt and a solution of a proton source is spray dried to form a solid powder composition A 1.5 M sodium nitrite feed solution (Feed Solution 1) was prepared by dissolving the required amount of sodium nitrite in deionized water. A 1 M citric acid feed solution adjusted to pH 4 (Feed Solution 2) was prepared by dissolving the required amount of citric acid in deionized water and adjusting the pH to 4 with 10 M aqueous sodium hydroxide. The pH of the solutions was measured using a Mettler Toledo Seven Compact pH meter.

[0169] 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-cure silicone L / S 14 tubing) connected using a Y-fitting and a single Masterflex peristaltic pump, and the feed solutions were combined just before spraying. A standard Buchi cyclone and collection pot was installed to collect the product.

[0170] The feed solution was subjected to spray drying in two batches under the following conditions: [Table 1]

[0171] Both batches were then vacuum dried for 24 hours using an Edwards Super Modulyo freeze dryer set at 25°C. Particle size distribution measurements were then performed on both batches 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 pressurized air at a pressure of 3.00 bar and depressurized air at 60 bar. Measurements were performed in triplicate.

[0172] The resulting particle size distribution measurements were as follows: [Table 2] VMD = Volume Average Particle Size

[0173] Example 2: Nitrite and proton source are spray dried separately and then blended to form a solid composition A solution of 1.5 M sodium nitrite was prepared by dissolving the required amount of sodium nitrite in deionized water. A solution of 1 M citric acid adjusted to pH 5.6 was prepared by dissolving the required amount of citric acid in deionized water and adjusting the pH to 5.6 with 10 M aqueous sodium hydroxide. The pH of the solution was measured using a Mettler Toledo Seven Compact pH meter.

[0174] These feed solutions were subjected to separate spray drying using a Buchi B290 spray dryer under the following conditions: [Table 3]

[0175] All batches were then vacuum dried for 24 hours using an Edwards Super Modulyo freeze dryer set at 25°C. Particle size distribution measurements were then performed on the three batches 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 pressurized air at a pressure of 3.00 bar and depressurized air at 60 bar. Measurements were performed in triplicate.

[0176] [Table 4]

[0177] The spray dried nitrite solids (component 2A) and spray dried citric acid solids (pH 5.6) (component 2C) were then blended in a Turbula T2F mixer at 46 rpm for 20 minutes in a ratio of 9:1 citrate solids:nitrite solids w / w to provide the powder composition of Example 2.

[0178] Example 3: Micronization of a Nitrite Solid with a Proton Source Solid to Produce a Solid Powder Composition Sodium nitrite, citric acid, and trisodium citrate were combined together 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.

[0179] The blend was micronized using an Attritor M3 fluid energy mill at a venturi pressure of 8 bar and a milling pressure of 2 bar. The blend was fed directly into a hopper at a target feed rate of approximately 2 g / min. The resulting powder (Example 3) was collected in a single collection jar under low humidity (20% RH).

[0180] Particle size distribution measurements were then performed 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 performed using pressurized air at 3.00 bar and depressurized air at 60 bar. Measurements were performed in triplicate.

[0181] The particle size distribution measurements obtained were as follows: [Table 5] VMD = Volume Average Particle Size

[0182] Reference Example 4: Separate Micronization of Nitrite and Proton Source Followed by Blending to Produce a Solid Composition Sodium nitrite was micronized using an Attritor M3 fluid energy mill at a venturi pressure of 8 bar and a milling pressure of 2 bar. Sodium nitrite was fed directly into the hopper at a target feed rate of approximately 2 g / min. The resulting powder (Compound 4A) was collected in a single collection jar under low humidity (20% RH).

[0183] Citric acid and trisodium citrate were combined together in the following weight proportions: 16.51% and 83.49%, respectively. The mixture was blended using a Turbula T2F mixer at 47 rpm for 10 minutes.

[0184] The blend was micronized using an Attritor M3 fluid energy mill at a venturi pressure of 8 bar and a milling pressure of 2 bar. The blend was fed directly into a hopper at a target feed rate of approximately 2 g / min. The resulting powder (Example 4B) was collected in a single collection jar under low humidity (20% RH).

[0185] Micronized nitrite solids (component 4A) and micronized citric acid solids (component 4B) were then blended in a Turbula T2F mixer at 46 rpm for 20 minutes in a ratio of 9:1 citrate solids:nitrite solids w / w to provide the powder composition of Reference Example 4.

[0186] NOx generation Examples 1A, 2, 3, and 4 were loaded into APTAR Unidose nasal spray (https: / / www.aptar.com / products / pharmaceutical / uds / ) supported on a rig 30 cm above a Petri dish (9.8 mm diameter) containing agarose with Hank's balanced salt solution and pH indicator (phenol red). Figure 1 shows the pattern of powder deposition due to local pH modification by the particles at the site of particle deposition.

[0187] Immediately after application, the plates were transferred to a sealed chamber and oxides of nitrogen (NOx) were measured over a 15-minute period by selected ion flow tube mass spectrometry (SIFT-MS). All powders, regardless of their manufacturing method, generated nitric oxide. However, over the 15-minute period, differences were observed in the total amount of NOx generated among the four powders.

[0188] It should be noted that agarose is buffered at a neutral to slightly alkaline pH, which should inhibit the reaction, but the particles can overcome this buffering effect for short periods of time and can negate the buffering effect in localized areas. The table below and Figure 2 show the cumulative generation of NO in Examples 1A, 2, 3, and 4. Experimental results are normalized for the % nitrite in the powder by the cumulative NO / nmole per mg of nitrite.

[0189] [Table 6]

[0190] Coated solid powder composition Example 5: Particles coated with hydrophobic materials DPPC or mesoporous silica Example 1B was blended with mesoporous silica in a 1:1 w / w ratio using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5A.

[0191] Example 1B was blended with DPPC in a 1:1 w / w ratio using a Turbula T2F mixer at 46 rpm for 20 minutes to provide the powder composition of Example 5B.

[0192] Example 3 was blended with mesoporous silica in a 1:1 w / w ratio using a Turbula T2F mixer at 46 rpm for 20 minutes to obtain the powder composition of Example 5C.

[0193] Example 3 was blended with DPPC in a 1:1 w / w ratio using a Turbula T2F mixer at 46 rpm for 20 minutes to provide the powder composition of Example 5D.

[0194] Example 6: PLGA-coated particles A PLGA RG 502 H solution was prepared by dissolving PLGA (1.5 g) in DCM (approximately 30 mL) to form a clear, colorless solution. Example 1B (1.5 g) was added to this solution with stirring to form a 1:1 w / w Feed Suspension 6A as a visually uniform white suspension.

[0195] Similarly, a separate PLGA RG 502H solution was prepared by dissolving PLGA (1.5 g) in DCM (approximately 30 mL) to form a clear, colorless solution. Example 3 (1.5 g) was added to this solution with stirring to form a 1:1 w / w Feed Solution 6B as a visually uniform white suspension.

[0196] The feed suspension was subjected to spray drying using a Buchi B290 spray dryer according to the method described above. The spray drying parameters are summarized below.

[0197] [Table 7]

[0198] Sample vials were placed horizontally in individual weigh boats in a low-humidity environment (28% RH). The caps were removed and the openings were covered with foil containing holes (punched with a needle). The samples were transferred to an Edwards Super Modulyo freeze dryer set at 25°C and subjected to vacuum drying for 24 hours (maximum observed vacuum pressure was approximately 0.1 mbar). After vacuum drying, the samples were transferred to a low-humidity (approximately 24% RH) environment and blanketed with nitrogen. The vials were then sealed with parafilm and placed in foil pouches with desiccant for storage at 2-8°C.

[0199] Particle size distribution measurements were then performed 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 pressurized air at a pressure of 3.00 bar and depressurized air at 60 bar. Measurements were performed in triplicate.

[0200] The particle size distribution measurements obtained were as follows: [Table 8] VMD = Volume Average Particle Size

[0201] Example 7: NOx Emissions of Coated Particles An aliquot (30 mg) of the powder sample was placed in a 60 mm Petri dish. A cellulose filter paper (50 mm diameter) was placed on top of the sample and gentle pressure was applied. 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 humidified air was drawn through the chamber at 650 ml / min for 30 minutes. The airflow from the outfeed was analyzed by Single Ion Flow Tube Mass Spectrometry (SIFT-MS).

[0202] [Table 9] TIFF2025535358000011.tif218146

[0203] Biological evaluation of the composition of the solid powder Example 8: Evaluation of the efficacy of four formulations against Pseudomonas aeruginosa Petri dishes containing Nutrient Agar (NA, available from AcuMedia) were prepared and allowed to stand. An inoculum of Pseudomonas aeruginosa (ATCC 9027) was prepared in phosphate-buffered saline (PBS, Sigma-Aldrich) at a concentration of 1x10 5 CFU mL -1 The inoculum was serially diluted to a final concentration of 0.1%. 100 mL of the inoculum was pipetted onto NA plates, spread, and allowed to dry at room temperature for 15 minutes. The lid was removed from the inoculated agar plate, and the open plate was placed inside an Aptar Unidose nasal spray can.

[0204] An Aptar delivery device containing the powder of either Example 1A, Example 3, Reference Example 4, or Example 2 was attached to an Aptar nasal spray device, and the powder (approximately 50 mg dose) was sprayed onto an agar plate. The table below shows the example used for each formulation.

[0205] [Table 10]

[0206] After 5 seconds, the lids were replaced on the agar plates, and the agar plates were incubated at 37°C ± 2°C for 16 hours. After incubation, the plates were photographed. For every plate, three biopsy punches were taken from a 2x2 cm area in the center of the agar plate. Bacteria were removed from each biopsy using a sterile cotton swab moistened with PBS, and any cells were suspended in 10 mL of PBS, then sonicated for 5 minutes, serially diluted, and plated onto NA. A negative control plate not exposed to the sprayed powder and a positive control plate to which 1 mL of bleach was added were also tested simultaneously. All tests were performed in quintuplicate.

[0207] For each test item, three replicates were randomly selected, and DNA was extracted from 400 μL of each biopsy using the DN easy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. Samples were eluted in AE buffer to a final volume of 100 μL.

[0208] For each extraction, qPCR was performed in triplicate using the QuantiNova Pathogen and IC kit (Qiagen) according to the manufacturer's instructions. Individual reaction tubes contained each primer at a final concentration of 16 μM and 5 μM of labeled probe.

[0209] The cycling conditions were as follows: 50°C for 10 minutes, 95°C for 2 minutes, 95°C for 5 seconds, 55°C for 30 seconds, and 72°C for 1 minute, for a total of 35 cycles. Each assay run was validated with a positive control (Pseudomonas aeruginosa) and a negative control (RNase-free water). Data were analyzed using Q-Rex software (Qiagen), and Cq values ​​were obtained from a predefined threshold. For each sample, the mean Cq value was calculated as 1x10 2 ~10 8 CFU mL -1 Compare with a standard curve in the established range of 10 CFU mL -1 It was calculated as follows.

[0210] Table 1: 1x10 cells after treatment with formulations 1, 2, 3, 4, and bleach compared to the untreated negative control. 5 CFU mL -1 Mean recovery and reduction of Pseudomonas aeruginosa from three biopsy punches taken from the center of nutrient agar plated with 1000 mg of ...

[0211] [Table 11] SD=standard deviation, CFU=colony forming unit, N / A=not applicable, *=p<0.05, **=p<0.01, ***=p<0.001

[0212] Biopsies taken from negative control plates showed an average of 7.44 ± 0.17 Log Pseudomonas aeruginosa. 10 CFU mL -1 A mean recovery rate of 3.52 ± 3.12 and 1.36 ± 2.13 Log of P. aeruginosa was observed from biopsies taken from formulations 2 and 3. 10 CFU mL -1 No viable P. aeruginosa was recovered from biopsies taken from plates containing formulations 1 and 4 or the positive control.

[0213] Table 2: 1x10 cells after treatment with formulations 1, 2, 3, 4, and bleach compared to the untreated negative control. 5 CFU mL -1Molecular quantification of Pseudomonas aeruginosa in biopsy punches taken from nutrient agar plated with HCl.

[0214] [Table 12] SD = standard deviation; CFU = colony forming units; # = quantification was below the limit of detection; ~ = quantification of positive control sample was performed at N=1, therefore standard deviation could not be calculated; N / A = not applicable; ** = p<0.01; *** = p<0.001

[0215] After treatment with powders of Formulation 1 and Formulation 4, 1x10 5 CFU mL -1 A significant reduction in the recovery of viable P. aeruginosa from biopsies taken from nutrient agar seeded with an inoculum of 0.01% was observed. No viable P. aeruginosa was observed when compared to the untreated negative control. Molecular quantitation reflects recovery from colony counts.

[0216] Example 9: Effect of powdered compositions on human umbilical vein endothelial cell (HUVEC) sprouting in a spheroid-based cellular angiogenesis assay 10x concentrated stock solutions / suspensions of Examples 1B and 6A were prepared in basal medium (without supplements and FCS) by vortexing and pipetting, after which semi-log dilution series were prepared in the same medium.

[0217] [Table 13]

[0218] endothelial cells Cells: HUVEC, primary human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany), passage 3–4 Morphology: Adhered and growing in a single layer like cobblestones Culture medium: Endothelial cell growth and basal medium (ECGM / ECBM, PromoCell) Subculture: Split 1:3; approximately 1 x 10 every 3-5 days 4 cells / cm 2Seed out with Incubation: 37℃, 5% CO2 Doubling time: 24-48 hours Storage: Approximately 1 x 10 in 70% medium, 20% FCS, 10% DMSO 6 Freeze in cells / ampoules Origin: Pooled donor human umbilical vein

[0219] Test Method Experiments were performed using a modified version 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 HUVECs into a hanging drop onto a plastic dish and allowing the spheroids to aggregate overnight. Next, 50 HUVEC spheroids were seeded into 0.9 ml of collagen gel, pipetted into each well of a 24-well plate, and allowed to polymerize. Preincubated test samples were added after 30 minutes by pipetting 10x working solutions (100 μl) onto the polymerized gel (see Table 1 for final assay concentrations). Plates were incubated at 37°C for 24 hours and fixed by adding 4% PFA (Roth, Karlsruhe, Germany).

[0220] quantitative The sprouting intensity of HUVEC spheroids treated with the test samples was quantified using an image analysis system, which measured the cumulative sprout length (CSL) per spheroid. Photographs of single spheroids were taken using an inverted microscope and digital imaging software—NIS-Elements BR3.0 (Nikon). The spheroid photographs were then uploaded to the Wimasis website for image analysis. The cumulative sprout length of each spheroid was measured using the image analyzer WimSprout. The average cumulative sprout length of 10 randomly selected spheroids was analyzed as an individual data point. The mean and SD values ​​for each triplicate data were converted to % of the reference control.

[0221] result Figure 3 shows the CSL versus the reference controls of Examples 1B and 6A. The effect of Example 1B (particles subjected to spray drying without coating) is small compared to the reference control. In contrast, the PLGA-coated particles of Example 6A show a significant dose-dependent effect compared to the reference control. This indicates that the coated particles provide a local environment that allows for the acidification of nitrite despite being in a substantially neutral environment.

[0222] 4 shows a schematic diagram of a wound dressing 100 of the present invention having a backing layer 102, a nitric oxide-producing layer 106, and a removable protective layer 108. The wound dressing 100 may be sealed before use. When the wound dressing 100 is needed, the wound dressing 100 may be removed from any packaging. The removable protective layer 108 may be removed from the wound dressing 100 to expose the nitric oxide-producing layer 106. The wound dressing may be applied to a subject's wound by placing the nitric oxide-producing layer 106 on the subject's wound. Optionally, water may be added to the exposed nitric oxide-producing layer 106 before applying the wound dressing.

[0223] The adhesive section 104 may adhere to the subject, adhering the wound dressing to the subject. The adhesive section and backing layer 102 may help adhere the wound dressing to the subject. Figure 4 is not to scale.

[0224] Example 10: NO release from a one-component wound dressing of carboxymethylcellulose (CMC) containing a nitrite source and a proton source (FIG. 5) Carboxymethylcellulose-based fabric (120 g / m 2 A powder (20 g / m) containing a nitrite source and a proton source was added to a powder (20 g / m) containing a nitrite source and a proton source using a high-intensity AC electric field as in Example 1A. 2 ) was impregnated.

[0225] The amount of nitric oxide released was measured by applying one of the dressings to a laboratory wound model and analyzing the generated gaseous nitric oxide using selected ion flow tube mass spectrometry (SIFT-MS). A schematic diagram of the apparatus used is shown in Figure 7.

[0226] A laboratory wound model was used, consisting of a 5 cm diameter shallow cylindrical cup with an infeed and an outfeed, placed within a heated stainless steel plate (30°C) at a 2° inclination angle toward the infeed. Two 4.9 cm diameter cellulose filter papers pre-saturated with sodium chloride solution (0.9% w / v) were placed within the cup to create a surrogate "wound bed."

[0227] The infeed was connected to a syringe pump that injected sodium chloride solution (0.9% w / v) at a rate of 0.4 ml / h for the duration of the test. A 5x5 cm square piece of one piece of dressing was cut from the main sample and weighed. The wound material was placed on the "wound bed". To prevent leakage during testing, the sample was placed on a 10x10cm blank carboxymethylcellulose fabric (120g / m 2 The plate was surrounded by a frame (SFM Ltd, Mercury) and a window of approximately 5x5cm was cut out from the centre.

[0228] A 10x10 cm stainless steel container with a mesh grid across its surface was placed on top of the coating with the mesh grid facing downwards and in contact with the coating to act as a weight. The measurement chamber (1100 ml plastic box) with infeed and outfeed for SIFT-MS was placed on top of the stainless steel plate of the laboratory wound model, completely covering the dressing, surrounding fabric frame, and stainless steel container. The plastic box was weighted with a weight of approximately 1 kg.

[0229] The outfeed of the plastic box was connected to a tube that was then connected to a Dreschel bottle containing 2 mM sodium hydroxide solution, followed by a Dreschel bottle containing silica beads, and finally to the inlet of the SIFT-MS. Air was drawn through the system at approximately 30 ml / min and analyzed by the SIFT-MS. The NO release profile over 2000 min is shown in Figure 5.

[0230] Example 11: NO release from a multi-layer composite coating with one component nitric oxide generating layer containing a nitrite source and a proton source (FIG. 6) Highly absorbent powder (136g / m 2 A powder (20 g / m2) containing a nitrite source and a proton source was applied to an absorbent wound pad made of polyester fibers (basis weight, Freudenberg, M1520) using a high-intensity AC electric field as in Example 1A. 2 ) was impregnated.

[0231] One dressing was applied to a laboratory wound model and the amount of nitric oxide released was measured by analyzing the evolved gaseous nitric oxide by selected ion flow tube mass spectrometry (SIFT-MS).

[0232] A laboratory wound model was used, consisting of a 5-cm-diameter shallow cylindrical cup with an infeed and an outfeed, placed within a heated stainless steel plate (30°C) at a 2° inclination angle toward the infeed. Two 4.9-cm-diameter cellulose filter papers pre-saturated with sodium chloride solution (0.9% w / v) were placed within the cup to create a surrogate "wound bed."

[0233] The infeed was connected to a syringe pump, which infused sodium chloride solution (0.9% w / v) at a rate of 0.4 ml / h for the duration of the test. A 5x5 cm square piece of one dressing was cut from the main sample and weighed. A 5x5 cm square piece of absorbent wicking material (20 g / m² polypropylene, Daltex® Absorb, Don & Low Ltd) was laminated to the wound contact surface of this sample.

[0234] A laminate dressing was placed on the "wound bed". To prevent leakage during testing, the sample was placed on a blank carboxymethylcellulose fabric (120 g / m 2 A 10x10cm frame was placed around the perimeter and a window of approximately 5x5cm was cut out of the centre (SFM Ltd, Mercury). A 10x10cm stainless steel container with a mesh grid across its surface was placed on top of the dressing with the mesh grid facing downwards, in contact with the dressing and acting as a weight. A measurement chamber (1100 ml plastic box) with infeed and outfeed for SIFT-MS was placed on top of the stainless steel plate of the laboratory wound model, covering the entire dressing, the surrounding fabric frame, and the stainless steel container.

[0235] The plastic box was weighted with a weight of approximately 1 kg. A tube was connected to the outfeed of the plastic box, which was then connected to a Dreschel bottle containing 2 mM sodium hydroxide solution, followed by a Dreschel bottle containing silica beads, and finally to the inlet of the SIFT-MS. Air was drawn through the system at approximately 30 ml / min and analyzed by the SIFT-MS. The NO release profile over 2000 min is shown in Figure 6.

Claims

1. A wound dressing for treating a wound, comprising a nitric oxide generating layer for generating nitric oxide by acidifying nitrite, wherein the nitric oxide generating layer comprises a solid powder nitrite component and a solid powder proton source component, wherein the solid powder nitrite component and the solid powder proton source component are as follows: a. a blend of one or more individual particles containing nitrite and one or more individual particles containing a proton source; b. one or more individual particles each containing nitrite and a proton source; c. an agglomerate of particles comprising one or more individual particles containing nitrite, one or more individual particles containing a proton source, and optionally including a binder; d. an agglomerate of particles, comprising one or more individual particles, containing nitrite and a proton source, and optionally containing a binder; or e. Combinations thereof A wound dressing provided by

2. 10. The wound dressing of claim 1, wherein the nitric oxide producing layer comprises a dry wound dressing substrate.

3. 3. The wound dressing of 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. 4. The wound dressing of claim 2 or 3, wherein at least a portion of the components of the dry proton source form part of the matrix of the dried wound dressing.

5. 5. The wound dressing of claim 2, wherein the dry proton source component comprises a solid powder proton source component, and further wherein a portion of the dry proton source component forms part of the substrate of the dry wound dressing.

6. A wound dressing according to any one of claims 2 to 5, wherein the substrate of the dry wound dressing consists of woven or nonwoven fibres.

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

8. 8. The wound dressing according to claim 1, wherein the water content of the nitric oxide producing layer is 10% or less, 5% or less, 2% or less, or 1% or less, based on the weight of the nitric oxide producing layer.

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

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

11. 10. The wound dressing of claim 1, wherein one or more individual particles or particle agglomerates are blended or coated with an excipient that affects the rate at which water penetrates the particles and / or the rate at which nitric oxide is formed from the particles.

12. 12. The wound dressing of claim 11, wherein the excipient that influences the rate at which water penetrates the particles is a hydrophobic material such as a polyol, a phospholipid, magnesium stearate or colloidal silica, and / or the excipient that influences the rate at which water penetrates the particles is a material that sequesteres nitric oxide or a precursor of nitric oxide, such as a thiol, an alcohol, an amine or an amide.

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

14. The blending of one or more individual particles containing nitrite with one or more individual particles containing a proton source can be accomplished by: (a) micronizing the nitrite solids together with the proton source solids; or (b) micronizing the two solids together with the proton source solids; (i) subjecting a solution containing nitrite to spray drying or freeze drying; (ii) subjecting a solution containing a proton source to spray drying or freeze drying; and (iii) blending the solids produced in steps (i) and (ii).

13. The wound dressing of claim 11 or claim 12, wherein the wound dressing is formed by either

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

16. A wound dressing according to any preceding claim, comprising one or more further dry layers adjacent to the nitric oxide generating layer.

17. 17. The wound dressing of any one of claims 1 to 16, further comprising one or more additional layers adjacent to the nitric oxide producing layer, with the proviso that the water content of any layer adjacent to the nitric oxide producing layer is 5% or less, 2% or less, or 1% or less, based on the weight of the layer adjacent to the nitric oxide producing layer.

18. 18. A wound dressing according to any preceding claim, comprising an antimicrobial agent.

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

20. 20. The packaged wound dressing of claim 19, wherein the low-moisture permeable packaging comprises one or more low-moisture permeable materials (e.g., aluminum foil) at the packaging wall and / or may be sealed.

21. 21. A packaged wound dressing according to claim 19 or 20, wherein the packaging atmosphere within the packaged wound dressing has a low moisture content initially upon packaging and / or includes a pack insert which sequesters moisture in the package.

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

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

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

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

Images