Antimicrobial compositions

Abstract

Powder compositions are described which comprise enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer. The composition comprises a first set of particles interspersed with a second set of particles. The first set of particles comprises the enzyme and the polymer, but substantially none of the substrate. The second set of particles comprises the substrate, but substantially none of the enzyme.

Description

[0001] Antimicrobial Compositions

[0002] This invention relates to compositions, such as powder compositions, for generating hydrogen peroxide, and their use in treating infections and wounds.

[0003] Honey has been used for treatment of microbial infections since ancient times. In recent years there has been a resurgence of interest in the therapeutic efficacy of honey, particularly in the area of wound healing. Clinical trials have shown that honey is an effective broad-spectrum antimicrobial agent which is effective against common wound-infecting organisms, such as Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Escherichia coli, and is effective against antibiotic-resistant strains of bacteria. As a natural product, honey also offers an attractive alternative to drug-based treatments.

[0004] Many different types of honey have antimicrobial activity. This activity is attributed largely to osmolarity, pH, hydrogen peroxide production and the presence of phytochemical components.

[0005] The applicant has appreciated that the antimicrobial effects of honey can be greatly enhanced and controlled by adding glucose oxidase to honey, and that compositions comprising honey and added glucose oxidase are applicable in the treatment of a number of infections, and notably in the treatment of infections caused by biofilms (see WO 2015 / 166197, WO 2016 / 083798 and WO 2016 / 124926).

[0006] However, because honey is a natural product, its composition can vary greatly depending on its source. For example, the difference in antimicrobial potency among honeys can be more than one hundred-fold, depending on the geographical, seasonal and botanical source of the honey, as well as the harvesting, processing and storage conditions. Consequently, the antimicrobial efficacy may also vary depending on the type of honey used. Furthermore, honey may also contain other components, such as allergens e.g. trace amounts of pollen, which may cause adverse reactions when applied to certain subjects and make it unsuitable for certain pharmaceutical applications. There is also considerable variability in physical characteristics such as viscosity and colour.

[0007] Honey is sticky and can be difficult to apply and remove from a patient. Honey may also require processing such that it is in a suitable form for application to subjects, which can add cost and complexity to the production process. Such processing may include creaming or pasteurisation.

[0008] Consequently, there is a desire to provide improved compositions which provide enhanced antimicrobial efficacy compared to honey, and which also overcome some of honey’s disadvantages. There is also a desire to provide compositions with improved stability. The applicant has formulated synthetic liquid and gel compositions which ma improvements over honey-based compositions, such as those disclosed in WO 2020 / 193993 and WO 2021 / 186165. However, although liquids and gels may be advantageous for certain applications, they may not be optimal for other applications. For example, powders may be more advantageous in certain circumstances. For instance, powders may be more readily applied to dressing materials to form antimicrobial dressings. In certain circumstances, powders could be applied directly to a wound.

[0009] Superabsorbent powder compositions comprising superabsorbent polymer are disclosed in WO 2019 / 077335. However, it is desired to provide powders that are not necessarily superabsorbent, and which have improved stability. For example, it is desired to provide dry compositions, such as powders, which maintain the ability to generate hydrogen peroxide at effective levels and which maintain their consistency, minimising clumping or aggregation, after being stored for extended periods of time.

[0010] Maintaining stability of powder compositions that are able to generate hydrogen peroxide is particularly challenging. The applicant has found that powder compositions comprising glucose oxidase and glucose exhibit a significant decrease in the ability to generate hydrogen peroxide even after storage for 60 days in a 99.9% nitrogen atmosphere. This suggests that maintaining stability may be more complex than simply minimising exposure to atmospheric water and oxygen, to prevent background generation of peroxide, and the degeneration of the enzyme. The applicant has, however, formulated compositions with improved stability.

[0011] In a broad sense, the invention concerns compositions which include enzyme that is able to convert a substrate to release hydrogen peroxide. Preferably, such compositions are able to generate hydrogen peroxide on addition of water. Such compositions may also include substrate for the enzyme.

[0012] The invention also concerns methods of making such compositions that are able to generate hydrogen peroxide. For example, this may include combining enzyme that is able to convert a substrate to release hydrogen peroxide and substrate for the enzyme.

[0013] Preferably, the composition is a dry composition. According to the invention there is provided a dry composition, comprising enzyme that is able to convert a substrate to release hydrogen peroxide; polymer; and optionally substrate for the enzyme.

[0014] Preferably, the composition is a solid composition. According to the invention there is provided a solid composition, comprising enzyme that is able to convert a substrate to release hydrogen peroxide; polymer; and optionally substrate for the enzyme. Examples of dry, solid compositions includes granular compositions or powdt herein to a “powder” may be used interchangeably with a “granular composition”. Dry compositions may also include films. Compositions of the invention may thus not be liquids or gels.

[0015] Most preferably, the composition is a powder composition, or a dry powder composition. So, according to the invention there is provided a powder composition, comprising enzyme that is able to convert a substrate to release hydrogen peroxide; polymer; and optionally substrate for the enzyme.

[0016] Preferably compositions of the invention are able to generate hydrogen peroxide on contact with water. So, compositions may comprise substrate for the enzyme, and / or they could comprise precursor substrate.

[0017] References herein to “enzyme” encompass one or more enzymes. For example, in some embodiments, compositions of the invention may comprise a plurality of enzymes that are able to convert a substrate to release hydrogen peroxide. For example, compositions may comprise two enzymes, such as glucose oxidase and galactose oxidase. In some embodiments, compositions of the invention may comprise only one enzyme that is able to convert a substrate to release hydrogen peroxide. For example, the only enzyme in the composition that is able to convert a substrate to release hydrogen peroxide may be glucose oxidase.

[0018] Preferably, the enzyme is a purified enzyme. The term “purified enzyme” is used herein to include an enzyme preparation in which the enzyme has been separated from at least some of the impurities originally present when the enzyme was produced. Preferably impurities that have been removed or reduced include those that would otherwise interfere with the ability of the enzyme to convert the substrate to release hydrogen peroxide.

[0019] Preferably, the enzyme is at least 95% pure. Even more preferably, the enzyme is at least 98% pure. Most preferably, the enzyme is at least 99% pure.

[0020] The enzyme may have been produced by recombinant or non-recombinant means, and may be a recombinant or non-recombinant enzyme. The enzyme may be purified from a microbial source, preferably from a non-genetically modified microbe.

[0021] The level of purity of the enzyme may be selected as appropriate depending on the intended use of the composition. For medical use, a medical grade or medical device grade of purity should be used. For pharmaceutical use, a pharmaceutical grade of purity should be used. The total amount of all the hydrogen peroxide-generating enzyme in composi invention may be 0.0005% to 0.5% (by weight), 0.001 % to 0.2% (by weight), 0.001% to 0.1 % (by weight), or 0.0005% to 0.05% (by weight).

[0022] If the composition includes more than one enzyme that is able to convert a substrate to release hydrogen peroxide, at least one of those enzymes may be present in an amount of 0.0005% to 0.5% (by weight), 0.001% to 0.2% (by weight), 0.001 % to 0.1 % (by weight), or 0.005% to 0.05% (by weight).

[0023] The amount of enzyme may vary depending on the desired level of hydrogen peroxide production on dilution, or the particular application of the composition. In some applications, the amount of enzyme may be higher. For example, if the composition (e.g. a powder) is to be used to coat or impregnate a dressing material, higher enzyme concentrations may be desirable to compensate for a lower coating density.

[0024] So, in some examples, the total amount of hydrogen peroxide generating enzyme in compositions of the invention may be 0.5 % to 10% (by weight), 0.5% to 7.5% (by weight), or 0.5% to 5% (by weight).

[0025] If the composition includes more than one enzyme that is able to convert a substrate to release hydrogen peroxide, at least one of those enzymes may be present in an amount of 0.5 % to 10% (by weight), 0.5% to 7.5% (by weight), or 0.5% to 5% (by weight).

[0026] A suitable amount of enzyme can be readily determined by a person of ordinary skill in the art, if necessary using a well diffusion assay, to determine the extent of hydrogen peroxide release for different amounts of enzyme. The amount of enzyme used may be selected so as to produce a composition for generating antimicrobial activity that is equivalent to a selected phenol standard (for example a 10%, 20%, or 30% phenol standard).

[0027] Compositions of the invention may comprise at least 1 unit, and preferably up to 1500 units, of the enzyme per gram of the composition. A “unit” is defined herein as the amount of enzyme (e.g. glucose oxidase) causing the oxidation of 1 micromole of substrate (e.g. glucose) per minute at 25 degrees centigrade at pH 7.0.

[0028] Preferably, the enzyme is, or comprises, an oxidoreductase enzyme. Examples of oxidoreductase enzymes include glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase, amino acid oxidase, or mannose oxidase.

[0029] Preferably, the oxidoreductase enzyme is glucose oxidase and the substrate for the oxidoreductase enzyme is glucose. In some embodiments, a composition according to the invention comprises rr for example at least 30 units, at least 50 units, or at least 100 units, and suitably less than 685 units, for example 100-500 units, of enzyme (e.g. glucose oxidase) per gram of the composition. In other embodiments of the invention, a composition according to the invention comprises at least 500 units, for example 500-1000 units, or 685-1000 units, of enzyme (e.g. glucose oxidase) per gram of the composition.

[0030] Compositions of the invention may only be able to generate hydrogen peroxide at a significant level following dilution by water. Addition of water may thus initiate hydrogen peroxide production. Consequently, compositions of the invention should not comprise sufficient free water to allow the enzyme to convert the substrate.

[0031] Addition of water, which may include contact with wound exudate, may result in sustained release of hydrogen peroxide at a specific level or concentration for a sustained period of time. The level may be affected by the amount of enzyme in the composition. The level of hydrogen peroxide production may be chosen so that the level is not too high, and thus cytotoxic, but is not too low and thus ineffective.

[0032] Compositions of the invention may provide for sustained release of hydrogen peroxide at a level of 0.1 mM to 20 mM for a period of at least twenty four hours, preferably at least 72 hours, following dilution of the composition. The level may be 0.5 mM to 10 mM . The level may be 1 mM to 7.5 mM .

[0033] In compositions of the invention, the water activity (aw) may be 0.4 or less, or 0.3 or less. A low water activity may be advantageous in preventing microbial proliferation, and it may be advantageous in minimising hydrogen peroxide production prior to activation by dilution.

[0034] Water activity is typically measured using a hygrometer, such as a resistive electrolytic hygrometer, a capacitance hygrometer or a dew point hygrometer. Measurement of water activity would typically take place at ambient temperature, such as normal temperature and pressure. Measurement of water activity may take place according to ISO 18787:2017.

[0035] Preferably, there is less than 10 ppm, less than 6 ppm, or less than 3 ppm hydrogen peroxide in compositions of the invention. Most preferably, there is substantially no hydrogen peroxide, trace amounts of hydrogen peroxide or no detectable hydrogen peroxide. For example, hydrogen peroxide is preferably not detectable using a hydrogen peroxide test strip, such as a Quantofix® peroxide test stick (Sigma Aldrich, UK). For example, hydrogen peroxide may present at a level less than 1 ppm or at a level less than 0.5 ppm. Hydrogen peroxide may be at a level less than 0.1 ppm. Compositions of the invention could feasibly contain low levels, or trace amoi

[0036] Typically, there would be 1% or less (by weight) water in the composition. Preferably, there is substantially no water. Compositions of the invention may be manufactured by lyophilisation, for example, which would remove water from the compositions. So, the composition may be a lyophilised composition.

[0037] References herein to “substrate” encompass one or more substrates. For example, in some embodiments, compositions of the invention may comprise a plurality of substrates. In some embodiments, compositions of the invention may comprise only one substrate. For example, the only substrate present may be glucose.

[0038] Preferably, the substrate is a purified substrate. The term “purified substrate” is used herein to include a substrate which has been separated from at least some of the impurities originally present when the substrate was obtained or produced. The purified substrate may be obtained from a natural source or may be synthetically produced. The purified substrate may be a processed, extracted, or refined substrate (i.e. a substrate in which impurities or unwanted elements have been removed by processing). Preferably, the purified substrate is at least 90%, 95%, or 99% pure (mass purity). Preferably the substrate is pharmaceutical grade.

[0039] In particular embodiments, the substrate is, or comprises, sugar. The term “sugar” is used herein to refer to a carbohydrate with the general formula Cm(H2O)n. The purified sugar may be obtained from a natural source (for example a processed, extracted, or refined natural sugar), or be synthetically produced. The sugar is preferably at least 90%, 95%, or 99% pure (mass purity). The sugar is preferably a pharmaceutical grade sugar. The sugar may be a monosaccharide or a disaccharide, preferably a monosaccharide. The sugar may include, for example purified D-glucose, hexose, or D-galactose. For example, the purified sugar may be medical grade, medical device grade, or pharmaceutical grade D-glucose, hexose, or D- galactose. The sugar may be an anhydrous sugar. For example, the glucose may be anhydrous glucose.

[0040] Instead of or in addition to the substrate, the composition may comprise a precursor substrate. Any disclosure herein which relates to the substrate, such as amounts and purity, may also apply to the precursor substrate.

[0041] For compositions of the invention which comprise a precursor-substrate, the composition may comprise one or more enzymes for converting the precursor-substrate to the substrate for the enzyme. However, in some embodiments, the precursor-substrate may not necessarily be converted to the substrate enzymatically. For example, for some precursor substrates, addition of water may be sufficient for conversion. Alternatively or additionally, compositions of the invention may comprise non-enzymatic catalysts. Compositions which comprise a precursor-substrate may comprise a first enz convert the substrate to release hydrogen peroxide, and a second enzyme that is able to convert the precursor-substrate to the substrate for the first enzyme.

[0042] The precursor-substrate is preferably a carbohydrate, such as a polysaccharide, or a sugar e.g. a disaccharide, or sugar derivative. For example, the precursor-substrate may be sucrose, the first enzyme may be glucose oxidase and the second enzyme may be invertase. In another example, the precursor-substrate may be maltose, the first enzyme may be glucose oxidase and the second enzyme may be maltase.

[0043] Compositions of the invention which comprise a precursor-substrate may comprise an enzyme (preferably a purified enzyme) that is able to convert the substrate to release hydrogen peroxide, and at least two enzymes (e.g. second and third enzymes, preferably purified enzymes) that are able to convert the precursor-substrate to the substrate for the first enzyme. For example, the precursor-substrate may be starch, the first enzyme may be glucose oxidase and the second and third enzymes may be amylase and maltase. For example, the precursorsubstrate may be cellulose, the first enzyme may be glucose oxidase and the second and third enzymes may be cellulose and beta-glucosidase.

[0044] In a preferred embodiment, the enzyme is at least 95% pure, and the substrate is at least 95% pure (all with reference to mass purity).

[0045] In another preferred embodiment, the enzyme is at least 98% pure, and the substrate is at least 98% pure (all with reference to mass purity).

[0046] In another preferred embodiment, the enzyme is at least 99% pure, and the substrate is at least 99% pure (all with reference to mass purity).

[0047] Compositions of the invention, or components of compositions of the invention, may be pharmaceutical grade. The term “pharmaceutical grade” is used herein to refer to include reference to a purity standard for a reagent that has been established by a recognized national or regional pharmacopeia (e.g., the U.S. Pharmacopeia (USP), British Pharmacopeia (BP), National Formulary (NF), European Pharmacopoeia (EP), or Japanese Pharmacopeia (JP)).

[0048] Preferably, there is 10% or less, by weight, of the substrate in compositions of the invention. There may be 15% or less by weight, of the substrate. There may be 10% or less, by weight, of the substrate. There may be less than 5%, by weight, of the substrate, e.g. about 3%, by weight, of the substrate.

[0049] The total amount of all substrate in the composition may be at least 1 %, by weight, of the substrate. For example, compositions of the invention may have 1 % to 10%, by weight, of the substrate, preferably 1 to 5% by weight of the substrate. The total amount of : example be about 3%, by weight. Although low amounts of total substrate may be preferred, higher amounts of substrate are also contemplated. For example, the total amount of substrate may be from 1% to 25%, by weight, of the composition.

[0050] If a composition of the invention comprises more than one substrate, at least one of those substrates may be at least 1%, by weight, of the composition. For example, at least one of the substrates may be 1 % to 10%, by weight, 1 to 5%, or about 3% by weight of the composition. At least one of the substrates may be from 1 % to 25%, by weight, of the composition.

[0051] Therefore, according to the invention, there is provided a powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer, wherein the total amount of substrate in the composition is less than 5%, by weight.

[0052] In compositions of the invention, the total amount of all sugar (e.g. monosaccharides and disaccharides) is preferably 10% or less by weight. For example, if a composition comprises glucose and fructose, the total amount of fructose and glucose may not exceed 10%, by weight, of the composition. Preferably, the total amount of sugar is 7.5% or less, by weight. In one example, the total amount of sugar in compositions of the invention is 1% to 10% by weight. In one embodiment, the total amount of sugar in the composition is 1 to 7.5% by weight. In another embodiment, the total amount of sugar in the composition is 1 to 5%, by weight. Most preferably, the total amount of sugar in the composition is less than 5%, by weight.

[0053] Therefore, according to the invention, there is provided a powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer, wherein the total amount of sugar in the composition is less than 5%, by weight.

[0054] Compositions of the invention preferably do not include fructose. Fructose is hygroscopic and is quicker to absorb moisture and slower to release it to the environment than sugars such as sucrose and glucose. Fructose is an effective humectant and can retain moisture for extended periods of time. This may lead to instability because compositions including fructose may lead to greater water absorption, and increased hydrogen peroxide production, during storage. It may also lead to an inferior powder consistency. For example, it may increase clumping or aggregation of the powder.

[0055] Preferably, compositions of the invention do not include any sugar other than glucose. Compositions of the invention may not include any sugar derivative. The term is used herein to refer to a sugar that has been modified by addition of one or more substituents other than a hydroxyl group. Sugar derivatives, thus encompass amino sugars, acidic sugars, deoxy sugars, sugar alcohols, glycosylamines and sugar phosphates. For example, sugar derivatives may include glucose-6-phosphateglucosamine, glucoronate, gluconate, galactosamine, glucosamine, sialic acid, deoxyribosefucose, rhamnose glucuronic acid, polyols (e.g. sorbitol, erythritol, xylitol, mannitol, lactitol and maltitol) and sucralose.

[0056] Compositions of the invention may not include sugars or sugar derivatives that have a high solubility in water, for example a solubility which is greater than glucose. Glucose has a solubility of 90g / 100g water at 20°C and 1 atm. So, compositions of the invention may not include a sugar or sugar derivative with a solubility greater than or equal to 100g / 100g water at 20°C and 1 atm, greater than or equal to 200g / 100g water at 20°C and 1 atm, greater than 300g / 100g water at 20°C and 1 atm.

[0057] Compositions of the invention may not include a sugar or sugar derivative that is more hygroscopic than glucose.

[0058] Polymer in compositions of the invention may be any medically acceptable polymer, such as any Food and Drug Administration-approved (FDA-approved) polymer.

[0059] Advantageously, the applicant has appreciated that high amounts of polymer in the composition may lead to improved stability.

[0060] References herein to “polymer” encompass one or more polymers. For example, compositions of the invention may comprise a plurality of polymers with distinct chemical structures, which are distinct from one another other than just by their molecular weight. For example, structurally distinct polymers may comprise structurally distinct monomer units.

[0061] The total amount of all polymer in the composition may be greater than 50%, by weight, of the composition. The total amount of all polymer may be greater than 75%, by weight, of the composition. The total amount of all polymer may be at least 80%, by weight, of the composition. The total amount of all polymer may be at least 85%, by weight, of the composition. The total amount of all polymer may be at least 90%, by weight, of the composition. The total amount of all polymer may be at least 95%, by weight, of the composition.

[0062] Therefore, according to the invention, there is provided a powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer, wherein the total amount of polymer in the compositior 75% by weight.

[0063] If a composition of the invention comprises more than one polymer, at least one of those polymers may be at least 5%, by weight, of the composition, at least 10%, by weight, of the composition or at least 20%, by weight, of the composition. For example, If the composition includes two polymers with a combined weight % of more 80, the first polymer may have a weight % of 25, whilst the second polymer may have a weight % of 55.

[0064] Compositions of the invention may include different types of the same polymer with the same molecular structure, such as the same monomer. For example there may be different molecular weights of the same polymer, such as polyethylene glycols with differing molecular weights.

[0065] Preferably, the polymer comprises a water-soluble polymer, which is soluble in water at normal temperature and pressure (NTP).

[0066] Examples of suitable water-soluble synthetic polymers include polyacrylic acid, polyethylene oxide, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, poly (isopropyl acrylamide) and poly(cyclopropyl methacrylamide).

[0067] Preferably, the polymer comprises a synthetic polymer.

[0068] Preferably, the polymer comprises a non-ionic polymer.

[0069] Preferably, the polymer comprises a biocompatible polymer.

[0070] Preferably, the polymer comprises a biodegradable polymer.

[0071] Preferably, the polymer comprises a polymer with a carbon chain backbone, such as a vinyl polymer.

[0072] Preferably, the polymer comprises a polymer with a hetero chain backbone, such as a polyether.

[0073] Preferably, the polymer comprises a plasticising polymer.

[0074] Preferably, the polymer comprises a semi-crystalline polymer, which is semi-crystalline at NTP.

[0075] Preferably, the polymer comprises a thermoplastic polymer.

[0076] The polymer may comprise a polymer with a glass transition temperature (Tg) of 30eC or higher, 50eC or higher, or 60eC or higher. Such polymers may afford a degree of hardness or granularity to the powder. Polymers with low Tg may not provide the desirabh hardness. There are a variety of thermal and mechanical analytical techniques that can be used to measure Tg. Most notably these include: Differential Scanning Calorimetry, Dynamic Mechanical Analysis and Thermomechanical Analysis. Differential Scanning Calorimetry may be undertaken according to ISO 11357. A preferred example of a polymer with a Tg of at least 50eC or more is polyvinyl alcohol. Other examples of polymers with Tg of at least 50eC include polyacryilic acid, polvinyl pyrrolidone and polyacrylamide.

[0077] The polymer may comprise a polymer with a Tg which is less than 50eC, preferably less than 30eC, more preferably less than 25eC. Polymers with such Tg values, such as polyethylene glycol, or polypropylene glycol may have plasticising properties.

[0078] Preferably, the polymer comprises polyvinyl alcohol. Polyvinyl alcohol may be present in the composition in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight. Polyvinyl alcohol may be present in an amount of 50 to 90% by weight, or 55 to 80% by weight.

[0079] The polyvinyl alcohol may have a molecular weight of 20,000 to 75,000, such as 25,000 to 60,000, e.g. 31 ,000 to 50,000.

[0080] The polyvinyl alcohol may be at least 80% hydrolysed, preferably at least 85% hydrolysed.

[0081] Preferably, the polymer comprises polyethylene glycol. Polyethylene glycol may be present in the composition in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight. Polyethylene glycol may be present in an amount of 10 to 50% by weight, or 15 to 45% by weight.

[0082] Preferably the polymer comprises polyvinyl alcohol and polyethylene glycol. Polyvinyl alcohol and polyethylene glycol may each be present in an amount of at least 10% by weight or at least 20% by weight. The amount of polyvinyl alcohol may be more than the amount of polyethylene glycol.

[0083] Polyethylene glycol in compositions of the invention may have a molecular weight of 10000 or less (i.e. PEG 10000), 7500 or less, or 5000 or less (i.e. PEG 5000). The polyethylene glycol may have a molecular weight of at least 2000 (i.e. PEG 2000), or at least 3000 (i.e. PEG 3000).

[0084] The polyethylene glycol may have a molecular weight of 2000 to 10000, 2000 to 7500 or 3000 to 5000.

[0085] The polymer may comprise one or more water-insoluble, synthetic polymers. These may include polyesters, such as aliphatic polyesters. Examples may include polylactic acid, poly(lactic-co-glycolic acid) or polycaprolactone. The polymer may comprise one or more natural polymers. The one or more r may comprise a protein-based polymer such as albumin, collagen or gelatin. The one or more natural polymers may comprise a polysaccharide-based polymer such as agarose, alginate, carrageenan, chitosan, cyclodextrins, dextran, hyaluronic acid, polysialic acid, starch or cellulose.

[0086] The polymer may comprise one or more cellulose-based polymers, such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose hydroxypropyl cellulose, hydroxypropyl methyl cellulose or cellulose acetate phthalate, or microcrystalline cellulose.

[0087] The polymer may comprise a starch-based polymer, such as sodium starch glycolate.

[0088] The polymer may comprise a hydrocolloid, such as alginic acid, carrageenan, chitosan, hyaluronic acid or pectinic acid.

[0089] The polymer may comprise a co-polymer. The co-polymer may be a graft co-polymer, such as Polyethylene glycol / polyvinyl alcohol (PEG / PVA).

[0090] A graft co-polymer may combine properties of the constituent polymers used to form the graft co-polymer.

[0091] In one preferred example, the composition comprises a first polymer and a second polymer. The composition may thus comprise a blend of first and second polymer. The applicant has appreciated that certain beneficial properties may be afforded by a polymer blend including the first and second polymer. The specific blend may assist in stabilisation, such as maintaining the ability of the composition to generate effective levels of hydrogen peroxide for an extended period after the composition has been made. The specific blend may also impart a desirable texture to the composition, for example the first polymer may impart hardness to enable easier milling or granulation. The second polymer may act a plasticiser.

[0092] The first polymer is preferably a water-soluble, synthetic polymer. The first polymer preferably has a Tg of at least 30eC, at least 50eC, more preferably at least 60eC. The first polymer is preferably a vinyl polymer, more preferably polyvinyl alcol

[0093] The second polymer is preferably a water-soluble synthetic polymer. The second polymer preferably has a Tg of less than 50eC, more preferably less than 30eC, even more preferably less than 25eC.

[0094] The first polymer preferably has a Tg which is higher than the second polymer.

[0095] The second polymer is preferably a polyether, more preferably polyethylene glycol. Polyethylene glycol, for example, has been found to highly compatible with hydrogen peroxide generating enzymes, such as glucose oxidase, and has a particularly beneficial effect on maintaining stability.

[0096] The first polymer may be present in an amount greater than the second polymer. For example, the first polymer may be present in an amount of 50 to 90%, by weight, preferably 55 to 85%, by weight.

[0097] The second polymer may be present in an amount 10 to 50%, by weight, preferably 15 to 45% by weight.

[0098] Therefore, according to the invention, there is provided a powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer, wherein the polymer comprises a first water-soluble, synthetic polymer and a second water-soluble, synthetic polymer, the first polymer having a Tg of 50eC or higher and the second polymer having a Tg less than 50eC, preferably wherein the first polymer comprises a vinyl polymer and the second polymer comprises a polyether polymer, more preferably wherein the first polymer is polyvinyl alcohol and the second polymer is polyethylene glycol.

[0099] Preferably, compositions of the invention comprise less than 10%, less than 5% or less than 0.5%, by weight, superabsorbent polymer (SAP). Even more preferably, the composition preferably comprises substantially no SAP. SAPs are polymers that can absorb and retain extremely large amounts of liquid relative to their own mass. Inclusion of SAPs may thus have an adverse effect on powder consistency, by promoting aggregation by absorption of atmospheric water.

[0100] SAPs are usually made from hydrophilic polymers containing anionic water-holding groups, such as carboxylic acid groups. Commonly, SAPs are produced from acrylic acid, its salts, or acrylamide. SAPs are preferably cross-linked, with the degree and type of cross-linking affecting its properties. For example, low-density cross-linked SAPs generally have higher water-retention capacity and swell to a larger degree, but may have a softer and stickier gel formation. High cross-link density polymers may exhibit lower water retention provide a firmer gel that is able to retain its shape more readily. SAPs may be classified according to the charge in the cross-linked chains. For example, SAPs may be non-ionic, ionic, amphoteric or zwitterionic. In some preferred embodiments, the SAPs are anionic.

[0101] Excluded from compositions of the invention may thus include one of more SAPs selected from sodium polyacrylate; hydrolysed cellulose-polyacrylonitrile; a starch-polyacrylonitrile copolymer; a cross-linked co-polymer of maleic anhydride, such as ethylene maleic anhydride copolymer; cross-linked carboxymethyl cellulose; polyvinyl alcohol co-polymer; and cross-linked polyethylene oxide; polysaccharide-based SAPs; and poly(amino acid)-based.

[0102] Typically a SAP has an absorption capacity or swelling capacity (which may also be termed free-absorbency capacity, free swelling capacity, fluid retention capacity or water-retention capacity) of at least 10 g of water, or aqueous solution, per gram of polymer (i.e. 10g / g). So, compositions of the invention may exclude polymers with such an absorption capacity. Compositions of the invention may exclude SAPs with an absorption capacity of at least 15 g / g, at least 20 g / g, at least 30 g / g, at least 50 g / g, at least 100 g / g at least 200 g / g, at least 500 g / g, or at least 1000 g / g. Absorption capacity may also be termed centrifuge retention capacity. The absorption capacity of a water-absorbing material, such as a SAP, can be measured by methods known to the skilled person. In one method of measuring absorption capacity, the ‘tea bag’ method, an amount of the material is placed into a tea bag (acrylic / polyester gauze with fine meshes) and the bag is dipped in an excess amount of water or saline solution for one hour to reach equilibrium swelling. The excess solution is removed by hanging the bag until no more liquid drops off. The tea bag is then weighed and the swelling capacity is calculated as follows:

[0103] (Wi - Wo) / Wo

[0104] Wi is the weight of the swollen sample and Wois the original weight of the material.

[0105] Water-retention capacity may be measured in deionised water. Alternatively, water-retention capacity may be measured in a saline solution, such as a 0.9 wt% saline solution.

[0106] In another method to measure absorption capacity, the centrifuge method, 0.2g (Wi), of the material is placed into a bag (60 x 60 mm) made of non-woven fabric. The bag is dipped in 100 mL of saline solution (0.9% by weight) for half an hour at room temperature. It is taken out, and then excess solution is removed by a centrifugal separator for 3 minutes at 250g, then the weight of the bag (W2) is measured. The same stages are carried out with an empty bag and the weight of the bag (Wo) is measured. The water-absorbing capacity is measured as follows:

[0107] (W2-W0-Wi) / Wi This test (NWSP 241 .0R2) is standardised by the European Disposables and Association (EDANA). Testing may be done in accordance with ISO 17190-6. Absorption capacity may be assessed at NTP.

[0108] Preferably, the compositions of the invention include a salt. Including a salt may be beneficial during the process of manufacturing the composition and help maintain stability. For example, the salt may help protect the enzyme during freezing and / or drying. It may, for instance, protect during lyophilisation.

[0109] The salt may be present in an amount of 10% or less, preferably 7.5% or less, more preferably 5% or less, by weight. For example, the salt may be present in an amount of about 3%, by weight.

[0110] The composition may comprise a buffer, for example a buffer salt. Examples of buffer salts include: PBS, TBS, Bis-Tris. PBS may be preferred because it may provide superior biocompatibility.

[0111] If the composition is in the form of a powder, the particle size may be adjusted to optimise the composition according to its intended application. For example, the smaller the particle size, the more easily it may become solubilised or absorb fluid. However, it may be undesirable for the particle size to be too small such that a significant amount becomes aerosolised upon dispensing.

[0112] The powder may have a mean particle size of 3000 pm or less. The powder may have a mean particle size of 2000 pm or less. The powder may have a mean particle size of 1000 pm or less. The powder may have a mean particle size of 500 pm or less.

[0113] The powder may have a mean particle size of 50 pm or more. The powder may have a mean particle size of 100 pm or more. The powder may have a mean particle size of 200 pm or more.

[0114] For example, the powder may have a mean particle size of 50 to 3000 pm. The powder may have a mean particle size of 100 to 2000 pm. The powder may have a mean particle size of 100 to 1000 pm.

[0115] The powder may contains less than 10% of particles with a size of 1000 pm or more. The powder may contain less than 10% of particles with a size of 50 pm or less.

[0116] The powder may have a modal particle size of 3000 pm or less. The powder may have a modal particle size of 2000 pm or less. The powder may have a modal particle size of 1000 pm or less. The powder may have modal particle size of 500 pm or less. The powde modal particle size of 50 pm or more. The powder may have a modal particle size of 100 pm or more. The powder may have a modal particle size of 200 pm or more. For example, the powder may have a modal particle size of 50 to 3000 pm. The powder may have a modal particle size of 100 to 2000 pm. The powder may have a modal particle size of 100 to 1000 pm.

[0117] The powder may have a median particle size of 3000 pm or less. The powder may have a median particle size of 2000 pm or less. The powder may have a median particle size of 1000 pm or less. The powder may have median particle size of 500 pm or less. The powder may have a median particle size of 50 pm or more. The powder may have a median particle size of 100 pm or more. The powder may have a median particle size of 200 pm or more. For example, the powder may have a median particle size of 50 to 3000 pm. The powder may have a median particle size of 100 to 2000 pm. The powder may have a median particle size of 100 to 1000 pm.

[0118] Particle size may be analysed using laser diffraction. Laser diffraction may employ the Mie or Fraunhofer model. Particle size may be measured in accordance with ISO 13320:2020. Particle size is conventionally reported in terms of diameter irrespective of the actual particle shape; commonly the equivalent spherical diameter. Particle size results may be represented in accordance with ISO 9276-1 and ISO 9276-2. The median particle size may be a mass median diameter.

[0119] Preferably, compositions of the invention are substantially free of catalase.

[0120] Preferably, compositions of the invention are substantially free of peroxidase, such as lactoperoxidase.

[0121] Preferably, compositions of the invention are substantially free of lactoferrin.

[0122] Preferably, compositions of the invention are substantially free of zinc oxide.

[0123] Preferably, compositions of the invention are substantially free of honey.

[0124] Preferably, compositions of the invention are substantially free of antioxidant.

[0125] Compositions of the invention may comprise a haemostat or a blood clotting agent. For example, compositions of the invention may comprise a coagulation factor. Potential coagulation factors include fibrinogen or thrombin. Artificial blood clotting agents could be included in compositions of the invention. Examples of such agents include carriers, such as albumin carriers, to which fibrinogen-binding peptides are immobilised. Compositions of the invention are preferably sterile. Compositions of the inve sterilised by any suitable means. Preferably compositions of the invention have been sterilised by irradiation. Irradiation may be achieved by gamma, electron beam or X-ray. Electron beam may be preferred. The Applicant has found that compositions can retain glucose oxidase activity (and, therefore, the ability to release hydrogen peroxide on dilution) following sterilisation by exposure to gamma irradiation or electron beam irradiation.

[0126] A suitable level or dose of irradiation (e.g. electron beam irradiation) may be 10-100 kGy, preferably 10-50 kGy, more preferably 15-40 kGy, such as 20-40 kGy, or 20-25 kGy. Advantageously, it has been found that compositions of the invention may be adequately sterilised using lower doses than required for SurgihoneyRO®. SurgihoneyRO® is typically sterilised with a dose of 35-70 kGy.

[0127] Compositions of the invention may be in a container or sachet. The container may assist in maintaining the sterility of the composition. Preferably, the container is sealed or airtight. The container may have a removable and / or replaceable cap or seal. The container is preferably opaque.

[0128] A composition of the invention may be provided with a dressing material. The dressing material may be coated with the composition. Suitable dressing materials include gauzes, bandages, tissues, films, gels, foams, hydrocolloids, alginates, hydrogels, or polysaccharide pastes, granules, beads or tulle. It may comprise carboxymethylcellulose. The composition may be present together with a wound-dressing matrix, such as a collagen or collagenglycosaminoglycan matrix. The dressing material may be a fabric. Compositions in combination with a dressing are preferably sterile, and may be sterilised using irradiation, e.g. gamma irradiation or electron beam irradiation.

[0129] The dressing material may be impregnated with the powder. The powder may adhere to a surface of the dressing material.

[0130] When water is added to compositions of the invention, a liquid or a gel, such as a hydrogel, may be formed. The polymer may absorb water to form the gel.

[0131] Consequently, according to the invention, there is provided a gel, or hydrogel, obtained, or obtainable, by adding water to a composition of the invention. The gel may thus generate hydrogen peroxide because it contains sufficient free water to allow the enzyme to convert the substrate.

[0132] So, according to the invention, there is provided a composition in the form of a gel comprising an enzyme, a substrate for the enzyme, polymer and water. The enzyme, substrate and polymer may be in any form as described herein. The gel preferably includes the form of a buffer salt, as described herein.

[0133] Compositions of the invention can be used to treat any microbial infection that can be treated by hydrogen peroxide. Examples include infection caused by gram positive bacteria, gram negative bacteria, acid-fast bacteria, viruses, yeasts, parasitic or pathogenic micro-organisms or fungi. For example, infections caused by the following micro-organisms may be treated: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Propionibacterium acnes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophytics, Beta haemolytic Streptococci Group A or B, Campylobacter coli, Campylobacter jejuni, Methicillin Resistant Staphylococcus Aureus (MRSA), Methicillin Sensitive Staphylococcus Aureus (MSSA), Botrytis cinerea, Mycobacterium tuberculosis, Cryptosporidium, Plasmodium, Streptococcus pyogenes, Streptococcus zooepidemicus and Toxoplasma.

[0134] There is further provided according to the invention a composition of the invention for use in the prevention or treatment of a microbial infection, for example a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm. So, there may be provided a composition of the invention for use in the prevention or treatment of a microbial infection that comprises a biofilm or a microbe that is capable of forming a biofilm. The biofilm may comprise bacteria, fungi and / or viruses.

[0135] There is also provided according to the invention use of a composition of the invention in the manufacture of a medicament for the prevention or treatment of a microbial infection, for example a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm.

[0136] The invention also provides a method of preventing or treating a microbial infection, for example a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm, wherein the method comprises administering an effective amount of a composition of the invention to a site of the infection.

[0137] According to the invention there is also provided use of a composition of the invention to prevent or inhibit microbial growth.

[0138] There is also provided according to the invention a composition of the invention for use as a medicament.

[0139] Compositions of the invention may be used to treat animals. Compositions of the invention may be used to treat humans. There is further provided according to the invention a composition of the invei prevention, treatment, or amelioration of a microbial infection.

[0140] The invention also provides use of a composition of the invention in the manufacture of a medicament for the prevention, treatment, or amelioration of a microbial infection.

[0141] There is further provided according to the invention a method of preventing, treating, or ameliorating a microbial infection, which comprises administering a composition of the invention to a subject in need of such prevention, treatment or amelioration. The subject may be a human or animal subject. Compositions of the invention may be topically administered.

[0142] There may be provided compositions of the invention for use in the treatment of a microbial infection that comprises a biofilm.

[0143] According to a preferred aspect of the invention, a composition of the invention may be used in a method of wound care, including the treatment of a wound, or the treatment or management of wound sepsis.

[0144] The wound may be an acute wound, chronic wound, surgical wound (for example, a Caesarean wound), chronic burn, or an acute burn. A composition of the invention may be used in the prophylactic prevention of wound sepsis. If a storage-stable composition of the invention is used, it will be appreciated that this may be diluted by liquid present at the wound site, which thereby leads to the release of hydrogen peroxide by the diluted composition.

[0145] There is also provided according to the invention a composition of the invention for treatment of a wound. There is also provided a method of treating a wound, which comprises administering a composition of the invention to a subject in need of such treatment. There is also provided a use of a composition of the invention in the manufacture of a medicament for the treatment of a wound.

[0146] Compositions of the invention may be used to treat chronic wounds or wounds that are critically colonized. The term “critically colonized” is often used to refer to a wound that has reached a critical point at which bacteria begin to negatively affect the wound and begin to elicit signs of their presence. A critically colonized wound may indicate the presence of a biofilm. A bacterial load of greater than 105organisms / gram of tissue is often accepted as impeding wound healing (Siddiqui AR, Bernstein JM (2010) Chronic wound infection: Facts and controversies. Clinics in Dermatology 28: 519-26; Edmonds, M., & Foster, A. (2004). The use of antibiotics in the diabetic foot. Am J Surg, 187(5A), 25S-28S. Consequently, compositions of the invention may be used to treat wounds that have a bacterial load of greater than 105organisms / gram of tissue. Compositions of the invention may be administered to a patient, such as placed on the wound of a patient, for a period of at least 24 hours or 48 hours or, more preferably, 72 hours.

[0147] There is further provided according to the invention use of a composition of the invention in the manufacture of a medicament for treatment of a wound.

[0148] There is also provided according to the invention a method of treating inflammation, which comprises administering a composition of the invention to a site of inflammation.

[0149] There is also provided according to the invention a composition of the invention for treatment of inflammation.

[0150] There is further provided according to the invention use of a composition of the invention in the manufacture of a medicament for treatment of inflammation.

[0151] There is also provided according to the invention a method of stimulating tissue growth, which comprises administering a composition of the invention to a site in need of such stimulation.

[0152] There is also provided according to the invention a composition of the invention for stimulating tissue growth.

[0153] There is further provided according to the invention use of a composition of the invention in the manufacture of a medicament for stimulating tissue growth.

[0154] There is also provided according to the invention a method of debriding a wound, which comprises administering a composition of the invention to a wound in need of debridement.

[0155] There is also provided according to the invention a composition of the invention for debriding a wound.

[0156] There is further provided according to the invention use of a composition of the invention in the manufacture of a medicament for debriding a wound.

[0157] There is also provided according to the invention a method of deodorising a wound, which comprises administering a composition of the invention to a wound in need of deodorising.

[0158] There is also provided according to the invention a composition of the invention for deodorising a wound. There is further provided according to the invention use of a composition of tf manufacture of a medicament for deodorising a wound.

[0159] A composition of the invention may be provided with instructions for use of the composition. For example, a composition of the invention may be packaged as a kit with the instructions.

[0160] The present invention also concerns methods of making compositions of the invention.

[0161] So, according to the invention, there is provided a method of preparing a composition, such as a composition of the invention as described above, comprising: forming a solution comprising polymer and enzyme that is able to convert a substrate to release hydrogen peroxide; and drying the solution to form a dried mixture comprising the enzyme and the polymer.

[0162] Optionally, the method may comprise adding the substrate in dried form (such as powder form), to the dried mixture. However, the composition may not necessarily include substrate for the enzyme. For example, the composition could be manufactured and sold separately from the substrate, possibly as an intermediate product. A user could, for instance, add substrate at the point of use. Preferably, however, compositions of the invention do comprise substrate, and so it is preferable that the substrate is added in dried form to the dried mixture.

[0163] Contacting the enzyme with polymer may advantageously coat or protect the enzyme and help to stabilise the enzyme.

[0164] Compositions of the invention may thus comprise granules or particles of substrate interspersed with granules or particles comprising enzyme and polymer. This may assist in separating enzyme and substrate and prevent premature generation of hydrogen peroxide. The enzyme may thus be suspended in, or coated with, polymer. The polymer may form a matrix within which enzyme is dispersed or suspended.

[0165] Compositions of the invention may be heterogenous powders. The compositions may thus comprise a first set of particles or granules which include the enzyme but which do not include the substrate, and a second set of particles or granules which include the substrate but which do not include the enzyme. This may differ from homogeneous powders in which each particle or granule comprises both enzyme and substrate. The first and second set of particles or granules may be mixed in the powder. The first set of particles may comprise polymer. The second set of particles may not comprise polymer.

[0166] Therefore, according to the invention, there is provided a powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide, substrate for the enzyme, and polymer, wherein the powder comprises a first set of particles which include the enzyme but which do not include the substrate, and a second set of particles substrate but which do not include the enzyme. Preferably, the first set of particles comprise polymer. The second set of particles may not comprise polymer.

[0167] Preferably, the solution is an aqueous solution. For this reason, it is preferable that the polymer is water-soluble.

[0168] The solution preferably comprises a salt. For example, the salt may comprise a buffer salt. The buffer may comprise PBS, TBS or Bis-Tris. PBS may be preferred.

[0169] The solution may have an ionic strength of 5 to 100 mM, preferably 10 to 75 mM, more preferably 25 to 50 mM. Advantageously, such ionic strengths may maintain stability of the composition.

[0170] The method may comprise a milling or grinding step to form a powder. The dried mixture may be milled before the substrate is added. The substrate may then be added in powder form. Alternatively, the substrate may be added to the dry mixture and then milled.

[0171] Preferably, the solution is dried by lyophilisation to form the dried mixture. The solution may be stored at freezing conditions, preferably at a temperature of less than -60eC, such as around - 80eC for at least 24 hours before being lyophilisation.

[0172] The method may comprise a sterilisation step, to form a sterile composition, optionally wherein the sterilisation step comprises exposuree to gamma or electron beam radiation. Sterilisation preferably occurs after milling.

[0173] Preferably, the method includes adding a purified enzyme. The enzyme may have a mass purity at least 95%, preferably at least 98%.

[0174] Preferably, the method includes adding a purified substrate. The substrate may have a mass purity of at least 95%, preferably at least 98%.

[0175] Specific examples are now described, with reference to the accompanying drawings in which:

[0176] Figure 1 shows the ability of various compositions (Blend 1 , see Example 1 ) to generate hydrogen peroxide on dilution, on the day of manufacture (DOM), after 28 days, and after 56 days;

[0177] Figure 2 shows the ability of compositions to generate hydrogen peroxide on dilution, 4.5 months after manufacture; Figure 3: Experimental plan (a) for determining hydrogen peroxide production of synthetic reactive oxygen powder formulations containing 0.2, 0.1 , and 0.05% glucose oxidase after 0, 10 and 18 days of storage at room temperature (21 °C) (b). Zone of inhibition produced by treating Staphylococcus aureus with 0.2% GOx synthetic reactive oxygen powder formulations after 0, 10 and 18 days of storage at room temperature (21 °C) (c). Controls of glucose and water were used both eliciting no reaction. n=3, error bars represent standard deviation; and

[0178] Figure 4. Experimental plan (a) for determining hydrogen peroxide release from 0.2% synthetic reactive oxygen powder formulated with fructose in place of glucose, stored for 0, 1 , 3 and 7 days after which glucose was added to the formulation (b). Measurements were taken 24 hours after the addition of glucose.

[0179] Example 1 - PVA / PEG (Blend 1 )

[0180] Materials

[0181] Dulbecco’s Phosphate buffered saline (Gibco, DPBS) was supplied by Fisher scientific, UK. Deionised (DI) water was created using a Purite Fusion water purification system. Anhydrous D-Glucose was supplied by Thermo Scientific Chemicals, UK. Glucose oxidase (GOx) was supplied by BBI Solutions, UK. Poly (ethylene glycol) with a molecular weight (M.W.) of 4000 (PEG) was supplied by Sigma Aldrich, UK. Poly (vinyl alcohol), 87.0-89.0% hydrolysed with a M.W. of approximately 31 ,000-50,000 (PVA) was supplied by Thermo Scientific Chemicals, UK. All materials were used as delivered with no further purification or modifications made.

[0182] Methods

[0183] DPBS was first diluted to an ionic concentration of 50 mM to create a working solution (W- PBS). A 10% PVA solution was then formed in W-DPS. This was achieved by continually stirring and heating the solution at 80°C for 1 hour or until full dissolution was observed. Once fully dissolved the PVA solution was allowed to cool to room temperature (RT). Separately, a 10% PEG solution was also formed in W-DPS by mixing at RT. A third solution was then created, also at RT, and consisted of 0.05% GOx in W-DPS. The 10% PEG solution was then combined with the GOx solution in a 24:1 ratio and mixed thoroughly. This combined PEG-GOx solution was then added to the cooled PVA solution, whilst stirring, at a ratio of 1 :3 until mixed.

[0184] This final polymer-enzyme solution was then frozen at -80°C for 24 hours. Lyophilisation was then conducted over 5 days using a ScanVac CoolSafe (Scientific laboratory supplies, UK) and drying was performed under high vacuum (4 x 10-4mbar) conditions with the condenser temperature set at -110°C. Once lyophilised the polymer-enzyme material was then milled and combiner such that the final composition of the powder was as follows:

[0185] 50 PBS B. GP (50 mM PBS buffer)

[0186] Constituent % (by weight)

[0187] PVA 70.50

[0188] PEG 23.50

[0189] GOX 0.0047

[0190] Salts 2.99

[0191] Glucose 3.01

[0192] Total 100

[0193] The methodology was repeated, but with variation, to create alternative powders as follows.

[0194] 25 PBS B. GP (25 mM PBS buffer)

[0195] Constituent % (by weight)

[0196] PVA 71 .57

[0197] PEG 23.86

[0198] GOX 0.0048

[0199] Salts 1 .52

[0200] Glucose 3.05

[0201] Total 100

[0202] 50 TBS B. GP (50 mM TBS buffer) 25 TBS B. GP (25 mM TBS buffer)

[0203] Constituent % (by weight) Constituent % (bv weiaht)

[0204] PVA 69.99pvA71.30

[0205] PEG 23.33 PEG 23.77

[0206] GOX 0.0047GQX0.0048

[0207] Salts 3.69 Salts 1 .88

[0208] Glucose 2.99 Glucose 3.042

[0209] Total 100 Total -| go 50 B-T B. GP (50 mM Bis-Tris buffer) 25 B-TB. GP (25 mM Bis-Tris

[0210] PVA 65.98 PVA 69.17

[0211] PEG 21.99 PEG 23.055

[0212] GOX 0.0046

[0213] GOX 0.0044

[0214] Salts 4.82

[0215] Salts 9.20

[0216] Glucose 2.95

[0217] Glucose 2.82 Total 100

[0218] Total 100

[0219] GP (no buffer)

[0220] PVA 72.67

[0221] PEG 24.22

[0222] GOX 0.0048

[0223] Glucose 3.10

[0224] Total 100

[0225] The powders are stored at room temperature under normal atmospheric conditions. Testing of hydrogen peroxide production is conducted using Amplex red reagents (See Example 2).

[0226] Example 2 - Amplex Red Assay

[0227] Materials

[0228] Invitrogen Amplex Red Glucose / Glucose oxidase Assay Kit (Ref A22189) o 5x Reaction Buffer (this is replaced with self-prepared 1x PBS - Gibco) o Blue top tube - Amplex red reagent o Green top tube - DMSO o Yellow top tube - Horseradish Peroxidase o Red top tube - Hydrogen peroxide used for hydrogen peroxide assay standard o Amber bottle - powdered glucose

[0229] Analytical Balance

[0230] 96-well plate reader

[0231] Statistical software (e.g. Matlab)

[0232] Computer

[0233] Eppendorf heating block

[0234] Vortex mixer

[0235] Micropipette o P1000 o P100 o P10 8 Channel pipette o 50|il

[0236] Small spatula

[0237] Eppendorf rack

[0238] Reagent reservoirs

[0239] Pipette tips

[0240] Eppendorf Tubes

[0241] 96-well plates

[0242] Ultrapu re water

[0243] Reagent / Stock Preparation

[0244] To be prepared just prior to the reaction and plate reading:

[0245] Prepare 1 OmM stock solution of Amplex Red reagent: o Once the Amplex Red reagent and DMSO have been warmed to room temperature, dissolve the contents of the Amplex Red reagent with 60pl DMSO.

[0246] Prepare the PBS / Reaction Buffer solution: o Gibco 10x PBS is to be diluted to 1 x using deionised water.

[0247] Prepare 10U / ml horseradish peroxidase stock (HRP) o Dissolve the contents of the vial in 1 ml 1 x PBS. Divide the HRP into single-use aliquots and store at <-20°C.

[0248] Prepare 10OU / ml Glucose Oxidase (GOx) stock o Dissolve the contents of the tube in 1 ml 1x PBS. To be frozen immediately, up to 5 freeze thaw cycles has been shown to have no impact on the results. However, if used without freezing there is -10% increased activity.

[0249] Prepare a 400mM (72mg / ml) glucose stock: o Dissolve required amount of glucose in volume of 1 x PBS to achieve concentration.

[0250] Prepare a 20mM H2O2 working solution: o Dilute the 3% stock solution into the appropriate volume of 1 x PBS (22.7pL of 3.0% H2O2 into 977pL of 1 X PBS). Once the H2O2 has been diluted, it becomes unstable and should be used promptly. o

[0251] For use when glucose is not present in powder formulation

[0252] Prepare a 0.125% glucose solution in 1x PBS

[0253] Activated H2O2 Assay 1 ) Using an analytical balance and a small spatula, weigh out 200mg of RO- bijou tubes.

[0254] 2) Add 4.8 mL of 1 x PBS or 0.125% glucose solution (if glucose is not present in the powder) to each bijou, maintaining a steady speed that can be replicated when sampling.

[0255] 3) Vortex each sample to ensure homogeneity.

[0256] 4) Transfer 500 pl of sample from each bijou into an Eppendorf and dilute with 500 pl of 1 x PBS.

[0257] 5) At the required time, make serial dilutions of 1 :100, 1 :1000 in PBS quickly to prevent H2O2 build up.

[0258] 6) Load 50pl of each of the samples in triplicate into a 96-well plate, making note of which wells contain which sample and dilution.

[0259] 7) Prepare the H2O2 standard curve: a. Dilute the H2O2 stock using 1 x PBS to create a 1 OmM solution b. Subsequently dilute the 1 OmM H2O2 solution 1 :100 (1 Opl into 990pl ultrapure water) to achieve a 100pM solution and create the following standard curve

[0260] 8) Load 50pl of the samples and standard curve samples

[0261] 9) Prepare a working solution of 1 OOpM Amplex Red reagent (use immediately):

[0262] 10) Mix the solution through inversion of the tube and transfer the solution into a reagent reservoir.

[0263] 11 ) Using a multichannel pipette, transfer 50pl of the working solution into each of the reaction wells.

[0264] 12) Allow to develop for 5 minutes and read the plate using the plate reader at 570nm. Example 3 - Storage Stability

[0265] The hydrogen peroxide-generating capabilities of the powders from Example 1 and Example 2 was then assessed at various time periods after formulation, following storage, according to the protocol in Example 2.

[0266] The results comparing powders from Example 1 are shown in Figure 1 .

[0267] It can be seen how the ability to generate high levels of hydrogen peroxide is maintained after storage for an extended period of time, and that even further improved stability is demonstrated for powders which include buffer salts. For powders which include buffer salts, there is no significant decrease in the level of hydrogen peroxide produced between the day of manufacture (DOM) to 56 days after manufacture.

[0268] The powders from Example 1 incorporating PBS buffer salts were tested for their ability to generate hydrogen peroxide over even longer periods of time. The results are shown in Figure 2. No significant reduction in the ability to generate hydrogen peroxide was observed after 4.5 months of storage.

[0269] Example 3 - Blend 2

[0270] The materials are as indicated in Example 1 .

[0271] Methods

[0272] DPBS was first diluted to an ionic concentration of 25 mM to create a working solution (W- PBS). A 10% PVA solution was then formed in W-DPS. This was achieved by continually stirring and heating the solution at 80°C for 1 hour or until full dissolution was observed. Once fully dissolved the PVA solution was allowed to cool to room temperature (RT). Separately, a 10% PEG solution was also formed in W-DPS by mixing at RT. A third solution was then created, also at RT, and consisted of 0.05% GOx in W-DPS. The 10% PEG solution was then combined with the GOx solution in a 34:1 ratio and mixed thoroughly. This combined PEG-GOx solution was then added to the cooled PVA solution, whilst stirring, at a ratio of 35:65 until mixed.

[0273] This final polymer-enzyme solution was then frozen at -80°C for 24 hours. Lyophilisation was then conducted over 5 days using a ScanVac CoolSafe (Scientific laboratory supplies, UK) and drying was performed under high vacuum (4 x 10-4mbar) conditions with the condenser temperature set at -110°C. Once lyophilised the polymer-enzyme material was then milled and combiner such that the final composition of the powder was as follows:

[0274] 50 PBS B. GP (50 mM PBS buffer)

[0275] Constituent % (by weight)

[0276] PVA 61.10

[0277] PEG 32.90

[0278] GOX 0.0047

[0279] Salts 2.99

[0280] Glucose 3.01

[0281] Total 100

[0282] The methodology was repeated, but with variation, to create an alternative powders as follows.

[0283] 25 PBS B. GP (25 mM PBS buffer)

[0284] Material %

[0285] PVA 62.03 10

[0286] PEG 33.4

[0287] GOX 0.0048

[0288] Salts 1 .52

[0289] Glucose 3.05

[0290] Total 100

[0291] 15

[0292] Blend 2 demonstrates greater inelastic properties, making it more amenable to milling, to potentially allow smaller particle sizes.

[0293] Glucose and Glucose-fructose powders containing GOx

[0294] Three formulations were created with 0.2, 0.1 and 0.05% GOx incorporated into powder systems containing only glucose, fructose and glucose-fructose.

[0295] The ability of these systems to produce and release hydrogen peroxide immediately postproduction was tested (Figure 3a). it was found that levels of H2O2 rose steadily over a 72-hour period where they peaked, producing concentrations of 2.44 ± 0.09, 2.10 ± 0.08 and 1 .96 ± 0.05 mmolg-1respectively. After 72 hours, the concentration of hydrogen peroxide reduced slightly to 2.09 ± 0.02, 1 .59 ± 0.05, 1 .47 ± 0.06 mmolg-1respectively after 7 days. Powders were then stored at room temperature (21 °C) for 10- and 18-days following production, before reassessment of each powder’s hydrogen peroxide producing capability. After 10 days of storage, it was found that the level of hydrogen peroxide produced was significantly reduced. The release profile was like that measured initially post formulation, however after hydrogen peroxide concentrations were reduced by 43.7 (1 .38 ± 0.6 mmolg-1), 69.2 (0.65 ± 0.3 mmolg-1) and 77.6% (0.44 ± 0.49 mmolg-1) respectively. This phenomenon continued and after 18 days of storage concentrations of hydrogen peroxide produced were only 0.86, 0.95 and 0.05% of the original value tested immediately postproduction (Figure 3b). Further confirmation of a decrease in efficacy was also prevalent in the results from bacterial zones of inhibition tests. The ability for the 0.2% GOx synthetic formulations to inhibit the growth of S. aureus was assessed immediately postproduction, and then again after 10 and 18 days, complimentary to that of the hydrogen peroxide assay. It was found that the zone produced initially were large (24.7 ± 1 .5 mm) that decreased to (11 .3 ± 1 .5 mm) after 10 days and after 18 days no zone of inhibition was detected (Figure 3c). Controls of both glucose and water produced no capacity to inhibit bacteria inferring that activity is due to the presence of GOx and the enzymatic reaction that occurs.

[0296] Further testing was conducted to better understand the capacity reduction of synthetic powders to produce hydrogen peroxide. Glucose in 0.2% Gox formulations was replaced with inert fructose, to prevent the enzymatic oxidation reaction from occurring. These formulations were stored for a week at room temperature (21 °C) and tested for hydrogen peroxide release at a 24- hour time point on days 0, 1 , 3 and 7 (0.2% fructose control) (Figure 4a). To assess if the presence of glucose during storage caused the decrease in efficacy noted in Figure 3, glucose was added to the 0.2% fructose formulations immediately before assessing the hydrogen peroxide production at a 24-hour time point on days 0, 1 , 3 and 7 (0.2% fructose-glucose). As expected without the presence of glucose the 0.2% fructose control indicated negligible concentrations of hydrogen peroxide over the 7 days of storage. A decreasing trend however was noted when assessing the capacity of 0.2% fructose formulations to produce hydrogen peroxide when glucose is added prior to testing. A 24-hour time point taken after 7 days of storage, produced 0.35 ± 0.02 mmolg-1of hydrogen peroxide. This was significantly less than that measured post-formulation where hydrogen peroxide concentrations were 0.68 ± 0.05 mmolg-1indicating a trend of a reducing capacity to produce hydrogen peroxide over the storage period (Figure 4b).

[0297] Examples of the invention are defined in the following numbered clauses.

[0298] 1 . A powder composition, comprising enzyme that is able to convert a substrate to release hydrogen peroxide; and polymer.

[0299] 2. The composition according to clause 1 , which is a powder or a granular composition.

[0300] 3. The composition according to clause 2, comprising particles in which enzyme is suspended in, or coated with, the polymer. 4. The composition according to any preceding clause, which is a powder or a granular composition comprising a first set of particles interspersed with a second set of particles, the first set of particles comprising enzyme and polymer, but substantially no substrate, and the second set of particles comprising substrate but substantially no enzyme, and optionally substantially no polymer.

[0301] 5. The composition according to clause 4, wherein the first set of particles comprise the enzyme suspended in, or coated with, the polymer.

[0302] 6. The composition according to any preceding clause, which is able to generate hydrogen peroxide on contact with water.

[0303] 7. The composition according to any preceding clause, comprising substrate.

[0304] 8. The composition according to any preceding clause, in which the total amount of the polymer is greater than 50%, by weight, greater than 75%, by weight, or at least 90%, by weight, of the composition.

[0305] 9. The composition according to any preceding clause, in which the substrate is less than 10%, by weight, or less than 5%, by weight, of the composition.

[0306] 10. The composition according to any preceding clause, wherein the total amount of sugar is 10% or less, by weight, or less than 5%, by weight, of the composition.

[0307] 11 . The composition according to any preceding clause, which is substantially free of fructose.

[0308] 12. The composition according to any preceding clause, wherein the polymer comprises a plurality of distinct polymers.

[0309] 13. The composition according to any preceding clause, wherein the polymer comprises a water-soluble polymer.

[0310] 14. The composition according to any preceding clause, wherein the polymer comprises a synthetic polymer.

[0311] 15. The composition according to any preceding clause, wherein the polymer comprises a non-ionic polymer. 16. The composition according to any preceding clause, wherein the poly biodegradable polymer.

[0312] 17. The composition according to any preceding clause, in which the polymer comprises a semi-crystalline polymer.

[0313] 18. The composition according to any preceding clause, in which the polymer comprises a polymer with a glass transition temperature of 50eC or higher.

[0314] 19. The composition according to any preceding clause, in which the polymer comprises a polymer with a glass transition temperature of less than 50eC, preferably less then 20eC.

[0315] 20. The composition according to any preceding clause, in which the polymer comprises a polymer with an organic carbon chain backbone, optionally a vinyl polymer.

[0316] 21 . The composition according to any preceding clause, in which the polymer comprises a polymer with a heterochain backbone.

[0317] 22. The composition according to any preceding clause, in which the polymer comprises a polyether.

[0318] 23. The composition according to any preceding clause, in which the polymer comprises one or more of polyacrylic acid, polyethylene oxide, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and polyacrylamide.

[0319] 24. The composition according to any preceding clause, in which the polymer comprises polyvinyl alcohol.

[0320] 25. The composition according to clause 24, in which the polyvinyl alcohol is present in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight. .

[0321] 26. The composition according to any preceding clause, in which the polymer comprises polyethylene glycol.

[0322] 27. The composition according to clause 26, in which the polyethylene glycol is present in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight.. 28. The composition according to any preceding clause, comprising less weight, superabsorbent polymer, preferably substantially no superabsorbent polymer.

[0323] 29. The composition according to any preceding clause which does not include a polymer which has a water absorption capacity of at least 10 g / g.

[0324] 30. The composition according to any preceding clause comprising salt.

[0325] 31 . The composition according to clause 30, wherein the total amount of salt present in the composition is less than 10% by weight, preferably less than 5% by weight.

[0326] 32. The composition according to clause 30 or clause 31 , wherein the salt comprises a buffer.

[0327] 33. The composition according to clause 32, wherein the buffer comprises PBS, TBS or Bis-Tris, preferably wherein the buffer comprises PBS.

[0328] 34. The composition according to any preceding clause, wherein the enzyme comprises glucose oxidase and the substrate comprises glucose.

[0329] 35. The composition according to any preceding clause, which has a water activity of 0.3 or less.

[0330] 36. The composition according to any preceding clause comprising less than 1 %, by weight, of water.

[0331] 37. The composition according to any preceding clause, which comprises substantially no hydrogen peroxide.

[0332] 38. The composition according to any preceding clause, comprising less than 10 ppm, less than 6 ppm or less than 3 ppm hydrogen peroxide.

[0333] 39. The composition according to any preceding clause which is sterile.

[0334] 40. The composition according to any preceding clause, wherein there is 0.001 % to 10%, by weight, of the enzyme in the composition, or 0.0025% to 5%, by weight, of the enzyme in the composition.

[0335] 41 . The composition according to any preceding clause comprising substantially no honey. 42. The composition according to any preceding clause, wherein the com powder or is granular, with an average particle size of 3000 pm or less.

[0336] 43. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 2000 pm or less.

[0337] 44. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 1000 pm or less.

[0338] 45. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 500 pm or less.

[0339] 46. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 50 pm or more.

[0340] 47. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 100 pm or more.

[0341] 48. The composition according to any preceding clause, wherein the composition is a powder or is granular, with an average particle size of 200 pm or more.

[0342] 49. The composition according to any of clauses 39 to 48, wherein the average is a mean.

[0343] 50. The composition according to any of clauses 39 to 48, wherein the average is a mode.

[0344] 51 . The composition according to any of clauses 39 to 48, wherein the average is a median.

[0345] 52. A wound dressing which comprises a dressing material for dressing a wound, and a composition according to any preceding clause.

[0346] 53. A package or container comprising a composition as defined in any of clauses 1 to 51 , which is air-tight or hermetically sealed.

[0347] 54. A composition according to any of clauses 1 to 51 , for use as a medicament.

[0348] 55. A composition according to any of clauses 1 to 51 , for use in prevention, treatment or amelioration of a microbial infection.

[0349] 56. A composition according to any of clauses 1 to 51 , for use in treatment of a wound.

[0350] 57. A method of preparing a composition, optionally wherein the composition is as defined in any of clauses 1 to 51 , comprising: forming a solution comprising polymer, and enzyme that is able to coi to release hydrogen peroxide; drying the solution to form a dried mixture comprising the enzyme and the polymer; and optionally adding substrate in dried form, to the dried mixture.

[0351] 58. The method according to clause 57, wherein the solution is an aqueous solution.

[0352] 59. The method according to any of clauses 57 or clause 58, wherein the solution comprises a salt.

[0353] 60. The method according to any of clauses 57 to 59, wherein the salt comprises a buffer.

[0354] 61 . The method according to clause 60, wherein the buffer comprises PBS, TBS or Bis-Tris.

[0355] 62. The method according to clause 61 , wherein the buffer comprises PBS.

[0356] 63. The method according to any of clauses 59 to 62, wherein the solution has an ionic strength of 5 to 100 mM, preferably 10 to 75 mM, more preferably 25 to 50 mM.

[0357] 64. The method according to any of clauses 57 to 63, comprising milling to form the powder.

[0358] 65. The method according to clause 64, wherein the dried mixture is milled before the substrate is added.

[0359] 66. The method according to clause 65, wherein the substrate is added in powder form.

[0360] 67. The method according to any of clauses 57 to 66, wherein the solution is dried by lyophilisation.

[0361] 68. The method according to any of clauses 57 to 67, wherein the composition is formulated such that the total amount of polymer is greater than 50%, by weight, greater than 75%, by weight, or is at least 90%, by weight, of the composition.

[0362] 69. The method according to any of clauses 57 to 68, wherein the polymer comprises a plurality of distinct polymers.

[0363] 70. The method according to any of clauses 57 to 69, wherein the polymer comprises a water-soluble polymer. 71 . The method according to any of clauses 57 to 70, wherein the polymc synthetic polymer.

[0364] 72. The method according to any of clauses 57 to 71 , wherein the polymer comprises a non-ionic polymer.

[0365] 73. The method according to any of clauses 57 to 72, wherein the polymer comprises a biodegradable polymer.

[0366] 74. The method according to any of clauses 57 to 73, in which the polymer comprises a semi-crystalline polymer.

[0367] 75. The method according to any of clauses 57 to 74, in which the polymer comprises a polymer with a glass transition temperature of 50eC or higher.

[0368] 76. The method according to any of clauses 57 to 75, in which the polymer comprises a polymer with a glass transition temperature of less than 50eC, preferably less then 20eC.

[0369] 77. The method according to any of clauses 57 to 76, in which the polymer comprises a polymer with an organic carbon chain backbone, optionally a vinyl polymer.

[0370] 78. The method according to any of clauses 57 to 77, in which the polymer comprises a polymer with a heterochain backbone.

[0371] 79. The method according to any of clauses 57 to 78, in which the polymer comprises a polyether.

[0372] 80. The method according to any of clauses 57 to 79, in which the polymer comprises one or more of polyacrylic acid, polyethylene oxide, polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol and polyacrylamide.

[0373] 81 . The method according to any of clauses 57 to 79, in which the polymer comprises polyvinyl alcohol.

[0374] 82. The method according to clause 81 , in which the composition is formulated such that the polyvinyl alcohol is present in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight..

[0375] 83. The method according to any of clauses 57 to 82, in which the polymer comprises polyethylene glycol. 84. The method according to clause 83, in which the composition is formulated such that the polyethylene glycol is present in an amount of at least 10%, by weight, at least 25% by weight, at least 50% by weight, at least 75% by weight, or at least 90% by weight..

[0376] 85. The method according to any of clauses 57 to 84, wherein the composition is formulated such that there is less than less than 1%, by weight, of water in the composition.

[0377] 86. The method according to any of clauses 57 to 85, wherein the composition is formulated such that there is 10% or less, by weight, of sugar in the composition.

[0378] 87. The method according to any of clauses 57 to 86, wherein the composition is formulated such that there is 5% or less, by weight, of sugar in the composition.

[0379] 88. The method according to any of clauses 57 to 87, wherein the composition is formulated such that the water activity (aw) of the composition is 0.3 or less.

[0380] 89. The method according to any of clauses 57 to 88, wherein the composition is formulated such that the substrate is 10% or less, by weight, of the composition.

[0381] 90. The method according to any of clauses 57 to 89, wherein the composition is formulated such that the substrate is 5% or less, by weight, of the composition.

[0382] 91 . The method according to any of clauses 57 to 90, wherein the composition is formulated such that the substrate is at least 1 %, by weight, of the composition.

[0383] 92. The method according to any of clauses 57 to 91 , wherein the substrate comprises glucose and the enzyme comprises glucose oxidase.

[0384] 93. The method according to any of clauses 57 to 92, comprising a sterilisation step, to form a sterile composition, optionally wherein the sterilisation step comprises expose to gamma or electron beam radiation.

[0385] 94. The method according to any of clauses 57 to 93, wherein the enzyme is a purified enzyme.

[0386] 95. The method according to clause 94, wherein the enzyme has a mass purity at least 95%, preferably at least 98%. 96. The method according to any of clauses 57 to 95, wherein the substrj substrate.

[0387] 97. The method according to clause 96, wherein the substrate has a mass purity of at least 95%, preferably at least 98%.

[0388] 98. The method according to any of clauses 57 to 97, wherein the enzyme is added such that it is 0.001% to 10%, by weight, of the composition, or 0.0025% to 5%, by weight, of the composition. 99. The method according to any of clauses 57 to 98, in which substantially no honey is added.

[0389] 100. The method according to any of clauses 57 to 99, in which substantially no: zinc oxide, catalase, peroxidase, and / or lactoferrin is added.

[0390] 101. A gel, or hydrogel, obtained or obtainable by contacting the composition as defined in any of clauses 1 to 51 , with water.

Claims

Claims1 . A powder composition comprising enzyme that is able to convert a substrate to release hydrogen peroxide; substrate for the enzyme; and polymer, the composition comprising a first set of particles interspersed with a second set of particles, the first set of particles comprising the enzyme and the polymer, but substantially none of the substrate, and the second set of particles comprising the substrate but substantially none of the enzyme.

2. The composition according to claim 1 , wherein the first set of particles comprise the enzyme suspended in, or coated with, the polymer.

3. The composition according to any preceding claim, in which the total amount of the polymer is greater than 75%, by weight, of the composition.

4. The composition according to any preceding claim, in which the substrate is less than 5%, by weight, of the composition.

5. The composition according to any preceding claim, wherein the total amount of sugar is less than 5%, by weight, of the composition.

6. The composition according to any preceding claim, in which the polymer comprises polyvinyl alcohol.

7. The composition according to any preceding claim, in which the polymer comprises polyethylene glycol.

8. The composition according to any preceding claim, comprising less than 0.5%, by weight, superabsorbent polymer, preferably substantially no superabsorbent polymer.

9. The composition according to any preceding claim comprising substantially no peroxidase.

10. The composition according to any preceding claim comprising salt, preferably wherein the salt comprises a buffer.11 . The composition according to any preceding claim, wherein the enzyme comprises glucose oxidase and the substrate comprises glucose.

12. The composition according to any preceding claim which is sterile.

13. The composition according to any preceding clause comprising subst14. A wound dressing which comprises a dressing material for dressing a wound, and a composition according to any preceding claim.

15. A composition according to any of claims 1 to 13, for use as a medicament.

16. A composition according to any of clauses 1 to 13, for use in prevention, treatment or amelioration of a microbial infection.

17. A method of preparing a powder composition, the powder composition optionally as defined in any of claims 1 to 13, the method comprising: forming a solution comprising polymer, and enzyme that is able to convert a substrate to release hydrogen peroxide; drying the solution to form a dried mixture comprising the enzyme and the polymer; and adding substrate for the enzyme in powder form, to the dried mixture.

18. The method according to claim 17, wherein the solution is an aqueous solution.

19. The method according to claim 17 or claim 18, wherein the solution comprises a salt, preferably a buffer20. The method according to claim 19, wherein the solution has an ionic strength of 5 to 100 mM, preferably 10 to 75 mM, more preferably 25 to 50 mM.21 . The method according to any of claims 17 to 20, comprising milling to form the powder, optionally wherein the dried mixture is milled before the substrate is added.

22. The method according to any of claims 17 to 21 , wherein the solution is dried by lyophilisation.

23. The method according to any of clauses 17 to 22, comprising a sterilisation step, to form a sterile composition, optionally wherein the sterilisation step comprises expose to gamma or electron beam radiation.

24. The method according to any of claims 17 to 23, wherein the enzyme is a purified enzyme with a mass purity of at least 95%, and the substrate is a purified substrate with a mass purity of at least 95%.

25. A gel, or hydrogel, obtained or obtainable by contacting the compositi any of claims 1 to 13, with water.

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