DUAL DISPENSER CONTAINING WATER AND ANTIMICROBIAL COMPOSITION

The present invention relates to devices for dispensing compositions, particularly compositions that can provide antimicrobial activity.

The applicant has found that compositions that are able to release hydrogen peroxide at the site of a microbial infection are particularly effective at preventing or inhibiting the infection.

Surgihoney™ (also known as SurgihoneyRO) is a chemical engineered honey that has the ability to deliver variable and sustained doses of reactive oxygen species (ROS). Studies in vitro and in vivo have demonstrated Surgihoney's efficacy in eradication of infection. This has included drug resistant strains, such as methicillin-resistant S. aureus (MRSA) and vanomycin- resistant Enterococcus faecium {Dryden, M., Lockyer, G., Saeed, K., & Cooke, J. (2014).

Engineered Honey: In Vitro Antimicrobial Activity of a Novel Topical Wound Care Treatment. Journal of Global Antimicrobial Resistance, 2, 168-172). It was also shown to be effective against fungi and prevented or reduced the seeding of biofilms (Dryden, M., Halstead, F., & Cooke, J. (2015). Engineered Honey to Manage Bacterial Bioburden and Biofilm in Chronic Wounds. EWMA Free Paper Session: Infection and Antimicrobials). Engineered honeys, such as honey with added glucose oxidase, and related compositions, are described in WO

2015/166197 A1 , WO 2016/083798 A1 , WO 2016/124926 A1 and WO 2017/013448 A1.

At present, Surgihoney is available in the form of a sachet or tube for topical administration. However, the applicant has found that, in certain circumstances, administration of Surgihoney in these forms may be inconvenient. For example, applying a viscous composition evenly to a wound can be difficult and may require the applier to physically spread the composition on the wound.

The applicant has also appreciated that some wounds may be dry because they do not produce much exudate. Applying Surgihoney and related compositions directly to dry wounds may not be as effective as applying the composition to wounds that readily produce exudate.

In a broad sense, the invention concerns a dispensing device for dispensing a composition that is able to generate hydrogen peroxide. The composition may comprise an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme.

Alternatively or additionally, the composition may comprise a precursor-substrate that can be converted to a substrate for the enzyme. The composition may not comprise sufficient free water to allow the enzyme to convert the substrate. The composition may not comprise sufficient free water to allow the precursor-substrate to be converted to the substrate. According to the invention there is provided a dispensing device comprising: a first chamber having or containing a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not having sufficient free water to allow the enzyme to convert the substrate; and a second chamber having or containing water.

According to the invention there is provided a dispensing device comprising: a first chamber having or containing a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and a precursor-substrate that can be converted to a substrate for the enzyme, the composition not having sufficient free water to allow the precursor-substrate to be converted to the substrate or to allow the enzyme to convert the substrate; and a second chamber having or containing water.

According to the invention there is provided a dispensing device comprising: a first chamber having or containing a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not having sufficient free water to allow the enzyme to convert the substrate; and a second chamber having or containing water; and at least one outlet for dispensing the composition and the water from the device.

According to the invention there is provided a dispensing device comprising: a first chamber having or containing a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and a precursor-substrate that can be converted to a substrate for the enzyme, the composition not having sufficient free water to allow the precursor-substrate to be converted to the substrate or to allow the enzyme to convert the substrate; and a second chamber having or containing water; and at least one outlet for dispensing the composition and the water from the device.

The water may be distilled or deionised water or the water may be provided as an aqueous solution. For example, the water may be provided as a saline solution.

As the composition does not contain sufficient free water to allow the enzyme to convert the substrate (or for the precursor-substrate to be converted to the substrate), the composition, whilst it is present in the first chamber, may not be able to produce hydrogen peroxide. When the composition is dispensed from the device with the water, the water and the composition may mix to form a mixture in which the enzyme is able to convert the substrate to release hydrogen peroxide (and/or such that the precursor-substrate may be converted to the substrate). Consequently, dispensing the water and the composition on to a dry wound, for instance, may allow production of hydrogen peroxide at the wound site. Furthermore, a dispensing device may remove the necessity of direct physical contact with, for example, a wound site.

The skilled person would understand that if a composition of the invention does not comprise sufficient free water to allow the enzyme to convert the substrate, it may contain trace amounts of free water that may allow trace amounts, or substantially no hydrogen peroxide to be produced. For example, hydrogen peroxide may present at a concentration less than 1 ppm. Hydrogen peroxide may be present at a concentration less than 0.5 ppm. Hydrogen peroxide may be present at a concentration less than 0.1 ppm. Hydrogen peroxide may be present in the composition at a concentration of 120 μΜ or less, preferably 100 μΜ or less, more preferably 80 μΜ or less. Once the composition is diluted, hydrogen peroxide may then be generated at substantial concentrations.

Compositions used in devices of the invention may separate (or compartmentalise) water from the enzyme and the substrate, such that the enzyme and the substrate are not in contact with sufficient free water to allow the enzyme to convert the substrate. For example, the composition may be a double emulsion, in which, for instance, droplets containing the enzyme and the substance (but without sufficient free water to allow the enzyme to convert the substrate) may be dispersed within globules of oil, and the globules of oil may be dispersed within an aqueous phase (e.g. water).

In one embodiment, the composition and the water may be dispensed separately from the device. For example, the device may comprise a first outlet for the composition and a second outlet for water. The first outlet may comprise a first tube through which the composition can be transported from the first chamber to a first exit hole or aperture. The second outlet may comprise a second tube through which the water can be transported from the second chamber to a second exit hole or aperture. The device may be arranged to separately dispense the composition and the water under pressure. For example the device may be arranged to dispense both the composition and the water as a spray or atomised liquid.

In a preferred embodiment, the device is arranged such that the composition and the water are pre-mixed, i.e. mixed prior to dispensing from the device. For example, the device may comprise a mixing chamber having a first inlet for receiving the composition, a second inlet for receiving the water, and an outlet for dispensing a mixture of the composition and the water out of the mixing chamber. The first inlet may comprise a tube through which the composition from the first chamber can flow to the mixing chamber; the second inlet may comprise a tube through which the water from the second chamber can flow to the mixing chamber; and the outlet may comprise a tube through which the mixture from the mixing chamber can flow out of the device through an exit hole or aperture. Pre-mixing the composition and the water may be

advantageous because the addition of water to the composition may reduce the viscosity of the composition and make it easier to dispense from the device. This may be particularly advantageous if the device is arranged to dispense under pressure, such as a spray.

In one embodiment, the device is arranged to dispense a mixture of the composition and the water under pressure. For example the device may be arranged to dispense the mixture of the composition and water as a spray, such as a pump action or trigger spray, an atomiser or an aerosol.

In use, it may be possible to vary the amount or volume of the composition and/or the amount or volume of water that can be dispensed from the device. This may allow the viscosity of the mixture of the composition and water to be varied, whether the mixing occurs before dispensing from the device (i.e. pre-mixing) or after dispensing from the device. Consequently, devices of the invention may comprise a means for varying the amount or volume of the composition and/or the amount or volume of the water that can be dispensed from the device.

If the device dispenses the composition and the water separately, the first outlet and/or second outlet may comprise a variable restriction for restricting the volume of the composition and/or the volume of the water dispensed from the device.

Preferably, the device of the invention permits the pre-mixing of a variable amount or volume of the composition and/or a variable amount or volume of the water. Thus, it may be possible to vary the relative amounts or volumes of the composition and the water that mix in the mixing chamber. Varying the relative amounts or volumes may allow the user to control the properties of the mixture. For example, increasing the ratio of the volume of water to the volume of composition may reduce viscosity and alter the properties of the spray dispensed from the device. A reduced viscosity may result in a spray with finer droplets, for instance.

If the device pre-mixes the composition and the water before dispensing, the first inlet and/or second inlet may comprises a variable restriction for restricting the volume of the composition and/or the volume of the water entering the mixing chamber.

US Pat. No. 4,355,739 describes a spraying device which allows the ratio of the quantities of liquid components from the chambers to be varied. Another example of a device which allows variation of the relative voiumes of fluids entering a mixing chamber from two distinct chambers is shown in US pat. No. 5,152,461.

Alternatively, if there is no pre-mixing, the first outlet and second outlet may comprise a variable restriction for restricting the amount or volume of the composition and/or the amount or volume of the water dispensed from the device. The device may be configured or configurable to dispense, a composition: water volume ratio of 5:1 or lower. For example, the composition: water volume ratio may be 5:1 to 1 :1 ; 2:1 to 1 :1 or 1.5:1 to 1 :1.

The device may be configured or configurable to dispense, a composition: water volume ratio of 1 :5 or higher. For example, the composition: water volume ratio may be 1 :1 to 1 :5 1 :1 to 1 :2 or 1 :1 to 1 :1.5

Although it is preferable that the composition in the first chamber is a liquid, it is conceivable that it could be a powder. If the composition is a liquid, it is preferable that the composition comprises a non-aqueous or organic solvent. This may be particularly advantageous if the composition would otherwise be viscous. For example, in some embodiments the composition may comprise a honey-based composition. Honey, has a high viscosity and is sticky, which can make it difficult to incorporate into spraying devices. Therefore, the viscosity of honey may be reduced by using a non-aqueous solvent. The use of a non-aqueous solvent may mean that the viscosity can be reduced without using water, which would initiate hydrogen peroxide production and may affect the storage stability of the composition.

In compositions in devices of the invention comprising a non-aqueous solvent, the non-aqueous solvent may comprise (or consist of) a mixture of more than one non-aqueous solvent.

Reference herein to "non-aqueous solvent" thus includes one or more, or at least one, nonaqueous solvent. However, in some embodiments, the non-aqueous solvent may comprise (or consist of) only one non-aqueous solvent.

In compositions that comprise a non-aqueous solvent, the non-aqueous solvent may comprise an alcohol. The non-aqueous solvent may comprise ethanol, dimethyl sulphoxide, glycerol, ethylene glycol and/or propylene glycol.

Solubility parameters and viscosity parameters for various non-aqueous solvents are shown in the following table.

I Substance ] Viscosity i

Ethanoi i 1.04mPa.s j

I Dimethyl j 1.99mPa.s i

sulphoxide

[ Glycerol j 1412mPa.s

j Ethylene j 16.1 mPa.s i|

glycol

1 Propylene I 42mPa.s i

1 glycol

|i Water j 1.3mPa.s j

In preferred embodiments, non-aqueous solvents may be selected so that they have solubility parameters in the range of the non-aqueous solvents exemplified in the tables above. For example, 6 _t /MPa ¹ ' ⁷ may be from 26 to 50, such as 26.5 to 47.8. 8d/MPa ^1/2 may be from 15 to 19, such as 15.6 to 18.1. δρ/MPa ¹ ' ² may be from 8 to 16, such as 8.8 to 16. 5 _h /MPa ¹ ' ² may be from 10 to 45, such as 10.2 to 42.3.

The non-aqueous solvent may be selected depending on the desired viscosity. For example, if a greater viscosity is desired, glycerol may be preferred. A suitable viscosity for a composition of the invention may be 100 mPa.s. or less at 20°C. In some embodiments, a suitable viscosity may be 75 mPa.s. or less at 20°C. In some embodiments, a suitable viscosity may be 75 mPa.s. or less at 20°C.

In some embodiments of compositions comprising a non-aqueous solvent, the compositions may comprise at least 2% by weight of the non-aqueous solvent. In some embodiments of compositions comprising a non-aqueous solvent, the compositions may comprise at least 5% by weight of the non-aqueous solvent. In some embodiments of compositions comprising a non-aqueous solvent, the compositions may comprise at least 10% by weight of the nonaqueous solvent. In other embodiments, the composition may comprise at least 20% by weight of the non-aqueous solvent. In other embodiments, the composition may comprise at least 25% by weight of the non-aqueous solvent. In other embodiments, the composition may comprise at least 50% by weight of the non-aqueous solvent. In some embodiments, the composition may comprise at least 75% by weight of the non-aqueous solvent. In some embodiments, the amount of non-aqueous solvent in the composition may be 50-90%, by weight. In some embodiments, the amount of non-aqueous solvent in the composition may be 1-50, 5-50 or 10- 50%, by weight.

In some embodiments, the composition is not, or does not comprise, honey and glycerol.

Compositions in devices of the invention may be in the form of an emulsion. Examples of emulsions are disclosed in WO 2017/013448 A1 and WO 2018/091890 A1. The inventors have appreciated that emulsions may be particularly suitable for devices of the invention because application of shear and exposure to water can cause phase inversion, which initiates hydrogen peroxide production. Before application of shear and exposure to water, the emulsions may remain stable for long periods of time.

Consequently, compositions in devices of the invention may comprise a first phase (or first liquid, or first component) and a second phase (or second liquid, or second component), an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme. The first phase and the second phase may be immiscible. For example, the first phase may be less polar than the second phase. The first phase may be a non-polar phase such as a lipophilic phase or a hydrophobic phase e.g. an oil. The second phase may be a polar phase, such as an aqueous phase. The second phase may comprise a non-aqueous solvent. Droplets or micelles of the second phase may be dispersed within the first phase.

The lipophilic phase may be an oil. In some embodiments, the oil is selected from olive oil, corn oil, vegetable oil, sunflower oil or paraffin oil. In a preferred embodiment, the oil may be paraffin oii.

The second phase may comprise water and/or non-aqueous solvent. The enzyme and the substance of the composition may be dissolved in the water and/or non-aqueous solvent. Suitable non-aqueous solvents are described above. Glycerol may be a particularly preferred non-aqueous solvent for emulsions.

It is conceivable that, in some embodiments, the second phase may not comprise water or may comprise substantially no water. In such circumstances, the second phase may be described as non-aqueous. For example, the enzyme and the substance comprising a substrate for the enzyme may be dissolved in a non-aqueous solvent. The non-aqueous solvent may be immiscible with respect to the first phase e.g. lipophilic phase. In some embodiments, the enzyme that is able to convert a substrate to release hydrogen peroxide and the substance that includes a substrate for the enzyme may be contained within micelles dispersed within the first phase, e.g. lipophilic phase.

The composition may comprise an emulsifying agent (or emulsifier). examples of suitable emulsifying agents include: Surfactants: Sodium lauryl sulphate, Cetrimide, Cetomacrogol 1000, PEG 1000 monostearate, Triethanolamine stearate, Sodium stearate; Fatty amphiphiles: Cetostearyl alcohol, Cetyl alcohol, Stearyl alcohol, Glyceryl monostearate, Stearic acid, Phosphatidylcholine. Surfactants for use in compositions of the invention may include one or more of TWEEN (e.g. TWEEN 80), SPAN (e.g. SPAN 80), Poloxamer (e.g. Poloxamer 407) and Polyglycerol polyricinoleate (PGPR). A preferred surfactant may be Poloxamer, such as Poloxamer 407. Another preferred surfactant may be PGPR.

The ratio of the first phase to the second phase may be from 9:1 to 1 :9, 8:1 to 1 :8, 7:1 to 1 :7, 6:1 to 1 :6, 5:1 to 1 :5, 4:1 to 1 :4, 3:1 to 1 :3, or 2:1 to 1 :2 (v/v), for example from 4:1 to 1 :4.

The composition may comprise 5-95%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, or 90-95% (v/v) lipophilic phase, or first phase (including any emulsifying agent present).

The composition may comprise 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10% (v/v) lipophilic phase, or first phase (including any emulsifying agent present).

The composition may comprise 5-95%, 10-95%, 15-95%, 20-95%, 25-95%, 30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, or 90-95% (v/v) aqueous phase, or second phase.

The composition may comprise 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, or 5-10% (v/v) aqueous phase, or second phase.

The composition may comprise 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, or 1-10% (w/v) of the substance, for example a honey or a sugar substance. A sugar substance may comprise the substrate for the enzyme, e.g. a purified substrate for the enzyme. For instance, the sugar substance may comprise one or more sugars. The sugar substance may comprise glucose and fructose.

The composition may comprise 1-60%, 5-60%, 10-60%, 15-60%, 20-60%, 25-60%, 30-60%, 35-60%, 40-60%, 45-60%, or 50-60% (w/v) of the substance. The composition may comprise 10-60% (w/w) of non-aqueous solvent. In some embodiments, a composition of the invention may comprise 20-50% (w/w) of a non-aqueous solvent. In some embodiments, a composition of the invention may comprise 35-40% (w/w) of a non-aqueous solvent.

The composition may comprise 10-40 % (w/w) of the first phase, e.g. lipophilic phase (such as oil). The composition may comprise 20-30% (w/w) of the first phase, e.g. lipophilic phase (such as oil).

The composition may comprise 1-10% (w/w) emulsifier. The composition may comprise 1-5% (w/w) emulsifier. The emulsifier is preferably a surfactant.

The composition may comprise 0-50% (w/w) of a sugar substance (e.g. glucose and fructose). The composition may comprise 20-40% (w/w) of the substance. In some embodiments, the composition may comprise 25 to 35% (w/w) of the substance.

The composition may comprise 20-50% (w/w) of non-aqueous solvent, 20-30% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1 -5% (w/w) emulsifier and 20-40% (w/w) of the substance which comprises a substrate for the enzyme.

The composition may comprise 10-60% (w/w) of non-aqueous solvent, 10-40% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1-10% (w/w) emulsifier and 10-50% (w/w) of the substance which comprises a substrate for the enzyme.

The composition may comprise 35-45% (w/w) of non-aqueous solvent, 20-30% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1-5% (w/w) emulsifier and 25-35% (w/w) of the substance which comprises a substrate for the enzyme.

The composition may comprise 30-60% (v/v) solvent, such as a non-aqueous, polar solvent.

The composition may comprise 30-60% (v/v) first phase, such as a lipophilic phase (e.g. oil),

The composition may comprise 1-10% (v/v) emulsifier such e.g. surfactant. In some

embodiments, compositions of the invention may comprise 1 -5% (v/v) emulsifier.

The composition may comprise 30-70% or 40-60% (w/w) of the substance that comprises a substrate for the enzyme, for example honey.

The ratio of the first phase to the second phase in the composition may be≤1 :1 (v/v), for example 0.1-1 :1 (v/v). In some embodiments, the ratio of the first phase to the second phase is <0.6:1 (v/v), for example 0.1-<0.6:1 (v/v). In some embodiments, the ratio of the first phase to the second phase is <0.4:1 (v/v), for example 0.1-0.4:1 (v/v).

The first phase in the composition may be present at less than 60% (v/v) of the composition. In some embodiments, the first phase is present at 10% to less than 60% (v/v) of the composition. In some embodiments, the first phase is present at 10% to less than 50% (v/v) of the composition. In some embodiments, the first phase is present at 10% to less than 40% (v/v) of the composition. In some embodiments, the first phase is present at 10% to less than 30% (v/v) of the composition. In some embodiments, the first phase is present at 10% to less than 25% (v/v) of the composition.

The composition may comprise an emulsifier. In some embodiments, the emuisifier is present at up to 25% (v/v) of the composition, for example 1-25% (v/v) of the composition, 5-25% (v/v) of the composition, or 10-25% (v/v) of the composition. In some embodiments, the composition may comprise 1-5% (v/v) of emulsifier.

The ratio of the amount of the substance that includes a substrate for the enzyme to the volume of the second phase in the composition may be from 0.5:1 to 2:1 , for example 1 :1.

The amount of the substance that includes a substrate for the enzyme in the composition may be up to 70% (w/v) of the composition, for example 5-70% (w/v), 10-70% (w/v), 20-70% (w/v), or 30-70% (w/v), or up to 60% (w/v) of the composition, for example 5-60% (w/v), 10-60% (w/v), 20-60% (w/v), or 30-60% (w/v), of the composition.

Emulsions may comprise paraffin oil and PGPR<

Emulsions may comprise paraffin oil, PGPR and glycerol.

Emulsions may comprise an organic salt such as magnesium sulfate heptahydrate. Honey is a natural product, so 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.

Honey may 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. Furthermore, for certain pharmaceutical applications, it may be difficult to get regulatory approval for honey-based compositions.

Consequently, although compositions comprising unrefined natural substances, such as honey, may be used in devices of the invention, other compositions may be used which may provide some of the antimicrobial benefits provided by honey, but which also overcome some of its disadvantages.

The term "unrefined" is used herein to refer to substances that have not been processed into a pure form. Unrefined substances include substances that may have been concentrated, for example by drying or boiling.

The substance may include one or more substrates from a natural source (termed herein a "natural substance"). Examples of natural substances include substances from a plant source, including from sap, roots, nectar, flowers, seeds, fruit, leaves, or shoots.

If the composition does comprise an unrefined natural substance such as honey, it is preferable that the substance is pasteurised.

If the composition does comprise an unrefined natural substance, such as honey, it is preferable that the substance does not contain catalase activity or contains essentially no catalase activity.

If the composition does comprise an unrefined natural substance, such as honey, the substance may be creamed.

In compositions of devices of the invention that comprise an unrefined natural substance such as honey, the unrefined natural substance may account for at least 10% by weight of the composition. In a more preferred embodiment, the unrefined natural substance many account for at least 25 % by weight of the composition. In an even more preferred embodiment, the unrefined natural substance may account for at least 50% by weight of the composition. The unrefined natural substance may account for at least 75% by weight of the composition, such as at least 80% or at least 90% by weight of the composition.

Preferably, devices of the invention do not comprise compositions formed from unrefined natural substances, such as honey. For example, the compositions of devices of the invention may comprise a purified enzyme that is able to convert a substrate to release hydrogen peroxide; and a purified substrate for the enzyme (and/or a purified precursor substrate that can

1 ^■ } be converted to a substrate for the enzyme). Such compositions may be formed by mixing a purified enzyme with purified substrate for the enzyme (and or a purified precursor substrate).

Surprisingly, the applicant has found that compositions comprising a purified enzyme and a purified substrate can be more effective at killing microorganisms than known honey-based compositions that can generate hydrogen peroxide. The enzyme in compositions of devices of the invention may thus be a purified enzyme.

References herein to "enzyme" may refer to one or more enzyme. For example, in some embodiments, compositions of devices the invention may comprise a plurality of enzymes that are able to convert a substrate to release hydrogen peroxide. In some embodiments, compositions of devices the invention may comprise only one enzyme that is able to convert a substrate to release hydrogen peroxide.

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.

It may not always be necessary or desirable that the purified enzyme is at a high level of purity provided that the enzyme is able to convert the substrate to release hydrogen peroxide. In some circumstances, it may be desirable to use a relatively crude enzyme preparation.

Examples of suitable purity levels include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% pure (mass purity). Preferably, the enzyme is at least 90% pure. Even more preferably, the enzyme is at least 99% pure.

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.

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 may be used. For pharmaceutical use, a pharmaceutical grade of purity may be used.

In some embodiments the enzyme is an oxidoreductase enzyme. Examples of oxidoreductase enzymes that can convert a substrate to release hydrogen peroxide include glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase, and aminoacid oxidase. The corresponding substrates for these oxidoreductase enzymes are D-glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate and aminoacid, respectively.

The oxidoreductase enzyme may be glucose oxidase, and the substrate may be D-glucose.

A mixture of one or more oxidoreductase enzymes and one or more substrates for the oxidoreductase enzymes may be present in compositions of devices of invention.

The compositions may comprise sufficient enzyme and substrate to provide for sustained release of hydrogen peroxide at a specific level or concentration. The compositions may comprise sufficient enzyme and substrate to provide for sustained release of hydrogen peroxide at a level of less than 2 mmol/iitre for a period of at least twenty four hours, following dilution of the composition.

The compositions may comprise sufficient enzyme and substrate to provide for sustained release of at least 0.02, 0.03, 0.04, 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 1 or 1.5 mmol/litre hydrogen peroxide for a period of at least 24 hours, more preferably 48 hours.

So, in some embodiments, the compositions may comprise sufficient enzyme and substrate to provide for sustained release of 0.1 to 2 mmol/litre hydrogen peroxide for a period of at least 24 hours, more preferably 48 hours.

For example, in some embodiments, the compositions may provide for sustained release of hydrogen peroxide at a concentration of at least 2 ppm, at least 5 ppm, at least 10 ppm, at least 20 ppm or at least 50 ppm. In preferred embodiments, the level may be at least 2 ppm. In some embodiments, the concentration may be, at the most, 500 ppm, 200 ppm, 100 ppm, 50 ppm, 20 ppm or 10 ppm. In preferred embodiments, the level may be 20 ppm or less. In even more preferred embodiments, the level may be 10 ppm or less. For example, the concentration may be 10 to 500 ppm, 20 to 200 ppm or 50 to 100 ppm, 2 to 50 ppm, 2 to 20 ppm or 5 to 10 ppm. If the composition does not comprise sufficient free water to allow the enzyme to convert the substrate, hydrogen peroxide production may only occur once it has been diluted by water and there is sufficient free water to allow the enzyme to convert the substrate. Addition of water may thus initiate hydrogen peroxide production. Compositions may provide for sustained release of hydrogen peroxide for at least 1 hour, at least 12 hours, at least 24 hours, at least 2 days, or at least 4 days. Preferably, the level of hydrogen peroxide is sustained for at least 4 days. In preferred embodiments, the level of hydrogen peroxide is sustained at 10 to 500 ppm for at least 1 hour, at least 12 hours, at least 24 hours, at least 2 days, or at least 4 days. In other embodiments, the level of hydrogen peroxide is sustained at 50 to 100 ppm for at least 1 hour, at least 12 hours, at least 24 hours, at least 2 days, or at least 4 days. In other embodiments, the level of hydrogen peroxide is sustained at 2 to 50 ppm for at least 12 hours, at least 24 hours, at least 2 days, or at least 4 days. In other embodiments, the level of hydrogen peroxide is sustained at 5 to 10 ppm for at least 12 hours, at least 24 hours, at least 2 days, or at least 4 days. In some embodiments, compositions may provide for sustained release of 2 to 500 ppm hydrogen peroxide for at least 24 hours.

The compositions may comprise 750 to 2000 ppm of the enzyme. Compositions of devices the invention may comprise at least 500 ppm of the enzyme. The compositions may comprise 250 to 1500 of the enzyme.

The enzyme activity (for example, the glucose oxidase activity) may range, for example, from 1- 400 lU/mg, or 1-300 lU/mg, for example 250-280 lU/mg. The amount of enzyme used is likely to depend on several factors, including the desired use of the composition, the desired level of hydrogen peroxide release, and the desired length of time for hydrogen peroxide release. A suitable amount of enzyme can readily be 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. Suitable amounts of enzyme (such as glucose oxidase) may be from 0.0001 % to 0.5% w/w of the composition. 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).

Compositions of devices 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.

In some embodiments, the composition may comprise more than 15 units, 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, the composition may comprise at least 500 units, for example 500-1000 units, or 685-1000 units, of enzyme (e.g. glucose oxidase) per gram of the composition.

References herein to "substrate" or precursor-substrate" may refer to one or more substrates. For example, in some embodiments, the compositions may comprise a plurality of substrates or precursor-substrates. In some embodiments, the compositions may comprise only one substrate or only one precursor substrate.

The term "purified substrate" or "purified precursor-substrate" is used herein to include a substrate or precursor-substrate preparation in which the substrate or precursor-substrate has been separated from at least some of the impurities originally present when the substrate or precursor-substrate was obtained or produced. The purified substrate or precursor-substrate may be obtained from a natural source or may be synthetically produced. The purified substrate or precursor-substrate may be a processed, extracted, or refined substrate or precursor- substrate (i.e. a substrate or precursor-substrate in which impurities or unwanted elements have been removed by processing).

It may not always be necessary or desirable that the purified substrate or precursor-substrate is at a high level of purity provided that the enzyme is able to convert the substrate to release hydrogen peroxide. In some circumstances, it may be desirable to use a relatively crude substrate or precursor-substrate preparation. Examples of suitable purity levels include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% pure. Preferably the purity level is at least 90%, even more preferably at least 99% pure. However, in some embodiments, it may be desirable that the purified substrate or purified precursor-substrate is a medical grade, medical device grade, or pharmaceutical grade substrate or precursor-substrate.

In particular embodiments, the purified substrate or precursor-substrate is or comprises a purified sugar. The term "sugar" is used herein to refer to a carbohydrate with the general formula C _m (H20) _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 purified sugar may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% pure.

Preferably, the purity level is at least 90%. Even more preferably, the purity level is at least 99%. The purified sugar may be a medical grade, medical device grade, or pharmaceutical grade sugar. 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.

In particular embodiments, the enzyme and the substrate are purified, for example purified glucose oxidase and purified D-glucose, suitably medical grade, medical device grade, or pharmaceutical grade glucose oxidase and D-glucose.

For compositions which comprise a precursor-substrate, the composition preferably comprises one or more enzymes (preferably purified 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 enough for conversion. Alternatively or additionally, compositions of devices of the invention may comprise non-enzymatic catalysts.

So, compositions of devices of the invention which comprise a precursor-substrate may comprise a first enzyme that is able to 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.

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.

Compositions of devices 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 precursor-substrate may be cellulose, the first enzyme may be glucose oxidase and the second and third enzymes may be cellulose and beta-glucosidase.

In some embodiments, the composition may comprise both a substrate that can be converted by the enzyme to generate hydrogen peroxide, and a precursor-substrate that can be converted to the substrate.

The composition and water in the device of the invention, are preferably sterile. Sterilisation may occur by any suitable means. Preferably the composition and/or water compositions are sterilised by irradiation. 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 radiation or electron beam radiation. A suitable level of gamma irradiation is 10-70 kGy, preferably 25-70 kGy, more preferably 35-70 kGy. A suitable level or dose of irradiation (e.g. electron beam irradiation) may be 10-100 kGy, preferably 30-80 kGy, more preferably 50-80kGy. The dose may be greater than 35 kGy. The dose may be less than 80 kGy, for example 75 kGy or less. In one embodiment, the composition and/or water may be sterilised by irradiation that is not gamma irradiation. Since ozone has not been authorised by the US FDA for sterilisation of honey-based products for use in wound healing, compositions in devices of the invention preferably have not been sterilized by ozonation, and do not include ozone, or any components that have been subjected to sterilisation by ozonation. In particular, the compositions in devices of the invention should not comprise ozonized honey or ozonated oil.

The composition in the device of the invention is may be a medical grade or medical device grade composition. The composition in the device of the invention may be a pharmaceutical grade composition.

Compositions of devices of the invention preferably comprise essentially no catalase, or substantially no catalase.

Compositions of devices of the invention preferably comprises essentially no peroxidase, or substantially no peroxidase.

Compositions of devices of the invention preferably comprises essentially no zinc oxide, or contain substantially no zinc oxide.

Compositions of the devices of the invention may comprise an antioxidant.

The term "storage-stable" is used herein to mean that the composition can be stored at ambient temperature for at least several days, preferably at least a week, more preferably at least one or two months, whilst retaining the ability to generate antimicrobial activity following dilution of the composition. A preferred storage temperature is below 37°C, preferably 20-25°C. Preferably compositions are stored away from exposure to light.

Hydrogen peroxide is generally unstable at ambient temperature. The lack of sufficient free water in compositions of the devices of the invention prevents the enzyme converting the substrate to release hydrogen peroxide, and thus helps to maintain the stability of the composition for extended periods at ambient temperature. A storage-stable composition for use in devices of the invention may include some water provided that there is not sufficient free water to allow the enzyme to convert the substrate (or to allow the precursor-substrate to be converted to the substrate for the enzyme). Suitable amounts of water will vary depending on the precise components of the composition. However, typically, a storage-stable composition for use in devices of the invention preferably comprises less than 20% (by weight) total water content, for example, 10%- 19%, water. Compositions in devices the invention may comprise 12% or less (by weight) of water. Compositions in devices of the invention may comprise 10% or less (by weight) of water. Compositions in devices of the invention may comprise 5% or less (by weight) of water. Compositions in devices of the invention may comprise 3% or less (by weight) of water. Compositions in devices of the invention may have a water activity (a _w ) of 0.7 or lower. For example, compositions of the invention may have an a _w of 0.6 or lower, such as 0.5 or lower.

Preferably, the compositions in the devices of the invention comprise substantially no 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.

The compositions in the devices of the invention may comprise an additional component which is preferably a solute. References herein to "solute" may refer to one or more solutes. For example, in some embodiments, compositions of devices of the invention may comprise a plurality of solutes. In some embodiments, the composition may only comprise one solute. Preferably, the solute is soluble in water.

The solute may be distinct from the substrate, or in some examples, the substrate may be same as the solute. For example, the composition may comprise fructose and fructose oxidase: the fructose being both the solute and the substrate for enzyme. In another example, the substrate may be glucose and the solute may be fructose.

The solute is preferably purified, meaning that the solute has been separated from at least some of the impurities originally present when the solute was obtained or produced. The purified solute may be obtained from a natural source or may be synthetically produced. The purified solute may be a processed, extracted, or refined substrate (i.e. a solute in which impurities or unwanted elements have been removed by processing).

It may not always be necessary or desirable that the purified solute is at a high level of purity. In some circumstances, it may be desirable to use a relatively crude solute preparation. Examples of suitable purity levels include at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% pure. Preferably, the purity level is at least 90%. Even more preferably, the purity level is at least 99%. However, in some embodiments, it may be desirable that the solute is a medical grade, medical device grade, or pharmaceutical grade solute.

The solute may be a carbohydrate. The solute may be a polysaccharide. Preferably, the solute is a sugar or sugar derivative. More preferably, the solute is a sugar. Suitable sugars include oligosaccharides, disaccharides or monosaccharides. Preferably, the sugar is a disaccharide or a monosaccharide. In particularly preferred embodiments, the sugar is a monosaccharide. Suitable sugars may include fructose, glucose, galactose, sucrose and maltose. In a particularly preferred embodiment, the sugar is fructose.

The term "sugar derivative" is used herein to refer to a sugar that has been modified by addition of one or more substituents other than a hydroxy I 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.

The compositions in devices of the invention may comprise two or more solutes, as described herein. For example, compositions in devices of the invention may comprise two or more sugars or sugar derivatives. The composition may comprise a maximum of two solutes, e.g. two sugars or sugar derivatives; or a maximum of three solutes, e.g. three sugars or sugar derivatives.

For instance, the compositions in devices of the invention may comprise glucose, fructose and sucrose.

The solute preferably has 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. In a preferred embodiment, the solute has a solubility greater than or equal to 100g/100g water at 20°C and 1 atm, in a more preferred embodiment, the solute has a solubility greater than or equal to 200g/100g water at 20°C and 1 atm, in an even more preferred embodiment, the solute has a solubility greater than 300g/1 OOg water at 20°C and 1 atm.

A solute with a high solubility may be advantageous because a high concentration of solutes may provide a high osmolarity or osmotic strength. Compositions with a high osmolality or osmotic strength may assist with the antimicrobial efficacy of the composition because they may reduce the amount of water available for microbes or draw water away from microbes, and may assist in wound healing and wound debridement.

Fructose is a particularly preferred solute because it has a solubility of about 375g/100g water at 20°C and 1 atm. Consequently, the solute may be fructose.

In some embodiments, the solute with a solubility of at least 100g/1 OOg water at 20°C and 1 atm, at least 200g/100g water at 20°C and 1 atm or at least 300g/1 OOg water at 20°C and 1 atm, may be the purified substrate. So, for example, the composition may comprise fructose and fructose oxidase.

The composition may comprise only one sugar or sugar derivative which is both the solute and the substrate, and one enzyme for converting the substrate and generating hydrogen peroxide.

In some embodiments, the solute with the solubility of at least 100g/100g water at 20°C and 1 atm, at least 200g/100g water at 20°C and 1 atm or at least 300g/1 OOg water at 20°C and 1 atm, may be distinct from the purified substrate. For example, a composition of the invention may comprise glucose, glucose oxidase and fructose.

In preferred embodiments, the purified substrate is a sugar or sugar derivative {e.g. glucose) and the solute is a sugar or sugar derivative (e.g. fructose).

Preferably, the composition comprises at least two sugars or sugar derivatives (e.g. including glucose and fructose). The composition may comprise a maximum of two sugars or sugar derivatives (e.g. only glucose and fructose).

The compositions in the devices of the invention may comprise at least 5% by weight of sugars and/or sugar derivatives. Compositions in devices of the invention may comprise at least 10% by weight of sugars and/or sugar derivatives. Compositions in devices of the invention may comprise at least 25% by weight of sugars and/or sugar derivatives. Compositions in devices of the invention may comprise at least 50% by weight of sugars and/or sugar derivatives.

Compositions in devices of the invention may comprise 95% by weight or less of sugars or sugar derivatives. Compositions in devices of the invention may comprise at least 75% by weight or less of sugars and/or sugar derivatives. For example, compositions in devices of the invention may comprise 10% to 95% by weight sugars and/or sugar derivatives. Compositions in devices of the invention may comprise 25% to 75% by weight sugars and/or sugar derivatives. Compositions in devices of the invention may comprise 50 to 95% by weight sugars and/or sugar derivatives.

The compositions in devices of the invention may comprise 5 to 50% by weight of substrate for the enzyme (e.g. glucose) or 5 to 50% by weight of the precursor-substrate that can be converted to the substrate for the enzyme. For instance, the compositions of the devices of the invention may comprise 5 to 25% by weight of substrate for the enzyme (e.g. glucose) or 5 to 25% by weight of the precursor substrate that can be converted to the substrate for the enzyme

The compositions in devices of the invention may comprise 5 to 75% by weight of solute (e.g. fructose). For instance, the compositions may comprise 10 to 50% by weight of solute. The compositions in the devices of the invention may comprise at least 50% by weight of substrate and solute combined and at least 5% by weight of non-aqueous solvent. For example, the compositions may comprise 50% to 95% by weight of substrate and solute combined and 5% to 50% by weight non-aqueous solvent.

Compositions in devices of the invention may comprise at least 50% by weight of sugar and at least 5% by weight of non-aqueous solvent. For example, the compositions may comprise 50% to 95% by weight sugar and 5% to 50% by weight non-aqueous solvent.

Compositions in devices of the invention may comprise at least 5% by weight of substrate for the enzyme {or precursor-substrate) and at least 5% by weight of non-aqueous solvent. For example, compositions in devices of the invention may comprise 5 to 50% substrate (or precursor-substrate) and 5 to 50% by weight non-aqueous solvent.

The compositions in devices of the invention may comprise a buffer, or a component that may be capable of acting as act as a buffer in an aqueous solution. An example of a suitable buffer is a citric acid/NaOH buffer, such as a 50 mmol citric acid NaOH buffer. The compositions of devices of the invention may be buffered (or may be capable of being buffered in an aqueous solution) at a pH of 5 or less, e.g. 3 to 5 (such as about pH 4). Alternatively, compositions of devices the invention may be buffered (or may be capable of being buffered in an aqueous solution) at a pH greater than 5, e.g. 6 to 8 (such as about pH 7).

Compositions in devices of the invention may comprise a salt.

According to the invention there is provided a method, the method comprising applying a composition and water, the composition and water either being applied sequentially or simultaneously, the composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not comprising sufficient free water to allow the enzyme to convert the substrate.

According to the invention there is provided a method of dispensing from a device, the device comprising a first chamber comprising a composition, the composition having an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not comprising sufficient free water to allow the enzyme to convert the substrate; a second chamber comprising water; the method comprising: a) dispensing separately from the device, the composition from the first chamber and the water from the second chamber; or b) pre-mixing the composition from the first chamber and the water from the second chamber to form a mixture, and then dispensing the mixture from the device. According to the invention there is provided a method of dispensing from a device, the device comprising a first chamber comprising a composition, the composition having an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not comprising sufficient free water to allow the enzyme to convert the substrate; a second chamber comprising water; and at least one outlet for dispensing the composition and the water from the device; the method comprising: a) dispensing separately from the device, the composition from the first chamber and the water from the second chamber; or b) pre-mixing the composition from the first chamber and the water from the second chamber to form a mixture, and then dispensing the mixture from the device.

If the composition and water are dispensed separately (e.g. through distinct holes or apertures). The method may comprise dispensing the composition and the water concurrently or sequentially.

The compositions, water or devices of methods of the invention may be as described herein,

According to the invention, the composition comprising the enzyme and substrate may be applied first (e.g. to a wound site) and the water may be applied second. Alternatively, the water may be applied first and the composition comprising the enzyme and the substrate may be applied second. Altematively, the composition and the water may be applied concurrently or simultaneously.

Methods of the invention may comprise dispensing or applying on to a wound. The method may comprise dispensing on to a surface which is not on a human or animal body. The method may comprise dispensing on to, or in, a human or animal body. The method comprise treating a wound, treating inflammation, stimulating tissue growth, debriding a wound, deodorising a wound, which comprises administering a composition of the invention to a wound in need of deodorising, or treating, or ameliorating a microbial infection. The microbial infection may comprise a biofilm.

According to the invention there is also provided a kit. The kit comprises a) a first device comprising: i) a chamber, the chamber comprising a composition having an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not comprising sufficient free water to allow the enzyme to convert the substrate; and ii) an outlet for the composition, and separately, b) a second device comprising: i) a chamber, the chamber comprising water; and ii) an outlet for the water.

The composition, water or devices of the invention may be as described herein, A device according to the invention may be configurable such that the contents of the first chamber can be mixed with the contents of the second chamber, prior to dispensing from the device.

According to the invention there is provided a device comprising: a first chamber comprising a composition, the composition having an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substrate for the enzyme, the composition not having sufficient free water to allow the enzyme to convert the substrate; and a second chamber comprising water; wherein prior to dispensing, the device is configurable from a first arrangement in which the composition cannot mix with the water, to a second arrangement in which the composition can mix with the water.

The device may be configurable from a first arrangement in which the composition in the first chamber cannot pass to the second chamber and/or the water in the second chamber cannot pass to the first chamber, and a second arrangement in which the composition in the first chamber can pass to the second chamber and/or the water in the second chamber can pass to the first chamber. For example, the device may be configurable to provide an opening, such as an opening between the first chamber and second chamber, to permit mixing of the composition and the water. The device may comprise a barrier, seal or septum. In one example, there may be a barrier, seal or septum separating the first chamber and second chamber. In another example there may be a barrier, seal or septum separating both the first and second chambers from a mixing chamber. The barrier, seal or septum may be openable, for example it may be breakable or frangible. The mixing of the water and the composition may occur manually, for example, by a user applying pressure, such as by massaging and/or shaking the device, to open the barrier, seal or septum.

The barrier, seal or septum may be positioned between the first chamber and the second chamber. It may comprise a wall separating the first chamber and the second chamber.

The device may be a sachet or a pouch. The sachet or pouch may be manufactured from a plastics or polymer material. The sachet or pouch may be flexible or deformable to enable a user to squeeze the sachet or pouch. Squeezing the sachet or pouch may allow breaking of the barrier, seal or septum between the first chamber and the second chamber. The barrier, seal or septum may be configured to break or rupture at a predetermined pressure, for example if a user applies sufficient pressure to a wall of the first and/or second chamber. Squeezing the sachet or pouch may allow the diluted composition to be dispensed from the device.

Although the device may have an outlet, such as a nozzle, to allow the diluted composition to be dispensed from the device, it may not be necessary. This is because a user may manually create an outlet once the composition and the water have mixed. An outlet may be created, for instance, by cutting, piercing or tearing a wall of the first and/or second chamber. The outlet may be formed by breaking a seal. In some embodiments, the device may thus not contain a nozzle or other such pre-formed dispensing means.

An example of a suitable mixing pouch for use in a device of the invention is provided by burstpouch.com (a division of Eastern States Packaging, Inc., 27 Glen Street, Suite 6, Stoughton, MA 02072 USA). In this example, a frangible membrane seal is installed across the pouch separating a first compartment from a second compartment, requiring deliberate pressure from a user to burst the frangible seal, allowing components of both compartments to mix. Such mixing pouches are available both with or without dispensing tips.

The composition and the water may form a gel or viscous liquid upon mixing.

Embodiments of the invention are now described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows a trigger spray device according to an embodiment of the invention;

Figure 2 is a graph showing the effect of compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to SurgihoneyRO™, at various concentrations;

Figure 3 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH

4.03) on the growth of planktonic MRSA, at various concentrations;

Figure 4 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (unbuffered) on the growth of planktonic MRSA, at various concentrations;

Figure 5 is a graph showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) (buffered at pH

7.04) on the growth of planktonic MRSA, at various concentrations;

Figure 6 is a table showing the effect of sterile and non-sterile compositions for use in the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations;

Figure 7 shows the effect of compositions for use in the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to

SurgihoneyRO, at various concentrations; Figure 8 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

Figure 9 shows a schematic drawing of a pouch comprising a frangible septum positioned between a first chamber and a second chamber.

Exa m pie 1 - SP ray a b I e D ey i ce

A device 1 as shown in Figure 1 , has a first chamber 3 and a second chamber 7. In the first chamber is a composition 5 comprising 50% by weight SurgihoneyRO™ (a commercially- available antimicrobial product formed from pasteurised honey with added glucose oxidase) and 50% by weight glycerol. In the second chamber is deionised water 9.

A first tube 11 connects the first chamber 3 to a mixing chamber 15 in a neck 14 of the device, and a second tube 13 connects the second chamber to the mixing chamber. The mixing chamber is connected to a nozzle 17, which defines an outlet hole (not shown). A trigger 19 is located on the neck of the device.

In use. the user operates the trigger 17, which draws the composition 5 from the first chamber 3 through the first tube 11 to the mixing chamber 15, and simultaneously draws the deionised water 9 from the second chamber 7 through the second tube 13 to the mixing chamber 15. The composition and the water mix in the mixing chamber to form a mixture and the mixture is ejected through the hole defined in the nozzle 17, as a spray. The composition, having been diluted by the water to form the mixture, produces hydrogen peroxide.

( ate® k s i ¾$ SynthetteRP)

Samples with batch number "RO" contain no glucose oxidase.

Samples with batch number "R01 " contain 50 ppm glucose oxidase.

Samples with batch number "R02" contain 1000 ppm glucose oxidase.

A. pH 4.03 buffered samples

Batch no NB01 p43RO

Non sterile

Description

Non sterile base buffered saccharide solution.

A2. Batch no NB01 p43RO

Sterile

Description Sterile base buffered saccharide solution

A3. Batch no NB01p44RO1

Non sterile

j Material [ Weight fraction

I Fructose 152.0%

I Glucose ^* Wo ~~~ j 50 mMol Citric acid/NaOH buffer pH 4.03 ] Ϊ 7.0%

Description

Non sterile base buffered R01 saccharide solution.

Batch no NB01 p44RO1

Sterile

Description

Sterile base buffered R01 saccharide solution A5. Batch no NB01p44RO2

Non sterile

Description

Non sterile base buffered R02 saccharide solution. A6. Batch no N B01 p43R02

Sterile

iption Sterile base buffered R02 saccharide solution Unbuffered samples

B1. Batch no NB01 p51 RO

Non sterile

Description

Non sterile base buffered saccharide solution.

Batch no NB01p51RO

Sterile

Description Sterile base buffered saccharide solution

B3, Batch no B01 p51 R01

Non sterile

Ϊ Material \ Weight fraction

Description

on sterile base buffered R01 saccharide solution.

B4. Batch no NB01 p51 R01

Sterile

Description

Sterile base buffered R01 saccharide solution

B5. Batch no NB01 p51 R02

Non sterile

Description

Non sterile base buffered R02 saccharide solution.

B6. Batch no NB01 p51 R02

Sterile

Sterile base buffered R02 saccharide solution

C. pH 7.04 buffered samples C1. Batch no NB01 p57RO

Non sterile

I ^" Material Weight fraction i Fructose | 52.0%

I Gk _j COSe

I 50 mMol Citric acid/NaOH buffer pH 7.04 I 17.0%

Description

Non sterile base buffered saccharide solution.

C2. Batch no NB01 p57RO

Sterile

Description

Sterile base buffered saccharide solution

C3, Batch no NB01 p57RO1

Non sterile

Description

Non sterile base buffered R01 saccharide solution.

C4. Batch no NB01 p57RO1

Sterile

Description

Sterile base buffered R01 saccharide solution C5. Batch no NB01 p57RO2

Non sterile

Material Weight fraction

Fructose

Glucose 31.0%

50 m oV Citric acid/NaOH buffer pH 7 4 17.0%

Description

Non sterile base buffered R02 saccharide solution.

C6. Batch no NB01 p57RO2

Material Weight fraction

Fructose 52.0%

Glucose 31.0%

50 mMol Citric acid/NaOH buffer pH 7.04 17.0%

Description

Sterile base buffered R02 saccharide solution

MIC and MBC were assessed for the R01 samples (containing 50 ppm glucose oxidase) and compared to SurgihoneyRO™ (also containing 50 ppm glucose oxidase). See Andrews J. M, Journal of Antimicrobial Chemotherapy (2001 ) 48, suppl. S1, 5-16.

The results are shown in Figures 2 to 6.

The results show that, like SurgihoneyRO, synthetic compositions containing glucose, glucose oxidase and fructose are able to inhibit microbial growth.

Out of all of synthetic compositions, the synthetic composition buffered at pH7.04 had the most effective MIC. Sterilised compositions were more effective than non-sterilised compositions, and synthetic composition buffered at pH7.04 synthetic had the most effective MBC when compared to other synthetic compositions and even when compared to SurgihoneyRO. FFiigguurreess 77 ((aa ttoo dd)) aanndd 88 sshhooww MMIICC aanndd MMBBCC rreessuullttss iinncclluuddiinngg SSuurrggiihhoonneeyyRROO ((RR0022)) ssaammpplleess aanndd ssyynntthheettiicc RR0022 ssaammpplleess..

A dispensing device 20 in the form of a flexible pouch formed from a plastics material (as shown in Figure 9) has a first compartment 3 and a second compartment 7. In the first compartment is a composition 5 comprising 50% by weight SurgihoneyRO and 50% by weight glycerol. In the second compartment is deionised water. The dispensing device comprises a frangible septum 22 positioned between the first compartment and the second compartment, and one outlet 21 in the form of a closed nozzle, which is in connection with the second compartment.

In use, a user applies pressure to the first compartment 3 and/or the second compartment 7. The resulting pressure causes the frangible septum 22 to rupture and open, allowing the composition and the water to mix and initiate hydrogen peroxide production. Mixing may be assisted by the user massaging the pouch. The user then opens the nozzle 21 and squeezes the pouch such that the diluted composition may be dispensed from the pouch to the desired site, such as a wound.