Antimicrobial Compositions

This invention relates to compositions for generating antimicrobial activity, particularly compositions that are able to generate hydrogen peroxide. The invention also relates to the use of such compositions for the treatment of oral and nasal conditions.

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.

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.

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).

Compositions described herein may be used to treat conditions that may not result from an infection by a pathogen, such as a bacterium, virus or fungus. Such conditions include oral conditions.

For example, aphthous ulcers, such as recurrent aphthous ulcers, may not result from infection by a pathogen. Aphthous ulcers are characterized by the repeated formation of benign and noncontagious mouth ulcers (aphthae) in otherwise healthy individuals The cause of aphthous ulcers is not completely understood, but is believed to involve a T cell-mediated immune response triggered by a variety of factors. Triggers may include nutritional deficiencies, local trauma, stress, hormonal influences, allergies, genetic predisposition or other factors

Another oral condition that may not result from infection of a pathogen is geographic tongue. This is an inflammatory condition of the mucous membrane of the tongue, usually on the dorsal surface.

Dentists typically find it difficult to treat such conditions. Surprisingly, compositions described herein have been found to work particularly well in treating such conditions. According to the invention there is provided a composition as described herein for use in the treatment of a condition that does not the result from an infection by a pathogen. The condition is preferably an oral condition.

According to the invention, there is provided use of a composition as described herein in the manufacture of a medicament for use in the treatment of an condition that does not the result from an infection by a pathogen. The condition is preferably an oral condition.

According to the invention, there is provided a method of treating an oral condition that does not result from an infection by a pathogen, which comprises administering a composition as described herein to a patient in need of such treatment. The condition is preferably an oral condition.

According to the invention, there is provided a composition as described herein for use in the treatment of aphthous ulcers (preferably recurrent aphthous ulcers) or geographic tongue.

According to the invention, there is provided use of a composition as described herein in the manufacture of a medicament for use in the treatment of aphthous ulcers (preferably recurrent aphthous ulcers) or geographic tongue.

According to the invention, there is provided a method of treating aphthous ulcers (preferably recurrent aphthous ulcers) or geographic tongue, which comprises administering a composition as described herein to a patient in need of such treatment.

Compositions may be administered topically. Compositions may be administered directly to the affected site in a subject's mouth.

In a broad sense, the invention concerns compositions comprising: an enzyme that is able to convert a substrate to release hydrogen peroxide; and a substance that includes a substrate for the enzyme. The invention concerns administering such compositions for the treatment of oral condition such as aphthous ulcers or geographic tongue.

The substance may be any substance that includes a substrate for the enzyme. Preferably the substance lacks catalase activity. Preferably the substance is an unrefined substance. 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. Preferably the substance includes 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. More preferably the substance is an unrefined natural substance.

In some embodiments, compositions may comprise an unrefined natural substance.

So, the invention concerns compositions which comprise an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional (i.e. added as a result of human intervention) to any enzyme activity able to convert the substrate to release hydrogen peroxide (referred to herein as "substrate conversion activity") that may be present in the substance. The invention also concerns use of such a composition in the treatment of oral conditions that may not result from infection by pathogens. The invention concerns treatment of aphthous ulcers (preferably recurrent aphthous ulcers) or geographic tongue.

Preferably the substance is a sugar substance. The term "sugar substance" is used herein to mean any substance that includes one or more sugars. The term "sugar" is used herein to refer to a carbohydrate with the general formula C _m (H ₂ 0) _n . Preferred sugars include

monosaccharides, such as D-glucose, hexose, or D-galactose. Preferably the sugar substance includes one or more sugars from a natural source (termed herein a "natural sugar substance"). More preferably the natural sugar substance is an unrefined natural sugar substance. The unrefined natural sugar substance may be (or be derived from) a natural sugar product. In a preferred embodiment, the unrefined natural sugar product is a honey. In some preferred embodiments, the honey is a honey that has been treated to remove or inactivate catalase activity. Alternatively, the unrefined natural sugar substance may be a processed natural sugar, such as a syrup or an inverted syrup.

Preferably the substance is a honey. The honey may preferably be a medical grade or medical device grade honey. In some embodiments, it is preferred that the honey is a honey that has been treated to remove or inactivate catalase activity originally present in the honey. According to a preferred embodiment of the invention, the substance is a pasteurised honey, and the enzyme is a glucose oxidase. According to preferred embodiments, the substance is a medical grade or medical device grade honey, and the enzyme is a medical grade or medical device grade enzyme, preferably glucose oxidase.

In some embodiments of the invention, the honey may be pasteurised. Pasteurisation of honey inactivates the catalase and glucose oxidase activity present in the honey. Optionally, the pasteurised honey may be filtered to remove any particles (such as wax particles and bee wings) that may be in the honey post-harvest. To form a storage-stable composition of the invention, a glucose oxidase is contacted with the pasteurised honey once it has cooled to a temperature (suitably 35-40°C) that will not inactivate the added glucose oxidase and at which the honey remains sufficiently liquid to facilitate mixing with glucose oxidase.

Honey can be pasteurised at a temperature that is sufficient for the heat inactivation of catalase activity. A suitable minimum temperature is from 60°C to 80°C. This temperature should be maintained preferably for at least two minutes.

In other embodiments of the invention, the honey may be unpasteurised.

According to some embodiments, the honey is a creamed honey. Creamed honey is a honey that has been processed to control crystallization. Creamed honey contains a large number of small crystals, which prevent the formation of larger crystals that can occur in unprocessed honey. A method for producing creamed honey was described in U.S. Patent 1 ,987,893. In this process, raw honey is first pasteurised, then previously processed creamed honey is added to the pasteurized honey to produce a mixture of 10% creamed honey and 90% pasteurised honey. The mixture is then allowed to rest at a controlled temperature of 14°C. This method produces a batch of creamed honey in about one week. A seed batch can be made by allowing normal honey to crystallize and crushing the crystals to the desired size. Large scale producers have modified this process by using paddles to stir the honey mixture while holding the mixture at 14°C. In alternative creaming methods, the pasteurisation step may be omitted, with the honey instead being slowly warmed to 37°C.

In some embodiments, the honey may not be creamed.

The substance itself may preferably lack an enzyme activity that is able to convert the substrate to release hydrogen peroxide (referred to as "substrate conversion activity"). Substrate conversion activity may be removed during processing or extraction of the substance, or inactivated before use of the substance in a composition of the invention. Substrate conversion activity may be inactivated by heat inactivation, for example by pasteurisation.

The additional enzyme is preferably 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. 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. Preferably the purity level is at least 90%. More preferably the purity level is at least 99%.

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 should be used.

Preferably 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 amioacid oxidase. The corresponding substrates for these oxidoreductase enzymes are D-glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate and aminoacid, respectively.

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

According to a preferred embodiment of the invention, the oxidoreductase enzyme is glucose oxidase and the substrate is D-glucose.

Preferably, the compositions of the invention lack catalase activity. Catalase is present in many plants and animals. Catalase activity may be removed during processing or extraction of the substance, or inactivated before use of the substance in a composition of the invention.

Catalase activity may be heat inactivated, for example by pasteurisation. A suitable temperature for heat inactivation of catalase activity is at least 60°C, 70°C, or 80°C, preferably for at least 2 minutes.

Compositions of the invention preferably lack peroxidase activity.

Compositions of the invention preferably lack zinc oxide or comprise substantially no zinc oxide. Compositions of the invention may comprise sufficient enzyme and substrate to provide for sustained release of hydrogen peroxide at a level of less than 2 mmol/litre for a period of at least twenty four hours, following dilution of the composition.

Compositions of the invention 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 one embodiment, 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.

Compositions of the invention may comprise sufficient enzyme and substrate to provide for sustained release of hydrogen peroxide at a specific level or concentration. For example, compositions of the invention 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 (e.g. if the composition is a dry or dried composition), 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, of the invention 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 of the invention may provide for sustained release of 2 to 500 ppm hydrogen peroxide for at least 24 hours.

Compositions of the invention may comprise 25 to 2000 ppm of the enzyme, for example 50 to 1000 ppm of the enzyme. Compositions of the invention may comprise 750 to 2000 ppm of the enzyme. Compositions of the invention may comprise greater than 500 ppm of the enzyme. Compositions of the invention may comprise 250 to 1500 ppm of the enzyme.

The enzyme activity (for example, the glucose oxidase activity) may range, for example, from 1 - 400 l U/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 amount of any catalase activity present in the substance, the amount of substrate present in the substance, 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 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, a composition according to the invention comprises 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, 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.

Preferably, compositions of the invention do not include sufficient free water to allow the enzyme to convert the substrate. Compositions of the invention may thus begin producing hydrogen peroxide on dilution.

In alternative embodiments, compositions of the invention may comprise sufficient free water to allow the enzyme to convert the substrate.

The invention concerns compositions comprising: an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance, and wherein the composition does not comprise sufficient free water to allow the enzyme to convert the substrate.

Compositions of the invention are preferably storage-stable. 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 a storage-stable composition 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 of the invention may include some water provided that there is not sufficient free water to allow the enzyme to convert the substrate. Suitable amounts of water will vary depending on the precise components of the composition. However, typically, a storage-stable composition of the invention preferably comprises less than 20% (by weight) total water content, for example, 10%-19%, water.

Compositions of the invention may comprise 12% or less (by weight) of water.

Compositions of the invention may comprise 10% or less (by weight) of water.

Compositions of the invention may comprise 5% or less (by weight) of water.

Compositions of the invention may comprise 3% or less (by weight) of water.

For compositions of the invention that do contain sufficient free water to allow the enzyme to convert the substrate, water may be present in an amount which is at least 20% by weight, or at least 30% by weight.

Preferably, compositions 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 (Signma Aldrich, UK). For example, hydrogen peroxide may be present at a level less than 1 ppm, or at a level of less than 0.5 ppm. Hydrogen peroxide may be at a level less than 0.1 ppm.

Compositions of the invention may be in a dry form or solid form. For example, compositions may be in the form of a powder. In compositions of the invention that are solutions or liquids, water may be present in an amount which is less than 20% by weight, but preferably greater than 10% by weight, more preferably greater than 15%, by weight. So, in some embodiments water may be present in an amount between 10 and 20%, by weight.

Compositions of the invention may comprise a buffer. An example of a suitable buffer is a citric acid/NaOH buffer, such as a 50 mMol citric acid/Na OH buffer. Compositions of the invention may be buffered at a pH of 5 or less, e.g. 3 to 5 (such as about pH 4). Alternatively, compositions of the invention may be buffered at a pH greater than 5, e.g. 6 to 8 (such as about PH 7).

Compositions of the invention may have a viscosity, such as a dynamic viscosity, of at least 5000 mPas at 20°C, more preferably at least 7500 at 20°C. Compositions of the invention may have a viscosity of 5000 to 20000 mPas at 20°C, more preferably 7500 to 12000 mPas at 20°C. A viscous solution or liquid may be afforded by high concentrations of sugars or sugar derivatives and may provide a similar viscosity to honey. A high viscosity may be beneficial in allowing a composition to remain in contact with a wound. A viscous solution or liquid may be afforded by the presence of, for example, non-aqueous solvents, polymers or hydrocolloid gelling agents as described herein.

A composition of the invention may comprise at least one suitable antimicrobial or

immunostimulatory component, excipient or adjuvant, or any other suitable component where it is desired to provide ability to generate antimicrobial activity. Preferably, however, the compositions do not include any antibiotic.

However, compositions of the invention may include an antibiotic. A suitable antibiotic may be co-amoxiclav. Administering compositions of the invention with an antibiotic such as co- amoxiclav may provide a synergistic effect, such as in the treatment of biofilms, e.g. biofilms comprising NTHi.

According to the invention there may be provided a composition as defined herein for use in treating an infection (such as an infection comprising a biofilm), wherein the composition is administered with an antibiotic. The infection may comprise Haemophilus influenza (e.g. non- typeable haemophilus influenza; NTHi). The antibiotic may be co-amoxiclav, although other antibiotics could be used. The administration may be Combined, Concurrent, or Sequential.

According to the invention, there is provided a kit comprising a composition of the invention and, separately, an antibiotic (e.g. co-amoxiclav). Compositions of the invention may comprise a polymer. The polymer in the composition may be any medically acceptable polymer, such as any Food and Drug Administration-approved (FDA- approved) polymer.

In some embodiments, the polymer may be a synthetic polymer. In some embodiments, the polymer is a natural polymer.

Optionally, the polymer is water soluble. The polymer may be soluble in an organic, or nonaqueous, solvent. The polymer may be soluble in a mixture of an aqueous and non-aqueous solvent. The polymer may be biodegradable or bioerodable. The polymer may be a co-polymer. In some embodiments, the polymer is selected from polyethylene oxide (or polyethylene glycol), polyvinyl alcohol and polyvinylpyrrolidone.

Other polymers may include poly(lactic-co-glycolic acid), polyglycolic acid, polylactic acid, polycaprolactone or polymeric surfactants. Another suitable polymer may be phosphino- carboxylic acid (PCA).

Further polymers may include polysaccharides such as cellulose (which includes derivatives such as hydroxypropyl methyl cellulose and hydroxypropyl cellulose), alginate, gelatin or cyclodextrins. Suitable polymers may also include chitosan or hyaluronic acid.

Compositions of the invention may comprise up to 50%, 25%, 10% or 5% by weight of the polymer. For example, the composition may comprise from 0.5 to 3% by weight of the polymer. Optionally, the polymer may be from 0.5 to 50% by weight of the composition.

Compositions of the invention may be electrospinnable.

According to the invention, there is also provided a method of producing a fiber, comprising electrospinning an electrospinnable composition of the invention.

According to the invention, there is provided a fiber, preferably a nanofiber, comprising: an enzyme that is able to convert a substrate to release hydrogen peroxide; and an unrefined natural substance that comprises a substrate for the enzyme, wherein the enzyme is additional to any enzyme that may already be present in the unrefined natural substance. The fiber preferably does not comprise sufficient free water to allow the enzyme to convert the substrate. The fiber may comprise one or more solutes as described herein.

According to the inventions, there is also provided a wound dressing or nanofibrous mat comprising one or more fibers of the invention. The electrospmnable composition may be a solution. The solvent in the solution may be, or may comprise, water. The solvent may comprise an aqueous and/or non-aqueous solvent, such as an organic solvent.

The electrospinnable composition may comprise one or more electrospinnable components. Any electrospinnable component which facilitates formation of a fiber or dressing according to the invention, may be suitable. Preferably, the one or more electrospinnable component is an electrospinnable polymer. In some embodiments, the polymer may be a synthetic polymer. In some embodiments, the polymer is a natural polymer. In some embodiments, the

electrospinnable polymer is selected from polyethylene oxide, polyvinyl alcohol and polyvinylpyrrolidone. Other polymers may include polycaprolactone or phosphino-carboxylic acid (PCA).

The electrospinnable component is preferably biocompatible. Optionally, the electrospinnable component is water soluble. The electrospinnable component may be soluble in an organic, or non-aqueous, solvent. The electrospinnable component may be soluble in a mixture of an aqueous and non-aqueous solvent. Suitable non-aqueous solvents may be, or may comprise, glycerol, dimethyl sulphoxide, ethylene glycol or propylene glycol.

The electrospinnable composition may comprise up to 50%, 25%, 10% or 5% by weight of the electrospinnable component. Optionally, the electrospinnable component may be from 1 to 50% by weight of the composition. The electrospinnable composition may comprise up to 30%, 20% or 10% by weight of the unrefined natural substance.

It will be appreciated that the relative amounts of the electrospinnable component and the solvent may be varied to alter the properties of the fibers.

A fiber that has been formed from the electrospinnable composition may comprise up to 80% by weight of the unrefined natural substance. The fiber may comprise 20% or more, by weight, of the electrospinnable component.

Compositions of the invention may comprise salt. Alternatively, the salt may be provided in a kit separately from the rest of the composition. Accordingly, there is also provided according to the invention a kit comprising: a composition for generating anti-microbial activity that comprises an enzyme that is able to convert a substrate to release hydrogen peroxide, and a substance that includes a substrate for the enzyme; and, separately, a salt. The kit may further include instructions, for example, for mixing of the components of the kit, and their use to treat a microbial infection. The salt may be provided in dry form, or in aqueous solution. The salt may comprise sodium chloride.

The composition in dry form may comprise a ratio of the composition for generating antimicrobial activity to the salt, for example, of from 1 :2 to 5: 1 , or from 2:3 to 3:2.

The composition may comprise, for example, 1-99%, 1 -80%, 1-70%, 1 -60%, 1-50%, 1 -40%, 1- 30%, 1 -20%, or 1 -10%, by weight, of the composition for generating anti-microbial activity.

The composition may comprise, for example, 1-99%, 1 -80%, 1-70%, 1 -60%, 1-50%, 1 -40%, 1- 30%, 1 -20%, or 1 -10%, by weight, of the salt.

The composition may be provided as an aqueous mixture. The aqueous mixture may be an isotonic or hypertonic mixture. The aqueous mixture may comprise, for example, 0.1-20% w/v salt, suitably 0.25-10%, 0.25-10%, 0.25-5%, 0.25-3%, 0.5-10%, 0.5-5%, or 0.5-3%, for example 0.9% w/v salt. The aqueous mixture may comprise, for example, 1 -300%, 1-250%, 1-200%, 1 - 150%, 1 -100%, 1 -50%, 1-40%, 1 -30%, 1-20%, or 1-10% w/v of the composition for generating anti-microbial activity. The aqueous mixture may comprise, for example, 10-300%, 10-250%, 10-200%, 10-150%, 10-100%, 10-50%, 10-40%, 10-30%, or 10-20% w/v of the composition for generating anti-microbial activity. The aqueous mixture may comprise, for example, 50-300%, 50-250%, 50-200%, 50-150%, or 50-100% w/v of the composition for generating anti-microbial activity. The aqueous mixture may comprise, for example, 0.1-20%, 0.1 -10%, 0.1 -5%, 0.1-1 % w/v of sodium bicarbonate.

Compositions of the invention, for example those comprising salt, may be used as a nasal douche, for example to prevent or treat nasal microbial infection, sinusitis, rhinitis, CRS, nasal allergy, cold or flu symptoms, congestion, or dryness. The compositions of the invention may be used to prevent or treat a microbial infection, for example a microbial infection that comprises a biofilm, or a microbe that is capable of forming a biofilm. The microbial infection that comprises a biofilm may be a nasal microbial infection, or the microbe that is capable of forming a biofilm may be part of a nasal microbial infection.

To treat certain conditions, e.g. nasal infections such as CRS, a composition of the invention may be diluted such that the composition is at a particular treatment concentration. Certain treatment concentrations may be optimal for different conditions. For example, it has been found that compositions of the invention which contain 1000 ppm of glucose oxidase, 52 wt. % fructose, 31 wt. % glucose and 17 wt.% 50 mMol Citric acid/NaOH buffer pH 7.04 may be optimal when forming an aqueous solution at a concentration of 71 g/l, particularly for treatment of microbes such as MRSA and MSSA, and biofilms which include such microbes.

Compositions of the invention may thus be particularly effective against MRSA and MSSA. Consequently, it may be beneficial to dilute compositions of the invention such that they form a solution of concentration of at least 30 g/l, such as from 30 g/l to 150 g/l. For example, the concentration of solution formed may be 50 to 100 g/l, such as 65 to 75 g/l.

The concentration of hydrogen peroxide produced in aqueous solutions formed by compositions of the invention may be at least 10 μΜ, such as 10 to 50 μΜ, for example 20 to 30 μΜ. The concentration may be maintained for at least an hour, preferably at least 2 hours, more preferably at least 10 hours and even more preferably at least 24 hours.

According to the invention, there may be provided a method of forming an antimicrobial solution comprising diluting a composition of the invention in an aqueous solution such that there is sufficient free water to allow the enzyme to convert the substrate. The method may comprise diluting the composition to form a solution which contains at least 30 g/l of the composition. The method may comprise diluting the composition to form a solution which contains 30 g/l to 150 g/l, 50 to 100 g/l or 65 to 75 g/l of the composition. According to the invention, there may also be provided a solution obtained or obtainable by this method. In the solution, the hydrogen peroxide may be present at a concentration of 10 to 50 μ , preferably 20 to 30 μΜ. The hydrogen peroxide may be present at a concentration of at least 10μΜ. The hydrogen peroxide may be present at a concentration less than 100 μΜ. The concentration may be maintained for at least 1 hour, preferably at least 2 hours, more preferably at least 10 hours and even more preferably at least 24 hours.

According to the invention there may be provided a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, an unrefined natural substance that includes a substrate for the enzyme, and sufficient free water to allow the enzyme to convert the substrate, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance, wherein hydrogen peroxide is present in a concentration of 10 to 50 μΜ, preferably 20 to 30 μΜ. The hydrogen peroxide may be present at a concentration of at least 10μΜ. The hydrogen peroxide may be present at a concentration less than 100 μΜ. The concentration may be maintained for at least 1 hour, preferably at least 2 hours, more preferably at least 10 hours and even more preferably at least 24 hours.

There may be provided compositions or solutions of the invention for use in the treatment of a microbial infection that comprises a biofilm. The microbial infection may comprise Haemophilus influenza (e.g. non-typeable Haemophilus influenza), RSA or MSSA. The compositions or solutions may be for use in the treatment of chronic rhinosinusitis. To use a composition or mixture of the invention as a nasal douche, it may be poured into one nostril and allowed to run out through the other, while the mouth is kept open to breathe, using gravity as an aid. Alternatively, some form of positive pressure may be applied to facilitate rinsing. For example, bottles made of flexible plastic, optionally with special tips to fit the nostril, can be squeezed to exert positive pressure of the mixture flowing through the sinuses while the mouth is kept open at all times in order to breathe and prevent snorting the liquid down the throat. Irrigation machines that utilize electric motor-driven pumps are also available. Some nasal irrigation systems that apply pressure have an anti-backwash valve to prevent used saltwater solution from flowing back into the nasal cavity.

Compositions or mixtures of the invention may be provided in a neti pot, a container used to administer nasal douche. Neti pots are typically made of metal, glass, ceramic or plastic. They rely on gravity, along with head positioning and repeated practice in order to rinse the outer sinus cavities. Typically they have a spout attached near the bottom, sometimes with a handle on the opposite side.

Compositions of the invention may comprise a non-aqueous solvent. In compositions of the invention that comprise a non-aqueous solvent, the non-aqueous solvent may comprise ethanol, dimethyl sulphoxide, glycerol, ethylene glycol or propylene glycol. Preferably, the nonaqueous solvent is or comprises glycerol. Glycerol may act as a humectant and so

compositions comprising glycerol may assist in softening or moisturising dry skin .

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

Substance Viscosity

Ethanol 1 .04mPa.s

Dimethyl 1 .99mPa.s

sulphoxide

Glycerol 1412mPa.s

Ethylene 16.1 mPa.s

glycol

Propylene 42mPa.s

glycol Water 1.3mPa.s

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 below. For example, 8 _t /MPa ^1/2 may be from 26 to 50, such as 26.5 to 47.8. 8 _d / Pa ^{1 2} may be from 15 to 19, such as 15.6 to 18.1. 5 _p /MPa ^{1 2} may be from 8 to 16, such as 8.8 to 16. 5 _h / Pa ^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.

In preferred embodiments of compositions comprising a non-aqueous solvent, the compositions may comprise at least 10% by weight of the non-aqueous 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. The amount of aqueous solvent may be varied depending on the intended application of the composition. For example, sprayable compositions, or compositions for use with an antibacterial wipe may comprise higher levels of the non-aqueous solvent, such that the compositions have a lower viscosity. In some embodiments, the amount of non-aqueous solvent in the composition may be 50-90%, by weight.

For some applications, it may be desirable to have compositions that comprise lower amounts of non-aqueous solvent, such as compositions for use in forming wound dressings.

Consequently, some compositions may comprise a maximum amount of non-aqueous solvent. The maximum amount of non-aqueous solvent in the composition may be 50% by weight or less. In some embodiments, the amount of non-aqueous solvent in the composition may be 1 - 50, 5-50 or 10-50%, by weight.

If compositions of the invention are to be used to coat a substrate, such as a fabric, the weight of the composition is preferably at least 100g per square metre of the substrate. In other embodiments, the weight of the composition may be at least 200g per square metre of the substrate. In other embodiments, the weight of the composition may be at least 300g per square metre of the substrate.

Compositions of the invention may comprise an amount of water that may typically be expected to permit the enzyme to convert the substrate. For example, compositions of the invention may comprise greater than 20% by weight of water, or greater than 30% by weight of water.

However, in some embodiments, this water may not be available, or free, to allow the enzyme to convert the substrate because the non-aqueous solvent may bind or lock in the water. A nonaqueous solvent may thus act as a humectant. The non-aqueous solvent or humectant may reduce the water activity (a _w ) of the composition.

Compositions of the invention may thus comprise a humectant. In some embodiments, the humectant is not a non-aqueous solvent. However, in preferred embodiments, the humectant is a non-aqueous solvent.

Viscous compositions may limit its range of applications. Inclusion of both a non-aqueous solvent or humectant, in the composition may reduce the viscosity of the composition, and less viscous compositions may be beneficial if, for example, the composition is to be readily sprayable. 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 50 mPa.s. or less at 20°C.

The relative amounts of the humectant or non-aqueous solvent, and the additional water, in a composition of the invention, may be selected such that the composition does not comprise sufficient free water to allow the enzyme to convert the substrate. When such a composition is contacted with even more water, for example if the composition is diluted or if the composition comes into contact with fluid from a wound, there may be sufficient free water for the enzyme to convert the substrate and produce hydrogen peroxide.

In compositions of the invention, the water activity (a _w ) may be less than 0.6, preferably less than 0.5.

In compositions of the invention comprising humectant or non-aqueous solvent, the amount of humectant or non-aqueous solvent may be at least 30% by weight. In other embodiments, the amount of humectant or non-aqueous solvent may be at least 40% by weight. In some embodiments, the amount of humectant or non-aqueous solvent may be at least 50% by weight. The amount of humectant or non-aqueous solvent may be 75% or less, by weight. The amount of humectant or non-aqueous solvent may be 60% or less by weight. In some embodiments, the amount of humectant or non-aqueous solvent may be 30-75% or 40-60% by weight.

Compositions of the invention may comprise a haemostatic agent or blood clotting agent. For example, compositions of the invention may comprise one or more blood coagulation factors, such as fibrinogen or thrombin. Compositions of the invention may comprise other naturally- occurring haemostats such as chitin. Alternatively, or additionally, compositions of the invention may comprise a synthetic haemostatic agent, such as an agent comprising carriers to each of which a plurality of fibrinogen binding peptides are immobilised. Such agents may include those described in WO 2008/065388 and WO 2015/104544, which are able to form a biogel on contact with fibrinogen, and in the absence of thrombin.

Compositions of the invention may comprise a lipophilic phase and an aqueous phase.

A composition of the invention may be in the form of a colloid or a suspension.

The term "colloid" is used herein to refer to a homogeneous non-crystalline substance consisting of large molecules or ultramicroscopic particles of one substance dispersed through a second substance. Colloids include gels, sols, and emulsions. The particles do not settle, and cannot be separated out by ordinary filtering or centrifuging like those in a suspension.

The term "suspension" is used herein to refer to a mixture in which small particles of a substance are dispersed throughout a liquid. If a suspension is left undisturbed, the particles are likely to settle to the bottom. The particles in a suspension are larger than those in either a colloid or a solution.

A composition of the invention may be in the form of an emulsion. The term "emulsion" is used herein to refer to a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible. An emulsion of the invention may be an oil and water emulsion, in particular an oil-in-water emulsion, or a water-in-oil emulsion. The composition may be a micro-emulsion.

The invention concerns compositions comprising: 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 an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance.

The first phase and the second 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 invention concerns compositions comprising: a lipophilic phase; an aqueous phase; an enzyme that is able to convert a substrate to release hydrogen peroxide; and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance.

The invention concerns compositions comprising: an oil; an emulsifier; an enzyme that is able to convert a substrate to release hydrogen peroxide; and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the unrefined natural substance.

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.

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. The compositions may not comprise sufficient water to allow the enzyme to convert the substrate.

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.

In some compositions, the composition may be in the form of a double emulsion. For example, droplets containing 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 dispersed within globules of a lipophilic phase (e.g. oil globules) and globules may be dispersed within an aqueous phase. Such a double emulsion may be termed a water-in-oil-in-water type (W/O/W) emulsion.

A composition of the invention may further comprise an emulsifying agent (or emulsifier).

Emulsions can be stabilized by adsorption of surface active agents (emulsifying agents) at the emulsion interface. Emulsifying agents lower the interfacial tension to maintain the droplets in a dispersed state. An emulsifying agent has a hydrophilic part and a lipophilic part. It is possible to calculate the relative quantities of an emulsifying agent(s) necessary to produce the most physically stable emulsions for a particular formulation with water combination. This approach is called the hydrophilic-lipophilic balance (HLB) method ("The HLB SYSTEM a time-saving guide to emulsifier selection" ICI Americas Inc., Wilmington, Delaware 19897, 1976, revised 1980). Each emulsifying agent is allocated an HLB number representing the relative properties of the lipophilic and hydrophilic parts of the molecule. High numbers (up to a theoretical number of 20), indicates an emulsifying agent exhibiting mainly hydrophilic or polar properties, whereas low numbers represent lipophilic or non-polar characteristics. According to the HLB System, all fats and oils have a Required HLB. Emulsions with optimal performance can be yielded by matching the HLB requirement with the emulsifying agent's HLB value. For an oil-in-water emulsion, the more polar the oil phase the more polar the emulsifying agent(s) must be. For example, to emulsify Soybean Oil, which has a Required HLB of 7, according to the HLB system, it would be necessary to use an emulsifying agent, or blend of emulsifying agents, with an HLB of 7 ± 1 . The HLB of emulsifying agents can be calculated or determined through trial and error.

Thus, the lipophilic phase of a composition of the invention may require an emulsifying agent of a particular HLB number in order to ensure a stable product. The lipophilic phase of a composition of the invention may comprise an oil or a wax. Examples of oils and waxes (by their International Nomenclature of Cosmetic Ingredients, INCI, name) for use in a lipophilic phase of a composition of the invention (with their respective Required HLBs) include the following:

Aleurites Moluccana Seed Oil [7] Grape (Vitis Vinifera) Seed Oil [7]

Almond Oil NF [6] Hybrid Safflower (Carthamus Tinctorius) Oil [9]

Anhydrous Lanolin USP [10] Isopropyl Myristate [1 1 .5]

Apricot Kernel Oil [7] Isopropyl Palmitate [1 1 .5]

Avocado (Persea Gratissima) Oil [7] Jojoba (Buxus Chinensis) Oil [6.5]

Babassu Oil [8] Lanolin [10]

Beeswax [12] Macadamia (Ternifolia) Nut Oil [7]

Borage (Borago Officinalis) Seed Oil [7] Mangifera Indica (Mango) Seed Butter [8] Brazil Nut Oil [8] Mineral Oil [10.5]

C12-15 Alkyl Benzoate [13] Myristyl Myristate [8.5]

Cannabis Sativa Seed Oil [7] Olive (Olea Europaea) Oil [7]

Canola Oil [7] Oryza Sativa (Rice Bran) Oil [7]

Caprylic/Capric Triglyceride [5] Peanut Oil NF [6]

Carrot (Daucus Carota Sativa) Seed Oil [6] Petrolatum [7]

Castor (Ricinus Communis) Oil [14] PPG-15 Stearyl Ether [7]

Ceresin [8] Retinyl Palmitate [6]

Cetearyl Alcohol [15.5] Safflower (Carthamus Tinctorius) Oil [8] Cetyl Alcohol [15.5] Sesame (Sesamum Indicum) Oil [7]

Cetyl Esters [10] Shea Butter (Butyrospermum Parkii) [8] Cetyl Palmitate [10] Soybean (Glycine Soja) Oil [7]

Coconut Oil [8] Stearic Acid [15]

Daucus Carota Sativa (Carrot) Root Stearyl Alcohol [15.5]

Extract [6]

Diisopropyl Adipate [9] Sunflower (Helianthus Annus) Oil [7]

Dimethicone [5] Sweet Almond (Prunus Amygdalus Dulcis) Oil [7]

Dog Rose (Rosa Canina) Hips Oil [7] Theobroma Cacao (Cocoa) Seed Butter [6]

Emu Oil [8] Tocopherol [6]

Evening Primrose Oil [7]

In some embodiments, the lipophilic phase of a composition of the invention comprises a beeswax.

In some embodiments, the lipophilic phase is 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 olive oil. In another preferred embodiment, the oil may be paraffin oil.

Water-in-oil emulsifying agents for use in compositions of the invention may have an HLB value in the range 3-6. Oil-in-water emulsifying agents for use in compositions of the invention may have an HLB value in the range 8-18. Examples of emulsifying agents (by their INCI name) for use in compositions of the invention (with their HLB numbers) include the following:

Calcium Stearoyl Lactylate [HLB = 5.1 ± 1] Oleth-20 [HLB = 15.3 ± 1 ]

Ceteareth-20 [HLB = 15.2 ± 1] PEG-100 Stearate [HLB = 18.8 ± 1 ]

Cetearyl Glucoside [HLB = 1 1 ± 1] PEG-20 Almond Glycerides [HLB = 10 ± 1 ] Ceteth-10 [HLB = 12.9 ± 1] PEG-20 Methyl Glucose Sesquistearate

[HLB = 15 ± 1]

Ceteth-2 [HLB = 5.3 ± 1 ] PEG-25 Hydrogenated Castor Oil

[HLB = 10.8 ± 1 ]

Ceteth-20 [HLB = 15.7 ± 1] PEG-30 Dipolyhydroxystearate [HLB = 5.5 ± 1] Cocamide MEA [HLB = 13.5 ± 1 ] PEG-4 Dilaurate [HLB = 6 ± 1 ]

Glyceryl Laurate [HLB = 5.2 ± 1] PEG-40 Sorbitan Peroleate [HLB = 9 ± 1] Glyceryl Stearate [HLB = 3.8 ± 1] PEG-60 Almond Glycerides [HLB = 15 ± 1 ] Glyceryl Stearate (and) PEG-100 Stearate PEG-8 Laurate [HLB = 13 ± 1]

[HLB = 1 1 ± 1]

Glyceryl Stearate SE [HLB = 5.8 ± 1 ] PEG-80 Sorbitan Laurate [HLB = 19.1 ± 1] Glycol Distearate [HLB = 1 ± 1] Polysorbate 20 [HLB = 16.7 ± 1]

Glycol Stearate [HLB = 2.9 ± 1] Polysorbate 60 [HLB = 14.9 ± 1]

lsoceteth-20 [HLB = 15.7 ± 1 ] Polysorbate 80 [HLB = 15 ± 1]

lsosteareth-20 [HLB = 15 ± 1 ] Polysorbate 85 [HLB = 1 1 ± 1]

Lauramide DEA [HLB = 15 ± 1] Sodium Stearoyl Lactylate [HLB = 8.3 ± 1 ] Laureth-23 [HLB = 16.9 ± 1] Sorbitan Isostearate [HLB = 4.7 ± 1 ]

Laureth-4 [HLB = 9.7 ± 1 ] Sorbitan Laurate [HLB = 8.6 ± 1 ]

Lecithin [HLB = 4 ± 1] Sorbitan Oleate [HLB = 4.3 ± 1 ]

Lecithin [HLB = 9.7 ± 1 ] Sorbitan Sesquioleate [HLB = 3.7 ± 1 ]

Linoleamide DEA [HLB = 10 ± 1 ] Sorbitan Stearate [HLB = 4.7 ± 1] Methyl Glucose Sesquistearate Sorbitan Stearate (and) Sucrose Cocoate

[HLB = 6.6 ± 1 ] [HLB = 6 ± 1 ]

Oleth-10 [HLB = 12.4 ± 1 ] Sorbitan Trioleate [HLB = 1.8 ± 1 ]

Oleth-10 / Polyoxyl 10 Oleyl Ether NF Stearamide E A [HLB = 1 1 ± 1]

[HLB = 12.4 ± 1 ]

Oleth-2 [HLB = 4.9 ± 1 ] Steareth-2 [HLB = 4.9 ± 1 ]

Oleth-20 [HLB = 12.4 ± 1 ] Steareth-21 [HLB = 15.5 ± 1]

In some embodiments, an emulsifying agent of a composition of the invention comprises a lecithin.

Emulsifying agents include ionic or non-ionic surfactants, and lipophilic fatty amphiles (for example, fatty alcohols or fatty acids). Non-ionic surfactants may be preferred since they may be less irritating to skin that anionic or cationic surfactants.

Other 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.

Examples of commercial emulsifying waxes include: Emulsifying wax BP (Cetostearyl alcohol, sodium lauryl sulphate), Emulsifying wax USNF (Cetyl alcohol, polysorbate), Cationic emulsifying wax BPC (Cetostearyl alcohol, cetrimide), Glyceryl monostearate S.E. (Glyceryl monostearate, sodium stearate), Cetomacrogol emulsifying wax BPC (Cetostearyl alcohol, cetomacrogol 1000), Polawax (Cetyl alcohol, non-ionic surfactant), Lecithin

(Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid).

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 07) and Polyglycerol polyricinoleate (PGPR). A preferred surfactant may be Poloxamer, such as Poloxamer 407. Another preferred surfactant may be PGPR.

Surfactants may include a surfactant polymer, or co-polymer. For example, a suitable surfactant may be a triblock copolymer consisting of a central hydrophobic block flanked by two hydrophilic blocks.

Compositions of the invention may comprise non-aqueous solvent. The non-aqueous solvent may be a polar solvent, such as a solvent with a dielectric constant of greater than 15. The nonaqueous solvent may be an organic solvent. For example, the solvent may be, or may comprise, glycerol, dimethylsulphoxide, propylene glycol or polyethylene glycol. The non- aqueous solvent may be immiscible with respect to the first phase e.g. the lipophilic phase (such as oil).

In a preferred embodiment, the non-aqueous solvent may be, or comprise, glycerol.

In some embodiments, compositions of the invention may comprise micelles, preferably reverse micelles. Within each micelle may be the enzyme and the substance (which may comprise an unrefined natural substance, such as honey), and outside of the micelle may be the first phase, e.g. the lipophilic phase (such as oil). Within each reverse micelle there may also be water and/or non-aqueous solvent. Within each micelle there may not be sufficient water for the enzyme to convert the substrate.

Compositions of the invention may comprise further components which may assist in reducing coalescence. Coalescence describes the situation in which two or more droplets, or micelles, combine to form a single droplet, or micelle. In order to reduce or prevent, coalescence, the strength of the interfacial film, i.e. the interface between the lipophilic phase and the aqueous phase, may be strengthened. This may be achieved, for example, by increasing the surfactant concentration, including an amphiphilic polymer, and/or by adding an alcohol, such as an aliphatic alcohol with 5-7 carbon atoms.

Compositions of the invention for topical application may be in the form, for example, of a cream, a lotion, or a lip balm.

The term "cream" is used herein to refer to a semi-solid emulsion of oil-in-water, or water-in-oil, for topical use. Oil-in-water (o/w) creams are composed of small droplets of oil dispersed in a continuous aqueous phase, and water-in-oil (w/o) creams are composed of small droplets of water dispersed in a continuous oily phase. Oil-in-water creams are less greasy and more easily washed off using water. Water-in-oil creams are more moisturising as they provide an oily barrier which reduces water loss from the outermost layer of the skin.

The term "cream" may also refer to a semi-solid emulsion in which droplets of a first phase are dispersed in a continuous second phase, or in which droplets of a second phase are dispersed in a continuous first phase. 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. The second phase may comprise water and/or non-aqueous solvent. 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. The non-aqueous solvent may be immiscible with respect to the first phase e.g. lipophilic phase. The term "lotion" is used herein to refer to a liquid suspension or emulsion for topical application. A lotion may comprise finely powdered, insoluble solids held in suspension by suspending agents and/or surface-active agents, or an emulsion (particularly, an oil-in-water emulsion) stabilized by one or more surface-active agents. A lotion has lower viscosity than a cream.

The term "lip balm" is used herein the refer to a wax-like substance applied topically to the lips of the mouth to moisturize and relieve chapped or dry lips. Lip balm may include, for example, beeswax or carnauba wax, camphor, cetyl alcohol, lanolin, paraffin, and petrolatum, among other ingredients.

The ratio of the lipophilic phase to the aqueous phase, or the ratio of the first phase to the second phase, in a composition of the invention 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.

A composition of the invention 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).

Alternatively, a composition of the invention 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).

A composition of the invention 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.

Alternatively, a composition of the invention 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.

A composition of the invention may comprise 1-60%, 1 -50%, 1-40%, 1 -30%, 1-20%, or 1-10% (w/v) of the substance, for example a honey.

A composition of the invention 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, for example a honey.

A composition of the invention may comprise 1-1500 units, 15-1500 units, 30-1500 units, 50- 1500 units, 100-1500 units, 1 -<685 units, 15-<685 units, 30-<685 units, 50-<685 units, 100- <685 units, 500-1000 units, 685-1000 units, or 100-500 units, of the enzyme, preferably glucose oxidase, per gram of the composition.

A composition of the invention may comprise no more than 85% water, for example no more than 80%, 70%, 60%, 50%, 40%, 30%, or 20% water, or less than 20% water, for example 10- 19% water. A composition of the invention may comprise less than 20% (w/w). A composition of the invention may comprise less than 15% (w/w) water. A composition of the invention may comprise less than 12% (w/w) water.

A composition of the invention 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.

A composition of the invention 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).

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

A composition of the invention 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.

A composition of the invention 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.

A composition of the invention 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.

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

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

A composition of the invention may comprise 1-10% (v/v) emulsifier such e.g. surfactant. The ratio of the first phase to the second phase in a composition of the invention 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 a composition of the invention 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.

A composition of the invention may comprise an emulsifier. In some embodiments, the emulsifier 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.

A composition of the invention may be an emulsion. In particular embodiments, a composition of the invention is an emulsion that comprises reverse micelles. The reverse micelles may be formed by the second phase.

In some embodiments of a composition of the invention, the enzyme and the substrate is dissolved in the second phase.

In particular embodiments of the invention, the first phase is, or comprises paraffin oil.

In particular embodiments of the invention, the second phase is, or comprises glycerol.

In particular embodiments of the invention, the emulsifier is, or comprises Polyglycerol polyricinoleate (PGPR).

In some embodiments, a composition of the invention is a cream. Typically, the viscosity of an emulsion used as a cream will be higher than that of an emulsion used as a spray. A cream may be formed by including a viscosity-increasing agent, such as a thickener or gelling agent (for example a hydrocolloid) in the composition.

Hydrocolloids are a heterogeneous group of hydrophilic, long-chain polymers (polysaccharides or proteins) characterised by their ability to form viscous dispersions and/or gels when dispersed in water (Saha and Bhattacharya, J Food Sci Technol, 2010, 47(6):587-597). The extent of thickening varies with the type and nature of the hydrocolloid. Some provide low viscosities at a fairly high concentration, but most provide a high viscosity at a concentration below 1 %. The viscosity of hydrocolloid dispersions arises predominantly from non-specific entanglement of conformationally disordered polymer chains. Hydrocolloids that can be used as thickening agents (referred to herein as hydrocolloid thickeners) include starch, modified starch, xanthan, galactomannans (such as guar gum, locust bean gum, and tara gum), gum Arabic or acacia gum, gum karaya, gum tragacanth, konjac maanan, and cellulose derivatives such as carboxymethyl cellulose, methyl cellulose, and hydroxypropylmethyl cellulose.

Some hydrocolloids are able to form gels, consisting of polymer molecules cross-linked to form an interconnected molecular network immersed in a liquid medium. A Theological definition of a gel is a viscoelastic system with a 'storage modulus' (G') larger than the 'loss modulus' (G") (de Vries 2004, Gums and stabilizers for the food industry, vol 12. RSC Publ, Oxford, pp 22-30). Hydrocolloids form gels by physical association of their polymer chains through hydrogen bonding, hydrophobic association, and cation-mediated cross-linking. Gelling-type hydrocolloids (or hydrocolloid gelling agents) include alginate, pectin, carrageenan, gelatin, gellan, agar, modified starch, methyl cellulose and hydroxypropylmethyl cellulose.

Gelation of hydrocolloids can occur by different mechanisms: ionotropic gelation, cold-set gelation and heat-set gelation (Burey et al. 2008, Crit Rev Food Sci Nutr 48:361-377).

lonotropic gelation occurs via cross-linking of hydrocolloid chains with ions, typically a cation- mediated gelation process of negatively-charged polysaccharides. Examples of hydrocolloids that can form gels by ionotropic gelation include alginate, carrageenan and pectin, lonotropic gelation can be carried out by either diffusion setting or internal gelation. In cold-set gelation, hydrocolloid powders are dissolved in warm/boiling water to form a dispersion which forms a gel on cooling. Agar and gelatin form gels by this mechanism. Heat-set gels require the application of heat to gel (for example, curdlan, konjac glucomannan, methyl cellulose, starch and globular proteins).

Thus, in some embodiments, a composition of the invention comprises a viscosity-increasing agent, such as a thickener or gelling agent, for example a hydrocolloid. In particular embodiments, the hydrocolloid is, or comprises, a polysaccharide or a protein. The hydrocolloid may be a hydrocolloid thickener, such as starch, modified starch, xanthan, a galactomannan (such as guar gum, locust bean gum, and tara gum), gum Arabic or acacia gum, gum karaya, gum tragacanth, konjac maanan, or a cellulose derivative, such as carboxymethyl cellulose, methyl cellulose, or hydroxypropylmethyl cellulose.

In other embodiments, the hydrocolloid is, or comprises a cross-linked hydrocolloid, for example a cross-linked polysaccharide, such as cross-linked alginate, pectin, carrageenan, gelatin, gellan, agar, agarose, modified starch, or a cellulose derivative, such as methyl cellulose or hydroxypropylmethyl cellulose.

The hydrocolloid may be cross-linked by any suitable method, for example including the methods for gelation of hydrocolloids described above: ionotropic gelation, cold-set gelation and heat-set gelation. In particular embodiments, molecules of the hydrocolloid are cross-linked by cations (for example calcium ions) as a result of ionotropic gelation of a hydrocolloid gelling agent. Examples of hydrocolloid cross-linked by cations that may be present in a composition of the invention include alginate, carrageenan or pectin.

In particular embodiments, a composition of the invention includes cross-linked alginate, for example alginate cross-linked by calcium ions. Alginate can form gels without prior heating because sodium alginate is soluble in cold water.

Cross-linked alginate may be formed from sodium alginate and calcium ions (for example, provided by calcium chloride). In some embodiments, water may be used as solvent to dissociate the calcium ions. However, since this could potentially activate production of hydrogen peroxide by the enzyme and the substance that includes a substrate for the enzyme, and limit the stability of the composition, it may be preferred to use a non-aqueous solvent to dissociate the calcium ions, such as ethanol or acetic acid.

We have appreciated that glycerol may be used to bind free water. This property allows water to be used to dissolve the alginate, provided sufficient glycerol is present to prevent premature release of hydrogen peroxide from the enzyme and the substance that includes a substrate for the enzyme.

Compositions of the invention may be sterilised by any suitable means. Preferably compositions of the invention have been 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 irradiation or electron beam irradiation. A suitable level of gamma irradiation is 10-70 kGy, preferably 25-70 kGy, more preferably 35-70 kGy. Alternatively, compositions of the invention may be sterilised by electron beam radiation.

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, compositions of the invention may be sterilised by irradiation that is not gamma irradiation.

According to the invention, there is provided a composition which comprises an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance; wherein the composition is sterile and has been sterilised by irradiation, preferably gamma irradiation or electron beam irradiation. According to the invention, there is also provided a method which comprises irradiating a composition comprising an enzyme that is able to convert a substrate to release hydrogen peroxide, and an unrefined natural substance that includes a substrate for the enzyme, wherein the enzyme is additional to any enzyme activity able to convert the substrate to release hydrogen peroxide that may be present in the substance; preferably wherein irradiating comprises exposure to gamma radiation or electron beam radiation.

Since ozone has not been authorised by the US FDA for sterilisation of honey-based products for use in wound healing, compositions for use according to 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, compositions for use according to the invention should not comprise ozonized honey or ozonated oil.

Preferred compositions for medical use according to the invention are sterile, single use compositions.

Sterilised compositions for use according to the invention that are stored away from exposure to light are expected to retain stability for at least six months. For example, such compositions may be packaged in high-density polyethylene/low-density polyethylene (HDPE/LDPE) tubes or in polyester-aluminium-polyethylene (PET/AI/PE) sachets.

A composition of the invention is preferably a medical grade or medical device grade composition.

The composition may be in the form of a solid or semi-solid preparation. Examples of solid or semi-solid preparations include capsules, pellets, gel caps, powders, hydrogels, pills, pillules, or globules. Alternatively, the composition may be in the form of a liquid preparation. Examples of liquid preparations include a syrup, paste, spray, drop, ointment, cream, lotion, oil, liniment, or gels. A typical gel includes an alcoholic gel such as an isopropanol, ethanol, or propanol gel, or a hydrogel.

A composition of the invention may be in a form suitable for administration to a human or animal subject. Suitable forms include forms adapted oral administration. Forms suitable for oral administration include a capsule, pellet, gel cap, pill, pillule, globule, lozenge, dental floss, toothpaste, mouthwash, dissolvable film strips. If a storage-stable composition is used, this may be diluted by liquid present at the site of administration (for example, by saliva for oral administration, or by exudate from a wound), leading to release of hydrogen peroxide at the administration site.

Compositions of the invention may be used to treat animals. Compositions of the invention may be used to treat humans. Statements and claims herein that are in the general format "[Composition X] for use in the treatment of [Condition Y]" may also be worded in the alternative format: "Use of [Composition X] in the manufacture of a medicament for the treatment of [Condition Y]"; or "A method of treating [Condition Y], which comprises administering [Composition X] to a subject in need of such treatment.

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

Figure 1 shows an optical microscopy images of reverse micelles in an emulsion containing Surgihoney™;

Figure 2 shows that SurgihoneyRO influences the balance of T-helper cell (Th) subsets by altering the expression of gatekeeping genes. Gene-expression profiles for Th lineage gatekeeping genes were analysed in nasal epithelial cells treated with 10g/L or 100g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous β-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. ^* p≤ 0.05, ^** p≤ 0.001 , ^*** p≤ 0.001 , ^**** p≤ 0.0001 ;

Figure 3 shows that SurgihoneyRO induces a Thi7 response in nasal epithelial cells. Gene- expression profiles for Th related cytokines were analysed in the nasal epithelial cells treated with 10g/L or 100g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative realtime PCR. The fold change was calculated based on normalisation to the expression of the endogenous β-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. ^* p≤ 0.05, ^** p < 0.001 , ^*** p≤ 0.001 , ^**** p < 0.0001 ;

Figure 4 shows that SurgihoneyRO can modulate the host immune response to invading pathogens in nasal epithelial cells. Gene-expression profiles for (a) matrix metalloprotease ( MP) related genes and (b) toll-like receptor (TLR) genes were analysed in nasal epithelial cells treated with 10g/L or 10Og/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous β-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p≤ 0.05, ** p < 0.001 , *** p < 0.001 , **** p≤ 0.0001 ; Figure 5 shows that SurgihoneyRO induces a Thi7 response in mast cells. Gene-expression profiles for Thi7 related cytokines were analysed in HMC-1 treated with 10g/L or 10Og/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous β-actin housekeeping gene. Each point shows the mean fold change in gene expression for 6 independent experiments. The dotted line for y=0 represents the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. ^* p≤ 0.05, ^** p≤ 0.001 , ^*** p≤ 0.001 , ^**** p≤ 0.0001 ;

Figure 6 shows fluorimetric measurements of hydrogen peroxide. The concentration of hydrogen peroxide production was measured over a time course of 24 hours. The kinetics of hydrogen peroxide production by both SurgihoneyRO and Acacia honey were measured without the presence of cells (a), with nasal epithelial cell line (b) and with mast cell line (c). The data represents the mean with the error bars showing standard deviation of the mean for at least 3 independent experiments;

Figure 7 shows that hydrogen peroxide treatment influences the balance of T-helper cell (Th) subsets by altering the expression of the gatekeeping genes in the same way as

SurgihoneyRO. Gene-expression profiles for Th lineage gatekeeping genes were analysed by RT-PCR. (a) shows GAT A3 gene expression in the nasal epithelial cells, (b) shows the expression of Thi7 related cytokines for the nasal epithelial cells and (c) for the mast cells. Both cell lines were treated either with or without hydrogen peroxide (0-400μ ) for 1 hour. The fold change calculated was based on normalisation to the expression of the endogenous β-actin housekeeping gene. Each point shows the mean fold change in gene expression for 5 independent experiments. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p < 0.05, ** p < 0.001 , *** p≤ 0.001 , **** p≤ 0.0001 ;

Figure 8 shows that hydrogen peroxide treatment can modulate anti-microbial and host immune response to invading pathogens in both nasal epithelial cells and mast cells. Gene-expression profiles for P related genes were analysed in (a) nasal epithelial cells and (b) mast cells treated with various concentrations of exogenous hydrogen peroxide (0-400μΜ) for 1 hour using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous β-actin housekeeping gene. For each treatment group the mean fold change in gene expression for 5 independent experiments is shown. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. ^* p≤ 0.05, ^** p≤ 0.001 , ^*** p≤ 0.001 , ^**** p < 0.0001 ;

Figure 9 shows analysis of gene-expression of IL10 in the nasal epithelial cells treated with various concentrations of exogenous hydrogen peroxide (0-400μΜ) for 1 hour (a); with 10g/L or 100g/L of SurgihoneyRO for 5 minutes, 1 hour and 2 hours using quantitative real-time PCR. The fold change was calculated based on normalisation to the expression of the endogenous β- actin housekeeping gene. Each point shows the mean fold change in gene expression for 5 independent experiments. The dotted line for y=0 representing the untreated control group to which gene expression in the treatment groups were normalised and compared using an unpaired two tailed T test. *p < 0.05, ** p≤ 0.001 , *** p≤ 0.001 , **** p≤ 0.0001 ;

Figure 10 is a graph showing the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to Surgihoney™, at various concentrations.

Figure 1 1 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 4.03) on the growth of planktonic MRSA, at various concentrations;

Figure 12 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (unbuffered) on the growth of planktonic MRSA, at various concentrations;

Figure 13 is a graph showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose (buffered at pH 7.04) on the growth of planktonic MRSA, at various concentrations;

Figure 14 is a table showing the effect of sterile and non-sterile compositions of the invention comprising glucose, glucose oxidase and fructose, on the MIC and MBC of planktonic MRSA, at various concentrations;

Figure 15 shows the effect of compositions of the invention comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MRSA, compared to

SurgihoneyRO, at various concentrations;

Figure 16 shows the effect of SyntheticRO on the MIC and MBC of planktonic MRSA, compared to SurgihoneyRO, at various concentrations;

Figure 17 shows the effect of SyntheticRO comprising glucose, glucose oxidase and fructose (SyntheticRO) on the growth of planktonic MSSA isolate;

Figure 18 (a and b) compares SyntheticRO with SurgihoneyRO using planktonic MRSA and MSSA, and Figure 18c is a table showing the MICs of a composition of the invention compared to SurgihoneyRO. Planktonic MRSA and MSSA in vitro cultures were grown in the presence of the respective compositions for 18 hours, then the absorbance (OD595) measured and compared to untreated cultures (n=6);

Figure 19 shows 48 hour RSA (a&c) and SSA (b&d) in vitro biofilms treated with

SyntheticRO+ (with enzyme) and SyntheticRO- (without enzyme) for 24 hours (n=5), with viability measured by cfu enumeration (a&b) and biomass measured by absorbance [OD595] (c&d);

Figure 20 shows 48 hour MRSA and MSSA biofilms treated with 71 g/l SyntheticRO+ and SyntheticRO- for 24 hours and then imaged using confocal microscopy and LIVE/DEAD staining (b), with maximum biofilm height measured and compared to untreated controls (a);

Figure 21 shows 48 hour biofilms formed by individual MRSA (a; n=7) and MSSA (b; n=5) clinical isolates treated with 71 g/l synthetic RO+ and viability measured by cfu enumeration;

Figure 22 shows the result of Surgihoney treatment of in vitro non-typeable H. influenza biofilms. (a) NTHi in vitro planktonic cultures were grown in the presence of SurgihoneyRO or Acacia for 18 h then growth assessed by measurement of absorbance (OD595). (b) 48 hour in vitro NTHi biofilms were treated with SurgihoneyRO or Acacia for 2 h and biofilm viability measured by cfu enumeration, (c) Fluorimetric measurements of H2O2 production by different SurgihoneyRO and Acacia concentrations at 2 h. (d) 48 hour in vitro NTHi biofilms treated with HBSS adjusted to pH6.3 (equivalent pH to 71 g/L SurgihoneyRO) for 2 h and biofilm viability measured by cfu enumeration *P≤0.05; **P<0.01 ; and

Figure 23 Shows a comparison between SurgihoneyRO and co-amoxiclav in the treatment of non-typeable H. influenza biofilms. (a) 48 hour in vitro NTHi biofilms were treated with 71 g/L SurgihoneyRO and 300/60 pg.ml co-amoxiclav alone, and in combination for two hourswith viability measured by cfu enumeration. Confocal microscopy was performed on (b) untreated 48 h NTHi biofilms, biofilms treated for 2 hours with (c) 300/60 μg.ml co-amoxiclav and d) 71 g/: SurgihoneyRO. Biofilms were imaged using Live/Dead staining to visualize ive cells (green fluorescence) and dead cells (red fluorescence). *P<0.05.

Surgihoney may also be referred to as Surgihoney™, SurgihoneyRO or SurgihoneyRO™. Compositions of the invention, such as compositions which comprise purified glucose, puridied fructose and glucose oxidase may be referred to as SyntheticRO, synthetic honey compositions or synthetic compositions.

Specific Examples

Example 1 - Treatment of Aphthous ulcers and Geographic tongue with Surgihoney Surgihoney was used to treat two patients with geographic tongue and recurrent aphthous ulcers. Surgihoney™ was applied topically and held over the affected areas three times a day for as long as possible before swallowing. This was continued for a week.

In both cases, the geographic tongue resolved in 48 hours. In one case, the ulcers did not progress, although this case had further aphthous ulcers 10 days after stopping treatment with Surgihoney. In the other case, the subject reported that the ulcers were not as bad as usual and no further ulcers were reported.

Example 2 - Surgihoney™ emulsions

Preparation

10g Surgihoney was dissolved in 10ml of glycerol. 10ml of paraffin oil was then added to a Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5° C. After 2 minutes, 10ml of Surgihoney-glycerol solution was added dropwise. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

Optical Microscopy

Optical microscopy revealed that the emulsion contained reverse micelles which encapsulated Surgihoney. Such micelles can be observed in Figure 4. The average micelle diameter was found to be 178 μητ

Hydrogen peroxide tests

Hydrogen peroxide stick tests (Purchased from Sigma Aldrich (Quantofix®)) were used to detect hydrogen peroxide in the emulsion. The tests were carried out before and after addition of water, and showed that before addition of water, the emulsion produced no hydrogen peroxide, and after water was added, the emulsion tested positive for hydrogen peroxide. A positive test was indicated by a colour change to blue.

Stability Test

The emulsion maintained its capacity to generate hydrogen peroxide after storage at ambient conditions for at least four weeks.

Spray Test

The emulsion was added to a pump-action spray bottle and was found to be sprayable. Example 3 - Effects of different parameters on stability of Surgihoney emulsions The effects of changing the Surgihoney emulsion preparation method described in Example 2, one parameter at a time, were investigated. The changes and their effects are summarised below. i) Proportion of the oil phase to the Surgihonev-glvcerol phase

Oil volumes greater than 10ml, and less than 10ml, were tested. The emulsion was found to be more stable when a lower volume of oil was used compared to the volume of the Surgihoney- glycerol phase. When the volume of the oil was less than 6ml, the emulsion was found to separate by less than 3% in total volume over 72 hours. A volume of 4ml allowed a separation of just 1.3% of the total volume over 72 hours. This stability is far greater than that of the method described in Example 2, which provided an emulsion with a separation of 9.4% of total volume over the same time period. ii) Volume of PGPR

PGPR volumes up to 4ml, and less than 1 ml, were tested. The emulsion was more stable when a higher amount of PGPR was used. At a volume of 4ml, PGPR provided greater stability than with use of lower volumes, and far greater stability than that of the emulsion described in Example 2. iii) Shear rate

Shear rates from 1000 1/s to 3000 1/s were tested. The emulsion was more stable when a higher shear rate was applied. A shear rate of 3000 1/s produced the most stable emulsion. Separation of only 4.6% of the total volume over 72 hours was observed for emulsion prepared at this shear rate, compared with a separation volume of 9.4% of the total volume over the same time period for emulsion prepared as described in Example 2. iv) Temperature

Temperatures from 20°C to 40°C were tested. There was no noticeable trend regard the stability of the emulsions as temperature was increased. However a temperature of 40°C produced the most stable emulsion. Separation of only 3.1 % of the total volume over 72 hours was observed for this emulsion. v) Length of shear

Shear times of 20 minutes and 30 minutes were tested, in addition to that used in the preparation method described in Example 2. However, there was no significant difference produced by extending the shear time.

Order of reagent addition The effect of changing the order in which the reagents are added to the rheometer was tested. The effect of adding all of the components before starting the rheometer was compared with the effect of adding the Surgihoney-glycerol and oil components first, then adding the PGPR after 1 -2 minutes. The most stable emulsion was formed when the PGPR was added last. The resulting emulsion provided a separation volume of 2.8% of the total volume over 120 hours. vii) Concentration of Surgihoney dissolved in glycerol

The following ratios of Surgihoney (g) to glycerol (ml) were tested: 1 g: 1 ml; 0.5g: 1 ml; 2g: 1 ml. The ratio that produced the most stable emulsion was 1 g: 1 ml, the same ratio used in the preparation method described in Example 2. viii) Sodium chloride

When sodium chloride is dissolved in the polar layer of the emulsion , it increases the polarity of this layer. It also forms electrostatic interactions with the lipid layer of the emulsion. The electrostatic interactions and increased polarity could improve stability and reduce coalescence. However, addition of sodium chloride (1 g, 2g or 4g) was not found to influence the stability of the emulsion.

The effects of the changes are summarised in the table below:

Emulsion SH Glycerol Paraffin PGPR Shear Temp. Order of Stability

(g) (ml) Oil (ml) (ml) rate (°C) addition (% total

(1 /s) vol. after

72 hrs)

Ex 7 10 10 10 1 2000 37.5 Oil, then 9.4

PGPR, then

SH/glycerol

Ex 8 (i) 10 10 6 1 2000 37.5 Oil, then 2.8

PGPR, then

SH/glycerol

Ex 8 (i) 10 10 4 1 2000 37.5 Oil, then 1 .3

PGPR, then

SH/glycerol

Ex 8 (ii) 10 10 10 4 2000 37.5 Oil, then 2.7

PGPR, then

SH/glycerol

Ex 8 (iii) 10 10 10 1 3000 37.5 Oil, then 4.6

PGPR, then

SH/glycerol Ex 8 (iv) 10 10 10 1 2000 40.0 Oil, then 3.1

PGPR, then

SH/glycerol

Ex 8 (vi) 10 10 10 1 2000 37.5 SH/glycerol 2.8 ^*

and oil,

then PGPR

* (after 120 hrs)

Example 4 - Surgihoney™ emulsions with high stability

The results from the changes described above were used to design a further method of preparing Surgihoney emulsion. This method is described below.

Preparation

10g Surgihoney was dissolved in 10ml of glycerol. 4, 6, 8, or 10ml of Paraffin oil was then added to the rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 10ml of Surgihoney-glycerol solution was then added to the rheometer. The rheometer was then started under the following conditions; Shear rate 3000 1/s, Temperature 40°C, gap 4000μηΊ, Run time 10 minutes. After 1 minute 4ml of PGPR (Polyglycerol polyricinoleate) was then added. Once a total of 10 minutes had elapsed the emulsion was transferred from the rheometer to a container.

All of the formulations were found to be highly stable, with a slight increase in stability observed as the volume of paraffin oil used was increased.

Example 5 - Surgihoney™ Cream Formulation

1.5g of Surgihoney was dissolved in 1 .5ml of glycerol. 1 g of sodium alginate was then dissolved in the Surgihoney-glycerol solution. Next 10ml of Paraffin oil was added to the Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. 1 ml of PGPR (Polyglycerol polyricinoleate) was then added. The rheometer was then started under the following conditions; Shear rate 2000 1/s, Temperature set at 37.5°C, gap 4000pm, Run time 10 minutes. After 2 minutes, 1 .5ml of the Surgihoney-alginate and glycerol solution was added to the rheometer. After 3 minutes 8ml of calcium chloride solution was added dropwise to the rheometer. Once a total of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier to a container.

Example 6 - Non-aqueous Surgihoney™ Cream Formulation

The method described in Example 10 uses water to dissociate calcium chloride into its ions. This could potentially activate the Surgihoney to produce hydrogen peroxide, and limit the stability of the cream formulation. However, we have appreciated that calcium chloride can be dissociated using non-aqueous solvents, such as ethanol or acetic acid. We have also appreciated that glycerol is able to bind to free water. This property allows water to be used to dissolve the alginate, provided sufficient glycerol is present to prevent premature release of hydrogen peroxide.

The method described below uses ethanol as a solvent for calcium chloride, and glycerol to bind free water in the alginate solution.

1 g of sodium alginate is dissolved in 15ml water. Next 30ml glycerol is added to the alginate solution and mixed. Then 30g Surgihoney is then dissolved in the solution. 10ml of Paraffin oil is then added to the rheometer (TA Instruments AR-G2) which has a Jacket Peltier and vane geometry attached. 10ml of Surgihoney solution is then added to the rheometer. The rheometer is then started under the following conditions: Shear rate 3000 1 /s, Temperature 40°C, gap 4000pm, Run time 10 minutes. After 1 minute 4ml of PGPR (Polyglycerol polyricinoleate) is added. After 2 minutes 8ml of non-aqueous calcium chloride solution (1 M Calcium chloride in ethanol) is added dropwise to the rheometer. Once a total of 10 minutes has elapsed, the emulsion is transferred from the rheometer to a container.

Summary of emulsion formulations described above.

Emulsio SH (g) Glycer Pa raff i PGP NaAlg(g Shea Tern Order of n/cream ol (ml) n Oil R )/CaCI ₂ ( r rate P- addition

(ml) (ml) ml) (1/s) (°C)

Ex 7 10 10 10 1 2000 37.5 Oil, then PGPR, then

SH/glycerol

Ex 9 10 10 4, 6, 8, 4 3000 40.0 Oil and

or 10 SH/glycerol, then PGPR Ex 10 1.5 1.5 10 1 1/8 (aq) 2000 37.5 Oil and PGPR, then

SH/glycerol/Na Alg, then CaCI ₂

Ex 1 1 30 30 10 4 1/8 3000 40 Oil and

(non-aq) SH/glycerol/Na

Alg(aq), then PGPR, then CaC (non-aq)

Example 7 - Treatment with electron beam irradiation

Samples of activated Acacia honey were prepared. Three glass sample vials were each filled with 10g of the activated honey. Each of the samples was tested by a Labtek PER100 peroxide test stick to determine hydrogen peroxide generation from the rate of colour generation. Samples were sterilised with electron beam irradiation at a dose of about 75 kGy and then tested for hydrogen peroxide generation.

Non Sterile Activated Acacia Honey Samples: Average measured rate of peroxide production 0.540 ppm/s standard Deviation 0.062.

Sterile Activated Acacia Honey Samples: Average measured rate of peroxide production 0.353 ppm/s standard Deviation 0.008

The honey samples maintained the ability to produce hydrogen peroxide after sterilisation with electron beam irradiation.

Example 8 - Immunomodulatory effects of SurgihoneyRO Treatment on Epithelial Cells

A human nasal epithelial cell line was used to investigate the immediate effect of

SurgihoneyRO (also known as Surgihoney) on the integrity and function of the epithelium. In addition, a human mast cell line was used as a representative leukocyte to investigate immunological responses to SurgihoneyRO. Mast cells were selected because of their function as sentinel cells, being located just underneath the epithelium in tissues. Mast cells have close contact to the external environment, for example being found in the nasal mucosa, where they are ideally placed to participate in the early recognition of pathogens - by acting as immune effectors and modulatory cells with an essential role in linking innate and adaptive immunity in the host's defence against pathogens such as bacteria. Another reason for using mast cells relates to findings of intracellular bacteria in mast cells (Hayes et al., J Allergy Clin /mmt/no/ 2015;135(6):1648-51). One of the objectives of this treatment would be to develop its use to target intracellular bacteria which act as a reservoir constantly seeding bacteria into the extracellular environment and therefore promoting ongoing inflammation and development of chronicity.

One of the objectives of the study was to determine whether treatment with SurgihoneyRO could elicit an immunomodulatory effect to the host. Figure 2 demonstrates the effects of SurgihoneyRO on the expression of four main gatekeeping genes for T cell differentiation. SurgihoneyRO treatment causes a shift in the Th balance toward T i2 and Thi7 polarisation for nasal epithelial cells. The results show this as there is no observed significant increase in the expression of T-bet across the time course with either concentration of SurgihoneyRO.

However there is a dose and time dependent increase in the expression of GAT A3 this difference being significant at all time points (p≤0.05) when treated with 100g/L. At 2 hours, there is a significant increase in expression by 2.74 fold with 100g/L and 2.19 fold with 10g/L of SurgihoneyRO.

Figure 2 also shows that there is a dose and time dependent decrease in FOXP3 expression with significance at both 1 hour (0.34 fold decrease, p≤0.05) and 2 hours (0.60 fold decrease, p≤0.0001 ) when treated with 100g/L SurgihoneyRO. The nasal epithelial cells display an increase in the expression of the Th gatekeeping gene RORTC. When treating with 100g/L SurgihoneyRO for 2 hours, the epithelial cells had a significant 1.89 fold increase in RORyC expression with p≤0.05. Data showed no effect of cell treatment with matching concentrations (1 Og/L and 100g/L) of the non-engineered base honey (Acacia) for all genes studied (data not shown).

The polarisation of Thi 7 is further supported by the changes in gene expression of the Thi7 related cytokines interleukin 22 (IL22) and interleukin 23 receptor (IL23R) (Figure 3). There is a time and dose dependent increase in the expression of IL22; increasing from 0.22 to 2.05 fold (p<0.05) and 1.67 (p≤0.01) across the time points with 100g/L and 0.86 fold to 1 .33 and 1.38 (p<0.05) with 10g/L respectively. Furthermore, there is a time and dose dependent increase in IL23R expression but only when treating with 100g/L; increasing from 2.74 fold (p≤0.001 ) to 8.54 fold (p≤0.05) and finally 14.28 fold (p<0.05) across the time course.

Having illustrated the ability of SurgihoneyRO to influence Thi7 responses, we wanted to investigate whether the treatment had the capacity to mediate host responses to invading pathogens. Matrix metalloproteinases ( MPs) are known to be secreted by activated epithelial cells. In addition to matrix degrading and wound healing properties, MMPs also play an important role in host defence responses against infectious agents. MMP7 is involved in the activation of antimicrobial defensin, releasing mature TNF and chemokines to tackle infections. M P9 can regulate immune responses and attract lymphocytic cell migration which facilitates tissue remodelling resulting from cleavage of extracellular matrix.

Figure 4a demonstrates a time and dose dependent upregulation of both MP7 and M P9 resulting in significant fold increases following treatments with both 10g/L and 100g/L of SurgihoneyRO. The expression of MMP7 significantly increased to a fold change of 4.56 (p<0.01) with 10g/L and 8.04 (p<0.05) with 100g/L at 2 hours. M P9 also significantly increased to 1 .37 fold (p≤0.01 ) with 10g/L and 3.73 fold (p≤0.05) with 100g/L. There were also significant increases in the expression of both defensin 5 and TNFa. There was a mean increase in defensin 5 expression of 0.9 fold (p≤0.01 ) and TNFa expression increased by 4.24 fold (p<0.05) following 2 hours of treatment with 100g/L of SurgihoneyRO. This is direct evidence to support the hypothesis that SurgihoneyRO has the ability to boost the host response to invading pathogens (Figure 4a).

An essential function of the early innate immune response to pathogens is played by Toll-Like Receptors (TLRs) whose recognition of pathogen-associated microbial patterns (PAMPs), and endogenous danger-associated molecular patterns (DAMPs) initiates downstream signalling cascades resulting in the production of pro-inflammatory and effecter cytokines that have a direct effect on the adaptive immune response. Figure 4b shows an increase in the gene expression of two TLRs (TLR2 and TLR4) following the treatment of nasal epithelial cells with SurgihoneyRO (Figure 4b). There is a highly significant (p≤0.001) mean increase of 1 .85 fold in expression of TLR2 when treating with 100g/L SurgihoneyRO at 2 hours.

We then studied the response of the mast cell line (HMC-1) to SurgihoneyRO treatment. In parallel to the effects seen with nasal epithelial cells, there is also an increase in the expression of Thi7 related cytokines following SurgihoneyRO treatment. When treating with 100g/L SurgihoneyRO for 2 hours, mast cells displayed significant increases of 1 .89 fold in RORTC expression (p≤0.01), 1 .35 fold in IL22 expression (p≤0.05) and 0.61 fold in IL23R expression (p≤0.05) respectively (Figure 5).

Example 9 - Hydrogen Peroxide Production by SurgihoneyRO

In addition to the potential immunomodulatory effects of SurgihoneyRO, we quantified the production of hydrogen peroxide by SurgihoneyRO for the concentrations used in this study (10g/L and 100g/L). The aim of this experiment was to validate whether the immunomodulatory effects observed previously were as a result of endogenous hydrogen peroxide production by SurgihoneyRO.

Figure 6a shows that the release of hydrogen peroxide by SurgihoneyRO is significantly higher than that of the non-engineered base honey (Acacia). The production of hydrogen peroxide peaks between 2 and 6 hours at which time it is roughly 12 fold higher for 100g/L than that for 10g/L. Following the exponential increase in the production, the level of hydrogen peroxide remains constant until 24 hours which was the last time point measured.

The study evaluated the production of hydrogen peroxide when the SurgihoneyRO and Acacia honey were used to treat both cell lines. It showed that the maximum amount of hydrogen peroxide produced by 100g/L SurgihoneyRO in the presence or absence of epithelial cells (Figure 6b) or mast cells (Figure 6c) was around 400 u , peaking at 2 hours. There was no significant difference whether there are any cells present. The presence of cells within the culture made no significant difference suggesting that SurgihoneyRO is the main source of hydrogen peroxide in the cell culture.

Example 10 - Immunomodulatory Effects of Exogenous Hydrogen Peroxide Treatment

Having established the concentration of hydrogen peroxide production by SurgihoneyRO in Figure 6, we wanted to know whether the concentration of hydrogen peroxide produced in the cell culture is able to induce immunomodulatory changes. In this case, we treated cells with an appropriate concentration range of pure hydrogen peroxide, from 0 - 400u .

The effect of the treatment with SurgihoneyRO correlated to that with a comparable concentration of pure hydrogen peroxide. Figure 7a shows that similar to the treatment with SurgihoneyRO, exogenous hydrogen peroxide elicits a dose dependent increase in GATA3 expression in nasal epithelial cells, with a significant fold increase of 0.92 (p≤0.5) when treating with 400μΜ hydrogen peroxide for 1 hour (Figure 7a).

A dose dependent increase in the expression of RORyC was also detected following exogenous hydrogen peroxide treatment, resulting in a mean increase in expression of 14.57 fold (p≤0.05) for the nasal epithelial cells and 2.83 fold (p≤0.05) for the mast cells with 400μΜ hydrogen peroxide.

These changes are also associated with increases of IL22 and IL23R for both cell lines (Figures 7b and 7c). With the nasal epithelial cells there is a steady increase in the expression of IL22 from 0.90 fold with 40μΜ to 1 .42 fold with 200μΜ and 4.55 fold with 400μΜ (p<0.001), and with IL23R the fold change in expression increases from 0.45 to 1 .20 (p<0.05) and 1 .87 (p<0.001 ) across the concentration range. With the mast cells there is an increase in expression of IL22 from 0.60 fold with 40μΜ to 1.25 with 200μΜ (p<0.05) and 1 .81 with 400μΜ (p<0.01 ), and with IL23R the fold change in expression increases from 0.47 to 0.58 and 1 .05 (p<0.05) across the concentration range.

The treatments with exogenous hydrogen peroxide with epithelial cells and mast cells also caused a dose dependent increase in the expression of the anti-microbial MMP7 and MP9 (Figures 8a and 8b). However there is only statistical significance with the expression of MMP9 for the nasal epithelial cells (1 .47 fold with 400μΜ, p≤0.01 ) and MMP7 for the mast cells which have an increase of 3.07 fold (p<0.05) and 3.31 fold (p<0.05) with 200μ and 400μΜ respectively.

Example 11 - A protective anti-inflammatory effect of SurgihoneyRO and Exogenous Hydrogen peroxide

Exogenous hydrogen peroxide treatment can directly induce a protective anti-inflammatory effect on epithelial cells by increasing the expression of IL10. Figure 9a shows a significant increase in expression of the anti-inflammatory cytokine IL10 to 4.63 fold (p≤0.05) in the nasal epithelial cells treated with 400μΜ hydrogen peroxide. In concordance with these effects, we also demonstrated that there is a time dependent increase in the expression of the protective and anti-inflammatory cytokine IL10; increasing from a fold change of 0.01 to 2.65 (p<0.05) and 4.73 (p≤0.05) across the time points with 100g/L and -0.81 to 3.01 (p≤0.05) and 4.85 with 10g/L respectively (Figure 9b). This increase in IL10 expression also correlated with the increase in GAT A3 expression in the nasal epithelial cells supporting the shift towards the Th2 lineage.

These data support a protective effect of both SurgihoneyRO and hydrogen peroxide and a shift in the balance towards the Th _∑ lineage specifically for the nasal epithelial cells.

Taken together, these experiments clearly demonstrate an immunomodulatory effect of SurgihoneyRO treatment in both nasal epithelial and mast cells with a shift towards a Th ₂ and Thi response, as well as anti-microbial and innate immunity responses. These effects could be mediated by the hydrogen peroxide production of SurgihoneyRO. It has been shown

SurgihoneyRO has anti-inflammatory and wound healing effects on skin. In our study, we showed that these effects could be mediated through the upregulation of the anti-inflammatory cytokine IL10. These data shed new insights into the immunomodulatory properties of

Surgihoney RO.

Example 12 - Synthetic Honey Compositions

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. pH 4.03 buffered sampl

Batch no NB01 p43RO

Non sterile

Material Weight fraction

Fructose 52.0%

Glucose 31 .0% 50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

Description

Non sterile base buffered saccharide solution.

A2. Batch no NB01 p43RO

Sterile

Description Sterile base buffered saccharide solution

Batch no NB01 p44RO1

Non sterile

Description

Non sterile base buffered R01 saccharide solution.

A4. Batch no NB01 p44RO1

Sterile

Description

Sterile base buffered R01 saccharide solution

Batch no NB01 p44RO2

Non sterile

Description

Non sterile base buffered R02 saccharide solution.

Batch no NB01 p43RO2

Sterile

Material Weight fraction

Fructose 52.0%

Glucose 31 .0%

50 mMol Citric acid/NaOH buffer pH 4.03 17.0%

GOX enzyme N/A Description Sterile base buffered R02 saccharide solution

B. Unbuffered samples

B1. Batch no NB01 p51 RO

Non sterile

Description

Non sterile base buffered saccharide solution.

Batch no NB01 p51 RO

Sterile

Description Sterile base buffered saccharide solution

B3. Batch no NB01 p51 RO1

Non sterile

Description

Non sterile base buffered R01 saccharide solution.

Batch no NB01 p51 RO1

Sterile

Description

Sterile base buffered R01 saccharide solution

Batch no NB01 p51 RO2

Non sterile

Description

Non sterile base buffered R02 saccharide solution. Batch no NB01 p51 RO2 Sterile

Description

Sterile base buffered R02 saccharide solution pH 7.04 buffered sampl

Batch no NB01 p57RO

Non sterile

Description

Non sterile base buffered saccharide solution.

Batch no NB01 p57RO

Sterile

Description

Sterile base buffered saccharide solution

Batch no NB01 p57RO1

Non sterile

Description

Non sterile base buffered R01 saccharide solution.

Batch no NB01 p57RO1

Sterile

Description

Sterile base buffered R01 saccharide solution Batch no NB01 p57RO2

Non sterile

Description

Non sterile base buffered R02 saccharide solution.

C6. Batch no NB01 p57RO2

Description

Sterile base buffered R02 saccharide solution

Example 13 - Efficacy of Synthetic honey compositions against planktonic MRSA

MIC and MBC were assessed for the R01 samples (containing 50 ppm glucose oxidase) and compared to Surgihoney™ (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 10 to 14.

The results show that, like Surgihoney, 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 Surgihoney.

Figures 15 (a to d) and 16 show MIC and MBC results including SurgihoneyR02 samples and synthetic honey R02 samples. pH 7.04 formulations were tested against a planktonic MSSA isolate. Figure 17 shows the results obtained. The synthetic R02 product was selected for further investigation. Figure 18 (a, b and c) show SyntheticRO (pH7.04) compared to SurgihoneyRO using planktonic phenotype. RO- indicates a product lacking enzyme activity.

Example 14 - Evaluation of Surgihoney™ and Synthetic Honey compositions on chronic rhinosinusitis-related mucosal bacterial strains of Staphylococcus aureus

SurgihoneyRO™ and synthetic RO (pH 7.04; R02) were tested on both the in vitro planktonic phenotype and established biofilms of clinical MRSA and MSSA isolates. Biofilm viability was assessed by colony forming unit (cfu) enumeration and biomass assessed, by measurement of absorbance. Data were validated using confocal microscopy.

• Materials and methods

Bacterial strains and growth conditions

S. aureus strains were sub-cultured from frozen stocks onto Colombia blood agar (CBA) plates (Oxoid, UK) and incubated for 18 h at 37°C and 5% CO ₂ , following which colonies were resuspended in Brain Heart Infusion (BHI) broth and grown to mid-exponential phase for experiments.

Planktonic assays

Flat-bottomed 96-well plates (Fisher Scientific, UK) were inoculated with ~1 .0 x 10 ⁶ planktonic bacteria per well (grown in supplemented BHI). SurgihoneyROTM and the synthetic products (RO+ and RO-) were prepared in BHI and added to wells at final concentrations of 6 g/L to 383 g/L. BHI alone was added in place of treatments for untreated controls. Cultures were incubated at 37oC and 5% CO ₂ for 18 hours then turbidity measured by absorbance (OD595) using an EZRead 400 spectrophotometer (Biochrom; n=6).

Biofilm assays

Mid-exponential planktonic cultures were used to inoculate individual wells of untreated 6-well polystyrene plates (-1.0 x 10 ⁸ planktonic bacteria per well; Corning Incorporated, USA).

Cultures were incubated at 37°C and 5% CO ₂ for 48h, replacing spent media with fresh BHI at 24 h. Prior to treatment spent media was removed and biofilms washed twice with Hanks' balanced salt solution (HBSS; Gibco, UK). Biofilms were treated with SurgihoneyRO, RO+ or RO- (all prepared in HBSS) at final concentrations of 7 to 142 g/L. HBSS alone was added in place of treatments for untreated controls. Biofilms were incubated at 37°C and 5% C02 for 24 h, following which the treatments were removed and biofilms washed twice with HBSS to remove unattached cells. Biofilms were resuspended in 1 ml HBSS by cell scraping and briefly vortexing, then serially diluted onto Columbia blood agar. Plates were incubated at 37°C and 5% CO2 and colony forming units (CFUs) enumerated (n=5). To assess the total biofilm biomass 100μΙ_ of the resuspended biofilms was diluted 10-fold in BHI and the turbidity measured by absorbance (OD595) using a Jenway 6300 spectrophotometer.

Confocal Microscopy

Mid-exponential planktonic cultures were used to inoculate 35 mm untreated glass-bottom CellView cell culture dishes (~1 .0 x 108 planktonic bacteria perwell; Greiner Bio One, UK). Cultures were incubated at 37°C and 5% CO ₂ for 48 h, replacing spent media with fresh BHI at 24 h. Media was removed, biofilms washed twice with HBSS, then treated with 71 g/L RO+, 71 g/L RO-, or HBSS alone (untreated control) for 24 h at 37°C and 5% C0 ₂ . Treatments wereremoved and biofilms washed twice with HBSS before staining with a

LIVE/DEAD Baclight Bacterial Viability Kit (Life Technologies, UK) as per manufacturer's instructions. Biofilms were examined using an inverted Leica SP8 confocal microscope using a 63x oil immersion lens with sequential scanning of 2μπι sections (Leica Microsystems, UK).

Statistical analyses

Statistical analyses of in vitro planktonic and biofilm data were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis multiple comparisons tests. Comparative data with a P value of≤0.05 were considered as statistically significant.

• Results

Treatment of established 48 h MRSA and MSSA biofilms using the same isolates revealed that treatment with the RO+ product for 24 h reduced biofilm viability in both instances (Figure 19 a&b). A log-fold reduction in viability was observed when treating MRSA biofilms with 53 and 71 g/L (p=0.0079), and a 2-log reduction when treating with 142 g/L (p=0.0159). In comparison, only a log-fold reduction was observed when treating MSSA biofilms with 36-142 g/L (p<0.05).

Treatment with RO- had no effect on viability of biofilms formed by either of the strains tested (Figure 19 a&b). Treatment of MRSA biofilms with either 36-71 g/L RO+ or RO- for 24 h also resulted in a significant increase in overall biofilm biomass (p≤0.05), whilst all concentrations tested (7-142 g/L) significantly increased the biomass of MSSA biofilms (p<0.05).

Confocal microscopy was used to validate the biofilm viability and biomass data. Both 48h established MRSA and MSSA biofilms demonstrated a reduction in viability and an increase in maximum biofilm thickness when treating with the 71 g/L RO- and RO+ products (Figures 20 a&b). The MRSA biofilm maximum height increased from 25.3 m to 36.3 m (RO-) and 45.3 μηι (RO+), whilst the MSSA biofilm increased from 19.66 Mm to 45.3 μηι (RO-) and 35.99 Mm (RO+). Finally, treatment of 48 h established biofilms formed by several clinical MRSA (n=7) and MSSA (n=5) isolates revealed that the viability of each biofilm was reduced with an average log-fold reduction observed within each group (Figure 21 a&b). Example 15 - Activity of Surgihoney™ against in vitro non-typeable Haemophilus influenza biofilms

• Materials and methods

Bacterial strains and growth conditions

Bacterial strains used in this study were isolated from nasopharyngeal swabs of children aged 4 years. NTHi was sub-cultured from frozen stocks onto Colombia agarwith chocolated horse blood (Oxoid, UK) and incubated for 18 h at 37°C and 5% CO ₂ , following which colonies were resuspended in Brain Heart Infusion (BHI) broth supplemented with 10 μg/ml Hemin and 2 μg/ml NAD, and grown to mid-exponential phase for experiments. Pseudomonas aeruginosa PA01 and a clinical methicillin resistant Staphylococcus aureus isolate were subcultured onto Colombia blood agar plates (Oxoid, UK) and grown in non-supplemented BHI. 65

Planktonic assays

Flat-bottomed 96-well plates (Fisher Scientific, UK) were inoculated with ~1 .0 x10 ⁶ planktonic bacteria per well (grown in supplemented BHI). SurgihoneyRO™ and the non-engineered base honey(Acacia)were both prepared in supplemented BHI and added to wells at final

concentrations of 6 g/L to 319 g/L. Supplemented BHI alone was added in place of treatments for untreated controls. Cultures were incubated at 37°C and 5% CO2 for 18 hours then turbidity measured by absorbance (OD595) using an EZRead 400

spectrophotometer (Biochrom; n=6). 75

Biofilm assays

Mid-exponential planktonic cultures were used to inoculate individual wells of untreated 6-well polystyrene plates (-1.0 x 10 ⁸ planktonic bacteria perwell; Corning Incorporated, USA). Cultures were incubated at 37°C and 5% CO ₂ for 48h, replacing spent media with fresh supplemented BHI (NTHi) at 24 h. Prior to treatment spent media was removed and biofilms washed twice with Hanks' balanced salt solution (HBSS; Gibco, UK). Biofilms were treated with SurgihoneyRO™ or Acacia (both prepared in HBSS) at final concentrations of 7 to 213 g/L. To assess the effect of pH biofilms were treated with HBSS adjusted to pH6.3 (the pH of 71 g/L SurgihoneyRO™ in HBSS). For adjuvant assays biofilms were treated with 300 g/mL amoxicillin and 60 μg/mL clavulanic acid (Co-amoxiclav). HBSS alone was added in place of treatments for untreated controls. Biofilms were incubated at 37°C and 5% CO2 for 2 h, following which the treatments were removed and biofilms washed twice with HBSS to remove unattached cells. Biofilms were resuspended in 1 ml HBSS by cell scraping and briefly vortexing the serially diluted onto Colombia agarwith chocolated horse blood (NTHi). Plates were incubated at 37oC and 5% C02 and colony forming units (c.f.u.) enumerated (n=4).

Confocal Microscopy

Mid-exponential planktonic cultures were used to inoculate 35 mm untreated glass-bottom CellView cell culture dishes (~1 .0 x 10 ⁸ planktonic bacteria perwell; Greiner Bio One, UK). Cultures were incubated at 37°C and 5% CO2 for 48 h, replacing spent media with fresh supplemented BHI at 24 h. Media was removed, biofilms washed twice with HBSS, then treated with 71 g/L SurgihoneyRO, 300/60 μg. ml co-amoxiclav or HBSS alone (untreated control) for 2 h at 37°C and 5% CO2. Treatments were removed and biofilms washed twice with HBSS before staining with a LIVE/DEAD Baclight Bacterial Viability Kit (Life Technologies, UK) as per manufacturer's instructions. Biofilms were examined using an inverted Leica SP8 confocal microscope using a 63x oil immersion lens with sequential scanning of 2 μηπ sections (Leica Microsystems, UK).

Hydrogen peroxide measurements

SurgihoneyRO™ and Acacia were prepared at a range of concentrations between 7 to 213 g/L in HBSS and incubated at 37°C and 5% CO2 for 2 h. Hydrogen peroxide production was then measured using a Fluorimetric Hydrogen Peroxide Assay Kit (Sigma-Aldrich, UK) as per manufacturer's instructions using an untreated flat-bottomed black 96-well plate (Greiner BioOne, UK). In brief, a standard curve was generated using known concentrations of H2O2, to which SurgihoneyRO™ and Acacia samples were compared, including a negative HBSS control. A master mix comprised of red peroxidase, horseradish peroxidase and assay buffer was added to each standard and sample well. Samples were incubated at room temperature and protected from light for 30 minutes afterwhich fluorescence was measured (excitation: 540 nm / emission: 590 nm; n=4).

Statistical analyses

Statistical analyses of in vitro planktonic and biofilm data were performed using one-way analysis of variance (ANOVA) and Kruskal-Wallis multiple comparisons tests. Comparative data with a P value of≤0.05 were considered as statistically significant.

• Results

SurgihoneyRO™ treatment reduces in vitro NTHi biofilm viability through increased H ₂ O ₂ generation

Treatment of established 48 h in vitro biofilms with 71 and 142 g/L SurgihoneyRO™ for 2 hours resulted in a 4-log and 3-log reduction in viability respectively (P≤0.05), whilst treatment with 213 g/L reduced viability 5-log (P<0.01 ; Fig. 22a). In comparison, treatment with the equivalent concentrations of Acacia had no effect on biofilm viability (P=0.75; Fig. 1 b). A dose- dependent increase in H2O2 levels in the surrounding media was also observed when treating with both Acacia and SurgihoneyRO™ (Fig. 22b). SurgihoneyRO™, however, produced significantly higher levels of H2O2 at all concentrations tested, ranging from 10.7 - 71.2 μΜ in comparison with 0.24 - 6.5 μΜ generated when treating with the equivalent concentrations of Acacia. Furthermore, these data indicate that the minimum concentration of H2O2 effective in reducing NTHi biofilm viability, as evidenced with 71 g/l SurgihoneyRO™, is approximately 14.2 to 25.7 μΜ (Fig. 22b). To account for a pH- mediated reduction in viability NTHi biofilms were also treated with HBSS adjusted to pH6.3, revealing no effect on biofilm viability (Fig. 22c).

SurgihoneyRO™ is more effective than co-amoxiclav in the treatment of NTHi biofilms

Following confirmation that 71 g/L SurgihoneyRO™ was effective in reducing NTHi biofilm viability the activity was compared to the conventional antibiotic co-amoxiclav, and also whether it could improve antibiotic efficacy when used as an adjuvant. Treatment of established 48 h in vitro biofilms with 71 g/L SurgihoneyRO™ for 2 hours resulted in a 5-log reduction in viability (P=0.029) whereas treatment with 300/60 g.ml co-amoxiclav had no effect on viability (P=0.343; Fig. 2a). Combined treatment, however, did not improve co-amoxiclav efficacy (Fig. 23a). Confocal laser scanning microscopy confirmed the reduction in biofilm viability of 48h NTHi biofilms following treatment with 71 g/L SurgihoneyRO™ for 2 hours, and also the ineffectiveness of co-amoxiclav (Fig.2b-d). The confocal micrographs also demonstrated that SurgihoneyRO™ treatment had no obvious effect on overall biofilm biomass or ultrastructure, with all biofilms being -70 Mm in maximum height (Fig. 23b-d).