WO2004064881A1

WO2004064881A1 - Skin treatment formulations

Info

Publication number: WO2004064881A1
Application number: PCT/GB2004/000179
Authority: WO
Inventors: Paul James Davis; Andrew John Austin
Original assignee: Insense Limited
Priority date: 2003-01-24
Filing date: 2004-01-21
Publication date: 2004-08-05

Abstract

A skin treatment formulation, e.g. in the form of an ointment or gel or a skin dressing, comprises a photocatalytic agent, e.g. flavine mononucleotide, and an electron donor substance, e.g. ethylene diamine tetra acetic acid, conveniently in the form of an agar gel (10). On exposure to light in the presence of oxygen (e.g. from the ambient atmosphere) the photocatalytic agent acts to cause conversion of oxygen to hydrogen peroxide. If this reaction occurs on or in the vicinity of skin, the hydrogen peroxide so produced can have a localised antibacterial effect. By controlling exposure to light, so hydrogen peroxide can be generated in situ in the vicinity of skin in a way that has not hitherto been possible.

Description

SKIN TREATMENT FORMULATIONS

Field of the Invention

This invention relates to skin treatment formulations, particularly skin dressings, for application to a part of a human or animal body for treatment of skin, and relates particularly (but not exclusively) to wound dressings for treatment of compromised skin, particularly skin lesions, i.e. any interruption in the surface of the skin, whether caused by injury or disease, including ulcers, burns, cuts, punctures, lacerations, blunt traumas etc. The invention also relates to a method of in situ generation of hydrogen peroxide.

Background to the Invention

There is a real need for improved active antimicrobial wound dressings and skin treatments (including those for burns), especially in the face of the many antibiotic resistant microbial strains that are so widespread today. Microbial contamination of skin, wounds and burns etc is an ever present problem. Once the integrity of the skin barrier is broken or compromised, the threat of microbial infection is greatly enhanced. A primary (but not the only) purpose of wound dressings is to restore the lost barrier integrity, thus preventing microbial colonisation and infection. This function can be aided by antimicrobial substances added to the dressing, or placed in contact with the wound as mobile gels, creams or fluids. If the wound is already contaminated before the dressing is applied, there is a greater need for antimicrobial substances to sterilise the wound, or at least reduce the microbial load, thus helping the natural immune defences and promoting the healing process. At the same time, it is also important to ensure that appropriate concentrations of oxygen are maintained in the wound, whilst the healing process is under way. Many of these considerations also apply to burns, skin ulcers and sites of infection, including acne lesions and boils etc.

Although the provision of efficient antimicrobial activity in wound treatment is highly desirable, there is still a major unmet need for reliable, controllable, sustained delivery of effective antiseptic substances through the various phases of wound healing. The routine use of antibiotics is not acceptable because of the risk of inducing even more antibiotic resistance within the microbial pathogen population. Increasingly, antibiotic treatment is not appropriate because wounds, as well as intact skin, are often already colonised by resistant bacteria, such as the infamous MRS A strain of Staphylococcus aureus. Other antiseptic substances, such as silver or Triclosan are not as potent as ideally required and suffer from other problems, including skin discolouration (silver) and emerging resistance (Triclosan). Iodine is an efficient microbiocide but at high concentrations it is toxic to the body's own cells (epithelial cells, keratinocytes and white blood cells etc) which are needed to heal and reconstruct the wound. It is difficult to provide a sustained flux of these substances at an optimal level, and even more difficult to control the flux rate over an extended time to match the changing individual needs for antisepsis as a wound or other lesion progresses. The only way to control flux rates from a conventionally dosed wound dressing product is to change the dressing, replacing one with another having a different starting dose. In fact, dressings with a pre-dosed supply of anti-microbial substance must normally be changed frequently, yet this is expensive of nursing time and can interfere with the healing process itself.

Hydrogen peroxide (H₂O₂) is a known antimicrobial substance with many advantages. It is produced naturally in the body by white blood cells as part of the immune defence activities in response to infection. There are no known microbial evasion mechanisms by which microbes can escape its effects and it has a short lifetime, very rapidly breaking down to water and oxygen in the tissues. It therefore does not accumulate to dangerous levels. When it is to be applied topically (e.g. to treat acne), its effectiveness is enhanced by the fact that it readily penetrates the skin surface to reach underlying sites of infection. The oxygen liberated when hydrogen peroxide is decomposed in the wound can itself be helpful in the process of healing, by inhibiting anaerobic bacteria and supporting cells that are engaged in the healing process, (e.g. white blood cells, keratinocytes and epithelial cells etc).

As hydrogen peroxide is so beneficial, it has been used for many years as an anti-microbial substance for cleansing wounds of all kinds and as a biologically compatible general antiseptic. In particular, hydrogen peroxide-containing ointments are used, e.g. , for treatment of leg ulcers, pressure sores, minor wounds and infection. There are, however, problems associated with the use of hydrogen peroxide. Hydrogen peroxide solution is very unstable and is readily oxidised to water and oxygen; further, hydrogen peroxide at high concentration can be damaging to normal skin. It is very difficult or even impossible to use hydrogen peroxide as part of a pre-dosed wound dressing: it's instability would make for a product with an impossibly short shelf-life, and dosing at the point of application would still not provide a sustained delivery over a usefully prolonged period. When it is used in wound treatment (as described in the British Pharmacopoeia, for example) very high concentrations (typically 3%) are needed to achieve a powerful antimicrobial effect over a very short time interval. Even this type of short burst can be effective, because of the great effectiveness of hydrogen peroxide, but there is the further disadvantage that such high concentrations can be relatively damaging to host cells and can impede the healing process. For this reason, use of hydrogen peroxide tends to be restricted to initial clean-up and sterilisation of wounds. Even so, it is a natural defence substance, produced by the body's own cells (at lower concentrations) and it is increasingly recognised as an intercellular and intracellular messenger molecule, involved in cell to cell molecular signalling and regulation. Undoubtedly, hydrogen peroxide is potentially a very beneficial molecule, if it can be used at the right concentrations and in the appropriate time course.

US 4576817 discloses an absorbent pad or bandage comprising serum-activated oxidoreductase enzyme, e.g. glucose oxidase, for producing hydrogen peroxide on contact with serum. On contact with suitable body fluid, including serum and oxidisable substrate, the enzyme catalyses reaction of the substrate (e.g. β-D-glucose for glucose oxidase) with water and oxygen in the serum to produce hydrogen peroxide (and gluconic acid when using glucose oxidase). A pad comprising glucose oxidase is shown in Example 1 to have bacteriostatic properties.

In US 4576817, the pad or bandage may also include a peroxidatic peroxidase enzyme, e.g. lactoperoxidase, for interacting with hydrogen peroxide thus produced and an oxygen- accepting anion in the serum (e.g. iodide ions) to produce an oxidised anionic bacterial inhibitor (plus e.g. hypoiodite from iodide ions). Fibres comprising glucose oxidase and lactoperoxidase are shown in Example 2 to have bacteriostatic properties.

The present invention concerns an alternative approach to production of hydrogen peroxide, and possibly also substances produced by reaction with hydrogen peroxide.

Summary of the Invention

In one aspect the present invention provides a skin treatment formulation comprising a photocatalytic agent and an electron donor substance.

When exposed to light in the presence of oxygen, the photocatalytic agent acts to cause conversion of oxygen to hydrogen peroxide, with corresponding oxidation of the electron donor substance. If this reaction occurs on or in the vicinity of skin, the hydrogen peroxide so produced can have a localised antibacterial effect. By controlling exposure to light, hydrogen peroxide can be generated in situ in the vicinity of skin in a way that has not hitherto been possible. In a typical wound there is a plentiful supply of catalase from the body's own cells. This catalase instantly converts hydrogen peroxide to oxygen, so use of a formulation of the invention has the effect of supplying oxygen to the wound.

The arrangement responds rapidly to the presence or absence of light, rapidly starting or stopping production of hydrogen peroxide. The rate of production of hydrogen peroxide is directly proportional to the intensity of light, within limits, permitting ready control of hydrogen peroxide levels. The invention can thus provide a readily controlled source of active antimicrobial agent for skin treatment purposes.

The skin treatment formulation may be in the form of an ointment or gel, e.g. having an otherwise conventional composition suited to application to skin. Preferably, however, the formulation is in the form of a skin dressing.

In a preferred aspect the invention thus provides a skin dressing comprising a photocatalytic agent and an electron donor substance.

The formulation, e.g. dressing, is used by being located on the skin of a human or animal, e.g. over a wound or on a region of skin to be treated for cosmetic or therapeutic purpose, e.g. for treatment of acne or other skin conditions. The formulation should be kept away from light until required for use, e.g. by being sealed in a suitable light-tight enclosure or packaging. In use, the formulation requires access to oxygen, e.g. in ambient air. The formulation may be left exposed to the atmosphere in use or, more preferably, may include or be used in conjunction with an oxygen-permeable covering that may adhere to the skin of a human or animal subject, e.g. in known manner. At least part of the covering is of oxygen-permeable material e.g. polyurethane or polytetrafluoroethylene (PTFE) constructed sufficiently thinly to enable oxygen from ambient air to pass easily through the covering. The oxygen-permeable material may be in the form of a "window" set in an otherwise relatively oxygen-impermeable covering, e.g. of possibly thicker more dense material.

Exposure of the formulation, e.g. dressing, to light may be controlled and regulated in a number of different ways.

In a simple embodiment, the formulation includes or is used in conjunction with a covering (possibly constituted by the oxygen-permeable covering discussed above) at least part of which is transparent to light, e.g. with the covering including a light-transparent region or window, so light from the surroundings can pass through the covering to cause the hydrogen peroxide-generating reaction. Means are preferably provided for regulating passage of light through the covering. For instance, a light-opaque cover or shutter means (operable manually or by automated means) may be provided for occluding at least the light transparent part of covering. As a further possibility, the light transmission properties of the covering may be variable in known manner (e.g. electrically) so the covering is variably transparent in a controllable manner (again being operable manually or automatically, e.g. by electronic control means). Such embodiments may rely on ambient light or light from an associated light source to drive the hydrogen peroxide-generating reaction in a controllable manner.

As a further possibility the formulation, e.g. dressing, may include or be used in conjunction with an internal, dedicated light source such as one or more light emitting diodes (LEDs) or an electroluminescent polymer film, with associated power supply, e.g. batteries. In this case the photocatalytic agent and electron donor are shielded in use from light from the surroundings, e.g. by use of a light-opaque (but oxygen-permeable) covering over these components and the light source, so the only exposure to light is from the light source which can readily be precisely regulated (again being operable manually or by automated means). Such embodiments desirably include control means, e.g. an electronic controller, for controlling the light source and hence controlling the timing and rate of generation of hydrogen peroxide to suit requirements.

In embodiments using a power supply e.g. battery, and control means, e.g. electronic controller, these components are conveniently housed in a separate module, linked by wires, infra red connection etc to the light source etc. The module conveniently includes attachment means, such as a strap or self-adhesive strip, to facilitate attachment to a person or animal being treated in the vicinity of the area of skin to be treated.

The invention can be used to generate hydrogen peroxide as appropriate for treatment, either as a continuous flux at an appropriate rate, at varying rates, as intermittent pulses, or at a rate regulated in a feedback loop, responding to signs or symptoms detectable in or associated with the region being treated, e.g. local heat or redness. The invention can thus provide a controllable means for generating hydrogen peroxide in situ at a site of skin to be treated (e.g. at the surface of a wound, burn etc), possibly delivering a sustained flux of changing concentrations, matched to the changing needs and conditions of a particular region being treated.

Hydrogen peroxide levels, in dressings and formulations of the invention, in use, are suitably in the micromolar to low millimolar range.

The photocatalytic agent conveniently comprises flavine mononucleotide (FMN). Experiments have been successfully performed with formulations having FMN levels in the range 30 micromolar to 100 millimolar. This range is not exhaustive or limiting.

The electron donor substance conveniently comprises ethylene diamine tetra acetic acid (EDTA). Experiments have been successfully performed with formulations having EDTA levels in the range 25 millimolar to saturation (estimated at about 0.8M in warm solution and about 0.5M at room temperature). This range is not exhaustive or limiting.

As a further possibility, the photocalytic agent may comprise zinc oxide particles, e.g. in the form of quantum sized particles with an organic electron donor e.g. ascorbic acid, as described by Bahnemann et al J. Phys Chem 1987 91, 3789-3798.

A layer of barrier material is preferably included, to be located in use between the skin and photocatalytic agent and electron donor substance, with the layer for example forming part of the dressing of the invention, to prevent undesired migration of materials. Such a barrier has the effect, inter alia, of preventing possibly interfering substances such as catalase, iron ions etc. being taken up into the formulation from a wound site. Suitable barrier material includes e.g. a semi-permeable sheet or membrane e.g. of cellulose acetate or cellulose ester, such as one that is permeable only to molecules of molecular weight less than, say, 350 Da (possibly having a nominal molecular weight cut-off of 500 Da but with an actual limit of less than 350 Da). Suitable membranes include cellulose acetate membrane code Z368024 supplied by Sigma and Spectrum SpectraPor cellulose ester membrane code 131054 supplied by NBS Biologicals. (Spectrum and SpectraPor are Trade Marks).

The photocatalytic agent and electron donor substance are preferably present in the form of one or more gels, suitably hydrated gels, preferably together in a single hydrated gel.

The or each hydrated gel conveniently comprises a polyacrylamide gel, an agar gel or an alginate gel, e.g. formed from alginic acid cross-linked in known manner, e.g. by use of calcium chloride. Most cross-linked gels of this type form a biopolymer matrix that retains very high molecular weight reagents or particles such as ZnO particles within the gel, preventing escape of such materials from the point of use. For lower molecular weight ingredients such as FMN, EDTA and ascorbic acid, a barrier material such as described above is required to prevent escape. The gel may be in the form of beadlets, beads, slabs or extruded threads etc.

For a dressing, the gel may be cast around a mechanical reinforcing structure, such as a sheet of cotton gauze or an inert flexible mesh, e.g. to provide a structurally reinforced hydrogel layer or slab.

The photocatalytic agent and electron donor substances are conveniently contained within an enclosure of material that is impermeable to the photocatalytic agent and the electron donor substance but that is permeable to oxygen, water and hydrogen peroxide and at least part of which is transparent to light. Thus, a dressing in accordance with the invention may further comprise an enclosure such as a sachet or bag, preferably made from barrier material e.g. membrane-like material that is permeable only to molecules of MW less than about 350 Da and that is transparent to light. The contents of the enclosure can be in the form of a simple aqueous solution (optionally with buffer salts etc.), or as a viscous solution of gel formed by the presence of suitable gelling materials (e.g. polysaccharides or synthetic polymers). In another embodiment, the enclosure can be filled with aqueous liquid, with or without thickening/gelling polymers, having an appropriate pH and ionic content, but in which the photocatalytic ingredients are supplied within particles or (preferably) microcapsules (e.g. liposomes or polymerised liposomes) suspended or dispersed in the liquid. Such particles or microcapsules can also be used with slabs or patches formed from gels, of the general type discussed above and below. The enclosure is conveniently made of barrier material as discussed above.

The gel may be in the form of a shear-thinning gel, e.g. of suitable gums such as xanthan gum (e.g. available under the Trade Mark Keltrol), in this case preferably without a mechanical reinforcing structure. Such gums are liquid when subjected to shear stress (e.g. when being poured or squeezed through a nozzle) but set when static. Thus the gel may be in the form of a pourable component, facilitating production of a gel in the dressing. Such a shear-thinning gel may also be used in combination with a preformed, mechanically reinforced gel.

The reagents may be present in a gel in a number of possible forms, including in solution as free molecules. To improve efficiency of retention of the reagents in the gel, the reagents may be chemically conjugated to each other, chemically conjugated to other molecules (e.g. polyethylene imine), or incorporated in a solid support such as beads.

Gels of different types e.g. cross-linked alginate and shear-thinning, may be used together with a single formulation, e.g. dressing.

Formulations, e.g. dressings, in accordance with the invention (or components thereof) are suitably supplied in sterile, sealed, water-impervious, light-opaque packages, e.g. foil pouches.

Dressings in accordance with the invention can be manufactured in a range of different sizes and shapes for treatment of areas of skin e.g. wounds of different sizes and shapes. Appropriate amounts of reagents for a particular dressing can be readily determined by experiment.

The formulation, e.g. dressing, can be used to generate hydrogen peroxide in situ in the vicinity of the skin of a person or animal being treated, adjacent a site for treatment, with the hydrogen peroxide exerting known antimicrobial or oxygen delivering action as discussed above.

Alternatively, the hydrogen peroxide generated in this way may be used in a two stage arrangement (e.g. as disclosed in US 4576817), in conjunction with a peroxidatic peroxidase enzyme, e.g. lactoperoxidase, to produce reactive oxygen intermediates that have antimicrobial properties and that can therefore assist in skin treatment and promoting wound healing.

Peroxidase enzymes useful in the invention include lactoperoxidase, horseradish peroxidase, chloroperoxidase and myeloperoxidase, with lactoperoxidase currently being favoured. A mixture of peroxidase enzymes may be used.

The active species produced by the action of peroxidase are difficult to define, and will to some extent depend the particular peroxidase in question. For example, horse radish peroxidase works very differently to lactoperoxidase. The detailed chemistry is complicated by the fact that the products are so reactive that they rapidly give rise to other, associated products that are also very reactive but with a high localised effect, having very short lifetimes and very narrow range. It is believed that hydroxyl radicals, singlet oxygen and superoxide are produced, just as in the "oxidative burst" reactions identified in neutrophil and macrophage leukocytes of the human body, and in the well-known "Fenton" reaction, based on the catalytic effects of ferric ions. These reactive species will only be effective within the dressing, at the location in which they are generated.

The peroxidase enzyme, where used, is conveniently present in one or more hydrated gels, e.g. as discussed above. The antimicrobial efficiency of the system can be further enhanced by the inclusion of iodide ions, which can be oxidised to iodine by the action of hydrogen peroxide, especially in the presence of the enzyme lactoperoxidase. Thus, the formulation desirably includes a supply of iodide ions, e.g. potassium iodide or sodium iodide. As iodine is also relatively toxic to host cells in the wound (e.g. epithelial cells, keratinocytes, white blood cells) it may not be advantageous to generate iodine continuously at a high concentration throughout the time that the formulation is in use in contact with the skin. Thus, in a preferred embodiment, the supply of iodide ions, e.g. iodide salt, is provided in a relatively quick-release form in a membrane or gauze or other suitable layer, interposed between the skin surface and the hydrogen peroxide-producing part of the formulation. In this way, the hydrogen peroxide produced initially, in a first phase of activity, is substantially consumed in an iodine-generating reaction, exposing the skin (e.g. wound) to a surge of iodine, the duration of which can be controlled by the amount, release-rate and position of the iodide supply. Such an iodine surge can be very useful in quickly ridding a wound of a microbial burden, and its relatively short duration allows healing by minimising damage to growing cells and their repairing activity. Once the iodide has been consumed, the system automatically reverts, in a subsequent phase of activity, to the production of hydrogen peroxide which maintains sterility and kills invading bacteria near the skin, e.g. wound surface - In other embodiments, however, it may be desired for the source of iodide ions to be such as to provide, in use, a sustained flux of iodine (and/or hypoiodous acid) for release into a wound, in addition (and in proportion) to hydrogen peroxide.

The invention also includes within its scope a method of in situ generation of hydrogen peroxide for skin treatment, comprising locating in the vicinity of skin to be treated a photocatalytic agent and an electron donor substance; and exposing the reagents to light in the presence of oxygen, resulting in production of hydrogen peroxide.

The invention will be further described, by way of illustration, with reference to the accompanying drawings, in which: Figure 1 illustrates schematically one embodiment of a skin dressing in accordance with the invention;

Figure 2 illustrates schematically an experimental arrangement for demonstrating functioning of a skin dressing in accordance with the invention;

Figure 3 is a chronoamperotometry graph of current (in nA) versus time (in seconds) obtained using apparatus as illustrated in Figure 2;

Figure 4 is a further graph of current (in nA) versus time (in seconds) obtained using apparatus as illustrated in Figure 2, showing light-dependent hydrogen peroxide production;

Figure 5 is a schematic illustration of the apparatus of Figure 2 in a light-tight enclosure;

Figure 6 is a graph similar to Figure 4 obtained using the apparatus illustrated in Figure 5; and

Figure 7 is a graph of current (in μA) versus time (in minutes).

Detailed Description of the Drawings

Referring to the drawings, the skin dressing 10 illustrated schematically in Figure 1 is of layered construction and comprises a gel slab 12 containing in solution flavine mononucleotide (FMN) and ethylene diamine tetra acetic acid (EDTA); an LED array 14 on one side of slab 12 with associated miniaturised electronic controller and battery 16; an oxygen-permeable polytetrafluoroethylene (PTFE) cover 18 covering the LED array 14 and gel slab 12; and a sheet 20 of Sigma semi-permeable barrier material on the side of slab 12 remote from the LED array 14. The dressing is shown on an area of skin 22 of a human or animal to be treated, including a wound site 24. The dressing 10 is located over the wound site 24, with barrier sheet 20 in contact with the skin, and with the dressing being held in position by adhesion of peripheral parts of the cover 18 to surrounding skin (not shown in Figure 1). The dressing also includes in gel slab 12 a sensor 26 linked to the controller 16 for feedback control.

In use of the dressing, power is supplied from the battery to the LED array 14, resulting in illumination of the slab 12. This rapidly initiates photocatalytic production of hydrogen peroxide in the gel, by conversion of oxygen (from the ambient atmosphere), with FMN being converted to FMNH₂ and EDTA functioning as a sacrificial electron donor. The rate of production is proportional to the intensity of illumination, within limits. When the LED array is switched off, hydrogen peroxide production is rapidly terminated.

The dressing may be operated manually, with a user turning the LED array on or off as required. Alternatively, the controller 16 can be programmed to regulate automatically operation of the LED in accordance with one or more programs. For example, a program may produce a preset pattern of peroxide flux rates over time. Alternatively, a number of other programs may each produce a different, usually simpler, pattern of peroxide production rates, to be selected as appropriate by a user according to an ongoing assessment of treatment required e.g. based on wound type and state. As a further possibility, the controller 16 may respond to feedback signals from the sensor, varying hydrogen peroxide production rates and timing appropriately.

Typically, the dressing will be used to generate hydrogen peroxide to the extent that concentrations within the dressing will be in the micromolar to low millimolar range.

A typical example of a gel slab 12 was produced as follows:

Gels containing the active components were prepared in the dark, or under minimal illumination, by the following procedure: 1 % agar (Sigma product No. A9539) was dissolved in boiling deionised water. The agar solution was cooled to about 45 °C (just above the gelling temperature) and had dissolved therein 25mM FMN (Riboflavin 5'- monophosphate (sodium salt dihydrate), supplied by Fluka, code 83810) and 25mM EDTA (EDTA Na₂, supplied by Sigma, code E1644). The resulting warm, ungelled solution was poured over a cotton gauze (which acted as a support for the gel) and allowed to cool and set. Pads of approximately 1 cm x 1 cm were cut, a size sufficient to allow them to cover the end of the electrode. Pads were kept in the dark at all times, to prevent the initiation of hydrogen peroxide production.

Figure 2 illustrates an experimental arrangement used to demonstrate photocatalytic production of hydrogen peroxide, using a FMN and EDTA-containing gel slab 12 and semi-permeable barrier sheet 20 located on a proprietary screen-printed carbon electrode 30, the terminals of which are linked to a Perkin Elmer DLK-60 electrochemical analyser 32 constituting a peroxide sensing system. The semi-permeable barrier sheet 20 is either cellulose acetate membrane, 500 Da nominal molecular weight cut off 350 Da actual cut off, supplied by Sigma (code Z368024) or Spectrum SpectraPor cellulose ester membrane, 500 Da nominal molecular weight cut off, 350 Da actual cut off, supplied by NBS Biologicals (code 131054) (Spectrum and SpectraPor are Trade Marks).

Experiment 1

In an experiment using the arrangement of Figure 2, 30μl of contact solution (0.1M phosphate buffer pH 6.0 + 0.1M KC1) was applied to the upper surface of electrode 30. Onto this was placed the low molecular weight cut off membrane 20 (nominally 500 Da, actually 350 Da) (approx. 1.2 x 1.2 cm), ensuring no air was trapped beneath. A further 20 μl of contact solution was applied to the upper surface of the membrane 20, and the FMN + EDTA agar gel slab 12 (approx 1 x 1cm), with a formulation prepared as described above, was placed onto the membrane, thus simulating application to a wound site, with the electrode being the equivalent to the wound. The gel 12 was slightly smaller than the separating membrane 20, to prevent liquid contact via the edge. This assembly was carried out as quickly as possible to keep light exposure to a minimum. The whole reaction area was then enclosed in a blacked out dark chamber, to provide a dark environment and to maintain humidity.

A potential of 0.95N was applied to the electrode terminals, and a steady baseline reading from analyser 32 was established after 2-3 minutes. On exposure to light, by removal of the chamber, the FMΝ/EDTA gel acted to produce hydrogen peroxide through the reduction of molecular oxygen (from the ambient atmosphere) via the photocatalytic action of FMΝ, using EDTA as the sacrificial electron donor, as represented in Figure 2. In the arrangement of Figure 2, any hydrogen peroxide that was produced was oxidised at the electrode. The resulting current was accurately measured through electronic analyser circuits in analyser 32, providing a measure of hydrogen peroxide production. The system can detect hydrogen peroxide at concentrations down to 10 μM.

Results

Figure 3 is a chronoamperotometry graph of results obtained using the set up of Figure 2 (with special software in place of that normally used with the DLK-60 analyser), showing results for different concentrations of hydrogen peroxide and demonstrating the ability to detect concentrations as low as 10 μM.

Experiment 2

In a further similar experiment with the arrangement of Figure 2, the arrangement was exposed to different conditions of light and hydrogen peroxide production monitored. The results are shown graphically in Figure 4.

In this experiment, the dark chamber was initially rapidly removed and replaced with a transparent chamber (to maintain humidity) with a 20 Watt halogen lamp placed over the arrangement to provide a light source. When the lamp was switched on, hydrogen peroxide was produced, as monitored by analyser 32. The lamp was then switched off, and the dark chamber replaced. The production of hydrogen peroxide stopped, as indicated by the level trace in Figure 4. The dark chamber was again swapped for a transparent chamber and the arrangement left exposed to ambient light, whereupon slow production of hydrogen peroxide began. Finally the halogen lamp was again switched on, resulting in an increase in the rate of hydrogen peroxide, as indicated by the increased slope of the graph of Figure 4.

Results

Figure 4 shows the rapid change in peroxide production in response to changing light conditions. It should be noted that the software analysis program used produces an inverse graph.

Experiment 3

A further experiment was carried out using the apparatus of Figure 2, located in a slightly modified dark chamber, as illustrated in Figure 5. In this case, dark chamber 40 has a small hole 42 in the upper wall thereof, located above the gel 12. An LED 44 powered by a 6V battery (not shown) is located above the chamber with the bulb 46 of the LED 44 passing through the hole 42, for illuminating the gel 12. With this arrangement, the only light to which gel 12 can be exposed is light coming from the bulb 46 of the LED 44 when switched on.

The LED was switched on and off twice at time intervals, and the production of hydrogen peroxide over the course of the experiment monitored and displayed graphically as in Figure 4, with results in this case being given in Figure 6.

Results

As seen in Figure 6, a vigorous hydrogen peroxide production rate was detected after the LED was turned on, with hydrogen peroxide production ceasing when the LED was turned off. When illumination resumed, hydrogen peroxide production restarted. This demonstrates the rapid response of the system to changing light levels.

Experiment 4 - antibacterial activity

A 7cm length of pre-wetted dialysis tube (Spectrum Labs, code 131054, of 500 Da nominal molecular weight cut off, with a flat diameter of 16mm) was taken, and clamped shut at one end. Into the bag were placed 0.75ml 250mM solution of EDTA (ethylene diamine tetra acetic acid, supplied by Sigma, code E1644) and 0.25ml lOOmM FMN (flavin mononucleotide, supplied by Fluka, code 83810). Both reagents were dissolved in distilled/deionised water. The open end of the tube was then clamped shut.

Test plates consisted of nutrient agar No. 2 (supplied by Oxoid), cast to a depth of approximately 5mm. The plates were left to stand for a minimum of 18 hours. A culture of Staphylococcus aureus was prepared to an optical density of between 6-7. 200 microlitres of the suspension was applied to each plate, and spread in several directions to ensure an even spreading was produced. The plates were allowed to dry for at least 15mins before use.

Illumination apparatus was constructed by suspending 2 ultrabright blue LEDs (supplied by Maplins Electronics, code L53MBC, light output of 90mcd each, with a peak wavelength of 430nm), 1 cm above the reactant bag. The LEDs were wired in parallel, and powered by 3x Duracell AA-sized batteries.

One plate was set up as above, and the sealed dialysis tube containing the reactants laid onto the plate, in one movement, to prevent scraping the bacteria from the surface. The plate was wrapped in aluminium foil to prevent light ingress, and placed in an incubator maintained at 37°C. A second plate was set up as previously described, but not wrapped in foil. The plate was placed into the 37 °C incubator, and the illuminated LEDs placed over the top of the bag, at a height of 1 cm. The LEDs were positioned so that they were shining directly onto the bag. The plates were left for 24 hours before being examined. Results

Examination of the plates showed that the plate with no illumination had bacterial growth up to the very edge of the dialysis bag with no clear areas (no bacterial growth) or reduced growth zones visible. In contrast, the plate that had the LED illumination showed visible zones of inhibition extending approximately 3mm beyond the edges of the dialysis bag. The result clearly demonstrates that an antibacterial action had been produced under illumination with blue light. In the absence of blue light, there was no antibacterial action.

Experiment 5 - production of hydrogen peroxide from FMN and EDTA using blue light

Onto a horizontally positioned screen-printed carbon electrode (comprising reference, working and counter electrode areas), 30 μm of electrode buffer (lOOmM sodium phosphate pH6 + lOOmM potassium chloride) was placed, covering the reference, working and outer electrodes. A single layer of 500 Da nominal mw cut off dialysis membrane of approx. size 1 cm x 1.5 cm was placed onto the electrode buffer, ensuring that no air bubbles were trapped. Onto the membrane, 80 μl of reaction solution was placed (125mM FMN + 37mM EDTA). The apparatus was set up in a dark box to minimise external light activation. 2 blue ultrabright LEDs were suspended directly above the reactants, at a height of 1 cm. The LEDs were not powered. The electrode was connected to a Whistonbrook Technologies "Ezescan" (Ezescan is a Trade Mark) electrochemical analyser and control unit, and a potential of 1150 mN was applied. The baseline was allowed to stabilise. The reaction was initiated by powering up the LEDs, with 3 Duracell AA batteries. The FMN/EDTA was illuminated for 30 minutes. Data was recorded via the "Ezescan" system by the procedures recommended by the manufacturer..

Results The resultant trace can be seen in Figure 7. This shows that a steady baseline was obtained, with no light. When the LEDs were powered up, at around 3mins, a lag phase was seen, during which the chemistry was activated, H₂O₂ was produced, and then diffused through the membrane. The curve shows that a steady production of H₂O₂ was obtained, until a plateau was reached, which is probably due to the reactive chemicals being depleted. The potential of 1150mN was used, since at this applied voltage only H₂O₂ is oxidised on the electrode. Therefore the rise in current was directly related to the presence of H₂O₂. The results also show that H₂O₂ is clearly diffusible through the 500 Da membrane.

Claims

1. A skin treatment formulation comprising a photocatalytic agent and an electron donor substance.

2. A formulation according to claim 1, in the form of an ointment or gel.

3. A formulation according to claim 1, in the form of a skin dressing.

4. A formulation according to any one of the preceding claims, including or for use with an oxygen-permeable covering.

5. A formulation according to any one of the preceding claims, including or for use in conjunction with a covering at least part of which is transparent to light.

6. A formulation according to claim 5, including means for regulating passage of light through the covering.

7. A formulation according to any one of claims 1 to 4, including or for use with a light source with associated power supply.

8. A formulation according to claim 7, further including a light-opaque but oxygen- permeable covering.

9. A formulation according to claim 7 or 8, including control means for controlling operation of the light source.

10. A formulation according to any one of the preceding claims, wherein the photocatalytic agent comprises flavine mononucleotide.

11. A formulation according to any one of the preceding claims, wherein the electron donor substance comprises ethylene diamine tetra acetic acid.

12. A formulation according to any one of the preceding claims, including or for use with a layer of semi-permeable material for location in use between the skin and photocatalytic agent and electron donor substance.

13. A formulation according to any one of the preceding claims, wherein the photocatalytic agent and electron donor substance are contained within an enclosure of material that is impermeable to the photocatalytic agent and the electron donor substance but that is permeable to oxygen, water and hydrogen peroxide and at least part of which is transparent to light.

14. A formulation according to claim 13, wherein the enclosure comprises semi- permeable material that is permeable only to molecules of less than about 350 Da.

15. A formulation according to any one of the preceding claims, wherein the photocatalytic agent and electron donor substance are present in the form of a hydrated gel.

16. A formulation according to claim 15, wherein the gel comprises an agar gel.

17. A formulation according to any one of claims 1 to 15, wherein the gel is in the form of a shear-thinning gel.

18. A formulation according to any one of the preceding claims, further comprising peroxidase enzyme, preferably in a hydrated gel.

19. A formulation according to any one of the preceding claims, further including a supply of iodide.

20. A formulation according to any one of the preceding claims, packaged in a sterile, sealed, water-impervious, light opaque package.

21. A method of in situ generation of hydrogen peroxide for skin treatment, comprising locating in the vicinity of skin to be treated a photocatalytic agent and an electron donor substance; and exposing the reagents to light in the presence of oxygen, resulting in production of hydrogen peroxide.