AU2013211509B2 - Combination of an oxidant and a photoactivator for the healing of wounds - Google Patents

Abstract

There is provided a wound healing composition which comprises at least one oxidant, at least one photoactivator capable of actuating the oxidant and at least one healing factor chosen from hyaluronic acid, glucosamine and allantoin in association with a pharmaceutically acceptable carrier. In addition, a method of topically treating wounds using at least one oxidant and at least one photoactivator capable of activating the oxidant followed by illumination of said photosensitizer is disclosed.

Description

COMBINATION OF AN OXIDANT AND A PHOTOACTIVATOR FOR THE HEALING

OF WOUNDS

[0000] The present application is a divisional application of Australian Application No. 2009311234 which is incorporated in its entirety herein by reference.

BACKGROUND (a) Field [0001] The subject matter disclosed generally relates to a wound healing composition and method of treatments thereof. (b) Related Prior Art [0002] The process of wound repair is fundamental to the restoration of tissue integrity and function after operative procedures or traumatic injury. Delayed wound healing and dehiscence of operative wounds represent a significant clinical problem.

[0003] Photodynamic therapy using photoactive dyes such as Erythrosine B, Safranin O has been employed to destroy bacteria, as described in WO 05/032459 and WO 05/021094, both to Albrecht et al.. The photoactive dyes are employed to directly destroy bacteria. The compositions described in these patent applications lack oxidants and healing factors, and they are not employed for directly promoting wound healing.

[0004] US Patent No. 6,056,548 to Neuberger et al. describes a method of destroying bacteria in the oral cavity, and promotes bucal hygiene using photoactive dyes. This patent also describes using a bleaching agent, hydrogen peroxide, to photobleach and destroy the photoactive dye used for destroying bacteria. However, the compositions used do not mention healing factors and they are not employed for directly promoting wound healing. WO 08/013962 to Grafe et al. describe the use of a composition which comprises collagen and a photoactivatable molecule, temoporfin (mTHPC) for the in vivo cross linking of collagen to strengthen and stabilize the microstructure of a collagen scaffold. This patent also describes that composition displayed anti-microbial effect, and disinfects the treatment site and curbs microbial growth. However, these compositions do not contain - oxidants, or healing factors and therefore promote wound healing by strengthening the collagen scaffold formed and bacteria destruction.

[0006] Although destroying bacteria present in a wounded site is conducive to wound healing, it does not directly stimulate wound repair. Therefore, it would be highly desirable to be provided with a novel composition for the healing of skin damages and wounds in order to not only destroy bacteria, but also to improve and accelerate the healing process following the establishment of pathologic lesions, trauma or injury.

[0006a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0006b] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY

[0006c] According to a first aspect, the invention provides a method for wound healing comprising the step of: a) topically applying on a patient’s wound a composition comprising at least one oxidant; at least one photoactivator, wherein the at least one photoactivator is eosin Y; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin; and b) exposing the composition of step a) to actinic light for a time sufficient to cause activation of the at least one photoactivator.

[0006d] According to a second aspect, the invention provides use of at least one oxidant; at least one photoactivator; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin, wherein the at least one photoactivator is eosin Y, in the preparation of a medicament for wound healing.

[0006e] According to a third aspect, the invention provides use of at least one oxidant and at least one photoactivator; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin, wherein the at least one photoactivator is eosin Y and is activated by actinic light having a wavelength between 400 nm and 600 nm, in the preparation of a medicament for wound healing.

[0007] In accordance with one embodiment there is disclosed a wound healing composition which comprises at least one oxidant, at least one photoactivator capable of activating the oxidant, and at least one healing factor chosen from hyaluronic acid, glucosamine and allantoin, in association with a pharmaceutically acceptable carrier.

[0008] The oxidants may be chosen from hydrogen peroxide, carbamide peroxide and benzoyl peroxide.

[0009] The wound healing composition may further comprise at least one hydrophilic gelling agent.

[0010] The hydrophilic gelling agent may be chosen from glucose, modified starch, methyl cellulose, carboxymethyl cellulose, propyl cellulose, hydroxypropyl cellulose, carbopol® polymers, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum, pectin, gelatin.

[0011] The photoactivators may be chosen from a xanthene derivative dye, an azo dye, a biological stain, and a carotenoid.

[0012] The xanthene derivative dye may be chosen from a fluorene dye, a fluorone dye, and a rhodole dye.

[0013] The fluorene dye may be chosen from a pyronine dye and a rhodamine dye. JP^IJ The pyronine dye may he chosen from pyronine Y and pyranine S, piOiil The rhodamlne dye may be chosen from rhodamine 8, rhodamine G and rhodamine WT.

[9019] The fluorone dye may be chosen from fluorescein an! fiuoieseeln dehvaSve& [0917] The fluorescein derivative may be chosen from pbioxine 8, rose bengai, and merbromlne.

[0018} The fluorescein derivative may be chosen from eosin and erythrosine.

[0019] The azodye may be chosen from methyl vroiet, neutral red, para: red, piiranth, carmoisine, allura red AC, tartrazine, orange G, ponceau 4R, mefliyi red, and murexide-ammonium purpurate.

[0920] The biological stain may be chosen from saffranin 0( basic fuchsin, acid fuschin, 3,3' dihexyfocarbocyanine iodide, carmtnic acid, and indocyanine green.

[0021} The carotenoid may be chosen from crocetiru α-crocin (8,8-diapo-8,0-carotenoic acid), zeaxanfhine, lycopene, <**carotenes ^carotene, bixin, and fucoxanthine, [0022} The carotenoid may be present: in the composition m.te fhlxifre phosen from saffron re!::powd!r| annatto e>#aet: and brown algae extract..

[0923] The wound healing composition may further comprise at least one chelating agent. |0024| The chelating agent may be chosen from ethyienediammetetraaceiic add i£DTA) and ethylene glycol tetraacetic add (EGTA), p02S| The wound healing composition may further comprise at least one lipolysis^iSteuiadhgtelcfrfrv |p28| The lipoiysls simulating factor may be chosen from caffeine and paraxanthine.

[0027] in accordance with one embodiment, there is disclosed a method for wound healing which comprises the steps of a) topically applying on a patient's skin a composition comprising, .at least one oxidant, at least one photoactivator capable of activating the oxidant; and b) treating said stun of step a) to actinic light for a time sufficient for said photoacfevator to cause activation of said oxidant p92§] The method for wound healing may comprise exposing the skin to actinic light for a period of about 60 seconds to about 6 minutes, {002$] The method for wound healing may comprise exposing the sin to actinic light for a penod of about SO seconds to about 6 minutes per cm2 of an area to be treated, [0030] The method for wound healing may comprise exposing the skin to a source of actinic light that is in continuous motion over the area being treated.

[0031] The method for wound healing may comprise exposing the skin to actinic light that may be visible light having a wavelength between 400 nm and 600 nm.

[0032] The following terms are defined below.

[0033] The term “hydrophilic gelling agent” is intended to mean a material iiat thicitens and stabilizes liquid solutions, emulsions, and suspensions. Hydrophillic gelling agents dissolve in liquid and provide a structure giving the resulting gel an appearance of a solid matter, while being mostly composed of a liquid. Hydrophiliio gelling agents are very similar to thickeners.

[0034] The term “actinic light” is intended to mean light energy emitted from a specific light source (lamp, LED, or laser} and capable of being absorbed by matter (e.g. the photoactivator defined below) and produce an identifiable or measurable change when I interacts with it; as clinically ideriiiable change we can presume a change in the color of the photoaetivator used (e.g. from red to transparent), £Q035| The term "photoaetivator" is intended to mean a chemical compound capable of absorbing actinic light The photoaetivator readily undergoes photoexcitatiors and then transfers its energy to other molecules, thus enhancing or accelerating the dispersion of light, and enhancing or activating the oxidant present in the reaction mixture, £0830J The term "oxidant" is intended to mean a either a chemical compound that readiiy transfers oxygen atoms and oxidize other compounds, or a substance mat gains eiectrons in a redox chemical reaction. |0037ϊ The term "chelating agent" is intended to mean a chemscaiihat removes metai ions, such as iron, and holds them In solution. £0O38| The term "healing factor" is Intended to mean a compound mat promotes or enhances the healing or regenerative process of a tissue.

[0033} The term lipolysis" is intended to mean the process in which lipids are broken down into their constituent fatty acids. PJ4DJ The term "tin m of exposure to actinic light" is intended to mean the time a tissue, skin or wound is exposed to actinic light per application of actinic light, p041| The term "total time of exposure to actinic light” is Intended to mean the cumuiative time a tissue, skin or wound is exposed to actinic light after several application of actinic light. p042! The term "pharmaceutically acceptable earner" is intended to mean a preservative solution, a saline solution, an isotonic (about 0,9%) saiine solution, or about a 5% albumin solution, suspension, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic substances suitable for delivery to a patient may be included in tie compositions of the present invention, the compositions may be solutions, suspensions or any appropriate formulation suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques jOPpMtaj Features and advantages of the subject matter hereof will income more apparent In light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and ire description are to be regarded as illustrative in nature, and not es restrictive and the full scope of the subject matter is set forth in the claims.

Bill mmmmm ofthe drawings |0O441 Fig, 1 illustrates that Eosin Y does not affect cellular viability. Hep G2 cells were treated for 24 hours with increasing concentrations {0.001 to 100 μΜ) of Eosin Y, or left untreated (CTt). Sfaurosporine (STS) was used as a positive control inducing cellular mortality. Higher concentrations (0.5 and 1 mM) of Eosin Y could not be tested as the dye interfered with tine assay, (00453 Fig. 2 illustrates that Erythresine B does not affect cellular viability. Hep G2 ceils were treated for 24 hours with increasing; concentrations (0.OG1 to 100 μΜ) of Erythresine S, or left untreated (CTL), Sfaurosporine (STS) was used as a positive control inducing cellular mortality. Higher concentrations (0,5 and 1 mM) of Erythresine B could not be tested as the dye interfered with the assay, (00461 Fig. 3 illustrates that initial wound closure is improved following application of a; wound healing; composition. Rats (n~2 per group) with an excision wound were treated or not with a wound healing composition composing an oxidant (carbamide peroxide) and a photoactivator mixture: (eosin Y, erythresine B and Saffron Red Powder), Lozenges, treated animals; circles: untreated animais (controls). ¢0047} Fig A illustrates that wound closure is improved following application of wound healing compositions. Rats <n~2 per group) with an excision wound were treated or not with wound healing composition (A) comprising an oxidant (carbamide peroxide) and a photoactivator mixture (eosln Y, erythrosine 8), wound healing composition (8) comprising an oxidant (carbamide peroxide) and a photoactivator mixture (eosin Y, erythrosine 8 and Saffron Red powder) or wound healing composition (C) comprising an oxidant (carbamide peroxide) and a photoactivator mixture: (eosin Y: erythrosine B, Saffron Red powder and Indoeyanin green), $$4¾ Pig- 5 Illustrates Hat wound closure is improved following apgicalon of a wound healing composition, Rats (n~2 per group) writ an excision wound were treated or not will a wound healing composition comprising in oxidant (carbamide peroxide) and a photoactivator mixture (eosin Yv erythrosine 8). lozenges: treated animals: triangles: untreated animals (controls). fD04$| Fig. 6 Illustrates that wound closure is improved following application of a wound healing composition.

[0OS03 Fig. 7 illustrates that wound healing is improved toligwing application of a wound healing composition.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS £80511 In accordance with one embodiment, there is provided a wound healing composition and the method of use of the composition on a patient's skin or wound. This product accelerate healing and restoration of a wound. t0052J In accordance with another embodiment, there is provided a method of use of the composition that is a photodynamic technique whereby the composition is activated by light providing a beneficial effect on the skin or wound and promoting healing. 100533 The composition and method may be used to treat injuries to the different layers of the skin, including incisions, lacerations, abrasions. puncture rounds, penetrations wounds, gunshot wounds, contusions, hematomas end crushing injuries. Lesions to mucosae may also be treated with the composition of the present invention, which may be used for example: to treat pathological lesions of the oral mucosa, such as parfodortfis, ulcers, and coid sores (orofacial herpes), (0664] The composition comprises a number of active principles selected from groups of possible components. These various active principles each have their mechanism of action, P6S6] Ckidahts (0056] The composition composes oxidants as a source of oxygen radicals. Peroxide compounds are oxidants that contain the peroxy group (R-0-0-R), which is a chainlike structure containing two oxygen atoms, each of which is bonded to the other and a radical or some element. Suitable oxidants for preparation of the aotive medium Include, but are not limited to: p®6fj Hydrogen peroxide (H2Oa) is the starting material to prepare organic; peroxides. HA is a powerful qxidlMg agent, and the unique property of hydrogen peroxide is that it breaks down into wafer and oxygen and does not form any persistent, toxic residual compound. Hydrogen peroxide for use in this composition can be used in a gel, for example with 5% hydrogen peroxide, A suitable range of concentration over which hydrogen peroxide can be used in the present composition is from about 3.5% to about 5%. P8§8] Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxide or percarbamide) Is soluble in water and contains approximately 35% hydrogen peroxide. Carbamide peroxide for use in this composition can be used as a gei, for example with 18% carbamide peroxide that represents 5,6 % hydrogen peroxide. A suitable range of concentration over which urea peroxide can be used in the present composition Is from about 10% to about 16%. Urea peroxide brakes down to urea and hydrogen peroxide in a siow-release fashion that can be accelerated with heat or photochemical reactions The released urea [carbamide, <MHs)CO$}], Is highly soluble in water and is a powerful protein de nature ni It increases solubility of some proteins and enhances rehydration of the skin and/or mucosa. 10039] Benzoyl peroxide consists of two benzoyl groups [benzole acid with the H ef the carboxylic acid removed) Joined by a peroxide group. It is found in treatments for acne, in concentrations varying from 2.5% to 10%. The released peroxide groups are effective at killing bacteria. Benzoyl peroxide also promotes skin turnover and clearing of pores, which further contributes to decreasing bacienai counts and reduce acne. Benzoyl peroxide breaks down to benzoic acid and oxygen upon contact with skin, neither of which are toxic. A suitable range of concentration over which benzoyl peroxide can be used in the present composition is from about 2.5% to about 5%.

[0δδδ] inclusion of other forms of peroxides (e.g. organic or inorganic peroxides) should be avoided due to their increased toxicity and their unpredictable reaction with the photodynamic energy transfer, pb§1| Photoactivators:

The photoactivators transfer light energy to the oxidants. Suitable photoactivators can be fluorescent dyes (or stains), although other dye groups or dyes {biological and histological dyes, feed colorings, carotenoids) can also be used. Combining photoactivators may increase photo-absorbtion by the combined dye molecules and enhance absorption and photo-biomodulabon selectivity. This creates multiple possibilities of generating new photosensitive, and/or selective photoactivator mixtures.

[0053] An advantageous characteristic of a photoactivator is increased fluorescence. In the present invention, re-emission of light in the green to yellow spectrum would be advantageous, since it is a deep penetrating wavelength range, with deep absorption by the blood. This confers a strong increase on the blood flow, vasodilatation and angiokinetie phenomena. Suitable photoachvators Induce, but are not limited to; mm mrnmmmmm [8865] The xanthene denvafcve dyes have bean used and tested for a long time worldwide. They display low toxicity and increased fluorescence The xanthene group consists of 3 sub-groups that are·, a) the fluorenes; b) floor ones; and c) the rhodoies.

[0066] The fluorenes group comprises the pyronmes (e.g. pyronine ¥ and B) and the rhodamines (e.g. rhodamlne B, &amp; and VVT). Depending on the concentration used, both pyronmes and rhodamines may he toxic and their interaction with light may lead to Increased toxicity. Similar effects are known to occur for the rhedofa dye group. (0867] The fluorone group comprises the fluorescein dye and the Ρ«| Fluorescein is a fluorophore commonly used in microscopy with an absorption max. of 494 nm and an emission max. of 521 nm. The disodium salt of fluorescein Is known as DSC Yellow 8. it has very high fluorescence but phofodegrades quickly. In the present composition, mixtures of fluorescein wsth other photoactivators such as indocyanln green and/or saffron red powder will confer increased photoabsorption to these other compounds. (0888] Eosins group comprises Eosin Y (tetrabromofluoresceln, acid red 87, D&amp;C Red 22} with an abs max 514-518 nm, stains cytoplasm of ceils, collagen, muscle fibers and red blood cells intensely red, and Eosin B (acid red 91, eosin scarlet, dibromo-dlnitrofluoresceln). with the seme staining characteristics as Eosin Y. Eosin Y, eosin S. or a mixture of both can be used because of their sensitivity to the light spectra used: broad spectrum clue isght, blue to green light and green light, Their tissue and bioiirn staining properties and their low toxicity are also advantageous. Both eosin Y and eosin B stain red blood cells and thus confer to the composition of the present invention haemostatic (controls the flow or stops the flow of blood) properties as well as increase the selective targeting of light to the soft tissues of the lesion or wound during the application of the composition. (¢0701 Phioxine β (2,4,5,7 tetrabromo AS.SJ.ieiraGhiorofluoreseeln. ' D&amp;C Red 28, acid red 82) is a red dye derivative of fluorescein which is used for disinfection and detoxification of waste water through photooxidation, it has an abs. max. of 585-548 nro. it is also used as an intermediate for making phsibsehsilivte dyesand drugs. pfillj Erythrosine B (acid red 51, tetraiodofiuorescein) is a cherry-pink. #$-bssed fluorine feed dye used as a biological stain, and a bioiim and dental plague disclosing agent, with max. abs, 524-530 nm in aqueous solution, it is subject to photodegradation. Erythroslne is aiso used in some embodiments doe to its photosensitivity to the light spectra used and its ability to stain biofilms, inclusion of erythrosine should be favored when using the composition in deep pockets of infected or contaminated tissue, such as periodontal pockets in periodontal therapy . |D0?2| Rose Bengal (4,5,6,7 tetrachioro 2,4,5,7 tetraiodoffuorescein, acid red 84) is a bnght bluish-pink biological dye with an absorption max. of 544-549 nm, that has been used as a dye, biological stain and diagnostic aid. Also used in synthetic chemistry to generate singlet from triplet oxygen. (¢0731 Merbromine (mercurochrome) is an organo-mercuric dssodiuro sat of fluorescein with an abs, max of 508 nm. St Is used as an antiseptic.

Slips' (00753 The azo (or diazo-} dyes share the N-N group, caked azo the group. They are used mainly in analytical chemistry or as food colorings and are not fluorescent. Suitable azo dyes include: Methyl violet, neutral red, para red {pigment red 1), amaranth (Azorubine S), Carmoisine (azorubine, food red 3, acid red 14), allura red AC (FD&amp;C 40), tartrazine {FD&amp;C Yellow 5), orange G (acid orange 10), Ponceau 41 (food red 7), methyl red (acid red 2), mure)d|p-aiimonium purpurate. P876) liolggicai Malos; p077| Dye molecules commonly used in staining protocols for biological rrrsfenais can also be used as pbotoactivators. Suitable biological stains ineluiei |0078) Saffranin (Saffranin D, basic red 2) is also an azo-dye anbll used In histology and cytology. It Is a classic counter stain in a Gram stain protocol. £0079) Fuchsin {basic or acid} (rosanillne hydrochloride) is a magenta biological dye that can stain bacteria and has been used as an anilseplie; it has an abs. max,. 540-555 nm, |0030| 3,3’ diiexylocarbocyanine iodide (DsQC6) is a fluorescent dye used for staining cell’s endoplasmic reticulum, vesicle membranes and mitochondria. It shows photodynamic toxicity; when exposed to blue light, has a green fluorescence. 10081) Carminic acid (acid red 4, natural red 4) is a red glucosidal hydroxyanthrapurin naturally obtained from cochineal insects {808¾ indocyanin green (ICG) is used as a diagnostic aid for blood volume determination, cardiac output, or hepatic function. ICG binds strongly to red blood cells and when used in mixture with fluorescein, it increases the absorption of blue to green light. fW3 Carotenoids {0884) Carotenoid dyes can also act as photoaetivators, {0085} laffmn red powder is a natural carotenoid-containing compound. Saffron is a spice derived from crocus saiivus. it is charactertred by a bitter taste and ibdifbrm or hay-like fragrance; these are caused by the * compounds plcrocrocin and saffranal. it also contains the carotenoid dye crocin that gives its characteristic yeiiow-ned color. £008¾ iaffron contains more than 158 diiefent compounds many of them are carotenoids: mangicrocin, reaxanihine, lycopene, and various a-and β-carotenes, that show good absorption of light and beneficial biological activity. Also saffron can act as both a photon-transfer agent and a healing factor. Saffron color is primarily the result of a-crocin (8,8 diapo-l,# carotenoid add). Dry saffron red powder is highly sensitive to fluctuating pH levels and rapidly breaks down chemically in the presence of light and oxidizing agents. It is more resistant to heat. Data show that saffron has antiearcinogenic, Immunomodufafing and antioxidant properties, for absorbance, it Is determined for the erode specific photons nm (blue light), It has a deep red colour and fbnrts crystals with a melting point of 186°C, When dissolved in water it forms an orange solution. £008Tjj Crocetin is another compound of saffron mat was found to express an antllipldemic action and promote oxygen penetration in different tissues. More specifically it was observed ah increased oxygenation of the capillarifes. An increase of the oxygenation of muscles and cerebral cortex was observed and led to an increased survival rate in laboratory animals with induced hemorrhagic shock or emphysema. 1108¾ Anatto a spice contains: as main constituent (70*80%) die carotenoid blxin which displayed retevantantioxidative properties. pSSP) fhcaroiene, also displayed suitable characteristics, £0090) Fuccxanthine is «constituent of brown algae with a pronounced ability for photosensitization of red-ox reactions. £0091] Heating factors; mm Healing factors comprise compounds mat promote or enhance the healing or regenerative process of the tissues on the applicate siteof the composition. During the photoactivation of the composition, there is an increase of the absorption of molecules at the treatmerit site by the skin or fie mucosa, An augmentation in the blood flow at the site of treatment is c^Sived isr an extent period of time. An Increase in the lymphatic drainage and a possible change in fie osmotic equilibrium due to the dynamic interaction of the free radical cascades can be enhanced or even fortified with life ipetasioni of healing factors. Suitable Healing factors include, but are not limited 10; |O093| Hyaluronic acid (Hyaiuronan, hyaluronate): is a non-suifated glycosaminogtyea n, distributed widely throughout connective, epithelial and natiral tissues/itis g^e if the pHmary components of the extracellular matrix, and contributes significantly to cell proliferation and migration, Hyaiuronan is a major component of the skin, where it Is involved in tissue repair. While it is abundant in eidraceilular matrices. It contributes to tissues Hydrodynamics, movement and proliferation of cells and participates in a wide number of cell surface receptor interactions, notably those including primary receptor CD44. the Hyaiuronidases enzymes degrade Hyaiuronan. There are at least seven |yps of hyaiuronldaseriike enzymes in humans, several of which are tumor suppressors, the degradation product of hyaluronic acid, the oligosapiharides and the very-low molecular weight hyaluronic acid, exhibit pro-angiogenic properties. In addition, recent studies show that hyaiuronan fragments, but not the native high molecular mass of hyaiuronan, can Induce inflammatory responses in macrophages and dendritic ceiis in tissue injury. Hyaluronic acid ;s well suited to biological applicates targeting the skin. Due to its high biocompatibility, it is used to stimulate tissue regeneration. Current studies evidenced hyaluronic acid appearing in hie early stages of healing to physically create room for white blood cells that mediate the immune response. It is used in the synthesis of biological scaffolds for wound healing applications and In wrinkie treatment, $094} Glucosamine: is one of the most abundant monosaccharides in human tissues and a precursor in the biological synthesis of glycosllated; proteins and lipids. It is commonly used in the treatment of osteoarthritis. The common form of glucosamine used is its sulfate salt Glucosamine shows a number of effects including an anfcflammatory activity, stimulation of the synthesis of proteoglycans and the synthesis of proteolytic enzymes. A suitable range of concentration over which glucosamine can be used In the present compositlon is from about 1% to about 3%. jpJS|| Aiiantoin: is a diureide of glyosillc add It has keratolytsc effect, increases the water content of the extracellular matrix, enhances the desquamation of the upper layers of dead (apoptotic) skin ceils, and promotes skin proliferation and wound healing.

[609$| Also, saffron can act as doth a photon-transfer agent and a healing factor.

[0097| Chelating agents: p09ij Chelating agents can be included to promote smear layer removal in closed infected pockets and dlficuit to reach lesions; act as a: metal Ion quencher and as a buffer. Suitable chelating agents include,, but are not limited to: [0O9S| Ethylenediaminotetraacetic acid (EDTA): It is an aminoadd, used to sequester di- and trivalent metal ions. EDTA binds to metals via 4 carboxylate and 2 amine groups. EDTA forms especially strong complexes with yn(lil>, FefMI),. CuClII), Co{tll}, Prevents collection of the platelets and blood όΐφ fermaSon. if is used in the endodontictherapy as a smear layer mrnoval agent durtr^ instrumentation, ills used to butter solutions. fpreot Ethylene glycol teiraacetic add (EGTA) Is related to EOTA, but with a mush higher afinity for calcium than for magnesium ions. It Is useful for making buffer solutions mat resemble the environment Inside fylni cells and is often employed In dentistry, more specifically endodontics, in the removal of Smear layer.

[06101! ypolysls stimulating factors: |6611ί| Upolysis Simulating faototi can he Inotuded tor use of the composition in cosmetic applications, such as wrinkle treatment.

Caffeine, and the metabolic derivative of caffeine paraxanihine can increase in toe lipolysis process to releases glycerol and lady acids into fheif^d#»arn: [0100] Hydrophilic gelling agents [0101] The wound healing composition may also contain one or more hydrophilic gelling agent The hydrophilic gelling agent enhances the consistency of the composition and contributes to facilitating me application of the composition to the skin or wounded area Also, when used with hydrogen peroxide {H2G2}. it may contribute to the slow the release of the HsQs, and provide a more immediate reaction because pure can be used directly. Suitable hydrophilic gelling agent include, but are not limited to glucose, modified starch, methyl cellulose, carfeexymethyt cellulose, propyl cellulose, 'hydroxypropyl cellulose, oarbopol® polymers, atginlc acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum, pectin, and gelatin.

[0182] Use of the composition [0183] The inclusion of suitable photosensitive compounds and activation with a light source of a proper wavelength, leads to the acceleration in the breakdown process of the source of peroxide (the oxidant) and the other reactions that take place, via a photodynamic phenomenon. The included dyes are iiiuminated by photons of a certain wavelength and excited to a higher energy state When the photoactivators' excited electrons return to a lower energy state, they emit photons with a lower energy: level, thus causing the emission of light of a longer wavelength (Stokes shift), in the proper environment, much of this energy transfer is transferred to oxygen or the reactive hydrogen peroxide and causes the formation of oxygen radicals, such as singlet oxygen.

[0104] The singlet oxygen and other reactive oxygen species generated by the activation of the composition are thought to operate in a hermetic fashion. That is, a health beneficial effect is brought about by the low exposure to a normally toxic stimuli (e.g. reactive oxygen), by stimulating and modulating stress response pathways in ceils of the targeted tissues. Endogenous response to exogenous generated free radicals (reactive oxygen species) is modulated in increased defense capacity against the exogenous fimifadieais and induces acceleration of healing and regenerative processes. Furthermore, activation of the composition will also produce an antibacterial effect. The extreme sensitivity of bacteria to exposure to free radicals makes the composition of the present invention a cfe facia bactericidal composition. £O10S| Possible mechanism of action should be a fortified redox signaling phenomenon resulting in accentuated signal transduction process in which cells convert one kind of signal into another; activated "second messengers” induce a "signal cascade" beginning with a relatively small stimulus that elicits a iarge response via biologically monitored amplification of such signals , These complex mechanisms act possibly Involving angiogenic phenomena via growth factor activation. jpoif This method could be described as a form of photodynamic therapy. However, unlike other photodynamic techniques, where the photoactoactivators are incorporated in the tissue structure, in the present method, the photoactive material is in simple contact: with the tissue and acts when activated by light, as a "photodpamfc device” that chemically interacts with the tissue, Additionally, the actinic light penetrates the issue, and the light that is emitted by the photoacfcvator flight of a longer wavelength) is also absorbed by the tissue. £0107] Any source of actinic light can be used. Any type of halogen, LED or plasma arc lamp, or laser may be suitable, The primary characteristic of suitable sources of actinic light will be that they emit light in a wavelength tor wavelengths) appropriate for activating the one or more photoactivators present In the composition, in one embodiment, an argon laser is used, in another embodiment, a potassiunvtiiany! phosphate (KTP) laser (e.g. a GreenUghtlaser) is used. In yet another embodiment, a LED photocuring device is the source of the actinic light In yet another embodiment, the source of the actinic light is a source of visible tight having a wavelength between 400 and 600 nm. Furthermore, the source of actinic light should have a suitable power density. Suitable power density for non-coilimated light sources (LED, halogen or plasma lamps) are In the range from about 900 mW/crth to about 2000 mW/cm2 Suitable power density for laser light sources are in the range from about 0.5 mW/crrr to about 0 8 roW/cm2 [0108$ The duration of the exposure to actinic light will be dependent on the surface of the treated area, and on the type of lesion, trauma or injury that is being treated. The photoactivation of the composition may take place within seconds or even fragment of seconds, but a prolonged exposure period is beneficial to exploit the synergistic effects of the absorbed, reflected and reemitted Sight on the composition of the present invention and lie interaction with the tissue being treated, in one embodiment, the time of exposure to actinic light of the tissue, skin or wound on which the wound healing composition has been applied is a period between 80 second and 5 minutes, in another embodiment, the time of exposure to actinic light of the tissue, skin or wound on which the wound healing composition has been applied is a period between 80 seconds and 5 minutes per cm2 of the area to be treated, so that the total time of exposure of a 10 cm2 are would be between 10 minutes and 50 minutes, in yet another embodiment, the source of actinic light is in continuous motion over the treated area for the appropriate time of exposure, in yet another embodiment, multiple applications of the wound healing composition and actinic light are performed, in some embodiments, fhe tissue, skin or wound is exposed to actinic light at ieast two, three, four, five or six times in some embodiments, a fresh application of the wound healing composition is applied before exposure to actinic light. EXAMPLE! |0109| An exemplary wound heaiing composition was prepared by mixing the following component:

Ϊ011δ| The oxidant (4 ml) and healing factors (1,5 ml) were mixed and the combined with She photoactivators <1 ml). The resulting composition was applied to the skin if a wounded patient, and activated with actinic light provided by a LED photocunng device (blue light). The composition was removed foliowing treatment

MiiPtEil C01112 An second exemplary wound healing composition wll prepared by mixing the following components;

1011¾ The oxidant (4 ml) and healing factors (1.5 mL) were mixed and the combined with the photoactivators (1 mL). The resulting composition was applied to the skin of a wounded patient and activated with actinic light provided by a LED photocunng device (blue light). The: composition was: removed following treatment. p£$|| This second exemplary composition is using the fluorescein dye asm iMdechvator ie other dyes (indocyanine green and saffron red powder) present in die composition. The addition of a smalt amount of fluorescein to the indocyanine green and saffron red powder solution caused neemission of light at wavelengths that activated the other dye compounds and improved the treatment by increasing the established clinical absorpticn/reemission criteria. (0114J indocyanine green binds well to hemoglobin and helps the selective energy absorption by the tissues and also helps targeting these tissues with the generated free radical cascades. Also, this photoactivators mixture is able to render saffron red fluorescent which again improves both the photodynamic and bioatimulating phenomena. EXAMFLlili piiSJ The toxicity of the photoactivators Eosin Y and Erythrosine 8 was evaluated by measuring the cytotoxicity of these compounds on human ceils. Hep <32 human hepatocellular carcinoma ceils with an epithelial morphology were treated for 24 hours with increasing concentrations (0,001 to 100 μΜ) of Eosin Y or Erymmsine 8, and the cellular survival was evaluated. Increasing concentrations of either Eosin Y (Fig. 1) or Erythrosine B (Fig. 2) did not affect cellular Ability When compared to untreated celts, ifaumspohhe (STS) was used as a positive control for inducing cellular mortality and caused a dose-dependent effect (Figs, 1 and 2), Similar results were obtained by measuring cell death by release of iactate dehydrogenase (LDH). Therefore, neither Eosin Y or Erythrosine B caused increased cellular mortality,

EXAMPLE IV

PM (01173 Random skin Haps m rat were used to study the wound healing procedures., for evaiuating the benefits of ischemic and pharmacologic preconditioning methods on skin flap survival, applying blood flow assessment technologies on flaps, demonstrating the effects of vascular shunts and the studies on skin flap viability. The random skin flap model was used to study the effect of the composition of the present invention on skin flap survival and associated modelafiQns contributing to healing process, [01183 Excision wound of 1 cm in width by 2 cm in length were cut dorsaily onNhe midilne of tie back, 2 cm below tie inferior angle of fee scapulae- The skin was cut wife a surgical blade, the parinicufus camosus and a 0.5 cm layer subcutaneous to fee panniculus camosus was excised from fee wound edges. The wound was nextphotographed with an 8 mm by 8 him size marker. One gram of the wound healing composition was applied to fee ifeUftd (0.5 g/em2} and irradiated wife a blue LED light during 3 minutes. p11§| Excisionswere performed on rats (n~2 per group) as described above in Example IV, and fee excisions were treated or not with a single application of 1 gram of a wound healing composition comprising the oxidant (carbamide peroxide) and a photoactivator mixture containing eosin; Y, erythrosine B and saffron red powder. The excisions were irradiated wife a LED Sight (blue light) for 3 minutes. The percentage of wound closure was evaluated (Fig. 3) over a ten-day period following treatment. Animals treated with the composition showed a more rapid wound closure over the initial first three-day period following treatment

:'||A|l»LE VI

[01283 Excision were performed on rats (n~2 per group) as described above in Example IV, and fee excisions were treated or not with a single application of 1 gram of a wound heaiing compositions comprising; (A) fee oxidant (carbamide peroxide) and a photoactivator mixture containing eosin Y, and erythrosine B; (8) fee oxidant (carbamide peroxide) and a photoactivator mixture containing eosin Y, erythrosine 8 and saffron red powder; or (C) the oxidant (carbamide peroxide) and a photoactivator mixture containing eosin Y, erythrosine 8, saffron red powder and indocyanine green. The excisions were Irradiated with a LED light (bice Sight) for 3 minutes. The percentage of wound closure was evaluated daily for four days (Fig. 4} Animals treated with compositions (A) and (B) showed improved wound closure.....over the four-day period following treatment. The addition of indocyanine green in composition {Ο repressed the wound healing effect observed for compositions (A) and 1¾ EXAMPLE VI! f0l21J Excision were performed on rats (n«2 per group) as described above in Example IV, and the excisions wore treated or not with a single application of 1 gram of a wound healing composition comprising the oxidant (carbamide peroxide) and a photoactivator mixture containing eosin Y and eryihrosine 8. The excisions were irradiated with a LEO light (blue Sight) ter 3 minutes. The percentage of wound closure was evaluated (Fig. $) over a twelve-day period. Animals treated with tee composition showed a more rapid wound closure over the first seven-day period following treatment epLifti! fptill Using a template of 3cm by 9 cm (3x9) in pfexigias, a flap was traced on tee dorsal skin with a surgical markert taking as limits the inferior angles of the scapulae and the superior bones of pelvis. A pure random pattern flap with oranial base was out using sterile techniques and elevated through deep fascia, including superficial fascia, panniculus camosus, the subcutaneous flssbi and skin. To minimize wound contraction and simulating the human condition, a 0.5 cm subcutaneous layer of panniculus carnosus tom the wound edges was removed. During one hour, an impermeable barrier (e g. a silicone sheeting) was placed between tee flap and its donor site to eliminate the possibility of wound bed support. The sheet was then removed, tee flap returned to its original position and the flapedges were surgically closed using 4/0 nylon suture in an interrupted fashion. Immediately following flap closure, flap pedicle were coated with 13.5 g of gel formulation (0.6 g/cm®) and irradiated. The controls did not receive any treatment. Care was taken to distribute ointment evenly along fee entire flap. Cel formulation was prepared the same day of fee experiment, For Gel + Light group tee animals ware treated with gal formulation, the flap was irradiated for 3 minutes with a LED lamp.

EXAMPLE IX ϊ$!3ϊ!| Excision ware performed on rats (n~2 per group) as described above, and the elisions ware treated or not with the gel formulation, and irradiated with a LED light (blue light) asdeseribed in Example VIII. P124| The results demonstrate a direct correlation of necrosis from fluorescein injection and direct visualisation. Biopsies were evaluated for changes in histology. Data from the treated group demonstrate a clinically significant 15 times reduction in necrosis, (percent necrosis, mean, ii of 45.7 (±17.36) vs. 30,42 (±20.18), in the control and treatment groups respectively). Now referring to Fig. 7A, the clinical evaluation of necrosis following |ap surgery, in the control and treatment group shows that higher necrosis is observed on the control group versus the treatment group 1811111 Hematoxylin ami eosin staining of biopsies from tee control and treatment group (Fig. 78) reveal that greater vascular recruitment occurs in the treated group (see black arrows therein) Masson Trichrome staining for the assessment of collagen fibril deposition (Fig. 7C) at a 4GX magnification shows that new collagen deposition is occurring in the treatment group vs the control group. The photodynamic treatment using photoactivators and wavelength specific light aimed at Increasing the viability of the skin flap by stimulating the vascular recruitment of collaterals in Haps to improve the loco-regional state of the new wound, including tee formation of new collagen itereih. J0128| The embodiments and examples presented herein are illustrative of the general nature; if thi subject matter claimed and are not limiting, it will be understood by those skied in the art how these embodiments can be readily modified and/or adapted for various applications and in various ways without departing from the spirit and scope of the subject matter disclosed claimed. The claims hereof are to be understood to include without limitation all alternative embodiments and equivalents of the subject matter hereof. Phrases, wares end terms employed herein are illustrative and are not limiting. Where permissible by law, ail references cited herein are incorporated by reference in their entirety. It will be appreciated that any aspects of fee different embodiments disclosed herein may be combined in a range of possible alternative embodiments, and alternative combinations of features, all cf which varied combinations of features are fc be understood to form a part of the subject matter claimed.

Claims (10)

1. CLAIMS:

   1. A method for wound healing comprising the step of: a) topically applying on a patient’s wound a composition comprising at least one oxidant; at least one photoactivator, wherein the at least one photoactivator is eosin Y; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin; and b) exposing the composition of step a) to actinic light for a time sufficient to cause activation of the at least one photoactivator.
2. 2. The method according to claim 1, wherein the composition is exposed to actinic light for a period of about 60 seconds to about 5 minutes.
3. 3. The method according to claim 1, wherein the composition is exposed to actinic light for a period of about 60 seconds to about 5 minutes per cm2.
4. 4. The method according to any one of claims 1 to 3, wherein the actinic light is from a source that is in continuous motion over the wound.
5. 5. The method according to any one of claims 1 to 4, wherein the actinic light is visible light having a wavelength between about 400 nm and about 600 nm.
6. 6. The method of any one of claims 1 to 5, wherein the wound is an injury of the skin selected from incisions, lacerations, abrasions, puncture wounds, penetrations wounds, gunshot wounds, contusions, hematomas and crushing injuries.
7. 7. The method of any one of claims 1 to 5, wherein the wound is a lesion of the oral mucosa selected from periodontitis, oral ulcers, and cold sores.
8. 8. The method of any one of claims 1 to 5, wherein the composition is exposed to the actinic light at least two, three, four, five or six times, and wherein a fresh application of the composition is applied before exposure to the actinic light.
9. 9. Use of at least one oxidant; at least one photoactivator; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin, wherein the at least one photoactivator is eosin Y, in the preparation of a medicament for wound healing.
10. 10. Use of at least one oxidant and at least one photoactivator; and a healing factor chosen from hyaluronic acid, glucosamine and allantoin, wherein the at least one photoactivator is eosin Y and is activated by actinic light having a wavelength between 400 nm and 600 nm, in the preparation of a medicament for wound healing.