Abstract:
A method for reducing scarring comprises applying into a wound a composition comprising resveratrol. The wound was formed at most one day before the applying, and no part of the skin surface of the wound is more than 3 cm from uninjured skin.

Description:
BACKGROUND 
       [0001]    Wound healing is a complex process, and involves the regulation of numerous cellular functions including the interactions of fibroblasts/fibrocytes, osteoblasts, chondrocytes, endothelial cells, inflammatory cells, epithelial cells and smooth muscle cells, with the extracellular matrix. Normal healing results in scar formation in humans. However, it is well known that certain animals, and even the human fetus, are capable of regenerative healing of wounds which is indistinguishable from surrounding skin. 
         [0002]    Although the intricate details of wound healing are still being discovered, the process follows along a typical time line having four phases: 
         [0003]    Hemostasis Phase—This phase includes vasoconstriction lasting for the first 5-10 minutes after the injury. 
         [0004]    Inflammation Phase—This phase includes vasodilation and a cellular response by inflammatory macrophages, neutrophils and fibroblasts. Neutrophils undergo cannbilaization to produce transforming growth factor beta-1 (TGF-β1), which stimulates production of type I collagen (the mature collagen present in nomal skin) and stimulates fibroblast to myofibroblasts mediated by hyaluronic acid and epidermal growth factor receptor (EGFR). Bacteria, foreign particles and damaged cells are removed from the wound. Vasodilation starts at about 10 minutes after the initial injury, and the cellular response typically starts 30 minutes after the initial injury. Keratinocytes detach from the basement membrane and migrate to cover the exposed wound and connective tissue, and the wound clot is replaced with epithelial cells and granulation tissue (type III collagen). Differentiating keratinocytes also produce TGF-β1. The cellular response may last 7 to 8 days. 
         [0005]    Proliferation Phase—This phase includes re-epithelialization of the wound, fibroplasia, including collagen synthesis and wound contraction. During this phase skin cells multiply and spread, covering the wound. Re-epithelialization typically starts 24 hours after the injury. Fibroplasia typically starts in 3 to 4 days after the injury. Myofibroblasts (present in granulation tissue) express alpha-smooth muscle actin and are responsible for wound contraction, which typically starts 7 days after the injury. 
         [0006]    Remodeling Phase—This phase includes scar/collagen remodeling. The newly formed collagen matrix becomes cross linked and organized starting about 3 weeks from wound initiation and lasting as long as 1 year. 
         [0007]    Scar formation is a typical response for normal healing in humans. As compared with normal skin, a scar contains an overproduction of type III and type I collagens, and the mixture is disorganized. The scar itself is not very elastic and is of a different color than normal skin. The scar is also missing the layer of kertinocytes found on normal skin. Furthermore, depending on how deep was the original wound, the scar may be missing the normal underlying layers of muscle, fat, blood vessels, and many layers of the skin; these missing layers may result in the scar forming a depression compared to the level of the surrounding skin. 
         [0008]    Some animals are capable of scar free healing. In axolotls, there is a substantial reduction in neutrophil infiltration and a relatively long delay in production of new extracellular matrix during scar free healing. Studies with athymic nude mice indicate that up-regulation in metalloproteinase-9 (MMP-9) throughout the remodeling phase may contribute to scar free healing. Matrix metalloproteinases (MMP&#39;s) are a family of zinc dependent enzymes capable of degradation of extracellular matrix and are vital to the remodeling of the matrix and migration of cells. During normal human wound healing, MMP-9 degrades the type IV collagen of the basement membrane allowing keratinocytes to detach from the basement membrane and migrate to cover the exposed wound and connective tissue. 
         [0009]    Human oral healing of wounds results in little to no scar formation. Oral mucosal wounds show a robust early up-regulation of MMP-1, MMP-2 and MMP-9 at 3 days after the initial injury, as compared to skin wounds at 14 days after the initial injury. The human fetus, which also shows scar free healing, is surrounded by amniotic fluid which contains high molecular weight hyaluronic acid. High molecular weight hyaluronic acid is known to increase expression of MMP-2 and MMP-9. Although high molecular weight hyaluronic acid application at a wound site can reduce scarring, a scar is nevertheless still formed. 
         [0010]    Resveratrol (trans-3,4′,5-trihydroxystilbene), a stilbenoid, is a grape polyphenol present in various plants, some food products, red wine and grapes. Resveratrol has anti-inflammatory, anti-carcinogenic and anti-oxidant properties, and has been extensively studied. Huge interest in resveratrol was created when it was discovered that it was able to active the SIRT1 gene, a gene implicated in the life span extension associate with calorie-restricted diets. However, resveratrol is poorly absorbed when consumed as a dietary supplement, and is subject to metabolic degradation, and beneficial effect have been difficult to observe in human clinical studies. 
       SUMMARY 
       [0011]    In a first aspect, the present invention is a method for reducing scarring comprises applying into a wound a composition comprising resveratrol. The wound was formed at most one day before the applying, and no part of the skin surface of the wound is more than 3 cm from uninjured skin. 
         [0012]    In a second aspect, the present invention is a composition for reducing scarring by applying into a wound which was formed at most one day before the applying, and in which no part of the skin surface of the wound is more than 3 cm from uninjured skin, the composition comprising resveratrol. 
         [0013]    In a third aspect, the present invention is a use of resveratrol to prepare a composition for reducing scarring by applying into a wound, wherein the wound was formed at most one day before the applying, and no part of the skin surface of the wound is more than 3 cm from uninjured skin. 
     
    
     DETAILED DESCRIPTION 
       [0014]    The present invention makes use of the discovery that compositions containing resveratrol, when applied into a wound soon after the initial injury, will greatly reduce scarring. In some cases, compositions containing resveratrol may even eliminate scarring altogether. Also discovered is that these effects of resveratrol may be enhanced when combined with one or more additional active agents. 
         [0015]    It has been discovered that if a wound or incision is completely healed in less than 3 days, before fibroplasia begins, then no scar will be formed at the location of the wound or incision. Therefore composition containing resveratrol will allow for scar free healing when applied to wounds or incisions that do not have any injured or missing tissue which is more than 3 cm from uninjured tissue. Examples include almost all incisions purposefully created by a surgeon, because the surgeon is able to bring the edges of the skin at the location of the incision to well within 3 cm of each other. Preferably, no part of the skin surface of the wound is more than 3 cm from uninjured skin, more preferably no part of the skin surface of the wound is more than 2 cm from uninjured skin, even more preferably no part of the skin surface of the wound is more than 1 cm from uninjured skin, and most preferably no part of the skin surface of the wound is more than 0.5 cm from uninjured skin. 
         [0016]    Compositions containing resveratrol, either as the sole active agent or in combination with other active agents, is preferably applied to a wound or incision at any time from prior to formation of a wound or incision up until at most one day after the formation of a wound or incision; more preferably prior to formation of a wound or incision, up until at most 1 hour after the formation of a wound or incision; and most preferably prior to formation of a wound or incision, up until at most 10 minutes after the formation of a wound or incision. Preferably, only a single application of a composition containing resveratrol is used. For example, a composition containing resveratrol may be applied topically to an incision site, or injected below an incision site, then the skin may be cut, optionally followed by closing the incision; for example the deep structures which have been cut under the skin may be tied down using VICRYL™ (polyglactin 910) sutures, and then skin sutured or sealed using DERMABOND ADVANCED™ topical skin adhesive or NEW-SKIN® liquid bandage. Alternatively, a composition containing resveratrol may be applied to the incision or wound after it is formed, followed by closing the wound or incision as described above. 
         [0017]    Preferably, resveratrol is present in a composition at a concentration of at least 0.01 micromoles/liter, more preferably at a concentration of at least 0.10 micromoles/liter, and most preferably at a concentration of at least 0.50 micromoles/liter. Preferably, resveratrol is present in the composition at a concentration of at most 100 micromoles/liter, more preferably at most 40 micromoles/liter. Examples include 0.75, 0.80, 0.90, 1.0, 1.25, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.19, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, 10.0, 15.0, 20.0, 25.0, 30.0 and 35.0 micromoles/liter. Concentrations of resveratrol above 100 micromoles/liter appear to be cytotoxic to keratinocytes. 
         [0018]    In some forms, such as gels and pastes, the delivery medium limits contact with the surrounding tissue, the surrounding tissue rapidly degrades the resveratrol, and the tissue itself will absorb the resveratrol, resulting in a much lower effective concentration of resveratrol. In those cases, the concentration of resveratrol may optionally be increased 10 fold. In those cases, preferably resveratrol is present in a composition at a concentration of at least 0. 1 micromoles/liter, more preferably at a concentration of at least 1.0 micromoles/liter, and most preferably at a concentration of at least 5.0 micromoles/liter. Preferably, resveratrol is present in those compositions at a concentration of at most 1000 micromoles/liter, more preferably at most 400 micromoles/liter. Examples include 7.5, 8.0, 9.0, 10, 12.5, 15, 16, 17, 18, 19, 20, 21, 21.9, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300 and 350 micromoles/liter. 
         [0019]    Resveratrol has a very low solubility in water, however only that portion which is dissolved in water will exert its effects. Furthermore, if the resveratrol is applied dissolved in a hydrophobic medium, it may slowly diffuse into the surrounding aqueous medium, and undesirably extend the effective application time. Therefore, it is preferable that the resveratrol be applied as a solution in an aqueous medium. For ease of application in a clinical setting, preferably the aqueous medium is a gel, paste, foam, suspension or thickened solution. Examples include aqueous compositions containing hydroxypropyl methylcellulose, high molecular weight hyaluronic acid, polyethylene glycol, agar, dextrin, pectin, trehalose, xanthan gum, polyoxyethylene alkyl ethers, chitosan, guar gum and sodium alginate. Other vehicles, adjuvants and excipients, which are hydrophilic or have hydrophilic moieties, and are compatible which application into wounds, may also be used. Other pharmaceutically acceptable adjuvant, excipients and vehicles may also be included. 
         [0020]    Premeasured amounts of the compositions containing resveratrol may also be used. These are referred to as unit dosage forms, since each premeasured amount is intended to be used on a single patient for one or more application, all used at the same time. Examples include prefilled syringes, pouches, packets and tubes. Another example would be a tube or dispenser which may be used to form foam of its contents just prior to application, for example by shaking or using a foaming agent. A self-foaming tablet, which forms foam when placed into water, could also be used. The volume of material present in these unit dosage forms may be at least 0.1 to 100 ml, or 1 to 50 ml. including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 and 45 ml. 
         [0021]    Other active agents may be included, such as other activators of SIRT 1 ; HDAC 2  (a class I histone deacetylase) inhibitors, such as trichostatin A; agents which stimulate the production of certain growth factors such as EGF, FGF-10 and IGF-1; luteolin; tretinoin (all-trans retinoic acid); and high molecular weight hyaluronic acid. 
         [0022]    Although it is not known exactly how resveratrol reduces scarring, resveratrol does up-regulate and increase the expression of a variety of agents which are involved in wound healing. One possible explanation is that resveratrol causes the over-expression of MMP-9, interleukin-8 (IL-8) and SIRT1, and increases expression of EGFR on the keratinocyte membrane and nucleus. SIRT1 may then promote differentiation, motility and proliferation of keratinocytes, and deacetylation and inactivation of p53 protein thus inhibiting p53-dependent cell death from apoptosis in response to stress in human tenocytes (fibroblast-like tendon cells). SIRT1 may induce nitric oxide (NO) production, which inhibits class I HDAC 2 from blocking growth factors, including epithelial growth factor, keratinocyte growth factor 2, fibroblast growth factor 10 (FGF-10), and insulin-like growth factor 1 (IGF-1). SIRT1 may also decrease inflammation and apoptosis through a variety of mechanisms. IL-8 has a direct and profound stimulatory effect on the migration of keratinocytes, which is likely due via the PLC-gamma pathway, and furthermore IL-8 may recruit neutrophils. As noted above, MMP-9 degrades the type IV collagen of the basement membrane. EGFR may cause keratinocyte and fibroblast migration and may protect and repair tissue through nuclear DNA repair. Resveratrol may also inhibit NF-kB dependent proinflammatory and matrix degrading gene products induced by IL-113 and nicotinamide. 
       EXAMPLES 
     Example 1 
     In vivo Application of Various Compositions Containing Resveratrol in a Rat Model 
       [0023]    In this example, 15 Sprague Dawley Rats, 6-8 weeks old, will be placed randomly into 5 different groups, Study Groups 1-5, resulting in 3 animals per study group. An incision, 2 cm in length, will be made on both the right and left shoulder of each rat: the left side will be an untreated control, while the right side will be treated with the Compositions 1-5, with the Study Group number corresponding to the Composition number. 
         [0024]    The Compositions 1-5 are: (1) 0.5 g resveratrol in 1.0 cc aqueous hydroxypropyl methyl cellulose gel (resveratrol concentration=2.19 micromoles/liter); (2) 0.5 g resveratrol in 1.0 cc aqueous high molecular weight hyaluronic acid gel (resveratrol concentration=2.19 micromoles/liter); (3) 0.5 g resveratrol and 0.5 g tretinoin in 1.0 cc aqueous hydroxypropyl methyl cellulose gel (resveratrol concentration =2.19 micromoles/liter); (4) 0.5 g resveratrol and 0.5 g luteolin in 1.0 cc aqueous hydroxypropyl methyl cellulose gel (resveratrol concentration=2.19 micromoles/liter); and (5) resveratrol powder. 
         [0025]    After each incision is made, the resveratrol containing composition will be applied to the right incision just prior to closure using interrupted 5-0 nylon sutures. The left incision will also be closed using interrupted 5-0 nylon sutures. Each incision will be photographed and measurements will be taken, each day for 7 days. On the 4 th  day, serum blood samples will be taken for systemic absorption assay. On the 7 th  day, a punch biopsy will be taken from each test and control incision. 
         [0026]    Since each skin flap of the incisions is very close together, when the composition containing resveratrol is applied soon after the incision is made, the incision on the right shoulder heals before fibroplasia begins, so no scar is formed. This is in contrast to the otherwise identical incision on the left side, where no resveratrol is applied, which displays a typical scar. 
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