Title:  Reparatives for chemosurgery and laser (thermal) therapy

United States Patent:  6,630,442

Issued:  October 7, 2003

Inventors:  Hersh; Theodore (3201 Andrews Ct. NW., Atlanta, GA 30305)

Appl. No.:  781868

Filed:  January 10, 1997

Abstract

A composition of glutathione and selenium, as a selenoamino acid or selenium yeast extract and an epidermal growth factor in a topical carrier and method of using the composition to reduce and repair skin damage, resulting from aesthetic (exfoliation and chemical peels) and surgical (laser and other therapies) procedures and other chemical and thermal burns to the cutaneous tissues.

SUMMARY OF THE INVENTION

The present invention deals with a composition and method for reducing the cutaneous effects and complications of chemosurgery and laser therapy of induced skin damage. The composition comprises an effective amount of a glutathione and selenoamino acid since selenium is the co-factor of glutathione peroxidase. The combination can be in the form of a lotion, cream, ointment, gel, spray, balm, emulsion or cosmetic foundation and can also include the further endogenous antioxidants acetyl L carnitine, melanins and superoxide dismutase as well as secondary exogenous components to be discussed hereinafter, plus the epidermal and/or fibroblast growth factors to aid in the tissue repair process by inducing orderly epidermal cell growth and maturation.

DETAILED DESCRIPTION OF THE INVENTION

As previously noted, the present invention deals with glutathione (GSH), in combination with selenium and thiol compounds used topically to act as free radical scavengers reducing skin changes and epidermal growth factors for enhancing repair. It is proposed that the described active ingredients be employed in topical compositions. Topical carriers are employed which should be both non-irritating to the skin and which are suitable for delivering the active components to the skin. Further, suitable topical carriers should be those which do not inhibit the antioxidant activity of the active ingredients thus reducing the efficiency of the composition for protecting the skin from the effects of acute and chronic ultraviolet radiation. Further, such carriers must be of sufficiently high purity and sufficiently low toxicity to render them suitable for chronic topical administration to the skin and be free of bacterial contaminants.

Certain antioxidants, particularly the endogenous L-glutathione and superoxide dismutase, as well as the element selenium, a co-factor for the enzyme glutathione peroxidase, and thiol compounds such as L-cysteine, can be employed in suitable carriers such as lotions, solutions, creams, ointments, foundation products, balms, sprays, aerosols or gels to protect and to treat the overlying skin surface in dealing specifically with the effects of the various free radicals on biornolecules, lipids, and cell membranes. Moreover, specific cellular growth factors, such as epithelial (epidermal) and/or fibroblast growth factors in appropriate concentrations and delivery vehicles, will be incorporated to these reparative preparations for accelerating healing of these wounds with quality repair.

Without being bound to any particular theory, it is noted that reduced glutathione is employed in protecting cells and aerobic organisms against oxidative stress by itself being oxidized. Thus, glutathione must act in combination with other enzyme systems in order to be reduced so that it may renew its role as a free radical scavenger. GSH functions also coordinately with the enzyme glutathione peroxidase to break down hydrogen peroxide and lipid hydroperoxides. Glutathione peroxidase in the body requires selenium as a cofactor to exert its biologic antioxidant function. Selenium compounds have been shown to scavenge oxygen-centered radicals in vivo with reduced glutathione through glutathione peroxidase. It is believed that selenium-GSH peroxidase catalyzes toxic hydrogen peroxide in the presence of reduced glutathione. This reaction reduces glutathione to oxidized glutathione (GSSG). In turn, the GSSG is reduced back to GSH by the enzyme GSH reductase thereby maintaining abundant cellular GSH to scavenge free radicals anew.

It is further contemplated that the present composition, as a preferred embodiment may include acetyl L carnitine. This latter component further participates in protecting cells against lipid peroxidation by locally increasing the amount of antioxidizing agents of GSH and ubiquinol. L-carnitine, also known as gamma trimethyl amino-beta hydroxy butyrate or Vitamin Bt occurs naturally in the body. It is a normal endogenous intermediary metabolite which has been identified in all mammalian cells and in blood and urine. It has the function of transporting fatty acids and other acidulated compounds across inner mitochondrial membranes and of maintaining the acyl CoA/free CoA ratio between the mitochondria and the cytosol of the cells. Acetyl L carnitine is the acetyl derivative of L-carnitine and is also a naturally occurring substance in the body as it provides a transport mechanism for the acetyl groups created by the beta oxidation of fatty acids while concomitantly regenerating acetyl co-enzymes in the cytosol of the cell.

Of interest herein, acetyl L carnitine has been shown to have a scavenging effect on the free superoxide anion. This antioxidant activity coupled by acetyl L carnitine's effect of inducing an increase in reduced glutathione and reduced ubiquinone levels provides a stabilizing effect on membranes by decreasing membrane lipid peroxidation. The skin is a highly vascular organ, extracellularly very rich in polyunsaturated fatty acids and thus prone to the process of lipid peroxidation. Thus, reduced glutathione and acetyl L carnitine in a topical preparation will act somewhat synergistically; the former as a reparative antioxidant which itself becomes oxidized and better able to be regenerated locally in its reduced form by the metabolic functions of acetyl L carnitine and by acetyl L carnitine's ability to enhance mitochondrial energy production. This is accomplished by the latter's actions on lipid metabolism and by the resulting increase in cytochrome oxidase, the final enzyme in the cellular respiratory chain.

Further, glutathione and selenium act synergistically in vivo as they are both constituents of the same enzymatic system. GSH serves as a specific donor substrate while selenium, provided from alimentary sources or locally from topically applied preparations of selenoamino acids, selenium yeast extracts or selenoamino acid chelates, provides the prosthetic group of GSH peroxidase, during its synthesis. The glutathione and selenium antioxidant functions are intrinsically related since by keeping a peroxidase in action, the GSH and selenium, contribute to the removal of the dismutation product of free oxygen radicals, namely, hydrogen peroxide. In a broad sense, GSH and selenium modulate free radical chains initiated or sustained by hydro peroxides.

Selenium is used in the present invention for its role as an antioxidant as well as its anticarcinogenic and antimutagenic properties. Selenium is an essential trace element, and a cofactor and constituent of the enzyme glutathione peroxidase. Selenium preparations as a sulfide have been used as topical antiseborrheic detergents (Selsun.RTM.) and in veterinary medicine topically for eczemas and dermatomycoses.

Selenomethionine decomposes lipid peroxides and inhibits in vivo lipid peroxidation in tissues of vitamin E deficient chicks. Other selenoproteins also show a high degree of inhibition of lipid peroxidation in hepatic tissues of various species, thus concluding that in vivo selenium has antioxidant behavior.

Extensive inflammation in an acute phase organ response induced by "burns." Free radical production is associated with inflammation and increased circulating lipid peroxides have been reported in burn patients. These parameters have been measured by urinary malondialdehyde levels as an indicator of lipid peroxidation. Supplemental oral anti-oxidants, such as vitamin E, and the trace elements which are directly involved in free radical scavenging, including selenium, zinc and copper, have been shown to have beneficial clinical effects and shorten hospitalization. The former acts as part of glutathione peroxidase while the latter two elements act as part of ceruloplasmin and superoxide dismutase. These trace elements also have site-specific antioxidant actions and, thus, the need to administer these locally at the site of the burn injury including chemical and thermal therapies for improved and accelerated wound repair.

Selenium has also been shown to affect the immune system. Selenium supplementation as 70% selenomethione in patients with psoriasis with normal pretreatment selenium blood levels showed in increase in blood 40% post treatment, although skin levels of selenium dependent glutathione peroxidase were unchanged in both normal and psoriatic skin. A statistically significant increase in the number of CD4+ T-cells was noted in the reticular dermis of the psoriatic lesions.

In other human studies, topical selenomethionine was investigated for its ability to reduce the degree of acute damage to the skin by sunburn as induced experimentally by ultraviolet irradiation. Eight women were treated for two weeks with lotion vehicle and then with three concentrations of selenomethionine (0.002%, 0.02% and 0.05%). The researchers found that topical selenomethione was effective in protecting against acute UV damage to the skin, as measured by the minimal erythema dose, using a multipbrt solar ultraviolet simulator. Plasma levels of selenium in these volunteers remained unchanged, suggesting the protective effect of the selenomethionine was locally at the skin.

The same investigators, Burke, K et al., conducted similar experiments in rodents to determine whether oral and/or topical selenomethionine supplementation could reduce the incidence of acute and/or chronic damage to the skin. This included sunburn and pigmentation as well as the development of skin cancers, respectively. These controlled studies showed that the concentration of selenium in skin in areas of topical application of the lotion containing selenomethionine were greater than those of the experimental animals given comparable oral doses. The selenium concentration of untreated skin and liver tissue were similar to those of animals receiving the oral selenium compound. There was no evidence of selenium toxicity in any of the experimental animals. The mice treated with selenomethionine had significantly less skin damage by ultraviolet irradiation, as indicated by reduced inflammation and pigmentation and by later onset and lesser incidence of skin cancers.

Selenium has been shown to be an effective inhibitor of skin tumor promotion in rodent skin, but the mechanism is not precisely known. Perhaps the common inciting factor by the carcinogens is the generation of toxic radicals. Selenium, as the co-factor of the enzyme glutathione peroxidase, detoxifies hydrogen peroxide and hydroperoxides within cells. This selenium-glutathione complex may lower the level of potentially damaging peroxide radicals that are generated from various carcinogenic promoting chemicals.

Selenium functions as an antioxidant, as stated, also by its role as a cofactor for glutathione peroxidase, a group of water soluble enzymes which also catalyze the destruction of both aqueous and membrane-bound hydroperoxides. In dietary selenium deficiency, these enzyme levels are markedly decreased resulting in severe free radical damage to the tissues so involved. The other related antioxidant systems cannot make up for depressed local activity or selenium and selenium dependent enzymes. Selenium deficiency also occurs after such injuries as burns and needs to be supplemented in these states, thus, the importance of providing selenium in these topical antioxidant preparations, as well as ascertaining adequate nutritional supplements.

"Cell growth stimulating compounds or factors" have been described as natural or exogenous compounds which have a stimulating effect on the elaboration and growth of specific cell lines. Specifically, in regard to promoting epidermal growth, such as in skin tissue repair or wound healing, various factors have been identified as growth factors, including epithelial (epidermal) growth factor (EGF), fibroblast growth factor (FGF), tissue respiratory factor (TRF), transforming growth factor (TGF) and insulin-like growth factor (IGF).

In the present formulations using antioxidants and other dermal nutrients and reparative compounds, one or more cell growth stimulating factors in suitable amounts effective for stimulating the growth of cells which encompass or surround and are injured or are responsible for healing wounds may be incorporated in the preparation of the present creams, balms, lotions, solution or gels, or other cosmetic compositions. Skin cellular reparative functions of dermatologic injuries or lesions (sunburn, thermal burns, radiation and laser burns, chemosurgery, dermatoses, etc.) are included in the list of therapies as examples.

A further active expedient is the use of epidermal growth factor. Epidermal growth factor (EFG) is an endogenous substances for the development and maintenance of the epidermis and dermis. EGF is a protein that catalyzes the cutaneous healing process by promoting epidermal and epithelial cells to divide and grow. It induces mitoses, so that skin constantly produces and uses EGF, particularly when skin is damaged, such as in ultraviolet radiation and after surgery, and trauma for both healing and reduction of scar and keloid formation. When applied topically, EGF generates and replaces epithelial cells. EGF also promotes synthesis of proteins, accumulation of collagen and formation of blood vessels. Following sunlight injury and during the aging process, topical application of EGF replaces the existing low levels of dermal growth factors to achieve improvement in the quality of the skin, thereby reducing sagging skin and wrinkles. The antioxidants protect and repair damaged skin from free radicals while the growth factors to be used in combinations will promote epidermal ell renewal and thus ensue in repair of affected tissues.

Epidermal growth factor is a 53 amino acid polypeptide which stimulates messenger RNA, DNA and protein synthesis. In vitro, it stimulates keratinocyte division and in vivo epidermal cell regeneration.

Epidermal growth factor (EGF) may be derived from various sources and is commercially available from specialized suppliers. Human EGF (hEGF) may also be produced from genetically engineered yeast, using the technique described by Brake et al., Alpha-factor directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisae (Proc. Natl. Acad. Sci-fi. USA 81:4642, 1984). EGF may also be derived from the submandibular glands of mice, so-called murine EGF. This factor may be isolated by the method of Savage and Cohen, as published in the Journal of Biol. Chemistry. 247:7609-7611, 1972.

In experimental animals, when applied to granulating wounds EGF has been shown to increase both the concentrations of DNA associated with cutaneous injury and of collagen hydroxyproline. In addition, other studies have been shown that when locally applied to fresh wounds, EGF accelerated the early phases of wound healing, as well as the quality of the repair. In humans, immunocytochemical techniques reveal the presence of EGF receptors within the cytoplasm of normal epidermis. In vitro studies confirm that EGF is sequentially processed within the cell's endosome. These studies also suggest that to yield a mitogenic signal, cells need to be exposed to EGF for about five hours, which may help to explain the "lag phase" in wound healing, by first achieving increases of DNA synthesis.

Normal regeneration of the epidermis after injury for healing of wounds requires mitosis and migration of the epidermal cells from residual epidermal appendages within the wound as well as from the intact epithelium surrounding the injury. Acceleration of epidermal regeneration may be enhanced by various agents as well as by epidermal growth factor (EGF). In vitro, this mitogenic effect requires continuous exposure of target cells to EGF. In their studies, Brown et al. (J. Expt. Med. 163, May: 1319-1324, 1986) administered human recombinant EGF mixed with topical creams. These were applied twice daily to animals with either partial thickness burns or split thickness epidermal burns. They noted a highly significant increase in the healing process of these wounds, purportedly due to the creams providing more continuous exposure of residual epidermal cells to the recombinant EGF. In this manner, EGF may stimulate the cells' mitotic activities for faster wound healing and improving the quality of the skin repair. The present patent application provides the synergistic antioxidant to reduce free radical injury complex coupled with EGF in cream, lotion, gel or other topical preparations to provide the active ingredients locally in order to enhance the repair process, as in the aforementioned clinical and investigative observations.

Thus, after cutaneous injury, residual epithelial cells proliferate in an organized fashion to regenerate an intact epidermis. Superficial wounds which do not result in total skin loss but retain at least a portion of the dermal layer, heal primarily by this process of epidermal regeneration. Epidermal growth factor induces replacement of cells by inducing mitosis. Many experiments, animal and human studies, have positively shown the beneficial effect of EGF in the process of wound repair. These clinical situations include partial thickness burns, skin graft donor sites, and chronic skin ulcers. It is also of use in healing radiation skin burns, surgical scars and in the repair process of cosmetic surgeries and cutaneous chemical peels.

Also useful herein is a component known as Tissue Respiratory Factor (TRF). TRF is a live yeast cell derivative which has been used in over the counter pharmaceutical preparations since patented in the 1940's and more recently as an ingredient in cosmetics. It is commercially available (Brooks Industries, Biodynes-TRF, South Plainfield, N.J.) and purported to be a powerful internal moisturizer which refreshes dry and infirm skin. TRF was first used as an anti-hemorrhoidal product (Preparation H, Whitehall Laboratories). TRF is composed of low molecular weight glycosidic/peptide fractions, with a ratio of 1:3. The residual glycopeptide linkages are through the amino acid asparagine residues. Because TRF is prepared from live yeast cell derivatives, additional trace quantities of coenzymes, vitamins, amino acids and minerals, characteristic of yeast species, are available in these extracts, which enhance the therapeutic capabilities of TRF in these pharmaceutic/cosmetic preparations.

TRF has a maximum absorbance of 13.0-20.0; ultraviolet spectrophotometer of a 1% TRF filtered solution reads at 256-258 NM. It is available as a water soluble material for gels, emulsions, lotions and creams. TRF has been shown to promote wound healing through its ability to increase fibroblast synthesis of collagen and elastin fibers resulting in smoothing of the skin. TRF's moisturizing effect is accomplished by increasing uptake of moisture by nascent protein and increasing oxygen utilization in the skin. TRF has been used in the treatment of sunburned skin and has been preferred for decreasing pain and discomfort of sunburn damaged skin when compared to a topical post-sun product containing the local anesthetic benzocaine. Thus, TRF, as other growth factors, may be used in combination to these proposed antioxidant preparations as a preventive and prophylactic agent to photodamaged, burned, incised, irradiated or inflamed skin of diverse etiologies.

Yeast extracts with mineral glycopeptides, such as selenomethionine or zinc glycopeptide as well as tissue respiratory factor have already been mentioned as essential components to these preparations. An additional product which may be part of the active ingredients of these compositions are sulphur rich yeast extract compounds, also commercially available (example: Clariskin, R.I.T.A. Corporation, Woodstock, Ill.). This material is extracted from the cytoplasm of eukaryotic cells of Saccharbmyces cerevisiae. These sulphur rich yeast extracts purportedly aid in the diminution or elimination of dermatologic "brown age spots" by diverting the process of melanogenesis toward the synthesis of lighter colored pigments in the skin. The reduction of brown melanin production is accomplished through the sulphur rich antioxidant, glutathione, and the associated enzyme, glutathione reductase. The latter enzyme is present in this yeast depigmenting extract and its intracellular function is to reduce glutathione (GSSG) that has already been oxidized in its role as an antioxidant as reduced glutathione (GSH).

The local relative or excess of reduced glutathione (GSH) and other thiol compounds in these cosmetic preparations participate in metabolic reactions by diverting the syntheses of melanocytic pigments, inhibiting the dark pigments (eumelanin) in favor of the biosynthetic pathway toward the clear pigments (pheomelanin). In addition, the glutathione reductase, as aforementioned, participates in the enzymatic reaction toward production of GSH, thus enhancing the anti-free radical activity of these complex and synergistic compounds.

The skin's "brown spots" are a result of over production of melanin by a specific cell called melanocyte. Although these cells tend to decrease in numbers in the mid-third decade of life, research has revealed that they tend to accumulate in body areas exposed to solar radiation hence localized hyperpigmentation of these areas, particularly the face, neck and hands. Repeated exposure to the sun's ultraviolet rays and the inherent aging process of skin cause these "brown age spots" to appear on exposed skin surfaces. Thus, this patent application for use of synergistic compounds in promoting and enhancing locally the body's, especially the skin's, defense mechanisms to free radical species. Glutathione, as GSH, plus the co-factor selenium of the enzyme glutathione peroxidase, plus thiol compounds and glutathione reductase of this yeast extract complete the body's prime antioxidant components, the glutathione cycle. These preparations, plus other key antioxidants and skin repair factors are able to both prevent and chain break free radical reactions in tissues and cells, concomitantly helping repair the skin or heal the wound, created by chemosurgery, laser therapy, burns and complications of the various etiologies so enumerated.

Thiol rich yeast extracts also provide glutathione peroxidase and the sulphur groups to promote its synthesis and enhance the glutathione pathways.

Beta glucans may be derived from plant based polysaccarides and have been known to stimulate macrophages, thereby activating non-specifically the immune system. Polymers belonging to the beta 1-3 glucan family have been purified from extracts of yeast wall (Saccharomyces cerevisiae). Beta glucans protect the skin's immunocompetent Langerhans cells from an inhibitory effect from ultraviolet rays. Thus, beta glucans are photoprotective and also have free radical scavenging activities and anti-inflammatory properties. These polysaccharides have also been shown to stimulate collagen and are synergistic in their functions with ascorbic acid. Beta glucans are commercially available from a number of suppliers, including Alban Muller International, Emerson, N.J. Recommended doses of the concentrate in water and sorbitol are at 0.01 to 0.1 percent, until 2.0 percent for anti-inflammatory activity comparable to a 0.1% hydrocortisone preparation. The free radical scavenging activity of this preparation in powder form requires a concentration of 0.015% to 0.045% to inhibit hydroxyl radicals. Other beta glucan preparations are available for inclusion in these reparative and solar protective compositions, such as microat uvax, from Nurture, Inc., Missoula., Mont. and Betavera from Brooks Industries, South Plainfield, N.J.

As noted previously, the active ingredients described above can be incorporated in any suitable pharmacologically acceptable carrier which is suitable for topical administration to the human skin. As such, the pharmacologically acceptable carrier must be of sufficient purity and have sufficiently low toxicity to rerider it suitable for administration to a human noting that, typically, the carrier can represent up to 99.99% and typically from at least approximately 80% of the total composition.

Typical compositions for use herein include a wide variety of physical forms. These include, but are not limited to, solutions, lotions, creams, oils, gels, sticks, sprays, ointments, balms and pastes. Generally, such carrier systems can be described as being solutions, emulsions, gels, solids and aerosols.

Solvents are generally employed in the preparation of suitable topical compositions. Such solvents can either be aqueous or organic based and, in either case, the solvent must be capable of having dispersed or dissolved therein the above-described active components while not being irritating to the user. Water is a typical aqueous solvent while suitable organic solvents include propylene glycol, butylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol and mixtures thereof. Solvents can be included in the overall composition in amounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. It is noted that compositions of the present invention can be produced in the form of an emollient. A wide variety of suitable emollients are known and may be used herein. In this regard, reference is made to U.S. Pat. No. 5,296,500, the disclosure of which is incorporated by reference.

Alternatively, the present composition can be formulated as a lotion containing from about 0.01% to 10% of the above described active ingredients. Further, the product.can be formulated from a solution carrier system as a cream. A cream of the present invention would preferably comprise from about 0.1% to 15% and preferably from 1% to 5% of the above described active ingredients. Lotions and creams can be formulated as emulsions as well as solutions.

It is contemplated that as one embodiment, the active ingredients described above be used as a lotion or cream emulsion of the oil-in-water type or as a water-in-oil type, both of which being extremely well known in the cosmetic field. Multi-phase emulsions such as the water-in-oil type is disclosed in U.S. Pat. No. 4,254,105, the disclosure of which is incorporated herein by reference.

It is further contemplated that the active ingredients of the present invention be formulated from a solution carrier system as an ointment. An ointment may comprise a simple base of animal or vegetable oils or semi-solid hydrocarbons (oleaginous). Ointments may also comprise absorption ointment bases which absorb water to form emulsions. Ointment carriers may also be water soluble. An ointment may comprise from 1% to 99% of an emollient plus to about 0.1% to 99% of a thickening agent. Reference is again made to U.S. Pat. No. 5,296,500 and the citations contained therein for a more complete disclosure of the various ointment, cream and lotion formulations for use herein.

It is important to supply locally both glutathione and the synergistic antioxidants to restore epidermal glutathione levels and enhance the reparative antioxidant chain breaking reactions. It becomes imperative to prevent UV ray damage by prophylaxis with skin care (sun protection) products and appropriate clothing, plus the prevention of free radicals and their neutralization by locally applied chain-breaking antioxidant preparations, as proposed in the present application.

Claim 1 of 10 Claims

I claim:

1. A method for enhancing repair from free radical damage to skin as a result of treatment of the skin with exfoliants, chemosurgery or laser therapy comprising topically applying active ingredients in a suitable topical carrier to skin damaged by exfoliants, chemosurgery or laser therapy, said active ingredients comprising glutathione and selenoamino acid in amounts suitable for enhancing repair of skin from free radical damage.

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