FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF

2.

1. (A) UNITED TECHNOLOGIES UT AG
(B) Switzerland
(C) Burglistrasse 8, 8002 Zurich Switzerland

The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
[0001] The present invention relates to topical compositions comprising interleukin-1 alpha in combination with peptides and cosmetic methods thereof.
Background of the invention
[0002] Interleukin-1 alpha, which is also named IL-1F1, is a naturally occurring polypeptide with the sequence well-known from the art. Uniprot/Swiss-prot database #P01583. In norm, interleukin-1 alpha is produced by human epidermis on a constitutive basis. Gahring LC et al., J Clin Invest. 1985, 76(4):1585. Mizutani H, et al., J Clin Invest 1991, 87(3):1066. Hauser C et al., J Immunol. 1986, 136(9):3317. Upon stimulation with interleukin-1 alpha, dermal fibroblasts produce components of dermis, e.g. procollagen, elastin, and hyaluronic acid, as well as tools for collagen turnover, e.g. collagenase, inhibitor of the collagenase (TIMP), and inhibitor of procollagen transformation to collagen (PGE2). Duncan MR et al., J Invest Dermatol. 1989, 92(5):699. Postlethwaite AE et al., J Cell Biol. 1988, 106(2):311. Kähäri VM et al., Biochim Biophys Acta. 1987, 929(2):142. Thus, interleukin-1 alpha is a unique regulator that coordinates the dynamic in vivo process of dermis remodeling and collagen turnover. Along with the abovementioned, interleukin-1 alpha is a pro-inflammatory cytokine which may induce a sequence of undesirable processes in skin, e.g. edema and redness. It may limit the use of interleukin-1 alpha in cosmetic products. Thus, there is a great need in a safe and effective agent that can selectively reduce the pro-inflammatory activity of interleukin-1 alpha along with maintaining the interleukin-1 alpha activities related to the dermis remodeling.
[0003] Surprisingly, we found that artificial hexapeptides of a formula Tyr-X-Gly-Phe-Leu-Y, wherein X is independently Gly, L- or D-Ala; and Y is independently Asp or Glu are useful for selective reducing the pro-inflammatory activity of interleukin-1 alpha along with maintaining the interleukin-1 alpha activities related to the dermis remodeling. The structure of the abovementioned hexapeptides is artificial and, while they have similarity with structure of N-terminus of endogenous opioid peptides (beta-endorphin, enkephalins, or dynorphins), they contains uneven for opioid peptides acidic amino acid Asp or Glu at sixth position that determines distinct biological properties with opioid peptides. The structure of the hexapeptides is not the same with the structure of N-terminus hexapeptide sequence of endogenous beta-endorphin or dynorphins and the structure contains uneven for beta-endorphin or dynorphins amino acid Asp or Glu at sixth position, the position close to N-terminal conservative motif Tyr-Gly-Gly-Phe-, critical for a peptide binding to opioid receptors. The structure of the hexapeptides is not the same with the structure of pentapeptide enkephalins and contains additive acidic amino acid Asp or Glu. Due to the presence of uneven for opioid peptides Asp or Glu in their structure, the hexapeptides becomes acidic and charged negatively at physiological pH, contrary to the enkephalins and other opioid peptides. It produces critical change in the biological activity of the hexapeptides as compared to the biological activity of opioid peptides. In particular, unlike opioid peptides, hexapeptides do not manifest neuronal activity and predominantly observed effect of the hexapeptides is activation of microcirculation in skin.
[0004] The use of interleukin-1 alpha in medicinal applications is known from the art. For example, U.S. Patent No. 4,816,436 discloses a process for treating arthritis or inflammation with the use of intra-articular, intramuscular, intravenous, or intraperitoneal injections of interleukin-1 alpha; U.S. Patent No. 5,120,534 discloses a method for treating thrombocytopenia by administering interleukin-1 alpha or Asp36, Ser141-derivative of interleukin-1 alpha; U.S. Patent No. 5,534,251 discloses stabilized medicinal composition comprising Asp36, Ser141-derivative of interleukin-1 alpha; EP0391444 discloses a pharmaceutical composition comprising interleukin-1 alpha, and suitable for forming a parenterally administratable aqueous formulation; WO9116916, JP4018033, EP0482213, and ES2121782T disclose an antitumor composition containing the combination of interleukin-1 and gamma-interferon. WO/2006/097359 discloses a composition for treating keratin fibres with aminexil in combination with interleukin-1 alpha taken as NOS synthesis stimulator. WO/2005/091891 discloses a method of altering coloration of a tattoo-affected dermal region by interleukin-1. However, no published or disclosed in the art related to topical compositions comprising combination of interleukin-1 alpha with the artificial hexapeptides of the formula Tyr-X-Gly-Phe-Leu-Y, wherein X is independently Gly, L- or D-Ala; and Y is independently Asp or Glu.
[0005] US 2005/0036974 discloses the use of beta-endorphin and beta-endorphin fragments in cosmetic compositions for enhancing barrier function of skin, enhancing moisturizing the skin, strengthening resistance of the skin to stress, pollution, and enhancing brightness of skin. However, no published or disclosed in the art related to topical compositions comprising combination of interleukin-1 alpha with beta-endorphins or beta-endorphin fragments.
[0006] WO/2006/106164 discloses the use of enkephalins for the preparation of a cosmetic or dermatopharmaceutical composition reducing and/or eliminating facial wrinkles. However, no published or disclosed in the art related to topical compositions comprising combination of interleukin-1 alpha with enkephalins.
[0007] US Patent No 5,002,933 discloses a synthetic hexapeptide Tyr-D-Ala-Gly-Phe-Leu-Glu for the use in medicinal preparations for the safe and effective treatment of erosive and ulcerative lesions of the gastrointestinal tract. However, nothing is published or disclosed in the art related to topical compositions comprising combination of interleukin-1 alpha with the hexapeptide Tyr-D-Ala-Gly-Phe-Leu-Glu, or their derivatives, or salts thereof.
[0008] It is an object of the present invention to provide a topical composition comprising interleukin-1 alpha and hexapeptides of the formula Tyr-X-Gly-Phe-Leu-Y, wherein X is independently Gly, L- or D-Ala; and Y is independently Asp or Glu; and methods for the use of the composition.
Detailed Description of the Invention
[0009] The present invention provides a topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0010] In a preferred embodiment of the present invention, the hexapeptide is selected from the group consisting of:
Tyr-Gly-Gly-Phe-Leu-Asp (SEQ ID NO: 1);
Tyr-D-Ala-Gly-Phe-Leu-Asp (SEQ ID NO: 2);
Tyr-L-Ala-Gly-Phe-Leu-Asp (SEQ ID NO: 3);
Tyr-Gly-Gly-Phe-Leu-Glu (SEQ ID NO: 4);
Tyr-D-Ala-Gly-Phe-Leu-Glu (SEQ ID NO: 5);
Tyr-L-Ala-Gly-Phe-Leu-Glu (SEQ ID NO: 6).
[0011] In preferred embodiments of the present invention, the content of interleukin-1 alpha in said compositions is in the range from 10-7 to 10-4 wt. %.
[0012] In preferred embodiments of the present invention, the content of the hexapeptide in said composition is in the range from 10-4 to 10-1 wt. %.
[0013] In preferred embodiments of the present invention, said compositions further comprise a buffer at a concentration effective to maintain the pH of the composition at between about 4.0 to about 7.5. Examples of dermatologically or cosmetically acceptable buffers include, but are not limited to, phosphate buffer, acetate, citrate buffer, succinate buffer, and glycine buffer.
[0014] The structure of interleukin-1 alpha is well-known from the art and described in details, for example, in databases in public domain like Uniprot/Swissprot database under the number of P01583. As used herein, the term “interleukin-1 alpha” refers to a protein having the following amino acid sequence and structure (naturally occurring human interleukin-1 alpha), and biologically active analogues and derivatives thereof:
Ser-Ala-Pro-Phe-Ser-Phe-Leu-Ser-Asn-Val-Lys-Tyr-Asn-Phe-Met-Arg-Ile-Ile-Lys-Tyr-Glu-Phe-Ile-Leu-Asn-Asp-Ala-Leu-Asn-Gln-Ser-Ile-Ile-Arg-Ala-Asn-Asp-Gln-Tyr-Leu-Thr-Ala-Ala-Ala-Leu-His-Asn-Leu-Asp-Glu-Ala-Val-Lys-Phe-Asp-Met-Gly-Ala-Tyr-Lys-Ser-Ser-Lys-Asp-Asp-Ala-Lys-Ile-Thr-Val-Ile-Leu-Arg-Ile-Ser-Lys-Thr-Gln-Leu-Tyr-Val-Thr-Ala-Gln-Asp-Glu-Asp-Gln-Pro-Val-Leu-Leu-Lys-Glu-Met-Pro-Glu-Ile-Pro-Lys-Thr-Ile-Thr-Gly-Ser-Glu-Thr-Asn-Leu-Leu-Phe-Phe-Trp-Glu-Thr-His-Gly-Thr-Lys-Asn-Tyr-Phe-Thr-Ser-Val-Ala-His-Pro-Asn-Leu-Phe-Ile-Ala-Thr-Lys-Gln-Asp-Tyr-Trp-Val-Cys-Leu-Ala-Gly-Gly-Pro-Pro-Ser-Ile-Thr-Asp-Phe-Gln-Ile-Leu-Glu-Asn-Gln-Ala
The term thus includes interleukin-1 alpha which is chemically synthesized or expressed using recombinant protein expression systems that use, for example, E-coli or yeast as the host. A preferred interleukin-1 alpha is human interleukin-1 alpha expressed using a protein expression system.
[0015] As used herein, the term “analogue of interleukin-1 alpha” refers to an interleukin-1 alpha that contains one or more amino acid substitutions, deletions, additions, or rearrangements compared with human interleukin-1 alpha at sites such that the interleukin-1 alpha analogue still retains the in vivo biological activity of interleukin-1 alpha. Examples of interleukin-1 alpha analogues include Asp36-interleukin-1 alpha and Ser141-interleukin-1 alpha.
[0016] Interleukin-1 alpha derivatives include naturally occurring interleukin-1 alpha and interleukin-1 alpha analogues that are chemically or enzymatically derivatized at one or more constituent amino acids, including side chain modifications, backbone modifications, and N-and C- terminal modifications, by for example acetylation, acylation, hydroxylation, methylation, amidation, phosphorylation, pegylation, or glycosylation, and that retain the in vivo biological activity of interleukin-1 alpha. An example of an interleukin-1 alpha derivative is N6-myristoyl-Lys11-interleukin-1 alpha and HisTag-interleukin-1 alpha.
[0017] As used herein, the term "topical composition" refers to a composition that is applied topically to the human skin. In this respect, the term "skin" encompasses whole skin or any portion of the skin.
[0018] As used herein, the term “dermatologically acceptable vehicle” refers to one or more liquid, semi-solid, or solid diluents, which are compatible with interleukin-1 alpha, and are suitable for administration to any portion of the human skin without undue/unacceptable aesthetic effects, e.g., greasiness, color, odor, etc.. Examples of such carriers include, but are not limited to, distilled or deionized water, propyleneglycol, glycerol, and oil.
[0019] The compositions of the present invention can comprise optional ingredients. Such optional ingredients generally are used individually at levels from about 0.0005% to about 10.0%, preferably from about 0.005% to about 1.0% by weight of the composition..
[0020] Examples of suitable optional ingredients include, but are not limited to, depigmentation agents; reflectants; humectants; antimicrobial (e.g., antibacterial) agents; UV absorbers; anti-acne agents; anti-aging agents; anti-wrinkling agents, antiseptics; local anesthetics; wound healing promoters; deodorants and antiperspirants; skin emollients and skin moisturizers; tanning agents; skin lightening agents; antifungals; depilating agents; external analgesics; counterirritants; anti-diaper rash agents; make-up preparations; vitamins and nutrients such as thiamin, riboflavin, niacin, pantothenates, pyridoxine, folic acid, cobalamin, biotin, choline, inositol, ascorbic acid, lipoic acid, carnitine, and etc.; amino acids and their derivatives such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, praline, serine, taurine, threonine, tryptophan, tyrosine, valine; minerals such as boron, calcium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc; herbal extracts; retinoids; bioflavonoids; anti-oxidants; skin conditioners; hair lighteners; chelating agents; cell turnover enhancers; coloring agents; sunscreens and the like, and mixtures thereof.
[0021] Examples of suitable reflectants include, but not limited to, mica, alumina, calcium silicate, glycol dioleate, glycol distearate, silica, sodium magnesium fluorosilicate, and mixtures thereof.
[0022] Examples of suitable UV absorbers include, but not limited to, benzophenone, bomelone, butyl paba, cinnamidopropyl trimethyl ammonium chloride, disodium distyrylbiphenyl disulfonate, paba, potassium methoxycinnamate, and mixtures thereof.
[0023] Examples of suitable humectants include, but not limited to, water soluble liquid polyols selected from the group comprising glycerine, propylene glycol, hexylene glycol, butylene glycol, pentylene glycol, dipropylene glycol, and mixtures thereof. The humectant is preferably present in an amount of from about 0 percent to about percent, more preferably from about 0.5 percent to about 5 percent, based on the overall weight of the composition.
[0024] Suitable amino acid agents include amino acids derived from the hydrolysis of various proteins as well as the salts, esters, and acyl derivatives thereof. Examples of such amino acid agents nonexclusively include amphoteric amino acids such as alkylamido alkylamines, i.e. stearyl acetyl glutamate, capryloyl silk amino acid, caprylol collagen amino acids; capryloyl kertain amino acids; capryloyl pea amino acids; cocodimonium hydroxypropyl silk amino acids; corn gluten amino acids; cysteine; glutamic acid; glycine; hair keratin amino acids; hair amino acids such as aspartic acid, threonine, serine, glutamic acid, glycine, alanine, half-cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, cysteic acid, lysine, histidine, arginine, cysteine, tryptophan, citrulline; lysine; silk amino acids, wheat amino acids; and mixtures thereof
[0025] Examples of suitable proteins include, but not limited to, collagen, deoxyribonuclease, iodized corn protein; keratin; milk protein; protease; serum protein; silk; sweet almond protein; wheat germ protein; wheat protein; wheat protein, alpha and beta helix of keratin proteins; hair proteins, such as intermediate filament proteins, high-sulfur proteins, ultrahigh-sulfur proteins, intermediate filament-associated proteins, high-tyrosine proteins, high-glycine tyrosine proteins, tricohyalin, arginine-rich peptides like as oligoarginines (Arg)8, and mixtures thereof.
[0026] Examples of suitable antiperspirants and deodorants include, but not limited to, aluminium chlorohydrates, aluminium zirconium chlorohydrates, and mixtures thereof.
[0027] Examples of sunscreen agents include, but not limited to, titanium dioxide and zinc oxide.
[0028] Examples of suitable counterirritants include, but not limited to, camphor, menthol, methyl salicylate, peppermint and clove oils, ichtammol, and mixtures thereof.
[0029] Examples of suitable anti-aging agents include, but are not limited to, inorganic sunscreens such as zinc oxide; organic sunscreens such as octyl-methyl cinnamates and derivatives thereof; retinoids; vitamins such as vitamin C, vitamin B, and derivatives thereof; antioxidants including acid such as glycolic acid, citric acid, lactic acid, malic acid, mandelic acid, ascorbic acid, alpha-hydroxybutyric acid, alpha-hydroxyisobutyric acid, alpha-hydroxyisocaproic acid, atrrolactic acid, alpha-hydroxyisovaleric acid, ethyl pyruvate, galacturonic acid, glucopehtonic acid, glucopheptono 1,4-lactone, gluconic acid, gluconolactone, glucuronic acid, glucurronolactone, glycolic acid, isopropyl pyruvate, methyl pyruvate, mucic acid, pyruvia acid, saccharic acid, saccaric acid 1,4-lactone, tartaric acid, and tartronic acid; succinic acid or salts thereof; acids such as beta-hydroxybutyric acid, beta-phenyl-lactic acid, beta-phenylpyruvic acid; botanical extracts such as green tea, soy, milk thistle, algae, aloe, angelica, bitter orange, coffee, goldthread, grapefruit, hoellen, honeysuckle, Job’s tears, lithospermum, mulberry, peony, puerarua, rice, safflower, and mixtures thereof. Suitable amounts of anti-aging agents include, based upon the total weight of the composition, from about 0.01 percent to about 10 percent, and preferably from about 0.04 percent to about 5 percent.
[0030] Examples of suitable depigmentation agents include, but are not limited to, hydroquinone and it derivatives; vitamins such as niacin, vitamin C and its derivatives; extracts such as chamomile and green tea, and mixtures thereof.
[0031] Examples of skin lightening agents include, but not limited to, hydroquinone, catechol and its derivatives, ascorbic acid and its derivatives, and mixtures thereof.
[0032] The compositions of the invention are prepared by well-known procedures. Such procedures include, but are not limited to, mixing the interleukin-1 alpha with other ingredients of the composition in conventional manner. Guidance for the preparation of cosmetic or dermatological compositions of the invention can be found in " Remington: The science and practice of pharmacy" 20th ed. Mack Publishing, Easton PA, 2000 ISBN 0-912734-04-3 and " Encyclopaedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988 ISBN 0-8247-2800-9 or a newer edition. As well known to the skilled person, illustrative additives to dermatological compositions include, but is not limited to: ointment bases, solvents, buffering agents, pH-adjusting agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gel-forming agents, perfumes, and skin protective agents.
[0033] The compositions of the present invention can be formulated in a variety of forms including, but are not limited to, lotions, gels, creams, sprays, and solutions. The compositions of the invention are prepared by methods well-known from the art in accordance with accepted procedures in a variety of forms. Such forms include, but are not limited to, solution, lotion, gel, emulsion, spray, and cream.
[0034] Further, the present invention provides a cosmetic method of improving the appearance of wrinkled, lined, dry, aged, photodamaged, cellulite-affected, scar-affected, stretch mark-affected, and/or acne-affected skin, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0035] Further, the present invention provides a cosmetic method of skin conditioning, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0036] The term “skin conditioning” refers to the maintaining the human skin in a good condition, including regulating the skin condition, preventing or reducing visible and/or tactile discontinuities in skin, and/or protecting the skin as it is indicated in European cosmetic legislation. The visible and/or tactile discontinuities in skin, especially at the skin surface wherein such discontinuities are generally undesirable, may be induced or caused by internal and/or external factors, and include the signs of skin aging described herein. The term “regulating skin condition” includes delaying, minimizing and/or preventing visible and/or tactile discontinuities in skin; ameliorating, e.g., diminishing, minimizing and/or effacing, discontinuities in skin; as well as improving skin appearance and/or feel.
[0037] The methods of the present invention are particularly advantageous for maintenance of aged skin in good condition, including regulating signs of skin aging, more especially visible and/or tactile discontinuities in skin texture associated with aging. “Regulating the signs of skin aging” includes regulating one or more of such signs, e.g., lines, wrinkles or pores. As used herein, regulating such signs includes delaying, minimizing and/or preventing signs of skin aging; as well as ameliorating, e.g., diminishing, minimizing and/or effacing signs of skin aging.
[0038] “Signs of skin aging” include, but are not limited to, all outward visibly and tactilely perceptible manifestations as well as any other macro or micro effects due to skin aging. Such signs may be induced or caused by intrinsic factors or extrinsic factors, e.g., chronological aging and/or environmental damage. These signs may result from processes which include, but are not limited to, the development of textural discontinuities such as wrinkles, including both fine superficial wrinkles and coarse deep wrinkles, skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), scaliness, flakiness and/or other forms of skin unevenness or roughness, loss of skin elasticity (loss and/or inactivation of functional skin elastin), sagging (including puffiness in the eye area and jowls), loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration (including undereye circles), blotching, sallowness, hyperpigmented skin regions such as age spots and freckles, keratoses, abnormal differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the stratum corneum, dermis, epidermis, the skin vascular system (e.g., telangiectasia or spider vessels), and underlying tissues, especially those proximate to the skin.
[0039] Further, the present invention provides a cosmetic method of reducing wrinkles, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0040] Further, the present invention provides a cosmetic method of improving skin elasticity and firmness, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0041] Further, the present invention provides a cosmetic method of reducing cellulite severity, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0042] The term “cellulite” refers to visible and tactile irregularities of skin caused by irregularities of subcutaneous tissue, also is named as orange peel, gynoid lipodystrophy, and etc. Cellulite is frequently accompanied with an increase in regional fat at the cellulite-affected area and is caused by irregularity of dermis-hypodermis junction and fat protrusions to the lower dermis giving rise to visible irregularity at skin surface. The term “cellulite severity” refers to the extent of skin irregularity and can be estimated by a skilled artisan or instrumental methods as it described in public domain, for example, by Smalls LK et al., J Cosmet Sci, 2005, 56(2): 105. The term “reducing cellulite severity” means reducing visible or tactile irregularity of skin surface at cellulite-affected skin area.
[0043] Further, the present invention provides a cosmetic method of enhancing collagen and elastin production in skin, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0044] Further, the present invention provides a cosmetic method of reducing stretch marks, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0045] Further, the present invention provides a cosmetic method of reducing reducing and/or preventing scars, the method comprising the step of applying to the skin of a subject in need thereof the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
[0046] The term “scar” refers to any visible and/or tactile discontinues of a skin resulted from the undesirable overproduction of fibrous tissue during a wound healing process, accompanied with acne, trauma, and surgery including plastic surgery.
[0047] Further, the present invention provides use of the topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle for the preparation of a cosmetic product for skin conditioning, reducing cellulite severity, improving skin elasticity and firmness, reducing wrinkles, enhancing collagen and elastin production in skin, reducing stretch marks, and/or reducing or preventing scars.
[0048] In practicing the methods of the invention, an effective amount of the composition of the invention is topically applied to the skin, and is preferably left on the skin for a period of at least about 15 minutes, more preferably at least about 30 minutes, even more preferably at least about 1 hour, most preferably for at least several hours, e.g., up to about 12 hours. This method can be reapplied from 1 to about 5, preferably from 1 to 3 times per day. Typically, the effective amount of the composition is from about 1 gram to about 100 grams, preferably from about 1 gram to about 20 grams.
[0049] The following examples are presented to demonstrate the invention. The examples are illustrative only and are not intended to limit the scope of the invention in any way.
Example 1
[0050] This example demonstrates the preparation of the peptides of the present invention.
The peptides having an amino acid sequences of SEQ ID NO: 1 through 6 were prepared by solid-phase peptide synthesis (SPPS) technique using Applied Biosystems 430A Peptide Synthesizer following the instructions provided in the instruction manual supplying by the manufacturer.
Example 2
[0051] This example demonstrates the efficacy of hexapeptides of SEQ ID NO: 1 through 6 to inhibit pro-inflammatory action of interleukin-1 alpha.
Cultured human dermal fibroblasts were incubated with 100 ng/ml interleukin-1 alpha with or without of 0.1 mg/ml peptides SEQ ID NO: 1 through 6. Forty eight hours after, levels of interleukin-6 in incubation medium as an index of pro-inflammatory activity of interleukin-1 alpha were measured. Data are presented as mean ± SEM (n=5) and normalized to the levels of interleukin-6 in control cultures.
Treatment IL-6, a.u.
Control 1
Interleukin-1 alpha 27 ± 4
Interleukin-1 alpha plus peptide of:
SEQ ID NO: 1
SEQ ID NO: 2
SEQ ID NO: 3
SEQ ID NO: 4
SEQ ID NO: 5
SEQ ID NO: 6
8 ± 3*
2 ± 2*
5 ± 2*
11 ± 2*
4 ± 3*
8 ± 2*
*Differs significantly of Interleukin-1 alpha (P<0.05)
Example 3
[0052] This example demonstrates the efficacy of hexapeptides to inhibit undesirable effects of interleukin-1 alpha under topical applications.
A water solution of 1 µg/ml interleukin-1 alpha with or without of 100 µg/ml peptide of SEQ ID NO: 2 were applied to a skin surface of volunteers. Unlike the composition without the peptide SEQ ID NO: 2, the composition comprising the peptide SEQ ID NO: 2 does not induce any irritation-like feeling on the skin. Thus, the composition comprising the hexapeptides in combination with interleukin-1 alpha provides particularly advanced properties as compared to the composition without the hexapeptides.
Example 4
[0053] This example demonstrates the topical composition of the present invention.

Ingredient Content, wt.%
Interleukin 1 alpha
Peptide SEQ ID NO: 2
Phosphate buffer
Optional ingredients (fragrance, etc)
Distilled water 0.00001
0.01
qs to pH 5.5
3
to 100
The composition preparation: ingredients are mixed in the conventional manner to prepare the composition.
The method of the use of the composition: 1 ml of the composition is topically applied to the facial skin, and is preferably left on the skin for a period of at least about 15 minutes.
Example 5
[0054] This example demonstrates the cosmetic methods of the present invention.
The composition of example 4 was applied twice-a-day for eight weeks to the cellulite-affected skin region of thighs of fifteen women aged = 35 years with body fat index (BFI) = 25 (33.9 ±0.9).
Efficacy of the composition for reducing cellulite severity was assessed by measurements of irregularity of dermis and dermis-hypodermis junction surface by ultrasonography, using a Dermascan® device at 20-Mhz frequency. Smalls LK et al., Quantitative model of cellulite: three-dimensional skin surface topography, biophysical characterization, and relationship to human perception. J Cosmet Sci. 2005, 56(2):105-20. Along with, the efficacy of the composition for improving skin elasticity and firmness was assessed by a suction method, using a Cutometer®. The efficacy of the composition for keeping a skin in a good condition, or skin conditioning efficacy, was assessed by transepidermal water loss (TEWL), using a Tewameter®. The efficacy of the composition for enhancing collagen and elastin production was assessed by ultrasonography, using a Dermascan® device at 20-Mhz frequency. Results obtained at eight week of the treatment are presented in the Table below in percent of changes of mean to basal level (just before the treatment).
Measure Change
Dermis-hypodermis junction distance -25%
Number of dark pixels (fat depositions in dermis) -27%
Number of bright pixels (collagen plus elastin in dermis) +27%
Elasticity +15%
Firmness +21%
TEWL -18%
Thus, the example shows that topical applying the composition of present invention is effective for reducing severity of cellulite, enhancing collagen and elastin production in skin, improving skin elasticity and firmness, and keeping the skin in a good condition.
Example 6
[0055] This example demonstrates the cosmetic methods of the present invention.
The composition of example 4 was applied twice-a-day for eight weeks to the aging sign-affected skin regions of faces of ten women aged = 45 years. The efficacy of the composition for the reducing signs of aging skin was assessed by a battery of tests. Skin substructure (epidermis plus dermis thickness, dermis density, and collagen) was assessed by ultrasonography, using Dermascan® device with 20-Mhz frequency. Micro-wrinkles deepness and orientation were assessed by profilometry. Deep wrinkles were assessed by laser profilometry on silicon prints. The efficacy of the composition for improving skin elasticity and firmness was assessed by a suction method, using a Cutometer®. The efficacy of the composition for keeping a skin in a good condition, or skin conditioning efficacy, was assessed by transepidermal water loss (TEWL), using a Tewameter®. Results obtained at eight week of the treatment are presented in the Table below in percent of changes of mean to basal level (just before the treatment).

Measure Change
Epidermis plus dermis thickness +21%
Number of bright pixels (collagen plus elastin in dermis) +22%
Elasticity +18%
Firmness +29%
TEWL -21%
Micro-wrinkles:
Isotropy
Mean depth of furrows
+ 12%
- 24%
Deep wrinkles:
Mean wrinkle depth
Wrinkle volume
- 22%
- 19%
Thus, the example shows that topical applying the composition of present invention is effective for reducing wrinkles, enhancing collagen and elastin production in skin, improving skin elasticity and firmness, and keeping the skin in a good condition.
Example 7
[0056] This example demonstrates the cosmetic methods of the present invention.
Three women having the symmetric scars after the plastic surgery treated scars with micro-needle roller with or without (control) the step of applying the composition of the example 4 onto the scars. It was found that the applying the composition decreases the time for the scar reducing as compared to the only roller use. Thus, the example shows that topical applying the composition of present invention is effective for reducing scars.
Example 8
[0057] This example demonstrates the cosmetic methods of the present invention.
Three women having the stretch marks after the pregnancy applied the composition of example 4 onto the stretch marks for eight weeks. It was found that the applying the composition reduced stretch marks as compared to baseline. Thus, the example shows that topical applying the composition of present invention is effective for reducing stretch marks.

SEQUENCE LISTING

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 1
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 1
Tyr Gly Gly Phe Leu Asp
1 5

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 2
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 2
Tyr D-Ala Gly Phe Leu Asp
1 5

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 3
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 3
Tyr Ala Gly Phe Leu Asp
1 5

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 4
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 4
Tyr Gly Gly Phe Leu Glu
1 5

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 5
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 5
Tyr D-Ala Gly Phe Leu Glu
1 5

<110> PETROPAVLOV, POMYTKIN
<120> TOPICAL COMPOSITIONS COMPRISING INTERLEUKIN-1 ALPHA AND PEPTIDES AND COSMETIC METHODS THEREOF
<160> NUMBER OF SEQ ID NOS: 6
<210> SEQ ID NO: 6
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Artificial sequence
<223> OTHER INFORMATION: Synthetic
<400> SEQUENCE: 6
Tyr Ala Gly Phe Leu Glu
1 5

We Claim:-
1. A topical composition comprising interleukin-1 alpha and a hexapeptide of the general formula (I):
Tyr-X-Gly-Phe-Leu-Y (I)
wherein X is independently Gly, L- or D-Ala; Y is independently Asp or Glu; and a dermatologically acceptable vehicle.
2. The composition of claim 1, wherein the content of interleukin-1 alpha in said composition is in the range from 10-7 to 10-4 wt. %.
3. The composition of claim 1, wherein the content of the hexapeptide in said composition is in the range from 10-4 to 10-1 wt. %.
4. The composition of claim 1, which further comprises a buffer at a concentration effective to maintain the pH of the composition at between about 4.0 to about 7.5.
5. The composition of claim 1, wherein the peptide is Tyr-Gly-Gly-Phe-Leu-Asp (SEQ ID NO: 1).
6. The composition of claim 1, wherein the peptide is Tyr-D-Ala-Gly-Phe-Leu-Asp (SEQ ID NO: 2).
7. The composition of claim 1, wherein the peptide is Tyr-L-Ala-Gly-Phe-Leu-Asp (SEQ ID NO: 3).
8. The composition of claim 1, wherein the peptide is Tyr-Gly-Gly-Phe-Leu-Glu (SEQ ID NO: 4).
9. The composition of claim 1, wherein the peptide is Tyr-D-Ala-Gly-Phe-Leu-Glu (SEQ ID NO: 5).
10. The composition of claim 1, wherein the peptide is Tyr-L-Ala-Gly-Phe-Leu-Glu (SEQ ID NO: 6).
Dated this 13th day of August, 2011.