WO2001087292A2

WO2001087292A2 - Compositions for the preservation of collagen and elastin

Info

Publication number: WO2001087292A2
Application number: PCT/CA2001/000687
Authority: WO
Inventors: Stephen R. Smith; Edward A. Charter
Original assignee: Canadian Inovatech, Inc.
Priority date: 2000-05-15
Filing date: 2001-05-14
Publication date: 2001-11-22
Also published as: AU5997801A; WO2001087292A3

Abstract

This invention relates to a method for inhibiting the degradation of collagen and/or elastin. Specifically, the invention relates to the use of ovotransferrin, (also known as conalbumin) to inhibit specific proteases which degrade collagen or elastin. The collagen or elastin may be present in human skin or in cosmetics. The invention also relates to compositions comprising ovotransferrin.

Description

COMPOSITIONS FOR THE PRESERVATION OF COLLAGEN AND

ELASTIN

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to a method for inhibiting the degradation of collagen and/or elastin. Specifically, the invention relates to the use of ovotransferrm, (also known as conalbumin) to inhibit specific proteases which degrade collagen or elastin. The collagen or elastin may be present in human skin or in cosmetics. The invention also relates to cosmetic compositions comprising ovotransferrm.

References

The following publications are cited in this application:

Lower, E. (1987) "Elastin in cosmetics", D & Cl 41-46

Coapman and Schlitz, (1987) "Is Collagen Useful in Topically-applied Skin Care cosmetic Products?" Clin. Res. 35(3) 675A;

U.S. Patent No. 376 808, Pratt, Issued January 24, 1888;

U.S. Patent No. 1 566 271, Cesa, Issued December 22, 1925;

U.S. Patent No. 1 924 972, Beckert et al., issued August 29, 1933;

U.S. Patent No. 2 100 090, Sommer et al., issued November 23, 1937; U.S. Patent No. 5,460,832, Yamaguchi et al., issued October 24, 1995;

U.S. Patent No. 3,843,008, Herr, issued December 9, 1969;

U.S. Patent No. 4,661,340, Nagy nee Kricsfalussy et al., issued April 28, Segarin E., Cosmetics, Science and Technology, 2nd Edition, Vol. 1 (1972)

U.S. Pat. No. 3,755,560, Dickert et al., issued August 28, 1973;

U.S. Pat. No. 4,421,769, Dixon et al., issued December 20, 1983; Hermitte, R. (1991) "Selected Biological Extracts" Cosmetics and

Toiletries 106:53-60.

McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986)

U.S. Pat. No. 4,254,105, Fakuda, issued Mar. 3, 1981 U.S. Pat. No. 5,487,884, Bissett, issued Jan. 30, 1996;

U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985;

U.S. Pat. No. 4,552,872, Cooper et al., issued Nov. 12, 1985;

U.S. Pat. No. 4,557,934, Cooper, issued Dec. 10, 1985;

U.S. Pat. No. 4,130,667, Smith, issued Dec. 19, 1978; U.S. Pat. No. 3,989,816, Rajadhyaksha, issued Nov. 2, 1976;

U.S. Pat. No. 4,017,641, DiGiulio, issued Apr. 12, 1977; and

European Patent Application 0043738, Cooper et al., published Jan. 13, 1982.

All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art

Skin is subject to deterioration through dermatological disorders, environmental abuse (wind, air conditioning, central heating) or through the normal aging process. Consumers are increasingly seeking "anti-aging" cosmetic products which treat or delay the visible signs of aging skin such as wrinkles, lines, sagging, hyperpigmentation and age spots.

The skin can be roughly divided into the epidermis, the dermis and the subcutaneous tissue. Particularly the dermis, as a skin supporting tissue, plays a key role in the maintenance of skin homeostasis. The dermis is configured mainly of fibroblasts, which produce proteins such as collagen to form a constructive structure in these connective tissues.

Collagen, as found in tissues, is not water soluble but has a high water- binding capacity. Collagen is commonly used in topically-applied cosmetic products. Claims for such preparations include moisturization, skin-smoothing, and replacement of age-related loss of dermal collagen (Coapman and Schlitz, (1987) "Is Collagen Useful in Topically-applied Skin Care cosmetic Products?" Clin. Res. 35(3) 675 A). Collagen is degraded by the protease collagense, also known as gelatinase.

Elastin is also commonly present in the skin. Elastin is a highly cross- linked insoluble hydrophobic polymeric schleroprotein, a fibro-protein rich in non- polar amino acids. It is a pale yellow component of connective tissues (along with mucopolysaccharides) with load bearing fibers which are highly refractile and are isotropic and which branch and become fenestrated. Age related changes in the skin's mechanical properties are in part a function of the degradation of the elastin network. In cosmetics elastin can be useful in ameliorating the effects of dry chapped skin, acting as a natural moisturizer, increasing and improving the tension of the skin etc. (Lower, E. (1987) "Elastin in cosmetics", D & Cl 41-46) Elastin is degraded by the protease elastase. It has also been found that the elastin and collagen present in cosmetic compositions degrade during the shelf life of the cosmetic compositions because of the action of collagenase and elastase produced by microbial contaminants inadvertently present in the cosmetic composition. Furthermore, humans contain the proteases, collagenase and elastase, either naturally or as produced by microbes present on their skin. Therefore, upon application of a cosmetic composition comprising either collagen, or elastin to the skin, these products immediately begin to degrade, thereby reducing their effectiveness in the treatment of the skin.

Whole eggs have been used previously in cosmetics. Cosmetic compositions prepared from hen and quail egg are known [U.S. Patent Nos. 376 808; 1 566 271; 1 924 972; 2 100 090]. Whole eggs, egg whites and egg yolks, opened immediately, are undesirable because they are not easily stored and they are liable to quick decomposition since they constitute an excellent nutrient medium for microbes. Furthermore, the consistency, color, odor etc., do not comply with standard of current cosmetics.

Whole egg whites have been treated with proteases to generate an egg white hydrolysate [U.S. Patent No. 5,460,832]. Egg whites from which ovomucoid and ovomucin have been removed have been mixed with preservatives and plastisizers to form a cosmetic [U.S. Patent No. 3,843,008]. Whole quail egg whites have also been mixed into cosmetic formulations [U.S. Patent No. 4,661,340].

It would be advantageous to identify that component of egg whites which inhibits the degradation of elastin and collagen in the human skin and/or in cosmetics. The formulation of such a component in cosmetics would be advantageous.

SUMMARY OF THE INVENTION

The present invention involves a method of inhibiting the degradation of elastin or collagen by the action of elastase or collagenase by the use of ovotransferrin.

As a first aspect of the present invention there is provided a cosmetic composition comprising an effective amount of ovotransferrin or peptide derivatives thereof and a compatible cosmetic carrier.

The present invention also encompasses a cosmetic composition comprising an effective amount of ovotransferrin or peptide derivatives thereof substantially free of other egg proteins and a compatible cosmetic carrier.

The present invention also encompasses a composition comprising collagen or elastin and an inhibitory amount of ovotransferrin or peptide derivatives thereof. Also encompassed is a cosmetic composition comprising collagen or elastin, an inhibitory amount of ovotransferrin and a compatible cosmetic carrier.

In its method aspects, the present invention includes a method for inhibiting the degradation of elastin which method comprises combining with said elastin an inhibitory amount of ovotransferrin or peptide derivatives thereof. This invention is also directed to a method for inhibiting the degradation of collagen which method comprises combining with said collagen an inhibitory amount of ovotransferrin or peptide derivatives thereof.

This invention is also directed to a cosmetic method of providing at least one skin care benefit selected from inhibiting collagen degradation or inhibiting elastin degradation, which method comprises identifying the skin to be treated, applying to the skin a sufficient amount of a topical composition comprising an inhibitory amount of ovotransferrin or peptide derivatives thereof.

This invention is also directed to a method for inhibiting the degradation of elastin or collagen in a cosmetic composition which method comprises providing a cosmetic composition comprising elastin or collagen and adding an inhibitory amount of ovotransferrin to the cosmetic composition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 - 4 show the inhibition of elastase by different ovotransferrin compositions made by the method set forth in Example 1. OREW is the ovotransferrin composition. BE8201, BE8271 and BE8292 are different batches of the ovotransferrin preparation.

Figures 5 - 6 show the inhibition of gelatinase by different ovotransferrin compositions made by the method set forth in Example 1. OREW is the ovotransferrin composition. BE8431 and 12174-3 are different batches of the ovotransferrin preparation. DETAILED DESCRIPTION OF THE INVENTION

Briefly, the present invention involves a method of inhibiting the degradation of elastin or collagen by elastase and collagenase, respectively. The method involves the addition of ovotransferrin to the elastin or collagen. It has been found that the addition of an inhibitory amount of ovotransferrin to a composition comprising elastin or collagen and also containing elastase or collagenase will result in the substantial inhibition of the degradation of the elastin or collagen, respectively.

When discussing such methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

"Ovotransferrin" or "conalbumin" means any protein capable of acting like egg white ovotransferrin. It includes peptide or protein derivatives of ovotransferrin, such as derivatives arising from mutations, insertions or deletions, wherein the peptide or protein derivative has the activity of egg white ovotransferrin. Egg white ovotransferrin, also known as conalbumin, is a glucoprotein with a molecular weight of approximately 78,000 daltons. It has an isoelectric point of 6.1. It contains two lobes connected by an α-helix. Each lobe is homologous and can bind an Fe³⁺ ion. The iron-binding site in each lobe is situated between two sub-domains. The presence of bicarbonate ion may enhance the binding of iron to the molecule.

Skin care cosmetic products often contain natural ingredients such as elastin and collagen. "Elastin" is a highly cross-linked insoluble hydrophobic polymeric schleroprotein, a fibro-protein rich in non-polar amino acids such as valine, leucine, isoleucine and phenylalanine. It is a pale yellow component of connective tissues (along with mucopolysaccharides) with load bearing fibers which are highly refractile and are isotropic and which branch and become fenestrated, the strength of the fibers being about one-tenth that of collagen. Elastin is one of only a few polymeric substances which, in the presence of water, exists in a form with rubber-like extensibility and low modulas of elasticity. After collagen, elastin is the most abundant constituent of the connective tissues and on a molecular level it has been reported to be in part analogous to collagen, being built up on a triple- chain helical arrangement of amino acid residues. At least two types of elastin are distinguishable, type I elastin isolated from aorta or skin and type II elastin isolated from cartilage. Age related changes in the skin's mechanical properties being in part a function of the degradation of the elastin network.

Elastin may be prepared from bovine neck ligaments, from ox tendon, from cutaneous sources and from human aortas. Elastin is commercially available as unhydrolyzed pure and sterile (Lower, E. (1987) "Elastin in Cosmetics" D&C1 41-46).

"Elastase" is a protease of broad specificity with a molecular weight of about 25,000. It is capable of hydrolyzing proteins at N-terminal peptide bonds of aliphatic residues. This enzyme breaks down elastin, fibrin, hemoglobulin, albumin, soy proteins and casein.

Collagen as found in tissues is not water soluble, but has a high water- binding capacity. Collagen is a large molecule (mw 300,000) made up of three alpha helical peptide chains twisted together. Each chain contains roughly 1,200 amino acids, with a great proportion of the uncommon hydroxyproline. At the ends of the alpha chains there are non-helical portions, known as the telopeptides, where linkage between chains takes place.

Collagen derivatives are prepared in accordance with refined biochemical methods. The starting material is usually shredded corium collagen from young cattle that is treated with water and alcohol, under controlled temperature and pH, to eliminate undesirable materials and to avoid denaturation. Hermitte, R. (1991) "Selected Biological Extracts" Cosmetics and Toiletries 106:53-60. Collagen is commercially available.

"Collagenase" is a proteolytic enzyme capable of digesting native undenatured collagen. It is found in Clostridium bacteria culture filtrates.

Various proteases degrade elastin and collagen. These proteases are elastase (degrades elastin) and collagenase, also known as gelatinase (which degrades collagen). This degradation may occur during the shelf life of the cosmetic if the proteases contaminate the cosmetic. Furthermore, natural human skin, or microbes present on the skin produce these proteases and therefore, upon application of the cosmetic to the skin the elastin or collagen present in the cosmetic is rapidly degraded.

It has been found that an extract of egg white inhibits elastase and collagenase. This extract may be prepared by the process set forth herein. The extract is substantially pure ovotransferrin (or conalbumin).

The term "substantially free of other egg proteins" or "substantially pure" means that the ovotransferrin composition comprises at least about 90% ovotransferrin, more preferably at least 95% ovotransferrin and most preferrably at least about 95% ovotransferrin.

The term "effective amount" or "inhibitory amount" means that amount of ovotransferrin composition which is capable of inhibiting the degradation of collagen or elastin. At the lower end of the amount, the composition will have an amount of ovotransferrin composition of about 0.001 %, about 0.005%, about 0.01 %, about 0.02%, about 0.05% , about 1 %, about 2%, to about 4% . At the higher end of the amount, the composition will have an amount of ovotransferrin of about 5%, about 10% , about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60% , about 70%, about 80%, to about 90% . Preferably, the amount of ovotransferrin in a cosmetic composition will be from about 0.001 % to about 90%, more preferably from about 0.005% to about 50%, more preferably from about 0.01 % to 10% and most preferably from about 0.05% to about 5%.

The term "inhibition" means the degradation of either collagen or elastin by elastase or collagenase/gelatinase respectively is substantially reduced. Inhibition can be measured by methods known in the art such as the assays set forth herein. The degradation is inhibited if less that 50% of the collagen or elastin is degraded, more preferably, less than 60% is degraded, and most preferably less than 80% of the collagen or elastin is degraded.

It is understood that the ovotransferrin may be added to a composition containing either collagen or elastm as a measure to prevent degradation of the collagen or elastin should the composition become contaminated by microbes producing elastase or collagenase. The ovotransferrin may also be added to the composition suspected of containing elastase or collagenase prior to the addition of the collagen or elastin. The method of the present invention may be carried out by the application of the ovotransferrin composition or a cosmetic composition containing ovotransferrin, one or more times daily to skin which requires the treatment. In general, a small quantity of the cosmetic composition, for example, from 0.1 to 5 ml is applied to the skin from a suitable container or applicator and spread over and/or rubbed into the skin using the hands or fingers or a suitable device.

The cosmetic compositions of the present invention may include a number of compatible components typically found in cosmetic compositions. The term "compatible", as used herein, means that the components of the composition must be capable of being commingled with the ovotransferrin composition and with each other, in a manner such that there is no interaction which would substantially reduce the efficacy of the cosmetic composition for preventing degradation of elastin or collagen in the skin or in a cosmetic composition containing elastin or collagen.

The cosmetic compositions of the present invention contain a safe and effective amount of an acceptable carrier. The term "compatible topical carrier" or "compatible cosmetic carrier" encompasses both pharmaceutically-acceptable carriers and cosmetically-acceptable carriers, and it encompasses substantially nonirritating compatible components (either taken alone or in mixtures) which are suitable for delivering the ovotransferrin to the skin. These carriers must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for chronic topical administration to the skin of humans or lower animals. The term "safe and effective amount" of carrier means an amount sufficient to deliver the ovotransferrin to the skin but not so much as to cause any side effects or skin reactions, generally from about 50% to about 99%, preferably from about 90% to about 98%, of the composition. The cosmetic compositions of the present invention may be made into a wide variety of product types. These include, for example, lotions, creams, beach oils, gels, sticks, sprays, ointments, pastes, mousses and cosmetics. These product types may comprise either of two basic types of carrier systems, i.e., solutions and emulsions.

The cosmetic compositions of the present invention formulated as solutions typically include a pharmaceutically- or cosmetically-acceptable organic solvent. The terms "pharmaceutically-acceptable organic solvent" and "cosmetically-acceptable organic solvent" refer to an organic solvent which, in addition to being capable of having dispersed or dissolved therein the ovotransferrin, also possesses acceptable safety (e.g. irritation and sensitization characteristics), as well as good aesthetic properties (e.g., does not feel greasy or tacky). The most typical example of such a solvent is water. Examples of suitable organic solvents also include: mono- or polyhydric alcohols, e.g. ethanol, cetyl alcohol, octyl-dodecanol, oleyl alcohol, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, isopropanol, butanediol, and mixtures thereof; fatty acids, e.g. stearic acid or mysristic acid; alkali metal salts of fatty acids; fatty acid esters, e.g. propyl myristate or esters of oleic acid formed with fatty alcohols, etc.; paraffin oil, wax; vaseline, woolfat etc.

If the cosmetic compositions of the present invention are formulated as an aerosol and applied to the skin as a spray-on, a compatible propellant is added to a solution composition. Examples of propellants useful herein include the chlorinated, fluorinated and chloro-fluorinated lower molecular weight hydrocarbons. Other propellants useful in the present invention include lower molecular weight hydrocarbon mixtures (e.g., the mixture of butane, isobutane and propane known commercially as Propellant A46, made by Phillips Chemical Co., a subsidiary of Phillips Petroleum Company), ethers and halohydrocarbons such as dimethyl ether or dichlorodifluoromefhane alone or mixtures thereof with dichlorotetrafϊuoroethane. Mixtures of hydrocarbon and halohydrocarbon propellants and nitrous oxide may also be used. Nitrogen and carbon dioxide can also be used as propellant gases. They are used at a level sufficient to expel the contents of the container. A more complete disclosure of propellants useful herein can be found in Sagarin, Cosmetics Science and Technology, 2nd Edition, Vol. 2, pp. 443-465 (1972), incorporated herein by reference.

Alternatively, emollients may comprise the carrier system of the present invention formulated as a solution. An example of a composition formulated in this way would be a beach oil product. Such compositions contain from about 2% to about 50% of a pharmaceutically/cosmetically-acceptable emollient. As used herein, "emollients" refer to materials used for the prevention or relief of dryness, as well as for the protection of the skin. A wide variety of suitable emollients are known and may be used herein. Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972), incorporated herein by reference, contains numerous examples of suitable materials.

A lotion can be made from a solution carrier system. Lotions typically comprise from about 0.001 % to about 20%, preferably from about 0.002% to about 10% , of the ovotransferrin; from about 1 % to about 20%, preferably from about 5% to about 10%, of an emollient; and from about 50% to about 90%, preferably from about 60% to about 80% of water. Another type of product that may be formulated from a solution carrier system is a cream. A cream of the present invention would comprise from about 0.001 % to about 20% , preferably from about 0.002% to about 10%, of the ovotransferrin; from about 5% to about 50%, preferably from about 10% to about 20%, of an emollient, and from about 45% to about 85%, preferably from about 50% to about 75%, water.

Yet another type of product that may be formulated from a solution carrier system is 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. Examples of such ointment bases include, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases may be oil-in-water or water-in-oil emulsions. Ointment carriers may also be water soluble. Examples of such ointment carriers include glycol ethers, propylene glycols, polyoxyl stearates, and polysorbates. An ointment may also comprise from about 2% to about 10% of an emollient plus from about 0.1 % to about 2% of a thickening agent. Examples of suitable thickening agents include: cellulose derivatives (e.g., methyl cellulose and hydroxy propylmethyl cellulose), synthetic high molecular weight polymers (e.g., carboxy vinyl polymer and poly vinyl alcohol), plant hydrocolloids (e.g., karaya gum and tragacanth gum), clay thickeners (e.g., colloidal magnesium aluminum silicate and bentonite), and carboxy vinyl polymers. A more complete disclosure of thickening agents useful herein can be found in Segarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 72-73 (1972), incorporated herein by reference.

If the carrier is formulated as an emulsion, from about 1 % to about 10%, preferably from about 2% to about 5%, of the carrier system comprises an emulsifier. Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiers are disclosed in, for example, U.S. Pat. No. 3,755,560, issued Aug. 28, 1973, Dickert et al,; U.S. Pat. No. 4,421,769, issued Dec. 20, 1983, Dixon et al.; and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324 (1986); the disclosures of which are incorporated herein by reference. Preferred emulsifiers are anionic or nonionic, although the other types may also be used.

Single emulsion skin care preparations, such as lotions and creams, of the oil-in-water type and water-in-oil type are well-known in the cosmetic art and are useful in the present invention. Multiphase emulsion compositions, such as the water-in-oil-in-water type, as disclosed in U.S. Pat. No. 4,254,105, Fakuda et al., issued Mar. 3, 1981, are also useful in the present invention. In general, such single or multiphase emulsions contain water, emollients and emulsifiers as essential ingredients.

Triple emulsion carrier systems comprising an oil-in-water-in-silicone fluid emulsion composition as disclosed in U.S. Pat. No. 5,487,884, Bissett, issued Jan. 30, 1996 herein incorporated by reference, are also useful in the present invention. This triple emulsion carrier system can be combined with from about 0.001 % to about 20% , preferably from about 0.002% to about 10% , of the ovotransferrin to yield the pharmaceutical/cosmetic composition of the present invention.

Another emulsion carrier system useful in the pharmaceutical/cosmetic compositions of the present invention is a microemulsion carrier system. Such a system comprises from about 9% to about 15% squalane; from about 25% to about 40% silicone oil; from about 8% to about 20% of a fatty alcohol; from about 15% to about 30% of polyoxyethylene sorbitan mono-fatty acid (commercially available under the trade name Tweens) or other nonionics; and from about 7% to about 20% water. This carrier system is combined with from about 0.002% to about 10% of the ovotransferrin. Lotions and creams can be formulated as emulsions as well as solutions. Typically such lotions comprise from about 0.001 % to about 20% , preferably from about 0.002% to about 10%, of the ovotransferrin; from about 1 % to about 20%, preferably from about 5% to about 10%, of an emollient; from about 25% to about 75% , preferably from about 45% to about 95% of water; and from about 1 % to about 10%, preferably from about 2% to about 5%, of an emulsifier. Such creams would typically comprise from about 0.001 % to about 20% , preferably from about 0.002% to about 10%, of the ovotransferrin; from about 1 % to about 20%, preferably from about 5% to about 10%, of an emollient; from about 20% to about 80%, preferably from about 30% to about 70%, water; and from about 1 % to about 10%, preferably from about 2% to about 5%, of an emulsifier.

If the cosmetic compositions of the present invention are formulated as a gel or a cosmetic stick, a suitable amount of a thickening agent, is added to a cream or lotion formulation.

The cosmetic compositions of the present invention may also be formulated as makeup products such as foundations, or lipsticks. Foundations are solution or lotion-based with appropriate amounts of thickeners, pigments and fragrance. Lipsticks are composed essentially of an oil- wax base stiff enough to form a stick, with pigmentation dispersed therein.

The topical cosmetic compositions of the present invention may contain, in addition to the aforementioned components, a wide variety of additional oil-soluble materials and/or water-soluble materials conventionally used in topical compositions, at their art-established levels. Among the optional oil-soluble materials are nonvolatile silicone fluids, such as poly dimethyl siloxanes with viscosities ranging from about 10 to about

100,000 centistokes at 25 °C. These siloxanes are useful to enhance skin feel.

These optional oil-soluble materials may comprise up to about 20% of the total composition, preferably up to about 10%.

Various water-soluble materials may also be present in the compositions of this invention. These include humectants, such as glycerol, sorbitol, propylene glycol, alkoxylated glucose and hexanetriol, ethyl cellulose, polyvinyl alcohol, carboxy methyl cellulose, vegetable gums and clays such as Veegum.RTM. (magnesium aluminum silicate, R. T. Vanderbilt, Inc.); proteins and polypeptides; preservatives such as the methyl, ethyl, propyl and butyl esters of hydroxybenzoic acid (Parabens-Mallinckrodt Chemical Corporation), EDTA, methylisothiazolinone and imidazolidinyl ureas (Germall 115~Sutton Laboratories); and an alkaline agent such as sodium hydroxide or potassium hydroxide to neutralize, if desired, part of the fatty acids or thickener which may be present.

In addition, the topical compositions herein can contain conventional cosmetic adjuvants, such as dyes, opacifiers (e.g., titanium dioxide), pigments and perfumes.

In addition the cosmetic composition may contain surfactants. The surfactants can be of an anionic, cationic or nonionic character, e.g. sodium lauryl sulfate, polyoxyethylene fatty acid esters, sorbitan fatty acid esters, polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fatty acid esters, etc. The cosmetic compositions may further contain antiseptic agents such as boric acid, sorbic acid, benzoic acid, salicylic acid-hydroxybenzoic acid etc.

The cosmetic compositions may also contain light-protecting agents. As a light-protecting agent 2-hydroxy-4-methoxy-benzophenone-5-sulfonic acid can be used.

The cosmetic compositions of the present invention may also include a safe and effective amount of a penetration enhancing agent. By "safe and effective amount" is meant an amount sufficient to enhance penetration of the ovotransferrin into the skin but not so much as to cause any side effects or skin reactions, generally from about 1 % to about 5% of the composition. Examples of useful penetration enhancers, among others, are disclosed in U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985; U.S. Pat. No. 4,552,872, Cooper et al., issued Nov. 12, 1985; U.S. Pat. No. 4,557,934, Cooper, issued Dec. 10, 1985; U.S. Pat. No. 4,130,667, Smith, issued Dec. 19, 1978; U.S. Pat. No. 3,989,816, Rajadhyaksha, issued Nov. 2, 1976; U.S. Pat. No. 4,017,641, DiGiulio, issued Apr. 12, 1977; and European Patent Application 0043738, Cooper et al., published Jan. 13, 1982. U.S. Pat. No. 4,537,776 teaches a penetration-enhancing vehicle consisting essentially of a) N-(2-hydroxyethyl) pyrrolidone and b) a cell envelope disordering compound selected from methyl laurate, oleic acid, oleyl alcohol, monoolein, myristyl alcohol, and mixtures thereof, wherein component (a) and (b) are present in a ratio of (a):(b) of about 1:5 to about 500:1 by weight. U.S. Pat. No. 4,557,934 teaches a pharmaceutical composition comprising the penetration enhancing agent l-dodecylazacycloheptan-2-one, and a penetration enhancing diol or cycloketo compound selected from the group consisting of: 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, pyrrolidone; l-(2-hydroxyefhyl)-azacyclopentan-2-one, and mixtures thereof. U.S. Pat. No. 4,130,667 describes a penetration enhancer comprising:

(a) at least about 0.1 % by weight of a sugar ester selected from sucrose monooctanoate, sucrose monodecanoate, sucrose monolaurate, sucrose monomyristate, sucrose monopalmitate, sucrose monostearate, sucrose monooleate, and sucrose dioleate; and

(b) at least about 0.1 % by weight of a phosphine oxide compound selected from octyldimefhyl phosphine oxide, nonyl dimethyl phosphine oxide, decyl dimethyl phosphine oxide, undecyl dimethyl phosphine oxide, dodecyl dimethyl phosphine oxide, 2-hydroxy decyl dimethyl phosphine oxide, 2-hydroxy undecyl dimethyl phosphine oxide, and 2-hydroxy dodecyl dimethyl phosphine oxide.

Other conventional skin care product additives may also be included in the compositions of the present invention. For example, hydrolysates, primrose oil, jojoba oil, epidermal growth factor, soybean saponins, mucopolysaccharides, and mixtures thereof may be used.

Various vitamins may also be included in the compositions of the present invention. For example, Vitamin A, and derivatives thereof, Vitamin B₂, biotin, pantothenic, Vitamin D, and mixtures thereof may be used.

The compositions of the invention are prepared by admixing the components in an optional order of succession. The order of succession of the addition of the components is determined by the properties, particularly the solubility of the said components and the type of compositions to be prepared.

The following examples are offered to illustrate this invention, and are not to be construed in any way as limiting the scope of this invention. EXAMPLES In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined it has its generally accepted meaning. Unless otherwise stated, all temperatures are in degrees Celsius. g := grams mg = milligram μg = microgram

L = liter ml = milliliter μl = microliter μm = micron ppm = parts per million rpm = revolutions per minute

BSA bovine serum albumin

Example 1

Preparation of egg white extract

Raw egg white was diluted 3 parts water: 1 part egg white and the pH was adjusted to 5.5. The solution was clarified by filtering through Star filter press pads (Ahlstrom Paper Group Mt. Holly Springs, PA), followed by filtering through XE400 filter press pads (Carlson Filtration Ltd., Lancashire, England).

A streamline 200 Expanded Bed chromatography column was packed with Streamline SP resin (Amercham Pharmacia Biotech, Baie D'Urfe Quebec Canada) that was equilibrated to pH 5.5 using 20 mM citrate/phosphate buffer. The filtered solution was pumped onto the column using the method of expanded bed adsorption. Water was pumped through the column to remove unbound products. The bound proteins were eluted off the column using a salt buffer (20 mM citrate/phosphate buffer plus 1M NaCl at pH 5.5). The collected proteins were concentrated and dialyzed using tangential flow filtration across an ultra-filtration membrane.

The resulting product was clarified by filtering through a 0.5 μm. gradient density cartridge filter (Domnick-Hunter, Birley, England). The filtrate was then sterile filtered through a 0.2 μm PROPORE-PES membrane cartridge filter (Domnick-Hunter, Birley, England). The filtered product was lyophilized to dryness.

The dried solids content of the filtered product comprises greater than 90% ovotransferrin by weight. The product comprises greater than 90% ovotransferrin protein relative to total protein on a dry protein basis.

Example 2 Elastase Inhibition by Ovotransferrin

The ability of the ovotransferrin composition to inhibit the activity of elastase to degrade elastin was tested using a method similar to that provided by the Sigma Chemical Company, "Enzymatic Assay of Elastase" EC 3.4.21.36.

The ovotransferrin solution was diluted to an appropriate dilution in 100 mM Tris-HCl, pH 8.0 at 25 °C. A 4.4 mM solution of the reagent, N-succinyl, ALA-ALA-ALA-p-nitroanilide was made. Immediately before use an elastase solution containing 0.2 - 0.5 units/ml of elastase (Sigma Chemical Company, St. Louis, MO) in 100 mM Tris-HCl, pH 8.0 was made. 2.70 ml of ovotransferrin solution was mixed with 0.2 ml of the reagent solution in a test tube and incubated for 1 minute in a 25 °C water bath. 0.1 ml elastase solution was added, the solution was mixed and the change in absorbance at 410nm over 5 minutes was measured.

Calculation

units/ml enzyme = (ΔA 410nm Test - ΔA 410nm Blanks (3) (dfl

(8.8X0.1) 3 = total volume (in milliliters) of assay df = dilution factor 8.8 = millimolar extinction coefficient of p-nitroaniline at 410 nm at pH 8.0 0.1 = volume (in milliliters) of enzyme used.

One unit of enzyme will hydrolyze 1.0 micromole of succinyl-ala-ala-ala-p- nitroanilide per minute at pH 8.0 at 25 °C.

Figures 1-4 show the inhibition of elastase by the ovotransferrin solution made by the method of Example 1.

Example 3 Inhibition of Gelatinase by Ovotransferrin

The ability of the ovotransferrin composition to inhibit the activity of gelatinase to degrade collagen was tested using a method similar to that provided by Boehringer Mannheim for Gelatinase B 92 kD, type IV collagenase. 0.5 mU (5 μl) of 92 progelatinase (Roche Diagnostics, Laval, Quebec, Canada) was activated in 3-5 ml glass tubes for 10 minutes at 37 °C with 10 μl of trypsin solution [3 mg Trypsin dissolved in 5 ml of 20 mM Tris-HCl, pH 7,5, 100 mM NaCl, 5 mM CaClJ adjusted to a total volume of 110 μl with activation buffer [50 mMTris-HCl, pH 7,5, 0.5 M NaCl, 5 mM CaClJ. Activation was halted by adding 10 μl of aprotinin solution [3.2 mg aprotinin (Sigma) is dissolved in 2 ml of 20 mM Tris-HCl, ρH7.5, 100 mM NaCl, 5mMCaCl₂] and incubated for 10 min. at 37 °C.

100 μl of the DNP-peptide solution [dinitrophenol-tagged substrate peptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg was dissolved in 10 ml of 50 mM Tris- HCl, pH 7.5, 0.15 M NaCl, 5 mM CaCl₂, 0.2 mg/ml BSA ] was added to the activated enzyme and incubated for a further 15 minutes at 37 °C. During the reaction gelatinase cleaves the Gly-Ile bond thereby yielding a hydrophobic DNP- tagged tripeptide. The reaction was stopped by mixing with 500 μl of 1 N HCI. The reaction mix was extracted with 1.6 ml of ethylacetate: 1-butanol [1:0.15(v/v)] solution. To enhance phase separation, the mixture was centrifuged briefly. The extinction of the organic(upper) phase was measured at 366 nm wavelength.

Calculation activity U/ml = ΔA-V e-d-t-v

ΔA = adsorbance _sample - adsorbance _blank at 366 nm

V = volume of organic phase 1.6 ml v = volume of added enzyme 0.005 ml e₃₆₆ ⁼ 17.3 μmol^cm² d = width of cuvette (1 cm) t = reaction time: 15 min Figures 5 - 6 show the inhibition of gelatinase by the ovotransferrin madethod similar to that set forth in Example 1.

Claims

Claims:

1. A cosmetic composition comprising an effective amount of ovotransferrin and a compatible cosmetic carrier.

2. A cosmetic composition comprising an effective amount of ovotransferrin substantially free of other egg proteins and a compatible cosmetic carrier.

3. A composition comprising collagen or elastin and an inhibitory amount of ovotransferrin.

4. The cosmetic composition according to Claim 3, wherein the active ingredient is collagen.

5. The cosmetic composition according to Claim 3, wherein the active ingredient is elastin.

6. A method for inhibiting the degradation of elastin which method comprises contacting said elastin with an inhibitory amount of ovotransferrin.

7. A method for inhibiting the degradation of collagen which method comprises contacting said collagen with an inhibitory amount of ovotransferrin.

8. A cosmetic method of providing at least one skin care benefit selected from inhibiting collagen degradation or inhibiting the elastin degradation in the skin, which method comprises identifying the skin to be treated, applying to the skin surface a sufficient amount of a topical composition comprising an inhibitory amount of ovotransferrin.

9. A method for inhibiting the degradation of elastin or collagen in a cosmetic composition which method comprises providing a cosmetic composition comprising elastin or collagen, and adding an inhibitory amount of ovotransferrin to the cosmetic composition.