AU614832B2 - Storage-stable compositions and methods for treating keratinous tissue - Google Patents

Description

p( OPI DATE 22/09/89 APPLN. ID 33523 89 '\OJP DAT 9yJ 0 PC J N ER PCT/US89/U0873

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9 INTERNA .EATY (PCT) (51) International Patent Classification 4 A61K 7/00, 7/06, 7/04 A61K 7/48 (11) International Publication Number: WO 89/ 079301 Alal Publiio D September 1898.09 (43) International Publication Date: 8 September 1989 (08.09.89) (21) International Application Number: PCT/US89/00873 (22) International Filing Date: (31) Priority Application Numbers: 3 March 1089 (03.03.89) 164,009 223,295 (32) Priority Dates: (33) Priority Country: 4 March 1988 (04.03.88) 22 July 1988 (22.07.88) (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FI, FR (European pater't), GB (European patent), HU, IT (European patent), i', KR, LU (European patent), NL (European patent), NO, RO, SE (European patent), SU.

Published With international search report.

(71) Applicant: CIRO'S TOUCH, LTD. [US/US]; 51R Bissell Road, Lebanon. NJ 08833 (US).

(72) Inventor: ROTHMAN, John 51R Bissell Road, Lebanon, NJ 08833 (US).

(74) Agent: COLEMAN, Henry, D; Darcy, Donovan Coleman, 71 Broadway, Suite 2200, New York, NY 10006 (US).

(54) Title: STORAGE-STABLE COMPOS.TIONS AND METHODS FOR TREATING KERATINOUS TISSUE (57) Abstract This invention relates to novel storage stable activated protein containing compositions comprising reducing agents, for example ammonium thioglycollate, and optionally, oxidizing agents, for example, hydrogen peroxide and/or antioxidants, preferably volatile antioxidants. The compositions are useful for conditioning horny keratinous tissue of mammals such as human hair and nails, and the hooves and fur of animals to improve their strength and appearance. In addition, these compositions are useful for promoting hair and nail growth.

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WO 89/07930 PCT/US89/00873 STORAGE-STABLE COMPOSITIONS AND METHODS FOR TREATING KERATINOUS TISSUE This is a continuation-in-part application of United States Patent Application Serial No. 223,295, entitled "Storage Stable Compositions and Methods for Treating Keratinous Tissue", filed July 22, 1988, which is a continuation-in-part application of United States Patent Application Serial No. 164,009, entitled "Compositions For Treating Keratinous Tissue", filed March 4, 1988.

BACKgROUND OF THE INVENTION This invention relates to novel storage stable activated protein containing compositions comprising reducing agents, oxidizing agents and/or anti-oxidants. The compositions are useful for conditioning horny keratinous tissue of mammals such as human hair and nails, and the hooves and fur of animals to improve their strength and appearance. In addition, these compositions are useful for promoting hair growth.

United States Patent 4,438,102 describes compositions which are useful for promoting the growth of normal dermal and epidermal tissue, more specifically the compositions are useful to promote wound healing in the soft keratinous tissue of the epidermis. Several examples of wound healing are provided in the specification.

The compositions of the prior patent are described as containing defined percentages of thioglycollic acid, ammonium hydroxide, glycerine, citric acid, hydrogen peroxide, commercial gelatin, a lower alkanol, and a solvent such as acetone or diethyl ether. The commercial grade gelatins disclosed by the patent contain do not contain sufficient cysteinyl content for use in the present invention.

It is known to wave hair permanently by a first treatment with a reducing mixture of thioglycollic acid and ammonium hydroxide. The mixture may also contain a protein.

The hair is subsequently treated in a second step with an oxidizing agent such as hydrogen peroxide or sodium bromate.

The procedures are described, for example, in U.S. Patents 4,158,704; 3,95/,605; 3,842,848; and several others.

The reducing step is believed to break the disulfide bonds of cystine disulfide bridges in the hair to form WO 89/07930 PCT/US89/00873 2 sulfhydryl groups. The hair is shaped under tension or pressure in the presence of the reducing agent. In a subsequent step, the oxidizing agent is contacted with the hair while it is still under tension to oxidize the sulfhydryl groups and reform disulfide bonds with the hair in the new shape.

U.S. Patent 3,842,848 describes a method of bonding especially prepared hydrolyzed peptide products of keratinaceous materials to human hair. The process is effected by conducting the reducing step of permanent waving in the presence of the peptide products and, thereafter, in a second step, oxidizing.

It has now been discovered that activated protein compositions which are used to chemically bond to hair and other horny keratinous tissue may be formulated with reducing agents, oxidizing agents and/or anti-oxidant components to produce compositions for treating horny keratinous tissue.

Especially advantageous characteristics of these compounds include storage stability and the ability to effectuate a reduction and oxidation process to form covalent disulfide linkages without having to resort to two separate solutions.

Thus, the essential ingredients of the compositions of this invention are the reducing agent, the oxidizing agent, the protein and/or the antioxidant. Numerous additional components may also be included, for example water, bases, acids, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, coloring agents and perfuming agents.

It is a surprising result that compositions of the present invention may be formulated in such a way that activated proteinaceous material in combination with reducing agent and oxidizing agent may produce a composition which can activate natural keratinous tissue and bind activated protein to the activated natural keratinous tissue.

It is an object of the present invention to provide novel compositions containing at least one activated thiolcontaining protein in combination with a reducing agent and an oxidizing agent. It is an additional object of the present invention to provide storage stable compositions for treating horny keratinous tissue of mammals at least in part by for- WO 89/07930 PCT/US89/00873 3 mulating the activated thiol-containing protein in combination with an antioxidant.

It is a further object of the present invention to provide storage stable compositions for treating horny keratinous tissue which are capable of maintaining the activity of an activated protein in the presence of an oxidizing agent to promote the shelf-life of the composition, and which can promote the formation of disulfide linkages between the activated protein and the treated keratinous tissue in one composition.

It is an additional object of the present invention to provide a new method for conditioning horny keratinous tissue which includes reducing the horny keratinous tissue and oxidizing said tissue without having to apply two separate solutions to the treated tissue to perform the reducing and oxidizing steps.

BRIEF DESCRIPTION OF THE INVENTION The novel compositions of the present invention are aqueous compositions having a pH of from about 4.0 to about 9, preferably a pH of about 7 to about 8, and most preferably a pH of about 7.6 (physiological pH). Compositions of the present invention may comprise a reducing agent, an activated protein, and an oxidizing agent, in addition to other less essential components. Other embodiments of the present invention may comprise a reducing agent, an activated protein, and an antioxidant. The compositions containing an hntioxidant may also contain an oxidizing agent. In those compositions where an anti-oxidant is used in combination with an oxidizing agent, it is preferred that the antioxidant used is a volatile antioxidant and the oxidizing agent used is a non-volatile oxidizing agent.

The protein used is an activated protein. An activated protein is a protein which has been subjected to a reducing agent, for example a thiol-containing compound, which results in the breaking of disulfide bonds of cystine residues within the protein structure to produce free thiol or mercaptide groups on cysteine residues. The ability of a protein to bind to native keratin is believed to be related in part to WO 89/07930 PCT/US89/00873 4 the number of thiol or mercaptide groups on the protein which are free to bind to mercaptide or thiol groups on the native keratin. Also, it is believed that the ability of the composition to oxidize the keratin mercaptide and a proximal protein mercaptide enhances the probability of disulfide covalent bond formation. Production of disulfide covalent bonds may produce the strongest interaction between activated protein and keratinous tissue. Any promotion in growth related to the binding of protein to keratinous tissue would be expected to be of greatest duration where covalent bonding as opposed to electrostatic or ionic binding occurs.

The protein in the compositions of the present invention may react with and form chemical bonds with the hard keratin of human and animal hair, nails and skin, thus effecting decreased hair breakage, increased hair thickness, increased nail hardness, decreased nail splitting and delamination and attachment of moist hydrated proteins to skin. The compositions are therefore useful for treating human hair, nails and skin to chemically bond the activated protein. In addition, compositions of the present invention are useful to promote wound healing in mammals. The compositions may also function as topical pharmaceutical carriers and vehicles.

The amount of activated protein that bonds to the keratinous tissue will vary with the concentration of reducing agents in the composition and the number of activated thiol or mercapto groups in the activated protein and the keratinous tissue. The time that the reducing agent is in contact with the keratinous tissue is also important; the longer the keratinous tissue is in contact with the reducing agent, the greater will be likelihood of a protein-keratinous tissue covalent bond formation.

The treatment using compositions of the present invention is preferably initially conducted at ambient temperatures, about 20"C to about 35°C; however, higher temperatures may be used. The keratinous tissue may be treated over a period of time ranging from about 10 minutes to about 6 hours. The compositions of the present invention may WO 89107930 PCT/US89/00873 also be formulated as sustained or controlled release formulations for prolonging treatment beyond 6 hours. Reaction is effected by bringing the compositions of the present invention into contact with the keratinous substrate to be treated and allowing the treated tissue to dry. The time of contact may be varied at will.

In certain embodiments of the present invention employing a volatile antioxidant, it may be preferred to subject the treated hair or tissue to heat under a hair dryer or similar heat source to volatilize and remove the antioxidant.

By removing the antioxidant in this way, oxidation to promote covalent disulfide formation by the oxygen in ambient air may be promoted. Preferred compositions may employ non-volatile oxidizing agents which may promote oxidation after the volatile antioxidants are removed from the formulations.

In a first aspect of the present invention, exemplary embodiments comprise about 0.01 to about 12.0% by weight of an activated protein component, about 0.1 to about 15% by weight of a compatible reducing agent, about 0.001 to about 4.0% by weight of an oxidizing agent, and at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents and perfuming agents.

Alternative embodiments of the present invention may include an anti-oxidant instead of an oxidizing agent.

Exemplary compositions comprise about 0.01 to about 12.0% by weight of an activated protein component, about 0.1 to about by weight of a compatible reducing agent, about 0.01 to about 2.0% by weight of an antioxidant and at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents, preservatives and perfume agents.

Additional embodiments of the present invent n comprise about 0.01% to about 12.0% by weight of an activated protein component, about 0.1 to about 15% of a compatible reducing agent, about 0.001 to about 4.0% by weight of an _e II 1P_ WO 89/079930 PCT/US89/00873 6 oxidizing agent and about 0.01 to about 4.0% by weight of antioxidant, preferably a volatile antioxidant and at least one component selected from the group consisting of water, acids, bases, buffering agents, solvents, emulsifying agents or surfactants, thickeners, coloring agents, preservatives and perfume agents.

The compositions of the present invention are formulated to enhance the formation of free mercaptide or thiol groups in the protein and the keratinous tissue to maximize the probability that a free thiol in the protein and a free thiol in the keratinous tissue will form a covalent disulfide bond. The inclusion of an oxidizing agent in the same composition as the reducing agent and activated protein is designed to maximize covalent disulfide formation without using a second oxidizing solution.

The compositions of the present invention may be formulated as gels, creams, lotions, sprays or liquids of varying viscosities.

"ETAILED DESCRIPTION OF THE INVENTION In a first aspect of the present invention, exemplary compositions are comprised of an activated protein, a compatible reducing agent, an oxidizing agent and at least one component selected from the group consisting of water, bases, acids, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents and perfuming agents.

In this first aspect of the invention, the activated protein comprises about 0.01 to about 12% by weight of the composition, preferably about 1.0 to about 5.0% by weight for certain formulations and about 6 to 10% by weight for other applications where more concentrated formulations are found to be advantageous.

Activated proteins for use in compositions of the present invention are preferably exemplified by proteins which have sufficient cysteinyl content, ac least about 1 cysteine amino acid for every 200 amino acids in a peptide chain (approximately, at least about 0.5% by weight cysteine, preferably, at I-ast about 1.0% by weight cysteine and most ir SPSTUS 89/00873 IPEAMi 05 APR199 R04-005.001 7 preferably, at least about 5% by weight cysteine) to covalently bind to the keratinous tissue of hair, skin and nails to produce a durable permanent bond to keratinous tissue. By permanent bond we mean that the protein is not easily washed or rubbed off from the keratinous tissue and becomes as permanent as normal hair and nails. A large number of exemplary proteins may be used in the present invention and incluie keratin, food proteins, for example, casein, alpha and beta-lactalbumin, seed proteins, for example, soybean proteins, linseed protein, cotton seed protein, corn protein and peanut protein, among others, hemoglobin, insulin, myosin, zein, ovalbumin, trypsin, chymotrypsin, chymotrypsinogen, elastases, thrombins, plasminogen, fibrinogen/fibrin, lysozyme, papain, human serum albumin, heat coagulable mucoproteins isolated from cartilage, bones and skin, gamma globulin blood proteins, and a number of the blood factor proteins, including, for example, factor VIII, XII, IXa and Xa, among others. Of course, proteins which contain large numbers of cysteinyl residues are preferred, because these proteins would form the greatest number of covalent bonds with the keratinous tissue and thus, produce the greatest durability. It is to be noted that the commercial gelatins disclosed in U.S. Patent No. 4,438,102 contain at most trace cysteinyl residues and most commercial grade gelatins, including Grade A edible gelatin, do contain undetectable amounts of cysteine. Such gelatin proteins, which do not contain sufficient cysteinyl content to covalently bind to keratinous tissue in any significant way, to produce a permanent attachment of the protein to the keratinous tissue, are therefore not contemplated for use in the present invention.

Preferred proteins for use in the present invention include proteins containing high percentages by weight of cysteine, for example, ribonuclease TI, human serum albumin and gamma globulins. An especially preferred protein for use in the present invention is keratin, because of its particularly high cysteine content (about 12% to about 17% by weight of cysteine). Proteins are activated by being subjected to a reducing agent at a pH of about 9.0 or above for a

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WO 89/07930 PCT/US89/00873 8 time sufficient to produce free thiol group. This period is generally about 5 minutes up to about one hour. Activation periods of greater than one hour are less preferred because although such activation periods may marginally increase the amount of activated thiol groups in the protein, such periods may also result in hydrolysis of the protein into shorter, less advantageous peptide units. A number of globular proteins contain cysteinyl residues within hydrophobic pockets. To activate these proteins and expose cysteinyl groups which exist in hydrophobic pockets to the keratinous tissue, it may be necessary to subject the proteins to denaturation and activation so that an activated cysteinyl residue of the denatured protein may be placed in proximity to the cysteinyl residues in the keratinous tissue to promote covalent binding.

The proteins of the present invention are preferably activated in the presence of reducing agent separately before they are formulated with the other components, because the addition of components other than a reducing agent at a pH above about 9.0 may adversely affect the rate at which cystinyl disulfide bonds in the protein are converted to cysteinyl mercaptide groups This may lower the overall activity of the protein. However, although less preferred, it is possible to generate activated protein after formulation by simply exposing unactivated protein to reducing agents during storage below pH 7.0, provided that the cysteinyl content of the protein is sufficiently high to activate sufficient cysteinyl residues to promote covalent binding to keratinous tissue.

The activated protein is preferably a keratin, but any protein which contains sufficient cysteinyl content to promote covalent binding to activated keratinous tissue is contemplated for use in the present invention. A particularly preferred keratin is Kerasolt m from Croda Chemicals International, Chesire, England. The molecular weight of proteins useful in the present invention preferably varies between about 5,000 and 500,000 Daltons, and most preferably varies between about 120,000 and 130,000 Daltons.

Reducing agents which are useful to activate protein -7 WO 89/07930 PCT/US89/00873 9 in the present invention include sulfides, thiol-containing compositions including dithiothreitol, trithiohexitol, glutathione, cysteine, mercaptoethanol, thioglycerol, thioalkanoic acid and mercaptocarboxylic acid, for example, mercaptosuccinic acid, thiolactic acid and their pharmaceutically acceptable salts, among others, including thioglyollic acid and salts of thioglycollic acid. Preferred reducing agents for activating the protein are thioglycerol, cysteine, thiolactic acid and thioglycollic acid, and their pharmaceutically acceptable salts. An especially preferred reducing agent for use in the present invention is ammonium thioglycollate. It is preferred that the reducing agent for activating the protein should be the same as the biologically compatible reducing agent which is used in the final formulation of the invention. The use of strong biologically incompatible reducing agents to activate the protein are less preferred and may make the use of the protein more difficult because the reducing agent may have to be removed from the activated protein before formulation.

Compositions of the present invention also contain a compatible reducing agent in an amount equal to about 0.1 to about 15% by weight of the formulation. Preferred compositions contain about 3.0 to about 10% by weight of a compatible reducing agent. The amount of compatible reducing agent varies according to the therapeutic use for which the compositions are intended, but generally falls within the range of about 3.0 to about 10% by weight. A compatible reducing agent is an agent which reduces cystinyl disulfide linkages in keratinous tissue to produce free thiol or mercaptide groups and is compatible with biological and/or pharmaceutical systems. Compatible reducing agents which are contemplated for use in the present invention include mercaptoethanol, dithiothreitol, glutathione, cysteine and salts of thioglycollic acid. An especially preferred reducing agent is ammonium thioglycollate.

Compositions of the present invention may additionally comprise about 0.001 to about 4.0% by weight of an oxidizing agent. Preferred embodiments comprise about 0.1 to about I C WO 89/07930 PCT/US89/00873 of the oxidizing agent and most preferably comprise about to about 1.0% of the oxidizing agent. The oxidizing agent is included in compositions of the present invention to enhance oxidation and promote the formation of covalent disulfide bonds between activated protein and keratinous tissue.

Exemplary oxidizing agents include hydrogen peroxide (which may or may not be stabilized with known stabilizers, for example urea) and its salts including ammonium sulfate peroxide, urea peroxide, pyrophosphate peroxide, carbonate peroxide, organic peroxides including acetyl peroxide, benzoyl peroxide, among others, alkali metal perborates including sodium perborate, the alkali metal bromates including sodium and potassium bromate and sodium and potassium iodate. In embodiments of the present invention which do not include an antioxidant, hydrogen peroxide is the preferred oxidizing agent. In the embodiments which include hydrogen peroxide, it is preferred that the amount of hydrogen peroxide should be in an amount equal to about 0.001 to about 1.5% by weight of the composition and most preferably about 0.05 to about Where oxidizing agents other than hydrogen peroxide are used, a higher percentage by weight is usually used compared to hydrogen peroxide which has a high oxidation equivalent per unit weight.

In addition to the above-described ingredients, additional components may be added to the formulation to enhance the effects of the compositions. In addition to water, additional components may include bases, acids, buffering agents, solvents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents and perfuming agents.

Exemplary acids and bases are added to adjust the pH of the formulation to desired levels. Preferred acids include organic acids for example acetic acid, citric acid and tartaric acid, among others, and inorganic phosphoric acid including its salts such as the salts of mono- and dihydrogen phosphoric acid. The inorganic phosphoric acid salts may also be included in the formulations as buffering agents.

Preferred bases include organic amines, for example, WO 89/07930 PCT/US89/00873 11 monoethanolamine, triethanolamine, trimethylamine and triethylamine. Most preferred bases include ammonium hydroxide.

Buffering agents, for example the inorganic phosphoric acid salts indicated above as well as other buffering agents, for example the salts of organic acids such as acetic acid and citric acid may be included in the formulations of the present invention in amounts effective to maintain the pH of the formulation over time. Preferably, the amount of buffering agent is no more than about 1.5% by weight of the formulation and most preferably is less than 0.75% by weight. The pH of the formulation may be a factor in determining its stability and in maintaining the activity of certain components in the formulation, especially the activated protein and the compatible reducing agent. Thus, a buffering agent may be included within the formulation to maintain the pH at a relatively constant level over time.

To add homogeneity to and promote the solubility of the formulation, certain organic solvents may be included.

Among the solvents that may be useful in certain embodiments of the present formulation include water, soluble polar organic solvents for example, alkanols such as methanol, ethanol, propanol, butanol and carbonyl containing solvents for example acetone, butanone and the like, among others.

S WO 89/07930 PCT/US89/00873 12 Additional solvents include ethers and amines, for example diethyl or dipropyl ether and trimethyl or triethyl amine.

Trimethylamine and triethylamine may also be added as bases.

The solvent added to the formulation may enhance the solubility of certain components. Where liquid formulations are contemplated, it is sometimes advisable to add an organic solvent to promote the solubility of certain less polar components, which without the added organic solvent may be only marginally soluble in water resulting in formulations having more than one phase. The addition of the organic solvent may produce a uniform, homogeneous single phase.

Emollients may also be included, especially in lotions to produce a uniform, homgeneous single phase and provide other favorable characteristics. An especially preferred emollient for use in formulations of the present invention is PPG 15-sterol ether, which also may be added to the formulations of the present invention for its emulsifying characteristics.

An emulsifying agent or surfactant is often added to embodiments of the present invention to enhance the characteristics of the form.ulation, to promote the solubility of the protein and other components and the phase stability of the formulation. Such agents also provide detergent-like qualities to the formulations. Suitable surfactants or emulsifying agents may be nonionic, anionic or amphoteric. Nonionic emulsifying compositions include, for example the lower alkylene oxide condensation products of hydrophobic compounds, for example ethylene oxide condensation products with higher fatty acids, higher fatty alcohols or alkylated aromatic hydrocarbons, higher molecular weight polypropylene glycols, amide and amine condensation products of which N-bis (2hydroxyethyl)-lauramide is exemplary. Preferred nonionic emulsifying compositions include polyoxyethylene ethers including polyoxyethyleneisohexadecyl ether, for example Arlasolve 2 00 tm (available from ICI Americas, Wilmington Delaware), polyoxyethylenelaury'f ther, for example Brij 35 tm (ICI), polyoxyethylenestearyl ettier, for example Brij 72 tm, i WO 89/07930 PCT/US89/00873 13 and Brij 78 tm (ICI) and polyoxypropylenestearyl ether, for example PPG-15 stearyl ether (Arlamol E, ICI). Other exemplary emulsifiers include ethoxylated lanolin, for example, Lanogel 41 (Amerchol, Inc., Edison, Exemplary anionic surfactants include sulfuric acid esters of polyhydric alcohols, e.g. lauryl sulfate, cetyl sulfate, etc., higher fatty alcohol sulfates derived from coconut oil, hydloxy sulfonated higher fatty acid esters such as, higher fatty acid esters of 2,3-dihydroxy-propane sulfonic acid, higher fatty acid esters of low molecular weight alkylol sulfonic acids, oleic acid ester of isethionic acid, sulfated higher fatty acid alkylolamides such as ethanolamide sulfates, higher fatty acid amides of amine alkyl sulfonic acids, lauric amide of taurine, among others, and aromatic containing anionic synthetic surfactants. Exemplary amphoteric surfactants include the salts of N-alkyl compounds of betaamino propionic acid wherein the alkyl group is derived from a fatty acid such as a mixture of coconut oil fatty acids, among others.

It may be preferable to add an anti-foaming agent to certain compositions to promote homogeneity and prevent foaming from surfact-nt action. A preferred anti-foaming agent for use in embodiments of the present invention includes, for example, Dimethiconetm, available from Dow chemical Corp., Midland, Michigan.

Thickeners or gelling agents may be added to provide additional weight and a more viscous feel to the formulations.

Suitable thickening agents include polyvinyl pyrollidone, for example PVP K30 (GAF Charllotte, polyacrylates, carbomers, for example carboxyvinyl polymer such as Carbapol 940 (available from B.F. Goodrich, Cleveland, Ohio) polyoxyethylene stearyl ethers, for example, polyoxyethylene-2 stearyl ether such as Steareth 2 tm (ICI) and polyoxyethylenestearyl ether such as Steareth 20 tm (ICI), sodium alginate, carageenan, agar, ethoxylated polyvinyl alcohol, gums, for example methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, propylcellulose and hydroxypropylcellulose, acacia, tragacanth, guar, and quince, among others.

WO 89/07930 PCT/US89/00873 14 In compositions which are contemplated to be formulated as a gel or lotion, Isoseteth 20 tm (polyoxyethyleneisohexadecyl ether, ICI), and Steareth 2 tm and 20 tm are preferred for use as thickening agents. In compositions which are contemplated to be formulated as creams, preferred thickeners include Steareth 2 tm and Steareth 20 tm and the carbomer polymers, for example Carbopol 940 tm Preservatives are added for preventing microbial growth in the presence of pr-tein nutrients. Exemplary preservatives include benzoic acia analogs including, among others, sodium benzoate. Other presevatives include propyl and methyl paraben, Dowicil tm (Dow Chemical Corp., Midland, Mi.) and formaldehyde solution. An especially preferred preservative is Germaben IItm, available from Sutton Laboratories, New Jersey.

Coloring agents and perfume agents may also be added to enhance the characteristics of the formulations.

In another aspect of the present invention, exemplary compositions include an antioxidant instead of an oxidizing agent. These compositions comprise about 0.01 to about 10% by weight of an activated protein component, about 0.1 to about by weight of a compatible reducing agent, about 0.001 to about 2.0% by weight of an antioxidant, and at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents or surfactants, thickeners, coloring agents and perfume agents.

In compositions comprising an antioxidant, the antioxidant is included to promote the storage stability of the formulations. Exemplary antioxidants may include alphatocopherol, hydroxyquinone, unipherol, tocopherol ascorbate, lecithin, chlorophyll, ascorbylpalmitate, linseed oil, tongue oil, other natural product antioxidants such as the steam distillation extract of rosemary as disclosed in U.S. Patent No.

4,450,097, thiazoline carboxylate, dihydroquinolines, methyl gallate, propyl gallate, alkylaryl and diarylamines.

Certain chelating agents, for example, EDTA, may be employed to enhance the antioxidant effect of the above agents. The chelating agent may function to chelate any dis- WO 89/07930 PCT/US89/00873 solved metals which may be responsible for che in situ generation of oxygen. Preferably, the chelating agent comprises between about 0.001 to about 0.5% of the formulation and most preferably the chelating agent comprises no more than about 0.1% of the formulation.

Preferably, in compositions which employ an antioxidant, a volatile antioxidant is used. Volatile antioxidants provide the advantage of protecting the activated protein and reducing agent from oxidation during storage. In addition, because the antioxidants are volatile, after the compositions are placed on the treatment area and exposed to air or a heat source, the antioxidant will evaporate from the treatment area leaving the remaining protein and activated keratinous tissue to be air oxidized. Volatile antioxidants include voltile carbonyl containing compounds, hindered phenolic compounds, for example 2,4,6-trialkyl phenols, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), phydroxytoluene and p-hydroxyanisole.

A volatile antioxidant as used in preferred embodiments of the present invention is an antioxidant that volatilizes or evaporates from the treatment area under normal drying conditions. Non-volatile antioxidants, although useful in certain aspects of the present invention, are less preferred than are volatile antioxidants, which are added to formulations for their ability to stabilize the active ingredients over time while in storage and their ability to be removed from the treatment area under normal drying conditions.

Preferred volatile antioxidants include those that are more easily volatilized, will evaporate more quickly from the treatment surface.

Certain antioxidants may be formulated in combination with solvents including water. This may promote azeotrope formation and volatility of the antioxidant. Azeotrope formation with water or with other solvents may result in the antioxidant volatilizing at a temperature lower than normal.

Thus, by formulating the compositions with, for example, an alcoholic or other solvent, the volatilization of the antioxidant may be enhanced, resulting in an enhanced rate of WO 89/07930 PCT/US89/00873 16 oxidation of the treated keratinous tissue.

In preferred embodiments of the present invention, when an oxidizing agent is not included in the formulation, about 0.01% to about 2.0% of antioxidant is included in the formulations. Without the additional oxidizing agent, the antioxidant is included to prevent atmospheric oxygen or oxygen dissolved in the solution from deactivating the protein during storage. In compositions in which oxidizing agents are employed to promote the oxidation of free thiols or mercaptides to covalent disulfide bonds, the oxidizing agent comprises about 0.001% to about 4.0% by weight of an oxidizing agent and the antioxidant comprises about 0.01% to about of the formulation.

In formulations of the present invention comprising an antioxidant or an antioxidant and oxidizing agent, the formulations may additionally comprise acids, bases, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents and perfume agents as described for other embodiments of the present invention. In formulations comprising an antioxidant, a volatile antioxidant is preferred. In such formulations additional organic solvent may be added to promote the volatilization of the antioxidant. It is recognized that the choice of additives is made to avoid any interactions that may affect the activity of the the activated protein, reducing agent, oxidizing agent or antioxidant.

The compositions of the present invention are applied to the treatment area as a liquid, cream, gel or lotion by rubbing the compositions into the hair or other tissue to be treated. After the compositions have been in contact with the treated tissue for a time period of about 20 minutes to six hours, the treated area is then dried at ambient air temperature or preferably, at elevated temperatures under a hair dryer or other heat source.

In a method for treating the nails of humans and the hooves of animals, compositions of the present invention which do not contain an oxidizing agent or an antioxidant may be used. Thus, the treatment of the nails of humans and the WO 89/07930 PCT/US89/00873 17 hooves of animals comprises exposing the nails or hooves to a composition comprising about 0.01 to about 12% by weight of an activated protein as described hereinabove, preferably keratin, about 0.1 to about 15% by weight of a pharmaceutically compatible reducing agent as described hereinabove, preferably thioglycollic acid or ammonium thioglycollate and the remainder of the composition comprises at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents or surfactants, thickeners, preservatives, organic solvents, coloring agents and perfuming agents.

The following examples are provided to illustrate the present invention and should not be construed to limit the scope of the invention of the present application in any way.

WO 89/07930 PCT/US89/00873 18 EXAMPLE 1 Dilute Liquid Component Weight Percent Ammonium Thioglycollate Ph 9 3.27 A Kerasoltm 0.95 Propylene Glycol 0.05 Lanogel 4 1 tm 0.05 B Brij 35 tm 0.13 PVP-K30tm25% Sol. 0.22 Glycerine 0.16 Citric Acid Sol 0.04 Hyd. Peroxide 0.53 C Acetone 0.13 Isopropyl Alcohol 70% 0.53 Kerasol"m 1.90 Water Distilled 85.40 D Germaben IItm 0.93 E Fragrance 1.90 Arlasolvetm 3.79 F Procedure: Add a mixture of Component B to 1/2 of Component D at 100" F and mix for 5 minutes in a high speed blender.

After thoroughly mixed, add a mixture of Component C and mix for 5 minutes. Let the mixture cool and add the balance of component D to the mixture at room temperature and mix for 2 minutes. Prepare A by adding 9.64% of a 28% ammonia solution to 90.64% of a 60% ammonium thioglycollate water solution, pH should read 9; if not, add more ammonia solution. Add kerasoltm to ammonium thioglycollate solution and mix until well homogenized. Let stand for at least five minutes. This produces mixture A. Add A to the BDC mixture and then mix in a high speed blender until thoroughly mixed. Next, add component E to mixture ABCD until thoroughly mixed. Finally add components F to mixture ABCDE and mix at high speed until homogeneous.

WO 89/07930 PCT/US89/00873 19 EXAMPLE II Concentrated Liquid Component Weight Percent Ammmonium Thioglycollate pH 9 9.75 A Kerasol tm 2.82 Propylene Glycol 0.13 Lanagel 4 1 t 0.13 B Brij 35 tm 0.39 m 25% 0.64 Glycerine 0.47 Citric Acid 0.12 Hydrogen Peroxide 1.55 C Acetone 0.39 Isopropyl Alcohol 70% 1.68 Kerasolm 5.65 Water 67.81 D Germaben II t m 2.82 E Fragrance and 5.65 F Solubilizer (1:2) Procedure: Add a mixture of Components B to 1/2 of Component D at 100" F and mix for 5 minutes in a high speed blender.

After thoroughly mixed, add a mixture of Components C and mix for 5 minutes. Let mixture cool and add the balance of component D to the mixture at room temperature and mix for 2 minutes. Prepare A by adding 9.64% of a 28% ammonia solution to 90.64% of a 60% ammonium thioglycollate water solution. pH should read 9; if not, add more ammonia solution. Add kerasolt m to ammonium thioglycollate solution and mix until well homogenized. Let stand for at least five minutes. This produces mixture A. Add A to the BDC mixture and then mix in a high speed blender until thoroughly mixed. Next, add components E to mixture ABCD until thoroughly mixed. Finally add components F to mixture ABCDE and mix at high speed until homogeneous.

WO 89/07930 PCT/US89/00873 Example III Lotion Component Weight Percent Example II Concentrated 32,19 A liquid Non-Perfumed Moisturizing 45.85 B Lotion Carbopol 940 2% Sol 19.51 C Triethylamine 1.225

H

2 0 1l225 D Procedure: Add Component A to Component B (see below) and thoroughly mix. Then add Component C until a homogeneous mixture is made. Finally, adjust the pH of mixture ABC with the triethylamine/H 2 0 mixture to about Non-Perfumed Moisturizing Lotion Component Weight Percent Arlamol Etm 1.36 Brij 72 tm 5.23 Brij 78 tm 1.32 A Mineral Oil 11.64 Propyl Paraben 0.18 Water 77.71 Sodium EDTA 0.09 Anti-Foam Dimethiconetm 0.09 B Methyl Paraben 0.36 Propylene Glycol 1.36 Dowicil 200 tm 0.45 C Formaldehyde 0.21 D Procedure forNon-perfumed Moisturizing Lotion Component: Add A to B at 160"F mixing thoroughly. Mix and cool to 100*F.

.Add Components C and D, mixing thoroughly. Allow to cool to Use at T I C I L 21 WO 89/07930 PCT/US89/008'73 Component Non-perfumed Moisturi Lotion Component Carbopol 940 tm Triethanolamine Concentrated Liquid Example II Fragrance EXAMPLE IV Thin Cream Weight Percent zing 8.70 58.26 2.61 28.70 1.74 Procedure: Mix Components of A together and adjust pH with triethanolamine. Add Component B and thoroughly mix until homogeneous. Mix in fragrance until homogeneous.

EXAMPLE V-THICK CREAM Component Weight Percent Example II Concentrated 30.22

A

liquid Concentrated Non-Perfumed 9.16 B Moisturizing Lotion Carbapol 940 4% Sol 58.50

C

Fragrance 2.14%

D

Procedure: Add Component A to component B (see below) and thoroughly mix. Then add Component C until a homogeneous mixture is made. Add Component D and mixture until a homogeneous thick cream is formed.

WO 89/07930 PCT/US89/00873 22 Concentrated Non-Perfumed Moisturizing Lotion Component Weight Percent Arlamol E tm 2.33 Brij 72 tm 8.95 Brij 78 tm 2,26 A Mineral Oil 19.94 Prupyl Paraben 0.31 Water 63.15 Sodium EDTA 0.016 Anti-Foam Dimethiconetm 0o016 B Methyl Paraben 0.7 Propylene Glycol 2.33 Procedure for Concentrated Non-Perfumed Moisturizing Lotion Component: Add A to B at 160°F mixing thoroughly. Mix and cool to 80"F. Use at EXAMPLE VI Component Weight Percent Ammonium Thioglycollate 0.1 to Ammonium Hydroxide 0.005 to Keratin 0.01 to 10.0% Hydrogen Peroxide 0.001 to Water 83.0 to 99.884% Procedure: Mix the ammonium thioglycollate, ammonium hydroxide and a small amount of water to form a mixture with a pH of about 9 to 11. About half of the protein is added to this mixture which may be agitated or stirred for about minutes followed by the addition of water, hydrogen peroxide and the remaining protein. Alternatively, after the first half of the protein is added, the water, hydrogen peroxide and remaining protein may be added.

WO 89/07930 PCT/US89/00873 23 EXAMPLE VII Component Percent by Weight Ammonium Thioglycollate 0.1 to Ammonium hydroxide 0.05 to Hydrogen Peroxide 0.001 to Keratin 0.01 to 10.0% Glycerine 0.15 to 0.25% Citric Acid 0.095 to 0.29% Solvent 0.16 to 1.28% Water 81.18 to 99.434% Procedure: Mix the ammonium thioglycollate, ammonium hydroxide and a small amount of water to form a mixture with a pH of about 9 to 11. About half of the protein is added to this mixture which may be agitated or stirred for about minutes followed by the addition of water, hydrogen peroxide and the remaining protein. Alternatively, after the first half of the protein is added, the water, hydrogen peroxide and remaining protein may be added. The other ingredients are then added after the hydrogen peroxide and stirred or agitated sufficiently to produce a homogeneous composition.

EXAMPLE VIII Concentrated Stock Solution Component Weight Percent Purified Water 32.55 Propylene Glycol 0.14 A Lanogel 41 0.14 Brij 35 0.40 Purified Water 0.487 B 25% 0.163 Glycerine 99% 0.48 Citric Acid 5.88% 0.12 Solution Hydrogen Peroxide 1.55 C Acetone 0.40 Isopropyl Alcohol 99% 0.91 Kerasol1m 5.65 Water 41.62 D Germaben IItm 2.82 Ammonium pH 9.0 8.81 Thioglycollate E Ammonia Sol'n 0.94% Kerasoltm 2.82% WO 89/07930 PCT/US89/00873 24 Procedure: Step 1: In a separate tank agitate B (water) very strongly and sprinkle B (PVP K-30) onto the Vortex. Mix until PVP K-30 solution is complete. Step 2: Charge a mixing tank with water at 35-40'C. Add the A phase ingredient and mix thoroughly. Add the PVP K-30 solution and mix in well. Step 3: Mix C phase together in a plastic container. Warm to Add to step 2 and add the D phase. Step 4: In a plastic container, add ammonium thioglycollate and then add ammonia solution slowly to bring the pH to 9.0. Add the Kerasoltm. Mix this solution well and add it to the batch.

EXAMPLE IX Component Weight Percent Purified Water 30.53 PVP K-30 0.163 Lanogel 41 0.14 A Propylene Glycol 0.14 Brij 35 0.40 Essence of Pellitory 0.50% Essence of Elder 0.50% Glycerine 0.48% Citric Acid 5.88% 0.12 Hydrogen Peroxide 3% 1.55 B Acetone 0.40 Isopropanol 99% 0.91 Kerasoltm 5.65 Germaben IItm 2.82 Purified Water 36.24 C Dehyquart A 1.00 Ammonium pH 9.0 8.81 Thioglycollate

D

Ammonia 0.94% Kerasoltm 2.82 Arlasolve 200 t m 4.00% Fragrance 1.88 E Procedure: Agitate purified water rapidly with "lightnin" mixer and sprinkle PVP K-30 slowly onto the surface. Allow PVP to go into solution. Add remainder of A ingredients and mix in well. Add B ingredients individually and mix well after each addition. Add C ingredients individually and mix in well after each addition. In a separate container add WO 89/07930 PCT/US89/00873 ammonium thioglycollate and use ammonia solution to bring the pH to 9.0. Add kerasol t m and mix in very well. Add this D phase to the batch and blend it in very well. In a separate container heat Arlasolve 2 0 0 tm very gently to liquify. Add remaining E ingredients separately and mix very well. Add this to the batch and mix until the product is uniform.

EXAMPLE X Component Weight Percent Purified Water 41.91 PVP K-30 0.22 Lanogel 41 0.05 A Propylene Glycol 0.05 Brij 35 0.13 Essence of Rosemary 0.33% Essence of Pimpernil 0.33% Allantoin 0.34% Glycerine 0.16% Citric Acid 5.88% 0.04 Hydrogen Peroxide 3% 0.52

B

Acetone 0.13 Isopropanol 99% 0.30 Kerasoltm 1.88 Germaben IItm 0.94 Purified Water 42.81

C

Ammonium pH 9.0 2.95 Thioglycollate

D

Ammonia 0.09% Kerasoltm 0.94 Arlasolve 2 0 0 tm 4.00% Fragrance 1.88

E

Procedure: Agitate purified water rapidly with "lightnin" mixer and sprinkle PVP K-30 slowly onto the surface. Allow PVP to go into solution. Add remainder of A ingredients and mix in well. Add B ingredients individually and mix well after each addition. Add C ingredients individually and mix in well after each addition. In a separate container add ammonium thioglycollate and use ammonia solution to bring the L I WO 89/07930 PCT/US89/00873 26 pH to 9.0. Add kerasoltm and mix in very well. Add this D phase to the batch and blend it in very well. In a separate container heat Arlasolve 200 tm very gently to liquify. Add remaining E ingredients separately and mix very well. Add this to the batch and mix until the product is uniform.

EXAMPLE XI Component Weight Percent Purified Water 41.57 PVP K-30 0.22 Lanogel 41 0.05 A Propylene Glycol 0,05 Brij 35 0.13 Biotin 0.001 Elastin 0.001 Glycerine 0.16% Citric Acid 5.88% 0.04 Hydrogen Peroxide 3% 0.52 B Acetone 0.13 Isopropanol 99% 0.30 Kerasoltm 1.88 Germaben IIt a 0.94 Purified Water 44.28 C Ammonium pH 9.0 2.95 Thioglycollate

D

Ammonia 0.09% Kerasoltm 0.94 Arlasolve 200 t m 4.00% Fragrance 1.75 E Procedure: Agitate purified water rapidly with "lightnin" mixer and sprinkle PVP K-30 slowly onto the surface. Allow PVP to go into solution. Add remainder of A ingredients and mix in well. Add B ingredients individually and mix well after each addition. Add C ingredients individually and mix in well after each addition. In a separate container premix ammonium thioglycollate into purified water and use ammonia solution to bring the pH to 9.0. Add kerasoltm and WO °9/07930 PCT/US89/00873 27 this to the batch and mix until the product is uniform.

EXAMPLE XI Component Weight Percent Purified Water 41.57 PVP K-30 0.22 Lanogel 41 0.05 A Propylene Glycol 0.05 Brij 35 0.13 Biotin 0,001 Elastin 0.001 Glycerine 0.16% Citric Acid 5.88% 0.04 Hydrogen Peroxide 3% 0.52 B Acetone 0.13 Isopropanol 99% 0.30 Kerasoltm 1.88 Germaben II t m 0.94 Purified Water 44.28 C Ammonium pH 9.0 2.95 Thioglycollate D Ammonia 0.09% Kerasoltm 0.94 Arlasolve 200 t m 4.00% Fragrance 1.75 E Procedure: Agitate purified water rapidly with "lightnin" mixer and sprinkle PVP K-30 slowly onto the surface. Allow PVP to go into solution. Add remainder of A ingredients and mix in well. Add B ingredients individually and mix well after each addition. Add C ingredients individually and mix in well after each addition. In a separate container premix ammonium thioglycollate into purified water and use ammonia solution to bring the pH to 9.0. Add kerasoltm and mix in very well. Add this D phase to the batch and blend it in very well. In a separate container heat Arlasolve 200 tm very gently to liquify. Add remaining E ingredients separately and mix very well. Add this to the batch and mix until the product is uniform.

I- I-- WO 89/07930 PCT/US89/00873 EXAMPLE XII Concentrated Non-Perfumed Moisturizing Lotion Component Weight Percent Arlamol E 2.33 Brij 72 8.93 Brij 78 2.25 A Mineral Oil 70 19.89 Propylparaben 0.31 Purified Water 62,15 Disodium EDTA 0.16 Dimetbicone 0.16 B Methylparaben 0.70 Propylene Glycol 2.33 Germaben II 0.79 C Procedure: Charge main mixing kettle with B ingredients and heat while mixing to 80-85"C. In a separate container heat A ingredients to 80-85"C and mix until uniform. At 80-85"C add mixed A ingredients to mixed B ingredients while thoroughly mixing. Cool to 50-55"C. At 50-55"C add Germaben and blend in very well. Continue to cool to 30"C and use at this temperature.

EXAMPLE XIII Component Weight Percent Concentrated Non-Perfumed 8.85 A Moisturizing Lotion (Ex. XII) Purified Water 56.94 Elastin 0.001 B Biotin 0.001 Carbopol 940 2.36 Concentrated Stock Sol'n 29.23 C Example VIII Fragrance 1.75 D Coloring Agent 0.87 1- WO 89/'07930 PCT/US89/00873 29 Procedure: This is a 4% Carbopol dispersion. Measure water and agitate at high speed. Add elastin and then biotin and allow them to disperse. Add Carbopol 940 to the lip of the vortex and mix well until the dispersion is complete. Add component A to the mixing kettel at 25-30"C and add B phase from above and mix until uniform. Add component C to mixture of A and B and mix until uniform. Add fragrance and coloring agent.

EXAMPLE XIV Non-Perfumed Moisturizing Lotion Component Weight Percent Arlamol E 1,36 A Brij 72 5.21 Mineral Oil 70 11.60 Propylparaben 0.18 Purified Water 77.4 Disodium EDTA 0.10 Dimethicone 0.09 B Methylparaben 0.41 Propylene Glycol 1.36 Dowicil 200 0.05 C Purified Water 0.50 Formaldehyde 37% 0.2 D Germaben II 0.23 E Procedure: Heat A phase components to 70-75 C and mix until uniform. Charge main kettle with water and begin heating to 70-75°C. Add the remainder of phase B components and mix to dissolve the solids. Add, at 70-75°C, A phase to B phase while mixing. Blend well and cool to 35-40'C. Premix C phase and add to the batch when the solution is clear. Add D and E phases one at a time and mix in well. Cool to 25-30'C and use at this temperature.

T C_ I WO 89/0 ,930 PCT/US89/00873 Component Non-Perfumed Moisturiz Lotion (Ex. XIV) Purified Water Carbopol 940 Essence of Rosemary Essence of Althea Essence of Bilberry Essence of Jaborand Essence of Verbena Triethanolamine 99% Concentrated Stock Sol Example VIII Fragrance EXAMPLE XV Weight Percent ing 8.70 56.11 1.15 0.20 0.20 0.20 0.20 0.20 2.60 'n 28.70 1.74 Procedure: In a separate mixing tank agitate water at high speed and spring Carbopol 940 onto the vortex. Disperse Carbopol and add remaining B phase components. Mix until carbopol dispersion is complete. Charge mixing tank with A component and hold at 25-30"C. Add B mixture and blend until homogeneous. Add triethanolamine and mix in well until homogeneous. Add component D until homogeneous. Add component E and blend in well.

EXAMPLE XVI STORAGE STABLE COMPOSITION Component Activated Protein Reducing Agent Antioxidant Water, acids, bases buffering agents, emulsifying agents, thickeners, preservatives, organic solvents coloring agents, fragrance Weight Percent 0.1 to 12.0% 0.1 to 15.0% 0.01 to 79.0 to 99.79% Procedure: Form activated protein separately with solution containing reducing agent at a pH above about 9. Mix in remaining components until final mixture is homogeneous.

WO 89/07930 PCT/US89/00873 31 EXAMPLE XVII STORAGE STABLE COMPOSITION INCLUDING OXIDIZING AGENT Component Weight Percent Activated Protein 0.1 to 12.0% Reducing Agent 0.1 to 15.0% Oxidizing Agent 0.001 to Antioxidant 0.01 to Water, acids, bases 75.0 to 99.789% buffering agents, emulsifying agents, thickeners, preservatives, organic solvents coloring agents, fragrance Procedure: Form activated protein separately with solution containing reducing agent at a pH above about 9. Mix in remaining components until final mixture is homogeneous.

Examples XVIII and XIX Hair Growth A 32 year old New York City man with a history of hair loss, who had tried many formulations and had been using minoxidil four times a day for two years as prescribed by a physician with no results. Two weeks after beginning the formulation of Example I daily he noticed that his hair was thicker. Within a month he noticed new growth which became profound within 3 months.

A 63 year old Florida man with a long history of hair loss skeptically used the formulation of Example I on the top and front of his head, up to his greatly receded hairline.

Within 3 weeks there was visible new growth in areas that had not grown visible hair in 20 years.

Examples XX and XXI Hair Thickening and Strengthening A 27 year old male hairdresser from New York who noticed considerable hair loss due to breakage on his pillow, in his comb, in the drains, etc. began using the formulation of Example I daily. Within 3 weeks hiL estimates of the hair on his comb in the morning decreased from over 100 hairs to under 10 hairs. He no longer noticed hair on his pillow in the morning, and only a normal amount of hair in his drains.

WO 89/07930 PCT/US89/00873 32 A 31 year old New Jersey man with very thin fly-away hair began using the formulation of Example I daily, and within one month found his hair to be thicker, with more body, and stayed where he wanted it with much less fly-away.

Example XXII Nail Hardening A 42 year old New York man with chronically thin brittle nails began using the formulation of Example III on his nails daily. Within 1 month he reported his nails were considerably harder, less prone to breaking, and he could grow them longer; especially one nail which had been injured years ago and had developed a chronic thin spot. This thin spot persisted relative to the rest of his nails, but since all of his nails were harder and thicker it was no longer a problem.

In addition, the rate of nail growth was markedly faster, based upon the frequency with which he had to file his nails before and after treatment.

Examples XXIII-XXVIII Animal Coat Restorationof Acute and Chronic Conditions A thoroughbred used as a carriage horse developed back rubs on the withers and back from the tack. Daily application of the formulation of Example IV resulted in 80% restoration of the acute condition within 8 days despite continued application of the tack and carriage driving. The attending veterinarian estimated that this level of fur restoration should have taken 6-8 weeks in the absence of driving and the application of tack.

The formulation from Example IV was used on over 8 horses at a major show barn for coat deficits due to wounds, abrasions Lnd back rubs. In all instances, the deficits resolved at a vastly accelerated rate in the estimate of a highly experienced groom who travels with the U.S. Olympic Equestrian Team.

A horse with a chronic hairless spot on the head which was present when the horse was purchased over 2 years ago experienced coat restoration in that area following 10 days of daily application of the formulation of example IV.

A horse with chronic coat loss on the lower legs due WO 89/07930 PCT/US89/00873 33 to bandages experienced coat restoration following 3 weeks of daily application of the formulation from Example I.

A cat with a paralyzed forelimb which for years was continually dragging on the grcund experienced coat restoration within 1 month after beginning daily applications of the formulation of Example I.

Eyample XXVIII Coat Restoration of Chronic Condition A dog had a severe pyoderma with hair loss, oozing and bleeding, and itching resulting in self-inflicted lacerations of over 1 year duration. Suspected IgA deficiency. Two weeks of lincomycin almost a year earlier provided a slight positive effect but was discontinued due to toxicity. In late, 1987 thyroid supplementation was tried without results. Euthanasia was considered. In the middle of 1988, experimental treatment with the composition from Example X was initiated. Within 4 days all open sores scabbed over. Within 8 days all lesions healed with appreciable hair growth. Within 21 days the dog was approximately normal in appearance. Complete hair regrowth over the entire body has begun. After 3 months, the dog is completely restored. No more deficits, selfmutilation, etc. The treatment was stopped and the problem seems to be completely and permanently resolved. There has been no loss of hair since ceasing treatment.

Example XXIX Hoof Growth A mare with seedy toe, an anaerobic infection of the lamina was treated by excising the tissue over the infection and the infection was treated with the composition of Example XIII. After only one week of use the hoof wall appeared appreciably healthier and of better texture. The coronary band looked much better. She started using this composition on all of the hooves of her animals.

Example XXX Coat Restoration of Chronic Condition A horse had hip rubs of over 1 year duration which refused to heal. After using 1 ounce of the composition of Example XV the open wounds healed and grew hair.

SWO 89/07930 PCT/US89/00873 34 Example XXXI Grooming Daily misting gets dog coats into show condition in one to two weeks which would otherwise have taken four to six months. Beskie terriers looked '"fantastic" after one month using the composition of Example X. The coats became silky and shiny and the dogs pads were strengthened with no cracking.

Examples XXXII and XXXIII Grooming and Hoof Conditioning Use of the composition of Example X greatly facilitated the transition from winter to summer coats of horses following a gloomy spring. After two applications the coat transition was greatly improved. Use of the composition of Example XI for hoof conditioning resulted in a nice texture, not soft and spongey yet moist and resilient, yet not dry and brittle.

Example XXXIV Hoof Conditioning In a number of cases, the composition of Example XIII resulted in hoof reconditioning, and the strengthening of "shelly" hooves, and dry, brittle and/or cracking hooves.

Example XXXV Restoration of Chronic Coat Condition Photographer for a New Jersey newspaper used the composition of Example XV for treating hair loss on a biopsy for her Burmese mountain dog which had not grown hair at the site of biopsy for over a year and a half. After 3 days of treatment, the photographer reported preliminary regrowth of hair.

This invention has been described in terms of specific embodiments set forth in detail herein, but it should be understood that these are by way of illustration and the invention is not necesarily limited thereto. Modifications and variations will be apparent from the disclosure and may be resorted to without departing from the spirit of the inventions those of skill in the art will readily understand.

Accordingly, such variations and modifications are considered to be within the purview and scope of the invention and the following claims.

Claims (15)

1. A composition for treating keratinous tissue in mammals comprising: a) 0.01 to 12% by weight of a protein containing sufficient cysteinyl groups to covalently bind said protein to said keratinous tissue; b) 0.1 to 15% by weight of a reducing agent capable of reducing cystine to cysteine in said protein; c) 0.001 to 4.0% by weight of an oxidizing agent; and io d) 81.0% to 99.889% by weight of at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents, thickeners, solvents, preservatives, coloring age:its and perfuming agents.

2. The composition according to claim 1 wherein said:: protein is a keratin protein.

3. The composition according to claims 1 or 2 wherein said reducing agent is a salt of thioglycollic acid.

4. The composition according to claims 1-3 wherein said* reducing agent is ammonium thioglycollate.

5. The composition according to claims 1-4 wherein said.. composition further comprises a volatile antioxidant.

6. The composition according to claims 5 or 6 wherein said protein is a keratin protein, said reducing agent is ammonium thioglycollate and said oxidizing agent is hydrogen. peroxide.

7. A composition for treating keratinous tissue in mammals comprising: a) 0.01 to 12% by weight of a protein containing sufficient cysteinyl groups to covalently bind said protein. 3o to said keratinous tissue; b) 0.1 to 15% by weight of a reducing agent capable of reducing cystine to cysteine in said protein; c) 0.001 to 2.0% by weight of a volatile antioxidant; and d) 81.0% to 99.889% by weight of at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents, thickeners, solvents, preservatives, coloring agents and perfuming agents.

8. The composition according to claim 7 wherein said protein is a keratin protein. T- L I I J C 36

9. The composition according to claims 7 or 8 wherein said reducing agent is a salt of thioglycollic acid. The composition according to claims 7-9 wherein said reducing agent is ammonium thioglycollate.

11. The composition according to claims 7-10 wherein said composition further comprises an oxidizing agent.

12. The composition according to claim 11 wherein said protein is a keratin protein, said reducing agent is ammonium thioglycollate and said oxidizing agent is hydrogen peroxide.

13. A method of treating keratinous tissue to improve its appearance and strength by contacting the tissue with a composition comprising: a) 0.01 to 12% by weight of a protein containing sufficient cysteinyl groups to covalently bind said protein to said keratinous tissue; b) 0.1 to 15% by weight of a reducing agent capable of reducing cystine to cysteine in said protein; c) 0.001 to 4.0% by weight of an oxidizing agent; and d) 81.0% to 99.889% by weight of at least one component selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents, thickeners, solvents, preservatives, coloring agents and perfuming agents.

14. The method according to claim 13 wherein said protein is a keratin protein, said reducing agent is ammonium thioglycollate and said oxidizing agent is hydrogen peroxide. The method accorCing to claim 13 or 14 comprising drying said keratinous tissue after said contacting step.

16. A method of treating keratinous tissue to improve its o0 appearance and strength by contacting the tissue with a composition comprising: a) 0.01 to 12% by weight of a protein containing sufficient cysteinyl groups to covalently bind said protein to said keratinous tissue; b) 0.1 to 15% by weight of a reducing agent capable of reducing cystine to cysteine in said protein; c) 0.001 to 2.0% by weight of a volatile antioxidant; and d) 81.0% to 99.889% by weight of at least one component selected from tre group consisting of water, acids, bases,

37- buffering agents, emulsifying agents, thickeners, solvents, preservatives, coloring agents and perfuming agents. 17. The method according to claim 16 wherein said protein is a keratin protein and said reducing agent is ammonium thioglycollate. 18. The method according to claim 16 or 17 wherein said composition further comprises an oxidizing agent. 19. The method according to claim 16 or 17 comprising drying said keratinous tissue after said contacting step. o 20. A method of treating hu-n nails and animal hooves and claws to improve their appearance and strength and/or promote their growth by contacting the tissue with a composition comprising: a) 0.01 to 12% by weight of a protein containing sufficient cysteinyl groups to covalently bind said protein to said keratinous tissue; b) 0.1 to 15% by weight of a reducing agent capable of reducing cystine to cysteine in said protein; and c) 81.0% to 99.889% by weight of at least one component be selected from the group consisting of water, acids, bases, buffering agents, emulsifying agents, thickeners, solvents, preservatives, coloring agents and perfuming agents. 21. The method according to claim 20 wherein said protein is keratin. 22. The method according to claim 20 or 21 wherein said reducing agent is selected from the group consisting of thioglycollic acid and ammonium thioglycollate. o. DATED: 4 February, 1991 CIRO'S TOUCH, LTD. By their Patent Attorneys: PHILLIPS ORMONDE FITZPATRICK JM (vi I INTERNATIONAL SEARCH REPORT International Application No. PCT/US89/00873 I. CLASSIFICATION OF SUBJECT MATTER (if several classific3tion symbols apply. indicate all) 6 According to International Patent Classification (IPC) or to both National Classification and IPC IPC4: A61K 7/00, 7/ 06, 7/04, 7/48 _US CT,- 424/61 70, 95, 130; 514/21 II FIELDS SEARCHED Minimum Documentahon Searched 7 Classification System Classification Symools US. CL. 514/21; 424/61, 70, 95, 130 Documentation Searched other than Min-mum Documentaion to the Extent that such Documents are Included in the Fields Searched 8 III. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation ol Document, with indication, where appropriate, of the elevant passages 12 Relevant to Claim No '3 Y US, A, 4,438,102 (GANCI) 20 MARCH 1984 1-8 (21.03.84). (NOTE ENTIRE DOCUMENT, 22-24 i,,',iCULARLY COL. 1, LINES 30-35). Y US, A, 4,195,095 (SHEFFNER) 03 MARCH 1980 9-21 (03.03.80) (NOTE ENTIRE DOCUMENT, 25-33 PARTICULARLY COL. 4, LINES 42-50). Y JP, A, 57-016810 (MURAI, 28 JANUARY 1982 1-33 (28.01.82). Y FR, A, 2,522,657 (L'OREAL SA) 09 SEPTEMBER 1-33 1983 (09.09.83). Special categories of cted documents: 0 later document published after the international filing date document defining the general state of the art which is not oie roritu date and not i conflict with the appnlcar ion ut considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier docment but published on or after the international X" document of particular relevance: the claimed invention n cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another which is ited to esabcish the uabication date of another Y" document of particular relevance: the claimed invention citation or othr ecal reason (as specified) cannot be considered to involve an inventive step when the document referir.j to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 04 APRIL 19,. International Searching Authority Signature of Authorized Officer ISA'UJ SUSAN S. RUCKER Form PCT/SA/2 J (second sheet) (Rev. 11-87) L