US 5,545,402 AGrant
Skin Care Method and Composition
Issue Date:1996-08-13
•8 Claims
•4 Drawing Sheets
Abstract
A composition for topical application to the skin for alleviation or prevention of dry flaky skin condition, dandruff or acne comprising one or more stratum corneum thiol proteases. The composition may further comprise a mild reducing agent and/or an additional enzyme selected from glycosidases, other proteases, lipases and mixtures thereof. Optional additional active ingredients include sunscreens, lipids, hydroxy carboxylic acids and ketocarboxylic acids.
Metadata
Assignee
- Elizabeth Arden Co., Division of Conopco, Inc.
Inventor
- Allan Watkinson
Application Information
Application Number:US 3047229
Filing Date:1994-09-12
Priority Date:1993-09-15
Art Unit:188
Classifications
IPC:
A61K 3800A61K 3801A61K 3802A61K 3843
Field of Search:
42494.63
Patent Drawings (4 sheets)
Description
The present invention relates to compositions for application to the skin and their cosmetic and/or pharmaceutical use. In particular, the invention relates to compositions comprising stratum corneum thiol protease enzymes and their use in alleviating or preventing conditions involving abnormal desquamation by facilitating desmosomal degradation.
In normal, healthy epidermis the continuous production of new stratum corneum is balanced by a well-regulated shedding of corneocytes from the skin surface. Little is known about this desquamation process at the molecular level.
It has been shown by A. Lundstrom and T. Egelrud (J. Invest Dermatol, (1988) 91 340-343; Arch Dermatol Res (1990) 282 234-237; J. Invest Dermatol (1990) 94 216-220) that cohesion between cells in the stratum corneum is dependant on protein structures. These structures must be degraded before cell dissociation can occur.
Furthermore, evidence has been provided to show that cell dissociation is preceded by a degradation of the extracellular parts of desmosomes. (T. Egelrud (1992) European Journal of Dermatology 2 46-49).
It is thought that the process of desquamation involves proteolytic degradation of desmosomes, causing the cohesive links between the cells to break down and thereby allowing detachment of peripheral corneocytes.
Little is known about the proteases thought to be involved in the desquamatory process. One particular protease, the serine protease stratum corneum chymotryptic-like enzyme (SCCE) has been implicated as a putative desmosomal degrading enzyme (see A. Lundstrom and T. Egelrud, Acta Derm Venersol (Stockh) (1991) 71, 471-474).
The present inventors have found that the stratum corneum additionally contains thiol proteases having cathepsin-like substrate specificity, hereinafter referred to as stratum corneum thiol proteases, which may be involved in the process of cell dissociation (desquamation) and desmosomal degradation. These enzymes are therefore of interest in the treatment of conditions where the underlying aetiology indicates that assisting the processes of desmosomal degradation and/or desquamation would be beneficial.
Accordingly the invention provides a composition for topical application to the skin, comprising one or more stratum corneum thiol proteases.
As used herein, the term "stratum corneum thiol protease" means a thiol protease, or a pro-enzyme thereof, which in its active form exhibits similar inhibitor sensitivity to cysteine proteases such as cathepsin B and L. More specifically, the term "stratum corneum thiol protease" means a thiol protease, or a pro-enzyme thereof, which in its active form is inhibited by E64.
It will be appreciated that a pro-enzyme is an inactive form of the enzyme which may be activated by appropriate proteolytic cleavage to give the active form.
Suitable stratum corneum thiol proteases for use according to the present invention have apparent molecular weights of 34 kDa and 35 kDa, as determined by the method of comparing electrophoretic mobility with standard proteases on sodium dodecyl sulphate polyacrylamide gel electrophoresis.
Preferably the composition comprises 0.00001 to 50% more preferably 0.001 to 20% and even more preferably 0,001 to 0.1% by weight of the composition stratum corneum thiol proteases.
In a preferred aspect according to the invention, the compositions also comprise a mild reducing agent which has the effect of activating the enzyme. Suitable reducing agents include sulphides, thiol-containing compositions including dithiothreitol, trithiohexitol, cysteine or protein hydrolysates rich in cysteine, N-acetyl-cysteine, mercaptoethanol, thioglycerol, thioalkanoic acid and mercaptocarboxylic acid analogues, for example, mercaptosuccinic acid, thiolactic acid and their salts, thioglycollic acid and salts of thioglycollic acid and mixtures thereof. Preferred reducing agents are thioglycerol, cysteine, thiolactic acid, thioglycollic acid and their salts and mixtures thereof. Especially preferred is cysteine.
The reducing agent is conveniently present in an amount of from 0.00001% to 15%, preferably 0.01% to 10%, even more preferably 0.1% to 10% by weight of the composition.
Compositions according to the invention may further comprise an additional enzyme selected from glycosidases, other proteases, lipases or similar lipid modifying enzymes, ceramidases and mixtures thereof. Preferably the composition comprises 0.00001 to 50%, more preferably 0.001 to 20%, even more preferably 0.001 to 0.1% by weight of the composition of the additional enzyme.
Stratum corneum thiol proteases may be extracted from human or animal skin or callus by high salt solution (e.g. 2M NaCl), detergent or solvent extraction, and purified by chromatography or electrophoretic techniques. Recombinant stratum corneum thiol proteases may also be produced by biotechnological means by the over-expression of its gene in yeast, bacteria, plant or mammalian cells. Glycosidases, other proteases and lipases for inclusion in the compositions according to the invention may suitably be isolated from animal, plant, fungal or bacterial sources.
Typical glycosidases include neuraminidase, mannosidase, galactosidase, glucosidase, N-acetyl glucosaminidase and N-acetyl galactosaminidase. Preferably these may be isolated from plant sources including almonds, green coffee beans, and spinach, or may be obtained commercially.
Suitable additional proteases include bromelain, papain, chymotrypsin and chymotrypsin-like enzymes, stratum corneum chymotryptic-like enzyme, lysosomal cathepsin and cathepsin-like enzymes, alcalase, savinase, chymopapain, clostripain, endoproteinase Asp N, protease V.8, proteinase K, subtilisin, thermolysin, plasmin, pronase, and trypsin and trypsin-like enzyme. Preferably the protease may be isolated from plant sources including the seeds of wheat, barley, maize, oilseed rape, cocoa, linseed, illipe, shea nut, palm kernal, jojoba bean, pea, green bean, broad bean, soya bean and sunflower, and olives, papaya, pineapple, coconut, tomato and figs.
Lipases, or similar lipid modifying enzymes, may be isolated from plant, animal or bacterial sources. Suitable enzymes include lipolase, pancreatic lipases, phospholipases, ceramidase, aryl sulphatase, cholesterol esterase, candida rugosa OF360 lipase, humicola sp. lipase, pseudomonas sp. lipase and Candida antarctica A and B lipases.
Compositions according to the invention also comprise a vehicle to act as a dilutant, dispersant or carrier for the active ingredients in the composition, so as to facilitate their distribution when the composition is applied to the skin and/or hair. Preferably the vehicle is cosmetically and/or pharmaceutically acceptable.
Vehicles other than water can include liquid or solid emollients, solvents, humectants, thickeners and powders typically found in cosmetic formulations. Examples of each of these types of vehicle, which can be used singly or as mixtures of one or more vehicles, are as follows:
Emollients, such as stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmirate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, eicosanyl alcohol, behenyl alcohol, cetyl palmitate, silicone oils such as dimethylpolysiloxane, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, cocoa butter, corn oil, cotton seed oil, tallow, lard, olive oil, palm kernel oil, rapeseed oil, safflower seed oil, evening primrose oil, soybean oil, sunflower seed oil, avocado oil, olive oil, sesame seed oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum jelly, mineral oil, butyl myristate, isostearic acid, palmitatic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate;
Propellants, such as air, propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide;
Solvents, such as squalene, squalane, ethyl alcohol, methylene chloride, isopropanol, acetone, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulphoxide, dimethyl formamide, tetrahydrofuran;
Humectants, such as polyhydric alcohols including glycerol, polyalkylene glycols and alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxysorbitol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof.
Powders, such as chalk, talc, fullers earth, kaolin, starch, gums, colloidal silica sodium polyacrylate, tetra alkyl and/or trialkyl aryl ammonium smectites, chemically modified magnesium aluminium silicate, organically modified montmorillonite clay, hydrated aluminium silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate.
The vehicle will usually form from 10 to 99.9%, preferably from 50 to 99% by weight of the emulsion, and can, in the absence of other adjuncts, form the balance of the composition.
A particularly convenient form of the composition according to the invention is an emulsion, in which case an oil or oily material will normally be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophillic-lyophilic balance (HLB) of the emulsifier employed.
Compositions according to the invention can optionally comprise one or more oils or other materials having the properties of an oil.
Examples of suitable oils include mineral oil and vegetable oils, and oil materials, such as those already proposed herein as emollients. Other oils or oily materials include silicone oils, both volatile and non-volatile, such as polydimethyl siloxanes.
The oil or oily material, when present for the purposes for forming an emulsion, will normally form up to 90%, preferably from 10 to 80% by volume of the composition.
Compositions according to the invention may also optionally comprise one or more emulsifiers, the choice of which will normally determine whether a water-in-oil or an oil-in-water emulsion is formed.
When a water-in-oil emulsion is required, the chosen emulsifier or emulsifiers should normally have an average HLB value of from 1 to 6. When an oil-in-water emulsion is required, a chosen emulsifier or emulsifiers should have an average HLB value of >6.
Examples of suitable emulsifiers are set out below in Table 1 in which the chemical name of the emulsifiers is given together with an example of a trade name as commercially available, and the average HLB value.
The foregoing list of emulsifiers is not intended to be limiting and merely exemplifies selected emulsifiers which are suitable for use in accordance with the invention.
It is to be understood that two or more emulsifiers can be employed if desired.
The amount of emulsifier or mixtures thereof, to be incorporated in the composition of the invention, when appropriate is from 1 to 50%, preferably from 2 to 20% and most preferably from 2 to 10% by weight of the composition.
The compositions of the invention can also comprise water, usually up to 80%, preferably from 5 to 80% by volume.
Emulsifiers or surfactants in the form of silicone polymers may be incorporated into compositions of the present invention in place of or in addition to the optional emulsifier(s) already mentioned.
A particularly preferred silicone surfactant is cyclomethicone and dimethicone copolyol, such as DC 3225C Formulation Aid available from DOW CORNING. Another is laurylmethicone copolyol, such as DC Q2-5200, also available from Dow Corning.
The amount of silicone surfactant, when present in the composition will normally be up to 25%, preferably from 0.5 to 15% by weight of the emulsion.
Various other adjuncts conventionally found in cosmetic or pharmaceutical formulations may optionally be present in the compositions according to the present invention. They include preservatives, such as para-hydroxy benzoate esters; antioxidants, such butyl hydroxy toluene; humectants, such as glycerol, sorbitol, 2-pyrrolidone-5-carboxylate, dibutylphthalate, gelatin, polyethylene glycol, preferably PEG 200-600; buffers, such as lactic acid together with a base such as triethanolamine or sodium hydroxide; surfactants, such as glycerol ethers, waxes, such as beeswax, ozokerite wax, paraffin wax; plant extracts, such as Aloe vera, cornflower, witch hazel, elderflower, cucumber; thickeners; activity enhancers; colourants; and perfumes.
Various types of additional skin benefit ingredients may optionally be present in the compositions according to the present invention. These include sunscreens, hydroxy carboxylic acids and ketocarboxylic acids or esters thereof and lipids such as ceramides, pseudoceramides (synthetic ceramide-like structures), polyol fatty acid polyesters, sterols, phospholipids, galactosyldiacylglycerols, glycosphingolipids, fatty acids or esters thereof, and mixtures thereof.
Suitable sunscreens include those materials commonly used to block ultraviolet light and may include inorganic sunscreen materials, such as ultrafine titanium dioxide, or organic sunscreens such as p-aminobenzoic acid and esters thereof, ethylhexyl p-methoxycinnamate, 2-ethoxyethyl p-methoxycinnamate or butylmethoxydibenzoylmethane, and mixtures thereof. It will be appreciated that the amount of sunscreen employed will vary depending on the degree of protection required.
Suitable ceramides and synthetic analogues thereof which may be employed are disclosed in European patent application EP-A-587288 which is incorporated by reference herein. Preferred ceramides have the structure. ##STR1## where A represents --CH.sub.2 --; --CHOR.sub.5 --; --CH.dbd.CH-- or --CHOY--
R represents a linear or branched saturated or unsaturated, hydroxylated or non-hydroxylated aliphatic hydrocarbon group having from 1 to 49 carbon atoms or a subgroup (2).
R.sub.1 represents a linear or branched, saturated or unsaturated, hydroxylated or non-hydroxylated aliphatic hydrocarbon group having from 8 to 28 carbon atoms;
R.sub.2, R.sub.3 and R.sub.5 individually represent H, a phosphate residue or a sulphate residue;
R.sub.4 represents H, a phosphate residue, a sulphate residue or sugar residue;
a is an integer of from 1 to 49
b is an integer of from 10 to 98
m is 0 or 1
Y represents H or a residue of a C.sub.1-22 fatty acid having the general structure (3) ##STR2## where Z is --OH or an epoxy oxygen
x is an integer of from 0 to 20
y is an integer of from 0 to 40 and
z is 0 or an integer of from 1 to 4
Ceramides having the general structure (1) are naturally occurring and can be isolated from a suitable plant source or from animal tissue such as pig skin or neural tissue. Ceramides can also be synthesised.
Particular preferred examples of ceramides are ceramide-1, ceramide-2 and ceramide-3.
Pseudoceramides are preferably selected from pseudoceramides (i.e. synthetic ceramide like structures) having the general structure (4): ##STR3## where B represents --OCH.sub.2 -- or CHOH.
R.sub.6 represents a linear or branched, saturated or unsaturated, hydroxylated or non-hydroxylated aliphatic hydrocarbon group having from 1 to 49 carbon atoms or the subgroup (2).
R.sub.7 represents a linear or branched, saturated or unsaturated, hydroxylated or non-hydroxylated hydrocarbon group having from 8 to 28 carbon atoms.
R.sub.8 represents H, or a subgroup --(CH.sub.2).sub.c COOH, where c is an integer of from 1 to 6, or a subgroup having the structure (5). ##STR4## where X.sub.1, X.sub.2 and X.sub.3 each individually represent H, a C.sub.1-5 alkyl or a C.sub.1-5 hydroxyalkyl;
d is 0 or an integer of from 1 to 4
e is 0 or 1
n is 0 or 1 and
p is 0 or 1;
R.sub.9 represents H, a phosphate residue, a sulphate residue or a sugar residue.
Polyol fatty acid polyesters are fatty acid polyesters derived from any aliphatic or aromatic polyol which has at least 4 free hydroxyl groups, of which at least 60% of these free hydroxyl groups are then esterified with one or more fatty acids having from 8 to 22 carbon atoms. The polyol is preferably chosen from sugar polyols, which comprise mono-, di- and polysaccharides. Preferred polyol fatty acid polyesters are sucrose fatty acid polyesters where the ester is derived from lauric acid or natural oils, such as palm oil, palm kernal oil, soyabean oil, coconut oil, fish oil and mixtures thereof.
Sterols may conveniently be selected from cholesterol, provitamin D.sub.3, campesterol, stigmastanol, stigmasterol, 5-dihydrocholesterol, .alpha.spinasterol, palysterol, clionasterol, .gamma.-sitosterol, stigmastenol, sargasterol, avenasterol, ergostanol, sitosterol, corbisterol, chondrillasterol, poriferasterol, haliclonaseterol, neospongosterol, fucosterol, aptostanol, Ergostadienol, ergosterol, 22-dihydroergosterol, brassicasterol, 24-methylenecholesterol, 5-dihydroergosterol, dehydroergosterol, 14-dehydroergosterol, 24-dehydroergosterol, fungisterol, cholestanol, coprostanol, Zymosterol, 7-hetocholesterol, Lathosterol, 22-dehydrocholesterol, .beta.-sitosterol, cholestatrien-3.beta.-01, coprostanol, cholestanol, ergosterol, 7-dehydrocholesterol, 24 -dehydrocholest-adione-3.beta.-01, equilenin, equilin, estrone, 17.beta.-estradiol, Androst-4-ene-3.beta., 17.beta.-diol, dehydroepiandrosterone and mixtures thereof. Cholesterol is preferred.
Fatty acids are preferably essential fatty acids chosen from linoleic acid, .gamma.-linolenic acid, homo-.gamma.-linolenic acid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid, .alpha.-linolenic acid, timnodonic acid, hexaenoic acid and mixtures thereof.
Lipids, such as those described above, may conveniently be incorporated in compositions according to the invention in an amount of from 0.00001 to 50%, preferably from 0.001 to 10%, more preferably from 0.1 to 10% by weight of the composition.
Suitable hydroxy acids include .alpha.-hydroxy acids, .beta.-hydroxyacids, other hydroxycarboxylic acids and mixtures thereof. Preferably, the hydroxy acid is chosen from an .alpha.-hydroxy acid, or ester thereof, having the general structure:
wherein R.sub.a and R.sub.b are H, alkyl, aralkyl or aryl group of saturated or unsaturated, isomeric or non-isomeric, straight or branched chain or cyclic form, having 1 to 30 carbon atoms, and in addition R.sub.2 may carry F, Cl, Br, I, N, S, OH, CHO, COOH and alkoxy group having 1 to 9 carbon atoms; and mixtures thereof.
The alpha hydroxy acids may be present as a free acid or an ester form, or in a salt form with an organic base or an inorganic alkali. The typical alkyl, aralkyl and aryl groups for R.sub.1 and R.sub.2 include methyl, ethyl, propyl, isopropyl, butyl, pentyl, octyl, lauryl, stearyl, benzyl and phenyl, etc.
D, DL, or L stereoisomeric forms of an alpha hydroxy acid may be employed compositions. The L form is preferred.
Suitable alpha hydroxy acids which may be used include, but are not limited to, alpha hydroxy acetic acid (also known as "glycolic acid"), alpha hydroxypropionic acid (also known as "lactic acid"), alpha hydroxyoctanoic acid (also known as "alpha hydroxy caprylic acid"), alpha hydroxydodecanoic acid (also known as "alpha hydroxy lauric acid") and mixtures thereof.
Suitable esters include, but are not limited to, alkyl esters (for example, methyl, ethyl, propyl, pentyl, hexyl, octyl esters) and mono-, di- or triglycerides, or mixtures thereof.
Suitable salts of alpha hydroxy acids include but are not limited to sodium, potassium, ammonium, triethanolamine, calcium, lithium salts. The salts may be obtained commercially or they may be prepared by methods known in the art, e.g., neutralizing an alpha hydroxy acid with a suitable base, such as hydroxide bases of ammonium, potassium, sodium.
Conveniently, a mixture of alpha hydroxy acids may be employed. A suitable mixture comprises lactic acid, alpha hydroxy octanoic acid and alpha hydroxy lauric acid.
The preferred compositions according to the invention contain at least 60% of an alpha hydroxy acid in L-configuration, by weight of total alpha hydroxy acid.
The alpha hydroxy acid is suitably present in an amount of from 0.001% to 70%, preferably from 0.1% to 20%, most preferably from 1% to 10% by weight of the composition.
The keto acids can be chosen from .alpha.-keto acids, .beta.-keto acids and mixtures thereof. A particularly preferred .alpha.-keto acid is 2-keto octanoic acid.
Compositions according to the invention may also include chelating agents, particularly those having high affinity with zinc and/or magnesium ions.
Suitable chelating agents may conveniently be selected from aminocarboxylic acids or salts thereof, polyphosphoric acids or salts thereof, diphosphonic acids, salts of diphosphonic acids, tertiary amines, aminophosphonic acids, iminodiacetic acid derivatives, azines, hydroxyquinolines, and amino acid esters.
Examples of suitable chelating agents include but are not limited to ethylene diamine tetraacetic acid, a salt of ethylene diamine tetraacetic acid, sodium pyrophosphate, sodium tripolyphosphate, 8-hydroxyquinoline, DL-(Methylene) dinitrolo tetra acetic acids, trans-decahydronaphthylene-trans-2, 3-bis-iminodiacetic, aminophenyl methylene diphosphonic acid, ethylene-bis-N,N1-( 2,6-carboxyl) piperdine, adenosine triphosphate, L-cysteine methyl ester and 8-hydroxyquinoline.
Preferred chelating agents are EDTA and/or pyrophosphate, and/or 8-hydroxy quinoline due to their ready availability, excellent performance, relatively low cost, and safety in use.
The chelating agent is employed in the inventive compositions in an amount effective to enhance the activity of the enzyme. It will be appreciated that the precise amount will depend on the particular chelating agent used. Typically, the amount is in the range of 0.1 to 2%, preferably from 0.2 to 2% by weight of the composition.
Compositions according to the invention are useful in treating or alleviating conditions of the skip which are characterised by hyperkeratinisation, decreased rate of desquamation or abnormal desmosomal formation.
Accordingly, the invention provides the cosmetic or pharmaceutical use of a composition comprising one or more stratum corneum thiol proteases, particularly in the treatment of conditions where the underlying aetiology indicates that assisting desquamation and/or desmosomal degradation would be beneficial.
The invention further provides a method of treating skin comprising topically administering thereto a composition comprising one or more stratum corneum thiol proteases.
It will be appreciated that compositions according to the invention will primarily be of use in the treatment of established symptoms although prophylaxis is not excluded.
Compositions according to the invention are of use in treating or preventing diseases of the skin such as psoriasis, ichthyosis and acne. Compositions according to the invention are of particular interest in treating dry and/or flaky skin and in smoothing and enhancing the quality of skin. The compositions may also be used to alleviate dandruff.
It will be appreciated that the amount of the composition and the frequency of its application to the skin will depend on the condition of the patient.
In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to areas of the skin or scalp, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin or scalp using the hand or fingers or a suitable device.
The topical skin treatment compositions according to the invention may be formulated in conventional manner using one or more cosmetically and/or pharmaceutically acceptable carriers or excipients.
For example, the topical compositions of the invention may suitably be formulated as a lotion having a viscosity of from 4,000 to 10,000 mPas, a fluid cream having a viscosity of from 10,000 to 20,000 mPas or a cream having a viscosity of from 20,000 to 100,000 mPas, or above. The composition can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator or a propellant-driven aerosol device or a container fitted with a pump suitable for finger operation. When the composition is a cream, it can simply be stored in a non-deformable bottle or squeeze container, such as a tube or a lidded jar. The composition may be used for general lotions and creams, leave-on-creams, wash-off cleansers, face masks shampoos and bath oils.
The invention accordingly also provides a closed container containing an acceptable composition as herein defined.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an inhibition profile of selected stratum corneum thiol protease inhibitors as determined by gelatin zymography.
FIG. 2 shows the effect of varying the concentration of cysteine on the activation of selected thiol proteases.
FIG. 3 shows the effect of a range of thiol reagents on selected stratum corneum thiol proteases.
FIG. 4 shows the effect of thiol protease on stratum corneum dsc1 degradation.
EXAMPLES
Characterisation of Stratum Corneum Thiol Proteases
Human stratum corneum biopsys were obtained by tape stripping and the detached squames were extracted using 1M sodium chloride in 0.05M tris pH6.8, 0.1% (v/v) Triton X-100, 0.1% (w/v) sodium azide. The extract was then fractionated and the proteolytic activity detected using gelatin zymography.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) was performed using the method of Laemmli (Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970)) with 11% of polyacrylamide gels (C=5%) containing 0.2% (w/v) gelatin. The extracts were fractionated at 200V for approximately 1 h along with standard proteases of bovine trypsin (Sigma) and human liver cathepsin B (Novabiochem) and molecular weight markers. The gels were washed in 50 mM sodium acetate pH 6 containing 2.5% (v/v) triton X100 to remove SDS, followed by 50 mM sodium acetate pH 6, and then incubated in 50 mM sodium acetate pH 6 with and without 5 mM cysteine for 24 h at 37.degree. C. Proteolytic activity was determined after 0.1% (w/v) coomassie brilliant blue staining and quantified by scanning using an Epson GT8000 scanner and analyzed using Phoretix 1D gel analysis system. A unit of gelatinolysis was defined from the integrated optical density of the bands corresponding to a value of 1000.
Zymographs containing 0.2% gelatin revealed two closely migrating bands of gelantinolysis with apparent molecular weights of 34 and 35 KDa, when 5 mM cysteine was included in the incubation but not in its absence. Similarly, when casein was incorporated into the zymograph as an alternative substrate to gelatin, no proteolysis was detected at 34 and 35 KDa.
Inhibition Profile
Stratum corneum extracts were fractionated by SDS PAGE as above and the combined stratum corneum thiol protease activity determined by gelatin zymography, in the presence or absence of specific protease inhibitors with 5 mM cysteine for 24 h at 37.degree. C. The two proteases cannot be sufficiently resolved to be measured individually. The results obtained are presented in FIG. 1.
The concentrations of inhibitors used were 1 .mu.M E64, 10 .mu.M leupeptin, 10 .mu.M chymostatin, 10 .mu.M antipain, 1 .mu.M aprotinin, 2 mM PMSF, 5 mM EDTA and 1 .mu.M pepstatin.
Thiol protease specificity was shown by the total inhibition of activity with E64.
Thiol Activation of Stratum Corneum Thiol Proteases
The effect of varying the concentration of cysteine on the activation of the thiol proteases was investigated using gelatin zymography. The gels were incubated in the absence or presence of cysteine (0.0005 to 0.5M) and the combined activity determined. The results obtained are presented in FIG. 2.
The effect of a range of alternative thiol reagents on the combined activation of stratum corneum thiol proteases was similarly determined by gelatin zymography. The zymography gels were incubated with the thiol reagents at a concentration of 5 mM and the resultant values compared with cysteine control. The results obtained (FIG. 3) show that dithiothreitol (DTT) was equally effective as cysteine. Ethanthiol, thioglycerol and thioglycollate were approximately half as effective on a molar basis.
Desmosomal Degradation Assay
To assess the effect of the proteases on desmosomal degradation in stratum corneum, Superose 12 purified 34 and 35 kDa stratum corneum thiol proteases were incubated with chopped peelings from human sunburnt stratum corneum. The skin was incubated in 0.1M sodium acetate pH6, in the presence and absence of 5 mM cysteine, for 20 h at 37.degree. C. with the enzymes. An incubation containing both enzymes and the thiol protease inhibitor E64 (20 .mu.M) was also performed.
After incubation, the skin was washed in tris buffered saline containing 0.5% (v/v) Tween 20 and incubated for 1 h at 37.degree. C. with a desmosomal marker antibody (.alpha.48/46) raised against the 46 and 48 kDa N-terminal fragments of human desmocollin 1 (dsc 1) (Gift from Dr I King, N.I.M.R). This was followed by incubating for 1 h with an anti-rabbit IgG conjugated to FITC and the resulting fluorescence was detected by microscopy with a U.V. light source. The fluorescence was quantified from photographic negatives using an Epson GT8000 scanner coupled with Phoreti image analysis software.
The results obtained are presented graphically in FIG. 4. The levels of dsc1 are decreased significantly by the enzyme in the presence of 5 mM cysteine, indicating desmosomal degradation. This degradation was inhibited by E64 (20 .mu.M), a specific inhibitor of thiol proteases.
In order that the invention may be well understood, the following examples are given by way of illustration only.
Claims
I claim:
1. A topical composition comprising (i) a stratum corneum thiol cysteine protease which in its active form is inhibited by E64 and has an apparent molecular weight of 34 or 35 kDa when determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis as herein described; and (ii) a mild reducing agent selected from the group consisting of sulphides, thiol-containing compositions including dithiothreitol, trithiohexitol, cysteine or protein hydrolysates rich in cysteine, N-acetyl-cysteine, mercaptoethanol, thioglycerol. mercaptosuccinic acid, thiolactic acid and their salts, thioglycollic acid and salts of thioglycollic acid and mixtures thereof.
2. A composition according to claim 1 wherein the stratum corneum thiol protease is present in an amount of from 0.00001 to 50% by weight of the composition.
3. A composition according to claim 1 wherein the mild reducing agent comprises a thiol-containing compound.
4. A composition according to claim 1 wherein the reducing agent is cysteine.
5. A composition according to claim 1 wherein the reducing agent is present in an amount of from 0.00001 to 15% by weight of the composition.
6. A composition according to claim 1 further comprising an enzyme selected from glycosidases, other proteases, lipases and mixtures thereof.
7. A composition according to claim 1 further comprising one or more ingredients selected from sunscreens, hydroxycarboxylic acids, ketocarboxylic acids, ceramides, pseudoceramides, polyol fatty acid polyesters, sterols, phospholipids, galactosyldiacylglycerols, glycosphingolipids, fatty acids or esters thereof.
8. A method of relieving or ameliorating dry skin conditions, acne and dandruff which includes the topical application to the skin of a composition comprising (i) a stratum corneum thiol cysteine protease which in its active form is inhibited by E64 and has an apparent molecular weight of 34 or 35 kDa when determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis as herein described; and (ii) a mild reducing agent selected from the group consisting of sulphides, thiol-containing compositions including dithiothreitol, trithiohexitol, cysteine or protein hydrolysates rich in cysteine, N-acetyl-cysteine, mercaptoethanol, thioglycerol, mercaptosuccinic acid, thiolactic acid and their salts, thioglycollic acid and salts of thioglycollic acid and mixtures thereof.
Patent Citations (8)
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