MXPA97002920A - Compositions for skin care containing fatty acid amides, azoles and retinol or ester retinil - Google Patents

Abstract

The present invention relates to fatty acid amides in combination with azoles and either retinol or retinyl ester which result in a synergistic improvement in keratinocyte proliferation and synergistic inhibition of keratinocyte differentiation. The effects of retinol or retinyl esters in combination with fatty acid amides and azoles were analogous to treatment with retinoic acid

Description

COMPOSITIONS FOR SKIN CARE CONTAINING FATTY ACID AMIDES, Azoles and RETINOL OR RETINYL ESTER FIELD OF THE INVENTION The invention relates to skin care compositions containing fatty acid amides, azoles and retinol or retinyl ester.

BACKGROUND OF THE INVENTION Retinol (vitamin A) is an endogenous compound, which occurs naturally in the human body and is essential for the normal differentiation of epithelial cells. Natural and synthetic vitamin A derivatives have been extensively used in the treatment of a variety of skin diseases and have been used as skin repairing or renewing agents. Retinoic acid has been used to treat a variety of skin conditions, for example, acne, wrinkles, psoriasis, age spots and discoloration. See, for example, Vahlquist. A. et al., ".

Jnvest. Der atol. , Vol. 94, Holland D.B. and Cunliffe, W.J. (1990), pp. 496-498; Ellis, C.N. et al., "Pharmacology of Retinols in Skin ", Vasel, Karger, Vol. 3, (1989), pp 249,252; Lowe, N.J. et al.," Pharmacology of Retinols in Skin ", Vol. 3, (1989) pp. 240-248; PCT Patent Application No. - WO 93/19743. Retinol and retinyl esters, such as retinyl acetate and retinyl palmitate, are easier to formulate / stabilize than retinoic acid. Unfortunately, retinol and retinyl esters are less effective than retinoic acid in providing skin benefits. The present invention is based, in part, on the discovery that certain combinations of retinol or retinyl esters with fatty acid amides and azoles results in a synergistic improvement in the proliferation of keratinocyte and differentiation repression. The effects of the combination of a fatty acid amide with azole and either retinol or a retinyl ester were analogous to the effects of retinoic acid. This effect was not only greater than the effect of either retinol / retinyl ester with a fatty acid amide or retinol / retinyl ester with azole, but all three ingredients acted in synergy with each other to promote a retinoic acid response. In this way, a mixture of fatty acid amides with retinol or retinyl esters resembles retinoic acid, it is still easier to use than retinoic acid. Thornfeldt (U.S. Patent No. 5,057,501) describes a method for the treatment of papulosquamous and eczematous diseases with a composition containing a sesquiterpene compound and about 0.025% to about 35% of a monocarboxylic fatty acid, ester or amide. The compositions may also include a retinoid; Thornfeldt teaches that certain retinoids, mainly isotretinoin, tretinoin, etretin (all of which are stereoforms of retinoic acid) and etretinate (an ester of trimethoxyphenyl retinoic acid) have proven effective against papulosquamous diseases. PCT Application WO / 9325177 (Procter and Gamble) describes compositions for topical application to the skin, which contain a specific type of acyclic carboxamide cooler and may include retinoids such as retinoic acid and its derivatives (for example cis and trans). PCT Application WO / 9403156 (Rhone Poulenc) discloses a topical composition containing linoleic acid or a derivative as an active ingredient for the treatment and prophylaxis of impure skin (e.g., skin affected by pimples, pustules or pimples); the composition may also contain 0.025-0.1% by weight of tretinoin. European Patent Application No. 0 388 275 (Pierre Fabre Cosmetique) describes compositions for treating seborrhea containing alkyl carboxamide and a zinc salt which may be zinc retinoate. Klaus et al. (U.S. Patent No. 5,216,148) describes the use of specific complex carboxamides to treat and prevent neoplasms, dermatoses andaging of the skin Van Scott et al. (U.S. Patent No. 4,380,549) and Yu et al., (U.S. Patent No. 4,363,815) describe the treatment of acne, dry, scaly, laminate skin with a hydroxy acid or the amide thereof. EP 582,548 describes the use of N, N- (Cl 4 alkyl) lauramide, EP 559,304 discloses the use of an amide containing a hydrocarbyl chain of at least 25 carbon atoms as a skin softening agent. Beauquey et al. (U.S. Patent No. 5,308,551) describe a composition for washing and conditioning the skin containing, among other ingredients, an alkanolamide of 1-4 carbon atoms of a fatty acid of 8-16 carbon atoms. British Patent Specification No. 1,126,289 (Hoffman-La Roche) discloses a vitamin supply preparation containing alcohol of vitamin A or a vitamin A ester, an emulsifier and a solvent, which is selected from an alcohol or a dialkylated ida of a monocarboxylic acid (for example, N, N-diethyl acetamide, N, N-dimethyl acetamide or N, N-dimethyl formamide). A previously filed European Patent Application EP 0 742 005 (Unilever, priority date: May 8, 1995), published on November 13, 1996 (after the priority date of the present application), describes combinations of amides of fatty acid with retinol or retinyl esters. None of the aforementioned documents, however, mentions azoles. Compositions containing retinoids and azoles have been described. See, for example, Yusuf et al., CA 2,101,101, Cauwenbergh, United States Patent ,476,852 and Keyhani, PCT Patent Application WO 9505852.

These documents, however, do not mention the fatty acid amides. Also known are compositions containing azoles and fatty acid amides. These compositions, however, do not include any retinoids.

See, for example, WO 95/17175; EP 0 347,199; Patent of the United States No. 4,867,971; and United States Patent No. 5,348,736. The technique cited above does not disclose skin conditioning compositions based on synergistic combinations of three ingredients: a fatty acid amide, an azole and a retinol or a retinyl ester.

None of the aforementioned references addresses the need for an effective alternative for retinoic acid.

BRIEF DESCRIPTION OF THE INVENTION The present invention includes, in part, a skin conditioning composition containing: (a) from 0.001% to about 10% retinol or a retinyl ester; (b) from 0.001% to about 50% of an azole; (c) from 0.0001% to about 50% of a fatty acid amide, wherein the fatty acid contains at least 6 carbon atoms; and (d) a cosmetically acceptable vehicle.

The term "conditioning" as used herein, means the prevention and treatment of dry skin, photodamaged skin, appearance of wrinkles, age spots, aging skin, increased flexibility of the stratum corneum, and general increase in the quality of the skin The composition can be used in a cosmetic method to improve skin desquamation and epidermal proliferation and differentiation. The present invention also includes the use of the inventive composition for the manufacture of a medicament for the treatment of wrinkled, dry, scaly, aged, photodamaged skin and to treat skin disorders (e.g., acne or psoriasis).

The invention further provides a cosmetic method for improving the proliferation of keratinocyte and the differentiation in the skin, the method comprising applying to the skin the composition of the invention as described above. The presence of the fatty acid amide and an azole in the product of the invention substantially improves the yield of the retinol or of a retinyl ester, ie, a fatty acid amide in combination with the azole, substantially increases the capacity of the retinol or a retinyl ester to effect cell proliferation and differentiation. Fatty acid amide or azole has little or no effect to improve the benefit of the skin, when used alone; only a substantial increase in skin benefit can be observed when amide and azole are combined with retinol or a retinyl ester. In summary, the present invention is based, at least in part, on the discovery of the synergistic interaction between retinol or a retinyl ester, fatty acid amides and azoles. In a preferred embodiment of the invention, the amide is a C8-C24 fatty acid amide, and preferably a mono or di-alkanolamide of a Cg-C2 fatty acid and the azole is climbazole.

According to the present invention, by virtue of including an effective amount of a fatty acid amide and an azole to compositions containing retinol or a retinyl ester, the performance of the compositions is substantially improved. Alternatively, lower levels of retinol or a retinyl ester can be included in the composition containing the fatty acid amide and the azole to equal the yield of a similar formulation without the amide and the azole.

DESCRIPTION OF THE PREFERRED MODALITY All percentages are by weight of the final composition, unless otherwise indicated. The compositions of the invention contain, as a first essential ingredient, a compound selected from the group consisting of retinol or a retinyl ester. The term "retinol" includes the following isomers of retinol: all-trans-retinol, 13-cis-retinol, 11-cis-retinol, 9-cis-retinol, 3 -4-didehydro-retinol. Preferred isomers are all-trans-retinol, 13-cis-retinol, 3-4-didehydro-retinol, 9-cis-retinol. Most preferred is all-trans-retinol, due to its wide commercial availability. The retinyl ester is an ester of retinol. The term "retinol" has been defined in the above. The retinyl esters suitable for use in the present invention- are retinol esters of c? ~ c 0 '** ^ e Preference esters of C2-C2o And more preferably esters of C2 ~ C3 and c16' ^ a * 3ue are rn * ^ s commonly available Examples of retinyl esters include but are not limited to: retinyl palmitate, retinyl formate, retinyl acetate, retinyl propionate, retinyl butyrate, retinyl valerate, retinyl isovalerate, retinyl hexanoate, retinyl heptanoate, retinyl octanoate, retinyl nonanoate, decanoate of retinyl, retinyl undecanoate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadecanoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate , retinyl oleate, retinyl lactate, retinyl glycolate, retinyl hydroxy caprylate, retinyl hydroxylaurate, retinyl tartarate.The preferred ester for use in the present invention is selected from the group consisting of and retinyl palmitate, retinyl acetate and retinyl propinate, since these are the most commonly available, and therefore, the least expensive. Retinyl linoleate is also preferred due to its superior efficacy.

The retinol or retinyl ester is employed in the composition of the invention in an amount from about 0.001% to about 10%, preferably in an amount from about 0.01% to about 1%, most preferably in an amount of about 0.01% at approximately 0.5%. The second essential ingredient of the composition of the invention is a fatty acid amide. Preferably, the fatty acid amide contains at least 6 carbon atoms. Suitable fatty acids include saturated and unsaturated, straight or branched chain fatty acids. Suitable fatty acids preferably contain from 8 to 24 carbon atoms, preferably 12 to 20 carbon atoms and more preferably from 12 to 18 carbon atoms, since the longer chain fatty acid amides are more beneficial for condition the skin. In the most preferred embodiment of the invention, amides of essential fatty acids are used, since the essential fatty acids provide nutrition to the skin. Examples of essential fatty acids include, but are not limited to, linoleic, linolenic, arachidonic, gamma-linolenic, homo-gamma-linolenic and mixtures thereof. Linoleic acid is most preferred since it is also a precursor for ceramide.

Amides suitable for use in the present invention may be simple amides (ie, those containing a -CONH2 group), N-alkylamides, N, N-dialkylamides, mono-alkanolamides and di-alkanolamides. Suitable alkyl or alkanol groups contain from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms and more preferably from 1 to 8 carbon atoms. Preferred amides included in the present invention are mono and di-alkanolamides, particularly essential fatty acids. The alkanolamides are more commonly available than the alkylamides. The preferred fatty acid amides are selected from mono and di-ethanolamides of linoleic acid, palmitic acid and coconut oil. The amide is included in the compositions of the invention in an amount ranging from about 0.0001% to about 50%, preferably from 0.01% to about 10%, more preferably from about 0.1% to about 5%. The third essential ingredient of the compositions of the invention is an azole. The azoles used in the present invention have the formula I: wherein R- ^, R2 and R3 are independently selected from hydrogen; sulfhydryl group (SH), thiol or an alkyl containing 1-12 carbon atoms; an aryl group; an aryl group substituted with 1-5 halogen atoms; a heterocyclic group containing nitrogen and / or oxygen atoms; and mixtures thereof. The most preferred are climbazole, miconazole, bifonazole, econazole, clotrimazole. Also suitable for use in the present invention are 1, 2,4-triazole, octyltriazole, ketoconazole, itraconazole, fluconazole, terconazole, sulconazole, liazole, butoconazole and mixtures thereof. Azole is included in the composition of the invention in an amount from about 0.0001% to 50%, preferably from about 0.001% to about 10%, more preferably from 0.1% to about 5%.

Cosmetically acceptable Vehicle The composition according to the invention also comprises a cosmetically acceptable vehicle to act as a diluent, dispersant or vehicle for the active components in the composition, in order to facilitate its distribution when the composition is applied to the skin. Vehicles other than or in addition to water may include liquid or solid emollients, solvents, humectants, thickeners and powders. An especially preferred non-aqueous vehicle is a polydimethylsiloxane and / or polydimethylphenylsiloxane. The silicones of this invention can be those with viscosities ranging from about 10 to 10,000,000 mm2 / s (centistokes) at 25 ° C. Especially desirable are mixtures of silicones with a low and high viscosity. These silicones are available from the General Electric Company under the trademarks of Vicasil, SE and SF and the Dow Corning Company under the series 200 and 550. The amounts of silicone that can be used in the compositions of this invention vary from 5% to 95%. %, preferably 25% to 90% by weight of the composition. The cosmetically acceptable vehicle will usually form from 5% to 99.9%, preferably from 25% to 80% by weight of the composition, and can, in the absence of other cosmetic auxiliaries, form the remainder of the composition. Preferably, the vehicle is at least 80% by weight of water, by weight of the vehicle. Preferably, water comprises at least 50% by weight of the composition of the invention, more preferably 60 to 80% by weight of the composition.

Optional Skin Benefit Materials and Cosmetic Auxiliary An oil or an oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, greatly depending on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed. Preferred compositions of the invention include sunscreens. Sunscreens include those materials commonly used to block ultraviolet light. Illustrative compounds are the derivatives of PABA, cinnamate and salicylate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxy-benzophenone (also known as oxybenzone) can be used. Octyl methoxycinnamate and 2-hydroxy-4-methoxy-benzophenone are commercially available under the tradenames of Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen used in the emulsions can vary depending on the degree of protection desired from the sun's UV radiation.

Another preferred optional ingredient is selected from essential fatty acids (EFA), ie, those fatty acids that are essential for plasma membrane formation of all cells, in EFA deficiency of keratocytes that make the cells rproliferative. EFA supplementation corrects this. EFAs also improve lipid biosynthesis of the epidermis and provide lipids for epidermal barrier formation. The essential fatty acids are preferably chosen from linoleic acid, y-linolenic acid, homo-7-linolenic acid, columbinic acid, eicosa- (n-6, 9-13) -trienoic acid, arachidonic acid, Y-linolenic acid, timnodonic acid, hexaenoic acid and mixtures thereof. Emollients are usually incorporated into the cosmetic compositions of the present invention. The levels of such emollients may vary from about 0.5% to about 50%, preferably between about 5% and 30% by weight of the total composition. Emollients can be classified under general chemical categories such as esters, fatty acids and alcohols, polyols and hydrocarbons. The esters can be mono or di-esters. Acceptable examples of fatty di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate and dioctyl succinate. Chain fatty esters Branches acceptable include 2-ethylhexyl myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid esters include triisopropyl trilinoleate and trialuryl citrate. Acceptable straight chain fatty esters include lauryl palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred esters include caprylate / coconut caprate (a mixture of coconut caprylate and coconut caprate), propylene glycol myristyl ether acetate, diisopropyl adipate and cetyl octanoate. Suitable fatty alcohols and acids include those compounds having from 10 to 20 carbon atoms. Especially preferred are compounds such as cetyl, myristyl, palmitic and stearyl alcohols, and acids. Among the polyols that can serve as emollients are the straight and branched chain alkyl polyhydroxyl compounds. For example, propylene glycol, sorbitol and glycerin are preferred. Polymeric polyols such as polypropylene glycol and polyethylene glycol may also be useful. Especially preferred as penetration enhancers are butylene and propylene glycol. Illustrative hydrocarbons that can serve as emollients are those having hydrocarbon chains of 12 to 30 carbon atoms. The examples Specific include any mineral oil, petroleum jelly; squalene and isoparaffins. Another category of functional ingredients within the cosmetic compositions of the present invention are thickeners. A thickener will usually be present in amounts of 0.1 to 20% by weight, preferably from about 0.5% to 10% by weight of the composition. Illustrative thickeners are entangled polyacrylate materials, available under the trade name Carbopol from B.F. Goodrich Company. Gums such as xanthan, carrageenan, gelatin, karaya, pectin and locust bean gum can be used. Under certain conditions, the thickener function can be achieved through a material that also serves as a silicone or emollient. For example, silicone gums in an excess of 10 centistokes and esters such as glycerol stearate have a double functionality. Powders can be introduced into the cosmetic compositions of the invention. These powders include chalk, talc, Fuller's earth, kaolin, starch, smectite clays, chemically modified aluminum-magnesium silicate, organically modified montmorillonite clay, hydrous aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures of them.

Cosmetic compositions -other auxiliary minor components can also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers and perfumes. The amounts of these other minor auxiliary components may vary from 0.001% to 20% by weight of the composition.

Use of Composition The composition according to the invention is primarily intended to be a product for topical application to human skin, especially as an agent for conditioning and softening the skin, and to avoid or reduce the appearance of wrinkled or aged skin. During use, a small amount of the composition, for example from 1 to 100 ml, is applied to the exposed areas of the skin, from a suitable container or applicator and, if necessary, then extended over and / or rub on the skin using your hand or fingers or a suitable device.

Form and Product Packaging The composition for treating the topical skin of the invention can be formulated as a lotion, a cream or a gel. The composition can be packed in a suitable container to adapt its viscosity and intended use by the consumer. For example, a lotion or cream can be packaged in a bottle or a rotating ball applicator, or an aerosol device driven by a propeller or a container equipped with a pump suitable for operation with the finger. When the composition is a cream, it can simply be stored in a non-deformable bottle or compressible container, such as a tube or a container with a lid. The composition can also be included in capsules such as those described in U.S. Patent 5,063,507, incorporated herein by reference. The invention therefore also provides a closed container containing a cosmetically acceptable composition as defined herein. The following specific examples further illustrate the invention.

MATERIALS AND METHODS Cell culture; Human keratinocytes, isolated from neonatal foreskin were developed by treatment with trypsin in Dulbecco Modification Eagle (DME) Hams F12 medium (1: 1) / 10% fetal bovine serum in the presence of irradiated 3T3 mouse fibroblasts to establish the division of keratinocyte colonies. Cells were developed under the above conditions until their second passage and kept frozen for future use. The frozen second-pass keratinocytes were thawed and plated in the previous medium and developed for 5 days before they were switched to serum-free MCDB 153-based media, Clonetics keratinocyte growth medium (KGM). Corporation, San Diego, CA, containing 0.15 mM Ca, or keratinocyte serum free medium (KSFM) from GIBCO containing 0.09 mM Ca). On the seventh day; when the cells were 80-90% confluent, they were triptinized and plated in the serum free medium for several experiments.

Timidine test Incorporation of -3.H-Thymidine and Proliferation of Keratinocyte Incorporation of 3H-Thymidine by cultured keratinocytes was used as an assay for keratinocyte proliferation. Thymidine is one of the four deoxynucleosides, which are the monomeric units of DNA, the universal library of information genetics in the animal kingdom. Prior to the cell division of a somatic cell such as a keratinocyte, the complete genome of the cell undergoing cell division was replicated. This involves the large-scale synthesis of DNA by the cell and allows the daughter cells to receive identical copies of the genetic material. When 3H-thymidine is included in the keratinocyte culture medium, which are synthesized by DNA in the cell division preparation, then the labeled nucleoside is incorporated into the newly synthesized DNA. The degree of incorporation of 3H-thymidine into a population of cells is proportional to the rate of DNA synthesis by this population of cells and therefore an indication of their cell proliferation. The keratinocytes (which were cultured as described above) were placed in 24 well plates at a density of 40,000 cells per well in a 1 ml medium. After incubation for four days or until the cells were 60-70% confluent, the medium was changed. Test compounds were added (in triplicate) to the wells 24 hours after the medium change, and four hours later, lμCi3H-Thymidine in 50 μl of medium was added per well. The cells were incubated for an additional 24 hours. The medium was removed from the cells, 10% ice-cold trichloroacetic acid (TCA) was added and the plates were incubated on ice for 30 minutes. The cells were washed five times with 5% TCA and allowed to dissolve in 500 μl of 0. IM NaOH for at least one hour (usually overnight). The preparations were neutralized with 0. IM HCl; 50 μl of the cell preparation was used to determine the total protein content. Disintegrations per minute were determined (DPM) from the 3 H DNA labeling by liquid scintillation representing 900 μl of the cell preparation. The thymidine incorporation results were expressed as DPM protein / μg.

Transqlutaminase assay Transqlutaminase Assay and Keratinocyte Differentiation During the procedure of terminal differentiation in the epidermis, a thick layer of 15nm of protein was formed, known as the cornified envelope (CE) on the inner surface of the cell periphery. EC is composed of numerous different proteins, which have been co-entangled by the formation of isodipeptide bonds of N e- (7- glutamyl) lysine by the action of at least two different transglutaminases (TGases) expressed in the epidermis. TGase I is expressed in abundance in the differentiated layers of the epidermis, especially the granulated layer, but it is absent in the undifferentiated basal epidermis. In this way, TGase I is a useful marker of keratinocyte differentiation of the epidermis with high levels of TGase I indicating a more differentiated state. A TGase I analysis based on ELISA, using a TGase I antibody, was used to analyze the differentiation state of the keratinocytes cultured in the following examples. Keratinocytes were plated (cultured as described above) in plates with 96 wells at a density of 3,000 cells per well in a 200 μl medium. After incubation for four days, the medium was changed to a medium containing test compounds (six replicates per test). The cells were cultured for an additional 72 hours, after which the medium was aspirated and the plates stored at -70 ° C. The plates were removed from the freezer, and the cells were washed with PBS. 100 μl of sterile water was added and the cells were frozen fractured by freezing at -70 ° C, then thawed. The cells were incubated for one hour at room temperature (R / T) with PBS / 3% BSA (washing pH regulator, bovine serum albumin), then rinsed with a freshly prepared aliquot of a washing pH regulator . The cells were incubated with 50 μl of Monoclonal anti-human transglutaminase primary antibody (IgG) obtained from Amersham (mouse) diluted 1: 300 in a washing pH regulator for one hour, at 37 ° C, then rinsed twice with a washing pH regulator . Then, the cells were incubated with 50 μl of secondary antibody (Feb fragment, peroxidase-conjugated anti-mouse IgG obtained from Amersham) diluted 1: 200 in wash buffer for one hour at 37 ° C, then rinsed twice with washing pH regulator. The cells were incubated with substrate solution (4 mg of o-phenylenediamine and 3.3 μl of 30% H202 in 10 ml of 0. IM citrate pH regulator pH 5.0) for five minutes, R / T, in the dark (under an aluminum sheet). The reaction was stopped by the addition of 50 μl of 4N of H2SO4. The absorbance of the samples at 492nm was read on a plate reader. Out of the six replicates, four were treated with both antibodies, two were treated only with the secondary antibody (ie, to determine the background binding of Ab conjugated with enzyme). The TGase levels were determined by subtracting the antecedent of the readings of each treatment and determining ± s.d. medium for replicas exposed to both Ab.

DNA test The level of TGase-1 detected after the treatment of the cells can be influenced by the number of cells, that is, the larger the number of cells the higher the level of TGase-1 detected. The level of TGase-1 was normalized to a DNA content of the cells in the same well thus eliminating the variation due to differences in the number of cells. The quantification of DNA is a particularly useful indicator of the number of cells, including the number of keratinocyte cells, since each of the cells has all the attempts and purposes of an identical genome and therefore an identical amount of DNA. The total DNA content of a cell well is therefore directly proportional to the number of cells in that well. DNA quantitation was used to normalize the TGase data for the number of cells. Keratinocytes were plated in 96-well plates at a density of 3,000 cells per well in a 200 μl medium. After incubation for four days, the medium was exchanged for a medium containing test compounds (six replicates per test). The cells were cultured for an additional 72 hours, after which the medium was aspirated and the plates were stored for at least 1.5 hours at -70 ° C. The plates were removed of the freezer and the cells were fixed with a cold ethanol / acetone solution 1: 1 for 30 minutes. 100 μl / well of Hoechst dye (final concentration of 10 μg / ml) was added and this was incubated for 15 minutes, covered and then read in a fluorimeter (ex 360 nm and em 460 nm). The dye solution was removed and the wells were rinsed with PBS in a preparation for the TGase assay.

EXAMPLE 1 Retinoic acid is more effective than retinol to alter the state of keratinocyte differentiation A. The effect of incorporating soluble protein of μg of 3H-thymidine 24 hours after the addition of retinoic acid or retinol at various concentrations was examined. The results that were obtained are summarized in Table IA.

TABLE IT EFFECT OF RETINOIC ACID (RA) AND RETINOL (ROH) IN THE INCORPORATION OF TIMERINE FROM KERATINOCYTE n = 3 All tested retinoic acid concentrations, ie, 2.5 x 10 ~ 7 M, 2.5 x 10"8 and 2.5 x 10" 9M, significantly increased the proliferation of keratinocyte over ethanol control and each of the retinol treatments 2.5 x 10"7 M, 2.5 x 10_8M and 2.5 x 10 ~ 9M and performed it in a dose-dependent manner This is consistent with retinoic acid which has a greater stimulation effect on epithelial proliferation than retinol.

B. The effect on Transglutaminase levels was examined after the addition of retinoic acid and retinol. The results that were obtained are summarized in Table IB.

TABLE IB EFFECT OF RETINOIC ACID (RA) AND RETINOL (ROH) ON AN OUERATINOCYTE TRANSGLUTANIMASE LEVEL n = 3 All tested retinoic acid concentrations, ie, 2.5 x 10"7 M, 2.5 x 10" 8M and 2.5 x 10"9M, reduced keratinocyte differentiation over the control of ethanol and each of the retinol treatments and did so to a significantly greater extent than each of the retinol treatments of 2.5 x 10"7 M, 2.5 x 10" "8M and 2.5 x 10" 9M corresponding. The reduction in the transglutaminase level was dose dependent for both retinoic acid and retinol. This is consistent with retinoic acid that has a greater inhibitory effect on epithelial differentiation than retinol.

EXAMPLE 2 LINOLEOIL-DIETANOLAMINE (LINOEOIL-DEA). BIFONAZOLE AND RETINOL ACTING SINEROISTICALLY TO IMPROVE THE PROLIFERATION OF OUERATINOCITO AND TO INHIBIT THE DIFFERENTIATION A. The effect of incorporation of 3 H-thymidine / soluble protein μg was analyzed 24 hours after the addition of the test compounds, and the combined results of the three independent experiments were normalized to their respective ethanol controls. The results that were obtained are summarized in Table 2A.

TABLE 2A EFFECT OF RETINOL. BIFONAZOL AND LINOLEOIL-DEA ON THE INCORPORATION OF TIMIDINE OF KERATINOCYTE Average addition value p value p value p value p value Thyrnidine treatment / μg of s vs vs. (*, @ protein ± s.d. control 2.5x10 * 2.5x10"* (control) ROH RA Control 4368 ± 250 (100%) 0.105 0.008 • = 0.103 @ = 0.039 2? Cis? RA 55691248 1127%) 0.008 0.002 • = 0.158 F = 0.0B5 2. 5x1 * R? Tinol 4856 * 217 (11 1%) 0.105 0.038 • = 0.600 ® = 0.403 2. 5x1 s'M ROH + 10 * lM tADEA 50271 3W (1 15%) 0.103 0.600 0.158 @ = 0.936 2. 5x10? M ROH + iO ^ 5052 ± 202 (116%) 0.039 0.403 0.085 • = 0.936 Kfonazole 2.5x00"'M ROH + I0» M LADEA 56701 68 (130%) 0.011 0.029 0.142 • = 0.153 + lO'M Bitonazoi 0 = 0.048 n = 3 * = p value vs 2.5xlO? M ROH + IO ^ M LADEA @ = p value vs 2.5x10"'M ROH + I0'M Bifonazole 2. 5 x 10"9M retinoic acid significantly reduced the incorporation of keratinocyte thymidine to 27% with respect to ethanol control and to 16% with respect to the retinol treatment of 2.5 x 10" 9M. Both 2.5 x 10 ~ 9M retinol + 10"8M linoleoyl-DEA and 2.5 x 10" 9M retinol + ~ 9M of bifonazole had a marginal stimulatory effect on keratinocyte proliferation; on respect to retinol by itself. However, the combination of 2.5 x 10 ~ 9M of retinol + 10"8M of linoleoyl-DEA + 10" 9M of bifonazole significantly increased the proliferation of keratinocyte with respect to the ethanol treatments and 2.5 x 10"8M of retinol to a 30% and 19%, respectively The combination of 2.5 x 10 ~ 9M of retinol + 10 ~ 8M of linoleoyl-DEA + 10"9M of bifonazole also increased the proliferation of keratinocyte with respect to the treatment with 2.5 x 10" 9M of retinol + 10"8M linoleoyl-DEA and 2.5 x 10" 9M retinol + 10"9M bifonazole. Therefore, the fatty acid amides, bifonazole and retinol act synergistically to increase the proliferation of keratinocyte at levels that closely resemble the stimulatory effect of retinoic acid. B. The effect of normalized transglutaminase 1 (TG1) levels on a DNA content of the cells in response to a 72 hour treatment with test compounds was examined and is shown in Table 2B.

TABLE 2B EFFECT OF RETINOL, BIFONAZOLE AND LINOLEOIL-MEA IN THE OATERINUMITATE TGASE n = 6 2. 5 x 10"8M retinoic acid was very effective in suppressing keratinocyte TGl levels, ie at 13% of the control level, neither 2.5 x 10" 8M retinol nor 10 ~ 8M LAMEA + 10 ~ 8M of bifonazole had an inhibitory effect on the keratinocyte level of TG1. However, 2.5 x 10 ~ 8M of retinol + 10"8M of LAMEA + 10" 8M of bifonazole repressed the keratinocyte TG1 at 42% of control levels. The retinol, fatty acid amides and bifonazole, therefore, act synergistically to repress the differentiation of keratinocyte in a manner analogous to the effect of retinoic acid.

EXAMPLE 3 LINOLEOIL-DEA, CLIMBAZOL AND RETINOL SYNERGISTICALLY IMPROVING THE PROLIFERATION OF KERATINOCYTE AND THE INHIBITED DIFFERENTIATION A. The effect of linoleoyl-DEA-climbazole and retinol on 3 H-thymidine incorporation was examined. The results that were obtained are summarized in Table 3A TABLE 3A EFFECT OF RETINOL. CLIMBAZOL AND LINOEOIL-DEA IN THE INCORPORATION OF OUERATINOCYTE TIMIDINE n = 3 * = value p vs 2.5 x 10 * M ROH + 1Q M LADEA @ = value p vs 2.5 x lO ^ M ROH + 10 'M Clímbazol 2. 5 x 10"7M of retinoic acid significantly increased the incorporation of keratinocyte thymidine to 30% with respect to the ethanol control and to 28% with respect to the 2.5 x 10" 8M retinol treatment. Both 2.5 x ~ 8M retinol + 10"8M linoleoyl-DEA as 2.5 x 10 ~ 8M retinol + 10" 9M climbazole had a significant stimulating effect on the proliferation of keratinocyte with respect to control and retinol. However, the combination of 2.5 x 10"8M of retinol + 10" 8M of linoleoyl-DEA + 10 ~ 9M of climbazole significantly increased the proliferation of keratinocyte over the control as over 2.5 x 10 ~ 8M of retinol, treatments, to a 29% and 27%, respectively. More significantly, the combination of 2.5 x 10"8M retinol + 10 ~ 8M linoleoyl-DEA + 10" 9M climbazole also greatly increased keratinocyte proliferation in both 2.5 x 10"8M retinol + 10" 8M linoleoyl- DEA as 2.5 x 10 ~ 8M retinol + 10"9M climbazole at 17% and 20%, respectively.Therefore, retinol, linoleoyl-DEA and climbazole act synergistically to increase the proliferation of keratinocyte at levels that resemble closely related to the stimulatory effect of retinoic acid B. The effect of normalized transglutaminase 1 (TGl) levels on a DNA content of cells in response to a 72-hour treatment with test compounds was analyzed and is shown in Table 3B.

TABLE 3B EFFECT OF RETINOL. CLIMBAZOL AND LINOLEOIL-DEA ON LEVELS OF TGASE DE OUERATINOCITO n = 6 2. 5 x 10"7M retinoic acid was very effective in suppressing keratinocyte TGl levels, (at 29%) control level, while the dilution of 2.5 x 10_9M retinoic acid was not as affective but continued to inhibit the levels of TGl at 55% 2.5 x 10"9M retinol, 2.5 x ~ 9M of retinol + 10 ~ 8M of LADEA and 2.5 x 10"" 9M of retinol + 10"8M of climbazole did not have any inhibitory effect on the TGL level of keratinocyte, however, 2.5 x 10" 9M of retinol + 10"8M of LADEA + 10 ~ 8M of climbazole significantly suppressed keratinocyte TGl at 83% of control levels.This inhibition was significantly greater than the control, ROH alone, ROH + LADEA and ROH + climbazole indicating that all three ingredients, that is, ROH, LADEA and climbazole act synergistically to inhibit keratinocyte TGl levels.This effect was even greater when the climbazole concentration was increased to lOx, that is, 2.5 x 10"9M + 10" 8M of LADEA + 10"7M of climbazole, which resulted in this combination inhibiting TGl levels at 72% control. Retinol, fatty acid amides and climbazole, therefore, act synergistically to repress keratinocyte differentiation in a manner analogous to the effect of retinoic acid.

EXAMPLE 4 CLOTRIMAZOL, LINOLEOIL-MEA ("LAMEA") AND RETINOL THAT SYNERGISTICALLY IMPROVE THE PROLIFERATION OF OUERATINOCYTE The effect of incorporation of 3 H-thymidine / soluble protein μg 24 hours after the addition was examined of the test compounds and the results are shown normalized for the control in Table 4 TABLE 4 EFFECT OF RETINOL, Linoleoyl-MEA AND CLOTRIMAZOL ON THE INCORPORATION OF TIMERINE OF KERATINOCYTE n = 3 * = value p vs 2.5 x IO ^ M ROH + 10"" M LAMEA @ = p value vs 2.5 x 10"" M ROH + IO ^ M Clotrimazole 2. 5 x 10 ~ 9M retinoic acid significantly increased keratinocyte thymidine incorporation to 28%, over ethanol control and to 15% over treatment 2.5 x 10"9M retinol, both 2.5 x 10_9M retinol + 10" 8M of linoleoyl-MEA as 2.5 x 10 ~ 9M of retinol + 10 ~ 8M of clotrimazole had a stimulating effect on the proliferation of keratinocyte in the control, but this effect was not greater than retinol by itself. However, the combination of 2.5 x 10 ~ 9M retinol + 10"8M linoleoyl-MEA + 10 ~ 8M clotrimazole significantly increased the proliferation of keratinocyte both in the ethanol control and in the 2.5 x 10" 8M treatment. retinol, at 29% and 16%, respectively. More unexpectedly, the combination of 2.5 x 10"9M retinol + 10" 8M linoleoyl-MEA + 10"8M clotrimazole also significantly increased the proliferation of keratinocyte on both 2.5 x 10 ~ 9M retinol + 10" 8M linoleoyl- MEA as 2.5 x 10 ~ 9M retinol + 10"8M clotrimazole at 21% and 17%, respectively.The retinol, linoleoyl-MEA and clotrimazole therefore act synergistically to increase the proliferation of keratinocyte at levels that resemble closely related to the stimulatory effect of retinoic acid Examples 1-4 demonstrate that retinoic acid, in a dose-dependent manner, increased the incorporation of thymidine and reduced levels of transglutamine I in skin keratinocytes. In other words, retinoic acid increased keratinocyte proliferation and reduced keratinocyte differentiation. In Examples 1-4, the retinoic acid was used as a positive control and the reference compound against which the other compounds under analysis were compared. Retinol was significantly less effective than retinoic acid in inhibiting keratinocyte differentiation and completely ineffective in increasing keratinocyte proliferation. The unexpected results of Examples 1-4, however, were that the effect of retinol on cultured keratinocytes can be improved to levels reaching those of retinoic acid by combining retinol or retinyl ester with a fatty acid amide and an azole, although an azole and a fatty acid amide each exert little or no benefit. The results documented above show that the fatty acid amides in combination with azoles act synergistically with retinol or retinyl ester, both to increase keratinocyte proliferation and to reduce keratinocyte differentiation, resembling the effect of retinoic acid.

The unexpected result of this study was that the effect of retinol on cultured keratinocytes can be improved to levels approaching those of retinoic acid by combining retinol with a fatty acid amide and an azole. This effect was not only greater than the effect of either retinol + fatty amino acid or retinol + azole but the three ingredients act synergistically to each other to promote a retinoic acid type response. Examples 6-11 illustrate topical compositions according to the present invention. The compositions can be processed in a conventional manner. They are suitable for cosmetic use. In particular, the compositions are suitable for application to wrinkled, rough, dry, scaly, aged and / or UV-damaged skin to improve the appearance and feel thereof as well as for the skin health application to avoid or delay its deterioration.

EXAMPLE 6 This example illustrates a high internal phase of a water-in-oil emulsion incorporating the inventive composition.

* Brij 92 is polyoxyethylene (2) oleyl ether EXAMPLE 7 This example illustrates a water-in-oil cream that incorporates the inventive composition.

* Brij 56 is cetyl alcohol POE (10) Alfol 16RD is cetyl alcohol EXAMPLE 8 This example illustrates an alcohol lotion incorporating the composition according to the invention.

EXAMPLE 9 This example illustrates another alcohol lotion which contains the inventive composition, r? Retinol 0.15 Palmitoylmonoethanolam Ja 0.1 Climbazol Ethanol 40 Antioxidarote 0.1 Perfume CS Water for 100 EXAMPLE 10 This example illustrates a tanning cream incorporating the composition of the invention.

EXAMPLE 11 This example illustrates a non-aqueous skin care composition incorporating the inventive combination. 1 A dimethylsilicone polymer having a molecular weight of at least 50,000 and a viscosity of at least 10,000 centistokes at 25 ° C, available from GEC. 2 Pentamer of cyclic dimethylsiloxane, available from Dow Corning Corp. 3 Dimethylsiloxane tetramer, available from Dow Corning Corp.

Claims (9)

1. A skin conditioning composition characterized in that it comprises (a) from 0.001% to about 10% of a compound selected from the group consisting of retinol and a retinyl ester; (b) from 0.0001% to about 50% of an azole (c) from 0.0001% to about 50% of a fatty acid amide; and (d) a cosmetically acceptable vehicle.

2. The composition according to claim 1, characterized in that the fatty acid comprises from 12 to 18 carbon atoms.

3. The composition according to any of claims 1 or 2, characterized in that the amide is selected from the group consisting of N-alkanolamides, N, N-dialkanoylamides, and mixtures thereof.

4. The composition according to any of claims 1-3, characterized in that the retinyl ester is selected from the group consisting of linoleate of retinyl, retinyl palmitate, retinyl acetate, retinyl propionate and mixtures thereof. -

5. The composition according to any of claims 1-3, characterized in that the ingredient (a) is retinol.

6. The composition according to any of claims 1-5, characterized in that the azole is selected from the group consisting of climbazole, miconazole, bifonazole, clotrimazole, econazole.

7. A cosmetic method for conditioning the skin, the method is characterized in that it comprises applying topically to the skin, the composition according to any of claims 1-6.

8. A cosmetic method for treating the appearance of wrinkled, dry, rough, scaly, aged or photodamaged skin, characterized in that it comprises applying to the skin a composition according to any of claims 1-6.

9. A cosmetic method to resemble the effect on skin of retinoic acid, the method is characterized in that it comprises applying to the skin the composition according to claims 1-6.