Abstract:
An amide of a hydroxy fatty acid amide in combination with either retinol or retinyl ester resulted in a synergistic repression in keratinocyte proliferation. The effects of the retinol or retinyl esters in combination with hydroxy fatty acid amides were analogous to treatment with retinoic acid.

Description:
FIELD OF THE INVENTION 
     The invention relates to skin care compositions containing an amide of a hydroxy fatty acid and a retinoid, preferably 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 normal epithelial cell differentiation. Natural and synthetic vitamin A derivatives have been used extensively in the treatment of a variety of skin disorders and have been used as skin repair or renewal agents. Retinoic acid has been employed to treat a variety of skin conditions, e.g., acne, wrinkles, psoriasis, age spots and discoloration. See e.g., Vahlquist, A. et al., J. Invest. Dermatol., Vol. 94, Holland D. B. and Cunliffe, W. J. (1990), pp. 496-498; Ellis, C. N. et al., &#34;Pharmacology of Retinols in Skin&#34;, Vasel, Karger, Vol. 3,(1989), pp. 249-252; Lowe, N. J. et al., &#34;Pharmacology of Retinols in Skin&#34;, Vol. 3, (1989), pp. 240-248; PCT Patent Application No. WO 93/19743. It is believed that the use of retinol or esters of retinol would be preferred over retinoic acid. Retinol is an endogenous compound which occurs naturally in the human body and is essential for normal epithelial cell differentiation. Retinol is also considered much safer than retinoic acid. Esters of retinol hydrolyze in-vivo to produce retinol. Retinol and retinyl esters are considered safer than retinoic acid. Unfortunately, retinol and retinyl esters are less effective than retinoic acid at providing skin benefits. The present invention is based, in part, on the discovery that a combination of retinol or retinyl esters with amides of hydroxy fatty acids results in a synergistic inhibition in keratinocyte differentiation. The effects of hydroxy fatty acid amides combined with retinol or a retinyl ester were analogous to the effects of retinoic acid. Thus, a mixture of hydroxy fatty acid amides with retinol or retinyl esters mimics retinoic acid yet is easier and safer to use than retinoic acid. 
     Thornfeldt (U.S. Pat. No. 5,057,501) discloses a method for treatment of papulosquamous and eczematous diseases with a composition containing a sesquiterpene compound and from 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, namely isotretinoin, tretinoin, etretin (all of which are stereoforms of retinoic acid) and etretinate (an ester of trimethoxyphenyl retinoic acid) have proven efficacy against papulosquamous diseases. PCT Application WO/9325177 (Proctor and Gamble) discloses compositions for topical application to skin which contain a specific type of acyclic carboxamide coolant and may include retinoids such as retinoic acid and its derivatives (e.g., cis and trans). PCT application WO/9403156 (Rhone Poulenc) discloses a topical composition containing linoleic acid or a derivative as an active ingredient for treatment and prophylaxis of impure skin (e.g., skin affected by pimples, pustules, or comedones); the composition may also contain 0.025-0.1 wt. % of tretinoin. European Patent Application No. 0 388 275 (Pierre Fabre Cosmetique) discloses compositions for treating seborrhea containing alkyl carboxamide and a zinc salt which may be zinc retinoate. 
     Klaus et al., (U.S. Pat. No. 5,216,148) disclose the use of specific complex carboxamides for treating and preventing neoplasms, dermatoses, and aging of skin. Van Scott et al. (U.S. Pat. No. 4,380,549) and Yu et al., (U.S. Pat. No. 4,363,815) disclose treatment of acne, dry, flaky, scaly skin with a hydroxyacid or the amide thereof. EP 0 582 458 discloses use of N,N-(1,4C alkyl) lauramide. EP 0 559 304 disclose the use of an amide containing a hydrocarbyl chain of at least 25 carbon atoms as a skin smoothening agent. Beauquey et al. (U.S. Pat. No. 5,308,551) disclose a skin washing and conditioning composition containing, among other ingredients, a 1-4 C alkanolamide of a 8-16 C fatty acid. Great Britain Patent Specification No. 1,126,289 (Hoffman-La Roche) discloses a stock vitamin preparation containing vitamin A alcohol or a vitamin A ester, an emulsifier and a solvent which is selected from an alcohol or a dialkyl amide of a monocarboxylic acid (e.g., N,N-diethyl-acetamide, N,N-dimethyl acetamide or N,N-dimethyl formamide). The vitamin preparation has a very high vitamin content, i.e., the minimum concentration is 250,000 I.U. vitamin A/ml. Further, the amides disclosed in the &#39;289 application do not include or mention melinamide. 
     The art cited above does not disclose skin conditioning compositions based on synergistic combinations of hydroxy fatty acid amides with retinol or a retinyl ester. None of the art cited above addresses the need for an effective alternative to retinoic acid. 
     These and other objects of the invention will become more apparent from the detailed description and examples that follow. 
     SUMMARY OF THE INVENTION 
     The present invention includes, in part, a skin conditioning composition containing: 
     (a) from about 0.001% to about 10% of a retinoid selected from the group consisting of retinol, a retinyl ester, and retinoic acid; 
     (b) from about 0.0001% to about 50% of an amide of a hydroxy fatty acid; and 
     (c) a cosmetically acceptable vehicle. 
     The term &#34;conditioning&#34; as used herein means prevention and treatment of dry skin, photodamaged skin, appearance of wrinkles, age spots, aged skin, acne, skin lightening, psoriasis, atopic dermatosis, controlling sebum excretion, increasing stratum corneum flexibility, and generally increasing the quality of skin. The composition may be used to improve skin desquamation and cellular proliferation. 
     The presence of a hydroxy fatty acid amide in the inventive product substantially improves the performance of retinol or a retinyl ester, i.e., a hydroxy fatty acid amide substantially increases the ability of retinol or a retinyl ester to affect cellular proliferation. A hydroxy fatty acid amide has no or little effect on improving skin benefit when used alone; a substantial increase in skin benefit is only realized when a hydroxy fatty acid amide is combined with retinol or a retinyl ester. In short, the present invention is based, at least in part, on the discovery of synergistic interaction between retinol or a retinyl ester and a hydroxy fatty acid amide. 
     In a preferred embodiment of the invention, a retinoid is selected from the group consisting of retinol or a retinyl ester. According to the present invention, by virtue of including an effective amount of a hydroxy fatty acid amide into compositions containing retinol or a retinyl ester, the performance of the compositions is substantially improved. Alternatively, lower levels of retinol or a retinyl ester may be included in the composition containing a hydroxy fatty acid amide to equal the performance of a similar formulation without the amide. 
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The inventive compositions contain, as a first essential ingredient, a compound selected from the group consisting of retinol or a retinyl ester. 
     The term &#34;retinol&#34; 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. 
     Retinyl ester is an ester of retinol. The term &#34;retinol&#34; has been defined above. Retinyl esters suitable for use in the present invention are C 1  -C 30  esters of retinol, preferably C 2  -C 20  esters, and most preferably C 2 , C 3 , and C 16  esters because they are more 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, retinylheptanoate, retinyloctanoate, retinylnonanoate, retinyldecanoate, retinyl undecandate, retinyl laurate, retinyl tridecanoate, retinyl myristate, retinyl pentadecanoate, retinyl heptadeconoate, retinyl stearate, retinyl isostearate, retinyl nonadecanoate, retinyl arachidonate, retinyl behenate, retinyl linoleate, retinyl oleate, retinyl lactate, retinyl glycolate, retinyl hydroxy caprylate, retinyl hydroxy laurate, retinyl tartarate. 
     The preferred ester for use in the present invention is selected from retinyl palmitate, retinyl acetate and retinyl propionate, because these are the most commercially available and therefore the cheapest. Retinyl ester is also preferred due to its efficacy. 
     The retinoid is employed in the inventive composition in an amount of from about 0.001% to about 10%, preferably in an amount of from about 0.01% to about 1%, most preferably in an amount of from about 0.01% to about 0.5% . 
     The second essential ingredient of the inventive compositions is an amide of a hydroxy fatty acid. The structure of an amide of a hydroxy fatty acid is as follows: ##STR1## wherein R 1 , R 2  and R 4  each is independently selected from hydrogen or an aliphatic saturated or unsaturated, straight or branched hydrocarbon chain which may be hydroxylated, containing from 1 to 20 carbon atoms; 
     R 3  is --(CH 2 ) n  where n is an integer from 0 to 18; 
     Preferably, R 1 , R 2 , R 4  each is independently C 2  -C 20 , more preferably C 2  -C 15 , most preferably C 3  -C 13 . 
     Preferably the hydroxy acid amide is an amide of α-or β-hydroxy acid, i.e., n is 0 or 1. 
     The most preferred hydroxy fatty acid amides to be included in the inventive compositions are: lactamide-monoethanolamide, C 13  β-hydroxy acid amide (2-hydroxy-C 13  -amide), N-hydroxyethyl-2-hydroxy-C 16  amide, 12-hydroxy-N-(2-hydroxyethyl) octadecanamide, and monoethanolamide of castor oil. 
     The amide is included in the inventive compositions in an amount ranging from about 0.0001% to about 50%, preferably from about 0.01% to about 10%, most preferably from about 0.1% to about 5%. 
     Cosmetically Acceptable Vehicle 
     The composition according to the invention also comprises a cosmetically acceptable vehicle to act as a dilutant, dispersant or carrier for the active components in the composition, so as to facilitate their distribution when the composition is applied to the skin. 
     Vehicles other than water can include liquid or solid emollients, solvents, humectants, thickeners and powders. An especially preferred nonaqueous carrier is a polydimethyl siloxane and/or a polydimethyl phenyl siloxane. Silicones of this invention may be those with viscosities ranging anywhere from about 10 to 10,000,000 centistokes at 25° C. Especially desirable are mixtures of low and high viscosity silicones. These silicones are available from the General Electric Company under trademarks Vicasil, SE and SF and from the Dow Corning Company under the 200 and 550 Series. Amounts of silicone which can be utilized in the compositions of this invention range anywhere from 5 to 95%, preferably from 25 to 90% by weight of the composition. 
     The cosmetically acceptable vehicle will usually form from about 5 to about 99.9%, preferably from about 25 to about 80% by weight of the composition, and can, in the absence of other cosmetic adjuncts, form the balance of the composition. 
     Optional Skin Benefit Materials and Cosmetic Adjuncts 
     An oil or oily material may 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 hydrophilic-lipophilic balance (HLB) of the emulsifier employed. 
     Various types of active ingredients may be present in cosmetic compositions of the present invention. Various types of active ingredients may be present in cosmetic compositions of the present invention. Actives are defined as skin or hair benefit agents other than emollients and other than ingredients that merely improve the physical characteristics of the composition. Although not limited to this category, general examples include sunscreens, tanning agents. 
     Sunscreens include those materials commonly employed 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 trademarks, Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen employed in the emulsions can vary depending upon the degree of protection desired from the sun&#39;s UV radiation. 
     Another preferred optional ingredient is selected from essential fatty acids (EFAs), i.e., those fatty acids which are essential for the plasma membrane formation of all cells, in keratinocytes EFA deficiency makes cells hyperproliferative. Supplementation of EFA corrects this. EFAs also enhance lipid biosynthesis of epidermis and provide lipids for the barrier formation of the epidermis. The essential fatty acids are preferably chosen from linoleic acid, γ-linolenic acid, homo-γ-linolenic acid, columbinic acid, eicosa-(n-6,9,13)-trienoic acid, arachidonic acid, γ-linolenic acid, timnodonic acid, hexaenoic acid and mixtures thereof. 
     Yet another preferred optional ingredient is selected from azoles, e.g., climbazole, bifonazole, clotrimazole, ketoconazole, miconazole, econazole, itraconazole, fluconazole, terconazole, butoconazole, sulconazole, lionazole and mixtures thereof. 
     Emollients are often incorporated into cosmetic compositions of the present invention. Levels of such emollients may range from about 0.5% to about 50%, preferably between about 5% and 30% by weight of the total composition. Emollients may be classified under such general chemical categories as esters, fatty acids and alcohols, polyols and hydrocarbons. 
     Esters may be mono- or di-esters. Acceptable examples of fatty di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and dioctyl succinate. Acceptable branched chain fatty esters include 2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid esters include trilsopropyl trilinoleate and trilauryl citrate. Acceptable straight chain fatty esters include lauryl palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred esters include coco-caprylate/caprate(a blend of coco-caprylate and coco-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 such compounds such as cetyl, myristyl, palmitic and stearyl alcohols and acids. 
     Among the polyols which may serve as emollients are linear and branched chain alkyl polyhydroxyl compounds. For example, propylene glycol, sorbitol and glycerin are preferred. Also useful may be polymeric polyols such as polypropylene glycol and polyethylene glycol. Butylene and propylene glycol are also especially preferred as penetration enhancers. 
     Exemplary hydrocarbons which may serve as emollients are those having hydrocarbon chains anywhere from 12 to 30 carbon atoms. Specific examples include 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 anywhere from 0.1 to 20% by weight, preferably from about 0.5% to 10% by weight of the composition. Exemplary thickeners are cross-linked polyacrylate materials available under the trademark Carbopol from the B.F. Goodrich Company. Gums may be employed such as xanthan, carrageenan, gelatin, karaya, pectin and locust beans gum. Under certain circumstances the thickening function may be accomplished by a material also serving as a silicone or emollient. For instance, silicone gums in excess of 10 centistokes and esters such as glycerol stearate have dual functionality. 
     Powders may be incorporated into the cosmetic composition of the invention. These powders include chalk, talc, Fullers earth, kaolin, starch, smectite clays, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof. 
     Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers, perfumes and preservatives (e.g., imidazolidinyl urea, dimethyl imidazolidinone and diazolidinyl urea). Amounts of these materials may range anywhere from 0.001% up to 20% by weight of the composition. 
     Use of the Composition 
     The composition according to the invention is intended primarily as a product for topical application to human skin, especially as an agent for conditioning and smoothening the skin, and preventing or reducing the appearance of wrinkled or aged skin. 
     In use, a small quantity of the composition, for example from 1 to 5 ml, is applied to exposed areas of the skin, from a suitable container or applicator and, if necessary, it is then spread over and/or rubbed into the skin using the hand or fingers or a suitable device. 
     Product Form and Packaging 
     The topical skin treatment composition of the invention can be formulated as a lotion, a fluid cream, a cream or a gel. 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 capsule, 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 invention accordingly also provides a closed container containing a cosmetically acceptable composition as herein defined. 
    
    
     The following specific examples further illustrate the invention, but the invention is not limited thereto. Retinoids were obtained from Sigma. 
     MATERIALS AND METHODS 
     Cell Culture: 
     Human keratinocytes, isolated from neonatal foreskin by trypsin treatment were grown in Dulbecco Modification Eagle (DME) Hams F12 (1:1) medium/10% fetal calf serum in the presence of irradiated 3T3 mouse fibroblasts for establishing dividing keratinocyte colonies. Cells were grown under the above condition until their second passage and kept frozen for future use. Frozen second passage keratinocytes were thawed and plated into the above medium and grown for five days before they were switched to a serum-free MCDB 153-based medium keratinocyte growth medium (KGM) from Clonetics Corporation, San Diego, Calif. containing 0.15 mM Ca, or keratinocyte serum-free media (KSFM) from GIBCO containing 0.09 mM Ca). On day 7, when the cells were 80-90% confluent, they were trypsinized and plated in the serum-free medium for the various experiments. 
     TRANSGLUTAMINASE ASSAY 
     Transglutaminase Assay and Keratinocyte Differentiation 
     During the process of terminal differentiation in the epidermis, a 15 nm thick layer of protein, known as the cornified envelope (CE) is formed on the inner surface of the cell periphery. The CE is composed of numerous distinct proteins which have been cross-linked together by the formation of N .sup.ε -(γ-glutamyl) lysine isodipeptide bonds catalyzed 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 granular layer, but is absent in the undifferentiated basal epidermis. Thus TGase I is a useful marker of epidermal keratinocyte differentiation with high TGase I levels indicating a more differentiated state. An ELISA based TGase I assay, using a TGase I antibody, was used to assess the state of differentiation of the cultured keratinocytes in the examples that follow. 
     For Example 1, the following procedure was used: 
     Keratinocytes (cultured as described above) were plated in 96 well plates at a density of 3,000 cells per well in 200 μl media. After incubation for four days the media was changed to media containing test compounds (six replicates per test). The cells were cultured for a further 72 hours after which time the media was aspirated and the plates stored at -70° C. Plates were removed from the freezer, and the cells washed with PBS. 100 μl sterile water was added and the cells were freeze fractured by freezing at -70° C. then thawing. The cells were incubated for one hour at room temperature (R/T) with PBS/3% BSA (wash buffer, bovine serum albumin), then rinsed with a fresh aliquot of wash buffer. Cells were incubated with 50 μl of primary antibodies monoclonal anti-human transglutaminase mouse antibody (IgG) obtained from Biomedical Industries diluted 1:2,000 in wash buffer for one hour, 37° C. then rinsed two times with wash buffer. Cells were then incubated with 50 μl of secondary antibody (Fab fragment, peroxidase conjugated anti-mouse IgG obtaining from Amersham) diluted 1:4,000 in wash buffer for one hour at 37° C., then rinsed two times with wash buffer. Cells were incubated with substrate solution (4 mg o-phenylene diamine and 3.3 μl 30% H 2  O 2  in 10 ml 0.1 M citrate buffer pH 5.0) for five minutes, R/T, in darkness (under aluminum foil). The reaction was stopped by the addition of 50 μl 4N H 2  SO 4 . The absorbance of samples was read at 492 nm in the plate reader. Out of the six replicates, four were treated with both antibodies, two were treated only with the secondary antibody (i.e., to determine background binding of enzyme conjugated Ab). TGase levels were determined by subtracting background from the readings from each treatment and determining mean ±s.d. for the replicates exposed to both Ab. 
     For Example 2, the following procedure was used: 
     Keratinocytes (cultured as described above) were plated in 96 well plates at a density of 3,000 cells per well in 200 μl of cell culture media. After incubation for four days, the media was changed to media containing test compounds (six replicates per test). The cells were cultured for a further 72 hours after which time the media was aspirated and the plates stored at -70° C. After the plates were removed from the freezer, the cells were further freezed fractured by freezing and thawing and then washed 3× with PBS. The cells were incubated for one hour at room temperature (R/T) with TBS/5% BSA buffer. Cells were then incubated with 100 μl of monoclonal anti-human transglutaminase (IgG) mouse antibody (primary antibody) obtained from Biomedical Technologies Inc. diluted 1:2000 in TBS/1% BSA buffer for two hours at 37° C., and then rinsed six times with wash buffer (TBS/1% BSA/0.05% Tween-20). Cells were next incubated with 100 μl of Fab fragment, peroxidase conjugated anti-mouse IgG antibody (secondary antibody) from Amersham diluted 1:4,000 in wash buffer for two hours at 37° C. and then rinsed three times with wash buffer and three times with PBS. Cells were incubated with substrate solution (4 mg o-phenylene diamine and 3.3 μl 30% H 2  O 2  in 1O ml 0.1M citrate buffer, pH 5.0) for five minutes at R/T and in darkness (under aluminum foil). The reaction was stopped by the addition of 50 μl 4N H2SO4. The absorbance of samples was read at 492 nm in the plate reader. Out of the six replicates, four were treated with both antibodies, two were treated only with the secondary antibody (i.e., to determine the background binding of the enzyme conjugated antibody). Transglutaminase I levels were determined by subtracted background from the readings from each treatment and determining the mean s.d. for the replicates exposed to both antibodies. 
     DNA ASSAY 
     The level of TGase-1 detected after treatment of the cells could be influenced by cell number, i.e., the greater the number of cells the greater the level of TGase-1 detected. The level of TGase-1 was normalized to DNA content of the cells in the same well thus eliminating variation due to differences in cell number. DNA quantitation is a particularly useful indicator of cell number, including keratinocyte cell number, because each cell has to all intents and purposes an identical genome and therefore an identical quantity of DNA The total DNA content of a well of cells therefore is directly proportional to the cell number in that well. Quantitation of DNA was used to normalize the TGase data to cell number. 
     Keratinocytes were plated in 96 well plates at a density of 3,000 cells per well in 200 μl media. After incubation for four days the media was changed for media containing test compounds (6 replicates per test). The cells were cultured for a further 72 hours after which time the media was aspirated and the plates stored for at least 1.5 hours at -70° C. Plates were removed from the freezer and thawed for 30 minutes. 100 μl/well of Hoechst dye (1 μg/ml final concentration) 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 preparation for the TGase assay. 
     EXAMPLE 1 
     Retinoic acid is more effective than retinol at altering keratinocyte differentiation state 
     The effect on Transglutaminase levels normalized to DNA content of the cells after addition of retinoic acid and retinol was examined and the results are shown in Table 1. 
     
                                           TABLE 1__________________________________________________________________________ ##STR2##__________________________________________________________________________ n = 3 
    
     All concentrations of retinoic acid tested, i.e., 2.5×10 -7  M, 2.5×10 -8  M and 2.5×10 -9  M decreased keratinocyte differentiation over both the ethanol control and did so to a significantly greater extent than each of the corresponding 2.5×10 -7  M, 2.5×10 -8  M and 2.5×10 -9  M retinol treatments. The decrease in transglutaminase level was dose dependent for both retinoic acid and retinol. This is consistent with retinoic acid having a greater inhibitory effect on epithelial differentiation than retinol. 
     EXAMPLE 2 
     Amides of hydroxy fatty acids and retinol act synergistically to repress keratinocyte differentiation 
     The effect on transglutaminase 1 (TG1) levels normalised to DNA content of the cells was examined in response to a 72 hour treatment with the test compounds is shown in Table 2A. The amide was obtained from Quest International. C 13  β-hydroxy acid amide has the following structure: ##STR3## 
     
                                           TABLE 2A__________________________________________________________________________Effect of Retinol And C.sub.13 β-Hydroxy Acid Amide On KeratinocyteTGase/DNA                        p  p  p value                    p   value                           value                              vs 10.sup.-6           mean TGase/                    value                        vs vs C13-β-           DNA × 10.sup.5 ± s.d                    vs  10.sup.-7                           10.sup.-7                              hydroxy-acidTreatment       (% control)                    Control                        ROH                           RA amide__________________________________________________________________________Control         18.42 ± 3.88                    --  0.001                           0.001                              0.001           (100%)2.5 × 10.sup.-8 M RA           1.05 ± 1.05 (6%)                    0.001                        0.001                           -- 0.0012.5 × 10.sup.-8 M Retinol           14.62 ± 2.99 (79%)                    0.001                        -- 0.001                              0.00110.sup.-8 M C13-β-hydroxy-acid amide           18.53 ± 4.58                    0.875                        0.001                           0.001                              --           (101%)2.5 × 10.sup.-8 M ROH + 10.sup.-8 M C13-β-           11.36 ± 2.43 (62%)                    0.001                        0.001                           0.001                              0.001hydroxy-acid amide__________________________________________________________________________ n = 3 
    
     2.5×10 -8  M retinoic acid was very effective at repressing keratinocyte TG1 levels (to 6%) of control level. 2.5×10 -8  M retinol was less effective than retinoic acid (79%) and 10 -8  M C13 α-hydroxy-acid amide had no inhibitory effect on the keratinocyte TG1 level when used alone. However 2.5×10 -8  M retinol +10 -8  M C13 α-hydroxy-acid amide repressed keratinocyte TG1 to 62% of control levels. C13 α-hydroxy-acid amide and retinol therefore act synergistically to repress keratinocyte differentiation in an analogous manner to the effect of retinoic acid. 
     The effect on transglutaminase 1 (TG1) levels normalised to DNA content of the cells was examined in response to a 72 hour treatment with the test compounds is shown in Table 2B. &#34;Lactamide MEA&#34; is lactamide monoethanolamide. It was obtained from Croda Chemicals. Lactamide MEA has the following structure: ##STR4## 
     
                                           TABLE 2B__________________________________________________________________________Effect Of Retinol And Lactamide MEA On Keratinocyte Differentiation                         p  p  p value                     p   value                            value                               vs            mean TGase/                     value                         vs vs 10.sup.-6            DNA × 10.sup.5 ± s.d                     vs  10.sup.-7                            10.sup.-7                               lactamide-Treatment        (% control)                     Control                         ROH                            RA de-DEA__________________________________________________________________________Control          64.11 ± 2.91                     --  0.110                            0.002                               0.001            (100%)2.5 × 10.sup.-7 M RA            46.71 ± 7.83 (73%)                     0.002                         0.030                            -- 0.0492.5 × 10.sup.-7 M Retinol            58.47 ± 6.25 (91%)                     0.110                         -- 0.030                               0.05410.sup.-6 M lactamide-MEA            55.22 ± 2.43 (86%)                     0.001                         0.054                            0.049                               --2.5 × 10.sup.-7 M ROH + 10.sup.-6 M lactamide-            46.29 ± 6.79 (72%)                     0.001                         0.018                            0.930                               0.024MEA__________________________________________________________________________ n = 3 
    
     2.5×10 -7  M retinoic acid was effective at repressing keratinocyte TG1 levels (to 73%) of control level. 2.5×10 -7  M retinol and 10 -6  M lactamide-DEA were less effective at inhibiting keratinocyte TG1 level when used alone. However 2.5×10 -7  M retinol +10 -6  M lactamide-DEA repressed keratinocyte TG1 to 72% of control levels. Lactamide-DEA and retinol therefore act synergistically to repress keratinocyte differentiation in an analogous manner to the effect of retinoic acid. 
     Examples 1 and 2 demonstrate that retinoic acid, in a dose dependent manner, decreased keratinocyte differentiation. In Examples 1 and 2, retinoic acid was used as positive control and reference compound against which the other compounds under analysis were compared. Retinol was completely ineffective at decreasing keratinocyte differentiation. 
     The unexpected results of Examples 1 and 2, however, were that the effect of retinol on cultured keratinocytes can be enhanced to levels approaching those of retinoic acid by combining retinol or retinyl ester with an amide of hydroxy fatty acid --a compound which exerts little or no benefit on its own. The results documented above demonstrate that an amide of hydroxy fatty acid acts synergistically with retinol or retinyl ester, to decrease keratinocyte differentiation, mimicking the effect of retinoic acid. 
     EXAMPLE 3 
     This example illustrates a high internal phase water-in-oil emulsion incorporating the inventive composition. ##STR5## 
     EXAMPLE 4 
     This example illustrates an oil-in-water cream incorporating the inventive composition. ##STR6## 
     EXAMPLE 5 
     This example illustrates an alcoholic lotion incorporating the composition according to the invention. ##STR7## 
     EXAMPLE 6 
     This example illustrates another alcoholic lotion containing the inventive composition. ##STR8## 
     EXAMPLE 7 
     This example illustrates a suncare cream incorporating the composition of the invention: ##STR9## 
     EXAMPLE 8 
     This example illustrates a non-aqueous skin care composition incorporating the inventive combination. ##STR10## 
     It should be understood that the specific forms of the invention herein illustrated and described are intended to be representative only. Changes, including but not limited to those suggested in this specification, may be made in the illustrated embodiments without departing from the clear teachings of the disclosure. Accordingly, reference should be made to the following appended claims in determining the full scope of the invention.