COSMETIC COMPOSITIONS

Abstract: Cosmetic compositions contain at least one compound stimulating the neosynthesis of at least one important component of the dermo-epidermal junction.

Description: The present invention relates to new cosmetic compositions containing at least one compound stimulating the neosynthesis of laminins, and/or α2β1 integrin and/or collagen IV of the dermo-epidermal junction. Human skin is composed of three superimposed tissues: the hypodermis (the deepest), the dermis and finally the epidermis (the outermost). In order to fulfil its function as the first line of protection against aggressive influences (mechanical, chemical, microbiological), the epidermis has a highly differentiated structure: from the basal layer, the cells known as keratinocytes undergo a metabolic process of keratinisation which causes them to be transformed as they migrate towards the upper layers, finally becoming extremely resistant and relatively impermeable corneocytes which, with the epidermal lipids secreted by the keratinocytes of the granular layer, form the stratum corneum, the outermost layer of the epidermis, in contact with the external environment. Moreover, the epidermis contains other types of cells having important functions and a great capacity for communication: melanocytes, Langerhans' cells, Merckel cells. This process of great differentiation and these numerous intercellular communications require numerous nutrient components. The epidermis is not, however, irrigated by blood or lymph vessels. These nutrient components must therefore reach it from the dermis which is irrigated. Correct diffusion of these components across the dermo-epidermal junction (DEJ) which connects these two tissues is therefore required. This dermo-epidermal junction must also manage the diffusion of biochemical messengers between the two tissues. It has been shown that numerous substances released by each of these tissues interfere with the activity of the other (research by Pr.R.E. Burgeson, Smola H., Exp.Cell.Res. 1998 Mar 15; 239(2): 399-410). Finally, the DEJ must provide contact between these two tissues which are of a very different nature. The dermis is a tissue composed of a fundamental substance interspersed with a few cells (fibroblasts) and very numerous fibres (collagens and elastin), which gives the dermis an amorphous and compact structure though capable of stretching under a mechanical stress and then reverting to its initial size when the stress is released. The epidermis is a stratified type with a high density of contiguous, columnar cells. The DEJ is thus capable of ensuring cohesion between these two tissues with relatively incompatible mechanical properties when the skin is mechanically deformed. In order to fulfil this function, the DEJ takes the shape of highly pronounced waves. For all these reasons, the DEJ has a very important role from a metabolic and mechanical angle for the good health of the skin. It has been established that, during the ageing process, the quality of the DEJ tends to diminish: it becomes flatter and fulfils its "spring" function much less satisfactorily (B.Le Varlet et al. Journal of Investigative Dermatology Symposium Proceedings 3:172-179, 1998). The important role of the DEJ is also attested by the possibility of metabolic diseases when one of the components of the DEJ is absent or of poor quality. The case of bullous pemphigoid may be mentioned, in which the absence of an important component (laminin-5) leads to the formation of blisters: the epidermis becomes detached from the dermis and forms blisters which develop into sores which are difficult to heal. The structure of the DEJ is becoming increasingly better known: four layers can be distinguished, each of which has specific constituents and a very precise role (Allen J., Br.J.Dermatol. 1997 Dec; 137(6): 907-15), (M.Aumailley, Kidney Internal, Vol 47, Suppl.49 (1995), pp S-4-S-7). These important components include laminins, integrins and above all collagens, including mainly collagen IV. In order to combat slackening, loss of flexibility and tone, dull and/or faded appearance of the skin, some importance has been paid to cosmetic products for revitalising the components of the dermis and/or epidermis. A new trend involves stimulating the synthesis of the important components of the DEJ. By selecting active ingredients for this function by two specific tests (described below in the experimental part), the Applicant discovered that ursolic acid and oleanolic acid, which are not described in the literature for this type of properties, are in fact capable of stimulating the neosynthesis of collagen IV and that, moreover, the combination with a specific palmitoyl pentapeptide -Lys-Thr- Thr-Lys-Ser for this activity, increased their stimulating capacity in an unexpected synergistic manner. Moreover, this palmitoyl pentapeptide -Lys-Thr-Thr-Lys-Ser is capable of stimulating the synthesis of 2β1 integrin and laminin. Finally, the Applicant discovered that another active principle, a peptide extract of white lupin, known for its anti-elastase and anti-collagenase (anti- metalloproteinase) properties is also capable of stimulating significantly the synthesis of collagen IV. These active principles alone or in combination are therefore perfectly suitable for stimulating a whole series of functions of the skin, particularly the neosynthesis of important components of DEJ, such as laminins, α2β1 integrin and, in a synergistic manner, collagen IV. For this reason, the present application provides a cosmetic composition containing at least one compound stimulating the neosynthesis of at least one and preferably two, particularly three important components of the dermo- epidermal junction and more particularly laminins, and/or α2β1 integrin and/or collagen IV. Examples of compounds which stimulate the neosynthesis of important components of the dermo-epidermal junction include preferably ursolic acid, oleanolic acid, palmitoyl pentapeptide in which the pentapeptide has the formula: -Lys-Thr-Thr-Lys-Ser and peptide extracts of white lupin. Ursolic acid is a pentacyclic triterpene: 3β-hydroxyurs-12-en-28-oic acid. It is found in a large number of plants, particularly rosemary. It is generally associated in these plants, and in the commercially available extracts, with its isomer oleanolic acid (3-hydroxyolean-12-en-oic acid). These 2 products have many biological properties in common, as described by Jie Liu in "Journal of Ethnopharmacology 49 (1995) 57-68)": hepatoprotective, anti-inflammatory, anti- tumour, hypolipaemic, anti-atherosclerosis, anti-ulcer, antimicrobial, hypoglycaemic effects, a protective effect towards the toxicity induced by cyclophosphamide, anti-cariogenic and anti-fertility effects. Other publications detail certain properties such as inhibition of human leukocyte elastase (Ying, Biochem. J (1991) 277,521-526), the protective effect towards lipid lipoperoxidation (Balanehru, Biochemistry Intemat. Vol. 24, no. 5, July 1991 , 981- 990), inhibition of lipoxygenase and proliferation of HL60 leukaemia cells (Simon, Biochimica and Biophysica Acta, 1125 (1992) 68-72), and finally anti-viral activity (Serra, Pharmacological Research, Vo. 29, no. 4, 1994, 359-366). The mixtures of ursolic acid/oleanolic acid are generally in the form of a sodium salt, the respective proportions of the two ranging from 80 - 20 to 70 - 30 depending on the plant from which said mixtures are extracted, the purity of the mixture of the two ranging from 60% to 98%. Consequently, the compositions according to the invention may contain either or both of the acids. The peptide extract of white lupin (variety L. albus) contains pure peptides with a low molecular weight obtained from seeds from which the lipids have been removed according to a bioengineering process which removes polysaccharides. This process, which comprises the unit steps of solubilisation of the vegetable proteins, enzymatic hydrolysis and ultrafiltration, makes it possible to obtain peptides containing 5 to 6 units of amino acids, on average. This type of product is sold, in particular, by the company Expanchimie under the commercial name of "Actimp1.9.3®" and it is known for its inhibiting properties with respect to matrix metalloproteinases (MMP). The product is an aqueous solution which contains about 10% of dry substance; its pH is 6.5 to 7.5. Palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser is a mimetic of a fragment of Type I procollagen. It is obtained by peptide synthesis and then bound to a palmitic acid chain in order to improve its lipophilic properties and hence its penetration of the skin. Palmitoyl pentapeptide -Lys-Thr-Thr-Lys-Ser is sold by the company Sederma under the commercial name Matrixyl®, contained in a gel in a quantity of 100 ppm, the gel containing about 25% of water, about 20% of butylene glycol, about 1% of carbomer and about 0.5% of polysorbate 20. The commercial product is in the form of a whitish opalescent gel with a pH of 4 to 6, a density at 20 °C of 1.140 to 1.160 and with a refractive index at 25 °C from 1.425 to 1.445. Under preferred conditions of carrying out the invention, a composition as mentioned above contains ursolic acid and/or oleanolic acid and also palmitoyl pentapeptide in which the pentapeptide has the formula: -Lys-Thr-Thr-Lys-Ser. In the compositions according to the invention, ursolic acid may represent, for example, from 0.001% to 10%, particularly 0.01% to 5%, preferably from 0.05% to 2% and more particularly from 0.07% to 1% of the finished composition. Oleanolic acid may represent, for example, from 0.00025% to 2.5%, particularly 0.0025% to 1.3%, preferably from 0.0125% to 0.5% and more particularly from 0.0175% to 0.25% of the finished composition. Palmitoyl pentapeptide may represent, for example, from 0.1 ppm to 30 ppm, particularly 0.5 ppm to 20 ppm, preferably from 1 ppm to 15 ppm and more particularly from 2 ppm to 10 ppm of the finished composition. The peptide extract of white lupin may represent, for example, from 0.2% to 10%, particularly 0.5% to 5%, preferably from 0.7% to 4% and more particularly from 1 % to 3% of the finished composition. Compositions containing from 0.01% to 5% of ursolic acid/oleanolic acid combined with 0.1 ppm to 30 ppm of palmitoyl pentapeptide-Lys-Thr-Thr-Lys- Ser, and those containing from 0.2% to 2% of ursolic acid/oleanolic acid combined with 1 ppm to 10 ppm of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser may be mentioned more particularly. Particularly preferred combinations of active principles are combinations in the same composition of - ursolic acid and oleanolic acid, - ursolic acid and/or oleanolic acid and palmitoyl pentapeptide-Lys-Thr- Thr-Lys-Ser, - ursolic acid and/or oleanolic acid and peptide extract of white lupin, - ursolic acid and/or oleanolic acid, palmitoyl pentapeptide-Lys-Thr-Thr- Lys-Ser and peptide extract of white lupin. The compositions according to the invention are intended substantially for application to the skin. Advantageously, they will take the form of hydro-alcoholic gels or hydro-glycolic gels, water/oil or oil/water emulsions with or without emulsifiers, with or without lamellar phases, water/oil/water or oil/water/oil triple emulsions, microemulsions, mini-emulsions. The compositions forming the subject matter of the present invention have very attractive properties. In particular, they have remarkable properties for combating the effects of skin ageing. These properties are illustrated below in the experimental part. They justify the use of the compositions described above as cosmetic compositions. The compositions according to the present invention are used, for example, in the fight both to cure and prevent the effects of skin ageing. They also find application in face and body care for combating skin slackening, in slimming, for sensitive skins, mature skins and for greasy skins. The compositions according to the present invention are prepared by conventional methods. The active compound(s) may be incorporated in excipients normally used in cosmetic compositions intended for topical application, such as glycerol, sorbitol, stearates, PEG, silicones, cocoa butter, aqueous or non-aqueous vehicles, oil substances of animal or vegetable origin, paraffin derivatives, glycols, alcohols or oil acids, various moisturisers, dispersing agents or emulsifiers, preservatives, perfumes. The present invention also provides a process for the preparation of a composition described above, characterised in that the active principle(s) is (are) mixed, by inherently known methods, with acceptable excipients, particularly cosmetically acceptable excipients. Generally speaking, an aqueous phase and an oil phase will be prepared separately, then mixed at elevated temperature with stirring. The present application also provides the use of a compound which stimulates the neosynthesis of at least one and preferably two, particularly three important components of the dermo-epidermal junction and more particularly laminins, and/or α2β1 integrin and/or collagen IV for the treatment of skin and particularly for curing and preventing the effects of skin ageing, and a process for curing and preventing the effects of skin ageing in which an effective amount of a composition as described above is applied topically. The preferred conditions of using the compositions described above also apply to the other subject matter of the invention mentioned above. The examples which follow illustrate the present invention. Example 1 : Anti-wrinkle cream An anti-wrinkle cream for normal skins and combination skins was prepared as follows: The following oil phase was prepared: Cetyl alcohol and glyceryl stearate and PEG-7S stearate and ceteth-20 and steareth-20 4 g Jojoba oil 3 g Caprylic/capric triglycerides 3 g Jojoba esters 1 g Hydrogenated polyisobutene 2 g Cyclomethicone 5 g This mixture was heated to 70 °C and homogenised thoroughly. Moreover, the following aqueous phase was prepared: Purified water, quantum sufficit (q.s.) 100 g Tetrasodium EDTA 0.05 g Sorbitol 2 g Cellulose 1 g Silica beads enrobed with titanium dioxide and iron oxide 1 g Copolymer of sodium acryloyldimethyl taurate and isohexadecane and polysorbate 80 2 g All the ingredients of the aqueous phase were dissolved in water and the mixture heated to 70 °C. The homogeneous oil phase heated to 70 °C was poured slowly over the aqueous phase heated to the same temperature, with fairly vigorous stirring. Stirring was maintained for 10 minutes after the end of the introduction of the oil phase. The oil in water emulsion formed. The emulsion was then cooled with moderate stirring. When the temperature had reached 40 °C, the following were added successively with moderate stirring: Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser 3 g Extract of white lupin 1 g Ursolic acid/oleanolic acid, sodium salt Polyethylene glycol 400 (PEG 400) (10/90) 2 g Sodium hyaluronate 0.5 g Preservatives 0.15 g Perfume 0.35 g After homogenisation, the emulsion was cooled to 30 °C with slow stirring. A pink coloured emulsion with a pleasant consistency and suitable for normal and combination skins was obtained. Example 2: Cosmetic emulsion The following oil phase was prepared: Cetyl alcohol and glyceryl stearate and PEG-7S stearate and ceteth-20 and steareth-20 4 g Jojoba oil 4 g Caprylic/capric triglycerides 4 g Jojoba esters 1 g Hydrogenated polyisobutene 4 g Isocetyl stearate 4 g Cetyl ricinoleate 4 g Shea butter 1 g Silicone oil (phenyl trimethicone) 5 g Silicone oil (dimethicone) 2 g Dipentaerythrityl hexacaprylate/hexacaprate 3 g This mixture was heated to 70 °C and homogenised thoroughly. Moreover, the following aqueous phase was prepared: Purified water, quantum sufficit (q.s.) 100 g Tetrasodium EDTA 0.05 g Sorbitol 3 g Glycerol 4 g Glyceryl polymethacrylate/propylene glycol 5 g Cellulose 2 g Silica beads enrobed with titanium dioxide and iron oxide 1 g Copolymer of sodium acryloyldimethyl taurate and isohexadecane and polysorbate 80 1.25 g All the ingredients of the aqueous phase were dissolved in water and the mixture heated to 70 °C. The homogeneous oil phase heated to 70 °C was poured slowly over the aqueous phase heated to the same temperature, with fairly vigorous stirring. Stirring was maintained for 10 minutes after the end of the introduction of the oil phase. The oil in water emulsion formed. The emulsion was then cooled with moderate stirring. When the temperature had reached 40 °C, the following were added successively with moderate stirring: Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser 3 g Extract of white lupin 1 g Ursolic acid/oleanolic acid, sodium salt Polyethylene glycol 400 (PEG 400) (10/90) 2 g Sodium hyaluronate 0.1 g Preservatives 0.15 g Perfume 0.45 g After homogenisation, the emulsion was cooled to 30 °C with slow stirring. A pink coloured emulsion with a pleasant consistency and suitable for dry and very dry skins was obtained. Example 3: Cream of the oil/water type The following cream of the oil/water type was prepared: The oil phase was composed of: Dimethicone copolyol and caprylic/capric triglycerides 3 g Isodecyl neopentanoate 5 g Hydrogenated polyisobutene 5 g Caprylic/capric triglycerides 5 g Jojoba oil 5 g Cetyl alcohol 1 g Stearic acid 1 g Beeswax 1 g Glycerol stearate 0.5 g Silicone oil (dimethicone) 2 g This oil phase was heated to 70 °C and homogenised thoroughly. Moreover, the following aqueous phase was prepared: Purified water, quantum sufficit (q.s.) 100 g Tetrasodium EDTA 0.05 g Glycerol 3 g Sorbitol 2 g Carbomer 0.25 g Xanthan gum 0.2 g Biosaccharide gum-1 5 g Polyethylene glycol 400 2 g Methyl paraben 0.25 g Propyl paraben 0.15 g 10% Sodium hydroxide 0.5 g All the ingredients of the aqueous phase were dissolved in water and the whole mixture was heated to 70 °C. The oil phase, homogenised and heated to 70 °C, was poured slowly over the aqueous phase heated to the same temperature with fairly vigorous stirring. Stirring was maintained for 10 minutes after the end of the introduction of the oil phase. The oil in water emulsion formed. The emulsion was then cooled with moderate stirring. When the temperature had reached 40 °C, the following were added successively with moderate stirring: Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser 10 g Extract of white lupin 0.5 g Ursolic acid/oleanolic acid, ethoxydiglycol (10/90) 0.2 g Perfume 0.35 g After homogenisation, the emulsion was cooled to 30 °C with slow stirring. A white oily cream with a very cool feel was obtained. Example 4: Cosmetic compositions The following water/oil cream was prepared: The oil phase was composed of: Cetyl dimethicone copolyol 3 g Isodecyl neopentanoate 5 g Hydrogenated polyisobutene 1.5 g Caprylic/capric triglycerides 1.5 g Jojoba oil 1.5 g Dimethiconol behenate 1.5 g Copolymer of cyclomethicone and dimethicone 10 g This oil phase was heated to 60 °C and homogenised thoroughly. Moreover, the following aqueous phase was prepared: Purified water, quantum sufficit (q.s.) 100 g Sodium chloride 0.5 g Glycerol 2 g Sorbitol 2 g Polyethylene glycol 400 2 g Methyl paraben 0.25 g Propyl paraben 0.15 g All the ingredients of the aqueous phase were dissolved in water and the whole mixture heated to 60 °C. The oil phase, homogenised and heated to 60 °C, was poured slowly over the aqueous phase heated to the same temperature with fairly vigorous stirring. Stirring was maintained for 10 minutes after the end of the introduction of the oil phase. The water in oil emulsion formed. The emulsion was then cooled with moderate stirring. When the temperature had reached 40 °C, the following were added successively with moderate stirring: Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser 1 g Extract of white lupin 2 g Ursolic acid/oleanolic acid, ethoxydiglycol (10/90) 10% Perfume 0.35 % After homogenisation, the emulsion was cooled to 30 °C with slow stirring. A white, very oily cream was obtained, suitable for application at night. Example 5: Oil/water cream with lamellar phases The following oil/water cream with lamellar phases was prepared: The oil phase was composed of: Cetearyl alcohol and cetearyl glucoside 6 g Octyl stearate 2 g Beeswax 1.5 g Propylene glycol dicaprylate/dicaprate 2.5 g Jojoba oil 4 g Shea butter 4 g Silicone oil (dimethicone) 2 g This oil phase was heated to 80 °C and homogenised thoroughly. Moreover, the following aqueous phase was prepared: Purified water, quantum sufficit (q.s.) 100 g Tetrasodium EDTA 0.05 g Glyceryl polymethacrylate and propylene glycol 10 g Polyethylene glycol 400 2 g Methyl paraben 0.25 g Propyl paraben 0.15 g o-Cymen-5-ol 0.1 g All the ingredients of the aqueous phase were dissolved in water and the whole mixture heated to 80 °C. The oil phase, homogenised and heated to 80 °C, was poured slowly over the aqueous phase heated to the same temperature with fairly vigorous stirring. Stirring was maintained for 10 minutes after the end of the introduction of the oil phase. The oil in water emulsion formed. The emulsion was then cooled with moderate stirring. When the temperature had reached 40 °C, the following were added successively with moderate stirring: Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser 5 g Extract of white lupin 5 g Ursolic acid/oleanolic acid, sodium salt/ Polyethylene glycol 400 (20/80) 10% Perfume 0.35 % After homogenisation, the emulsion was cooled to 30 °C with slow stirring. A white, very oily cream with lamellar phases was obtained which may facilitate the penetration of the active principles and prolong hydration thereof. Pharmacological study Cytotoxicity All these products and mixtures below proved to be non cytotoxic in the useful concentrations under the conditions described in experiment 1. Experiment 1 : Effect of the products tested on the production of collagen IV, laminin and α2β1 integrin by human fibroblasts (immunoenzyme method of the immunoblot type). The following products and mixtures were tested: -P1 : 1 % or 2% gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser -P2: 1 % or 2% malt extract -P3: 2% or 3% yeast extract -P4: 0.0037% or 0.0075% ursolic acid/oleanolic acid, sodium salt -M1 : Mixtures P1 (2%) + P2 (2%) or P1 (1 %) + P2 (1 %) -M2: Mixtures P1 (2%) + P3 (3%) or P1 (1%) + P3 (2%) -M3: Mixtures P1 (2%) + P4 (0.0075%) or P1 (1 %) + P4 (0.0037%) -M4: Mixtures P2 (2%) + P3 (3%) or P2 (1 %) + P3 (2%) -M5: Mixtures P2 (2%) + P4 (0.0075%) or P2 (1 %) + P4 (0.0037%) -M6: Mixtures P3 (3%) + P4 (0.0075%) or P3 (2%) + P4 (0.0037%) - human TGFβ (transforming growth factor β (TGFβ, Sigma T7039) 10 ng/ml (final) Retinoic acid (RA. "all-trans retinoic acid", Sigma R2625)10 "8 M, known for its anti-ageing effect. The procedure was as follows: Normal human dermal fibroblasts (NHDF, R8PF2, used in the 8th passage) were seeded onto an MEM/M199 (Gibco) medium without serum. They were cultured to confluence at 37 °C and under 5% of CO 2 . They were then treated in triplicate respectively with the 12 preparations or mixtures, continuously for 72 hours. The cell lawns were observed at the end of the treatments and . the samples were frozen at -80 °C. At the end of the treatments, sodium dodecyl sulfate (SDS) (1 % final) was added to the culture media, and the cell and extracellular proteins were extracted for 30 min with stirring; extraction was completed with gentle sonication of all the samples. Immunoblotting was then carried out. The protein fractions were transferred onto nitrocellulose (Hybond, ECL, Amersham) using a MilliBlot module (Millipore, transfer under vacuum). Two nitrocelluloses were used for each antibody used (8 membranes in total, 45 spots per membrane). For each series, control wells (without cell) with culture medium containing the test products in the greatest concentrations were prepared (in order to detect whether the test product was recognised by either of the antibodies). Moreover, controls without primary antibodies were carried out (untreated cells). The membranes were saturated by incubation for 16 hours (4 °C) in a phosphate buffer PBS/0.05% Tween 20/5% skimmed milk (PBSTL). The strips of control wells without primary antibody were cut out for separate treatment (incubation with the peroxidase conjugate only). After washing, the specific antigen sites were labelled with primary antibodies (see table below) in the dilutions indicated (in PBSTL, see table below), for 1 hour at 37 °C. The primary antibodies fixed were revealed by an anti-immunoglobuiin-peroxidase conjugate. After extensive washing in PBS/0.05% Tween 20 (PBST), the peroxidase activity was detected by the ECL method (Enhanced ChemiLuminiscence, Amersham) on Kodak MP film. The images were captured on GelPrint 2000i (BioPhotonics Corp.); the densitometric analyses were obtained using One-D-Scan software (Scanalytics). The evaluation parameter was MTT hydrolysis. The various antibodies used were as follows: The raw data were transferred and processed using PRISM® software (Graph Pad Software). The inter-group comparisons were carried out by analysis of variance (ANOVA) using the DUNNETT multiple comparison test. Immunofluorescence The immunofluorescence controls were carried out on untreated cell lawns (references) fixed with methanol at -20 °C and dried. The principle, the reagents and the times were the same as for immunoblotting. The secondary antibodies were fluorescein-N-isothiocyanate conjugates (FITC) (see table below) instead of peroxidase conjugates. The cell nuclei were stained by incubation in a solution of 1 μg/ml Hoechst dye (bis-benzimide, Sigma B1155). The sections were observed under an epifluorescence microscope (NIKON, Diaphot 300). The images were captured using a COHU camera controlled by Visiolab 200 software. The results obtained are as follows: On the whole, retinoic acid (10 "8 M) and TGF β (10 ng/ml) did not stimulate (or stimulated very little) the production of the markers selected, whereas a relative efficacy of TGF β with respect to the production of collagen IV and laminin could be expected. Effect on actin Actin was used as a reference marker to show that the cell model used was valid. A relative fluctuation in the results was observed with the anti-actin monoclonal antibody. As a general rule, none of the products or mixtures showed any appreciable stimulation of actin expression (no significantly increased cell proliferation; the experiment was carried out to confluence). Moreover, the products and mixtures did not significantly reduce the production of actin, although malt extract (2% and 1%) and ursolic acid/oleanolic acid, sodium salt (0.0075%) apparently had a tendency to reduce the signal measured. Note interference with yeast extract: the product alone (without cell) produced a visible signal suggesting that a component of the product was recognised by the anti-actin monoclonal antibody. Effect on 2β1 integrin The gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser tested alone in the two selected concentrations led to an increase in the α2β1 integrin signal. The other products tested did not exhibit any significant (reproducible) increase in α2β1 integrin. Note a very strong signal obtained with yeast extract; this product contains components having epitopes that were recognised by one or other of the anti- integrin antibodies of the mixture used. It seems highly likely, therefore, that this product contains protein components of cell origin in a not inconsiderable quantity. The activity of this product on the production of α2β1 integrin cannot, therefore, be measured using the experimental conditions of this test. The mixtures containing malt extract, yeast extract and ursolic acid/oleanolic acid, sodium salt, did not show any significant activity. Effect on laminin The gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser tested alone in a concentration of 2% and 1% significantly stimulated the production of laminin by NHDF. Malt extract, yeast extract and ursolic acid/oleanolic acid, sodium salt, did not show any significant stimulation under these experimental conditions. The mixtures showed variable activities. Effect on collagen IV The measurements of relative production of collagen IV showed homogeneous results. The gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser strongly stimulated the production of collagen IV; moreover, all the mixtures containing the gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser stimulated the production of collagen IV by a factor of more than 2. Malt extract and yeast extract did not show any significant stimulation. Note that, as with laminin, yeast extract is not recognised by anti-collagen IV (no significant signal in the control of yeast extract without cells). 0.0075% Ursolic acid/oleanolic acid, sodium salt, showed a significant intensification of the signal (factor of 1.5); less stimulation (not significant) was observed at 0.0037%. The mixtures containing 0.0075% ursolic acid/oleanolic acid, sodium salt, had an activity of the same order as the product alone. The results obtained with the isolated products are shown in the table below (densitometric analysis, results in % relative to the untreated reference). The results obtained with the mixtures are shown in the table below. Conclusions: The gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser (P1) stimulated the production of the three selected markers of the dermo-epidermal junction. The activity of the product was particularly pronounced on collagen IV. Malt extract (P2) alone or in mixture did not show any particular activity. Yeast extract (P3) produced artefacts with the anti-actin antibody and above all with the anti-integrin antibody. The product alone or in mixture did not show any particular activity with respect to laminin and collagen IV. Ursolic acid/oleanolic acid, sodium salt (P4) stimulated the production of collagen IV. The effect observed was less pronounced than with the gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser; this effect is, however, apparently reproducible (reproduced with the mixtures containing ursolic acid/oleanolic acid, sodium salt). Experiment 2: Evaluation of the activation of the neosynthesis of collagen IV in the SkinEthic® reconstructed epidermis model. The following products were tested: - Placebo: PEG400/purifϊed water (10/90); named Po - Formulation 1 : 0.1% Ursolic acid/oleanolic acid, sodium salt, named F1 - Formulation 2: 3% Gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser, named F2 - Formulation 3: Mixture of 0.1% ursolic acid/oleanolic acid, sodium salt, and 3% gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser, named F3 - Formulation 4: 1 % Extract of white lupin, named F4, (F1 , F2, F3 andF4 were used in aqueous solution in 10% final of PEG 400) as follows: Reconstructed 13 day epidermis models of the Skinethic type (4 cm 3 ) were used, cultivated in Skinethic medium. The expression of the messengers α2β1 integrin and collagen IV was verified in the reconstructed epidermis models by the RT polymerase chain reaction method (RT-PCR). 20 epidermis models were treated with the formulations or the placebo applied topically in a quantity of 5 mg/cm 2 per epidermis (20 μl). Four epidermis models were treated with 20 μl for each product; for each treatment, two epidermis models were cultured for 18 h and extracted for analysis; the two other epidermis models of each batch were again treated and re-cultured for 48 additional hours before extraction (72 h treatment in total). The RNA were then analysed by Northern blotting and the proteins by Southern blotting. RNA analyses by Northern blotting The DNA probes below were prepared from fragments of cDNA produced during the preliminary steps, after reamplification and purification. Schematically, the antisense strand of each cDNA was amplified using the following reaction mixture (25 μl; final concentrations): 25 U/ml RedTaq (Sigma D4309); 2.5 μl buffer RedTaq 10x; 20 μM dTTP; 20 μM dCTP; 20 μM dGTP; 2 μM [α- 32 P]-dATP (15 μl, 150 μCi, 3000 Ci/mmole, Amersham PB10204); 1 ng/μl purified cDNA; 1 μM antisense primer (-). PCR conditions: 94 °C 2 min; then 30 times the sequence (95 °C 1 min, 55 °C 1 min, 72 °C 2 min), then 1 elongation cycle of 7 min at 72 °C then purification of the three probes over 3 Chroma Spin- 200 columns (Clontech S0269) according to the supplier's directions; purification monitored by liquid scintillation counting; collection of the fractions relating to each probe. Extraction of total RNA, electrophoresis and transfer At the end of the treatment, the epidermis models were separated from their boat and frozen immediately (-80 °C) in 1.25 ml of Tri-Reagent (TR, Sigma T9424) in an Eppendorf tube without RNase. Northern blotting was carried out as follows: - defrosting of the samples, extensive grinding and removal of solid particles (membrane support...) - extraction of the total RNA of each epidermis according to the methodology advocated by the supplier of Tri-Reagent (Sigma); the fraction containing the proteins was kept for separate analysis; - solubilisation of the extracted RNA in 50 μl of water without RNase (water-diEthylPyroCarbonate (DEPC) (Maniatis et al, 2nd. ed.) - verification of the quality and homogeneity of the samples of RNA after separation of an aliquot (2 μl) by electrophoresis in agarose/formaldehyde gel in the presence of ethidium bromide (Maniatis et al., 2nd. ed.). Observation under UV and images captured on GelPrint 2000i (BioPhotonics Corp.); - quantification of the RNA by measuring the absorption at 260 nm; adjustment of the RNA solutions to 1 μg/μl; - separation of each of the RNA samples over 4 agarose/formaldehyde gels (2 μg of RNA/well for actin and integrins; 5 μg/well for collagen IV); - transfer by capillarity (Maniatis et al.) onto Hybond-N nylon membranes (Amersham RPN1510N), 16 hours (one membrane per probe); covalent bonding of the RNA to the nylon by heating the dry membranes for 90 min at 80 °C. Hybridisations and analyses - prehybridisation of the individual membranes in 8 ml of ExpressHyb (Clontech S1135)/0.1 mg/ml "salmon testes DNA, Sigma D7656", denatured; prehybridisation at 68 °C, 20 min (3 hybridisation bottles; Stuart oven); - hybridisation for 16 hours at 68 °C, after addition of labelled probe; - 4 washes in 2 x saline sodium citrate (SSC) (Maniatis et al.) % SDS at 68 °C, for 30 min; 1 wash in 0.1 x SSC/0.5% SDS at 68 °C; 1 wash in 2 x SSC; - autoradiography of the membranes wrapped in Saran wrap at -80 °C on Kodak Biomax MS film. Images captured; - direct counting of the radioactivity of the spots using an Instantlmager (Packard Instruments). Analysis of the proteins by Southern blotting The proteins were repurified from the water/solvent interfaces derived from the purification protocol for the RNA. The total protein fraction was prepared as specified in the protocol for using Tri-Reagent. The proteins were finally dissociated in an SDS electrophoresis depot buffer (SDS PAGE); the final concentrations of SDS and 2-mercaptoethanol were 2%. The proteins were separated by SDS-PAGE (8% acrylamide gels) and transferred (electric transfer in a liquid medium) onto nitrocellulose (Hybond, ECL, Amersham). In each series, controls in the absence of primary antibodies were carried out. The membranes were saturated by incubation for 16 hours (4 °C) in a PBS buffer/0.05% Tween 20/5% skimmed milk (PBSTL). The strips of control wells without primary antibody were cut out for separate treatment (incubation with the peroxidase conjugate only). After washing, the specific antigen sites were labelled with the primary antibodies in the dilutions indicated in PBSTL (see table below) for 1 hour at 37 °C. The primary antibodies fixed were revealed by an anti-immunoglobulin-peroxidase conjugate. After extensive washing in PBST, the peroxidase activity was detected by the ECL method Enhanced ChemiLuminescence, Amersham) on Kodak MP film. The images were captured on GelPrint 2000i (BioPhotonics Corp.); the densitometric analyses were obtained using One-D-Scan software (Scanalytics). The table below lists the various antibodies used. The results obtained are as follows: Effects on transcription, Northern blotting The effects observed by Phosphorimager are quantified in % against the placebo. Actin (reference) The RNA of all the samples was quantified; the same amount of RNA was deposited for all the samples treated. Thus, in the measurement where the number of copies of actin messenger is regarded as stable and large in a normal cell, the quantity of actin messengers must be stable in the absence of profound modifications of the expression profile of the cell genes (hyperproliferation, cytotoxicity...). Apart from a few experimental fluctuations, the amount of actin messenger measured was relatively stable after 18 h of treatment; larger fluctuations were observed at time 72 h. The measurement of the expression of the various markers in each sample was related to the relative expression of actin in the same sample. α2 integrin The treatments did not show a very pronounced stimulating effect on the expression of α2 integrin messenger. Only formulation F1 showed a slight tendency, at time 72 h only, (+ 17% compared with the reference). β1 integrin The treatments did not show a very pronounced effect on the expression of βl integrin messenger. Formulation F1 had a tendency to stimulate expression at 18 h (+ 16% compared with the reference) and at 72 h (+ 12%). F2 also had a tendency to stimulate expression, at 72 h only (+ 12% compared with the reference). Collagen IV ( 2 chain) Formulations F1 and F4 had a tendency to stimulate expression at time 18 h (of the order of + 20% compared with the reference). All the treatments inhibited the expression of collagen IV at time 72 h; this effect is apparently linked with a great intensity of marking of collagen IV in the placebo, at 72 h. Effects on the quality of the protein markers, Western blotting The results are given in relative figures with respect to actin versus placebo, after densitometric analysis of the immunodetected bands. As a reminder: F3 = F1 + F2. The immunoenzyme analysis was carried out on repurified protein fractions and after separation by SDS-PAGE. Actin (reference) The actin band was perfectly clear and relatively homogeneous within each group of samples (18 h and 72 h; one gel per treatment time). 2β1 integrin Unlike the case of the other antibodies used, the mixture of anti-integrin antibodies generated considerable background making it difficult to interpret the results. It appears, however, that formulations F1, F2, F3 had a tendency to increase the intensity of the α2β1 signal. Collagen IV The epidermis extracts treated with F1 for 18 h had an intensity of marking of collagen IV greater than that of the references (+ 53% compared with the reference). The effect was less pronounced at time 72 h. These results confirm those obtained by Northern blotting. The treatments with F3 and F4, on the other hand, tended to stimulate the production of collagen IV after the double treatment of 72 h. Conclusions: In the Western blotting test, ursolic acid and palmitoyl pentapeptide both increase the neosynthesis of 2β1 integrin without having a synergistic effect between them. In this model, these two compounds each have a minor effect on the neosynthesis of collagen IV; on the other hand, at 72 hours, they have a considerable synergistic effect when they are used together. The peptide extract of white lupin, although having a negligible effect on the neosynthesis of α2β1 integrin, has a very good activity on the neosynthesis of collagen IV at 72 hours. Experiment 3: test with 3 compounds in mixture in comparison with the separate compounds, according to the same protocol as above and involving only the analysis of the expression of collagen IV. The following products were tested: - Placebo: named Po - Formulation 1: 0.1% Ursolic acid/oleanolic acid, sodium salt, named F1 - Formulation 2: 3% Gel of palmitoyl pentapeptide -Lys-Thr-Thr-Lys-Ser, named F2 - Formulation 3: Mixture of 0.1% ursolic acid/oleanolic acid, sodium salt, and 3% gel of palmitoyl pentapeptide-Lys-Thr-Thr-Lys-Ser, named F3 - Formulation 4: 1% Extract of white lupin, named F4 - Formulation 5 = F1 + F4 named F5 - Formulation 6 = F2 + F4 named F6 - Formulation 7 = F1 + F2 + F4 named F7 in aqueous solution in a 10% final concentration of PEG 400. Results (analysis by Western blotting compared with placebo): Conclusions: Of the 3 compounds, ursolic acid/oleanolic acid shows an increase in the neosynthesis of collagen IV compared with the placebo, whereas the mixture of the three compounds shows a significantly greater increase in the neosynthesis of collagen IV with an obvious synergistic effect. Experiment 4: A study was carried out on the effects of the composition of example 1 on a population of 40 Caucasian women aged 46 years, 3 of whom were of phototype II, 18 of phototype III and 19 of phototype IV and in relation to the skin type: 36 with combination skin and 4 with combination skin with a tendency to be greasy. The experimenters applied the composition of example 1 twice daily for 8 weeks in the usual quantity to the face as a whole, around the eyes, and to the neck. The investigator monitored the tolerance and carried out an evaluation of the efficacy, - skin impressions, - cutometric measurements, - macrophotographs, by way of illustration, - a self-assessment questionnaire completed by the volunteers. The test proceeded as follows: Initial state: SO - medical examination for inclusion, - verification of the criteria for inclusion or non-inclusion, - initial clinical assessment of the functional and physical signs of local tolerance and skin condition before treatment, - skin impressions of crow's feet, - cutometric measurements on the cheekbone - macrophotographs of crow's feet in 15 volunteers. After 4 weeks of treatment (28 days), S4: - intermediate clinical assessment of the functional and physical signs of local tolerance and efficacy, - skin impressions of crow's feet, - cutometric measurements on the cheekbone, - intermediate self-assessment by the volunteers. After 8 weeks of treatment (56 days), S8: - final clinical assessment of the functional and physical signs of local tolerance and efficacy, - skin impressions of crow's feet, - cutometric measurements on the cheekbone, - macrophotographs of crow's feet in 15 volunteers - final self-assessment by the volunteers. The results obtained are as follows: The examination of efficacy showed, at the end of 8 weeks of twice-daily application of the composition of example 1 : - a significant reduction in skin slackness, from the first month of treatment, - a reduction in the number of wrinkles, this being significant from the second month of treatment, - a significant reduction in the depth of these wrinkles from the first month, - a reduction in their length, this being significant during the second month. All the parameters analysed on the skin impressions of crow's feet showed a favourable development over the 8 weeks of treatment with, in particular, a statistically significant reduction in average roughness, a notable reduction in the maximum deviation, and a significant reduction in the proportion of furrows of moderate depth (class 2). The cutometric measurements revealed, in a statistically significant manner, a skin which was more taught, less subject to fatigue and had improved tone; these effects, which characterise an improvement in the firmness of the skin, are significant as from the first month of treatment. Finally, the self-assessment questionnaires reflected a very favourable perception of the composition of example 1 by a very large majority of the volunteers, both as regards its cosmetic qualities and its efficacy; it was considered to be effective or very effective both in terms of its anti-wrinkle effect and firming effect by 91% of the volunteers, and 87.5% of the volunteers would buy the product which they rated 8/10 on average. Experiment 5: The effects of the composition of example 2 were studied on a population of 34 Caucasian women with an average age of 52, 17 of whom were of phototype HI and 17 of phototype IV as regards skin type: 15 with a slightly dry skin and 19 with a moderately dry skin. The experimenters applied the composition of example 2 twice daily for 8 weeks in the usual quantity to the face as a whole, around the eyes and to the neck, and to the forearm. Clinical monitoring of the tolerance and an evaluation of the efficacy was carried out by - skin impressions, - cutometric measurements, - macrophotographs by way of illustration, - self-assessment questionnaire completed by the volunteers. The test proceeded as follows: Initial state, SO - examination for inclusion, - verification of the criteria for inclusion or non-inclusion, - initial clinical assessment of the functional and physical signs of local tolerance and skin condition before treatment, - skin impressions of crow's feet, - cutometric measurements on the cheekbone - macrophotographs of crow's feet in 15 volunteers. After 4 weeks of treatment (28 days), S4: - intermediate clinical assessment of the functional and physical signs of local tolerance and efficacy, - skin impressions of crow's feet, - cutometric measurements on the cheekbone, - intermediate self-assessment by the volunteers. After 8 weeks of treatment (56 days), S8: - final clinical assessment of the functional and physical signs of local tolerance and efficacy, - skin impressions of crow's feet, - cutometric measurements on the cheekbone, - final self-assessment by the volunteers. The results obtained are as follows: The anti-wrinkle effect was characterised clinically by a significant reduction in skin slackness, the quantity of wrinkles and their depth; these improvements were statistically significant from the first month of treatment. A reduction in the length of the furrows also commenced in a significant manner during the second month of treatment. All the parameters analysed on the skin impressions of crow's feet developed favourably during the 8 weeks of treatment with, in particular, a significant reduction in the length developed, the total number of furrows and the proportion of furrows of moderate depth, these developments occurring during the first 4 weeks of treatment. The cutometric measurements revealed, in a statistically significant manner, a skin which had better tone, was less subject to fatigue and more taught; the improvement in tone and the taughtening and "anti-stress" effects became apparent in a statistically significant manner from the 4th week of application. Finally, the self-assessment questionnaires reflected a very favourable perception of the product tested by a very large majority of the volunteers, both as regards its cosmetic qualities and its efficacy; it was considered to be effective or very effective as a moisturiser, anti-wrinkle and firming agent by 91% of the volunteers, and 82% of the volunteers would buy the product which they rated 8/10 on average.

Claims: CLAIMS 1. A cosmetic composition containing at least one compound stimulating the neosynthesis of at least one important component of the dermo-epidermal junction. 2. A cosmetic composition according to claim 1 , characterised in that said compound stimulates the neosynthesis of at least two, three important components of the dermo-epidermal junction. 3. A cosmetic composition according to one of claims 1 and 2, characterised in that the important components of the dermo-epidermal junction are selected from laminins, α2β1 integrin and collagen IV. 4. A cosmetic composition according to one of claims 1 to 3, characterised in that it contains ursolic acid or oleanolic acid. 5. A cosmetic composition according to one of claims 1 to 4, characterised in that it contains palmitoyl pentapeptide in which the pentapeptide has the sequence: -Lys-Thr-Thr-Lys-Ser. 6. A cosmetic composition according to one of claims 1 to 5, characterised in that it contains a peptide extract of white lupin. 7. A cosmetic composition according to one of claims 1 to 6, characterised in that it contains ursolic acid which represents from 0.001 % to 10% of the finished composition. 8. A cosmetic composition according to one of claims 1 to 7, characterised in that it contains oleanolic acid which represents from 0.00025% to 2.5% of the finished composition. 9. A cosmetic composition according to one of claims 1 to 8, characterised in that it contains palmitoyl pentapeptide which represents from 0.1 ppm to 30 ppm of the finished composition. 10. A cosmetic composition according to one of claims 1 to 9, characterised in that it contains peptide extract of white lupin which represents from 0.2%) to 10% of the finished composition. 11. A cosmetic composition according to one of claims 1 to 10, characterised in that it contains 0.01% to 5% of ursolic acid or oleanolic acid combined with 0.1 ppm to 30 ppm of palmitoyl pentapeptide. 12. A cosmetic composition according to one of claims 1 to 11 , characterised in that it contains 0.2% to 2% of ursolic acid or oleanolic acid combined with 1 ppm to 10 ppm of palmitoyl pentapeptide. 13. The use of a compound stimulating the neosynthesis of at least one important component of the dermo-epidermal junction such as laminins and/or α2β1 integrin and/or collagen IV in the fight to both cure and prevent the effects of skin ageing.

Inventors: BOBIER-RIVAL CARINE, FRUCTUS ALAIN, LE SQUER SYLVIE, MARCANTUANI CHRISTELLE, MATTA ANNE-MARIE, OGER PASCALE, BOBIER-RIVAL, CARINE, FRUCTUS, ALAIN, LE SQUER, SYLVIE, MARCANTUANI, CHRISTELLE, MATTA, ANNE-MARIE, OGER, PASCALE
Applicants: BOBIER RIVAL CARINE, BOOTS CO PLC, FRUCTUS ALAIN, LE SQUER SYLVIE, MARCANTUANI CHRISTELLE, MATTA ANNE MARIE, OGER PASCALE, BOBIER-RIVAL, CARINE, FRUCTUS, ALAIN, LE SQUER, SYLVIE, MARCANTUANI, CHRISTELLE, MATTA, ANNE-MARIE, OGER, PASCALE, THE BOOTS COMPANY PLC
Application year: 2001
Main classification: A61K7
IPC codes: A61K8, A61Q19