Cosmetic Composition

Abstract

Cosmetic active agents and methods are provided that are useful for reducing the number and / or size of pores in human skin.

Description

[Technical field]

[0001] The present invention relates to cosmetic active agents and methods useful for reducing the number and / or size of pores in human skin. [Background technology]

[0002] The desire to appear attractive is natural to the modern consumer, and although ideals of attractiveness change over time, it is widely accepted that the condition and appearance of our skin greatly influences how attractive we appear.

[0003] Today's consumers are offered a multitude of cosmetic products for the care of the skin. These products are generally in the form of creams and lotions, and contain water to moisturize the skin and fats and lipids to replenish the oils, exerting their effect on the outermost layer of the skin.

[0004] Pores are small openings in the skin through which sweat and sebum reach the surface of the skin. Pores are the openings of hair follicles. Sebaceous glands within each hair follicle secrete sebum, a lubricating oil, through the pores. Sebum production is important for skin health because it protects and moisturizes the skin. Facial pores are typically visible to the naked eye and range from about 250 to 500 micrometers in diameter. The normal size range depends on factors such as skin tone and age. Enlarged pores are pores that appear widened and are clearly visible to the naked eye.

[0005] While pore size is largely a genetic factor, excess sebum, or oil, production can also cause pores to appear enlarged as the oil mixes with skin waste and clogs the pores. Skin aging and reduced elasticity can also cause pores to appear enlarged. Other contributing factors include chronic acne, hormonal changes, sunburn, smoking, radiation dermatitis, and vitamin A deficiency.

[0006] Traditionally, large pores have been treated by unclogging pores through exfoliation and peeling, targeting underlying related causes such as increased sebum production and skin aging.

[0007] Large pores can be made to appear smaller by reducing or regulating sebum production and preventing skin aging. Exfoliation can also help unclog and reduce dilation. Retinoids and anti-aging solutions can be used to prevent skin aging and promote firmer, more elastic skin. Scrubs, cleansers, and face masks lightly exfoliate the skin, while chemical peels can also deeply exfoliate the skin and reduce sebum production.

[0008] Topical retinoids are vitamin A derivatives that help reverse aging and sun-induced skin changes by increasing skin thickness and elasticity and slowing collagen breakdown. Retinoids promote cell turnover and expel excess sebum from pores. However, retinoids can cause side effects such as burning, scaling, peeling, redness, and swelling, which can make them uncomfortable to use.

[0009] Chemical peels are solutions that remove and renew the outer layer of skin, acting as a much deeper exfoliant than face scrubs and cleansers. This exfoliation pushes sebum out of the pores, reducing their volume and therefore their appearance. Chemical peels have been proven effective in treating large pores. However, people with rosacea, darker skin, and sensitive skin may experience some irritation from using chemical peels. Summary of the Invention

[0010] Thus, there remains a need for methods to effectively treat large pores while avoiding skin irritation and other unpleasant side effects. The present invention provides in a first aspect a cosmetic active agent for treating large pores.

[0011] Cosmetic active agents include: (i) a mixture of mannose-6-phosphate and mannose, wherein the molar ratio of mannose-6-phosphate to mannose is 3:1 to 0.3:1; (ii) copper ions; (iii) a first amino acid selected from the group consisting of lysine, arginine, histidine, and mixtures thereof; (iv) a second amino acid selected from the group consisting of proline, aspartic acid, glutamic acid, and mixtures thereof; and (v) An acid selected from the group consisting of lactic acid and 2-pyrrolidone-5-carboxylic acid.

[0012] In a second aspect, the present invention provides a cosmetic composition comprising a cosmetic active agent and a cosmetically acceptable excipient. In particular, the present invention relates to a skin care composition.

[0013] In a third aspect, the present invention provides a method for reducing the number and / or size of pores in human skin, which comprises the step of applying a cosmetic active agent or cosmetic composition to the human skin, which is particularly useful for facial skin.

[0014] In a fourth aspect, the present invention provides a cosmetic method for reducing parakeratosis in human skin, which comprises the step of applying a cosmetic active agent or cosmetic composition to the human skin, which is particularly useful for facial skin.

[0015] In a fifth aspect, the present invention provides a cosmetic method for restoring epidermal integrity to human skin, comprising the step of applying a cosmetic active agent or cosmetic composition to human skin, which is particularly useful for facial skin.

[0016] In a sixth aspect, the present invention provides a method of promoting collagen synthesis in human skin, which comprises the step of applying to the human skin a cosmetic active agent or cosmetic composition, which is particularly useful for facial skin. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows HE staining of RHE, showing nuclei in the stratum corneum. [Diagram 2] FIG. 2 shows involucrin immunostaining in RHE mimicking parakeratosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Surprisingly, it has been found that the cosmetic active agents of the present invention are highly effective against enlarged pores, significantly reducing the pore area, while at the same time avoiding the undesirable side effects known from prior art active agents.

[0019] The cosmetic active agent of the present invention comprises several known components which interact in a synergistic manner to provide a previously unknown pore-shrinking effect, which has not previously been associated with any of the contained components, nor has there been any hint or suggestion in the prior art to this effect.

[0020] The cosmetic active agent of the present invention comprises a mixture of mannose-6-phosphate and mannose, wherein the molar ratio of mannose-6-phosphate to mannose is between 3:1 and 0.3:1. Cosmetic active ingredients comprising such mixtures have already been described in WO 2020 / 201185 in the context of anti-aging, the contents of which are incorporated herein by reference, in particular with regard to the synthesis of the ingredients.

[0021] The cosmetic active agent of the present invention may comprise D-mannose-6-phosphate, L-mannose-6-phosphate, or a mixture thereof. Preferably, it comprises D-mannose-6-phosphate. Similarly, the cosmetic active agent of the present invention may comprise D-mannose, L-mannose, or a mixture thereof. Preferably, it comprises D-mannose. Throughout this application, unless otherwise specified, the terms "mannose-6-phosphate" and "mannose" are intended to encompass both the D- and L-forms, as well as mixtures thereof.

[0022] In the cosmetic active agent of the present invention, mannose-6-phosphate can be present in any cosmetically acceptable form.For example, depending on pH, mannose-6-phosphate can be present in protonated form or salt form.Suitable counterions include, but are not limited to, monovalent cations, such as sodium, potassium, or ammonium; divalent cations, such as copper, zinc, calcium, magnesium, or manganese; or trivalent cations, such as aluminum; or mixtures thereof.Mannose-6-phosphate can also be mixed with one or more cosmetically acceptable positively charged substances, and can form salts with said cosmetically acceptable positively charged substances.

[0023] Throughout this application, unless otherwise specified, the term "mannose-6-phosphate" is intended to encompass not only the free form, but also the protonated form and cosmetically acceptable salts of mannose-6-phosphate, and mixtures thereof.

[0024] The cosmetic active agent of the present invention further comprises copper ions. Within the cosmetic active agent of the present invention, these copper ions are mainly or exclusively Cu 2+ However, during the preparation of cosmetic active agents, copper ions are 2+ ions and / or Cu + ions, the latter (part of which) is subsequently oxidized to Cu 2+ Ions may be formed.

[0025] The cosmetic active agent of the present invention further comprises a first amino acid and a second amino acid. Without being bound by theory, it is believed that the first amino acid and / or the second amino acid associate with the copper ion, possibly by ionic bonding and / or the formation of a complex-like structure.

[0026] Throughout this application, the term "amino acid" is intended to encompass not only the free form of an amino acid, but also its close derivatives, such as salts, esters, amides, N-acetylates, or hydroxamates.

[0027] The first amino acid used in the cosmetic active agent of the present invention has a basic side chain. In one embodiment, the first amino acid is selected from the group consisting of lysine, arginine, histidine, and mixtures thereof.

[0028] The second amino acid used in the cosmetic active agent of the present invention has an acidic side chain.Alternatively, the second amino acid may be proline.In one embodiment, the second amino acid is selected from the group consisting of proline, aspartic acid, glutamic acid, and mixtures thereof.

[0029] The cosmetic active agent of the present invention further comprises an acid. The acid may serve several purposes, such as adjusting pH and / or moisturizing properties. In one embodiment, the acid is selected from the group consisting of lactic acid, malic acid, succinic acid, fumaric acid, maleic acid, pyruvic acid, citric acid, gluconic acid, lactobionic acid, sorbic acid, tartaric acid, oxalic acid, 2-pyrrolidone-5-carboxylic acid, and mixtures thereof. Preferably, the acid is selected from the group consisting of lactic acid and 2-pyrrolidone-5-carboxylic acid.

[0030] The cosmetic active agents of the present invention are capable of significantly reducing the number and / or size of pores in human skin. The cosmetic active agents of the present invention are also capable of reducing parakeratosis, restoring epidermal integrity and promoting collagen synthesis in human skin. These effects are particularly advantageous on visible areas of the skin, such as the face.

[0031] Mannose-6-phosphate can be prepared from mannose by enzymatic phosphorylation.Suitable phosphorylation conditions are illustratively described in WO 2008 / 142155, and example 1 of WO 2020 / 201185 describes in detail the possible synthesis of mannose-6-phosphate.The disclosures of these two documents in this regard are incorporated herein by reference.

[0032] Enzymatic phosphorylation typically produces a mixture of mannose-6-phosphate and mannose. Depending on reaction time and other conditions, the conversion rate and thus the ratio of mannose-6-phosphate to mannose may vary. Therefore, preferably, reaction time and conditions are selected to directly obtain the desired ratio of mannose-6-phosphate to mannose. Alternatively, one or both of the products can be added or removed to adjust the ratio.

[0033] In one embodiment, the molar ratio of mannose-6-phosphate to mannose is between 2:1 and 1:1, more preferably between 1.9:1 and 1.1:1, in particular about 1.5:1. These ratios have been found to be particularly advantageous.

[0034] In one embodiment, the cosmetic active agent of the present invention comprises mannose-6-phosphate at a concentration of 30 to 220 mM, more preferably at a concentration of 60 to 170 mM, and most preferably at a concentration of about 120 mM.

[0035] In particular, the cosmetic active agent of the present invention may contain 0.5-6.0% by weight of mannose-6-phosphate sodium salt, more preferably 2.0-4.0% by weight of mannose-6-phosphate sodium salt, and most preferably about 3.0% by weight of mannose-6-phosphate sodium salt. Alternatively, the cosmetic active agent of the present invention may contain any other form of mannose-6-phosphate as described above in a corresponding amount.

[0036] In one embodiment, the cosmetic active agent of the present invention comprises 0.5-5.0% by weight mannose, more preferably 0.8-3.0% by weight mannose, and most preferably about 1.5% by weight mannose.

[0037] The copper ions present in the cosmetic active agent of the present invention may be in any suitable form during preparation of the cosmetic active agent, e.g., Cu 2+ and / or Cu + They may be provided in the form of salts. 2+ The counterion used in the copper salt may or may not be directly associated with the copper ion in the cosmetic active of the present invention after the cosmetic active is prepared.

[0038] Thus, in one embodiment, copper ion is provided as copper salt selected from the group consisting of copper sulfate, copper phosphate, copper carbonate, copper chloride, copper acetate, copper malate, copper succinate, copper fumarate, copper maleate, copper pyruvate, copper citrate, copper gluconate, copper glucuronate, copper lactobionate, copper sorbate, copper tartrate, copper oxalate, copper lactate, copper pyroglutamate, copper prophosphate, copper aspartate, copper glutamate and mixtures thereof, more preferably as copper sulfate.It has been found that these copper salts are particularly suitable for use in cosmetic compositions and allow the formation of stable products.

[0039] In one embodiment, the cosmetic active agent of the present invention contains copper ions at a concentration of about 10 mM to about 40 mM, more preferably about 12 mM to about 30 mM, and most preferably about 12.5 mM to about 25 mM. For example, the cosmetic active agent may contain copper ions at a concentration of 12.5 mM to 25.1 mM.

[0040] In one embodiment, the first amino acid comprises or consists of a lysine. In one embodiment, the second amino acid comprises or consists of a proline. Preferably, the first amino acid comprises or consists of lysine and the second amino acid comprises or consists of proline.

[0041] The cosmetic active agent of the present invention may comprise the first amino acid and the second amino acid in any suitable ratio.

[0042] In one embodiment, the cosmetic active agent of the present invention comprises a first amino acid and a second amino acid in a molar ratio of about 3:5 to about 5:2, more preferably about 9:10 to about 10:6, and most preferably about 93:100 to about 100:63. For example, the cosmetic active agent of the present invention may comprise lysine and proline in a molar ratio of about 0.071:0.119 to about 0.120:0.049, more preferably about 0.0886:0.0955 to about 0.0958:0.0608.

[0043] In one embodiment, the cosmetic active agent of the present invention comprises the first amino acid at a concentration of about 50 mM to about 120 mM, more preferably about 60 mM to about 100 mM, and most preferably about 61 mM to about 96 mM, for example about 78 mM.

[0044] In one embodiment, the cosmetic active agent of the present invention comprises the second amino acid at a concentration of about 50 mM to about 120 mM, more preferably about 60 mM to about 100 mM, and most preferably about 61 mM to about 96 mM, for example about 78 mM. The acid contained in the cosmetic active agent of the present invention may be used to adjust the pH of the cosmetic active agent to a cosmetically acceptable level.

[0045] Thus, in one embodiment, the cosmetic active of the present invention comprises an acid in an amount such that the pH of the cosmetic active is from about 3.8 to about 6.0, more preferably from about 4.5 to about 5.3. Those skilled in the art will know how to determine the amount required. The cosmetic active agents of the present invention may contain additional ingredients to support the desired effect or provide other benefits.

[0046] Thus, the cosmetic active agent of the present invention may optionally further contain other cosmetic active ingredients. Any cosmetic active ingredient commonly used in the preparation of cosmetic preparations for use on human skin can be used in the present invention.

[0047] The cosmetic active agent of the present invention may optionally further contain solvents, excipients and / or other adjuvants. Any solvents, excipients and / or other adjuvants commonly used in the preparation of cosmetic preparations for use on human skin can be used in the present invention.

[0048] In particular, the cosmetic active agent of the present invention may further comprise 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, and / or glycerol, which may, for example, act as preservatives.

[0049] Alternatively or in addition, the cosmetic active agent of the present invention may further comprise sodium phosphate and / or sodium hydroxide. By way of example, sodium phosphate can be used as a buffering agent.

[0050] In one embodiment, the cosmetic active agent of the present invention further comprises another amino acid, said amino acid being selected from the group consisting of glutamine, asparagine, glycine, hydroxyproline, serine, methionine, threonine, and mixtures thereof.

[0051] In a preferred embodiment, the cosmetic active agent of the present invention comprises: [Table 1]

[0052] The cosmetic active agents of the present invention are advantageously used in cosmetic compositions, especially skin care compositions.

[0053] Thus, in a further aspect, the present invention provides a cosmetic composition, in particular a skin care composition, comprising a cosmetic active agent as described above and a cosmetically acceptable excipient. Preferably, the cosmetic composition comprises a cosmetic active agent in the preferred embodiments outlined herein.

[0054] Typically, the cosmetic active agents of the present invention are used in cosmetic compositions at a concentration of about 0.1 to 5.0% by weight, more preferably about 0.5 to 3.0% by weight, for example about 1% by weight. The cosmetic compositions of the present invention include cosmetically acceptable excipients.

[0055] The cosmetic composition of the present invention, particularly the skin care composition, may contain one or more cosmetically acceptable excipients. Any excipients commonly used in the preparation of cosmetic preparations for use on human skin can be used in the present invention. Suitable excipients include, but are not limited to, ingredients that can affect the sensory properties, skin penetration, and bioavailability of the cosmetic active agent of the present invention. More specifically, they include liquids such as water, oil, or surfactants, which are of petroleum, animal, vegetable, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, etc.

[0056] The formulation for topical application to the skin may take any physical form. Illustratively, the cosmetic composition, and in particular the skin care composition, may be in the form of liposomal compositions, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, surfactant mixed micelles, surfactant-phospholipid mixed micelles, millispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which may be added to achieve a higher penetration of the cosmetic active agent of the present invention.

[0057] Cosmetic compositions, and in particular skin care compositions, can be prepared in any solid, liquid, or semi-solid form that is useful for topical or transdermal application. Thus, these preparations for topical or transdermal application include, but are not limited to, creams, numerous emulsions, including, but not limited to, oil-in-water and / or silicone emulsions, water-in-oil and / or silicone emulsions, water / oil / water or water / silicone / water emulsions, and oil / water / oil or silicone / water / silicone emulsions, micro-emulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, hydro-alcoholic solutions, hydro-glycolic solutions, hydrogels, liniments, ceras, soaps, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils, and sprays or aerosols (sprays), including leave-on and rinse-off formulations. To achieve the beneficial effects described herein, the cosmetic active agents or cosmetic compositions of the present invention are advantageously applied to the skin, especially to the skin of the face.

[0058] Throughout this application, the term "skin" refers specifically to human skin. In a further aspect, the present invention provides a method of reducing the number and / or size of pores in human skin.

[0059] One of the factors responsible for the formation of pores is the alteration of epidermal integrity associated with the irritation of parakeratosis, which leads to impaired epidermal regeneration and a massive accumulation of dead cells on the skin surface, blocking the pores.

[0060] It has been found that by applying the cosmetic active agent of the invention or the cosmetic composition of the invention to the skin, it is possible to reduce the pore area of ​​the skin, particularly the skin of the face.

[0061] It has further been found that parakeratosis can be alleviated by applying the cosmetic active agent of the present invention or the cosmetic composition of the present invention to the skin. It has further been found that by applying the cosmetic active agent according to the invention or the cosmetic composition according to the invention to the skin, the integrity of the epidermis can be restored.

[0062] It has further been found that collagen synthesis can be promoted by applying the cosmetic active agent of the invention or the cosmetic composition of the invention to the skin. These effects have been confirmed by in vitro, ex vivo, and clinical studies, as shown in the examples below.

[0063] The present invention also relates to the use of a cosmetic active agent or a cosmetic composition according to the invention for any of the above mentioned purposes. The present invention is further illustrated by the following non-limiting examples:

[0064] Example 1: Preparation of a cosmetic active agent The procedure for preparing 1.00 kg of a cosmetic active agent according to the invention is as follows: Into a stirred and temperature-controlled reactor, 125 g of demineralized water are introduced. The water is lightly stirred and the temperature is maintained between 18°C ​​and 28°C, preferably 23°C. 9.4g proline (C 5 H 9 NO 2; 115.13 g / mol; CAS: 147-85-3) was introduced into the reactor and thoroughly dispersed with gentle stirring. 2.84 g of copper sulfate (CuSO 4 ; 159.61 g / mol; CAS: 7758-98-7) was introduced into the reactor and thoroughly dispersed with gentle stirring. 11.9g of lysine (C 6 H 14 N 2 O 2 ; 146.19 g / mol; CAS: 56-87-1) was introduced into the reactor and thoroughly dispersed under gentle stirring to obtain a deep blue solution, the pH of which was 9.45 (values ​​in the range of 9.0-10.0, preferably 9.3-9.6, were considered acceptable). 27.6 g of mannose-6-phosphate (C 6 H 13 O 9 P; 260.14 g / mol; CAS: 3672-15-9) and 14.4 g of mannose (C 6 H 12 O 6 ; 180.16 g / mol; CAS 3458-28-4) were introduced into the reactor. These substances were added together in the form of an aqueous solution (a suitable mixture contains mannose-6-phosphate in a concentration ranging from 0.065 kg / kg to 0.098 kg / kg and mannose in a concentration ranging from 0.031 kg / kg to 0.047 kg / kg). In this example, an aqueous solution was used with a concentration of 0.0813 kg / kg of mannose-6-phosphate and 0.0424 kg / kg of mannose, so that the amount of solution added was 339.5 g. 14.9g lactic acid (C 3 H 6 O 3 ; 90.08 g / mol; CAS: 79-33-4) was introduced into the reactor and thoroughly dispersed under gentle stirring. The pH of the mixture was measured, giving a value of 3.25 (values ​​in the range of 2.9 to 3.6, preferably 3.1 to 3.4, were considered acceptable). The pH was adjusted to 4.65 with 2N sodium hydroxide (Sodium Hydroxide: CAS 1310-73-2) (values ​​in the range of 4.2 to 5.1, preferably 4.5 to 4.8 were considered acceptable). The weight of the mixture was adjusted to 750 g with demineralized water. The weight of the mixture was calculated by dividing the weight of 1,3-propanediol (C 3 H 8 O 2 ; 76.09 g / mol; CAS 504-63-2) was added to bring the weight to 1.00 kg. The mixture was stored in a stoppered sterile bottle at cool temperature (4°C–10°C).

[0065] The cosmetic active obtained by this method had the following composition and a pH of about 5.06: [Table 2]

[0066] Example 2: Skin care composition containing cosmetic active agents For the clinical study described in Example 9 below, the following compositions were prepared: [Table 3]

[0067] For the clinical study described in Example 10 below, the following compositions were prepared: [Table 4]

[0068] Example 3: Statistical analysis For the in vitro and ex vivo studies described below, all results are presented as the mean ± standard error of the mean (SEM) of three independent triplicates.

[0069] For all studies, the Shapiro Wilk test was used to verify whether the raw data followed the Gaussian law. For normally distributed data, means were compared using either unpaired or paired t-tests (≤2 groups) or one-way ANOVA followed by post-hoc tests (≥2 groups). For non-normally distributed data, the Wilcoxon test (paired) was used for paired data and the Kruskal-Wallis test followed by the Mann-Whitney U test (unpaired) was used for unpaired data. Results were judged to be significant as follows: # p<0.1, * p<0.05, ** p<0.01, *** p<0.001.

[0070] Example 4: Transcriptome study of keratinocytes material and method Normal human epidermal keratinocytes (NHEK) were seeded at 300,000 cells per well in 6-well plates. After 48 h of culture, NHEK were rinsed twice with phosphate-buffered saline (PBS) and left overnight in factor-free medium before stimulation.

[0071] The cells were stimulated with 0.5% of the cosmetic active agent of Example 1. 24 hours after stimulation, total RNA was extracted by the "TRIzol method" (Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagen). Cold Spring Harb Protoc. 2010 Jun; 2010(6): pdb.prot5439. doi: 10.1101 / pdb.prot5439. PMID: 20516177.). The quality of the RNA was controlled and reverse transcription was performed to obtain cDNA.

[0072] RT-qPCR was performed using 10 ng of cDNA per well on special plates designed to study the transcriptomic expression of different genes involved in epidermal biology in NHEKs. Gene expression results obtained in keratinocytes were normalized according to EIF2B1 (eukaryotic translation initiation factor 2B subunit alpha) and ABL1 (ABL proto-oncogene 1, non-receptor tyrosine kinase) housekeeping genes.

[0073] result Transcriptome analysis was performed by RT-qPCR on plates containing target genes involved in detachment, differentiation, and stem cell markers. Results are expressed relative to the untreated condition as a negative control and normalized by the average of the most stable housekeeping genes (EIF2B1 and ABL1).

[0074] A significant regulation of seven genes was observed upon treatment with the cosmetic active agent of the present invention. The results are shown in the table below: [Table 5]

[0075] Specifically, cosmetic actives significantly upregulated the KLK7 gene, which is involved in the skin desquamation process. They also reduced the expression of genes involved in differentiation, such as LOR, SPRR3, and CDSN. Finally, cosmetic actives upregulated CTNNB1, ITGA6, and ITGB1, which are involved in the stem cell niche.

[0076] These various effects suggest that the cosmetic active agent of the invention may provide clear benefits in situations of parakeratosis, where the stratum corneum accumulates due to insufficient skin renewal and desquamation processes.

[0077] Example 5: Evaluation of parakeratosis Cultivation and treatment of skin explants Reconstituted human epidermis (RHE) was cultured at an air-liquid interface and pretreated for 24 hours with 1% of the cosmetic active of Example 1. RHE was stressed for 48 hours with 2.5% oleic acid to induce epidermal changes mimicking parakeratosis in the presence or absence of 1% of the cosmetic active of Example 1. RHE was fixed in a formaldehyde solution. Fixed samples were dehydrated in successive baths of increasing concentrations of ethanol before embedding in paraffin.

[0078] Evaluation of parakeratosis Transverse sections were made using a microtome (5 μm thick, 2 sections per slide, 1 slide per RHE) and stored at room temperature until analysis. Tissue sections were deparaffinized and stained according to a standard protocol for HE staining: briefly, sections were stained with hematoxylin, rinsed, and stained with eosin. Sections were then rinsed and mounted in aqueous medium. To assess parakeratosis, the number of nuclei within the stratum corneum was counted at defined lengths of RHE sections.

[0079] Involucrin immunostaining Tissue sections were deparaffinized and incubated in unmasking solution at 95°C to optimize antigen-antibody interactions. Slides were cooled to room temperature in the same solution. After saturation with TBS-Tween-2%BSA (TRIS BASE solution containing Tween and 2% bovine serum albumin), tissue sections were incubated overnight in a primary antibody solution against the marker of interest (involucrin).

[0080] After washing, the binding sites recognized by the primary antibody were revealed by a secondary fluorescent antibody (GAR-568). The labeled tissue sections were mounted with ProlongR containing DAPI (4',6-diamidino-2-phenylindole) for nuclear staining. The labeled tissue sections were observed using a ZEISS 710 confocal microscope. Images were taken and processed with ZEN software (objective x20).

[0081] Parakeratosis evaluation results Figure 1 shows HE staining of RHE, showing nuclei within the stratum corneum. The number of nuclei counted is shown in the following table: Reduction in parakeratosis (number of nuclei in the stratum corneum) [Table 6]

[0082] As can be seen from Figures 1 and 2, treatment with oleic acid significantly increased the number of nuclei in the stratum corneum, a marker of parakeratosis. The placebo control did not improve this phenomenon, whereas 1% of the cosmetic active agent of the invention significantly reduced parakeratosis by 53%, as observed by the reduction in the number of nuclei.

[0083] Results of involucrin immunostaining FIG. 2 shows involucrin immunostaining in RHE mimicking parakeratosis, and the following table shows quantification of staining intensity. Limitation of hyperdifferentiation (% involucrin expression in parakeratotic conditions) [Table 7]

[0084] As can be seen from Figures 3 and 4, immunostaining for involucrin confirmed the restoration of epidermal integrity, as indicated by a -38% decrease in involucrin expression at 1% of the cosmetic active agent of the invention.

[0085] Example 6: Dermal fragility: a transcriptomic study of fibroblasts material and method Normal human dermal fibroblasts (NHDFs) were seeded at 300,000 cells per well in a 6-well plate. After 48 h of culture, 200 μM H 2 O 2 Treatment for 2 hours induced premature senescence compared to basal conditions. NHDFs were then rinsed twice with PBS and left overnight in FCS-free medium before stimulation.

[0086] Cells were divided into basal and premature senescence (H 2O 2 ) and stimulated with 0.5% of the cosmetic active agent of Example 1. 24 hours after stimulation, total RNA was extracted by the "Extract-all method". RNA quality was controlled and reverse transcription was performed to obtain cDNA.

[0087] RT-qPCR was performed using 10 ng of cDNA per well on special plates designed to study the transcriptome expression of different genes involved in dermal biology in NHDFs. Gene expression results obtained in fibroblasts were normalized according to PES1 (Pescadillo ribosome biogenesis factor 1) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) housekeeping genes in the basal state and HMBS (hydroxymethylbilane synthase) and GAPDH in the early senescent state.

[0088] result The results are shown in the following table: [Table 8]

[0089] In basal conditions, the cosmetic active agent of the present invention was found to significantly upregulate the COL3A1 and LOXL4 genes involved in the structure of the dermis through their role in the organization of collagen and elastin fibers, and also reduced the expression of genes involved in matrix degradation such as MMP1, MMP13, and MMP3.

[0090] After premature aging, the cosmetic active agent of the present invention also upregulated COL3A1, indicating an improvement in the structure of the dermis in this condition, and also protected the matrix from degradation by decreasing the expression of the MMP1, MMP13 and MMP8 genes.In addition, the cosmetic active agent of the present invention upregulated the ADAM2 gene, indicating a benefit regarding skin homeostasis.

[0091] These results demonstrated that the cosmetic active agent of the present invention can protect the dermis from degradation and can be an excellent active agent for protecting the skin from dermal fragility, a factor involved in pore formation.

[0092] Example 7: Evaluation of collagen stimulation: ex vivo stimulation following dermal vulnerability Cultivation and treatment of skin explants Fresh human skin explants were taken from a 31 year old donor. Skin explants were topically pre-treated for 3 days with 1% (v / v) of the cosmetic active of Example 1. The untreated condition was treated with the vehicle (distilled water).

[0093] Dermal attenuation was induced by enzymatic digestion overnight and skin explants were post-treated for 3 days. Culture medium was renewed every other day. After the end of the culture, RNA was extracted from the skin and reverse transcribed into cDNA. The mRNA expression of COL1A1, COL5A1, COL7A1, and COL17A1 was measured by semi-quantitative PCR with an Applied Biosystems 7300 Real Time PCR System. The mRNA expression levels were calculated and normalized to a reference gene (GAPDH).

[0094] result As can be seen from the table below, enzymatic collagen degradation results in a significant loss of collagen I, V, VII, and XVII, and topical application of 1% of the cosmetic active agent of the present invention was found to significantly restore collagen I, V, VII, and XVII by +31%, +31%, +45%, and +35%, respectively. [Table 9]

[0095] Example 8: Evaluation of collagen stimulation: in vitro organization of collagen fibers material and method Cell culture was performed with primary cells isolated from biopsies. Normal human dermal fibroblasts (NHDFs) were seeded in triplicate at 10,000 cells per well in 96-well black plates. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics at 37°C, 5% CO. 2 At the end of the incubation, NHDF was added to 200 μM H 2 O 2 Incubate at 37°C and 5% CO 2 for 2 hours, then rinsed twice with PBS and incubated for 72 hours in basal medium (DMEM medium without FCS) supplemented with 1% antibiotics containing 10 ng / ml TGF-β + 20 μg / ml vitamin C for 1% of the cosmetic active of Example 1 as a positive control for procollagen I synthesis.

[0096] At the end of the incubation, cells were fixed with 2% paraformaldehyde (PAF) solution for 5 min and then permeabilized with 2% PAF solution supplemented with 0.5% Triton X100 for 15 min. Nonspecific sites were saturated with 3% bovine serum albumin (BSA) and primary antibodies targeting collagen I and decorin were incubated overnight at 4 °C. The next day, cells were rinsed and incubated with secondary antibodies conjugated to Alexa Fluor 488 for collagen I and Alexa Fluor 568 for decorin. Fluorescence pictures and detection were taken automatically with the PICO tool (Molecular Devices).

[0097] result The results are shown in the following table: [Table 10]

[0098] 1% of the cosmetic active agent of the invention significantly stimulated collagen I synthesis in age-induced conditions, as previously shown with a dosage of procollagen I in fibroblast culture medium.

[0099] Decorin expression was also significantly improved in age-induced conditions, demonstrating the effect of the presence of 1% of the cosmetic active agent of the invention on collagen organization.

[0100] Example 9: Clinical evaluation: Effects on enlarged pores in Caucasian volunteers Panel Description The clinical study was carried out under double-blind and placebo-controlled conditions on a group of 33 volunteers aged 27-66 years (mean age 44 years), divided into two subgroups: 20-30 years (17 volunteers) and 50 years and older (16 volunteers). The 33 volunteers were divided into two groups with an even distribution according to age in order to perform an evaluation by age range as well as a comprehensive cohort evaluation.

[0101] Volunteers were recruited according to the inclusion criteria of having visible pores, lack of radiance in the skin, and a dull complexion. All subjects who participated in the study signed an informed consent at the start of the study. The study was conducted in accordance with and adhered to the principles of the Declaration of Helsinki.

[0102] Volunteers applied an active cream containing 1% of the cosmetic active agent of Example 1 to one side of the face and a placebo cream to the other side twice daily for 56 days. The compositions of these creams are described in Example 2 above. The reduction of enlarged pores was analyzed based on quantification of total pore area using VisioFace®.

[0103] Reduction of enlarged pores measured with VisioFace® The technique consists of acquiring high-resolution photographs of the 3 / 4 face in perfectly reproducible lighting conditions, cross-polarized and diffuse light.

[0104] Acquisition is performed with a high-resolution camera. The lens used is a Nikkor 60 mm equipped with filters. Illumination is provided by two flashlights. The flash head is equipped with a filter slot for mounting polarizing gel (HN32 Sarelec, France).

[0105] To obtain cross-polarized light, the filter of the camera lens is placed at 90° to the polarization of the filter of the flash: polarized light emitted by the flash and reflected by the skin of the face at the moment the picture is taken is "cut" by the camera filter, i.e. these reflections do not appear in the photograph.

[0106] To obtain a diffuse light photograph, the filter in the camera lens is rotated 45° compared to the cross-polarized position. The analysis is carried out by means of specific software developed by Spincontrol. After gradient correction and binarization of the photo, a threshold is used to remove the background.

[0107] Analyses are performed on the same specific regions at each time point of the study. The total area of ​​the pores (mm2) extracted from the photographs is then calculated and the reduction in pores after application of the product and over time is measured.

[0108] result The results for the comprehensive cohort are shown in Table 1, for volunteers aged 20–30 years in Table 2, and for volunteers over 50 years in Table 3.

[0109] In comprehensive cohort, the presence of 1% of the cosmetic active agent of the present invention was found to significantly reduce total pore area by -11.9%, -19% and -23.3% after 15, 28 and 56 days, respectively.Interestingly, placebo formulation had no effect on pore surface.Furthermore, active cream was shown to be significantly superior to placebo, i.e., a significant reduction in total pore area was observed after 15, 28 and 56 days, respectively, compared to placebo.

[0110] In the younger subgroup, the presence of 1% of the cosmetic active agent of the present invention was found to significantly reduce total pore area by -10.3%, -19.5%, and -24.3% after 15, 28, and 56 days, respectively.Interestingly, the placebo formulation had no effect on the surface of pores after 15 and 56 days.Furthermore, the active cream was shown to be significantly superior to the placebo, i.e., a significant reduction in total pore area was observed after 15, 28, and 56 days, respectively, compared to the placebo.

[0111] In the elderly subgroup, the presence of 1% of the cosmetic active agent of the present invention was found to significantly reduce total pore area by -13.6%, -18.7%, and -22.3% after 15, 28, and 56 days, respectively.Interestingly, the placebo formulation had no effect on the surface of pores.Furthermore, the active cream was shown to be significantly superior to placebo, i.e., a significant reduction in total pore area was observed after 15, 28, and 56 days, respectively, compared to placebo.

[0112] Overall, these results demonstrate that the cosmetic active agent of the present invention is significantly effective against enlarged facial pores, regardless of the age of the volunteers. Indeed, a significant reduction in total pore area was demonstrated in both younger and older subgroups, demonstrating that the active agent is effective against various causes of the formation of enlarged pores, including aging.

[0113] Example 10: Clinical evaluation: Effects on enlarged pores in Asian volunteers Panel Description The clinical study was a double-blind and placebo-controlled study conducted on 20 Asian males aged 35 years or older (mean age 42±10 years) divided into two groups, 10 volunteers applied the active cream and the other 10 volunteers applied the placebo cream of Example 2.

[0114] Volunteers were recruited according to the inclusion criteria of having visible enlarged pores on the face. All subjects who participated in the study signed an informed consent at the start of the study. The study was conducted in accordance with and adhered to the principles of the Declaration of Helsinki.

[0115] Volunteers applied either an active cream containing 1% of the cosmetic active of Example 1 or a placebo cream to the entire face twice daily for seven days. The reduction of enlarged pores was analyzed by ColorFaceR based on quantification of total pore area.

[0116] Reduction of enlarged pores measured with ColorFaceR Digital photographs of the face were taken at different times using ColorFaceR (Newtone), a 2D acquisition system that allows to take diverse and standardized photographs of the face. The device is equipped with a 24Mpixel captor. Different acquisition modes exist: ultraviolet (UV) images, unfiltered images, cross-polarized images, 45° standard images, and 60° standard images. ColorFaceR was used to analyze the pore surface at D0 and 7 days after treatment.

[0117] result The results are shown in Table 4 below. It was found that the total pore area was significantly reduced by -30.2% after just 7 days of use, while the placebo formulation showed a lower effect.In fact, after 7 days of application in Asian men, the active performance in reducing pore size was found to be significantly better than the placebo.

[0118] Thus, the cosmetic active agent of the present invention is effective against enlarged facial pores of Asian men. [Table 11] [Table 12]

[0119]

Table 13

Table 14

Claims

1. Cosmetic active agents, including: (i) a mixture of mannose-6-phosphate and mannose, wherein the molar ratio of mannose-6-phosphate to mannose is 3:1 to 0.3:1; (ii) copper ions; (iii) a first amino acid selected from the group consisting of lysine, arginine, histidine, and mixtures thereof; (iv) a second amino acid selected from the group consisting of proline, aspartic acid, glutamic acid, and mixtures thereof; and (v) An acid selected from the group consisting of lactic acid, malic acid, succinic acid, fumaric acid, maleic acid, pyruvic acid, citric acid, gluconic acid, lactobionic acid, sorbic acid, tartaric acid, oxalic acid, 2-pyrrolidone-5-carboxylic acid, and mixtures thereof.

2. 2. The cosmetic active agent according to claim 1, wherein the molar ratio of mannose-6-phosphate to mannose is between 2:1 and 1:1, more preferably between 1.9:1 and 1.1:1, in particular about 1.5:

1.

3. 2. The cosmetic active agent according to claim 1, comprising mannose-6-phosphate at a concentration of from 30 to 220 mM, more preferably at a concentration of from 60 to 170 mM, and most preferably at a concentration of about 120 mM.

4. 2. The cosmetic active agent according to claim 1, comprising 0.5 to 5.0% by weight of mannose, more preferably 0.8 to 3.0% by weight of mannose, and most preferably about 1.5% by weight of mannose.

5. 2. The cosmetic active agent of claim 1, wherein the copper ions are provided as a copper salt selected from the group consisting of copper sulfate, copper phosphate, copper carbonate, copper chloride, copper acetate, copper malate, copper succinate, copper fumarate, copper maleate, copper pyruvate, copper citrate, copper gluconate, copper glucuronate, copper lactobionate, copper sorbate, copper tartrate, copper oxalate, copper lactate, copper pyroglutamate, copper prophosphate, copper aspartate, copper glutamate, and mixtures thereof, more preferably as copper sulfate.

6. 10. The cosmetic active agent of claim 1, comprising copper ions at a concentration of about 10 mM to about 40 mM, more preferably about 12 mM to about 30 mM, and most preferably about 12.5 mM to about 25 mM.

7. 2. The cosmetic active agent according to claim 1, wherein the first amino acid comprises or consists of lysine and / or the second amino acid comprises or consists of proline.

8. 10. The cosmetic active agent of claim 1, comprising a first amino acid and a second amino acid in a molar ratio of about 3:5 to about 5:2, more preferably about 9:10 to about 10:6, and most preferably about 93:100 to about 100:

63.

9. 10. The cosmetic active of claim 1, comprising an acid in an amount such that the pH of the cosmetic active is from about 3.8 to about 6.0, more preferably from about 4.5 to about 5.

3.

10. 10. The cosmetic active agent of claim 1, further comprising another amino acid or a salt or ester thereof, wherein the amino acid is selected from the group consisting of glutamine, asparagine, glycine, hydroxyproline, serine, methionine, threonine, and mixtures thereof.

11. 10. The cosmetic active agent of claim 1, further comprising an additive selected from the group consisting of sodium hydroxide, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, 2,3-butanediol, 1,4-butanediol, glycerol, sodium phosphate, and mixtures thereof.

12. The cosmetic active agent of claim 1, comprising: Table 1

13. A cosmetic composition, in particular a skin care composition, comprising the cosmetic active agent of claim 1 and a cosmetically acceptable excipient.

14. A cosmetic composition, particularly a skin care composition, comprising the cosmetic active agent of claim 12 and a cosmetically acceptable excipient.

15. A method for reducing the number and / or size of pores on human skin, comprising the step of applying to human skin, in particular facial skin, a cosmetic active agent according to any one of claims 1 to 12 or a cosmetic composition according to claim 13 or 14.

16. 15. A cosmetic method for reducing parakeratosis in human skin, comprising the step of applying to human skin, in particular facial skin, a cosmetic active agent according to any one of claims 1 to 12 or a cosmetic composition according to claim 13 or 14.

17. A cosmetic method for restoring the epidermal integrity of human skin, comprising the step of applying to human skin, in particular facial skin, a cosmetic active agent according to any one of claims 1 to 12 or a cosmetic composition according to claim 13 or 14.

18. A method for promoting collagen synthesis in human skin, comprising the step of applying to human skin, in particular facial skin, a cosmetic active agent according to any one of claims 1 to 12 or a cosmetic composition according to claim 13 or 14.

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