Anti-aging cosmetic composition

Abstract

Cosmetic active agents comprising a mixture of mannose-6-phosphate and mannose are disclosed.

Description

[Technical field]

[0001] The present invention relates to active ingredients and methods useful for reducing the visible signs of aging in human skin. More specifically, the present invention relates to a cosmetic active agent comprising a mixture of mannose-6-phosphate and mannose, and its use for reducing wrinkles and age spots, as well as for structuring collagen fibers. [Background technology]

[0002] The desire to look attractive is natural to the modern consumer, and while ideals of what is attractive may change over time, we can all agree that the condition and appearance of our skin significantly impacts how attractive we look.

[0003] Today's consumers are offered a multitude of cosmetic products for the care of their skin. Generally, these products are in the form of creams and lotions, contain water to moisturize the skin, and fats and lipids to re-smooth it, and their effects extend to the outermost layer of the skin. The provision of effective cosmetic preparations that are useful in treating the causes of skin aging and thereby reducing the visible signs of aging remains an unmet need.

[0004] The structural framework of skin is called its extracellular matrix. This inner framework comprises a network of intermeshing polymers, such as collagen and elastin, in which skin cells are contained. It is responsible for the mechanical properties of skin, including firmness, elasticity, suppleness and resilience. The physical signs of skin aging reflect the condition of the skin matrix. More specifically, the weaker and less regular the matrix is, the more wrinkled, rough and saggy the skin tends to become.

[0005] The reticular dermis is the lowest layer of the dermis, lying below the papillary dermis, and is composed of dense irregular connective tissue and characterized by densely arranged collagen fibers. These protein fibers give the dermis its firmness, extensibility, and elasticity. The orientation of collagen fibers within the reticular dermis forms lines of tension called Langer's strips that are essential for wound healing and mechanical properties.

[0006] Mitochondria are unique organelles that power the cell, converting oxygen and nutrients into adenosine triphosphate (ATP), the chemical energy that enables the cell's metabolism. Mitochondria are able to synthesize proteins independently of the cell nucleus and cytoplasm. They play a crucial role in human evolution by supporting essential cell functions (energy source, protection of DNA, signaling cell replication, activating cell apoptosis, maintaining the cell's electrochemical integrity...).

[0007] A decrease in mitochondrial activity has been linked to normal cell aging. Aging and external stresses cause some damage to the mitochondria of dermal fibroblasts, leading to reactive oxygen species (ROS) production and insufficient energy production in the process of converting oxygen into energy. As a result, the gene expression pattern of dermal fibroblasts is altered, causing disruptions in collagen production, inflammation, disturbances in detoxification pathways (proteasomes) and oxidation of skin pigments (lipofuscin), which leads to extracellular matrix (ECM) disorder, premature skin aging and the appearance of age spots.

[0008] Changes in the collagen production pattern in the ECM, caused by any of the aforementioned phenomena, lead to disorganization of the reticular dermis, changes in the packing of the fibers, and a decrease in the mechanical properties of the skin. The complex hierarchical structure of collagen can be evaluated using atomic force microscopy (AFM). This technique involves scanning the surface of skin cells or skin sections with a sharp tip, which identifies and follows the relaxation of the support during the scan. This method therefore gives information about the biomechanical properties and topography of the skin under investigation.

[0009] There remains a need to provide methods and cosmetic actives that, when applied to the skin, effectively reverse or reduce undesirable signs of skin aging, such as age spots or wrinkles. It is known from the prior art that mannose-6-phosphate can have a positive effect on the appearance of the skin.For example, US 2012 / 0071425 A1 describes the use of mannose-6-phosphate to improve the macroscopic properties of the skin, for example, to reduce the redness of the skin. Furthermore, monosaccharides such as mannose or rhamnose are known to reduce the signs of skin aging (US 2010 / 0190727 A1). Summary of the Invention

[0010] However, the present inventors have surprisingly found that a mixture of mannose-6-phosphate and mannose in a particular molar ratio exhibits enhanced efficacy in reducing the signs of skin aging. The present invention provides a cosmetic active agent comprising a mixture of mannose-6-phosphate and mannose, the molar ratio of mannose-6-phosphate to mannose being between 3:1 and 0.3:1.

[0011] In this application, this mixture of mannose-6-phosphate and mannose in the specified ratio is sometimes also referred to as "mannose-6-phosphate complex". Thus, this term is meant to have the same meaning as "the cosmetic active agent of the present invention" or "a mixture of mannose-6-phosphate and mannose" or other similar terms. The cosmetic active agents of the present invention are capable of significantly reducing the signs of skin aging, such as wrinkles and age spots.

[0012] 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. Likewise, 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 meant to encompass both the D- and L-forms, as well as mixtures thereof.

[0013] In the cosmetic active agent of the present invention, mannose-6-phosphate may be present in any cosmetically acceptable form. For example, depending on the pH, mannose-6-phosphate may be present in protonated form or in 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 may be mixed with one or more amino acids, such as lysine or arginine, and / or with any other cosmetically acceptable positively charged substance(s), and may form a salt with the amino acid(s) and / or the cosmetically acceptable positively charged substance(s). Preferably, mannose-6-phosphate is present as sodium or lysine or arginine salt or salt of a divalent cation.

[0014] Throughout this application, unless otherwise specified, the term "mannose-6-phosphate" is meant to encompass both the protonated form of mannose-6-phosphate and any cosmetically acceptable salts, as well as mixtures thereof.

[0015] Preferably, the molar ratio of mannose-6-phosphate to mannose is from 2:1 to 1:1, more preferably from 1.9:1 to 1.1:1, especially about 1.5:1. The mixture of mannose-6-phosphate and mannose may be used in pure form or in combination with other active ingredients and / or excipients.

[0016] Thus, in one embodiment, the cosmetic active agent consists at least essentially of a mixture of mannose-6-phosphate and mannose. Preferably, the cosmetic active agent of the invention contains mannose-6-phosphate in a concentration between 30 and 220 mM, more preferably between 60 and 170 mM, and most preferably at a concentration of about 120 mM.

[0017] In particular, the cosmetic active agent of the present invention may contain 0.5-6.0 wt% of mannose-6-phosphate sodium salt, more preferably 2.0-4.0 wt% of mannose-6-phosphate sodium salt, and most preferably about 3.0 wt% of mannose-6-phosphate sodium salt. Alternatively, the cosmetic active agent of the present invention may contain corresponding amounts of mannose-6-phosphate in any of the other forms described above.

[0018] Preferably, the cosmetic active agent of the present invention comprises 0.5 to 5.0 wt% mannose, more preferably 0.8 to 3.0 wt% mannose, and most preferably about 1.5 wt% mannose.

[0019] In certain embodiments, the cosmetic active agent of the present invention further comprises glycerol and / or sodium phosphate. Alternatively or in addition, the cosmetic active agent of the invention may further comprise 1,2-propanediol and / or water. In particular, glycerol may be replaced by 1,2-propanediol in concentrations up to 30% or by water.

[0020] In a preferred embodiment, the cosmetic active agent of the present invention further comprises: 2.0~4.0wt% Mannose-6-phosphate sodium salt 0.8~3.0wt% Mannose Up to 0.2wt% Sodium phosphate 50wt% glycerol qsp 100wt% water

[0021] The cosmetic actives of the present invention may be at any suitable pH for cosmetic use, such as a pH of 1.5 to 9.0, more preferably a pH of 3.0 to 6.5, and most preferably a pH of 4.5 to 6.0, such as a pH of about 5.

[0022] The cosmetic preparation of the present invention is advantageously used in cosmetic compositions, especially in skin care compositions. Thus, in a further aspect, the present invention provides a cosmetic composition, and especially a skin care composition, comprising a cosmetic active agent as described above and a cosmetically acceptable excipient. More specifically, the present invention provides an anti-aging skin care composition.

[0023] In a further aspect, the present invention provides a method for reducing the signs of ageing in the skin, comprising the step of applying to the skin, in particular to facial skin, a cosmetic active agent according to the invention or a cosmetic composition according to the invention.

[0024] It has been found that it is feasible to reduce facial skin wrinkles by applying the cosmetic active agent of the invention or the cosmetic composition of the invention to the skin. It has further been found that it is feasible to reduce age spots by applying the cosmetic active agent of the invention or the cosmetic composition of the invention to the skin. It has further been found that it is feasible to structure the collagen fibres by applying to the skin a cosmetic active agent according to the invention or a cosmetic composition according to the invention.

[0025] The cosmetic active agent of the present invention allows: - Reprogramming mitochondrial energy metabolism in old cells, - Promotes the remodeling of the dermal matrix, - Strengthening the dermal epithelial junction (DEJ), - reorganizing the dermal matrix to resemble that of younger skin; - Significantly reduces visible age spots, - Visibly reduce the appearance of crow's feet and - Reduce neck wrinkles and reshape the face.

[0026] These effects have been confirmed by in vitro, ex vivo and clinical studies, as described in the examples below.

[0027] In particular, it has been found that the cosmetic active agents of the invention increase ATP synthase activity and citrate synthase activity, correlating with a reactivation of cellular metabolism and mitochondrial biogenesis (Example 3). At the same time, the cosmetic active agent according to the invention does not promote protein glycation which is involved in age-related disorders (Example 4).

[0028] It has further been found that the cosmetic active agents of the invention stimulate genes controlling extracellular matrix components, matrix aggregation and fibroblast proliferation (Example 5), which leads to improved dermal remodeling.

[0029] Treatment with the cosmetic active agent of the invention also leads to an increase in type V collagen, Fibulin-1 and Tenascin-C expression (Example 6). These results demonstrate a significant improvement in dermal structure and organization.

[0030] These results were confirmed by ex vivo studies, which demonstrated that the cosmetic active agents of the invention have a strong influence on the dermis and its organization and remodeling (Example 7).

[0031] Collagen fibers in the dermis of young individuals are typically highly cohesive and well In contrast to mature skin, which typically exhibits a coarser, less organized collagen fiber network, mature skin typically exhibits a coarser, less organized collagen fiber network. Treating mature skin with the cosmetic active agent of the present invention results in significantly improved collagen fiber organization and increased cohesion between them (Example 8).

[0032] Proteins in skin are modified through oxidation, glycation and carbamylation. These modified proteins are constantly eliminated by proteasome activity. However, during skin aging, proteasome activity slows down, resulting in the accumulation of modified proteins. As a result, various skin disorders such as pigmented spots, senile spots and invisible spots appear. It has now been found that the cosmetic active agent of the present invention can stimulate proteasome activity (Example 9).

[0033] UV irradiation increases the amount of oxidized proteins in the epidermis and dermis. Interestingly, the cosmetic active agent of the present invention not only prevents this increase, but also significantly reduces the amount of oxidized proteins (Example 10).

[0034] Further in vitro comparison of the mixture of mannose-6-phosphate and mannose according to the invention (also referred to as "mannose-6-phosphate complex") with mannose and mannose-6-phosphate for their effect in reducing mitochondrial mass showed a significantly lower effect in reducing mitochondrial mass than the mixture, suggesting a synergistic biological effect of the mixture in reducing the signs of cellular aging (Example 11).

[0035] Clinical studies have confirmed that the number and size of visible age spots can be significantly reduced (Example 13). Furthermore, the cosmetic active agents of the present invention significantly improve collagen density and organization (Example 13).

[0036] The cosmetic active agent of the invention also produces a significant reduction in crow's feet (Example 13). Last but not least, the cosmetic active agent of the present invention is able to significantly reduce neck wrinkles, both in terms of volume and depth (Example 14).

[0037] Specific uses of the cosmetic actives of the present invention include, but are not limited to, anti-aging serums or creams, anti-aging products for the reduction of deep wrinkles, intensive treatments for facial rejuvenation, anti-wrinkle products for nasolabial folds, anti-tick neck products, anti-crow's feet products, anti-age spot serums, skin care for mature skin, and derma cosmetics.

[0038] Mannose-6-phosphate (M6P) is a metabolic product in humans that allows cells to produce energy and perform normal functions. It is an essential component for promoting glycolysis (promoting energy production) in cells. The cosmetic active agents of the present invention can be prepared from plant sources using green chemistry. Mannose-6-phosphate can be prepared from mannose by enzymatic phosphorylation.Suitable phosphorylation conditions are illustratively described in WO 2008 / 142155 A2, the contents of which are incorporated herein by reference in this respect.The following Example 1 details the feasible synthesis of mannose-6-phosphate.

[0039] Enzymatic phosphorylation typically provides a mixture of mannose-6-phosphate and mannose. Depending on reaction time and other conditions, the conversion and thus the ratio of mannose-6-phosphate to mannose may vary. Therefore, reaction time and conditions are preferably selected to directly obtain the desired ratio of mannose-6-phosphate to mannose. Alternatively, it is feasible to adjust the ratio by adding or removing one or both products.

[0040] Preferably, the cosmetic active agent of the present invention comprises 0.5 to 5.0 wt% mannose, more preferably 0.8 to 3.0 wt% mannose, and most preferably about 1.5 wt% mannose. The molar ratio of mannose-6-phosphate to mannose in the cosmetic active agent of the invention is from 3:1 to 0.3:1, more preferably from 2:1 to 1:1, most preferably from 1.9:1 to 1.1:1, and especially about 1.5:1. The concentration of mannose-6-phosphate in the cosmetic active agent of the present invention is preferably 30 to 220 mM, more preferably 60 to 170 mM, and most preferably about 120 mM. The concentration of mannose in the cosmetic active agent of the present invention is preferably 0.5-5.0 wt%, more preferably 0.8-3.0 wt%, and most preferably about 1.5 wt%.

[0041] The above ratios and concentrations have been found to provide optimal anti-aging activity to the skin. 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 may be used in the present invention.

[0042] 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 may be used in the present invention. In particular, the cosmetic active agent of the present invention may further comprise glycerol, which may act as a preservative. Alternatively or in addition, the cosmetic active agent of the present invention may further comprise sodium phosphate, which may illustratively be used as a buffering agent.

[0043] In a second aspect, the present invention provides a cosmetic composition comprising a cosmetic active agent of the present invention, especially in the preferred embodiments outlined herein. Typically, the cosmetic active agents of the present invention are used in cosmetic compositions at a concentration of about 0.1-5.0 wt%, more preferably at a concentration of about 0.5-5.0 wt%, illustratively at a concentration of about 3 wt%. More specifically, the present invention provides skin care compositions, particularly anti-aging skin care compositions. The cosmetic compositions of the present invention typically further comprise a cosmetically acceptable excipient.

[0044] The cosmetic composition of the present invention, and especially 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 may 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 are not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoquinol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, etc.

[0045] Preparations for topical application to the skin may take any physical form. Illustratively, cosmetic compositions, and in particular skin care compositions, 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.

[0046] Cosmetic compositions, and especially skin care compositions, may be prepared in any solid, liquid, or semi-solid form 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, sprays, or aerosols (sprays), including leave-on and rinse-off formulations.

[0047] To obtain the beneficial effects described herein, the cosmetic active agent or cosmetic composition of the invention is advantageously applied to the skin, in particular to the skin of the face. Throughout this application, the term "skin" refers especially to human skin. The present invention therefore also provides a method for reducing the signs of ageing in the skin, comprising the step of applying to the skin a cosmetic active agent or a cosmetic composition of the invention, in particular to facial skin.

[0048] The benefits of the cosmetic active agents and cosmetic compositions of the present invention are particularly beneficial on areas of the skin that are visible, exposed, and that commonly exhibit signs of aging, such as wrinkles or age spots, including, but not limited to, the face, neck, neckline, hands, and forearms. The present invention also relates to the use of a cosmetic active agent or a cosmetic composition according to the invention for reducing the signs of ageing on the skin.

[0049] This use includes, inter alia, the reduction of wrinkles, the reduction of age spots, and / or the structuring of collagen fibres. The invention is further illustrated by the following non-limiting examples: [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows the results of ATP synthase activity in Example 3. [Diagram 2] FIG. 1 shows the results of citrate synthase activity in Example 3. [Diagram 3] 1 is an atomic force microscope photograph in Example 8. [Figure 4] FIG. 1 shows a SAXS profile in Example 8. [Diagram 5] FIG. 1 shows protein oxidation levels in Example 10. [Figure 6] FIG. 1 shows the results after 1 hour of treatment in Example 11. [Figure 7] FIG. 1 shows the results 48 hours after treatment in Example 11.

[0051] Example 1: Preparation of D-mannose-6-phosphate General Mannose-6-phosphate was obtained by reacting mannose with condensed phosphates, especially pyrophosphate, in the presence of acid phosphatase from Shigella flexneri. Mannose-6-phosphate was purified by removing excess condensed phosphates and released phosphates, optionally followed by removal of residual mannose.

[0052] Acid phosphatase from Shigella flexneri. The gene for acid phosphatase from Shigella flexneri (commonly named PhoN-Sf) was ordered and synthesized by DNA 2.0, now ATUM (Newark, CA, USA) (UniProt accession number O50542). The gene was cloned into pET-26b(+) and the expression vector was subsequently transformed into E. coli BL21 (DE3) cells.

[0053] PhoN-Sf was expressed using autoinduction medium (BG Fox and PG Blommel, Curr Protoc Protein Sci 2009, doi:10.1002 / a471140864.ps0523s56; reference 71491, Novagen, Merck KGaA, Darmstadt, Germany). Cells were harvested by centrifugation and the supernatant was discarded. PhoN-Sf enzyme was isolated as follows: cells were lysed using a PandaPlus 2000 homogenizer (GEA Niro Soavi, Parma, Italy), cell debris was removed by centrifugation and ultrafiltration (GE Healthcare, MW cut off 750 kDa), and the enzyme contained in the filtrate was used either directly or after concentration (vacuum evaporation at 30°C).

[0054] PhoN-Sf enzyme activity was measured spectrophotometrically by measuring the dephosphorylation of 4-nitrophenyl phosphate (p-NP) via the release of p-nitrophenol. The reaction medium contained 104 mM sodium citrate buffer pH 4.80, 5.8 mM magnesium chloride, 0.11 mM zinc chloride, 1.15 mM p-NPP, correctly diluted enzyme (activity ranging from 0.1 to 0.8 U / mL). The mixture was incubated at 30 °C for regular time intervals, and 100 μL of reaction medium was mixed with 200 μL of 0.5 N NaOH (reaction quenching). A volume of 250 μL was then introduced into the wells of a 96-well microplate and the absorbance was measured at 405 nm.

[0055] One unit (U) of phosphatase activity corresponds to the amount of enzyme that releases 1 micromole of p-NP per minute under the assay conditions.

[0056] Mannose-6-phosphate synthesis The enzymatic reaction was carried out as follows: - D-mannose, 0.15 or 0.225M; - disodium pyrophosphate, pH 4.15: 0.33 or 0.50M, respectively; - PhoN-Sf enzyme: 0.20U / mL, - Magnesium and zinc chloride salts: 0.50 and 0.1 mM, respectively; - Temperature: 30°C; moderate stirring; - Interval: 22 and 28 hours, respectively.

[0057] Purification and quantification When it was decided to stop the reaction, the pH of the reaction medium was adjusted to 2.0 with an appropriately selected acid. Residual disodium pyrophosphate and released phosphate were removed by sequential addition of magnesium and ammonium salts to bring the pH close to 9.5. The formed precipitate containing pyrophosphate, phosphate, magnesium and ammonium was removed by filtration. Magnesium and ammonium were removed using a cation exchange resin, and finally the pH of the solution was adjusted using concentrated sodium hydroxide to obtain D-mannose-6-phosphate sodium salt with pH values ​​ranging from 4.0 to 7.0.

[0058] The content of D-mannose-6-phosphate and D-mannose was measured using the K-MANGL enzymatic kit from Libios (Pontcharra sur Turdine, France). The complete kit indicates the amount of D-mannose-6-phosphate and D-mannose, whereas the modified kit, in which ATP has been removed, allows only the concentration of D-mannose-6-phosphate to be measured; the difference between both values ​​gives the concentration of D-mannose.

[0059] The above process provides a mixture of D-mannose-6-phosphate and D-mannose in a molar ratio of about 1.5:1. Optionally, D-mannose and D-mannose-6-phosphate can be separated using an anion exchange resin. The cosmetic active used for the study described below contained 105.05 mmol / kg D-mannose-6-phosphate sodium salt and 87.42 mmol / kg D-mannose. It had a pH of 4.78.

[0060] Example 2: Skin care composition containing mannose-6-phosphate For the clinical studies described below (Examples 12 and 13), the following compositions were prepared: [Table 1] This corresponds to Test Composition 2 referred to in Example 4.

[0061] Example 3: Mitochondrial energy metabolism Cell Model Normal human epidermal keratinocytes NHEK (adult single donor, ref C-12003, PromoCell) were cultured in Keratinocyte Growth Medium 2 (PromoCell, ref C-20011) according to the manufacturer's recommendations. - Lot 415Z005.2 (Breast, single donor, female, Caucasian, 26 years old) => "Young" - Lot 409Z012.1 (Breast, single donor, female, Caucasian, 51 years old) => "mature"

[0062] Compounds and treatments tested Test composition 1 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 at 0.5% and 0.1% (v / v) in keratinocyte growth medium 2 (KGM2; culture medium). The cells were treated the day after seeding and incubated for 72 hours with 0.1% or 0.5% of Test Composition 1. The untreated condition, i.e., 72 hours of incubation without any active, was used as the baseline. In early cell passages, young donor cells were found to grow more rapidly than mature donor cells.

[0063] ATP synthase activity Cells were seeded in 6-well plates at 150'000 cells / well (young donors) or 180'000 cells / well (mature donors). After treatment, cells were harvested by trypsination, counted on a Kova slide and kept at 4°C until measurement. For each run, 400'000 cells were permeabilized and incubated in 96-well plates with 0.5 mM ATP, 0.5 mM NADH, 9 UI PK / LDH (pyruvate kinase / lactate dehydrogenase), 2 mM PEP (phosphoenolpyruvate) and 10 μM antimycin A in Tris buffer (pH 8). Activity was measured in real time by a spectrofluorometer (Tecan Infinite 200; DO at 340 nm). Results were expressed as percentage of ATP synthase activity after normalization with untreated cells (100% activity) and oligomycin A (1 μM; 0% activity).

[0064] Citrate synthase activity Cells were seeded at 40'000 cells / well in 24-well plates. After treatment, cells were harvested by trypsination, counted on a Kova slide and kept at 4°C until measurement. 30'000 cells were then permeabilized and incubated in 96-well half-area microplates with 150 μM 5,5'-dithio-bis 2-nitrobenzoic acid, 300 μM acetyl-CoA and 1 mM oxaloacetate in 10 mM Tris (pH 8). Enzyme activity was measured in real time by a spectrofluorometer (Tecan Infinite 200; DO at 415 nm). Results were expressed as percentage of citrate synthase activity after normalization with untreated cells (100% activity).

[0065] calculation Calculations were first performed for each simplicate, and the average inhibition rate for each simplicate was calculated. Effective Concentrations 50 and 20: Calculations were performed for each triplicate using nonlinear regression in Graphpad Prism 4. EC50 and EC20 were calculated with reference to positive and negative controls.

[0066] Percentage of ATP synthase activity: %Activity=100×(Slope オリゴマイシン -Slope 試料 ) / (Slope オリゴマイシン -Slope 未処置 ) Slope: The slope of the curve (change in optical density as a function of time).

[0067] Percentage of citrate synthase activity: %Activity = 100 × (ΔOD 背景 -ΔOD 製品 ) / (ΔOD 背景 -ΔOD 未処置 ) ΔOD: change in optical density as a function of time

[0068] result Test Composition 1 was found to significantly increase ATP synthase activity after 72 hours of incubation at 0.1% and 0.5% in each donor. The results are shown in Figure 1. The efficacy was more pronounced in the mature donors, indicating an impact on the reactivation of cell metabolism in the damaged state. Indeed, the efficacy was 2.9 times more pronounced in the mature donors compared to the untreated state.

[0069] Test composition 1 was also found to increase mitochondrial biogenesis via citrate synthase. The results are shown in Figure 2. The effect is comparable in young and old donors, showing an increase in citrate synthase activity after 72h incubation at 0.1% and 0.5%. These results prove that the cosmetic active agent of the present invention is capable of reactivating mitochondrial biogenesis.

[0070] Example 4: Advanced glycation end products (AGEs) production Treatment of skin explants Skin explants from a 45-year-old donor were stabilized for 4 hours at 37°C / 5% CO2 in DMEM medium supplemented with antibiotics (penicillin 100 U / mL and streptomycin 100 μg / mL) and 5% fetal calf serum. For Example 4, Test Composition 2 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 into an emulsion at 4% (v / v). Test Composition 2 corresponds to the cosmetic composition described in Example 2. The explants were treated with Test Composition 2 for 48 hours. For comparison, the explants were treated with a placebo composition that contained everything except the mixture of D-mannose-6-phosphate and D-mannose. The three explants per condition (untreated at T0, treated with Test Composition 2, and treated with placebo composition) were then immediately frozen in liquid nitrogen for storage before cutting.

[0071] Immunostaining of AGEs Frozen skin explants were placed in Tissue-Tek® and cut with a cryostat. Sections had a thickness of 10 μM. Slides were stored at −20° C., dried, and then fixed with acetone. Specific regions were blocked; primary antibodies against AGEs were incubated at room temperature, as well as secondary antibodies conjugated to Alexa-type fluorochromes. Nuclei were labeled with DAPI (Roche), a DNA-specific fluorescent marker. After each incubation, sections were washed three times. Finally, the slides were mounted with mounting medium and observed under a fluorescence microscope (Olympus CK40) equipped with filters adapted for the different fluorescent dyes. Images were taken with a ×40 lens.

[0072] Fluorescence quantification Fluorescence quantification was performed using ImageJ software. For all images without DAPI, fluorescence of the total area of ​​the section was measured. Shapiro Wilk test was performed and conditions were compared using Student's test.

[0073] result Test composition 2 was found not to induce an increase in AGEs in skin explants after topical application for 2 days. As a result, the cosmetic active agent of the invention does not promote protein glycation (which is involved in age-related disorders) in skin explants.

[0074] Example 5: Dermal matrix reconstruction: transcriptome analysis Normal human dermal fibroblasts (NHDFs) from a 28-year-old donor were seeded at 300,000 cells per well in 6-well plates in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FCS (fetal calf serum). After 48 h of culture, NHDFs were rinsed twice with PBS and rested overnight in DMEM medium without FCS before stimulation. For Example 5, Test Composition 3 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 at 4% (v / v) in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 10% FCS (fetal calf serum).

[0075] Cells were stimulated with test composition 3 in DMEM medium without FCS for 6 hours. After 6 hours of stimulation, total RNA was extracted by phenol-chloroform extraction. RNA quality was controlled and reverse transcription was performed to obtain cDNA. RT-qPCR was performed with 10 ng of cDNA per well in specific plates designed to study the transcriptome expression of different genes involved in the biology of the dermis of NHDFs. Gene expression results obtained in fibroblasts were normalized according to the housekeeping genes POLR2A (RNA polymerase II, subunit A) and PES1 (pescadillo ribosomal biogenesis factor 1). Results were expressed after normalizing the data to the untreated condition.

[0076] result Transcriptome analysis was performed by RT-qPCR in dermal plates with 95 targeted genes involved in extracellular matrix, remodeling, antioxidant enzymes and stress defense, neurotrophin pathway, cell proliferation, DNA repair and stem cell markers. Results were compared using untreated conditions as a negative control and expressed normalized to the average of the most stable housekeeping genes (POLR2A and PES1). The results demonstrated a strong effect on dermal reconstruction and remodeling via upregulation of CTGF, CYR61 and TGFB1. Indeed, these three genes code for proteins involved in regulating tissue remodeling, regulating collagen synthesis and stimulating fibroblast proliferation.

[0077] More precisely, Test Composition 3 is CTGF(+80% *** ) and CYR61 (+84%) ***The RNA expression of various genes involved in the extracellular matrix composition, such as CTGF, connective tissue growth factor, is a regulator of collagen synthesis, and its upregulation confirms the improvement of the extracellular matrix structure. CYR61 (Cysteine-rich angiogenic inducer 61) encodes an extracellular matrix component that aids and stimulates dermal fibroblast migration, promoting dermal regeneration and reconstruction. TGFB1 (Transforming growth factor beta 1) expression (+18% ** ) also demonstrates that dermal remodeling occurs due to increased fibroblast proliferation. In conclusion, the cosmetic active agent of the present invention has demonstrated biological activity at the dermal level by stimulating genes controlling extracellular matrix components, matrix aggregation and fibroblast proliferation, which results in improved dermal remodeling.

[0078] Example 6: Dermal matrix reconstruction: immunohistochemistry assay NativeSkin® skin explants, human normal skin biopsies embedded in a solid and nutrient-rich matrix, were used. The epidermal surface was kept in contact with air to allow topical application. The donor was a 59-year-old woman undergoing abdominoplasty surgery. For Examples 6, 7, 8, and 9, Test Composition 4 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 in PBS (phosphate buffered saline) at 4% (v / v). Test Composition 4 was contacted with NativeSkin® by topical application and was removed and renewed daily for seven days, and results were compared to untreated skin explants.

[0079] Skin explants were fixed in formalin and embedded in paraffin. Biomarkers of interest - type V collagen, Fibulin-1 and Tenascin-C - were detected by immunofluorescence. Biomarker expression was analyzed by fluorescence microscopy (DM5000B - Leica Microsystems); 10 images were acquired per biomarker. For each photograph, fluorescence was quantified using imaging software (ImageJ). Regions of interest were demarcated and total fluorescence intensity was calculated relative to the total area of ​​interest. Values ​​were compared to the control to express activating or inhibiting effects. Statistical analysis was performed for each data by Student's test (p<0.05 * , p<0.005 ** and p<0.0005 *** ).

[0080] result Type V collagen is a fibrillar collagen. It is a minor component of the dermal matrix that associates with collagen type I fibers. Type V collagen expression was increased by 17% (p value < 0.001) with 4% test composition 4.

[0081] Fibulin-1 is a component of the extracellular matrix. It is associated with the dermal-epidermal junction (DEJ) but is expressed at very low levels. Test composition 4 increased the expression of Fibulin-1 by +13% (p-value<0.01).

[0082] Tenascin-C is a matrix protein involved in cell cohesion at the dermal-epidermal junction and is located in the basal layer. Test composition 4 at 4% increased the expression of Tenascin-C by +51% (p-value < 0.001), making it possible to maintain the cohesion of the dermal-epidermal junction. These results provide strong evidence for significantly improving dermal mechanics through overexpression of key proteins that play a role in dermal structure and mechanics.

[0083] Example 7: Dermal Matrix Reconstruction: Ex Vivo Morphological Analysis For Examples 6, 7, 8, and 9, Test Composition 4 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 in PBS (phosphate buffered saline) at 4% (v / v). Immunohistochemical assays were performed on skin explants treated in the same conditions as described in Example 6 from the same donor, aged 59 years, used for immunohistochemical staining.

[0084] Sections of 5 μm were performed and the slices obtained were stained with Masson's trichrome stain, which allows the detection of collagen fibers and muscle tissue in histological slices. Hematoxylin stains nuclei purple, fuchsin ponceau stains muscle fibers, red blood cells, cytoplasm, and elastic fibers pink, and vertreumiere specifically stains collagen fibers. Staining analysis was performed on a LEICA® DFC 280 and several representative photographs of the slices were taken. The immunostained photographs were qualitatively analyzed to describe the status of the different skin compartments.

[0085] result Skin explants treated with Test Composition 4 were found to exhibit similar epidermal morphology as the untreated control. At the dermal level, the papillary dermis showed identical morphology to the control for both collagen and elastic fibers, while the reticular dermis showed increased density with Test Composition 4 compared to the untreated condition. These findings indicate that the cosmetic active agent of the invention has a strong effect on the dermis and its organization and remodeling: the density of the reticular dermis is increased by 36%, the dermal-epidermal junction becomes more distinct and the epidermis is more cohesive.

[0086] Example 8: Dermal matrix remodeling: collagen fiber organization (AFM and SAXS analysis) Human skin explants with an average diameter of 12 mm (± 1 mm) were prepared for each abdominoplasty from two different donors: - Young donor: 30 year old Caucasian female - Mature donor: 65 year old Caucasian female

[0087] The explants were maintained in BEM culture medium (BIO-EC's Explants Medium) at 37° C. in a humidified, 5%-CO2 atmosphere. For Examples 6, 7, 8, and 9, Test Composition 4 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 in PBS (phosphate buffered saline) at 4% (v / v). Test Composition 4 was applied topically on a basis of 2 μl per explant (2 mg / cm 2 ) and spread using a small spatula.

[0088] For the mature donors, test composition 4 was applied on D0, D1, D2, D3, D5, D6 and D7. For comparison, control explants from both young and older donors were cultured for 8 days without any special treatment except for renewal of the culture medium. Culture medium was half-refreshed (1 mL) on D1, D4 and D6 for young donors and half-refreshed (1 mL) on D2, D3 and D6 for mature donors. On D0, explants were harvested from batch T0 and frozen at -80°C. On D8, one explant of every batch was harvested and treated in the same manner as in T0. Frozen samples were cut into 20 μm thick sections using a Leica CM 3050 cryostat and then mounted on coverslips provided by Novitom and placed on Superfrost® plus silanized glass slides. The samples were cryofixed after the procedure. 20 μm thick cold-cut cryosections were deposited on glass slides and stored at −80° C. They were allowed to return to room temperature 24 h before viewing.

[0089] After treatment, samples were cryofixed, and 20-μm-thick cold-cut cryosections were attached to glass slides and stored at −80°C. They were allowed to return to room temperature for 24 h before observation. Sections were observed in tapping mode with a tip having a radius of curvature of approximately 10 nm (corresponding to the resolution of the assembly) on a Bruker Multimode 8 microscope equipped with a Nanoscope V (Bruker) controller. * Images of 512 pixels were acquired at a scan rate of 1 Hz. At three different points in the deep dermis, separated by more than 100 μm, * Three successive 3 μm images were acquired. The conditions for observing the zone were that it was relatively flat and free of significant irregularities that could damage the AFM tip or lever. Images were then processed with Gwyddion software. For confirmation, SAXS analysis was performed on the same sample. Cryofixed samples were allowed to return to room temperature for 8 hours before viewing.

[0090] SAXS data collection: - Beamline ID02 (ESRF, France) - X-ray beam energy: 12.46keV - Mode 7 / 8 multi-bunch - Beam size on sample: 150(h)×100(v)μm2 - Sample detector distance: 4m - 2D detector - Exposure time per pattern: 1 second - T=22℃, RH40%

[0091] At least 20 diffraction patterns were collected at different positions per sample. The data were processed with the SAXSutilities software developed by the ESRF. The diffraction profiles presented in this report were obtained by 360° angular integration of the 2D diffraction signal and subtraction of the average diffraction signal from the mica support.

[0092] result Atomic force microscopy (AFM) revealed strong differences in the collagen organization in the dermis between young and mature donors: indeed, collagen fibers are highly cohesive, well organized and parallel in young donors, whereas mature donors have a much coarser and less organized collagen fiber network. Treatment of mature skin with Test Composition 4 for 8 days resulted in improved organization of the collagen fibers and increased cohesion between them (Figure 3).

[0093] Small angle X-ray scattering (SAXS) measurements can be used to correlate the quality of the collagen fiber organization with the quantification data. Indeed, the more cohesive and well-organized the collagen fiber network is, the stronger and narrower the peaks observed in the SAXS measurements. SAXS profiles confirmed significant differences between young and mature donors: collagen fibers from mature donors were less organized and cohesive than those from young donors, confirming the presence of collagen fiber disorder associated with aging. Treatment of mature skin explants with Test Composition 4 for 8 days led to a strong improvement in the SAXS profile, indicating a better mechanism than in young skin (Figure 4). The SAXS measurements thus make it possible to confirm the findings obtained by the AFM studies. The cosmetic active agent of the invention thus has a strong influence on the matrix remodelling by reorganising the collagen fibres: it improves the cohesion of the collagen fibres and the dermis up to 4.8 times in mature skin compared to untreated and up to 2.1 times in untreated young skin.

[0094] Example 9: Proteasome activity Skin explant treatment NativeSkin® skin explants, human normal skin biopsies embedded in a solid and nutrient-rich matrix, were used. The epidermal surface was kept in contact with air to allow topical application. The donor was a 59-year-old woman undergoing abdominoplasty surgery. For Examples 6, 7, 8, and 9, Test Composition 4 was prepared by diluting the mixture of D-mannose-6-phosphate and D-mannose obtained according to Example 1 in PBS (phosphate buffered saline) at 4% (v / v). Test Composition 4 was contacted with NativeSkin® by topical application and was removed and renewed daily for seven days, and results were compared to untreated skin explants.

[0095] Protein extraction At the time of analysis, the standard OxiProteomics protein extraction method was adapted for the skin explant samples to extract and prepare the samples for analysis. An internal positive control was generated and included in the analysis (C+). Extracted proteins were quantified by the Bradford method and divided into equal amounts for analysis.

[0096] Carbonyl score analysis After extraction and solubilization, protein in the samples was quantified by the Bradford method using calibrated BSA as the standard (Bradford M. Anal. Biochem., 72, 248 (1976)). Absorbance was measured in triplicate at 595 nm and protein concentrations were determined by interpolation to a BSA standard curve. After labeling the carbonylated proteins with a fluorescent probe, the proteins were resolved on a 4-20% gradient SDS-PAGE. The proteins were fixed on the gel, and the carbonylated proteins were verified by fluorescence scanning. Total proteins were post-stained with SyproRuby. Densitometric analysis of protein bands was performed using ImageJ analysis software (NIH, USA).

[0097] Density histograms and lane profile plots were obtained from each sample for both carbonylated and total proteins. Carbonylated protein signals were normalized with the total protein signal for each sample to obtain a carbonyl score.

number

[0098] result The results demonstrate the activity of protecting proteins from oxidative damage in three donors by reducing the carbonyl score, which is directly proportional to the proteasome activity. Thus, the cosmetic active agent of the present invention induces a significant improvement of proteasome activity of 20%.

[0099] Example 10: Oxidized protein production Treatment of skin explants and UV irradiation Skin explants from a 45-year-old donor were stabilized for 4 hours at 37°C / 5% CO2 in DMEM medium supplemented with antibiotics (penicillin 100 U / mL and streptomycin 100 μg / mL) and 5% fetal calf serum. Explants were pretreated for 24 hours, irradiated with UVA (8 J / cm2) and UVB (0.2 J / cm2), and treated again with active formulation, placebo or positive control for 24 hours. Three explants per condition were then immediately frozen in liquid nitrogen for storage prior to sectioning.

[0100] Immunostaining of oxidized proteins Frozen skin explants were placed in Tissue-Tek® and cut with a cryostat. Sections had a thickness of 10 μM. Slides were stored at −20° C., dried, and then fixed with acetone. Sections are treated with DNPH (2,4-dinitrophenylhydrazine), which interacts with carbonyl groups of oxidized proteins. Specific regions were blocked; for immunostaining of carbonylated proteins, primary antibodies against DNP (dinitrophenyl) and secondary antibodies conjugated with Alexa Fluor 970 dyes were used. Nuclei were labeled with DAPI (Roche), a DNA-specific fluorescent marker. After each incubation, sections were washed three times.

[0101] Fluorescence quantification Fluorescence quantification was performed using ImageJ software. For all images without DAPI, fluorescence of the total area of ​​the section was measured. Shapiro Wilk test was performed and conditions were compared using Student's test.

[0102] result UV radiation increases the amount of oxidized proteins in the epidermis and dermis. Treatment with placebo further enhanced this increase: 35% more oxidized protein compared to irradiated controls. Interestingly, the addition of the active ingredient to the placebo not only prevented this increase, but also significantly reduced the amount of oxidized proteins by 19% compared to the irradiated control (UV). Thus, when the skin was irradiated with UV, the amount of oxidized proteins was significantly reduced by 40% in the presence of the active compared to the placebo (Figure 5). These results demonstrate that the cosmetic active agents of the present invention result in a significant reduction of oxidized proteins.

[0103] Example 11: Effects on mitochondrial mass material and method The mannose-6-phosphate complex used in this example had the following composition: 0.11097 mmol mannose-6 phosphophosphate (31.307 mg, molecular weight: 282.12 g / mol, for the monosodium form) and 0.07030 mmol mannose (12.663 mg, molecular weight: 180.16 g / mol) in 1 g of aqueous solution.

[0104] Normal human dermal fibroblasts were isolated from a skin biopsy from a breast reduction surgery on a 17-year-old female. 10'000 cells were seeded per well in black 96-well plates in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) supplemented with 10% FCS (fetal calf serum, Biowest) and 1% antibiotics (Sigma Aldrich) and cultured for 72 hours in an incubator at 37°C in a humid atmosphere containing 5% CO2. The cells were then rinsed twice with PBS (phosphate buffered saline) and incubated overnight in the same incubator in DMEM medium without FCS. The following day, the cells were treated with (i) mannose, 20 mM, (ii) mannose-6-phosphate, 20 mM, or (iii) the above-mentioned mannose-6-phosphate complex, 20 mM (n=3), in the presence of insulin (100 nM). One hour and 48 hours after treatment, MitoTracker TM Green FM (ThermoFisher) was added directly to each well to a final concentration of 200 nM, and the plate was incubated in the dark in an incubator for 15 minutes. The cells were then rinsed twice with PBS, and each well was filled with 200 μl of PBS. TM The fluorescence intensity of Green FM was measured using a microplate reader (excitation wavelength: 488 nm / emission wavelength: 525 nm).

[0105] result In this study, mitochondrial mass in cells was measured using MitoTracker TM The fluorescence intensity emitted by Green FM was used for the determination. Without being bound by theory, it is believed that during cell starvation, a highly condensed mitochondrial network is formed, which suggests an increase in mitochondrial mass. Mitochondrial mass also increases if the mitophagy process is not activated to eliminate aged and altered mitochondria that accumulate in cells. It is believed that oxidative stress in cells is a consequence of this accumulation, which in the long term may lead to cell death. Thus, mitophagy is an important process for cell physiology, and more particularly, maintaining the mitophagy / mitochondrial biogenesis balance is essential to maintain cells in an optimal state.

[0106] In this example, MitoTracker was stained 1 hour after treatment with (i) mannose, (ii) mannose-6-phosphate, and (iii) mannose-6-phosphate complex, each at 20 mM. TM A significant decrease in the Green FM specific signal was observed by -51%, -53% and -79%, respectively (Figure 6). After 48 hours of treatment, a significant decrease in the signal for (i) mannose, (ii) mannose-6-phosphate and (iii) mannose-6-phosphate conjugate at 20 mM, respectively, by -37%, -60% and -82%, respectively, was also observed (Figure 7).

[0107] These results showed a significant reduction in mitochondrial mass in cells, suggesting the reactivation of mitophagy with each active ingredient, thus preventing oxidative stress in cells from aged and altered mitochondria and maintaining the cells in an optimal state. More surprisingly, the mannose-6-phosphate complex was found to be significantly more efficient: 2.5-fold and 2.4-fold after 1 hour, and 3.6-fold and 2.3-fold after 48 hours compared to mannose and mannose-6-phosphate alone, respectively, thus indicating a synergistic biological effect of the mannose-6-phosphate complex in reducing mitochondrial mass.

[0108] Example 12: Cosmetic compositions used for clinical studies In the clinical studies described in Examples 13 and 14 below, the following active and placebo, respectively, cosmetic compositions were used (INCI formulations; see also Example 2):

[0109] Actives: Aqua / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, Ceteth-20, Steareth-20, Isodecyl Neopentanoate, Mannose-Phosphate Sodium Salt, Mannose, Phenoxyethanol, Methylparaben, Propylparaben, Ethylparaben, Dimethicone, Fragrance, Benzyl Salicylate, Linalool, D-Limonene

[0110] Placebo: Aqua / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, Ceteth-20, Steareth-20, Isodecyl Neopentanoate, Phenoxyethanol, Methylparaben, Propylparaben, Ethylparaben, Dimethicone, Fragrance, Benzyl Salicylate, Linalool, D-Limonene

[0111] Example 13: Anti-aging study of the facial area (clinical study) Panel Description A double-blind and placebo-controlled clinical study was conducted in 22 volunteers (ages 50 to 70 years, mean age: 59 ± 4.9 years). The volunteers presented with crow's feet and nasolabial folds and age spots on their faces. Volunteers applied the cosmetic cream twice a day to their face for days 28 (D28) and 56 (D56): active was applied to one side of the face and placebo was applied to the other side (Example 12). Collagen density was analyzed using a SIAscope and signs of aging such as wrinkles and age spots were analyzed by VISIA analysis.

[0112] VISIA CR analysis Digital photographs of the face were taken on D28 and D56 with repositioning on D0 using a Visia CR 2.3 from Canfield Imaging Systems. Repositioning control was performed directly on the data processing screen with overlay visualization of the images at each capture. VISIA allows taking photographs with different types of illumination and extremely fast image capture. The series of photographs taken under multispectral imaging and analysis allows capturing visual information that influences the appearance of the skin.

[0113] Collagen analysis by SIAscope SIAscopy is an intradermal spectroscopic analysis that can visualize the distribution of chromophores in the skin: melanin, hemoglobin and collagen, up to 2 mm beneath the skin. A SIAscope, a portable scanning device connected to Siametrics software, was used.

[0114] Once in contact with the skin, the SIAscope shines light onto the skin. Some of the light is reflected and scattered from the surface; the rest is transmitted to the top layer of the skin. Some of the light is absorbed by melanin in the epidermis, then passes into the dermis and is absorbed by hemoglobin in the blood vessels. Scattering also occurs in the dermis when the light interacts with collagen, which returns some of the light to the surface. By interpreting the combinations of wavelengths accepted by the SIAscope, Siametrics can then create SIA scans; these are created with reference to a built-in, proprietary mathematical model of skin optics.

[0115] Age Spot Analysis Results During aging, different types of age spots are observed. This study focuses on visible age spots. Visible age spots encompass all small visible age spots found on the skin. Visible age spots are due to cellular senescence and impaired detoxification systems associated with aging. The number of visible age spots was analyzed 28 and 56 days after application. After 28 days, a significant 2.1-fold reduction in the number of visible age spots was observed compared to D0 and placebo ( * p<0.05). The efficacy of the active was also demonstrated after 56 days by a significant reduction in the number of visible dark spots compared to placebo (-1.9%, * p<0.05) was maintained. Considering the total area of ​​spots, after 28 days, D0 (-5.6%, * p<0.05) and placebo (-2.3%, * The same trend of significant reduction in total area of ​​visible spots was also observed compared to control group (p<0.05).

[0116] Collagen density and structure measurement results Collagen density and organization was measured after D28 and D58 of application using an SIAscope. After 28 days, a significant improvement in collagen density was observed compared to D0 and placebo. Compared to placebo, active had a 3.8-fold (p<0.05 * ) leading to a significant increase in collagen organization. After 56 days, similar results were obtained with a significant improvement in collagen density compared to D0 and placebo. Active increased collagen organization 2.2-fold more than placebo (p<0.05). * ) improved. These clinical results demonstrate a positive impact on collagen density and mechanics.

[0117] Results of eye corner wrinkle analysis Focusing on crow's feet, the active was found to produce a slight improvement compared to placebo after D28 of treatment (-4.6% vs. -3.0%). After 56 days of application, this effect increased as shown by a significant reduction of -11.5% compared to placebo.

[0118] Example 14: Anti-aging study in the neck area (clinical study) Panel explanation A double-blind and placebo-controlled clinical study was conducted in 39 volunteers (ages 45 to 75 years, mean age: 56±6.2 years) who presented with wrinkles in the neck area. Volunteers applied a cosmetic cream containing 4% active or a placebo cream (Example 12) twice daily to the entire face and neck for 28 (D28) and 56 (D56) days. Neck wrinkles were analyzed for wrinkle volume and number using the AEVA-HE® analysis.

[0119] Neck wrinkle analysis using AEVA-HE® The AEVA-HE® system measures the effectiveness of cosmetic products without touching the skin. For this study, an AEVA-HE® system with 250 sensors was used to measure the depth, length and number of wrinkles. Based on a fringe projection unit that uses light associated with stereoscopic vision, the AEVA-HE® system provides high-resolution 3D scans. Volunteers were placed on the bench of the VisioTOP-500, placed in a precise and stable position and repositioned between different measurement times.

[0120] Self-assessment In addition, a self-assessment questionnaire was administered to each volunteer in D56. Each volunteer was asked how they felt about the products they had used.

[0121] result Neck wrinkle volume was analyzed using 3D reconstructions from AEVA-HE®. After 28 days of application, a significant reduction of -25.9% was observed with active compared to D0, whereas treatment with placebo had no significant effect. After 56 days, the active group showed a significant sustained reduction in wrinkle volume by -26.3%, while the placebo group showed no significant effect.

[0122] In addition, neck wrinkle depth was also analyzed after 28 and 56 days of application. After 28 days, a significant reduction in neck wrinkle depth was observed by -11.4% compared to D0, whereas the placebo only reduced it by -3.20% compared to D0. After 56 days, there was a significant reduction of -10.2% compared to D0, and the efficacy of the active was maintained, whereas the placebo showed no significant effect.

[0123] These results demonstrate that the cosmetic active ingredients of the present invention are able to significantly reduce neck wrinkles in terms of both volume and depth. A self-assessment analysis after 56 days of application revealed the following: - 84% of volunteers were convinced of the effectiveness of the active at the end of the study (vs. 60% for placebo); and - 74% of volunteers thought their skin was firmer after applying active (vs. 45% for placebo).

Claims

1. A cosmetic active agent comprising a mixture of mannose-6-phosphate and mannose, the molar ratio of mannose-6-phosphate to mannose being between 3:1 and 0.3:1, and the concentration of mannose-6-phosphate being between 30 and 220 mM.

2. 2. The cosmetic active agent of claim 1, wherein the molar ratio of mannose-6-phosphate to mannose is from 2:1 to 1:

1.

3. 3. A cosmetic active agent according to claim 1 or 2, which does not contain any other cosmetic active ingredients.

4. A cosmetic active agent according to any one of claims 1 to 3, comprising mannose-6-phosphate in a concentration of from 60 to 170 mM.

5. A cosmetic active agent according to any one of claims 1 to 4, comprising 0.5 to 5.0 wt% mannose.

6. A cosmetic active agent according to any one of claims 1 to 5, further comprising glycerol and / or sodium phosphate.

7. A cosmetic active agent according to any one of claims 1 to 6, comprising: 【Table 1】

8. A cosmetic composition comprising a cosmetic active agent according to any one of claims 1 to 7 and a cosmetically acceptable excipient.

9. The cosmetic composition according to claim 8, which is for skin care.

10. A method for reducing the signs of ageing in the skin, comprising the step of applying to the skin a cosmetic active agent according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

11. The method of claim 10, wherein wrinkles are reduced.

12. 12. The method of claim 10 or 11, wherein age spots are reduced.

13. The method according to any one of claims 10 to 12, wherein the collagen fibres are structured.

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