COSMETIC COMPOSITION CONTAINING PROTOZOA

Abstract

[0001] The present invention relates to a cosmetic composition for skin care comprising protozoa, in particular protozoa of the amoebic genus Willaertia.

Description

Title of the invention: COSMETIC COMPOSITION COMPRISING PROTOZOA technical field

[0001] The present invention relates to a cosmetic composition for skin care comprising protozoa, in particular protozoa of the amoebic genus Willaertia. Previous technique

[0002] The skin is an important organ of the human body because it constitutes the first mechanical, chemical, and biological barrier against external aggressions and participates in thermoregulation. The skin is composed of three main layers: the epidermis, the superficial and thin layer; the dermis, the thicker layer; and the hypodermis, the deepest layer.

[0003] The outer layer, the epidermis, is mainly composed of keratinocytes, melanocytes, and comeocytes, as well as collagen and elastin fibers that contribute to maintaining cell organization and the skin's elasticity and firmness. The dermis is a connective tissue primarily composed of the extracellular matrix produced by fibroblasts, the main dermal cell population.

[0004] The skin can undergo various forms of alteration, including the formation of fine lines and wrinkles, a natural process due to the slowing of skin regeneration and the deterioration of the extracellular matrix. This alteration, primarily due to age, is accelerated by exposure to UV rays, which denature the DNA of skin cells and promote the formation of age spots due to the proliferation of melanocytes. Other factors such as diet, pollution, stress, and skin hydration can also contribute to this alteration.

[0005] This skin aging can be mitigated by the application of cosmetic products that induce: - skin regeneration through improvement in the quality and abundance of the skin's extracellular matrix, - a slowing down of skin cell death, - an improvement in cell renewal, - protection of the skin against UV rays.

[0006] The cosmetics market offers a multitude of anti-aging, moisturizing, regenerating, photo-aging, and anti-wrinkle products. This segment is a rapidly growing industry that has been challenged in recent years by due to the origin and impact of its ingredients and manufacturing techniques. By focusing on reducing carbon dioxide emissions and improving product biodegradability, many conventional ingredients are being rejected to meet more stringent regulations and consumer demands.

[0007] Biotechnology is one of the solutions to support and stimulate more sustainable growth for the cosmetics industry. Indeed, many functional compounds from microorganisms such as bacteria, fungi or algae have been developed in recent years, offering benefits for the skin thanks to their emulsifying, antimicrobial or antioxidant properties (Prabhuddha L. Gupta et al. Nat. Prod. Bioprospect. 9, 267-278 (2019)).

[0008] A current trend is to offer cosmetic compositions combining several cosmetic effects. For example, compositions that are both moisturizing and regenerating. The solution to achieving such a result is to increase the number of active ingredients in the composition.

[0009] However, the multiplication of active ingredients presents some drawbacks: - the different active ingredients can pose formulation problems; - the multiplication of active ingredients sensitizes the skin and poses tolerance problems for the most sensitive and atopic skin; - these principles are generally of synthetic and chemical origin; and - the cost of composition is considerably increased.

[0010] There is therefore a need to bring to market cosmetic compositions based on natural cosmetic principles that provide rapid and satisfactory results. It is also necessary to develop effective cosmetic compositions without having to use a large number of ingredients. Summary

[0011] In this context, the inventors unexpectedly demonstrated that protozoa, and in particular protozoa of the amoebic genus Willaertia, strongly induce the expression of genes involved in cell renewal, the slowing of cell death, and protection against oxidative stress and ultraviolet (UV) radiation in epithelial cells, and more moderately induce wound healing and protection of the skin against infectious agents. Using a 3D skin model, they also showed that protozoa protect the skin from intrinsic and extrinsic aging, particularly that related to age and UV exposure. Thus, these results demonstrate the potential of using protozoa for skincare.

[0012] A cosmetic composition is proposed for skin care, preferably for preventing or reducing one or more signs of skin aging. comprising protozoa of the amoebic genus Willaertia and one or more acceptable cosmetic active ingredients and excipients. Preferably, the protozoa belong to the species Willaertia magna, and preferably, said protozoa correspond to the strain registered under number PTA 7824 with the ATCC. In a preferred embodiment, said protozoa are in lysed form.

[0013] In a particular embodiment, said acceptable cosmetic excipients are selected from the group consisting of: water, an aqueous component, a fatty substance, a surfactant, a colorant, a texturizing agent, a preservative, a humectant, an emulsifier, an antioxidant, a perfume, and a solvent. The composition may be in the form of a lotion, solution, suspension, emulsion, foam, balm, oil, cream, gel, liquid soap, or solid soap.

[0014] According to another aspect, the use of the composition described above is proposed as an anti-aging agent, preferably to prevent or reduce one or more signs of skin aging, in particular skin aging related to ultraviolet exposure, preferably by increasing the thickness of the epidermis, the differentiation of the stratum granulosum or stratum corneum and / or by increasing the quality and quantity of the extracellular matrix present in the skin, in a further preferred embodiment, to promote skin regeneration, slow down cell death in the skin or protect against oxidative stress such as that induced by exposure to ultraviolet radiation.According to this disclosure, the sign(s) of skin aging may be chosen from the group consisting of: the appearance of wrinkles or fine lines on the skin, sagging of the skin, loss of elasticity, firmness or density of the skin.

[0015] According to another aspect, protozoa of the amoebic genus Willaertia are proposed for use to protect the skin against infectious agents in a subject, preferably said protozoa belonging to the species Willaertia magna, more particularly to the strain filed under number PTA 7824 at the ATCC or for use for skin healing, preferably said protozoa belonging to the species Willaertia magna, more particularly to the strain filed under number PTA 7824 at the ATCC. Brief description of the drawings

[0016] Other features, details and advantages will become apparent from reading the detailed description below and from analyzing the accompanying drawings, in which: Fig. 1

[0017] [Fig. 1] shows the expression levels of the TLR2, HAS, and FBL5 genes in human epithelial cells (ATCC CCL-2) treated with Willaertia amoeba lysate (PTA-7824) (lot 22.LYO.040) at different dilutions. The control corresponds to level of expression in untreated cells. Fig. 2

[0018] [Fig. 2] shows the expression levels of the BCL2, PIWI1L, FOXO1, and SGK1 genes in human epithelial cells (ATCC CCL-2) treated with Willaertia amoeba lysate (PTA-7824) (lot 22.LYO.040) at different dilutions. The control corresponds to the expression level in untreated cells. Fig. 3

[0019] [Fig.3] shows the histological analysis of hematoxylin, phloxine and saffron (HPS) staining of untreated (control) young (left) or aged (right) 3D skin models. Scale bar: 50 µm. Fig. 4

[0020] [Fig.4] shows the histological analysis of hematoxylin, phloxine and saffron (HPS) staining of untreated (control left) aged 3D skin models or skin treated with Willaertia amoeba lysate (PTA-7824) at two concentrations. Scale bar: 50 µm. Fig. 5

[0021] [Fig. 5] shows the HPS histological analysis (A) and Warthin Starry staining (B) of untreated, unexposed (left) and exposed (right) ultraviolet (UV) 3D skin models. Scale bar: 50 pm. Fig. 6

[0022] [Fig. 6] shows the HPS histological analysis (A) and Warthin Starry staining (B) of untreated (left) and UV-exposed 3D skin models treated with Willaertia amoeba lysate (PTA-7824) at two concentrations. Scale bar: 50 µm. Description of the implementation methods

[0023] The applicant unexpectedly demonstrated that protozoa, and in particular protozoa of the amoeba Willaertia genus, strongly induce (2 to 5 times more than the control) the expression of genes involved in skin regeneration, the slowing of cell death in the skin, and protection against oxidative stress, particularly against the effects of ultraviolet radiation. They also moderately induce expression in wound healing and protection of the skin against infectious agents (less than twice that of the control). More specifically, in a 3D skin model, the applicant showed that protozoa of the amoeba Willaertia genus reduce several signs of skin aging, particularly those related to ultraviolet exposure.

[0024] Cosmetic composition

[0025] In a first aspect, the present application relates to a cosmetic composition for skin care, in particular for treating or preventing non- skin pathology, preferably to prevent or reduce one or more signs of skin aging, said composition comprising protozoa of the amoebic genus Willaertia.

[0026] Protozoa are motile heterotrophic protists that ingest their food by phagocytosis. Amoebae are among the free-living forms of protozoa.

[0027] Preferably, the protozoa used belong to the species Willaertia magna.

[0028] More preferably, the protozoa used in the context of the invention correspond to the strain filed on August 21, 2006, under number PTA 7824 with the ATCC, this strain having been filed in the names of the CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) - 3 rue Michel Ange - 75794 PARIS CEDEX 16 / France and UNIVERSITE LYON 1 CLAUDE BERNARD - 43 Boulevard du 11 Novembre 1918 - 69622 VILLEURBANNE Cedex / France. Said filed strain is also described in the publication of international application PCT WO2008 / 043969.

[0029] The cosmetic composition of the present application preferably comprises a lysate of protozoa of the amoebic genus Willaertia as described above.

[0030] A lysate commonly refers to a material obtained following the destruction or dissolution of biological cells by a process of cell lysis, for example, induced by physical, chemical, or biological agents, thereby releasing the intracellular biological constituents naturally contained within the cells of the microorganism in question. The lysate can be obtained by any method well known to those skilled in the art, such as high-pressure homogenization or grinding.

[0031] In particular, protozoa of the amoebic genus Willaertia (e.g., ATCC PTA-7824) are cultured in bioreactors, harvested by centrifugation to remove the culture medium, and then lysed by mechanical grinding with a high-pressure or propeller homogenizer. In a particular mode, the lysate can then be dehydrated by freeze-drying, spray drying, or any other drying process.

[0032] Preferably, the cosmetic composition comprises less than 1% (w / v) of dehydrated lysate. The cosmetic composition of this application may also include at least one acceptable cosmetic excipient.

[0033] By "acceptable cosmetic excipient" is meant a substance which has no cosmetic or biological effect as such, but which is useful for the incorporation of the active agent into the cosmetic composition, for shaping said composition, or for its stability over time.

[0034] The excipient is advantageously chosen from solvents, co-solvents (ethanol, glycerol, benzyl alcohol), thickeners, diluents, antioxidants, colorants, chemical or physical radiation filters UVA and UVB type sunscreens such as synthetic filters associated or not with filters made of particles capable of mitigating the effects of this radiation on the skin, self-tanning agents, pigments, fillers, preservatives, perfumes, humectants, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, trace elements, film-forming polymers, chemical or mineral filters, moisturizing agents or thermal waters, natural polymers such as polysaccharides or polypeptides, cellulose derivatives, or synthetic polymers, and optionally at least one other cosmetic active agent.

[0035] The cosmetic composition according to the invention advantageously comprises an aqueous phase and can advantageously be in all forms normally used for topical application, in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water or water-in-oil emulsion, an aqueous gel, or in any other form for topical application.

[0036] This composition may be more or less fluid and have the appearance of a cream, ointment, milk, lotion, solution, suspension, emulsion, balm, oil, serum, paste, foam, gel, or liquid or solid soap. It may optionally be applied to the skin in the form of an aerosol or by means of a patch, mask, or applicator.

[0037] The cosmetic composition of the present application may also include other active ingredients which may be chosen in particular from moisturizing agents such as hyaluronic acid or glycerol, smoothing agents or anti-wrinkle agents such as adenosine, natural or synthetic polymer complexes producing tightening films on the skin at the time of application to the skin, or even anti-aging actives, more particularly plant extracts obtained by processes known to those skilled in the art and which act on biological targets of the superficial or deep layers of the skin in particular, the epidermis, and the dermis, to maintain the elasticity of the skin, produce a plumping effect, ensure the renewal or repair of tissues, the maintenance of the skin barrier or acting on the skin tone.It can also include brightening or whitening agents that act on age spots, particularly on the hands.

[0038] Cosmetic use

[0039] The inventors in this application have shown that protozoa of the amoebic genus Willaertia can prevent or reduce one or more signs of skin aging and can be used as an anti-aging agent.

[0040] By "anti-aging agent" is meant a cosmetic agent having a biological activity enabling the prevention or reduction of one or more signs of skin aging.

[0041] Skin aging is a non-pathological alteration of the skin, that is to say a normal physiological process, involving intrinsic factors (genetics, hormones, diseases...) and extrinsic factors (UV, stress, climatic aggressions, pollution...), and which intensifies during an individual's life due to a decline in their defense system and their increasing need for antioxidants.

[0042] Changes at the level of the stratum corneum, the epidermis, the epidermal junction and the dermis vary according to the age of the individual and are manifested by "visible signs" of aging, which progress with age, more or less quickly depending on the individual and his environment (intrinsic and extrinsic factors).

[0043] Skin aging, whether it results from a normal phenomenon of senescence or is accentuated by an external factor such as exposure to UV radiation, involves dysfunctions of cell differentiation and / or renewal resulting in atrophy of all the layers of the skin.

[0044] From a histological point of view, we observe, among other things, a decrease in the quality of the dermis with a loss of consistency of the extracellular matrix, a decrease in the thickness of the epidermis and an alteration of the basal layer containing the proliferating keratinocytes.

[0045] From an aesthetic point of view, these alterations result in a change in the appearance of the skin and its mechanical properties: the skin is less smooth and may show fine lines, which can lead over time to the formation of deep wrinkles. The skin also exhibits a loss of elasticity, density, and firmness, which can lead to tissue sagging and an accentuation of wrinkles. Skin aging can also lead to the appearance of age spots.

[0046] In particular, the inventors have shown that the treatment of aged or UV-exposed skin with protozoa, in particular protozoa of the amoebic genus Willaertia, makes it possible to increase the thickness of the epidermis, to increase the terminal differentiation of the stratum granulosum and / or the stratum corneum, thus improving the skin barrier function, and to increase the quality and quantity of the extracellular matrix present in the skin, thus improving the elasticity, density and firmness of the skin.

[0047] In view of these results, protozoa, in particular protozoa of the amoebic genus Willaertia as described above, therefore find applications in the cosmetic field as an anti-aging agent to prevent or reduce one or more signs of skin aging, in particular aging related to ultraviolet exposure, preferably by increasing the thickness of the epidermis, the differentiation of the stratum granulosum or the stratum corneum and / or by increasing the quality and quantity of the extracellular matrix present in the skin.

[0048] Thus, protozoa, in particular protozoa of the amoebic genus Willaertia as described above, can be used to prevent or reduce skin laxity, improve skin elasticity, firmness or density, or prevent or reduce skin wrinkles and fine lines.

[0049] In particular, the inventors have shown that protozoa, especially protozoa of the amoebic genus Willaertia, can induce the expression of the following genes in epithelial cells: HAS (for "hyaluronan synthase 1", Gene ID: 3036, updated September 7, 2023) involved in hyaluronic acid synthesis, BCL2 (for "BCL2 apoptosis regulator", Gene ID: 596, updated September 17, 2023) involved in inhibiting cell death, P1W1L1 (for "piwi-like RNA-mediated gene silencing 1", Gene ID: 9271, updated September 7, 2023) involved in cell renewal, and FOXO1 (for "Forkhead box 01", Gene ID: 2308, updated September 17, 2023) and SGK1 (for “serum / glucocorticoid regulated kinase 1, Gene ID: 6646, updated September 17, 2023) involved in protection against the effects of ultraviolet and oxidative stress.

[0050] Thus, in a preferred embodiment, the present application relates to the use of the cosmetic composition as described above for: - to promote skin regeneration, preferably by improving cell renewal, in particular by inducing the expression of the P1W1L1 gene in skin cells, - reduce cell death in the skin, particularly by inducing the expression of the BCL2 gene in skin cells, - to protect the skin against oxidative stress such as exposure to ultraviolet radiation, in particular by inducing the expression of the FOXO1 and SGK1 genes in skin cells.

[0051] The terms "skin cell regeneration" or "skin regeneration" in this application refer to the process by which epidermal cells continually renew themselves to maintain the protective and aesthetic function of the skin. This process involves the formation of new cells in the basal layer of the epidermis, their migration to the skin surface, their keratinization, and their desquamation. The average duration of the skin cell renewal cycle is 28 days, but this can vary depending on age, skin type, and environmental factors. Skin cell regeneration is essential for maintaining the health, hydration, elasticity, and radiance of the skin.

[0052] For the purposes of this application, "cell death in the skin" means the process of apoptosis that contributes to skin homeostasis by regulating the renewal of epidermal cells. Excessive apoptosis can lead to cell loss, a decrease in epidermal thickness, and a reduction in production. of collagen and elastin and an increase in wrinkles and spots.

[0053] For the purposes of this application, "oxidative stress" refers in particular to stress resulting from the toxic effects of dicarbonyl compounds (e.g., glyoxal and methylglyoxal) on proteins. More generally, oxidative stress is generated by the normal functioning of our body and by external elements (pollution, UV radiation), and is characterized by the production of reactive oxygen species (ROS) that can react with all cellular components, particularly proteins and DNA, leading to their modification by oxidation.

[0054] An additional object according to the invention is a cosmetic process for preventing or reducing one or more signs of skin aging in an individual, comprising administering a cosmetically effective amount of protozoa, in particular protozoa of the amoebic genus Willaertia as described above, preferably topically, to said individual.

[0055] Protozoa are typically applied to the skin to be treated, for example on the face, neck, chest or hands. Preferably, the protozoa are present in a cosmetic composition as described above, and are therefore applied in the form of a cosmetic composition, for example in the form of a cream.

[0056] Therapeutic use

[0057] The inventors have also shown that protozoa, in particular protozoa of the amoebic genus Willaertia, can induce in human epithelial cells the expression of the following genes: FBL5 (for "F-box and leucine rich repeat protein 5", Gene ID: 26234, updated on September 7, 2023) involved in wound healing and TLR2 (for "toll like receptor 2", Gene ID: 7097, updated on September 7, 2023) involved in anti-infectious immunity.

[0058] Thus, in another aspect, the present application relates to protozoa, in particular protozoa of the amoebic genus Willaertia as described above for use in skin healing, in particular by inducing the expression of the FBL5 gene in skin cells or for use in protecting the skin against infectious agents, in particular by inducing the expression of the TLR2 gene in skin cells in a subject.

[0059] Skin healing is the physiological process of tissue repair that is triggered following skin injury, whether accidental, surgical, or pathological. Skin healing involves several successive stages: coagulation, inflammation, proliferation, and remodeling. The goal of skin healing is to restore the integrity, function, and appearance of the skin, but it can be hindered by individual, environmental, or therapeutic factors. Skin healing can result in the formation of a scar, which is the final outcome. tissue repair. The scar can be normal, hypertrophic, atrophic or keloid, depending on its morphology, color, texture and relief.

[0060] Skin immunity is the skin's ability to defend itself against infectious agents and external aggressions by mobilizing physical, chemical, and biological protective mechanisms. Skin immunity relies on the mucocutaneous barrier, which consists of the epidermis, the mucosal epithelium, and associated secretions. Skin immunity also involves cells and molecules of the immune system, which recognize and eliminate foreign or harmful elements. Skin immunity is essential for maintaining the health, function, and appearance of the skin, but it can be impaired by individual, environmental, or therapeutic factors.

[0061] The term "subject" or "patient" should be understood in the context of the invention as any living being belonging to the animal kingdom. Preferably, "subject" is understood to mean a human being.

[0062] The present disclosure also consists of the use of protozoa, preferably protozoa of the amoebic genus Willaertia as previously described, for the preparation of a drug for use in skin healing or for protecting the skin against infectious agents.

[0063] In another particular embodiment, the present disclosure is a method for skin healing or for protecting the skin against infectious agents in a subject comprising administering a therapeutically effective amount of protozoa, preferably protozoa of the amoebic genus Willaertia as previously described in said subject.

[0064] In the context of disclosure, a therapeutically effective amount refers to a dose sufficient to promote skin healing, in particular by restoring the integrity, function, appearance of the skin, by reducing the duration of the inflammatory phase, or refers to a dose sufficient to protect the skin against infectious agents, by decreasing the risks of infection by microorganisms.

[0065] The effective dose is determined and adjusted according to factors such as the composition used, the physical characteristics of the individual concerned such as sex, age and weight, concomitant medication, and other factors, which persons competent in the medical field will recognize.

[0066] The composition according to this disclosure may be included in a pharmaceutical composition, optionally in combination with a pharmaceutically acceptable excipient.

[0067] The term "pharmaceutical composition" should be understood as any composition intended to treat a subject. A "pharmaceutically acceptable excipient" refers to a vehicle that does not produce an undesirable, allergic, or other reaction. Adverse reaction when administered to a mammal, particularly a human, as the case may be. A pharmaceutically acceptable carrier or excipient means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid of any type. Examples

[0068] The following examples illustrate the invention but are not intended to be limiting.

[0069] Example 1: Determination of the biological activity of Willaertia magna Lysate (strain ATCC PTA-7824)

[0070] 1. Amoeba lysate

[0071] The Willaertia magna amoeba lysate (strain ATCC PTA-7824) was stored at -20°C.

[0072] 2. Cytotoxicity tests

[0073] - Materials and methods

[0074] Cytotoxicity analysis is performed on cultured human lymphocyte cells (ATCC CCL-240).

[0075] Briefly, 1.103 lymphocyte cells / well (48-well plate, flat bottom) are cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with fetal bovine serum (10%) and penicillin and streptomycin (5000U / mL, 5000pg / mL respectively), at 37°C, and under a 5% CO2 atmosphere, with or without dilutions of the amoeba lysate.

[0076] Every 24 hours, lymphocyte viability is assessed by Trypan blue exclusion assay (1 / 2 dilution). The count is performed on a Kova Glasstic counting slide using a Leica DMI 3000B optical microscope. The results are expressed as a percentage (%) of viability (n=3, mean + / - deviation).

[0077] The amoeba lysate was diluted 1 / 10, 1 / 100, 1 / 500, 1 / 1000 (successive dilution method) in RPMI 1640 medium supplemented with fetal calf serum (10%) and penicillin and streptomycin (5000U / mL, 5000pg / mL respectively).

[0078] - Results

[0079] [Tables 1] Time (hour) Average Standard deviation Control 0 100.00 0.00 24 99.63 0.42 48 0.77 98 8 7 100.00 0.76 0.00 96 99.80 0.27 120 99.40 0.80 Dilution 1 / 10 0 100.00 0.00 24 0.00 0.00 48 0.00 0.00 7? 0.00 0.00 96 0.00 0.00 120 0.00 0.00 Dilution 1 / 100 100.00 0.00 24 99.83 0.22 48 93.13 2.24 7? 86.00 1.13 96 71.90 1.13 120 60.83 5.46 Dilution 1 / 500 0 100.00 0.00 24 99.97 0.04 48 100.00 0.00 72 98.83 1.56 96 99.37 0.58 120 100.00 0.00 Dilution 1 / 1000 0 100.00 0.00 24 100.00 0.00 48 99.90 0.13 77 99.20 1.07 96 100.00 0.00 120 99.43 0.76

[0080] [Table 1] : % mean viability (n=3, mean +1- standard deviation) for a given dilution of the amoeba lysate as a function of time.

[0081] The amoeba lysate shows no toxicity for a dilution of 1 / 500 and 1 / 1000.

[0082] 3. Induction test of genes of interest

[0083] Target gene induction analysis is performed on human epithelial cells (ATCC CCL-2) cultured in Dulbecco's Modified Eagle Medium (DMEM) (GibCo-BRL) supplemented with fetal calf serum (10%) and penicillin and streptomycin (5000U / mL, 5000pg / mL respectively), at 37°C, and under a 5% CO2 atmosphere.

[0084] Briefly, 1106 human epithelial cells / wells are cultured in DMEM medium supplemented with fetal calf serum (10%) and penicillin and streptomycin (5000U / mL, 5000pg / mL respectively), at 37°C, and under a 5% CO2 atmosphere.

[0085] Human epithelial cells are stimulated or not with amoeba lysate (dilution 1 / 100, 1 / 500, 1 / 1000) for 8 hours at 37°C, and under a 5% CO2 atmosphere.

[0086] Total RNAs are extracted according to the Qiagen RNAeasy protocol, quantified by nanodrop (260 nm), and normalized to 100 ng / pL (stored at -20°C). During the quantification of total RNAs, the 260 / 280 nm ratio is defined. This ratio allows for the detection of nucleic acid contamination by proteins. Its value varies between 2.0 and 2.2 for total RNA. The 260 / 280 ratio must be between 2.0 and 2.2. Only samples exhibiting the above-mentioned ratio were used for the analyses.

[0087] The mRNAs contained in the total RNA samples are converted to cDNA by reverse transcription using a single-stranded 18-mer oligonucleotide primer, oligo(dt) 18, and then stored at -20°C. Quantitative PCR is performed on a Bio-Rad CFX96, using the Syber-Green method and primers specific to the target genes of interest. The results are expressed as a multiplicative factor (Fc for "fold change") according to the formula below, averaged (n=3, mean + / - standard deviation).

[0088] Fc = 2A(-AACt) with AACt = (Ct target gene - Ct constitutive gene)^ - (Ct target gene - Ct constitutive gene)control

[0089] The constitutive gene or control gene is the GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase and the target genes are: TLR2 (immunity), TGF-beta (cell multiplication, anti-inflammation), RUNX1 (cell multiplication, tissue cohesion, cell differentiation), P1W1LI (cell renewal), SGK1 (UV protection, anti-oxidative stress, photoaging), FOXO1 (UV protection, anti-oxidative stress, photoaging), BCL2 (anti-cell death), HMMR (hyaluronic acid receptor, cell mobility), HAS (hyaluronic acid synthesis), COLA1 (collagen synthesis), FBLS (wound healing).

[0090] [Tables2] Target Gene Average Condition Heart Rate Deviation BCL2 1100 0.61 0.29 BCL2 1 / 500 0.88 0.35 BCL2 1 / 1000 2.05 0.84 COLA1 1 / 100 0.77 0.42 COLA1 1 / 500 0.93 0.25 COL Al 1 / 1000 0.56 0.15 FBL5 1 / 100 1.66 0.06 FBL5 1 / 500 1.64 0.23 FBL5 1 / 1000 0.81 0.08 FOXO1 1 / 100 0.69 0.18 FOXO1 1 / 500 2.52 1.11 FOXO1 1 / 1000 0.75 0.20 HAS 1 / 100 0.94 0.10 HAS 1 / 500 1.80 0.78 HAS 1 / 1000 1.21 0.42 HMMR 1 / 100 1.07 0.43 HWIR 1 / 500 1.07 0.28 HMMR 1 / 1000 1.03 0.41 PIWÜL 1 / 100 2.61 1.25 PIWI1L 1 / 500 5.18 PI WI1L 1 / 1000 4.82 0.83 RUN 1 / 100 0.96 0.46 RUN 1 / 500 0.91 0.24 RUN 1 / 1000 0.43 0.06 SGK1 1 / 100 1.15 0.55 SGK1 1 / 500 2.59 0.70 SGK1 1 / 1000 0.88 0.23 TGFb 1 / 100 0.62 0.26 TGFb 1 / 500 1.04 0.37 TGFb 1 / 1000 0.97 0.26 TLR2 1 / 100 0.71 0.29 TLR2 1 / 500 1.56 0.90 TLR2 1 / 1000 0.91 0.34 [Table 2]: The results present the Fc (n=3, mean + / - standard deviation) as a function of dilution. Fold changes greater than or equal to 1.5 are indicated in bold.

[0091] A reading of [Table 2] indicates that the 7 genes: BCL2 (anti-cell death), FBL5 (wound healing), FOXO1 (UV protection, anti-oxidative stress, photoaging), SGK1 (UV protection, anti-oxidative stress, photoaging), HAS (hyaluronic acid synthesis), P1W1L1 (cell renewal), TLR2 (immunity) are positively regulated (Fc greater than or equal to 1.5) by treatment with amoeba lysate, and therefore may be of cosmetic interest.

[0092] For the remainder of the analysis, genes whose expression was increased by at least 1.5 times by treatment with the amoeba lysate were considered. These genes were separated into two categories, in order to carry out a more detailed analysis: genes whose expression increased between 1.5 and 1.99, and genes whose expression increased by 2 or more.

[0093] - Genes whose increased expression is between 1.5 and 1.99.

[0094] Treatment with amoeba lysate moderately stimulates, in human cells, the expression of the following cellular functions: wound healing (FBL5), hyaluronic acid synthesis (HAS), anti-infectious immunity: viruses and bacteria (TLR2) ( [Fig.1]).

[0095] - Genes whose increased expression is greater than or equal to 2.

[0096] Treatment with the amoeba lysate strongly stimulates the expression of the following cellular functions in human cells: protection against cell death (BCL2), cell renewal (PFWILF), UV protection, anti-oxidative stress, and photoaging (FOXO1 and SGK1). The measured expressions are 2 to 5 times higher than in the control ([Fig. 2]). The maximum expression of protection against cell death (BCL2) is observed at a 1 / 1000 dilution of the amoeba lysate. The maximum expression for the other three genes is observed at a 1 / 500 dilution of the amoeba lysate.

[0097] Conclusion

[0098] The Willaertia magna amoeba lysate (strain ATCC PTA-7824) is non-cytotoxic from a 1 / 500 dilution.

[0099] Amoeba lysate moderately stimulates the expression of cellular healing functions, hyaluronic acid synthesis, and anti-infectious immunity (viruses and bacteria) which are of cosmetic interest.

[0100] The amoeba lysate strongly induces the expression of the following important cellular functions in cosmetics: protection against cell death, cell renewal, and UV protection, anti-oxidative stress and anti-photoaging. The measured expressions are 2 to 5 times higher compared to the control.

[0101] The amoeba lysate can therefore be used for the following cosmetic purposes: - protection against cell death of skin cells, - cell renewal of skin cells, - protection against the effects of UV, - anti-oxidative stress, - protection against photo-aging.

[0102] Example 2: Evaluation of the effect of a Willaertia magna amoeba lysate (ATCC strain PTA-7824) on a 3D skin model aged (intrinsic aging) or exposed to UV radiation (extrinsic aging)

[0103] 1. Materials and methods

[0104] 1.1 Isolation of skin cells

[0105] The human skin tissue was collected in accordance with the principles of the Declaration of Helsinki and its use was declared to the French Ministry of Research (declaration no. DC-2020-4346). Written informed consent was obtained from the donor in accordance with the French Bioethics Law of 2014 (Law 94-954 of 29 July 1994).

[0106] Primary cultures of human fibroblasts, keratinocytes, and melanocytes were established from a biopsy of healthy skin obtained from an infant (< 5 years) or an adult (> 40 years). Keratinocytes (normal human epidermal keratinocyte: NHEK) and melanocytes (normal human epidermal melanocyte: NHEM) from normal human epidermis and dermal fibroblasts (normal human dermal fibroblast: NHDF) were isolated from human skin as previously described (Germain L. et al, 1993).

[0107] 1.2 Preparation of cosmetic products

[0108] The Willaertia magna amoeba lysate (strain ATCC PTA-7824) was stored at -20°C. Before being tested on a 3D skin model, the amoeba lysate underwent cytotoxicity tests to verify cell viability at the recommended concentration.

[0109] 1.3 3D Reconstructed Skin and Treatment

[0110] - Intrinsic aging

[0111] The LabSkin 3D model (LabSkin Creations) of reconstructed full-thickness skin was obtained by culturing NHDF (> 40 years) in a matrix composed of collagen, glycosaminoglycans and chitosan (LabSkin matriXTM) for 21 days under cell culture conditions optimized for the neosynthesis of the extracellular matrix (ECM), as previously described (Dos Santos M. et al., 2005, Tissue Engineering, 2005 May-Jun; 11(5-6)).

[0112] The NHEKs were then seeded on the upper part of the dermis-equivalent constructs and raised to the air / liquid interface to allow the formation of the epidermal compartment.

[0113] From day ten until the end of culture on day 42, the 3D skin samples were systemically treated with Willaertia amoeba lysate (PTA-7824) at 0.2E-0.3% or 0.1E-0.3%. The treatments were repeated at each medium change, i.e., 3 times per week.

[0114] Equivalent skin samples harvested on day 42 of total cell culture were immediately fixed in 4% neutral buffered formalin (Diapath) for 24 hours and embedded in paraffin or in an OCT (Optimal Cutting Temperature) compound and frozen at -80°C, for histological analysis and immunohistological analysis, respectively.

[0115] For each cell culture condition and each analysis, three copies of 3D skin equivalents were produced.

[0116] - Extrinsic aging

[0117] The LabSkin 3D model (LabSkin Creations) of fully reconstructed skin was obtained by culturing NHDFs (< 5 years old) in a matrix composed of collagen, glycosaminoglycans, and chitosan (LabSkin matriXTM) for 21 days under cell culture conditions optimized for ECM neosynthesis, as previously described (Dos Santos M. et al., 2005, Tissue Engineering, 2005 May-Jun;1 1(5-6)). NHEK and NHEM cells were then seeded on top of the equivalent dermal constructs and raised at the air / liquid interface to allow the formation of the pigmented compartment of the epidermis.

[0118] From day 10 until the end of culture on day 42, the 3D reconstructed skin samples were systemically treated with Amoeba lysate as follows: 0.45E-0.3% or 0.2E-0.3%. The treatments were repeated at each medium change, i.e., 3 times per week.

[0119] On day 42, the tissue constructs were exposed to 100 mJ / cm2 UVB.

[0120] Equivalent skin samples taken on day 42 of the total cell culture were immediately fixed in 4% neutral buffered formalin (Diapath) for 24 hours and embedded in paraffin or in an OCT compound and frozen at -80°C, for histological and immunohistochemical analysis, respectively.

[0121] For each cell culture condition and each analysis, three copies of 3D skin equivalents were produced.

[0122] 1.4 Histological analysis

[0123] - Staining with hematoxylin, phloxine and saffron

[0124] To evaluate the overall skin structure of the samples, hematoxylin-phloxine-saffron (HPS) staining was performed. 5 µm paraffin sections were prepared from each condition. After deparaffinization and rehydration, the samples were stained with HPS. After rinsing, the sections were dehydrated before slide mounting with a hydrophobic mounting medium.

[0125] - Warthin-Starry coloring

[0126] To assess melanin synthesis, Warthin-Starry silver staining was performed. After dewaxing and rehydration, the sections were incubated with a 6% silver nitrate solution for 20 minutes, following the instructions in the supplier's kit (Merck). Silver development was stopped by a water wash step. The sections were then incubated with a hydroquinone mixture. and gelatin. This step allowed the development of melanin granules in dark brown to black and the counter-staining of equivalent skin sections in yellow to orange.

[0127] 1.5 Image Acquisition

[0128] HPS- and Warthin-Starry-stained specimens were observed using an Axioskop 2 Plus optical microscope (Zeiss), and images were captured using a DS-Ri 1 CCD camera (Nikon) and NIS-Elements software (Nikon). The 16-bit images were saved in an uncompressed image file format (TIFF). Nine representative images were captured for each condition in the same manner.

[0129] 1.6 Statistical Analysis

[0130] The data were evaluated using a one-way Student's t-test, and statistically significant differences are indicated by asterisks as follows: *P < 0.05, **P < 0.01 and ***P < 0.001.

[0131] 2. Results

[0132] Intrinsic aging

[0133] 2.1.1 Histological analysis of the untreated 3D aging skin model.

[0134] The histological analysis of the 3D model of untreated young or aging skin is presented in the ([Fig.3]).

[0135] The overall structure of the reconstructed aged skin is consistent at the epidermal and dermal levels, with a multilayered epidermis, well differentiated from the basal layer to the stratum corneum, firmly anchored over the dermal equivalent filled with fibroblasts and a newly synthesized extracellular matrix (ECM). The dermo-epidermal junction appears cohesive and regular. However, compared to the young skin model, aging affects the epidermal and dermal compartments. The extracellular matrix is ​​less dense and abundant and is disorganized, which impacts tissue elasticity and hydration. The thickness of the epidermis is reduced, with fewer layers of living cells. Furthermore, terminal differentiation is disrupted, with less stratum granulosum and stratum corneum, which impacts barrier function ([Fig. 3]).

[0136] 2.1.2 Histological analysis of a 3D model of aging skin treated with lysate of amoeba

[0137] The histological analysis of the 3D aging skin model treated with Willaertia amoeba lysate (PTA-7824) is presented in ([Fig. 4]). Compared to the untreated aged condition, treatment with Willaertia amoeba lysate (PTA-7824) at 0.2E-0.3% increased epidermal thickness.

[0138] Furthermore, there was a recovery of terminal differentiation after treatment compared to the untreated condition. Regarding the dermal compartment, treatment with Willaertia amoeba lysate (PTA-7824) improved quality and abundance of the extracellular matrix ([Fig.4]). Similar effects were observed with treatment with 0.1E-03% amoeba lysate.

[0139] 2.2 Intrinsic aging

[0140] 2.2.1 Histological analysis of a 3D model of untreated skin in UVB exposure conditions

[0141] The histological analysis of the 3D model of pigmented skin exposed to UVB is presented in ([Fig.5]).

[0142] The overall structure of reconstructed skin exposed to UVB is consistent at the epidermal and dermal levels, with a multilayered epidermis, well differentiated from the basal layer to the stratum corneum, firmly anchored to the top of the dermal equivalent filled with fibroblasts and newly synthesized ECM. The dermo-epidermal junction appears cohesive and regular. However, compared to the unexposed condition, UVB stress impacts both the dermal and epidermal compartments. The extracellular matrix is ​​less dense, less abundant, and disorganized, which affects tissue elasticity and hydration. The epidermal thickness is reduced, with fewer layers of living cells, impacting barrier function ([Fig. 5].A).

[0143] With regard to Warthin Starry staining, UVB stress stimulates melanocytes to produce melanin pigments compared to the non-UVB-exposed state ([Fig.5].B).

[0144] The histological analysis of the 3D model of pigmented skin exposed to UVB and treated with the Willaertia amoeba lysate (PTA-7824) is presented in the ([Fig.6]).

[0145] Compared to untreated and UVB-exposed conditions, treatment with Willaertia amoeba lysate (PTA-7824) 0.45E-0.3% improves the quality and abundance of the ECM. Furthermore, the treatment induces an increase in epidermal thickness ([Fig. 6].A). Similar effects are observed with treatment using Willaertia amoeba lysate (PTA-7824) at 0.2E-0.3%.

[0146] Regarding Warthin Starry staining, treatment with 0.45E-03% Willaertia amoeba lysate (PTA-7824) induces moderate melanocyte stimulation compared to the untreated and UVB-exposed conditions. No difference was observed between the untreated and UVB-treated conditions after UVB stress ([Fig. 6].B).

[0147] Conclusions

[0148] Overall, the histological results demonstrated beneficial effects of Willaertia amoeba lysate (PTA-7824) on the intrinsic and extrinsic aging of the 3D reconstructed skin model. Reference to deposited biological material

[0149] In this application, reference is made to the following biological material and strain: - identification reference "Amoeba Willaertia magna: Will.C2c.Maky" and registration number "PTA-7824" deposited at the American Type Culture Collection (ATCC), in the United States, on August 21, 2006 by CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS) - 3 rue Michel Ange - 75794 PARIS CEDEX 16 / France and UNIVERSITE LYON 1 CLAUDE BERNARD - 43 Boulevard du 11 Novembre 1918 - 69622 VILLEURBANNE Cedex / France.

Claims

Claims

1. Cosmetic composition for skin care, preferably for preventing or reducing one or more signs of skin aging comprising protozoa of the amoebic genus Willaertia and at least one acceptable cosmetic excipient.

2. Composition according to claim 1, characterized in that the protozoa belong to the species Willaertia magna, preferably said protozoa correspond to the strain deposited under number PTA 7824 at the ATCC.

3. Composition according to claim 1 or 2 characterized in that said protozoa are in lysed form.

4. Composition according to one of claims 1 to 3, comprising one or more acceptable cosmetic excipients chosen from the group consisting of: water, an aqueous component, a fatty substance, a surfactant, a colorant, a texturizing agent, a preservative, a humectant, an emulsifier, an antioxidant, a perfume and a solvent.

5. Composition according to one of claims 1 to 4 in the form of a lotion, solution, suspension, emulsion, mousse, balm, oil, cream, gel, liquid soap or solid soap.

6. Use of a cosmetic composition according to one of claims 1 to 5 as an anti-aging agent, for treating or preventing a non-pathological alteration of the skin, preferably for preventing or reducing one or more signs of skin aging, in particular skin aging linked to exposure to ultraviolet rays, preferably by increasing the thickness of the epidermis, the differentiation of the stratum granulosum or the stratum corneum and / or by increasing the quality and quantity of the extracellular matrix present in the skin.

7. Use of a cosmetic composition according to claim 6 for promoting skin regeneration, slowing cell death in the skin or protecting against oxidative stress such as that induced by exposure to ultraviolet radiation.

8. Use of a cosmetic composition according to one of claims 6 or 7 in which the sign(s) of aging of the skin are chosen from the group consisting of: the appearance of wrinkles or fine lines on the skin, sagging of the skin, loss of elasticity, firmness or density of the skin.

9. Protozoa of the amoebic genus Willaertia for use in to protect the skin against infectious agents in a subject, preferably said protozoa belonging to the species Willaertia magna, more particularly to the strain deposited under number PTA 7824 at the ATCC.

10. Protozoa of the amoebic genus Willaertia for use in skin healing, preferably said protozoa belonging to the species Willaertia magna, more particularly to the strain deposited under number PTA 7824 at the ATCC.

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