• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Composition comprising hyaluronic acid of high molecular weight, inulin and alpha-glucan oligosaccharide. The invention relates to a composition comprising from 0.001 to 0.1% (w/w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts, from 0.01 to 5% (w/w) inulin, and from 0.01 to 5% (w/w) of alpha-glucan oligosaccharide. The invention also relates to the topical use of this composition for the preparation of a composition for the treatment and/or prevention of cutaneous affections of special skins selected from the group consisting of skins with excess fat and imperfections, hyper-reactive sensitive skins and excessively dry or desquamated skins. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2638714A1
    申请号:ES201630505
    申请日:2016-04-21
    公开日:2017-10-23
    发明作者:Aurora Del Carmen GARRE CONTRERAS；Susana MEZQUITA REGUEIRO；Maialen ELIZARI GALAR
    申请人:Laboratorios Cinfa SA；
    IPC主号:
    专利说明:

    5
    10
    fifteen
    twenty
    25
    30
    35
    DESCRIPTION
    Composition comprising hyaluronic acid of high molecular weight, inulin and oligosaccharide alpha-glucan
    The present invention relates to the field of dermatology and cosmetics, in particular to compositions comprising prebiotic compounds and high molecular weight hyaluronic acid for the treatment of special skins that suffer frequent conditions such as atopic dermatitis, seborrheic dermatitis, acne, cutaneous hypersensitivity, eczema, xerosis, erythrosis or rosacea.
    STATE OF THE TECHNIQUE
    Human skin, with a surface area of approximately 17,000 cm2, is one of the largest organs of the human body; Its outermost layer is formed by a stratified epithelium, the epidermis. It is very hydrophobic, relatively waterproof and very resistant. Being located at the interface between the environment and vital organs, the skin has the ability to maintain an effective barrier against the loss of body fluids and protection against physical and chemical damage.
    In addition to the physical barrier, the skin is an immunological barrier, whose main constituents are: antimicrobial peptides, which can be induced both in inflammatory lesions and in the absence of inflammation, receptors such as TLRs that participate in the recognition of microbial components by the subsequent activation of signaling pathways that result in the expression of proinflammatory cytokines and interferons, and activated T and B lymphocytes, representatives of adaptive immunity.
    The barrier function of the skin is complemented by the colonization of its surface by the cutaneous microflora. It is composed of a limited number of species, mainly Gram positive, resident and transitory. Residents refer to species that are viable and that reproduce, for example, Propionibacteria (Propionibacterium acnes, P. avidum and P.granulosum), coagulase-negative staphylococcus (Staphylococcus epidermidis), micrococci, corinebacteria and acinetobacteria. Transient species refer to species
    5
    10
    fifteen
    twenty
    25
    30
    35
    Contaminants with little or no capacity for growth and reproduction, such as Streptococcus, Staphylococcusaureus, Escherichia coli and Pseudomonas spp. When the cutaneous microflora is in equilibrium, the resident species occupy a space that prevents colonization by pathogenic microorganisms.
    In healthy skin, chemical, microbiological and mechanical aggressions to the skin have no general consequences, since their protective mechanisms are able to easily compensate for such disturbances. However, even in the case of non-pathological deviations from the norm, for example, as a result of damage due to wear or irritations caused by the environment, injuries, sun damage, skin aging etc., the protection mechanism deteriorates on the surface of the skin. The disruption, for various reasons, of the skin barrier can also cause a deterioration of the innate immunity of the skin. Both conditions, the disruption of the barrier and the deterioration of immunity, underlie the appearance of most diseases and skin conditions.
    In the case of pathologically sensitive skin, the barrier damage is present a priori. The consequence is the increase in the permeability of the corneal layer and the insufficient protection of the skin against the loss of hygroscopic substances and water. This type of skin reacts excessively to normally well tolerated aggressions, such as a sudden change in temperature, wind, fno, certain dermocosmetic products or a very strong emotion. Clmically these are unstable skins, which alternate episodes of normalcy with others of irritation with redness, dryness and even flaking and rashes.
    In skins with excess fat, the disruption of the cutaneous barrier contributes to acne proliferation or seborrheic dermatitis. Acne is the most common skin disease, being very common in adolescents and young people. Its etiology is multifactorial being the main factors in the production of acne: 1- The increase in sebum production and hyperplasia of the sebaceous gland; 2- Abnormal peeling of keratinocytes; 3- The presence of Propionibacterium acnes; and 4- The inflammation. Seborrheic dermatitis causes scales to form, ranging from white to yellowish, in greasy areas such as the scalp, face or inside the ear. It can occur with or without skin redness and, although the causes are unknown, it is related to the irritation caused by a fungus called
    5
    10
    fifteen
    twenty
    25
    30
    35
    malassezia, which colonizes in very fatty cutaneous areas, and causes micro-inflammations.
    Another very common condition in which the cutaneous barrier is affected is atopic dermatitis. More and more frequent in children, especially in babies, outbreaks of atopic dermatitis usually begin with a simple redness in the skin that causes itching. Then the skin becomes dry and rough and sometimes begins to suppurate before the blisters form. Many people have a genetic predisposition for atopic dermatitis. Those who are affected by this disease have a very reactive immune system. In addition, the atopic skin shows extreme constitutional dryness. The allergens of our environment can penetrate deeply into the lower layers of the epidermis as a result of the modification of the cutaneous barrier, stimulating the immune system, which reacts in an exaggerated way causing the appearance of the clinical symptoms of atopic dermatitis.
    The treatment of the previous cutaneous conditions, and others associated, is mainly based on the application of moisturizing creams and ointments whose effectiveness is relative or even poor in many cases. In particular, preparations based on hyaluronic acid enjoy great popularity. Hyaluronic acid is able to retain water in a percentage equivalent to thousands of times its weight. That is why it is used successfully for the hydration of the epidermis. Additionally, the ability of hyaluronic acid to reconstitute the fibers that support the skin tissues, giving a better shape to the skin, has been described. The fact that this compound is already present in significant amounts naturally in the skin contributes to its good reputation as a dermocosmetic ingredient in moisturizing and restorative creams.
    However, the contribution of these remedies to repair the barrier function in the pathologies or cutaneous conditions mentioned above is not entirely effective and, in addition, is very limited in time. For this reason, the use of active pharmaceutical compounds such as antibiotics (in the case of acne) or corticosteroids (in the case of atopic dermatitis) is frequent, whose continued use is not convenient, which makes them not recommended for the treatment of the conditions of interest, which, by their etiology, are repeated frequently or last for prolonged periods of time. That is why there is a need to provide new compositions that
    5
    10
    fifteen
    twenty
    25
    30
    35
    seek good long-term efficacy and without contraindications in the treatment of conditions such as atopic dermatitis, seborrheic dermatitis, acne, cutaneous hypersensitivity, etc.
    EXPLANATION OF THE INVENTION
    The inventors have developed a composition comprising prebiotics and high molecular weight hyaluronic acid that is effective in the treatment of skin conditions whose origin underlies a disruption of the skin's barrier function. This composition can be used for prolonged periods of time and does not present any contraindication.
    Thus, a first aspect of the invention relates to a composition comprising: from 0.001 to 0.1% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts, from 0.01 to 5% (w / w) of inulin, and 0.01 to 5% (w / w) of alpha-glucan oligosaccharide.
    The concentrations referred to refer to percentages by weight (% w / w), or what is the same, grams of product per 100 g of the total composition. The combination of active components is particularly suitable to cope with conditions such as atopic dermatitis, seborrheic dermatitis, acne, cutaneous hypersensitivity, rosacea, xerosis, eczema, erythrosis, couperose, psoriasis and other conditions in which a disruption of function underlies skin barrier. These conditions are common in special skins that can be divided into three large groups: 1- skins with excess fat and imperfections, which have a tendency to acne and seborrheic dermatitis; 2- hyper reactive sensitive skin, which has a tendency to erythrosis, couperose and rosacea; and 3- excessively dry or scaly skin, with a tendency to atopic dermatitis, psoriasis, eczema or xerosis.
    As demonstrated in the examples of the present application, the topical application of the compositions of the invention repairs the barrier function of the skin and restores the balance of the cutaneous microflora. All this results not only in an improvement of the symptoms of the conditions that affect these sensitive skin (pruritus, eczema, inflammation, peeling) but also prevents their appearance and affects
    5
    10
    fifteen
    twenty
    25
    30
    35
    about the pathological mechanisms of these conditions. The effectiveness of the combination of assets of the composition of the invention, without the need to add another type of assets whose long-term use is discouraged, is surprising, allowing its application to a large majority of the population (including the elderly, patients with different pathologies ^ as, children, pregnant women and babies) due to their absence of contraindications and side effects.
    Thus, a second aspect of the invention relates to the use of the composition defined in the first aspect for the preparation of a topical composition for the treatment and / or prevention of cutaneous conditions of special skins selected from the group consisting of skins with excess fat and imperfections, sensitive hyper-reactive skin and excessively dry or peeling skin. This aspect can be reformulated as the composition defined in the first aspect for topical use in the treatment and / or prevention of skin conditions of special skins selected from the group consisting of skins with excess fat and imperfections, sensitive hyper-reactive skins and skins. excessively dry or peeling
    The invention also relates to a method for the treatment and / or prevention of skin conditions of special skins selected from the group consisting of skins with excess fat and imperfections, sensitive hyper-reactive skin and excessively dry or flaky skin comprising the administration. topic of an effective amount of the composition defined in the first aspect together with topically acceptable vehicles and / or excipients, to a subject in need thereof
    DETAILED EXPLANATION OF THE INVENTION
    The invention relates to a composition comprising hyaluronic acid of high molecular weight or any of its pharmaceutically or cosmetically acceptable salts, inulin and oligosaccharide alpha-glucan with reparative effect of the cutaneous barrier and restoring the balance of the cutaneous flora that is effective in the treatment of conditions that affect special skin.
    Hyaluronic acid (CAS No .: 9004-61-9) is a glycosaminoglycan polysaccharide consisting of dimeros of N-acetylglucosamine and glucuronic acid. In humans it is found in large quantities in the joints, cartilage and skin. its
    5
    10
    fifteen
    twenty
    25
    30
    Incorporation into cosmetic and dermatological products has been known for many years, mainly fulfilling a moisturizing function in topical creams and ointments (Becker L. et al, International Journal of Toxicology 2009, vol. 28, No. 4S, p. 5-67 ). In these types of compositions for topical use they often contain a pharmaceutically or cosmetically acceptable salt of hyaluronic acid, in particular sodium hyaluronate (CAS No .: 9067-32-7) or potassium (CAS No .: 31799-91-4) . The beneficial properties for hyaluronic acid skin are extensible to its pharmaceutically or cosmetically acceptable salts, in particular to its sodium and potassium salt.
    Hyaluronic acid (or hyaluronate) of high molecular weight refers to one having a molecular weight equal to or greater than 300 KDa, for example 300 to 5000 KDa, in particular equal to or greater than 500 KDa, for example 500 to 2000 KDa, more particularly from 1000 to 2000 KDa. High molecular weight hyaluronic acid forms semi-permeable viscoelastic gel-like films that moisturize the skin. The larger the molecular size, the greater the aggregation and entanglement of the molecules, and therefore, the greater the viscoelastic film associated with the surface of the skin. Due to its high molecular weight, this hyaluronic acid does not penetrate deeper than the cracks between the skin peeling cells. This component can be obtained from commercial sources.
    Inulin and alpha-glucan oligosaccharide are carbohydrates considered prebiotic, that is, compounds not digestible by the acids and enzymes of the human gastrointestinal tract but that stimulate the growth and activity of bacteria beneficial to the flora, in the case of present invention, of the cutaneous flora. Inulin (CAS No .: 9005-80-5) is a polysaccharide composed of fructose units that are extracted from the roots, tubers and rhizomes of certain phanerogam plants (Burdock, chicory, dandelion, yacon, etc.). The alpha-glucan oligosaccharide (CAS No: 27707-45-5) is, as the name implies, an oligosaccharide composed of sucrose and maltose dimeros. It is obtained from sucrose and maltose by enzymatic synthesis with glycosyltransferase enzyme. This component is also commercially available.
    In a particular embodiment, the composition comprises:
    5
    10
    fifteen
    twenty
    25
    30
    35
    0.005 to 0.05% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts, 0.05 to 1% (w / w) of inulin, and 0.05 to 1% (w / w) of alpha-glucan oligosaccharide.
    In another particular embodiment, the composition of the invention comprises:
    0.07 to 0.03% (w / w) of high molecular weight hyaluronic acid or its salts
    pharmaceutically or cosmetically acceptable,
    0.1 to 0.6% (w / w) inulin, and
    0.05 to 0.15% (w / w) of alpha-glucan oligosaccharide
    In another particular embodiment, the composition comprises:
    0.01% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts,
    0.4% (w / w) inulin, and
    0.1% (w / w) of alpha-glucan oligosaccharide.
    In another particular embodiment the composition contains high molecular weight sodium or potassium hyaluronate.
    Preferably the compositions of the invention are compositions for topical use. Said topical compositions also contain topically acceptable carriers and / or excipients. By "topically acceptable vehicles and / or excipients" is meant compounds that are suitable for topical administration, which may be pharmaceutical or cosmetic excipients and vehicles generally known in the state of the art. Thus, the compositions of the invention may be cosmetic or pharmaceutical topical compositions and contain cosmetically or pharmaceutically acceptable carriers and / or excipients. Such excipients and vehicles include, but are not limited to, moisturizing agents, emollients, emulsifiers, solvents, thickeners, pH regulating agents, antioxidants, preservatives, vehicles, or mixtures thereof. The excipients and / or vehicles used have an affinity for the skin, are well tolerated, stable and are used in an adequate amount to provide the desired consistency, and ease of application. Additionally, the compositions may contain other components such as fragrances, dyes and other components known in the state of the art for use in
    5
    10
    fifteen
    twenty
    25
    30
    35
    topical formulations Pharmaceutically and cosmetically acceptable excipients and vehicles for topical use are known in the state of the art of cosmetics and pharmacy. In a particular embodiment, the topical compositions of the invention are cosmetic compositions and comprise cosmetically acceptable carriers and / or excipients.
    The topical compositions of the invention can be applied to the skin and lips in the form of: a gel, cream, lotion, emulsion, ointment, a non-ionic vesicular dispersion, aerosol, gel cream, gel cream, suspension, dispersion, powder, bar solid, wipe, poultice, foam, spray, oil, liquid, stick, a polyurethane patch, a silicone patch, a solution or any other form that is known in the technique of cosmetics and pharmacy.
    The compositions of the invention may additionally incorporate other active ingredients. Non-limiting examples of active agents suitable for use in the compositions of the present invention are: sunscreen agents, skin soothing agents, antioxidants, vitamins, minerals, anti-seborrheic agents, vasodilators, melanogenesis inhibitors, anti-allergens, antifungals, antiseptics, analgesics, metric oxide synthase inhibitors, insect repellents, self tanning agents, skin penetration promoters, skin refreshing agents, chelating agents, dyes that include varnishes and pigments that may be untreated or chemically treated in the surface to improve wettability or some other property, pearling agents, emollients, agents to stimulate the synthesis of dermal or epidermal macromolecules and / or to prevent their degradation, agents to stimulate fibroblast proliferation and / or to stimulate keratinocyte differentiation , muscle relaxants, stress agents, anti agents pollution and / or free radical sequestrants, anesthetics, antineoplasics, antivmcos, hypopigmentation agents, immune system boosting agents, immune system suppressing agents, anti-inflammatory agents, insect repellents, matting agents, photostabilizing agents, preservatives, protective agents skin and staining agents.
    The topical compositions of the present invention can be prepared according to procedures well known in the state of the art. Appropriate excipients and / or vehicles, and their quantities, can be easily determined by
    5
    10
    fifteen
    twenty
    25
    30
    35
    those skilled in the art according to the type of formulation that is prepared and to the regions of the human skin to which said composition should be administered. Thus, the person skilled in the art may select and / or avoid auxiliary substances that could be suitable or unsuitable for use in different regions of the skin, for example, face, neck, arms, legs, etc. The particular combination of components in the composition is largely determined by chemical compatibility. Particularly, such a selection takes into account that the components do not interfere with the effect of urea and isoleucine.
    For example, the composition according to the invention may contain:
    In the case of an emulsion:
    Oils (vegetable or minerals), waxes and fats (long chain alcohols) up to 30%
    Silicones, also volatiles (cyclopentasiloxane, dimethicone) up to 20% Moisturizers (glycerin, propylene glycol, PEG) up to 20%
    Ethanol (alcohol, alcohol denat.) Up to 10%
    Other active ingredients (vitamins, antioxidants, plant extracts) up to 10% Loading agents (talc, silica, nylon powder) up to 5%
    Ultraviolet filters up to 5%
    Anionic, amphoteric and non-ionic emulsifiers and surfactants (PEG stearate, ceteareth) up to 5%
    Preservatives and antimicrobials up to 2%
    Dyes up to 2%
    Perfume up to 1%
    Water q.s. 100
    In the case of a tonic solution or solution:
    Ethanol or solubilizers (alcohol, denat alcohol, water) up to 70%
    Moisturizers (glycerin, PEG) up to 25%
    Plant extracts up to 10%
    Oils (vegetable or mineral) up to 5%
    Silicones, also volatiles (cyclopentasiloxane, dimethicone) up to 5%
    Charging agents (silica, nylon powder) up to 5%
    Other ingredients (vitamins, ultraviolet filters) up to 5%
    5
    10
    fifteen
    twenty
    25
    30
    35
    Anionic, amphoteric and non-ionic emulsifiers and surfactants (sorbitan stearate) up to 5%
    Thickeners (carbomer) up to 2%
    Preservatives and antimicrobials up to 2%
    Perfume up to 1.5%
    Dyes up to 1%
    Water q.s. 100
    In the composition of the invention, the film that forms the high molecular weight hyaluronic acid or hyaluronate houses the prebiotic compounds providing better hydration, barrier repair and effectively releasing the prebiotic compounds so that these favor the restoration of balance. of the cutaneous flora, avoiding or eradicating the excessive growth and colonization of non-beneficial microorganisms such as E. coli, S. aureus, Candida albicans or even Propionibacterium acnes. This results in an obvious improvement in conditions that, such as acne, atopic dermatitis, etc., affect special skin whose barriers are damaged. The synergy that is established between high molecular weight hyaluronic acid, inulin and oligosaccharide alpha-glucan is particularly beneficial for the prevention and / or treatment of skin conditions of special skin selected from the group consisting of skins with excess fat and imperfections, sensitive hyper-reactive skin and excessively dry or peeling skin. A particular embodiment refers to the topical use of the composition of the first aspect of the invention for the preparation of a cosmetic topical composition for the treatment and / or prevention of skin conditions of special skins selected from the group defined above.
    In a particular embodiment of the invention, skin conditions are characterized in that a disruption of the skin's barrier function underlies them. In another embodiment, the cutaneous conditions are characterized in that in them there is also an alteration of the cutaneous flora and / or a loss of immunity. Preferably, the treatment of these conditions includes repairing the cutaneous barrier function and restoring the balance of the cutaneous flora.
    Conditions that affect sensitive skin within the meaning of the present invention are: atopic dermatitis, seborrheic dermatitis, acne, hypersensitivity
    5
    10
    fifteen
    twenty
    25
    30
    Cutanea, rosacea, psoriasis, facial erythrosis, and facial couperose. The above list is not limiting, there may be other conditions not mentioned that are characterized because they underlie a disruption of the skin's barrier function and, sometimes, the alteration of the cutaneous flora and the loss of immunity, which would also be included in the scope of the present invention.
    In a particular embodiment, the skin condition is selected from atopic dermatitis and acne.
    Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "understand" includes the case "consists of". For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.
    BRIEF DESCRIPTION OF THE DRAWINGS
    FIG. 1. Percentage of resazurine compared to the Healthy Control group throughout the duration of the study (7 days) in cut injury.
    FIG. 2. Resazurin percentage compared to the Healthy Control group throughout the duration of the study (7 days) in chemical burn.
    FIG. 3. Histological sections taken with hematoxylin-eosin (H&E). A) 10x Cut Group; B) 10x cutting and treatment group; C) 20x cutting and treatment group.
    FIG. 4. Histological cuts taken with H&E. A) 10x Healthy Control Group; B) Chemical burn group and 10x treatment; C) Chemical burn group 10x.
    5
    10
    fifteen
    twenty
    25
    30
    35
    FIG. 5. Cellular count of S. aureus over 48 hours in a saline medium. Lmea blue; microorganism incubated without composition C. Lmea roja; microorganism incubated with composition C.
    FIG. 6. Normalized comparison between both experimental groups of S. aureus in saline medium over 48 hours. The values are calculated considering the Control Group as 100% of the microbial population.
    FIG. 7. Cellular count of P. acnes over 48 hours in a saline medium. Lmea blue; microorganism incubated without composition C. Lmea roja; microorganism incubated with composition C.
    FIG. 8. Normalized comparison between both experimental groups of P. acnes in saline medium over 48 hours. The values are calculated considering the Control Group as 100% of the microbial population.
    FIG. 9. Cellular count of M. kristinae over 48 hours in a saline medium. Lmea blue; microorganism incubated without composition C. Lmea roja; microorganism incubated with composition C.
    FIG. 10. Standardized comparison between both experimental groups of M. kristinae in saline medium over 48 hours. The values are calculated considering the Control Group as 100% of the microbial population.
    FIG. 11. Cell count of L. pentosus over 48 hours in a saline medium. Lmea blue; microorganism incubated without composition C. Lmea roja; microorganism incubated with composition C.
    FIG. 12. Normalized comparison between both experimental groups of L. pentosus in saline medium over 48 hours. The values are calculated considering the Control Group as 100% of the microbial population.
    FIG. 13. Gram staining in situ on epidermis. A) S. aureus B) S. aureus + composition C.
    5
    10
    fifteen
    twenty
    25
    30
    35
    FIG. 14. Gram staining in situ on epidermis. A) P. acnes B) P. acnes + composition C.
    FIG. 15. Gram stain in situ on epidermis. A) M. kristinae B) M. kristinae + composition C.
    FIG. 16. Gram staining in situ on epidermis. A) L. pentosus B) L. pentosus + composition C.
    EXAMPLES
    The following examples demonstrate the reparative / regenerative capacity of the skin barrier function and the ability to maintain / restore the balance of the cutaneous flora of the composition of the invention. The different trials of this study were carried out in organotypic cultures of human skin explants.
    In order to assess the reparative / regenerative capacity of the skin barrier function of the composition of the invention, the study was carried out in skin explants of a patient before two types of skin wounds; chemical burn and cut. To assess the ability to maintain / restore the balance of the cutaneous flora, the study was carried out in skin explants of a single patient where four microorganisms present in the skin microbiota were cultured in the epidermis.
    1. Studies carried out:
    1.1. REPAIR / REGENERATIVE CAPACITY OF BARRIER FUNCTION (Study 1)
    An injury was made to the epidermis either by chemical corrosion (hydrochloric acid) or by cutting (scalpel). The injured explants were treated with the composition of the invention for a total of 7 days after the injury, with an application every 12 hours. The studies carried out in these explants were the following:
    1.1.1. Feasibility study and cell metabolism
    5
    10
    fifteen
    twenty
    25
    30
    35
    The metabolic status and viability of human skin explants were studied using fluorimetric study with resazurin.
    1.1.2. Histological study
    Through the hematoxylin-eosin staining, a macroscopic study of the state of the dermis and epidermis in the different skin explants was carried out.
    1.2. MAINTENANCE CAPACITY / RESTORATION OF THE FLORA BALANCE (Study 2)
    Four different microorganisms were grown, normally present in the skin microbiota: Staphylococcus aureus, Propionibacterium acnes, Micrococcus kristinae and Lactobacillus pentosus. The microorganisms were incubated with the composition of the invention for 48 hours, both in human skin explants and in low saline medium in organic matter. The studies carried out in these explants were the following:
    1.2.1. Cell count of microorganisms
    Counting of the microorganisms present in saline medium under organic load where the increase or decrease of the different organisms in the presence and absence of the compound under study was assessed.
    1.2.2. Gram stain in situ on the epidermis
    Realization of Gram stain in different skin explants. To check the growth and expansion of the different microorganisms planted on the explants in the presence or absence of the compound under study
    2. Materials
    Composition under study (composition of the invention): a composition composed of 0.01% (w / w) of hyaluronic acid of high molecular weight, 0.4% was studied
    5
    10
    fifteen
    twenty
    25
    30
    35
    (w / w) inulin, and 0.1% (w / w) alpha-glucan oligosaccharide. This composition will be referred to hereafter as composition C. The hyaluronic acid of DSM (Hyaluronic acid-BT, product code 50 3346 2) was a pure sodium hyaluronate powder of molecular weight around 1.6 MDa. Inulin and alpha-glucan oligosaccharide were provided through the product Biolin P (Gova Ingredients). This product consists of a powder that contains about 80% (w / w) inulin and about 20% (w / w) of alpha-glucan oligosaccharide.
    Composition C was prepared according to the study to be performed by suspending the components at the concentrations indicated above in culture medium of Dulbecco Modified Eagle (Dulbecco's Modified Eagle's Medium) DMEM (barrier repair activity study) or in PBS phosphate buffer (recovery study of the microbial flora).
    The means and solutions used in the experimental phase are indicated below:
    Eagle Medium Modified by Dulbecco. Sigma D-5546.
    Penicillin / streptomycin. Gibco L5L4O-L22.
    Get out of Hank's Sigma H-2387.
    PBS phosphate buffer (10x). Roche 11 666 789001.
    Abdominal explants of human skin, discs of 10 mm diameter Resazurina. Sigma R7OL7.
    IO% formalin solution. Sigma HT501128.
    DPX, Means of inclusion for histology. Sigma 4458L Hematoxylin. Sigma H9627.
    Eosina Sigma 861006.
    Gram Stain Kit. BD 212539.
    Man, Rogosa and Sharpe (MRS) agar. Thermo Scientific PO0231.
    RCM (culture medium for reinforced clostridia). Thermo Scientific CM0L49.
    PCA (plate count agar). Thermo Scientific CM0463.
    3. Experimental groups
    The following groups were included in the barrier repair activity study:
    5
    10
    fifteen
    twenty
    25
    30
    35
    1. Healthy Control Group: 4 skin explants without being injured.
    2. Cut Group: 4 skin explants with deep incisions.
    3. Cut Group + Composition C: 4 skin explants with incisions and post-treated with Composition C.
    4. Chemical burn group: 4 skin explants injured by hydrochloric acid.
    5. Burn group + composition C: 4 explants of skin with abrasion and post-treated with composition C.
    In the study of maintenance / restoration of the balance of the flora, a single trial was carried out, the groups studied in this task were the following:
    1. S. aureus group. 4 skin explants incubated with Stophylococcus aureus for 48 hours.
    2. Group S. aureus + composition C. 4 explants incubated with Staphylococcus aureus and composition C for 48 hours.
    3. P. acnes group. 4 skin explants incubated with Propionibacterium acnes for 48 hours.
    4. Group P. acnes + composition C. 4 explants incubated with Propionibacterium acnes and composition C for 48 hours.
    5. Group M. Kristinae. 4 skin explants incubated with Micrococcus kristinae for 48 hours.
    6. Group M. kristinae + composition C. 4 explants incubated with Micrococcus kristinae and composition C for 48 hours.
    7. L. pentosus group. 4 skin explants incubated with Lactobacillus pentosus for 48 hours.
    8. Group L. pentosus + composition C. 4 explants incubated with Lactobacillus pentosus and composition C for 48 hours.
    The density of microorganisms in all experimental groups was between 105 and 106 cfu / ml (cfu = colony forming units).
    4. Methods
    4.1 RESAZURINE TEST
    5
    10
    fifteen
    twenty
    25
    30
    35
    Resazurin dye (7-hydroxy-3H-phenoxazin-3-one 10-oxide) is widely used as an indicator of cell viability in proliferation and cytotoxicity assays. The assay is based on the fact that viable and metabolically active cells reduce resazurin to resorufin (fluorescent compound), which is released into the culture medium. This conversion is intracellular, facilitated by mitochondrial, microsomal and cytosolic oxidoreductases. In an event such as chemical or physical abrasion, where there is a loss of cell proliferation and viability, the cells that make up the epithelial tissue lose the ability to reduce resazurin. The resazurin reduction ratio being directly proportional to the number of viable cells present. Therefore, resazurin reduction will be a direct measure of the metabolic capacity of skin cells.
    Every 24 hours, skin explants were incubated with 6 ^ M resazurin for at least 1 hour. Subsequently, 100 µl of supernatant was taken from each sample and transferred to a 96-well plate. The resorufin produced was measured by fluorimetry at an excitation wavelength of 530-560 nm and an emission wavelength of 590 nm.
    4.2. HISTOLOGICAL PROCESSING
    4.2.1. Fixation and inclusion in paraffin
    The skin explants for morphological studies were incubated in 10% formalin for a maximum of 24-48 hours. The samples were kept in a refrigerator at 4 ° C. Once the sample was fixed, it was included in paraffin according to the following protocol: First, the pieces were dehydrated by immersion in alcohols of increasing graduation: 50 ° alcohol (1 hour 30 minutes) , 70 ° alcohol (1 hour 30 minutes), two passes in 96 ° alcohol of 1 hour 30 minutes each and 3 passes in 100 ° alcohol of 1 hour 30 minutes each. Subsequently, two passes were carried out in methyl benzoate. To end the inclusion in liquid paraffin in a 60 ° oven. With the samples in liquid paraffin, the realization of the paraffin molds was carried out by means of a paraffin dispenser. After processing, the blocks were cut in the microtome. 5 non-serial cuts of 5 ^ m thickness of each block were made, which were well stained with Hematoxylin-Eosin, or with orcein.
    5
    10
    fifteen
    twenty
    25
    30
    35
    4.2.2. Hematoxylin-Eosin staining (H&E)
    To have the cuttings of the samples included in paraffin, they were first kept in the oven for 2 hours at 60 ° C and subsequently dewaxed in citrosol (2 passes of 10 minutes each). They were rehydrated by dipping (one minute long) in decreasing graduation alcohols: absolute alcohol, 96 ° alcohol (2 passes), 70 ° alcohol and finally washed in running water.
    In the H&E stain the cuts were introduced into Harris Hematoxylin for 3 and a half minutes and washed in running water. Subsequently the cuts were introduced in alcoholic eosin for 20 seconds and washed again with running water. Dehydration was carried out in several 10-second passes each: in 70 ° alcohol, in 96 ° alcohol, in absolute alcohol (2 times) and in 50% absolute alcohol with citrosol. Finally they were clarified in citrosol (2 bathrooms of 10 minutes). Finally all the cuts were mounted with DPX histological mounting medium.
    4.3. MICROORGANISM CELL CULTURES
    The different microbial strains: Staphylococcus aureus ATCC 6538,
    Propionibacterium acnes ATCC 6919, Micrococcus kristinae ATCC 27570 and Lactobacillus pentosus ATCC 8041. The defrosting and planting process of the different strains was carried out following the steps indicated by the distributor. Both S. aureus and M. kirstinae were grown at 37 ° C in PCA medium. L. pentosus was grown at 37 ° in MRS medium. And finally P. acnes was grown at 37 ° C under anaerobic conditions in RCM culture medium.
    4.4. CONTROL GROWTH IN SALINE
    In order to determine the effect of the composition C on the different microorganisms, a sowing of said microorganisms was carried out in saline medium in the absence or presence of the composition. The different microorganisms were seeded in the saline medium with or without composition C at a cell density of 5 x 104 microorganism / ml. To subsequently keep two daily counts of these microbial populations for 48 hours.
    5
    10
    fifteen
    twenty
    25
    30
    35
    The different cell counts were carried out through serial dilutions (1/10) of the mother matrix where the microorganisms were seeded, until reaching a density of 102-105 cfu / ml (based on turbidity patterns). To subsequently sow 100-50 ^ l of these dilutions in agar plates (with the medium and conditions indicated in section 4.3.). After 24 hours, the counting of the colonies present in said plates was made. For the count to be representative, each plate must have between 30 and 300 cfu.
    4.5. GRAM IN SITU STATION ON HUMAN SKIN EXPLANENTS
    On human skin explants, 10 ^ l of saline solution was added in the absence and presence of composition C, with an approximate amount of microorganisms of 107 cfu / ml. After 48 hours of incubation of the different microorganisms on the skin explants at 37 ° C and 4% CO2, except for Propionibacterium acnes that was incubated under anaerobic conditions, Gram staining was carried out on the human skin explants following the process indicated by the commercial house BD. The explant was incubated with violet crystal for 1 minute to subsequently add the stabilizer and a bleaching agent to remove the remaining violet crystal on the sample.
    5. Results and discussion
    5.1. Study 1: Patient number 1: Caucasian woman, 53 years. Abdominal transplant
    In this study, skin explants were injured either by cutting or by abrasion with hydrochloric acid. After the injury, the explants were kept in culture for seven more days and the viability of the tissue (Resazurine) and its histological study were analyzed.
    5.1.1 VIABILITY AND METABOLIC CAPACITY ANALYSIS
    Resazurin toxicity test values are calculated considering the Healthy Control Group (healthy skin explants untreated or irradiated) as 100% resazurin concentration.
    5
    10
    fifteen
    twenty
    25
    30
    35
    Figure 1 shows the percentage of Resazurin with respect to the Healthy Control group over the duration of the study (7 days) in cut injury. The results indicated a better recovery of the skin's metabolic status in injured explants treated with composition C than in untreated injured explants. Also, from day 4 to 4, a metabolic activity was observed above the control values.
    Figure 2 shows the percentage of Resazurin with respect to the Healthy Control group over the duration of the study (7 days) in chemical burn. The results observed in explants injured by chemical abrasion indicated an improvement in the metabolic activity and health of the major skin in injured explants and treated with composition C with respect to untreated injured explants. Once the area under the curve was calculated, it was also verified that the recovery rate of the explants injured by chemical burn was three times higher when it was incubated with the composition C.
    The results in both cut injury and chemical abrasion indicate that composition C has reparative capacity and also improves the metabolic activity of the skin.
    5.1.3. HISTOLOGICAL STUDY
    The samples of the different experimental groups were fixed and processed for subsequent staining and histological study. Samples were taken after 2 days, 4 days and 7 days after the different injuries occurred in order to carry out a complete follow-up of the evolution of the different explants. Hematoxylin-eosin staining was carried out to carry out a morphological study of the tissue state of skin explants.
    In skin damaged by cutting the regenerative capacity was evident in the closure of the dermis, as shown in Figure 3.
    In skin damaged by chemical abrasion, the regenerative capacity was evident by almost completely maintaining the structure of the skin, without the presence of interstitial edema and dermis restructuring (see figure 4).
    5
    10
    fifteen
    twenty
    25
    30
    35
    In short, composition C has a very high regenerative activity in injured skin, with a metabolic activity being observed above healthy explants, as well as a regeneration in the histological structure. Additionally, composition C in explants with chemical burn improves the metabolic state and viability of the skin up to three times faster than without treatment.
    5.2. Study 2: Patient number 2: Caucasian woman, 52 years. Abdominal Explant 5.2.1. PROCARIOTA CELLULAR ACCOUNTING IN SALINE MEDIUM WITH LOW LOAD
    organic
    In this task the sowing of the different microorganisms under study was carried out in a saline medium with low organic load in the presence and absence of the composition C. A cell count was performed twice every 24 hours in a total of 48 hours of study .
    Figure 5 describes the cellular viability of Staphylococcus aureus over 48 hours in a saline medium in the presence or absence of the compound under study. Figure 6 shows the normalized cell count for this same microorganism considering the saline medium without composition C as 100% of the bacterial population. The results indicate that the microbial cultures of S aureus incubated with the compound under study had a lower percentage of cells than the culture of said bacterial community incubated in saline medium that did not hatch with the composition C.
    Figures 7 and 8 show the viability of P. acnes over 48 hours in saline medium in the presence or absence of composition C. In microbial cultures of P. acnes incubated with composition C a lower percentage of cells was observed that the culture of said bacterial community incubated in saline medium without the composition.
    Figures 9 and 10 show the viability of M. kristinae over 48 hours in saline medium in the presence or absence of composition C. The data obtained in the cell count of M. kristinae indicated that the compound favored said bacterial community.
    5
    10
    fifteen
    twenty
    25
    Figures 11 and 12 show the viability of L. pentosus over 48 hours in saline medium in the presence or absence of composition C. The data obtained in the cell count of L. pentosus, as observed in M. Kristinae indicated that the compound favored the growth of said organism.
    In short, the results show that composition C has a clear beneficial activity to restore or maintain the balance of the microbial flora of the skin, appreciating an increase in microbial populations beneficial to the surface of the skin such as Micrococcus kristinae and Lactobacillus pentosus while that potentially pathogenic microorganisms such as Staphylococcus aureus or Propionibacterium acnes are negatively affected by the presence of the composition.
    5.2.2. GRAM IN SITU STATION ON THE EPIDERMIS OF DIFFERENT SKIN EXPLANENTS
    In this study, the different microorganisms were incubated on the epidermis of human skin explants for 48 hours and subsequently carried out a Gram stain in situ on the epidermis of the different experimental groups. The results are shown in Figures 13-16.
    The results in human skin explants corroborated the results obtained in saline medium. Thus, an increase of the beneficial communities, M. kristinae and L. pentosus, on the explants in the presence of the compound under study was confirmed, while potentially pathogenic communities such as S. aureus and P. acnes showed considerable decreases in the presence of the composition.
    权利要求:
    Claims (15)
    [1]
    5
    10
    fifteen
    twenty
    25
    30
    35
    1. Composition comprising:
    0.001 to 0.1% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts, 0.01 to 5% (w / w) of inulin, and 0.01 to 5% (w / w) of alpha-glucan oligosaccharide.
    [2]
    2. The composition according to claim 1 comprising:
    0.005 to 0.05% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts, 0.05 to 1% (w / w) of inulin, and 0.05 to 1% (w / w) of alpha-glucan oligosaccharide.
    [3]
    3. The composition according to claim 2 comprising:
    [0]
    0.01% (w / w) of high molecular weight hyaluronic acid or its pharmaceutically or cosmetically acceptable salts,
    [0]
    0.4% (w / w) inulin, and
    [0]
    0.1% (w / w) of alpha-glucan oligosaccharide.
    [4]
    4. The composition according to any one of claims 1-3 containing high molecular weight sodium or potassium hyaluronate.
    [5]
    5. The composition according to any of claims 1-4 which is a topical composition and further comprises topically acceptable vehicles and / or excipients.
    [6]
    6. The composition according to claim 5 containing:
    Oils, waxes and / or fats up to 30%;
    Silicones up to 20%;
    Moisturizers up to 20%;
    Ethanol up to 10%;
    Other active ingredients up to 10%;
    Loading agents up to 5%;
    Ultraviolet filters up to 5%;
    5
    10
    fifteen
    twenty
    25
    Emulsifiers and / or surfactants up to 5%;
    Preservatives and / or antimicrobials up to 2%;
    Dyes up to 2%;
    Perfume up to 1%; and Water to reach 100%
    [7]
    7. The composition according to any of claims 4-6 which is a gel, a cream, an emulsion, a lotion, an ointment, a polyurethane patch, a silicone patch or a solution.
    [8]
    8. Use of the composition defined in any of claims 1-4 for the preparation of a topical composition for the treatment and / or prevention of skin conditions of special skins selected from the group consisting of excess fat skin and imperfections, skin sensitive hyper-reactive and excessively dry or peeling skin.
    [9]
    9. Use according to claim 8 wherein the skin conditions are characterized in that a disruption of the skin's barrier function underlies them.
    [10]
    10. Use according to claim 9, wherein the skin conditions are selected from the group consisting of atopic dermatitis, seborrheic dermatitis, acne, skin hypersensitivity, rosacea, psoriasis, xerosis, eczema, facial erythrosis, and facial couperosis.
    [11]
    11. Use according to claim 10, wherein the skin condition is selected from atopic dermatitis and acne.
    [12]
    12. Use according to claim 9 wherein the treatment comprises repairing the cutaneous barrier function and restoring the balance of the cutaneous flora.
    类似技术:
    公开号 | 公开日 | 专利标题
    US20210330691A1|2021-10-28|Methods of using cosmetic compositions comprising exopolysaccharides derived from microbial mats
    ES2143452T3|2005-05-16|COSMETIC COMPOSITION THAT CONTAINS AT LEAST A POLISACARIDE FROM BACTERIA OF HYDROTHERMAL ORIGIN.
    KR20110015531A|2011-02-16|Compositions and methods for skin care
    CN103961283B|2016-09-07|A kind of anti-acne repairs lyophilized formulations and preparation and application thereof
    ES2626612T3|2017-07-25|Composition comprising hydroxyapatite and a calcium salt to reinforce the barrier function of the skin and / or semi-mucous membranes
    KR20090005839A|2009-01-14|Cosmetic composition comprising seaweeds extracts
    CN110051602B|2021-11-26|Skin moistening, moisturizing and repairing emulsion and preparation method thereof
    KR20090038648A|2009-04-21|A composition using the cosmetic containing chamaecyparis obtusa oil for a pimpled skin
    ES2638714B1|2018-09-11|COMPOSITION THAT INCLUDES HIALURONIC ACID OF ELEVATE MOLECULAR, INULIN AND ALPHA-GLUCANO OLIGOSACÁRIDO WEIGHT
    JP2022515237A|2022-02-17|Topical formulation for delivery of microbial-derived materials
    ES2772126T3|2020-07-07|Cosmetic use of keuin
    CN111329803A|2020-06-26|Whitening essence and preparation method thereof
    ES2663574B1|2019-01-21|COMBINATION WITH REGENERATIVE FUNCTION OF THE SKIN BARRIER
    KR101252468B1|2013-04-16|Cosmetics composition for skin troubles
    BR112013027489B1|2019-10-29|cosmetic use of e-polylysine |
    JP6805399B2|2020-12-23|Skin care agent containing plant-derived lactic acid bacteria
    US20220008490A1|2022-01-13|Composition for the prevention and treatment of skin diseases
    KR102279878B1|2021-07-20|Cosmetic composition for improving acne
    RU2309750C2|2007-11-10|Therapeutic and cosmetic agent and preparation for therapy of damaged external tissues
    EP3954359A1|2022-02-16|Topical cosmetic composition and uses thereof
    KR102336699B1|2021-12-08|Agent for inhibiting decomposition of hyaluronic acid comprising sodium 2-mercaptoethane sulfonate, and the composition comprising the same
    ES2666306T5|2021-06-09|Topical composition comprising an association of at least one blue algae extract with at least one alpha-hydroxy acid or one of its salts
    CN111467359A|2020-07-31|Composition for resisting photoaging, preparation and application thereof
    BG3227U1|2019-08-30|Cosmetic composition
    CN109620745A|2019-04-16|A kind of anti-oxidant and anti-facial essence composition just to decline
    同族专利:
    公开号 | 公开日
    ES2638714B1|2018-09-11|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20100074851A1|2006-08-01|2010-03-25|Auriga International S.A.|Cosmetic or pharmaceutical composition containing hyaluronic acid|
    GB2472379A|2009-07-15|2011-02-09|Oskia Skincare Ltd|Topical cosmetic formulation comprising MSM, a vitamin and a carbohydrate|
    FR3009962A1|2013-08-28|2015-03-06|Herasens Lab|COMPOSITIONS BASED ON PREBIOTICS AND EXTRACTS OF ORCHIOID CURCULIGO AND USES THEREOF|
    CN103520082A|2013-10-10|2014-01-22|深圳市金因生物技术有限公司|Anti-aging composition containing epidermal growth factor and preparation method thereof|
    法律状态:
    2018-09-11| FG2A| Definitive protection|Ref document number: 2638714 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180911 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201630505A|ES2638714B1|2016-04-21|2016-04-21|COMPOSITION THAT INCLUDES HIALURONIC ACID OF ELEVATE MOLECULAR, INULIN AND ALPHA-GLUCANO OLIGOSACÁRIDO WEIGHT|ES201630505A| ES2638714B1|2016-04-21|2016-04-21|COMPOSITION THAT INCLUDES HIALURONIC ACID OF ELEVATE MOLECULAR, INULIN AND ALPHA-GLUCANO OLIGOSACÁRIDO WEIGHT|
    [返回顶部]