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    • 专利摘要:
    The subject of the invention is an active ingredient obtained from Ophiopogon japonicus for use in topical application to the skin in the treatment of atopic dermatitis in humans, in particular to reduce the rate of relapse of the Atopic dermatitis, decrease the severity score of atopic dermatitis (SCORAD), reduce the intensity and frequency of outbreaks of atopic dermatitis, decrease the importance of atopic dermatitis eczema and improve the quality index of life of patients with atopic dermatitis and their families.
    公开号:FR3046541A1
    申请号:FR1650228
    申请日:2016-01-12
    公开日:2017-07-14
    发明作者:Jean Paufique
    申请人:Societe Industrielle Limousine dApplication Biologique SA SILAB；
    IPC主号:
    专利说明:

    Active ingredient obtained from Ophiopogon japonicus
    FOR THE TREATMENT OF ATOPIC DERMATITIS
    The present invention relates to the treatment of atopic dermatitis.
    Atopic dermatitis, also known as atopic eczema, is a chronic skin disease of genetic and environmental origin. It is the most common dermatosis in children, and can appear from the first months of life. It reaches 20% of children under 7, is still around 18% in children 7 to 16 years, and 3% of adults are still affected. The notion of chronicity and evolution with relapses is important.
    The prevalence (number of patients in the general population) of atopic dermatitis has tripled in 30 years in industrialized countries. This prevalence is increasing especially with the environment, lifestyle changes and increased hygiene in our industrialized societies. It is therefore an emerging problem of public health.
    Whatever the age, the appearance of atopic dermatitis is identical, with dry skin due to lack of protective hydrolipidic film and patches of eczema, red, pruriginous, thickened (lichenified) with possible oozing.
    It is a complex multifactorial disease that makes treatment difficult. All symptoms of pathogenesis of atopic dermatitis (dry skin, red spots, itching, rash and swelling, in periods of crisis and lull) are associated mainly with the destruction of the skin barrier on Injured areas, but also uninjured areas, due to the weak synthesis of differentiation proteins such as cytokeratin 10, involucrine, loricrin and fillaggrin.
    The symptoms of atopic dermatitis are also characterized by over-expression of pro-inflammatory molecules such as interleukins (IL-) 4, 8, 13 and lymphopoietin stromal thymus (TSLP). In this vicious circle, the inflammatory response impacts on the synthesis of filaggrin. In the presence of Th2 cytokines, the differentiated keratinocytes significantly reduce the expression of the FLG (filaggrin) gene. The atopic inflammatory response is the central factor increasing the destruction of the cutaneous barrier function.
    Two groups of genes involved in atopic dermatitis have been identified: genes coding for the immune system and genes encoding epidermal structure proteins.
    The majority of existing products intended to treat the atopic skins, act on only one of the factors of the disease: either by limiting the inflammation: it is essentially of topical corticosteroids (for example, the dexamethasone, synthetic circle of inflammation, or by improving the skin barrier: these include emollients that clog the altered skin barrier to mechanically block hydration in the skin.
    There are also ingredients derived from plants, but these act essentially as anti-inflammatory or as a restructuring agent of the skin barrier. The objective of the present invention is to provide an active ingredient obtained from a plant capable of acting on various factors of atopic dermatitis. For this purpose, the invention provides an active ingredient obtained from Ophiopogon japonicus for use, in topical application to the skin, in the treatment of atopic dermatitis. Ophiopogon japonicus is a perennial, low and abundant herbaceous species with a rhizome of the family Liliaceae.
    It is cultivated as a covering ornamental plant with a tuberous rhizome with many uses in traditional Chinese medicine. The species is native to Japan and Korea, and is also cultivated in Vietnam and China, including Sichuan, Zhejiang and Hubei provinces. It is called Lily of the Valley in France, and Mondo grass, Fountain plant, Monkey grass or Dwarf lilyturf in English. The tuberous root has a length of a few centimeters; it is light yellow to yellow-brown outside, with longitudinal wrinkles. Its scent is weak, and its taste slightly sweet and mucilaginous.
    In traditional Chinese medicine, the root is known to nourish the lungs and the yin, to nourish the stomach and produce fluids, to remove heat from the heart and to soothe the spirit. The main therapeutic indications are dry coughs, sore throats, insomnia, irritability, constipation and diphtheria, according to the 16th edition of the Japanese Pharmacopoeia.
    The roots of Ophiopogon japonicus come from cultivated fields in China and Vietnam. Ophiopogon japonicus extracts are known in cosmetics, in particular in patent FR2930729 as a moisturizer by acting on the NMF level and on the formation of tight junctions of the layers. But moisturizing ingredients are not necessarily effective on the different markers of atopic dermatitis, and surprisingly, according to the invention, an active ingredient obtained from Ophiopogon japonicus can treat the multiple factors of atopic dermatitis. It decreases relapse rate, severity, intensity and frequency of attacks of atopic dermatitis, decreases the importance of eczema and improves the quality of life of patients and families.
    Its mode of action is based on both markers of inflammation and genes related to inflammation, markers of differentiation and genes associated with differentiation. It thus makes it possible to consolidate the epidermal construction and the integrity of the cutaneous barrier. Other features and advantages will emerge from the description in detail of the invention which follows, with reference to the appended figures:
    FIG. 1A represents the image visualized on a microscope of a section of normal reconstructed epidermis (corresponding to the result of Table 15 - Normal ER - Control, general morphology +++),
    FIG. 1B shows the image visualized on a microscope of a section of inflamed reconstructed epidermis (corresponding to the result of Table 15 - REI - Control, general morphology),
    FIG. 1C represents the image visualized on a microscope of a section of inflammed reconstructed epidermis treated topically with an active ingredient according to the invention (corresponding to the result of Table 15 - REI - Example 1 at 1%, general morphology + +)
    FIG. 1D represents the image visualized on a microscope of a section of inflammed reconstructed epidermis presenting an altered barrier (corresponding to the result of Table 15 - REBAI - Control, general morphology -),
    FIG. 1E represents the image visualized on a microscope of an inflammed reconstructed epidermal section having an altered barrier treated topically with an active ingredient according to the invention (corresponding to the result of Table 15 - REBAI - Example 1 at 1% , general morphology ++),
    FIG. 2A represents the image visualized under a microscope of a section of normal reconstructed epidermis labeled with a fluorescent probe (lucifer yellow) (corresponding to the result of Table 16 - Normal RE - Control, measurement of penetration of lucifer yellow: 4 )
    Figure 2B shows the microscopically visualized image of a section of inflamed reconstructed epidermis with a marked altered barrier with a fluorescent probe (lucifer yellow) (corresponding to the result of Table 16 - REBAI -Layout, lucifer penetration measurement yellow: 85)
    FIG. 2C represents the image visualized under a microscope of a section of inflammed reconstructed epidermis presenting an altered barrier treated topically with an active principle according to the invention, labeled with a fluorescent probe (lucifer yellow) (corresponding to the result of Table 16 - REBAI - Example 1 at 1%, penetration measure of lucifer yellow: 51). Definitions
    By "active principle" or "active" or "extract" within the meaning of the invention means at least one molecule, preferably a set of molecules having an effect on skin cells.
    By "Active ingredient obtained from Ophiopogon japonicus" within the meaning of the invention means any molecule or mixture of molecules obtained from Ophiopogon japonicus. It may be native molecules of the plant or molecules obtained by any type of transformation of the native molecules of the plant for example by hydrolysis. Preferably, it is a hydrolyzate.
    By "hydrolyzate" is meant any extract derived from Ophiopogon japonicus, obtained with a process comprising at least one step of enzymatic or chemical hydrolysis of Ophiopogon japonicus, preferably at least one enzymatic hydrolysis step. "Ophiopogon japonicus" means all or part of the plant. It can be the whole plant or part of the plant. Preferably it is tubers of Ophiopogon japonicus.
    By "oligosaccharides" is meant oligomers formed of a number of monosaccharides by glycosidic bond, the number of monosaccharide units being less than 25 units. By "polysaccharides" is meant polymers consisting of several monosaccharides bonded to each other by osidic bonds, the number of monosaccharide units being greater than 25 units. "Atopic skin" means the skin of a person suffering from atopic dermatitis.
    DETAILED DESCRIPTION OF THE INVENTION The invention therefore relates to an active ingredient obtained from Ophiopogon japonicus for use in the treatment of atopic dermatitis in humans. The active ingredient is applied topically to the skin to act on all the parameters of atopic dermatitis. It is used on atopic skin to: decrease the rate of relapse, decrease the severity score of atopic dermatitis (SCORAD), reduce the intensity and frequency of relapses, decrease the importance of eczema, improve the quality of life index of patients and families.
    These effects have been evaluated by dermatologists in particular on a panel of children with atopic skin.
    These efficacies on the factors of atopic dermatitis are explained since the active principle according to the invention acts both by: decreasing the inflammation of the cutaneous cells, regulating the differentiation of cutaneous cells, - promoting the epidermal construction, restoring the integrity the barrier function of the skin.
    Indeed, the active ingredient according to the invention is capable of acting both: on the markers of inflammation of the cutaneous cells: in particular it decreases the content of TSLP and IL-8 in these cells, - on the genes associated with skin cell inflammation: in particular, it normalizes the expression of the NELL2 gene and of the Tenascine C gene in these cells, on skin cell differentiation markers: in particular, it increases the filaggrin in these cells, on the genes associated with skin cell differentiation: in particular, it stimulates the expression of the FLG gene (filaggrin) and the LOR gene (Loricrin) and decreases the expression of the TGM1 gene (transglutaminase) in these cells, on the morphological construction of the epidermis, on the integrity of the epidermal barrier.
    These different efficiencies can be demonstrated on any model of atopic skin, in particular on the specific reconstructed epidermis models such as REI (reconstructed epidermis inflamed) and REBAI (inflammed reconstructed epidermis with an impaired skin barrier) as described in publication P Rouaud-Tinguely et al. "From the morphological to the transcriptomic characterization of a three-dimensional compromised in vitro model mimicking otopic dermatitis" British Journal of Dermatology (2015) 173, ppl006-1014.
    The active principle obtained from Ophiopogon japonicus useful according to the invention in the treatment of atopic dermatitis is preferably an active ingredient obtained from Ophiopogon japonicus comprising sugars. Even more preferentially, it comprises fructosans, and more particularly it comprises at least 57% of fructosans by weight relative to the weight of the total sugars of the active ingredient, still more preferably at least 80%. Fructans are polysaccharides composed of fructose and glucose.
    The sugars contained in the active principle are preferably constituted by 45 to 80% fructose, 20 to 50% glucose and 0 to 5% galactose. These sugars can be in the form of monomers, oligomers and polymers. Mostly, the sugars contained in the active principle are oligo and polysaccharides of molecular weight less than 400 kDa in the form of fructans. Thus, preferably the active ingredient according to the invention comprises oligo and polysaccharides of molecular weight less than 400 kDa in the form of fructans, representing at least 57% by weight of the sugars present in the active principle. According to a particularly suitable variant, the active ingredient is obtained from tubers of Ophiopogon japonicus.
    According to one embodiment, the active ingredient useful according to the invention is in the form of a powder, in particular a light-colored powder, and has at least one of the following characteristics, preferably all: a dry matter content between 900 and 1000 mg / g, a sugar content of between 500 and 800 mg / g, ie at least 50% of sugars by weight relative to the weight of solids.
    The solids content can be measured by passing the oven at 105 ° C. of a sample until a constant weight is obtained.
    The overall sugar content can be determined by the DUBOIS method on a range of fructose (Dubois M. et al., Analytical Chemistry, 28, 3, 350-356, 1956).
    The characterization of the molar mass of the carbohydrates present in the active principle of the present invention can be carried out by HPLC method and the determination of simple sugars by ionic liquid chromatography.
    The molar masses of the carbohydrates are evaluated by comparing the retention times of the peaks detected in the samples of the active ingredient with the retention times of previously injected standards.
    The operating conditions are preferably as follows: - Apparatus: Agilent 1100 HPLC Seriates - Columns: PL aquagel-OH columns C60, C40, C30 with pre-column of the same characteristics
    Elution mode: isocratic
    Mobile phase: buffer NaNCh 0.3M + NaH2PO4-2H20 0.01M pH7 Detection wave lengths: UV 254nm and 280nm The active principle according to the invention is preferably an active ingredient obtained in aqueous medium from tubers of Ophiopogon japonicus. By "obtained in an aqueous medium" is meant a medium containing mainly water, or a basic aqueous or acidic medium. In particular, it is not an oil or an essential oil.
    Preferably, the active ingredient according to the invention is a hydrolyzate of Ophiopogon japonicus, more preferably an enzymatic hydrolyzate.
    According to a particularly suitable variant, the active ingredient according to the invention is a hydrolyzate of Ophiopogon japonicus tubers, preferably an enzymatic hydrolyzate.
    In particular, the active ingredient useful according to the invention can be obtained by the implementation of the following steps: solubilization of powder of Ophiopogon japonicus (preferably tubers) in water at a rate of at least 50 g / l, at least one enzymatic hydrolysis of the sugars, separation of the soluble and insoluble phases, for example by decantation, enzymatic inactivation by thermal treatment of the soluble phase. The enzymatic inactivation may be followed by one or more filtration and / or concentration steps. The active ingredient can be obtained in liquid form or in powder form by atomization or lyophilization. Preferably it is atomized, in the presence of an atomization adjuvant maltodextrin type, and used in powder form.
    The parameters of the various steps must be adjusted in order to obtain active ingredients having the characteristics of the invention, in particular the presence of fructans having a molecular weight of less than 400 kDa.
    The active ingredient according to the invention is preferably used in a dermatological composition, this composition comprising a dermatologically acceptable medium. These are compositions in different galenic forms, suitable for topical administration to the skin.
    These compositions may especially be in the form of oil-in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil) which may be optionally microemulsions or nanoemulsions, or in the form of solutions, suspensions, hydrodispersions, aqueous gels or powders. They can be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam, or in solid form.
    It is preferably a cream, a gel or an ointment.
    It may be dermatological compositions comprising at least 0.1% of active principle obtained from Ophiopogon japonicus, according to the present invention, preferably between 0.1 and 1%.
    These compositions comprise, in addition to the active agent, a physiologically acceptable and dermatologically acceptable medium, that is to say which does not cause feelings of discomfort that are unacceptable to the user, such as redness, tightness or tingling.
    The compositions according to the invention may contain, as dermatologically acceptable adjuvant, at least one compound chosen from: oils, which may be chosen in particular from silicone oils, linear or cyclic, volatile or nonvolatile; waxes, such as ozokerite, polyethylene wax, beeswax or carnauba wax, silicone elastomers, surfactants, preferably emulsifiers, whether they are nonionic, anionic or cationic or amphoteric, - thickeners and / or gelling agents, - humectants, such as polyols such as glycerin, - organic filters, - inorganic filters, - dyes, preservatives, fillers, - tensors, - sequestering agents , and their mixtures, without this list being limiting.
    Examples of such adjuvants are cited in particular in the CTFA Dictionary (International Cosmetic Ingredient Dictionary and Handbook published by the Personal Care Product Council). Of course, those skilled in the art will take care to choose any additional compounds, active or non-active, and their amount, so that the advantageous properties of the mixture are not, or not substantially, impaired by the addition envisaged.
    Dermatological compositions comprising an active ingredient according to the invention can therefore be used to treat atopic dermatitis.
    In order to illustrate these cosmetic effects on the skin, the following examples with their test results are presented.
    Examples
    Example 1 Active Principle According to the Invention
    The active substance derived from tubers of Ophiopogon japonicus is characterized as follows: - Light beige powder, - Dry matter: 960mg / g - Sugar content: o Monosaccharides: 8% relative to the dry matter o Oligo and polysaccharides: 88% relative to the dry matter - Mineral ash content: 2% relative to the dry matter - Protein content: 2% relative to the dry matter
    Example 2a: Dermatological composition in the form of cream
    The formula is composed of a commercial cream Diprobase® Cream from Merck in which was added the active ingredient according to the invention of Example 1.
    Table 1
    Example 2b: Dermatological composition in the form of cream
    The formula is composed of a commercial cream Diprobase® Cream from Merck in which was added the active ingredient according to the invention of Example 1.
    Table 2
    Example 2c: Dermatological composition in the form of comfort cream The formula is composed of a commercial cream Diprobase® Cream from Merck in which was added the active ingredient according to the invention of Example 1.
    Table 3
    EVALUATION OF EFFICACY ON THE PARAMETERS OF ATOPIC DERMATITIS
    In vivo tests The in vivo efficacy of an active ingredient obtained from Ophiopogon japonicus was tested in a study on children with atopic skin.
    The inclusion criteria are: - Children aged 4 months to 4 years with moderate to moderate atopic dermatitis, - Subjects having had at least one seizure in the last 2 months, - Subjects having received corticosteroid treatment to treat atopic dermatitis before the study, - Subjects not using an antibiotic, anti-histamine or calcineurin inhibitor for 3 weeks, - Subjects not having other dermatological conditions, or chronic or progressive disease.
    This randomized study of 2 months consists of a simple blind study of about 100 subjects, having been treated either with a placebo formula or with a formula containing the active ingredient according to the invention at 0.3%. (composition of Example 2c).
    The subjects applied the product 2 times a day on the whole body.
    The evaluation criteria for effectiveness are the Infant's Dermatitis Quality Index (IDQOL), the Dermatitis Family Impact Questionnaire (DFI), and Scoring Atopy Dermatitis (SCORAD). These criteria were evaluated by a dermatologist doctor at OJ, J30 and J60.
    The protocol of visits is presented in Table 4 below:
    Table 4 The dermal exposure (EDS) of atopic children to the active principle according to the invention is determined according to the following formula: SED = [QA (g / d) × 1000 mg / g × C (%) / 100 × DAp (% ) / 100] / body weight SED: Systemic Exposure Dosage QA: Quantity of product applied by the dermal route C: Concentration of the substance studied in the finished product on the area of application DAp: Dermal absorption expressed as a percentage of the dose of test supposed to be applied in real conditions
    The exposed children are between 4 months and 4 years old. For the calculation of exposure, the target population is the infant population. In order to maximize the value of exposure, infants represent the most sensitive population because of a greater surface area / body weight ratio than that of adults or children.
    The amount of product applied dermally is 1.1 g / day for a body surface area of 2200 cm 2 for a body product and a weight of 3.4 kg. Dermal absorption of the active ingredient is not fixed, a value of 50% will be retained, according to the recommendations of the SCCS 9th revision (September 2015).
    It is considered that the active ingredient will be used at a dose of 0.3% in the finished cosmetic product, which equates to a concentration of 0.03% in the finished product. SEDnurn = = [1.1 x 1000 x 0.03% / 100 x 50% / 100] / 3.4 = 0.048 mg / kg bw / day o Effect on relapse rate
    The relapse rate was analyzed for both groups after 60 days. It is presented in Table 5 below.
    Table 5
    It is found that under the conditions of the study, after 60 days of twice-daily treatment on the whole body, the rate of relapse with the treatment according to the invention is half as important as with the placebo treatment. O EFFECT ON THE GRAVITY SCORE OF ATOPIC DERMATITIS (SCORAP)
    The SCORAD or severity score of atopic dermatitis was created and validated in 1990 by a group of experts: the European Task Force of Atopy Dermatitis. It is a reference tool for monitoring and evaluating the pathology by doctors. The SCORAD is defined by the analysis of different items: average area of atopic lesions, erythema, edema / papules, oozing / scabs, excoriation, lichenification, non-lesion dryness, pruritus, insomnia. The SCORAD analysis was performed by the dermatologist in patients who did not observe a relapse during the study (60% of patients treated with placebo, 79% of patients treated with AP).
    The results are shown in Table 6:
    Table 6
    It was found that under the conditions of the study, after 60 days of twice-daily treatment of the whole body, the decrease in the severity score of atopic dermatitis observed by the dermatologist is -13% for the placebo and -57% for the formula containing an active ingredient obtained from Ophiopogon japonicus.
    It is found that under the conditions of the study, after 60 days of treatment, the atopic dermatitis severity score of the group treated with the active ingredient obtained from Ophiopogon japonicus is lower than that of the placebo-treated group. The analysis of the different SCORAD items was also done after 30 days and after 60 days is shown in Table 7:
    Table 7
    A significant improvement is observed in particular on the evaluation parameters of erythema and pruritus. o General evaluation of dermatologist treatments Both treatments are evaluated by dermatologists on several criteria: softening effect, reduction of the intensity of outbreaks, reduction of the frequency of outbreaks, good product tolerance and overall product satisfaction.
    The results after 30 days and 60 days of treatment are presented in the following Table 8:
    Table 8
    It was found that under the conditions of the study, after 30 days of treatment, the general evaluation performed by dermatologists on the group treated with the active ingredient obtained from Ophiopogon japonicus is generally better than that of the group treated with placebo.
    This effect continues after 60 days of treatment; the active principle obtained from Ophiopogon japonicus shows a softening effect, reduces the intensity of relapses, the frequency of relapses, significantly higher than those observed in the group treated with placebo. It is overall satisfactory. o Effect on quality of life
    Atopic dermatitis is a chronic disease that affects the quality of life and requires daily care. One of the important follow-ups of the evolution of atopic dermatitis focuses on the parameters related to quality of life: importance of eczema, depreciation of the quality of life index of the child (IDQoL), depreciation of the Family Quality of Life Index (DFIQ), a total index of quality of life.
    These parameters were evaluated in patients who had no relapse during the study (placebo n = 26 and PA n = 32).
    The results after 30 days and 60 days of treatment are shown in the following Table 9.
    Table 9
    Under the conditions of the study, after 30 days of treatment, an improvement in QoL quality of life was observed for the group treated with the active ingredient obtained from Ophiopogon japonicus.
    This effect continues after 60 days of treatment and the improvement is significantly visible on the parameters:
    Importance of eczema: 89% reduction - Improvement of the child's quality of life index (IDQoL): 59% reduction - Improvement of the family quality of life index (DFIQ): reduction of 51% - Improvement of the total index of the quality of life (total index QoL): reduction of 58%. o General evaluation of the treatments by the patients The two treatments are evaluated by the patients on several criteria: delayed effect of the relapse, daily comfort, product well tolerated, pleasant use of the product, pleasure to use the product, satisfactory product, effectiveness of the product.
    The results after 30 days and 60 days of treatments are presented in the following Table 10:
    Table 10
    Under the study conditions, after 30 days of treatment, the overall assessment of patients on the active ingredient group obtained from Ophiopogon japonicus was generally better than that of the placebo group.
    This effect continues after 60 days of treatment; the active ingredient obtained from Ophiopogon japonicus is significantly satisfactory and effective compared to placebo.
    In conclusion, dermatologists consider that the active principle obtained from Ophiopogon japonicus according to the invention has demonstrated a softening effect, reduced the intensity and frequency of outbreaks. It is therefore a globally satisfactory product. It reduces the rate of relapse in atopic children, and decreases significantly by 25% after 60 days of treatment SCORAD index of children with atopic.
    The patients consider that the active ingredient according to the invention brings a feeling of comfort. They perceive a general efficiency and consider the product as globally satisfactory.
    Above all, the active ingredient according to the invention reduces the importance of eczema and improves the quality of life of children and their families.
    In vitro tests
    For the implementation of in vitro tests, two specific reconstructed epidermal (RE) models were used. These are those described in the publication of the British Journal of Dermatology (2015) 173,1006-1014).
    The in vitro model of 3D reconstructed epidermal inflammation (REI) mimics the inflammation of atopic dermatitis.
    The in vitro 3D model of an inflamed reconstructed epidermis with an impaired barrier (REBAI) mimics the inflammation of atopic dermatitis on an altered epidermis.
    These in vitro 3D models REI and REBAI were made as follows.
    Normal human keratinocytes are grown in monolayers with a specific medium. The REI and REBAI models are obtained by inoculation of normal human keratinocytes onto culture inserts. The cells are cultured in complete medium for several days.
    For the REI model
    At the 14th day of RE culture, the inflammation is caused by the addition of an inflammatory cocktail (Poly l / C + TNFa + IL-4 + IL-13) for 2 additional days.
    For the REBAI model
    On the 15th day of RE culture, the ERs are treated topically with a solution of SDS (sodium dodecyl sulfate), in order to obtain a significant alteration of the cutaneous barrier. Inflammation is caused by the addition of an inflammatory cocktail (Poly l / C + TNFa + IL-4 + IL-13) in the culture medium for an additional day.
    These two models are compared to a normal model of reconstructed epidermis (RE) that has not been treated by inflammatory cocktail or SDS. o Effect on markers of inflammation
    This study consists in evaluating the effect of an active ingredient obtained from Ophiopogon japonicus on the markers of inflammation (IL-8 and TSLP) on a REI model and on a REBAI model.
    The active ingredient was added in systemic treatment, ie at 0.10 and 0.25% directly in the culture medium of the ERs.
    Amanaka et al (2011) and Stalder et al (2014) have explained in their respective publications (The role of cytokines / chemokines in the pathogenesis of atopic dermatitis, Fragility of epidermis and its consequence in dermatology) that atopic skin is characterized mainly by overexpression of pro-inflammatory molecules such as IL-4, IL-8, IL-13 and thymic stromal lymphopoeitin (TSLP).
    The inflammatory response in the REI and REBAI 3D models is evaluated by measuring the content of interleukin IL-8 and thymic stromal lymphopoietin (TSLP) released in the tissue subnapeants.
    Subnants of REI and RE are collected and stored at -20 ° C. Secretions of IL-8 and TSLP are measured using the Elisa kit.
    The results are compared to a normal RE model, which has not been in contact with the inflammatory cocktail.
    The results are shown in Table 11 below.
    Table 11
    It is found that the inflammatory cocktail indeed causes an inflammatory effect (release of TSLP and IL-8) in the REI model.
    It is also found that the inflammatory cocktail indeed causes an inflammatory effect (IL-8 release) in the REBAI model.
    The active substance obtained from Ophiopogon japonicus has no negative effect on the normal RE model, it does not cause inflammation.
    On the other hand, in the REI model, the active ingredient obtained from Ophiopogon japonicus makes it possible to reduce the inflammation caused by decreasing the level of TSLP by 25% and the IL-8 content by 21%.
    Similarly, in the REBAI model, the active ingredient obtained from Ophiopogon japonicus reduces the inflammation caused, by decreasing the IL-8 content by 24%. The use of an active ingredient obtained from Ophiopogon japonicus therefore makes it possible to limit the inflammation on the injured areas and on the non-injured areas of the atopic skin. o Effect on the genes associated with inflammation The study consists of evaluating the effect of an active ingredient obtained from Ophiopogon japonicus on the genes associated with inflammation.
    The active ingredient was added in systemic treatment, ie at 0.10 and 0.25% directly in the culture medium of the ERs.
    In 2011, Kamsteeg identified in its publication Type 2 Helper T-Cell Cytokines Induce Morphology and Molecular Features of Atopy Dermatitis in Human Skin Equivalent, that the key genes associated with inflammation are carbonic anhydrase II (CAII) and factor equivalent. Neutral epidermal growth (NELL2) in atopic skin lesions compared with psoriasis.
    The protocol of the present study is described following.
    The total RNAs of the ERs are extracted by an RNeasy kit. Quantification and quality evaluation of the isolated RNAs is performed using a NanoDrop spectrophotometer and an Agilent bioanalyzer.
    The fluorescence of the signals is detected by a specific scanner and the image analysis is performed by software. The analysis of biological processes is carried out on the transcriptomic data of the DAVID database. 1730 genes from the 20,000 genes of the REI model were overexpressed compared to the normal RE model, and 2,086 genes were under-expressed.
    Four pathways include these 3816 genes: cell adhesion, cell migration, inflammation and epidermal cell differentiation. The genes (CXCL8, CA2, NELL2, TLR2, TLR3, NOTCH, STAT2, SPINK5, PLAUR and ILIA) associated with inflammation are overexpressed in the REI.
    The results obtained are shown in Table 12 below.
    Table 12
    It is noted that the inflammatory cocktail indeed causes a significant inflammatory effect (over-expression of the NELL2 Tenascine C genes) in the REI model.
    The active ingredient obtained from Ophiopogon japonicus, in the REI model, makes it possible to reduce the expression of NELL2 and that of Tenascine C. In particular the active principle tested at 0.5% makes it possible to reduce the expression of NELL2 by 70%. and 38% that of Tenascine C. The use of an active ingredient obtained from Ophiopogon japonicus thus makes it possible to limit the expression of the genes of inflammation characteristic of atopic dermatitis.
    O EFFECT ON DIFFERENTIATION MARKERS The study consists in evaluating the effect of an active principle obtained from Ophiopogon japonicus on differentiation markers on the REI model and on the REBAI model.
    All symptoms of atopic dermatitis are associated with disruption of the skin barrier function of atopic patients. Several publications, such as Agrawal 2014, Skin Role Defects in Atopia Dermatitis, and Feingold 2014, Role of lipids in the formation and maintenance of the cutaneous permeability barrier, explain that syntheses of the differentiation proteins (loricrin, involucrine and filaggrin) are sub- expressed in atopic skin. The impact of the inflammatory response of the reconstructed epidermis is evaluated on a differentiation marker, filaggrin.
    After 17 days of culture of the inflammed RE, the epidermis are fixed, dehydrated with 4% paraformaldehyde and included in paraffin. 4pm sections are made using a microtome. Morphological analysis of the sections is done by HE staining (hematoxylin eosin). Immunohistofluorescence analysis of filaggrin, loricrin and involucrine is performed after incubation of a primary antibody and a secondary antibody. The visualization is performed on a microscope coupled to an image analysis system. Quantitative analysis is performed with MatLab® software.
    The results are compared to a normal RE model, which has not been in contact with the inflammatory cocktail.
    The active principle of Example 1 was added in systemic treatment, ie at 0.10 and 0.25% directly in the culture medium of the ERs.
    The results obtained are shown in Table 13 below.
    Table 13
    It is found that the inflammatory cocktail does indeed cause a limitation of differentiation (inhibition of filaggrin synthesis) in the REI model and in the REBAI model.
    The active ingredient has no effect on the normal RE model, it does not increase the differentiation marker.
    On the other hand, in the REI model, the active principle obtained from Ophiopogon japonicus makes it possible systemically to maintain the synthesis of differentiation markers. In particular tested at 0.25%, it allows systemically, to increase the synthesis of filaggrin by 45%.
    In the REBAI model, the active principle obtained from Ophiopogon japonicus allows, systemically, to maintain the synthesis of differentiation markers. In particular tested at 0.25%, it allows systemically to increase the synthesis of filaggrin by 32%. The use of an active ingredient obtained from Ophiopogon japonicus in systemic treatment therefore makes it possible to increase the synthesis of differentiation markers on atopic skin. o Effect on the genes associated with differentiation The study consists in evaluating the effect of an active ingredient obtained from Ophiopogon japonicus according to the invention on the genes associated with differentiation.
    The active ingredient was added in systemic treatment, ie at 0.10 and 0.25% directly in the culture medium of the ERs.
    In 2005, Sugiura indicated after analysis of 23,000 cutaneous biopsy genes from patients with atopic dermatitis compared to controls, that 10 genes show significant differences between patients and controls. Important modifications are the under-regulation of the genes encoding loricrin and filaggrin. Large-scale DNA microarray analytesis of atopic lesions shows of an epidermis differentiation gene cluster in the alternative pathway and lack of protective gene expression in the cornified envelope.
    In 2014, Zhang completed in his publication Screening for key genes associated with atopic dermatitis with DNA microarrays, that the key genes associated with atopic dermatitis are related to differentiation: loricrin (LOR), keratin 17 (KRT17), small repetition rich in proline (SPRRs) and involucrine (IVL).
    The protocol of the study is described following.
    The total RNAs of the ERs are extracted by an RNeasy kit. Quantification and quality evaluation of the isolated RNAs is performed using a NanoDrop spectrophotometer and an Agilent bioanalyzer.
    The fluorescence of the signals is detected by a specific SureScan Microarrays scanner and the analysis of the images is performed by software. The analysis of biological processes is carried out on the transcriptomic data of the DAVID database. 1730 genes from the 20 000 genes of the infested RE model were overexpressed compared to the normal RE model, and 2086 genes were under-expressed.
    Four pathways include these 3816 genes: cell adhesion, cell migration, inflammation and epidermal cell differentiation:
    The genes (TGM1, S100A8, S100A9 and KLK7) associated with differentiation are overexpressed in the inflammed RE.
    The genes (FLG, LOR, KRT1, KRT10 and ITGA2) also associated with differentiation are under-regulated in the inflammed RE.
    The results are shown in Table 14 below:
    Table 14
    It is found that the inflammatory cocktail indeed causes a limitation of the differentiation (under expression of the FLG gene, Filaggrin and the LOR gene, Loricrin and overexpression of the TGM1 gene, transglutaminase 1) in the REI model. The use of an active ingredient obtained from Ophiopogon japonicus thus makes it possible to increase the expression of the Filaggrine and Loricrin genes, and to reduce the expression of the Transglutaminase 1 gene, associated with differentiation. o Effect on epidermal construction The study consists in evaluating the effect of an active ingredient obtained from Ophiopogon japonicus on the epidermal construction on the REI model and on the REBAI model.
    Epidermal construction in inflammed 3D models is evaluated by morphological analysis of epidermal sections after HE (hematoxylin-eosin) staining.
    After 17 days of RE culture, the epidermis are fixed, dehydrated with 4% paraformaldehyde and included in paraffin. 4pm sections are made using a microtome. The morphological analysis of the sections is done by HE staining. The visualization is performed on a microscope coupled to an image analysis system.
    The active ingredient was added topically, at 0.5 and 1% in a cosmetic formula on the epidermis. The placebo formula has also been tested.
    The results are shown in Table 15 below and in Figures IA to 1E.
    Table 15 +++ means normal reconstructed epidermis, compact epidermal layers ++ means altered reconstructed epidermis, less compact epidermal layers + means altered reconstructed epidermis, much less compact epidermal layers - means highly altered reconstructed epidermis, non-compact epidermal layers or spongy appearance of the epidermis
    It is found that the inflammatory cocktail indeed causes a significant disturbance of the overall morphology of the reconstructed epidermis. It appears altered and spongy in the control of the inflammed RE models.
    In contrast, the active ingredient obtained from Ophiopogon japonicus used topically can protect the general morphology of ER in inflammed RE models. The use of an active ingredient obtained from Ophiopogon japonicus consequently makes it possible, in topical application, to improve the general morphology of the epidermis on atopic skin. o Effect on the integrity of the epidermal barrier The study consists of evaluating the effect of an active ingredient obtained from Ophiopogon japonicus according to the invention on the integrity of the epidermal barrier on the REBAI model.
    One of the tests to visualize the integrity of the barrier function is performed using a fluorescent probe, the dye lucifer yellow. The more intense the coloring, the greater the epidermal barrier.
    After several days of RE culture, lucifer yellow are deposited on reconstructed epidermis. After incubation at 37 ° C, the epidermis are rinsed with PBS buffer and fixed with PFA. 4pm sections are then made using a microtome.
    The visualization of the integrity of the cutaneous barrier is carried out on a microscope coupled to an image analysis system. The thickness of the epidermis and the penetration of lucifer yellow were measured on histological sections.
    The active ingredient was added topically, at 0.5 and 1% in a cosmetic formula on the epidermis. The placebo formula has also been tested.
    The results are shown in Table 16 below and in Figures 2A-2C.
    Table 16
    The degradation of the integrity of the epidermal barrier is very important on the REBAI model.
    It is found that the active ingredient obtained from Ophiopogon japonicus makes it possible to protect the integrity of the epidermal barrier in the REBAI model. The use of an active ingredient obtained from Ophiopogon japonicus therefore makes it possible, in topical or systemic application, to protect the integrity of the epidermal barrier of atopic skin.
    权利要求:
    Claims (18)
    [1" id="c-fr-0001]
    1. Active ingredient obtained from Ophiopogon japonicus for use in topical application to the skin in the treatment of atopic dermatitis in humans.
    [2" id="c-fr-0002]
    2. The active principle for its use according to claim 1 on atopic skins.
    [3" id="c-fr-0003]
    3. Active principle for use according to one of the preceding claims, for reducing the relapse rate of atopic dermatitis.
    [4" id="c-fr-0004]
    4. Active ingredient for use according to one of the preceding claims for reducing the severity score of atopic dermatitis (SCORAD).
    [5" id="c-fr-0005]
    5. Active ingredient for use according to one of the preceding claims for reducing the intensity and frequency of outbreaks of atopic dermatitis.
    [6" id="c-fr-0006]
    6. Active ingredient for use according to one of the preceding claims, to reduce the importance of eczema atopic dermatitis.
    [7" id="c-fr-0007]
    7. The active principle for its use according to one of the preceding claims, for improving the quality of life index of patients with atopic dermatitis and their families.
    [8" id="c-fr-0008]
    8. Active principle for its use according to one of the preceding claims, for use in the treatment of atopic dermatitis by acting on the markers of inflammation of the skin cells, on the genes associated with the inflammation of the cells. dermal differentiation markers, the genes associated with skin cell differentiation, the morphological construction of the epidermis, and the integrity of the epidermal barrier.
    [9" id="c-fr-0009]
    9. Active ingredient for use according to one of the preceding claims, for use in the treatment of atopic dermatitis by acting: - by decreasing the content of TSLP and IL-8 in cutaneous cells, - by normalizing the expression of the NELL2 gene and of the Tenascin C gene in cutaneous cells, - by increasing filaggrin in cutaneous cells, - by stimulating the expression of the filaggrin gene and the loricrin gene in cutaneous cells, - by decreasing the expression of the TGM1 gene (transglutaminase 1) in cutaneous cells.
    [10" id="c-fr-0010]
    10. Active ingredient for use according to one of the preceding claims, characterized in that it comprises sugars.
    [11" id="c-fr-0011]
    11. Active ingredient for use according to one of the preceding claims, characterized in that it comprises at least 50% sugar by weight relative to the total weight of dry matter.
    [12" id="c-fr-0012]
    12. Active ingredient for use according to one of the preceding claims, characterized in that it comprises fructosans.
    [13" id="c-fr-0013]
    13. Active ingredient for use according to one of claims 10 to 12, characterized in that it comprises at least 57% of fructans by weight relative to the weight of total sugars of the active ingredient.
    [14" id="c-fr-0014]
    14. Active ingredient for use according to one of the preceding claims, characterized in that it is obtained from tubers of Ophiopogon japonicus.
    [15" id="c-fr-0015]
    15. Active ingredient for use according to one of the preceding claims, characterized in that it is a hydrolyzate of Ophiopogon japonicus.
    [16" id="c-fr-0016]
    16. Active ingredient for use according to one of the preceding claims, characterized in that it is an enzymatic hydrolyzate of Ophiopogon japonicus.
    [17" id="c-fr-0017]
    17. Active ingredient for use according to one of the preceding claims, in a dermatological composition of at least 0.1%.
    [18" id="c-fr-0018]
    18. Active ingredient for use according to one of the preceding claims, in a dermatological composition in the form of cream, gel or ointment.
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    EP3402502A1|2018-11-21|
    US20190060390A1|2019-02-28|
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    CN103736068A|2014-01-21|2014-04-23|杜艳华|Drug for treating vulvar pruritus|
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    法律状态:
    2017-01-27| PLFP| Fee payment|Year of fee payment: 2 |
    2017-07-14| PLSC| Publication of the preliminary search report|Effective date: 20170714 |
    2018-01-30| PLFP| Fee payment|Year of fee payment: 3 |
    2020-01-28| PLFP| Fee payment|Year of fee payment: 5 |
    2021-01-26| PLFP| Fee payment|Year of fee payment: 6 |
    2022-01-25| PLFP| Fee payment|Year of fee payment: 7 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1650228A|FR3046541B1|2016-01-12|2016-01-12|ACTIVE PRINCIPLE OBTAINED FROM OPHIOPOGON JAPONICUS FOR THE TREATMENT OF ATOPIC DERMATITIS|
    FR1650228|2016-01-12|FR1650228A| FR3046541B1|2016-01-12|2016-01-12|ACTIVE PRINCIPLE OBTAINED FROM OPHIOPOGON JAPONICUS FOR THE TREATMENT OF ATOPIC DERMATITIS|
    EP17711706.6A| EP3402502A1|2016-01-12|2017-01-12|Active ingredient obtained fromophiopogon japonicus|
    US16/069,265| US20190060390A1|2016-01-12|2017-01-12|Active ingredient obtained from ophiopogon japonicus for the treatment of atopic dermatitis|
    PCT/FR2017/050070| WO2017121965A1|2016-01-12|2017-01-12|Active ingredient obtained from ophiopogon japonicus, for the treatment of atopic dermatitis|
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