阅读说明：本技术 用于炎症性皮肤病的硫酸葡聚糖 (Dextran sulfate for inflammatory skin diseases ) 是由 M·F·阿里耶斯 S·普瓦尼 H·埃尔南德斯-皮容 于 2020-04-03 设计创作，主要内容包括：本发明涉及硫酸葡聚糖和含有硫酸葡聚糖的皮肤病学组合物或皮肤美容学组合物在治疗和/或预防炎症性皮肤病,特别是特应性皮炎中的用途。(The present invention relates to the use of dextran sulfate and of a dermatological or dermocosmetic composition containing dextran sulfate for the treatment and/or prevention of inflammatory skin diseases, in particular atopic dermatitis.)

1. Dextran sulfate, or a dermatologically or dermocosmetically acceptable salt thereof, for use in the treatment and/or prevention of inflammatory skin diseases, said dextran sulfate, or a dermatologically or dermocosmetically acceptable salt thereof, being obtained by:

fermentation of beets to obtain glucans, followed by

-sulphating dextran to obtain dextran sulphate, especially in the presence of magnesium sulphate, and

-optionally salifying to obtain a dermatologically or dermocosmetically acceptable dextran sulfate salt.

2. Dextran sulfate or its dermatologically or dermocosmetically acceptable salts for use according to claim 1, characterized in that it has a molecular weight of 2kDa to 5000 kDa.

3. Dextran sulfate or its dermatologically or dermocosmetically acceptable salts for use according to claim 1, characterized in that it has a molecular weight of 9kDa to 20 kDa.

4. Dextran sulfate, or the dermatologically or dermocosmetically acceptable salt thereof, for use according to any one of claims 1 to 3, characterized in that dextran sulfate is in the form of a sodium salt.

5. Dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof for use according to any one of claims 1 to 4, characterized in that said inflammatory skin diseases are selected from the group consisting of atopic dermatitis, eczema, psoriasis, rosacea, lichen planus, prurigo, seborrheic dermatitis and comedones.

6. Dextran sulfate, or the dermatologically or dermocosmetically acceptable salt thereof, for use according to any one of claims 1 to 4, characterized in that said inflammatory dermatological disease is atopic dermatitis.

7. Dermatological or dermocosmetic composition, for use in the treatment and/or prevention of inflammatory skin diseases, comprising, as active ingredient, at least one dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof, and at least one dermatologically or dermocosmetically acceptable excipient.

8. The dermatological or dermocosmetic composition for use according to claim 7, characterized in that dextran sulfate or the dermatologically or dermocosmetically acceptable salt thereof has a molecular weight of from 2kDa to 5000 kDa.

9. The dermatological or dermocosmetic composition for use according to claim 7, characterized in that dextran sulfate or the dermatologically or dermocosmetically acceptable salt thereof has a molecular weight of 9kDa to 20 kDa.

10. The dermatological or dermocosmetic composition for use according to any one of claims 7 to 9, characterized in that it comprises from 0.01 to 0.5% by weight, relative to the total weight of the composition, of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof.

11. The dermatological or dermocosmetic composition for use according to any one of claims 7 to 10, characterized in that the dextran sulfate is in the form of a sodium salt.

12. The dermatological or dermocosmetic composition for use according to any one of claims 7 to 11, characterized in that said inflammatory dermatosis is selected from the group consisting of atopic dermatitis, eczema, psoriasis, rosacea, lichen planus, prurigo, seborrheic dermatitis and comedones.

13. The dermatological or dermocosmetic composition for use according to any one of claims 7 to 11, characterized in that said inflammatory skin disease is atopic dermatitis.

14. The dermatological or dermocosmetic composition according to any one of claims 7 to 13, characterized in that it is in a suitable form for topical application.

Technical Field

The present invention relates to dextran sulfate or dermatologically or dermocosmetically acceptable salts thereof, as well as to dermatological or dermocosmetic compositions comprising the same, for their use in the treatment and/or prevention of inflammatory skin diseases, in particular atopic dermatitis.

Background

Skin diseases are skin and mucosal diseases characterized by unsightly clinical manifestations, such as redness and flaking plaques. Several pathologies are under the name of inflammatory skin diseases. Non-limiting examples include atopic dermatitis, eczema, psoriasis, rosacea, lichen planus, prurigo, seborrheic dermatitis, and acne. These skin diseases are usually caused by inflammatory phenomena and immune disorders.

Atopic dermatitis is a clinical manifestation of atopic skin. It is a chronic inflammatory skin disease that occurs in a genetically determined setting. It affects 15% to 30% of children and 2% to 10% of adults. Its prevalence in industrialized countries is increasing; over the past three decades, it has increased to two or even three times and is now considered a major public health problem. Atopic dermatitis is commonly associated with other atopic diseases, such as allergic rhinitis and asthma. This condition occurs most frequently in early childhood and is characterized by recurrent rashes over the course of several years. It develops in the form of an outbreak interspersed with spontaneous remission. Lesions are characterized by severe skin dryness associated with clinical manifestations of inflammation: papular, vesicular, scaly and very itchy erythematous rash. Histologically, like many other skin diseases, atopic dermatitis is characterized by infiltration of lymphocytes, monocytes, and eosinophils around small and capillary vessels; biochemically, it is characterized by the expression of cytokines, such as Thymic Stromal Lymphopoietin (TSLP), a major protein that causes atopic dermatitis-related inflammation. Furthermore, chemokines, in particular interleukin 8(IL8) and lipid mediators of inflammation, such as prostaglandin 6kF1 α (PG6kF1 α), have been shown to be closely associated with skin diseases in general, such as atopic dermatitis and chronic inflammatory diseases.

Eczema is an pruritic skin disease characterized by an inflamed skin with redness, blisters, scaling and itching. It may begin early in life; even in newborns. The affected individuals experience a period commonly referred to as an "eczematous outbreak" during which symptoms worsen. These bursts of varying duration are interspersed with periods of remission. Eczema is a genetic disease, but environmental factors (such as the presence of chemical irritants or stress) can affect its pathogenesis.

Psoriasis is a typical inflammatory disease characterized by the appearance of thick, flaky patches of skin. These plaques are present in different parts of the body, most commonly in the elbows, knees and scalp. This chronic disease progresses periodically with a remission phase. Psoriasis is very unpleasant and even painful when it occurs on the palms or soles or folds of the skin. Psoriasis is of various types, the most common form being plaque psoriasis or psoriasis vulgaris. Other forms are guttate psoriasis, erythrodermic psoriasis and pustular psoriasis.

Rosacea is a common chronic progressive inflammatory skin disease associated with vascular relaxation. It is a disease affecting facial small blood vessels. It often affects fair people and can have significant psychological and emotional consequences. The name of the disease refers to the characteristic color of the face during the period of the disease.

Lichen planus is an itchy rash that affects adults only. It is an inflammatory skin disease of unknown cause. This skin disorder affects the skin and mucous membranes as well as the hair and nails. These last two areas are usually in chronic progression and may lead to irreversible sequelae such as hair loss and nail destruction.

Prurigo is a severe itching of the skin with erythema and vesicular papules with signs of scratching flexibility. This is often an excess sensitivity to insect bites, which can last for an unusually long period of time, especially in young children.

Seborrheic dermatitis is a chronic inflammatory skin disease that affects areas rich in sebaceous glands, i.e., the scalp and face. This is due to the yeast malassezia that is present on the skin and grows in sebum. This condition develops in outbreaks due to stress, lack of sunlight and pollution.

Acne is a common skin disorder caused by inflammation of the pilosebaceous glands, mainly due to colonization by propionibacterium acnes (Cutibacterium acnes) in the infundibulum (dreno et al, JEADV 2018,32 (suppl 2) 5-14).

In the case of mild inflammatory skin diseases, emollients and keratolytics are suggested. The purpose of these treatments is to allow the patient to tolerate the lesions, but they usually only slow down the progression. For more severe conditions, anti-inflammatory drugs or corticosteroids that modulate skin inflammation have been used for many years. All of these treatments have serious side effects and sometimes place a significant burden on the patient. Due to the above-mentioned major side effects of the existing treatments of skin or scalp diseases caused by the activation state of the innate immunity of the skin and inflammatory epidermal reactions, there is a real need for new cosmetic active ingredients and new cosmetic compositions that can be used instead of or together with the treatment of said skin or scalp pathologies.

Disclosure of Invention

The present invention is directed to responding to these needs. Indeed, completely unexpected, the inventors have demonstrated that dextran sulfate has an interesting pharmacological activity for the treatment and prevention of inflammatory skin diseases, in particular atopic dermatitis.

Accordingly, a first object of the present invention relates to dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof for use in the treatment and/or prevention of inflammatory skin diseases.

Another object of the present invention relates to the use of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof for the preparation of a dermatological or dermocosmetic composition intended for the treatment and/or prevention of inflammatory skin diseases.

Another object of the invention relates to the use of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof for the treatment and/or prevention of inflammatory skin diseases.

Another object of the invention relates to a method for the treatment and/or prevention of inflammatory skin diseases, comprising administering to a human in need thereof an effective amount of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof.

In the context of the present invention, dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof is advantageously obtainable or obtained by:

fermenting sugar beets, in particular beet sugar, to obtain glucans, and then

-sulphating dextran to obtain dextran sulphate, especially in the presence of magnesium sulphate, and

-optionally salifying to obtain a dermatologically or dermocosmetically acceptable dextran sulfate salt, more particularly dextran sulfate sodium salt.

The dextran sulphate thus obtained or a dermatologically or dermocosmetically acceptable salt thereof advantageously has an average molecular weight of between 9kDa and 20 kDa.

Definition of

Within the meaning of the present invention, "prevention" refers to preventing the onset of a disease or disorder or one or more signs and/or symptoms.

The term "treatment" or "treating" inflammatory skin disease refers to reducing and/or inhibiting the development of an inflammatory skin disease and/or at least one symptom thereof.

In the present invention, "dermatologically or dermatologically acceptable" means useful in the preparation of dermatological or dermocosmetic compositions, which are generally safe, non-toxic, biologically or otherwise undesirable, and acceptable for dermatological or dermocosmetic use, particularly topical applications.

In the present invention, a "dermatologically or dermocosmetically acceptable salt" of dextran sulfate refers to a dermatologically or dermocosmetically acceptable base addition salt formed by the sulfate functional group (-OSO3H) of dextran sulfate, the acid proton of which is replaced by a metal ion, such as an alkali metal ion (e.g., Na or K), an alkaline earth ion (e.g., Mg or Ca), or an aluminum ion; or coordinating with pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, etc.; or with pharmaceutically acceptable inorganic base such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, etc. Advantageously, it is a sodium or potassium salt, preferably the sodium salt.

By "topical application" is meant application to the skin, mucosa and/or appendages.

Detailed Description

Dextran is a polysaccharide, more specifically a neutral polysaccharide without charged groups. It is a branched glucose polymer (dextrose). Advantageously, the polymer will comprise a backbone having alpha-1, 6 glycosidic linkages between the glucose monomers and branches formed by alpha-1, 2, alpha-1, 3 and/or alpha-1, 4 glycosidic linkages.

The glucan can be prepared by beet fermentation. It can be obtained from natural glucan by hydrolysis and purification of the glucan fractions of different molecular weights. It can also be prepared synthetically.

The dextran may be sulfated, especially in the presence of magnesium sulfate, to yield dextran sulfate.

Thus, dextran sulfate is a dextran whose at least part of the hydroxyl groups have been replaced by sulfate groups.

Dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof is advantageously prepared by:

fermenting sugar beets, in particular beet sugar, to obtain glucans, and then

-sulphating dextran to obtain dextran sulphate, especially in the presence of magnesium sulphate, and

-optionally salifying to obtain a dermatologically or dermocosmetically acceptable dextran sulfate salt, more particularly dextran sulfate sodium salt.

More particularly, dextran sulfate is in the form of a sodium salt and can advantageously be obtained or obtained by:

fermenting sugar beets, in particular beet sugar, to obtain glucans, and then

Sulfating dextran to dextran sulfate, especially by dissolution in acidified water, especially by formic acid, especially in the presence of magnesium sulfate,

salification to obtain the sodium dextran sulfate salt,

-purification, in particular by one or more of precipitation by dissolution in water and in ethanol, and

recovery of dextran sulfate sodium salt, in particular by centrifugation, drying (e.g. under vacuum) and grinding.

The physicochemical properties of dextran sulphate known in the prior art make it a good compound for cosmetic compositions, having good solubility in water and saline solutions and high stability in solutions with pH ranging from 4 to 10 at ambient temperature. Dextran sulfate is also described as having water absorbing properties, a protective effect against damage caused by free radicals (especially by topical application), a stabilizing effect of proteins or labile substances and hydration due to its excellent hydrophilicity. Biological properties, such as anticoagulation, inhibition of enzymes such as hyaluronidase, glucosidase, elastase or thrombin, or antiviral activity are also described.

Dextran sulfate may be synthetic or natural. It is to be understood that the dextran sulfate may be from any source.

Preferably, according to the invention, the dextran sulphate is present in the form of a sodium salt. The INCI name is dextran sulfate sodium, and the CAS number is 9011-18-1.

According to the invention, the dextran sulfate or dermatologically or dermocosmetically acceptable salt thereof advantageously has an average molecular weight of between 2kDa and 5000kDa, preferably between 4kDa and 1000kDa, more preferably between 5kDa and 100kDa, even more preferably between 9kDa and 20kDa, just as preferably the dextran sulfate or dermatologically or dermocosmetically acceptable salt thereof has a molecular weight of between 4kDa and 8 kDa.

Preferably, the dextran sulfate according to the present invention is provided by the company SAFIC ALCAN under the name Dextralip 10C and is in the form of a sodium salt.

Dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof can be used for the treatment and/or prevention of inflammatory skin diseases.

Preferably, the inflammatory skin disease is selected from the group consisting of atopic dermatitis, eczema, psoriasis, rosacea, lichen planus, prurigo, seborrheic dermatitis, and acne. Preferably, it is atopic dermatitis.

Another object of the present invention relates to a dermatological or dermocosmetic composition, for its use for the treatment and/or prevention of inflammatory skin diseases, comprising, as active ingredient, at least one dextran sulfate as defined above, or a dermatologically or dermocosmetically acceptable salt thereof, and at least one dermatologically or dermocosmetically acceptable excipient.

Another object of the present invention relates to the use of a dermatological or dermocosmetic composition comprising, as active ingredient, at least one dextran sulfate as defined above, or a dermatologically or dermocosmetically acceptable salt thereof, and at least one dermatologically or dermocosmetically acceptable excipient, for the treatment and/or prevention of inflammatory skin diseases.

Another object of the present invention relates to the use of a dermatological or dermocosmetic composition comprising, as active ingredient, at least one dextran sulfate as defined above, or a dermatologically or dermocosmetically acceptable salt thereof, and at least one dermatologically or dermocosmetically acceptable excipient, for the preparation of a medicament intended for the treatment and/or prevention of inflammatory skin diseases.

Another object of the present invention relates to a method for treating and/or preventing inflammatory skin diseases, comprising administering to a person in need thereof an effective amount of a dermatological or dermocosmetic composition comprising, as active ingredient, at least one dextran sulfate as defined above, or a dermatologically or dermocosmetically acceptable salt thereof, and at least one dermatologically or dermocosmetically acceptable excipient.

In a particular embodiment, the composition according to the invention is used for the treatment and/or prevention of an inflammatory skin disease selected from the group consisting of atopic dermatitis, eczema, psoriasis, rosacea, lichen planus, prurigo, seborrheic dermatitis and comedones.

Preferably, the composition according to the invention is for use in the treatment and/or prevention of atopic dermatitis.

In a particular embodiment, the dermatological or dermocosmetic composition according to the invention comprises from 0.01% to 1%, preferably from 0.01% to 0.5%, even more preferably from 0.02% to 0.3% by weight of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof, relative to the total weight of the composition. Advantageously, the dermatological or dermocosmetic composition according to the invention comprises 0.03% by dry weight of dextran sulfate or a dermatologically or dermocosmetically acceptable salt thereof, relative to the total weight of the composition.

The compositions according to the invention are advantageously intended for topical application, in particular on the skin.

The compositions according to the invention may therefore be presented in the known forms for topical application, i.e. in particular lotions, mousses, gels, dispersions, lotions, sprays, essences, balms (baumes), masks or creams.

Advantageously, it will be a cream or balm.

The invention also relates to dermatological or dermocosmetic compositions according to one of the embodiments of the invention, characterized in that they are present in a suitable form for topical application.

In addition to the sodium sulphate according to the invention, these compounds generally comprise a physiologically acceptable medium, generally based on water or solvents, such as alcohols, ethers or glycols. They may also contain surfactants, complexing agents, preservatives, stabilizers, emulsifiers, thickeners, gelling agents, humectants (humectants), emollients, trace elements, essential oils, fragrances, dyes, matte agents, chemical or mineral filters, humectants (agents), thermal spring water, or the like.

The following examples illustrate the invention without limiting its scope.

Examples

Example 1 Effect of dextran sulfate on prostaglandin PG6K in inflammation

Keratinocytes are the most common cells in the epidermis. In response to several extracellular factors present in the environment, the epidermis releases various bioactivating mediators, in particular bioactive lipids, prostaglandins and leukotrienes, which play an important role in the initiation and regulation of inflammatory skin reactions and are also involved in the regulation of immune responses.

Keratinocytes appear to be a good model for skin pharmacology studies. This cell model allows the determination in vitro of the ability of various compounds to modulate the production of these mediators produced by arachidonic acid metabolism.

In this study, the present inventors have specifically studied a prostaglandin, 6-keto-PGF 1 α (PG6KF1 α), which is a stable metabolite of prostacyclin. This metabolite is one of the major metabolites produced by stimulated keratinocytes and represents a regulation of the production of arachidonic acid metabolites (Dorris and Stokes Peebles, Mediators of inflammation, vol 2012, article ID 926968, page 9).

The objective of this study was to find potential anti-inflammatory activity by measuring the effect of dextran sulfate on prostaglandin PG6KF1 α release.

The cell line used in this study was the HaCat line (human keratinocytes).

Cells were cultured in DMEM (Dulbecco modified eagle's medium) supplemented with fetal bovine serum in 24-well plates at 37 ℃ in an environment of 5% CO 2. They were then preincubated for 60 minutes, still at 37 ℃, with the test product dissolved in water. The arachidonic acid pathway was stimulated for 5 hours. This is the stimulation phase; the stimulator was calcium ionophore A23187 (0.01% DMSO solution) used at 5. mu.M. In this study, two different dextran sulfates were tested, one being native dextran sulfate, from SAFIC ALCANMolecular weight 9000-20000Da, and from Welding GMBH&Synthesis of Co dextran sulfateSugar, molecular weight 4000-8000 Da. After 5 hours of stimulation, the culture supernatant of each well was aspirated, centrifuged at 3000 rpm, and then stored at-20 ℃, still in the same medium. Prostaglandin PG6KF1 α production in the culture supernatants was measured using the Euromedex Elisa kit according to the supplier's instructions. Statistical analysis was performed by ANOVA followed by Dunnett's post-test. Note that 3 independent experiments were performed. The results for each product and each dose are the average of the measurements made on 3 wells. Thus, a control group (without a stimulating agent) can quantify the production of basal PG6KF1 α. All other groups had a stimulation period, or no other product (the maximum production of PG6KF1 α was known), or in the presence of indomethacin (non-steroidal anti-inflammatory drug) used as reference product, or in the presence of dextran sulphate at different concentrations.

The results of PG6KF1 α activation (in PG/ml) are summarized in tables 1A and 1B below:

table 1A: effect of native dextran sulfate (Dextralip) having a molecular weight of 9000-20000Da on the production of PG6KF1 alpha

[ Table 1A ]

Conc: concentration; moy: average value; SEM: standard error of the mean; inh: inhibiting; indo: indomethacin.

Table 1B: effect of synthetic dextran sulfate having molecular weight of 4000 to 8000Da on production of PG6KF1 alpha

[ Table 1B ]

Conc: concentration; moy: average value; SEM: standard error of the mean; inh: inhibiting;

and NS: not significant.

Stimulation with calcium ionophore a23187 greatly stimulated the production of PG6KF1 α. Indomethacin is a nonsteroidal anti-inflammatory drug that inhibits this production by a factor of three, confirming the relevance of this test. The two dextran sulfates tested from 10 μ g/ml to 3mg/ml showed statistically significant inhibition of PG6KF1 α release. This generation in response to an increase in the concentration of synthetic dextran sulfate resembles a bell-shaped curve. Also, the concentration response effect cannot be clearly shown using native dextran sulfate.

The present inventors have demonstrated the efficacy of dextran sulfate in inflammation.

Example 2 Effect of dextran sulfate on NFKB Activity in inflammation

The aim of this study was to evaluate the potential soothing properties of dextran sulfate at the level of activation of the transcription factor NFKB stimulated by TNF α in human keratinocytes. The cell line used in this study was a HaCat line (human keratinocytes) stably transfected with a luciferase reporter gene.

These cells were cultured in 24-well plates under the same conditions as in example 1. They are then pre-incubated for 60 minutes with the product to be tested, which is added to the culture medium in the form of an aqueous solution. The stimulus TNF α (0.3ng/ml, diluted in medium) was added to the culture, followed by incubation at 37 ℃ for 5 hours. In this study, native dextran sulfate, from SAFIC ALCAN, was testedThe molecular weight is 9000-20000 Da. In this test, a positive control, dexamethasone (a synthetic glucocorticoid with anti-inflammatory and immunosuppressive effects, added to the culture medium in the form of an aqueous solution) was used, and the test was performed at 2 μ M in the culture medium. Note that 3 independent experiments were performed. Bright-GloTM reagent (which induces cell lysis and thus luciferase release) and its substrate (luciferin) were added prior to reading luminescence. Raw data were analyzed by Excel. The comparisons between groups were performed by one-way ANOVA,followed by Dunnett's post-test.

The results of NFKB activation (RLU) are summarized in table 2 below:

[ Table 2]

RLU relative light units; cont: comparison; conc: concentration; moy: average value; sem: standard error of the mean; inh: inhibiting; stim: stimulation of TNF α; dexa: dexamethasone; S.Dex: dextran sulfate; and NS: not significant.

TNF α stimulation (0.3ng/ml) did induce NFKB activation. Dexamethasone used as immunosuppressant inhibited this activation by more than 40% (p <0.05vs TNF α free), which validated the reliability of the test.

Dextran sulfate tested at 0.3 and 1mg/ml did not reduce NFKB activation. In turn, at a concentration of 3mg/ml, dextran sulfate significantly (p <0.01) reduced TNF α -induced activation of NFKB by 42%.

The inventors have thus demonstrated that dextran sulfate has soothing properties.

Example 3 Effect of dextran sulfate on skin inflammation (model of atopic dermatitis)

Atopic dermatitis or atopic eczema is a chronic inflammatory disease, progresses periodically, and has a remission stage. Atopic dermatitis lesions are particularly due to the activation of allergen-specific T cells. This immune response may be due to penetration of environmental allergens into the skin. Disruption of the skin barrier and thus dispersion of the allergen is associated with the induction of specific immune responses and eczematous lesions. Two complementary hypotheses have been proposed to explain the origin of the disease. The first hypothesis is that impairment of skin barrier function will allow allergens to enter and induce immune sensitization. The second hypothesis was that atopy is an immune system dysfunction, resulting in a beneficial imbalance of Th1/Th2 for Th2 and the production of allergen-specific IgE. For all of these reasons, atopic dermatitis is considered to be a complex disease involving multiple mechanisms.

In the present study, the effect of dextran sulfate according to the present invention on stimulated human keratinocytes was evaluated in a model simulating an atopic dermatitis environment.

Method

The study was performed on normal human epidermal keratinocytes cultured under standard conditions (37 ℃, 5% CO 2). The medium was standard medium (keratinocytes-SFM supplemented with 0.25ng/ml Epidermal Growth Factor (EGF), 25. mu.g/ml pituitary extract and 25. mu.g/ml gentamicin). The test medium without growth factors was identical.

To simulate an atopic dermatitis environment, keratinocytes were preincubated for 1 hour in medium with or without (control) test compound, vehicle (water) or positive control bafilomycin (macrolide family, concentration 30 nM). The dextran sulfate tested in this study was from SAFIC ALCANThe molecular weight is 9000-20000 Da. After pre-culture, keratinocytes were stimulated for 24 hours by a mixture of TLR ligands (Poly I: C and PamC3) and inflammatory cytokines (IL-4 and IL-13) added to the cells. Unstimulated control conditions were also run simultaneously. The cells were cultured for 24 hours.

The culture supernatant was collected, centrifuged and frozen at-80 ℃. TSLP and IL-8 were quantified by ELISA.

Three independent experiments were performed. Statistical analysis was determined by one-way ANOVA followed by Dunnett's post-test.

Results

TSLP produced by normal human epidermal keratinocytes measured at 24 hours is shown in table 3 below.

[ Table 3]

Moy: average value; sem: standard error of the mean; inh: inhibiting; p < 0.05; p <0.01vs stimulation.

Stimulation of keratinocytes by mixed reagents (Poly I: C, PamC3, IL-4 and IL-13) to mimic the atopic dermatitis environment did induce increased TSLP production at 24 hours. Bafilomycin used as a positive control significantly inhibited the production of TSLP. These results fully validate the pharmacological test.

The dextran sulfate according to the present invention almost completely inhibited TSLP production measured at 24 hours. In fact, TSLP production decreased by 90% at 3. mu.g/ml dextran sulfate and by 96% at 30. mu.g/ml (p <0.01 for both concentrations, compared to the stimulation conditions in Table 3). Conversely, a 10-fold lower dextran sulfate concentration does not induce a reduction in TSLP production. The inhibitory effect of dextran sulfate on TSLP production induced by the atopic dermatitis environment appears to be clearly concentration dependent.

Interleukin 8(IL-8) produced by normal human epidermal keratinocytes measured at 24 hours is shown in Table 4 below.

[ Table 4]

Moy: average value; sem: standard error of the mean; inh: inhibiting; p <0.01vs stimulation.

Stimulation of keratinocytes by the reagent mixture to mimic the atopic dermatitis environment induces a large production of IL-8. Bafilomycin (30nM) significantly inhibited IL-8 production (by more than 50%). All these results demonstrate pharmacological testing.

The dextran sulfate according to the present invention reduces IL-8 production induced by an atopic dermatitis environment in a concentration-dependent manner. The lowest dextran sulfate concentration tested was insufficient to inhibit IL-8 production (Table 2). In turn, at 3 μ g/ml, dextran sulfate significantly reduced IL-8 production by more than 80% (p <0.01 compared to stimulated conditions). At 30 μ g/ml, dextran sulfate completely inhibited IL-8 production (100% inhibition), indicating that dextran sulfate according to the present invention is very effective for reducing these selective cytokines in atopic dermatitis.

Conclusion of these studies

Thus, the present inventors have shown that normal human epidermal keratinocytes produce large amounts of TSLP and IL-8 after 24 hours of stimulation by mixed reagents (Poly I: C, PamC3, IL-4 and IL-13) that mimic the environment of atopic dermatitis. In this model, the inventors demonstrated that dextran sulphate according to the invention is very effective and able to prevent this release of inflammatory cytokines (TSLP, in particular, a key marker in the development of atopic dermatitis), demonstrating a protective role in this disease.

Example 4 evaluation of dextran sulfate in the Regulation of genes involved in keratinocyte differentiation and hydration Estimation of

The epidermis serves as a mechanical and chemical barrier for the body and plays an important protective role. Which ensures that an impermeable skin barrier function is maintained. Keratinocytes, the keratinocytes of the stratum corneum, together with the lipid matrix, largely ensure this function. However, deeper layers also help to build elements inherent in this function. The differentiation capacity of epidermal keratinocytes ensures the construction of a barrier with a selectively permeable function (Elias and Choi, Exp. Dermatol.14(10), p 719-726, 2005)).

The differentiation of keratinocytes is regulated spatially and temporally, from the deepest layers of the Skin (basement membrane with the least differentiation) to the stratum corneum (the last step in the differentiation of keratinocytes into keratinocytes) (Houben et al, Skin Pharmacol. physiol.20(3), p 122-132, 2007). From a cellular and molecular point of view, the formation of keratin filaments, the transformation of keratinocytes into keratinocytes, or "keratinization", and the formation of intercellular lipid junctions (moment) of the lamellar structure are mainly observed, ensuring the impermeability and function of the skin barrier.

In terms of proteins, epidermal differentiation is mainly focused on the development of structural cytoplasmic proteins, such as cytokeratins, which contribute to the structural integrity of the epidermis. Their expression varies according to the degree of maturation of keratinocytes. Basic keratin 1 and acidic keratin 10 are early markers of terminal differentiation of keratinocytes and are present in the basal layer of the epidermis. In this biological process which occurs subsequently, expression of other markers can proceed in the same manner as in the keratinocyte membranes such as involucrin, along with certain major enzymes (leading to structural proteins forming bridges between them), and keratinocyte lipids and Transglutaminase (TGM), such as TGM1 or 3(Houben et al, Skin pharmacol. physiol.20(3), p 122-.

The fibrous matrix present in keratinocytes is formed in the stratum granulosum during the transition phase between keratinocytes and keratinocytes. Loricrin is a structural protein containing glutamine and lysine residues that can adhere to other proteins in the cuticle coat. Basic filaggrin molecules produced from their precursor filaggrin precursors are stored in the hyaline keratocytes, bind to the cytokeratin filaments, and are then able to aggregate. The silk fibroin, which is degraded by caspase 14, is also a major source of several major components of the natural hydration factors in the stratum corneum. Other markers are specific for differentiated keratinocytes. claudine 4(CLDN4) is a tight junction protein. It is expressed mainly in the stratum granulosum and is increased during keratinocyte differentiation. Corneal Desmoplanin (CDSN) is expressed in keratinocytes. It is an essential protein of the keratinized granule, and its proteolysis is essential for desquamation. Kallikreins, such as KLK5 and KLK7, exhibit activity similar to chymotrypsin and play a role in proteolysis of the pre-desquamation intercellular cohesive structure, i.e. removal of the outermost layer of the stratum corneum.

At the same time, keratinocyte lipid synthesis and transport form the basis for the intercellular lipid junctions of keratinocytes, which are essential for the skin barrier, and are the final stages of terminal epidermal differentiation. This extracellular lipid matrix is the main barrier for transdermal transport of liquids and electrolytes (Feingold, j. lipid res.48, p 2531-. Thus, keratinocyte expression of a certain number of enzymes and lipid transporters is upregulated along with differentiation. The linker results from a balance between the three lipid substances, cholesterol, free fatty acids and ceramides. These lipids are derived from glucosylceramides, sphingomyelin, cholesterol and phospholipids produced in the spinous and granular layers. They are transported by the lamina, a secretory organelle, fused to the stratum granulosum and stratum corneum. In addition to these lipid precursors, the lamina contains a number of enzymes, including lipases, such as acid sphingomyelinase, β -glucocerebrosidase or phospholipase a2, as well as acid and neutral lipases. These enzymes are delivered to the extracellular space simultaneously with the lipid precursors, converting sphingomyelin to ceramide, glucocerebroside to ceramide, and phospholipids to free fatty acids and glycerol, respectively. SULT2B1 is a sulfotransferase cholesterol expressed in differentiated keratinocytes and is involved in the synthesis of cholesterol sulfate. A recent study also showed that cholesterol sulfate induces expression of filaggrin by increasing ROR α expression (Hanyu et al, biochem. Biophys. Res. Commun.428(1), p 99-104,2012).

Epidermal ceramides play a particular and important role and are important markers of skin barrier function. The expression and activity of enzymes that play a role in skin ceramide production are particularly increased when barrier function is altered in conjunction with changes in the degree of epidermal differentiation (Feingold, 2007). This is a special case of acid sphingomyelinase (aSmase) and β -glucose ceramidase, which are involved in the extracellular metabolism of skin ceramides. UGCG (UDP-glucose ceramide glucosyltransferase) is also involved in the synthesis of glucose ceramides. UGCG catalyzes the first glycosylation step in glycosphingolipid biosynthesis and is essential for the regular arrangement of lipids and proteins in the lamina and for maintaining the epidermal barrier (Jennemann et al, j. biol. chem.282(5), p 3083-. DGS2 (sphingolipid C4-hydroxylase/delta-4 desaturase) acts as both a sphingolipid C4-hydroxylase and a delta-4 desaturase; its dihydroceramide hydroxylase activity competes with the production of human skin phytoceramides.

The protein FABP-E (FABP5) that binds to epidermal fatty acids is a lipid transporter. FABP-E plays an important role in keratinocyte differentiation.

One of the functions of water in the stratum corneum is to activate the enzymatic hydrolysis reactions necessary for skin softness and normal desquamation (Rawlings and Matts J. invest. Dermatol.124(6), p 1099-1110, 2005). If the water content in the stratum corneum is below a critical level, the enzymatic reaction is disrupted, leading to adhesion of keratinocytes and accumulation of cells on the skin surface. This can cause significant dryness and itching, and the skin can flake off and exfoliate.

The barrier function of the skin also includes resistance to microorganisms. The epithelium plays a positive role in the innate defenses of the host. The skin antimicrobial system relies inter alia on the presence of certain surface lipids and certain constituent proteins which are increasingly expressed depending on the differentiation state of keratinocytes, such as RNAse7 or protease inhibitor 3. These proteins have antimicrobial activity. Innate immunity also has an adaptive component that relies on inducible secretion of antimicrobial peptides with direct antimicrobial activity. They play an important role as inflammatory mediators by affecting epithelial and inflammatory cells. Antimicrobial peptides are typically synthesized on top of the stratum spinosum and stratum granulosum layers, but they are active in the stratum corneum where they are released. The most studied skin antimicrobial peptides are the β -defensins and cathelicidins. Human β -defensins are the major antimicrobial peptides found in human epithelium, four of which have been identified in the skin, hBD 1-4. Although they belong to the same family, they are regulated by different pathways. Human β -defensin 2(hBD-2 or DEFB4A), a 4kDa peptide that binds heparin, is one of the major skin antimicrobial peptides.

Skin moisture content depends on two points, percutaneous moisturization of skin blood and epidermal retention, which are related to barrier function. However, the barrier to water loss is not a trivial matter. The normal exchange of water between the external and internal environment through the stratum corneum is known as trans-epidermal water loss (TEWL) and is inherent to the sensorless water loss (IWL).

Normal human keratinocytes were cultured for 48 h with dextran sulfate (2mg/ml) dissolved in the culture mediumThen (c) is performed. The dextran sulfate tested in this study was from Welding GMBH&Co; the molecular weight thereof is 4000-8000 Da. The effect of this compound was assessed by RT-qPCR techniques, analyzing 12 target genes selected for their importance in keratinocyte differentiation, antimicrobial defense and hydration. Calcium chloride (tested at 1.5 mM) was used as a reference compound (physiological agent inducing terminal epidermal differentiation). Cells were then harvested for analysis of target expression (mRNA) by real-time PCR. TriPure Isolation was used according to the supplier's instructions (Roche Life Science, Meylan, France)Extracting total RNA. They were analyzed using a Bioanalyzer 2100(Agilent Technologies, Les Ulis, France). mRNA is reverse transcribed into complementary DNA using oligo (dT) and Transcriptor reverse transcriptase. According to the instructions of the supplier (Roche)PCR was performed in the system. The PCR mixture used was SYBR green I. Results were obtained using Microsoft WindowsAnd (6) analyzing. The relative amount of each gene was calculated by normalization with two reference genes, RPL13A (ribosomal protein L13A) and TBP (TATA box binding protein).

Results

Since the expression of certain markers in control cells is very weak, the relative amounts may be very high and variable depending on the experiment.

The inventors showed that after 48 hours of culture, dextran sulfate tested at 2mg/ml induced significantly different keratinocyte lipid differentiation markers (induction of SULT2B1, FABP5, DGS2), multiple keratinocyte protein differentiation markers (induction of KLK7, FLG, TGM1, CASP14 and CLDN4), induction of antimicrobial peptide hBD2 (or DEFB4) and finally induction of hydration markers (FLG and CASP 14). All these results are shown in table 5 below.

[ Table 5]

Moy: average value; QR: relative amounts; sem: standard error of the mean; NV: is not verified; SULT2B 1: cholesterol sulfotransferase; FABP 5: epidermal fatty acid binding protein; DEGS 2: sphingolipid C4-hydroxylase/delta-4 desaturase; KLK 7: kallikrein type 7; FLG: a silk fibroin; CDSN: corneal catenin; TGM 1: transglutaminase 1; CASP 14: caspase 14; CLDN 4: (ii) claudine 4; DEFB 4A: beta-defensin 2.

The inventors thus show that dextran sulfate acts effectively by activating keratinocyte differentiation and by inducing expression of antimicrobial peptides. This dual role makes it possible to conclude that: dextran sulfate can significantly restore the skin barrier, enhance the skin's microbial defenses and prevent skin dehydration. The present inventors demonstrate that dextran sulfate is useful in the treatment and/or prevention of inflammatory skin diseases.