HK1193039A - Peptidyl arginine deiminase 1 and/or 3 activator compounds in the epidermis and uses thereof - Google Patents

Description

peptidyl arginine deiminase 1 and/or 3 activator compounds in the epidermis and uses thereof

Technical Field

The present invention relates to the field of molecules capable of activating peptidyl-arginine deiminase type I (or PAD 1) and/or peptidyl-arginine deiminase type III (or PAD 3), said molecules comprising theophylline acetate (alfylline) or a salt thereof; and to all the uses of said molecules in the cosmetic and therapeutic fields.

More particularly, the invention relates to the cosmetic use of theophylline acetic acid or a salt thereof as a skin moisturizer.

The invention further relates to the use of a dermatological composition comprising theophylline acetic acid or a salt thereof for treating dry skin suffering from skin diseases such as xerosis, ichthyosis, psoriasis, allergic dermatitis, hyperkeratosis and bullous ichthyosiform erythroderma.

Background

The skin consists of three compartments, the subcutaneous tissue (which is the deepest layer), the dermis and the epidermis. The epidermal layer is a keratinized malpighian (malpighian) epithelium, which protects the body from mechanical, chemical and biological damage, prevents moisture loss by limiting evaporation of moisture contained in the skin, and participates in photoprotection by absorbing a portion of ultraviolet rays. These vital functions are collectively referred to as "epidermal barrier function".

The epidermis is mainly composed of keratinocytes. These keratinocytes proliferate in the basal layer and then undergo a vector differentiation procedure to successively constitute the spinous layer, followed by the granular layer. Finally, during keratinization, they die and are transformed into keratinocytes. The accumulation of keratinocytes forms the outermost cellular layer of the epidermis, which is called the stratum corneum (cornue) or stratum corneum (stratum cornum). The stratum corneum is primarily responsible for epidermal barrier function due to its mechanical resistance, watertightness and its content of antimicrobial peptides and urocanic acid.

Keratinocytes lack nuclei and other organelles. They consist of a fibrous matrix that mainly contains keratin and filaggrin and is surrounded by a digestive protein shell (cornified envelope) that replaces the plasma membrane. Keratinocytes are connected to one another by means of a novel connecting structure, a keratinocyte-cell bridge (cornneodesmosomes). In the desquamation process, a process very finely controlled by many proteases and protease inhibitors, the most superficial keratinocytes detach from the skin after proteolysis of the intercellular bridges between the keratinocytes.

Physiologically, the stratum corneum contains 10% to 15% water. This moisturization (hydration) is essential for epidermal barrier and desquamation and must be maintained regardless of external moisture conditions. In fact, it is capable of making active a number of enzymes both inside (proteases, transglutaminase, PAD, etc.) and outside (proteases, lipases, glycosidases, etc.) the keratinocytes. It also has a plasticizing effect which enables the stratum corneum to retain its elasticity and its integrity after mechanical stress.

The decrease in the moisturizing effect of the stratum corneum, which is characterized by "dry" skin or xerosis, is manifested by a tight feel, an unpleasant tactile sensation, a scaly appearance, and a persistent surface fissures and a scaly appearance. On a molecular level, it induces a) a reduction in the degradation of desmosomal components (corneal desmin, desmoglein, and plaque proteins) and retention of intercellular bridges across the surface of keratinocytes and throughout the top layer of the epidermal stratum corneum, leading to hyperkeratosis and b) a change in maturation of intercellular lipids and corneal envelopes.

Xerosis can occur in any anatomical area and in case of high variations, for example under certain climatic conditions (cold, wind, dry, rapid and repeated passage from a cold and dry air-conditioning building to a hot and humid environment), under the action of psychological stress and chemical factors (alcohol, organic solvents, detergents, etc.; repeated washing, for example with certain soaps) or physical factors (ultraviolet rays). It can also occur in newborns (which are due to sudden passage from amniotic fluid in an aqueous medium into the air) and in the elderly (due to seasonal changes). But it can also be induced by exposure to sunlight.

Many skin diseases also cause interference of the skin barrier and dryness of the stratum corneum: ichthyosis, especially ichthyosis vulgaris (OMIM 146700), allergic dermatitis (OMIM 605803), and psoriasis (OMIM 177900).

Natural moisturizing Factors (FNHs) ensure the moisturizing effect of the stratum corneum, which enable the outermost layer of keratinocytes to retain moisture by acting against the dryness of the environment. The composition of FNH (which can represent up to 20% of the dry weight of the stratum corneum) is as follows:

free amino acids and derivatives 52.0

(containing pyrrolidone carboxylic acid 12.0)

Lactate 12.0

Sugar, organic acid, peptide 8.5

Urea 7.0

Chloride 6.0

Sodium 5.0

Potassium 4.0

Calcium, magnesium, phosphate 3.5

Ammonia, uric acid and glucosamine 1.5

Citrate and formate 0.5

More than 50% of these components correspond to free amino acids and certain derivatives thereof. In particular, pyrrolidone carboxylic acid (a spontaneous derivative of glutamine capable of representing up to 12% FNH) exerts a major hygroscopic effect. Uric acid (formed from histidine by the histidine enzyme) absorbs a portion of the uv-B radiation.

All these amino acids result directly from the degradation of filaggrin, a basic protein of 37kDa size, synthesized by granular keratinocytes in the form of larger (400 kDa) precursors (filaggrin). The essential components of such a transparent keratin particle are formed by repeating 10 to 12 silk fibroin subunits (depending on individual differences), each having a length of 327 amino acids and bound together by a 7-amino acid binding peptide. During the keratinization process, the profilaggrin is cleaved into basic filaggrin subunits. These filaggrin subunits synthesize the intermediate fibers of keratin (K1 and K10) and promote their aggregation and the formation of a fibrous matrix within keratinocytes. Then, in the stratum corneum, the filaggrin is deiminated, which causes it to dissociate from the intermediate fibers. It can then be completely degraded by calpain I, caspase 14 and bleomycin hydrolase, thereby producing the amino acid constituent of FNH. Deimination of the filaggrin is therefore a necessary, even limiting step in maintaining the moisturizing effect of the stratum corneum and the epidermal barrier function.

Deimination (or citrullination) is a post-translational modification catalyzed by the calcium-dependent enzyme family, PAD (e.c. 3.5.3.15). It corresponds to the conversion of an arginyl residue (positively charged) to a citrullinyl residue (charge neutral). There are five isoforms of PAD, encoded by five distinct genes (called PADI), that repopulate positions 1p 35-36 on the short arm of human chromosome 1. These are PAD1, PAD2, PAD3, PAD4 and PAD 6. However, PAD2 is ubiquitous, with other isoforms being expressed in a more restricted manner, depending on the tissue being analyzed. In particular, only PAD1, PAD2 and PAD3 were detected in normal human epidermis. Based on biochemical and physicochemical parameters, and because they are co-localized with filaggrin in the fibrous matrix within keratinocytes, PAD1 and PAD3 have been demonstrated to be the isoforms responsible for the deimination of filaggrin. Thus, if one wishes to target the production of amino acids by FNH, one must target these isoforms.

In addition, according to the adaptation mechanism, the production of FNH is regulated by catabolism of filaggrin according to the external humidity ratio. For example, in the last few days of embryonic development in rats, filaggrin accumulates in the entire height of the stratum corneum. A few hours after birth, it is proteolyzed in the outer part according to the same pattern as observed in adult skin. Maintaining neonatal rats in an environment with a relative humidity above 80% prevents activation of this degradation process without initiating protein synthesis. Similarly, when hairless mice were transferred from a moist environment to a normal environment, the ratio of free amino acids and the conductance of the skin surface (reflection of the moisturizing effect) were restored within 3 days.

For the purpose of reducing skin dryness, improving the moisturizing effect of the stratum corneum and treating xerosis or ichthyosis, cosmetic or pharmaceutical preparations containing moisture-absorbing activators are used for external application on the skin. These agents contain, for example, urea or lactic acid (other components of FNH). Generally, hydroxy acids have become one of the largest classes of compounds used in the cosmetic industry due to their moisturizing and "anti-aging" properties, but the most popular and most commonly used moisturizer is undoubtedly glycerol.

No matter how the formulation has been developed, it is clearly necessary to propose new active agents for the cosmetic industry and/or pharmacopoeia to improve the symptoms of dry skin. The aim of the present invention is precisely to promote the production of FNH by using novel compounds capable of acting on PAD1 and PAD3 to promote the natural moisturizing action of the stratum corneum. This can be applied particularly advantageously in the field of care and/or make-up products for the skin, lips, eyelashes, eyebrows, hair, scalp or nails of the body or face; the field of sun-screening or self-tanning products; hair products are in particular the field of products for dyeing, conditioning and/or caring for hair.

Disclosure of Invention

In fact, after significant research, the applicant has found, in a surprising and unexpected way: theophylline acetic acid or salts thereof, caffeine and theobromine (used alone or in combination) have the ability to increase the activity of PAD1 and/or PAD3, in particular the ability to deinimize their physiological substrates, filaggrin, to promote the natural moisturizing effect of the stratum corneum. The chemical formula of these molecules is specified below:

2- (1, 3-dimethyl-2, 6-dioxo-2, 3-dihydro-1H-purin-7 (6H) -yl) acetic acid

1,3, 7-trimethyl-1H-purine-2, 6(3H,7H) -dione

3, 7-dimethyl-1H-purine-2, 6(3H,7H) -dione

It has been demonstrated that the deimination reaction is divided into 5 successive steps:

i) nucleophilic attack of peptidyl-L-arginine C ζ by the thiol group of Cys of the active site of the enzyme;

ii) formation of a (hydrogen and salt) bond between the Asp of the active site and the substrate;

iii) cleavage of the bond between C ζ and Ν η 2 of peptidyl-L-arginine and release of ammonia;

iv) a second nucleophilic attack, this time through a water molecule; and

v) hydrolysis of the adduct formed at the end of the preceding reaction and release of the final deimination product (peptidyl-L-citrulline).

The present invention therefore relates to these 5 steps of activating a reaction catalyzed by PAD1 or PAD3 either by using the active agents as described above, alone or in combination.

Several disorders are characterized at the clinical level by deimination deletions, such as psoriasis and bullous ichthyosiform erythroderma congenitum (OMIM 113800). Similarly, we noted a decrease in the detection of deiminated proteins in the stratum corneum of patients with allergic dermatitis (see example 3 below). However, in the stratum corneum of patients with psoriasis, such as allergic dermatitis, PAD is expressed at levels similar to controls in both damaged and undamaged areas.

Accordingly, the present invention relates to the cosmetic use of a composition comprising an activator compound of PAD1 and/or PAD3 selected from theophylline acetic acid or a salt thereof, caffeine, theobromine and mixtures thereof as a skin moisturizer.

More particularly, the composition is intended to promote deimination of filaggrin in the epidermis.

The compounds according to the invention are also intended to promote the production of FNH (natural moisturizing factor) in the stratum corneum.

Thus, these activators of PAD1 and/or PAD3 are selected for use in promoting FNH production in the stratum corneum and for their use in moisturizing the epidermis (in particular the stratum corneum), in improving any form of skin dryness, or in enhancing epidermal barrier function and preventing signs of skin aging.

The active agent of the invention can be of any origin, i.e. isolated from a plant such as coffee or cocoa; they may be produced by microorganisms even if they are not naturally produced by the organism of any origin; or obtained by chemical synthesis.

In the context of the present invention, the expression "salt of theophylline acetic acid" refers to an organic or inorganic salt of theophylline acetic acid.

As organic salts that can be used according to the invention, mention may be made of those described in prior art FR 2639541. Preferably, it will be the triethanolamine salt.

As the inorganic salt of theophylline acetic acid, sodium salt, potassium salt or lithium salt may be mentioned.

In a particular embodiment of the invention, the theophylline acetate salt is formed by adding an organic or inorganic base as a neutralizing agent to a composition comprising theophylline acetic acid. Preferably, the organic base is triethanolamine and the inorganic base is NaOH.

The present invention also relates to a pharmaceutical and/or cosmetic composition comprising at least one PAD1 and/or PAD3 activator as defined above and in particular theophylline acetic acid or a salt thereof (alone or in combination with caffeine and/or theobromine) in combination with at least one pharmaceutically or cosmetically acceptable excipient.

Preferably, the composition may be of natural or synthetic origin, more preferably of synthetic origin.

Preferably, theophylline acetic acid is present in the composition in an amount of from 0.5% to 3% by weight based on the total weight of the composition in the case of a pharmaceutical composition, and from 0.1% to 1% by weight based on the total weight of the composition in the case of a cosmetic composition.

Preferably, the PAD1 and/or PAD3 activator compound used will be theophylline acetic acid or a salt thereof.

In a particular embodiment of the invention, theophylline acetic acid or a salt thereof is combined with caffeine and/or theobromine.

Considering the activator effect of theophylline acetic acid or a salt thereof on the PAD1 and/or PAD3 enzymes, it is able to increase the degradation of filaggrin in the stratum corneum and thus increase the production of amino acids comprising FNH, in particular glutamine-derived Pyrrolidone Carboxylic Acid (PCA). An increase in the assay of the acid indicates the moisturizing activity of these compounds.

Preferably, the composition according to the invention will be applied externally.

Preferably, the composition according to the invention will comprise at least one liquid fatty phase capable of comprising at least one compound comprising carbon, hydrocarbon, fluorine and/or silicone, volatile or non-volatile, alone or in a mixture, chosen from oils of mineral, animal, vegetable or synthetic origin and/or solvents.

In a preferred embodiment, the composition according to the invention will preferably be provided in the form of a care and/or make-up product for the skin, lips, eyelashes, eyebrows, hair, scalp or nails of the body or face; provided in the form of a sunscreen or self-tanning product; provided in the form of a hair product, in particular a coloring, conditioning and/or caring product for the hair.

Thus, another aspect of the invention relates to the therapeutic use of the active agents as described above, alone or in combination, to prevent and/or treat these deimination abnormalities and/or to promote the moisturizing effect of the stratum corneum and to ameliorate any form of pathological skin dryness.

In the context of the present invention, "pathological skin dryness" refers to any type of skin dryness which is either directly related to a skin condition or is due to a dermatological treatment of a skin condition.

In a particular embodiment of the invention, caffeine will be used as the sole therapeutically active ingredient in the composition.

In another preferred embodiment of the invention, theobromine will be used as the sole therapeutically active ingredient in the composition.

The composition according to the invention will be able to ensure the moisturizing action of the epidermis, in particular of the stratum corneum, the improvement of any form of skin dryness, the enhancement of the epidermal barrier function and the prevention of signs of skin ageing.

Moisturizing effect of the epidermis refers to improvement or maintenance of water balance of the epidermis.

Any form of improvement in skin dryness refers to any improvement in the moisturizing effect of the epidermis, and is particularly characterized by a lack of water in the stratum corneum, a film of water and fat (un film hydro lipid) that is too thin to be on the surface and no longer protects the skin, a lack of sebum.

Skin aging refers to the reduction of barrier function in addition to the action on the dermis and loss of tissue elasticity. Although there is little increase in the loss of water that is not perceived, clinical experience has shown that older people are more prone to dry skin than younger people in good health. First, this is explained by the alteration of the lipid barrier. The epidermal barrier is also more easily altered and the process of repair is slower. Raman spectroscopy techniques have been able to show the ability of the stratum corneum to retain water and the amount of FNH reduction with age (mainly in the most superficial layers).

Because of its remarkable mutagenic capacity, ultraviolet B-induced DNA (ADN, french publication) damage is one of the major causes of skin cancer pathogenesis. Most of them are absorbed by melanin, but one component of FNH (trans-urocanic acid, which has an average ratio of about 5. mu.g/cm in skin)²) A first light protecting barrier is ensured. This is a relatively effective natural sunscreen that was added to many cosmetics in the seventies of the 20 th century. In the stratum corneum, trans-urocanic acid is derived from the catabolism of filaggrin, as are the amino acids of most FNHs.

The invention therefore also relates to the cosmetic use of an active agent as described above, alone or in combination, for moisturizing the epidermis, to promote the natural production of trans-urocanic acid and to protect the skin against uv rays.

Preferably, the present invention relates to a composition for the treatment and prevention of skin diseases; it is used for treating xerosis, ichthyosis, psoriasis, allergic dermatitis, hyperkeratosis, and congenital bullous ichthyosiform erythroderma.

In a particular embodiment of the invention, the pharmaceutical composition according to the invention comprises at least one PAD1 and/or PAD3 activator compound consisting of theophylline acetic acid or a salt thereof in combination with caffeine and/or theobromine and at least one pharmaceutically or cosmetically acceptable excipient for therapeutic use in the treatment and prevention of skin diseases; can be used for treating xerosis, ichthyosis, hyperkeratosis, and congenital bullous ichthyosiform erythroderma.

The invention also relates to a method for the cosmetic treatment of keratinous materials, in particular the skin of the body or the face, the lips, the nails, the hair and/or the eyelashes, comprising applying to said materials a cosmetic composition according to the invention.

Drawings

In the context of the present invention, various biochemical analytical methods have made it possible to select several molecules that modulate the catalytic activity of PAD1 and PAD3, based on the specific ability of said molecules to deiminate silk fibroin. These results will be illustrated in the accompanying drawings, which show:

FIG. 1: analysis of the purified recombinant protein.

After purification, PAD1, PAD3 and recombinant human silk poly protein (Fil-His) were separated by polyacrylamide gel electrophoresis in the presence of SDS (PAGE-SDS), stained with coomassie blue or immunodetected as described herein. AHF11 (an anti-filaggrin monoclonal antibody) detected approximately 45kDa of non-deiminated Fil-His. Apparent molecular weights are indicated in kDa on the left.

FIG. 2: kinetics of deimination of recombinant human filaggrin (Fil-His) by PAD 1.

After incubation with PAD1, Fil-His was immunodetected with AHF11, an anti-filaggrin monoclonal antibody. Apparent molecular weights are indicated in kDa on the left side and incubation times in minutes (min) at the top of the insert. Note that the progressive change in migration from 45 to 66kDa was induced by deimination of the Fil-His of PAD 1.

FIG. 3: deimination of recombinant human silk poly protein (Fil-His) by PAD1 in the presence or absence of streptomycin.

As described herein, Fil-His was immunodetected by AHF11 (an anti-filaggrin monoclonal antibody) or by AMC (an anti-citrulline antibody) in the presence of 5mM streptomycin (str) or in the presence of 1% dimethyl sulfoxide (d) (solvent) after incubation with PAD 1. Apparent molecular weights are indicated in kDa on the left side and incubation times in minutes (min) at the top of the insert. Note that PAD1 was inhibited by streptomycin because the intensity of immunodetection by AMC was lower when incubation occurred in the presence of the molecule compared to incubation in the presence of solvent alone.

FIG. 4: deimination of recombinant human filaggrin (Fil-His) by PAD1 in the presence or absence of caffeine.

As described herein, Fil-His was immunodetected by AHF11 (an anti-filaggrin monoclonal antibody) or by AMC (an anti-citrulline antibody) after incubation with PAD1 in the presence of 50 μ M caffeine (caf) or in the presence of 1% dimethyl sulfoxide (d) (solvent). Apparent molecular weights are indicated in kDa on the left side and incubation times in minutes (min) at the top of the insert. Note that PAD1 was caffeine activated because Fil-His was more strongly deiminated in the presence of the molecule than in the presence of solvent alone.

FIG. 5: deimination of recombinant human silk poly protein (Fil-His) by PAD3 in the presence or absence of theobromine.

As described herein, Fil-His was detected immunologically by AHF11 (an anti-filaggrin monoclonal antibody) or by AMC (an anti-citrulline antibody) after incubation with PAD3 in the presence of 200. mu.M theobromine (tho) or in the presence of 1% dimethylsulfoxide (d) (solvent). Apparent molecular weights are indicated in kDa on the left side and incubation times in minutes (min) at the top of the insert. Note that PAD3 is activated by theobromine.

FIG. 6: deimination of recombinant human silk poly protein (Fil-His) by PAD3 in the presence or absence of theophylline acetic acid.

As described herein, Fil-His was detected immunologically by AHF11 (an anti-filaggrin monoclonal antibody) or by AMC (an anti-citrulline antibody) after incubation with PAD3 in the presence of theophylline acetate (ac) at a final concentration of 50 μ M (top) or 200 μ M (bottom) or in the presence of 1% dimethyl sulfoxide (d) (solvent). Apparent molecular weights are indicated in kDa on the left side and incubation times in minutes (min) at the top of the insert. Note that PAD3 was activated by theophylline acetic acid (particularly evident at the highest concentrations).

FIG. 7: deimination of recombinant human silk fibroin by PAD1 or PAD3 in the presence of caffeine, theobromine, and/or theophylline acetic acid, alone or in combination.

Fil-His was incubated for 5 minutes in the presence of PAD1 (A-B) or 60 minutes in the presence of PAD3 (C-D). As described herein, 312.5 μ Μ caffeine (Caf), theophylline acetic acid (Ac), or theobromine (Th é) was added to the reaction mixture, either alone (a and C) or in combination (B and D), prior to incubation. After incubation, Fil-His was immunodetected by anti-citrulline Antibody (AMC) and the immunodetection intensity was quantified using ImageJ software. Data are presented in bar graph form as the relative percentage of activity of each enzyme relative to a control (-) performed in the absence of active agent.

FIG. 8: PAD1, PAD3 and deiminated proteins by immunohistological analysis on sections of normal human skin or skin from patients with allergic dermatitis.

After chemical modification of citrulline, the deiminated protein was detected by anti-citrulline (AMC) immunoassay. Negative controls (Neg) were run in parallel. PAD1 and PAD3 were immunodetected by anti-PAD 1 and anti-PAD 3 antibodies as described above (example 3). The images presented the results obtained for eight patients (DA 1, 2 and 3) and three controls (Nor 1) [ bar =150 μm ].

FIG. 9: western blot analysis of deiminated proteins after external application of theophylline acetic acid in the form of triethanolamine or sodium salts on the surface of the reconstituted epidermis.

The epidermis was reconstituted for 24 hours by external treatment with control gel (1) or with theophylline acetic acid in the form of triethanolamine salt (2) or sodium salt (3) containing concentrations of 3% (2 and 3).

A. Total protein was separated by electrophoresis and immunodetected by anti-citrulline Antibody (AMC) and anti-actin antibody. Experiments were performed in triplicate (I, II and III). Apparent molecular weights are indicated in kDa on the left.

B. Immunoassay intensity was quantified using ImageJ software. The values obtained were normalized to actin. They are presented in bar graph form as relative percentage to the control performed in the absence of theophylline acetic acid.

Note that the immunodetection intensity of citrullinated protein was higher for the epidermis treated with the gel comprising theophylline acetic acid in salt form compared to the epidermis treated with the control gel.

FIG. 10: immunohistological analysis of deiminated proteins after external application of theophylline acetic acid in sodium salt form on the surface of reconstituted epidermis.

Frozen sections of untreated normal skin (a and D) and reconstituted epidermis treated with control gels (B and E) or gels containing theophylline acetic acid in the form of the sodium salt at a concentration of 3% (bar =150 μm) were subjected to immunohistological analysis using an anti-citrulline Antibody (AMC) modified with (a-C) or without (D-F; negative control) citrulline. Note that the intensity of stratum corneum staining was increased in the reconstituted epidermis treated with theophylline acetic acid (C) in the sodium salt form compared to the reconstituted epidermis treated with the control gel (B).

Detailed Description

Pharmacological evaluation

A/Heavy loadActivity assay of PAD1 and/or PAD3 of histone filaggrin

The method comprises the following steps:

1. production and purification of recombinant human filaggrin

A recombinant human filaggrin of 324 amino acid subunits was produced by fusion at the COOH terminus using the pET-41b vector (Merck, KGaA, Darmstardt, Germany) with a 6-histidine tag in E.coli BL21Codon plus (DE3+) -RIL strain (Stratagene, La Jolla, CA) ("GenBank" database accession: AF 043380).

For this purpose, the following oligonucleotide pairs were first used: 5'-CATATGCTATACCAGGTGAGCACTCATG-3' and 5'-CTCGAGCCCTGAACGTCCAGACCGTCC-3', and cDNA was amplified by PCR from mRNA extracted from human epidermis, and then purified and cloned into pCRII-Topo vector (Invitrogen, Carlsbad, Calif.).

Thus, the PCR product was flanked by an NdeI site and an XhoI site, and these two additional restriction sites enabled subcloning from the pCRII-Topo vector into the pET-41b expression vector. After confirmation by sequencing, a filaggrin subunit (hereinafter referred to as Fil-His) was produced and purified by nickel affinity chromatography according to protocols well known to those skilled in the art. The Fil-His recombinant protein thus purified was analyzed by PAGE-SDS (10% gel) and immunodetected with AHF11 anti-filaggrin monoclonal antibody. The purity was monitored by staining with coomassie blue, and the Fil-His protein had an apparent molecular weight of about 45kDa (see fig. 1). The concentration of the purified fractions obtained (in mg/ml) was determined using a standard range of bovine serum albumin using a NanoDrop1000 (Fisher Scientific, ilkirch, France).

2. Preparation of active recombinant human PAD1 and PAD3

PAD1 and PAD3, produced and purified according to protocols well known to those skilled in the art, were purchased from professor Hidenari Takahara (university of d' Ibaraki, Japon). The characteristics of the purified active human recombinant PAD1 and PAD3 are shown in the following table:

PAD

activity

Protein concentration

Specific activity

	(Unit/ml)	(mg/ml)	(Unit/mg)
				PAD1	206.0	0.696	296.0
PAD3	10.2	0.400	25.5

One unit is specified as the amount of PAD catalyzed by Bz-L-Arg-O-Et to form 1. mu. molBz-L-Cit-O-Et within 1 hour at 55 ℃.

After separation by PAGE-SDS, the purity of each enzyme was evaluated by staining with Coomassie blue (see FIG. 1).

3. Deimination of Fil-His by PAD1 and PAD3

At 50 ℃ in the presence of 40mU of PAD1 or PAD3 in deimination buffer (CaCl)₂10mM, dithiothreitol 5mM, Tris-HCl50mM pH 7.4) 25ng of Fil-His. After variable incubation times according to the isotype of PAD (typically, 2 to 5 minutes for PAD1 and 60 to 180 minutes for PAD 3), the reaction was quenched by addition of sample buffer. The proteins were then separated by PAGE-SDS and immunodetected by AHF11 anti-filaggrin monoclonal antibody (diluted to 1/5000) and AMC modified anti-citrulline antibody (Millipore, Mollsheim, France).

As a result:

1. evaluation of the deimination of Fil-His

When Fil-His was not deiminated, its apparent molecular weight in the denatured gel was approximately 45 kDa. After staining with coomassie blue or immunodetection with AHF11 antibody, it migrated as a single protein (see figure 1). Deimination of Fil-His induced a progressive increase in its apparent molecular weight from 45 to 66kDa (see FIG. 2). The form of 66kDa corresponds to the fully deiminated form, while the intermediate forms correspond to the more or less deiminated form. Thus, the extent of deimination of Fil-His correlates with its apparent molecular weight after separation by PAGE-SDS.

Fil-His was immunodetected by AMC antibody only after incubation with active PAD. The higher the intensity of its immunoassay, the higher its deimination degree (see FIG. 3).

2. Streptomycin inhibition PAD1

Before the deimination reaction starts, it is possible to assess the effect of the added molecules to the reaction mixture on PAD (activator or inhibitor) by changes in the migration of silk fibroin and/or changes in the intensity of detection by AMC antibodies. For example, inhibition of PAD1 by streptomycin could be demonstrated by this method (see figure 3). The intensity of immunodetection by AMC antibody bands of mass-66 kDa or between 45 and 60kDa was lower when the Fil-His3 or 5 min incubation was done with PAD1 in the presence of 5mM streptomycin compared to the incubation at all the same time points in the presence of solvent (1% dimethyl sulfoxide) alone.

B/PAD1 and/or PAD3 activators

The method comprises the following steps:

deimination by Fil-His of PAD1 or PAD3 in the presence of an active agent or combination of active agents

As described above (paragraph A.3), in the presence of 40mU of PAD1 or PAD3 at 50 ℃ in deimination buffer (CaCl)₂Deimination of Fil-His (25 ng) was performed in 10mM, dithiothreitol 5mM, Tris-HCl50mM pH 7.4) for 0, 3, 5, 60 and 180 minutes. The active agents in 1% dimethylsulfoxide solution, obtained at final concentrations of 50, 200 and 312.5 μm, or 1% dimethylsulfoxide alone (control), or a mixture of several active agents each diluted to 312.5 μm in 1% dimethylsulfoxide, was added to the reaction mixture prior to addition of the enzyme. In some cases, the active agent is diluted in water. Following incubation, the proteins of the reaction mixture were immunodetected using AMC antibodies. The immunoassay intensity was quantified by densitometry using NIH ImageJ software.

As a result:

1. activation of PAD1 by caffeine

Prior to deimination, Fil-His was immunodetected by AHF11 antibody as a band of about 45kDa, rather than by AMC antibody (see figure 4). After 3 and 5 min incubation in the presence of PAD1 and dimethyl sulfoxide (final 1%), the Fil-His moiety was deiminated by PAD 1: it was then immunodetected with AMC antibody as a band of size between 45 and-60 kDa. After incubation for 3 and 5 minutes in the presence of PAD1 and caffeine (finally 50 μ M in 1% dimethyl sulfoxide), the band was more intense for immunodetection and an additional band (migrating to 66 kDa) was detected with AMC antibody (see figure 4). This 66kDa protein corresponds to completely deiminated Fil-His. Quantitative analysis confirmed the results and showed that after incubation in the presence of caffeine, the intensity of the immunodetection of the band between 45 and 66kDa increased by 151% compared to the control.

Comparative results were obtained during the tests with caffeine dissolved and diluted in ultrapure water.

Thus, caffeine activates PAD 1.

2. Activation of PAD3 by theobromine

Fil-His had an apparent molecular weight of about 45kDa prior to deimination; Fil-His was immunodetected by AHF11 antibody, but not by AMC antibody (see fig. 5). After incubation for 180 min in the presence of PAD3 and dimethyl sulfoxide (1%), Fil-His was deiminated by the PAD3 moiety: it was then detected by AMC antibody immunoassay as a band of size between 45 and-66 kDa. After incubation for 180 minutes in the presence of PAD3 and theobromine (200 μ M in 1% dimethyl sulfoxide), the intensity of the bands immunodetected with AMC antibody was stronger (see figure 5). Quantification of the intensity of the immunoassay showed an increase of 182%.

Comparative results were obtained during the experiments with theobromine dissolved and diluted in ultrapure water.

Theobromine thus activates PAD 3.

3. Activation of PAD3 by theophylline acetic acid.

Prior to deimination, Fil-His was immunodetected by AHF11 antibody near 45kDa but not by AMC antibody (see figure 6). After incubation for 180 minutes in the presence of PAD3 and dimethyl sulfoxide (1%), the Fil-His moiety was deiminated by PAD 1: it was then immunodetected with AMC antibody as a band of between 45 and-60 kDa and a band of-66 kDa. After incubation for 180 minutes in the presence of PAD3 and theophylline acetic acid (200 μ M in 1% dimethyl sulfoxide), the intensity of the bands detected by AMC antibody immunoassay was significantly stronger (see figure 6). Quantification of the intensity of the immunoassay corroborates this result and shows a 225% increase.

Comparative results were obtained during the experiments with theophylline acetic acid dissolved and diluted in ultrapure water.

Thus, theophylline acetic acid activates PAD 3.

4. Evaluation of the Effect of mixtures of caffeine, theobromine and/or theophylline acetic acid on PAD1 and PAD3 Activity

A similar assay of Fil-His deimination by PAD1 (5 min incubation) and PAD3 (60 min incubation) was performed in the presence of each of the three active agents (caffeine, theobromine and theophylline acetic acid) or a mixture of two or three of these active agents. The active agent is dissolved and then diluted in ultrapure water. It was used in the assay at a final concentration of 312.5 μm. The intensity of the immunodetection by the AMC antibody was quantified as before (see FIG. 7).

The results obtained confirm that caffeine activates PAD1 and theobromine and theophylline acetic acid activate PAD 3. In addition, they demonstrated that the three active agents used alone activated PAD1 and PAD3 at comparable activation levels (see fig. 7A and 7C).

Addition of the active agents in pairs or three together did not induce an inhibitory effect. The same level of PAD1 activation was observed (see fig. 7B). The addition of two or three active agents appears to more significantly activate PAD3 (see fig. 7D).

Thus, caffeine, theobromine, and theophylline acetic acid, alone or in combination, activate PAD1 and PAD 3.

C/Deiminated proteins of the stratum corneum of an injured epidermis of the skin of a patient suffering from allergic dermatitisIs examined Reduction of measurement

The method comprises the following steps:

1. skin section

Biopsies of 3mm diameter were performed on damaged skin of 8 patients with allergic dermatitis (confirmed by the dermatologist of the Turutz Hospital) and healthy skin of 3 controls, which were fixed in formaldehyde and enclosed in paraffin. Each biopsy was serially sectioned (6 μm), deposited on Superfrost slides, deparaffinized with serial xylene and ethanol baths, rehydrated and stained with hematoxylin-eosin or analyzed by immunohistology.

2. Detection of deiminated proteins by immunohistology

For immunodetection of deiminated proteins, the protein was purified at 37 ℃, in modified buffer: 0.0125% FeCl₃2.3M of H₂SO₄1.5M of H₃PO₄Skin sections were incubated for 3 hours in 0.25% diacetylmonoxime and 0.125% antipyrine. Negative controls were systematically prepared by omitting diacetylmonoxime and antipyrine. After washing with water, sections were incubated with AMC reference antibody diluted to 1/500 and then aged in the presence of diaminobenzidine as peroxidase chromogenic substrate using the "ImpressReagent anti rabbitig PO" kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). Finally, a counterstain with hematoxylin was performed.

3. Detection of PAD1 and PAD3 by immunohistology

PAD1 and PAD3 were immunodetected using specific anti-PAD 1 and anti-PAD 3 (B3) anti-peptide antibodies diluted to 1/80 and 1/100, respectively, according to the protocol described above. Counter staining was performed with hematoxylin. A negative control was prepared by omitting the primary antibody.

As a result:

1. reduction of deiminated proteins in the epidermis of patients with allergic dermatitis

All samples that did not undergo chemical modification were negative. Skin samples from control individuals exhibited very strong and continuous staining with AMC antibodies penetrating the epidermal stratum corneum and on all keratinocyte layers. In contrast, skin samples from patients with allergic dermatitis exhibited significantly weaker and often discontinuous staining of the stratum corneum (see fig. 8).

In summary, deimination of proteins of the damaged epidermis was significantly reduced in patients with allergic dermatitis compared to healthy controls.

2. Expression of PAD1 and 3 in the epidermis of a patient with allergic dermatitis

For all samples of harvested allergic skin, PAD1 was detected in the cytoplasm of glial cells of the active layer of all epidermis, with higher intensity in the most differentiated keratinocytes. The same immunodetection properties were observed for normal skin samples, as is well known to those skilled in the art. The intensity of staining obtained on allergic skin and normal skin was the same.

In normal epidermis, PAD3 is detected primarily in the cytoplasm of granular keratinocytes, as is well known to those skilled in the art. In allergic skin of all patients, PAD3 was detected in the cytoplasm of several cell layers of the most differentiated keratinocytes. Generally, the staining intensity is comparable to that of normal epidermis (see fig. 8).

Thus, PAD1 and PAD3 are widely expressed in the epidermis of patients with allergic dermatitis.

Thus, the absence of these enzymes does not account for the reduction in protein deimination previously observed, but may instead be due to lower activity.

D/Increase of deiminated proteins of the stratum corneum after treatment with a gel comprising theophylline acetic acid

The method comprises the following steps:

1. reconstruction of the epidermis

Reconstruction of human epidermis at the gas-liquid interface at 0.33cm²Growth in size of (2) for 14 days. Then, the mixture was brushed at a rate of 5mg/cm²The pre-formulations containing the various active agents of (a) are uniformly applied to their surface. Treatment times of 24 hours were performed in triplicate at 37 ℃. The epidermis was then divided: half frozen in Tissue Tek, then 6 μm cryoprecipitate-sections for immunohistology; the other half was lyophilized at-80 ℃ for preparation of total protein extracts prior to Western blot analysis. Total proteins were extracted by boiling in sample buffer (0.175M Tris-HCl pH 6.8; 12.5% beta-mercaptoethanol, 7.5% SDS, 25% glycerol) and separated by SDS-PAGE (in 4-15% gradient gels).

2. Detection of deiminated proteins by immunohistology

Following incubation in modified buffer, deiminated proteins were immunodetected as described above. Negative controls were prepared systematically by omitting the modifications. After washing with water, sections were incubated with AMC anti-citrulline antibody diluted to 1/1000. Finally, a counterstain with hematoxylin was performed.

3. Detection of deiminated proteins by Western blot

Protein extracts clarified by centrifugation and separated by PAGE-SDS were detected by AMC anti-citrulline antibody as described above or by anti-actin antibody (Millipore, cloneMAB 1501) immunodetection. The immunoassay intensity was quantified as described above.

As a result:

1. increase of deiminated proteins in reconstituted epidermis treated with theophylline acetic acid in the form of triethanolamine or sodium salt.

Deiminated proteins with apparent molecular weights between 90 and 30kDa were detected in all extracts (see fig. 9A). One of them is filaggrin (arrow). The immunodetection intensity in the lane equivalent to the epidermis treated with the aqueous gel containing theophylline acetic acid in the form of triethanolamine salt or sodium salt was stronger (about 2.5 times) than that in the lane equivalent to the epidermis treated with the aqueous gel alone (fig. 9A and 9B).

2. Increase of deiminated proteins in the stratum corneum of reconstituted epidermis treated with theophylline acetic acid in sodium salt form.

All samples that did not undergo chemical modification were negative (see fig. 10D, 10E and 10F). Samples of reconstituted epidermis using AMC antibodies showed discontinuous staining throughout the stratum corneum. The immunoassay was more intense after treatment with theophylline acetic acid in the sodium salt form (see figure 10).

In conclusion, theophylline acetic acid, applied in salt form to the surface of the reconstituted epidermis in an aqueous gel, increases the deimination of the proteins of the stratum corneum and in particular of the filaggrin.

E、Preparation of an aqueous gel containing 3% theophylline acetic acid in sodium salt form

Adding 2% of water

Mode of operation

Step A: in water preheated to 70 ℃, theophylline acetic acid + NaOH addition was dispersed with stirring (1300 rpm): 70 deg.C

And B: phenoxyethanol + slowly sprinkled into the cellulose hydroxyethyl ether was added with stirring (500 rpm), and then the shear force was increased.

B was then added to A with stirring (1300 rpm over 10 minutes). Cooled and homogenized (1 hour at 900 rpm)

Final pH =7.16

And (3) macroscopic observation: viscous, transparent, colorless gel.

Examples of compositions

Several formulations according to the invention in the form of creams have the following composition (amounts are given in mass percentages relative to the total weight of the composition).

Advantageously, the formulation according to the invention has between 0.5% and 3% of theophylline acetic acid or a salt thereof, said percentages being adjusted according to the desired wetting capacity.

Example 1:

name (R)	Mass%	Function(s)
			Theophylline acetic acid	0.5 to 3	Moisture-retaining agent
Glycerol	15	Wetting agent
			Vaseline	8	Skin-moistening agent
Liquid paraffin	2	Skin-moistening agent
			Stearic acid	1.5	Emulsifier
Glyceryl monostearate	5	Emulsifier
			Cyclomethicone	1.5	Skin-moistening agent
Dimethicone	0.5	Skin-moistening agent
			Polyethylene glycol 600	5.0	Wetting agent
Triethanolamine	Proper amount of	Neutralizer pH =6.5
			Propyl p-hydroxybenzoate	Proper amount of	Preservative
Water (W)	Proper amount to 100.0

Example 2:

Claims (14)

1. Cosmetic use of a composition comprising theophylline acetic acid or a salt thereof as a skin moisturizer.

2. Use according to claim 1, characterized in that said composition is intended to promote deimination of filaggrin in the epidermis.

3. Use according to claim 1, characterized in that the composition is intended to promote the production of FNH (natural moisturizing factor) in the stratum corneum.

4. Use according to claim 1, characterized in that said composition is intended to moisturize the epidermis, in particular the stratum corneum, to improve any form of skin dryness, or to enhance the epidermal barrier function and prevent signs of skin aging.

5. Use according to claim 1, wherein the composition is intended to promote the natural production of trans-urocanic acid and to protect the skin against uv light.

6. Use according to claim 1, characterized in that the theophylline acetic acid or salt thereof is combined with caffeine and/or theobromine.

7. A cosmetic method of moisturizing skin comprising applying to the skin a cosmetic composition comprising theophylline acetic acid or a salt thereof.

8. An activator compound of PAD1 and/or PAD3 selected from theophylline acetic acid or a salt thereof, caffeine, theobromine and mixtures thereof for use as a medicament for treating dry skin suffering from a skin disease such as xerosis, ichthyosis, psoriasis, allergic dermatitis, hyperkeratosis and erythroderma bullosa.

9. The compound according to claim 8 for use in ameliorating any form of pathological skin dryness.

10. A dermatological or cosmetic composition comprising at least one activator compound of PAD1 and/or PAD3, said activator compound of PAD1 and/or PAD3 consisting of theophylline acetic acid or a salt thereof in combination with caffeine and/or theobromine, and at least one pharmaceutically or cosmetically acceptable excipient; for cosmetic or dermatological use according to one of claims 1 to 9.

11. Cosmetic composition according to claim 10, characterized in that the theophylline acetic acid or salt thereof represents from 0.1% to 1% by weight of the total weight of the composition.

12. The pharmaceutical composition according to claim 10, wherein the theophylline acetic acid or salt thereof is present in an amount of 0.5 to 3% by weight based on the total weight of the composition.

13. Composition according to one of claims 10 to 12, characterized in that it comprises at least one liquid fatty phase comprising at least one compound comprising carbon, hydrocarbon, fluorine and/or silicone, volatile or non-volatile, alone or in mixture, chosen from oils of mineral, animal, vegetable or synthetic origin and/or solvents.

14. Composition according to one of claims 10 to 13, characterized in that it is provided as a care and/or make-up product for the skin, lips, eyelashes, eyebrows, hair, scalp or nails of the body or face; provided as a sunscreen or self-tanning product; are provided as hair products, in particular for dyeing, conditioning and/or caring for the hair.