EP2237775A1

EP2237775A1 - Composition for topical use

Info

Publication number: EP2237775A1
Application number: EP09703782A
Authority: EP
Inventors: Jean Hilaire Saurat
Original assignee: Individual
Current assignee: THESAN PHARMACEUTICALS Inc
Priority date: 2008-01-23
Filing date: 2009-01-23
Publication date: 2010-10-13
Also published as: US20100324109A1; US9011885B2; EP2082736A1; US9463182B2; US9358220B2; US20150005373A1; US20150216840A1; WO2009093207A1; US20150094351A1

Abstract

The invention relates to the use, as a dermatological or cosmetic drug, of compounds capable of interacting in a transitional manner with the Ah R receptor (Aryl Hydrocarbon Receptor) as agents for modulating skin functions such as the sebaceous function, skin healing, the so-called dermatoporosis skin atrophy, oestrogenic deprivation, and anti-infectious defence, without inducing other toxic effects of the TCDD type. The compounds interacting with the Ah R receptor are selected so as to have a metabolism promoting the dissociation of these effects, in particular due to in situ production from a promoter and/or the in situ modulated metabolisation.

Description

COMPOSITION FOR TOPICAL USE

The present invention relates to a composition for topical use, intended for treating and / or preventing dysfunctions of cutaneous functions. The present invention more specifically relates to a pharmaceutical composition for topical use, intended for the treatment and / or prevention of dysfunctions of the sebaceous function, of cicatrization, of so-called "dermatoporosis" atrophy (Ref Dermatology 2007.215, 284-294). , oestrogenic depriation of the skin and anti-infectious defense.

There are already treatments for the malfunctions mentioned above, but which are however not free from defects. For example, as examples:

• It is known that a derivative of vitamin A, isotretinoin (or 13-cis retinoic acid), is active in the treatment of diseases such as hyperseborrhea and acne by inducing atrophy of the sebaceous glands. But this substance must be administered systemically and not topically, because topically it does not atrophy the sebaceous gland.

To facilitate the healing of skin wounds, it is known to protect the tissues during reconstruction, particularly by means of hydrocolloid dressings, but this technique provides a superficial mechanical protection without specifically stimulating the intrinsic processes of reconstitution of the tissues themselves, when There is a defect in the production of growth factors, especially vascular factors.

• It has been proposed to treat dermatoporosis with retinoids and fragments of hyaluronic acid, but the former may be irritating and the latter difficult to prepare in their size and optimal viscosity.

• In the case of estrogen deprivation of the skin, which contributes to its weakening and dermatoporosis, it is known to administer estrogens systemically, or topically. However, this contribution of exogenous hormones has the disadvantage of modifying the overall hormonal balance of the patient and, according to several publications, to increase the risk of the appearance of certain cancers.

• To reinforce the anti-infectious defense capacities in case of exposure or susceptibility to bacterial skin infections, it has already been proposed to use long-term antibiotic, but this method has the drawback of selecting resistant strains and not increasing the intrinsic defenses of the subject.

There remains an unmet need to develop new drugs against the aforementioned dysfunctions and in particular pharmaceutical compositions for topical use to avoid the disadvantages associated with systemic administration.

The compounds interacting with the AhR (Aryl Hydrocarbon Receptor) receptor, the prototype of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are for the most part xenotoxic, inducing various types of tissue lesions and others. manifestations of intoxication, dependent on the dose and the species. Among these manifestations of dioxin intoxication, the skin occupies a major place, partly because of the well-known expression of the AhR receptor by the epithelial and mesenchymal cells of the skin. This is why the therapeutic and / or preventive use of such compounds as active agents modulating cutaneous functions seems a priori excluded.

During human intoxication by TCDD, a complex syndrome is observed in the skin. The thorough study of this syndrome in humans and in experimental animals has led the applicant to observe the cellular and molecular mechanisms of constitution of these toxic lesions. Surprisingly, the Applicant has found some favorable effects of exposure to these toxic compounds, described below, and found ways to obtain them, in the absence of the toxic effects mentioned above, to modulate cutaneous functions. , especially the sebaceous function, the anti-infectious defense, the cicatrization, the atrophy of the skin called "dermatoporosis (Ref Dermatology 2007.215, 284-294)", and the estrogenic deprivation.

Consequently, the invention relates to AhR receptor agonist compounds and / or the proteins involved in its cellular activation pathways, these compounds acting in the context of the invention as agents modulating cutaneous functions, in particular sebaceous function, anti-infectious defense, scarring, so-called "dermatoporosis" atrophy, and estrogen deprivation of the skin.

According to a first aspect of the invention, the subject of the invention is a topical pharmaceutical composition intended to induce, in a transient manner, the activation of AhR cutaneous tissue, said composition comprising an active substance selected from metabolizable Agonist ligands of AhR, and from the precursors in situ of said ligands.

The toxicity of xenotoxic ligands of AhR, such as TCDD, appears mainly due to the fact that these substances are stored by the human organism, in whose tissues they have prolonged half-lives, sometimes of the order of 4 -12 years. As a result, TCDD molecules permanently released from storage sites such as fatty tissue to target tissues expressing the AhR receptor, such as the skin, continually activate the AhR signaling pathway, resulting in effects. pathological. Thus, unlike TCDD, through the use of defined metabolizable ligands of AhR, whose half-life duration in the body is evaluated in hours or days, the activation of cutaneous AhR topically results only in a beneficial activation of certain cutaneous functions, functions that the TCDD intoxication made it possible to identify for the first time in the man.

In this context, the active substance may be chosen from endogenous ligands of AhR: the applicant believes that there are very likely endogenous ligands, or exogenous including environmental, which provide a physiological function of the AhR signaling pathway. These ligands are essentially short-lived.

Thus, according to a ^2nd aspect of the invention, it relates to a composition for topical use, for inducing transiently AhR receptor activation of skin tissue, said composition comprising an active substance chosen from AhR half-life agonist ligands and in situ precursors of said ligands.

The ligands used in the context of the present invention preferably have half-lives in the human body of between 2 and 96 hours and, more specifically, and depending on the application, between 6 and 24 hours.

The present invention is therefore completely distinct, and even antinomic, from certain inventions of the prior art which have proposed the use of AhR receptor antagonist ligands, in order to prevent gene expression of the type that may be induced. by exposure to TCDD and other xeno-toxic AhR ligands. These inventions, described in particular in the documents WO2004 / 041758, WO2007 / 060256 and WO2007 / 128725, were based on extrapolations from in vitro experiments and the postulate that activation of the AhR pathway would be inherently harmful. On the contrary, the Applicant has found that activation in humans and experimental animals of the AhR pathway may have favorable effects for the treatment of diseases such as abnormalities of sebaceous function, scarring, the so-called "dermatoporosis" atrophy, the anti-infectious defense, and the estrogenic depriation of the skin.

The ligands according to the invention are chosen in that they preferably fulfill four criteria:

1. An AhR receiver activation capability.

2. A modulation capacity of a specific gene regulated by AhR.

3. A half-life in the short human body, preferably between 2 hours and 96 hours, and more specifically, and depending on the application, between 6 and 24 hours.

4. Preferably, a measurable positive effect on a recognized criterion of sebaceous hyperactivity, wound healing, dermatoporosis, bacterial colonization of the skin, and / or estrogenic deprivation.

The modulated gene may in particular be the CYP IA1 gene. This modulated gene may also be one of the many genes described herein as modulated by TCDD exposure and whose promoter expresses an AhR binding site. The Applicant considers that modulation of the CYPlAl gene is proposed as a paradigm based on current observations and knowledge; thus it does not exclude that the desired effects are obtained by AhR ligands not having as predominant effect to activate the expression of CYPlAl. The applicant further considers that shelf life should be a decisive element in dissociating beneficial effects / toxic effects by inducing a transient activation of AhR. The applicant, however, does not exclude that certain ligands whose half-life deviates from the numerical values indicated above may be endowed with this property of dissociation beneficial effects / toxic effects. Indeed the duration of activation of the AhR receiver in the skin may not depend exclusively on the half-life of the ligand in the body.

In general, the dissociation between beneficial effect according to the invention and toxic effects is obtained by stimulating the AhR receptor in a modulated and transient manner, that is to say close to the physiological action of natural endo / exogenous ligands. toxic. This dissociation can also be obtained by the formation of a ligand in situ from a precursor administered topically. Said active substance may be a precursor selected from the metabolic proligands of AhR ligands. Said active substance may also be an activatable precursor ligand AhR under the effect of a physical agent, especially under the effect of UV radiation, or any other biological process, such as metabolic activation at within a skin tissue by saprophytic or pathogenic flora.

The invention will be better understood by those skilled in the art by the following description of several specific applications, with reference to the corresponding examples and the accompanying figures, in which

• Figure 1 shows the disappearance of the sebaceous glands by exposure to toxic doses of oral TCDD in humans;

• Figure 2 illustrates the reproduction of this phenomenon of disappearance of the sebaceous glands by the topical application of TCDD in the mouse: Figure 2 shows the disappearance of the sebaceous glands of the skin of the ears of C57BL / 6 mice treated once a day during 45j by TCDD 2mcg / ml (bottom photo) compared to the control (top photo).

FIG. 3 illustrates the reduction of the sebaceous glands by topical application of 4 ligands according to the invention: FIG. 3 shows the reduction of the surface of the sebaceous glands in the ears of C57BL / 6 mice treated once a day for 45 days by different ligands according to the invention.

• Figure 4 shows the major inhibition of gene expression of key enzymes of sebaceous lipogenesis in humans exposed to TCDD by the analysis of the whole transcriptome (Affymetrix) in human skin exposed to TCDD: Genes lipo / sebogenesis enzymes are highly under expressed. The appendix in Figure 4 is a table that gives the symbols of genes whose expression is strongly repressed against controls in human skin exposed to TCDD. • Figures 5 and 6 show the healing times of superficial wounds in the absence and exposure to TCDD in humans. Figure 5 shows the percentage of healed area as a function of time of 4 dermabrasion in case of presence of TCDD in the skin in comparison with 4 controls (Cl -C4).

Figure 6 shows the average values calculated from the data of Figure 5. Thus the complete healing time is reduced from 9.3 (gray bars) to 5 days (black bars).

FIG. 7 shows the healing times of experimental wounds in a culture of human keratinocytes in vitro, treated with TCDD and with ligands according to the invention.

Figure 8 shows the expression of messenger RNAs of growth factors and their human skin receptors exposed to TCDD in vivo.

FIG. 9 shows the cicatrization of a human chronic wound on which a solution of Trp generating an AhR activation according to the invention has been applied for 10 days. The contraction of the chronic wound is noted; the arrows show the development of epithelial scarring with keratinocyte tongues migrating from the edges.

Figure 10a shows the induction in the skin of messenger RNAs of estrogen receptors and aromatase by exposure to TCDD in a human.

Fig. 10b shows the strong ectopic and focal expression of ER alpha receptor protein in pilosebaceous structures on a skin specimen of a subject exposed to TCDD.

• Figure 11 shows the concentration of estradiol (E2) in human skin exposed to TCDD, determined by Elisa on human skin fragments.

• Figure 12 shows the decrease in colonization by Staphylococcus sp. human skin exposed to TCDD (four-month data).

• Figure 13 shows the decrease in colonization of the skin of the human face by propionebacterium acnes in skin exposed to TCDD (four-month data).

• Figures 14 and 15 show the AhR receptor activation values in two cell types by different products in order to identify active substances that activate AhR but have a short half-life. Figure 14 shows the AhR activation of HEP G 2 cells by different ligands according to the invention. The values represent the percentage of activation compared to that obtained with TCDD O.OlμM, by the most active μ M concentration in the range tested for each substance. Figure 15 shows the AhR activation of keratinocytes by different ligands according to the invention. The values represent the percentage of activation relative to that obtained with the O.OIμM TCDD, by the most active μ M concentration in the range tested for each substance.

• Figure 16 shows the activation of AhR by different extracts of common bacteria from human skin in the presence of tryptophan.

FIG. 17 shows the induction of a specific biological activity, CYP1A1 by AhR activating ligands, according to the invention. The tests are carried out using the kit

"P450-GLO assay" from Promega.

With regard to the sebaceous glands, the useful effect of TCDD, within the meaning of the present invention, is the gradual decrease in the size of the glands, as well as the decrease in the expression of genes of the key enzymes of sebaceous lipogenesis .

This decrease is observed in humans morphologically (histology) after a period of 2 to 4 weeks after exposure and becomes complete to the 8 ^th week (Fig 1). Comparative data for normal human skin shown in Figure 1 were published by Montagna (1963).

A strong and focal expression of the CYPlAl enzyme (mRNA and immunohistochemistry) in the sebaceous glands and their hamartomatous metaplasia in MADISH is observed. This indicates a strong AhR dependent activation at the level of the sebaceous glands and demonstrates for the first time that this organ is a dependent AhR target, therefore that can be modulated by agonists of this receptor. This effect is not observed on the other cutaneous structures (hairs, sweat glands, differentiation of the epidermis), which led the applicant to consider that the effect on the sebaceous gland results from an inhibition of the differentiation of the skin. the sebaceous stem cell by the TCDD / AhR pathway. Thus our analysis of the human skin transcriptome exposed to TCDD indicates that the downstream molecular target of the TCDD / AhR / pathway could be a human blimt analogue, (a murine transcription factor that promotes the sebaceous stem cell ( 126, 597-609, 2006), which results in a very significant decrease in the enzymes of sebaceous lipogenesis, as shown by the analysis of the transcriptome of human skin exposed to TCDD (FIG. It should also be emphasized that the immunolistochemistry of human skin exposed to TCDD reveals a strong and focal expression of estrogen receptors in the sebaceous glands (Fig. 10b).

These data led the applicant to design a new approach for the modulation of sebaceous hypertrophies. This approach involves the activation of dependent AhR pathways in the sebaceous glands by agonists of this receptor. It involves the inhibition of sebaceous stem cell differentiation factors and the proliferation of sebocytes, the inhibition of sebaceous lipogenesis enzymes, as well as the local stimulation of estrogen production pathways and the local overexpression of estrogen receptors.

This effect of gradually decreasing the size of the sebaceous glands can be reproduced in animals by topical application of TCDD. This effect is specific because there is no atrophy in man of other adnexal structures such as hair and sweat glands. Thus, the present invention uses the effect of specific inhibition of the sebaceous gland by an AhR ligand for the prevention and treatment of diseases involving hypertrophy of these glands, such as hyperseborrhoea and acne, by inducing atrophy. sebaceous glands. This invention offers for this application a spectrum of activity potentially identical to that of oral isotretinoin, but topically. The invention also identifies the key enzymes of sebaceous lipogenesis as well as the factors that promote the sebaceous stem cell as a target of AhR activators, but also potentially other chemical modulators that may have therapeutic application. Therapeutic application against sebaceous gland tumors and their analogs, such as sebaceous carcinomas of Muir-Torre syndrome, is also contemplated.

The AhR ligands used to reproduce this effect are chosen on the basis of a reduction of at least 40% in the size and number of the sebaceous glands, without inducing toxic signs as well as a reduction of the expression of the enzymes. keys to sebaceous lipogenesis. The means for obtaining the effect on the sebaceous glands without inducing other toxic effects of TCDD type, especially the formation of hamartomatous structures (Metabolizing Acquired Dioxin Induced Skin Hamartomas, MADISH), consist in the use of compounds interacting transiently with the AhR receiver, so that the kinetics is favorable to this temporal dissociation between favorable effect and toxic effects. With regard to scarring and atrophy of the skin called "dermatoporosis (Ref Dermatology 2007.215.284-294)" the useful effect of the TCDD found by the applicant is manifested by an impressive speed of healing wounds. Thus, the Applicant has observed that epidermoid-dermal wounds whose average healing time is usually 10 days, were covered in 3 days. This effect is related to the induction by TCDD of several growth factors (Vascular Endothelial Growth Factor VEGF, Amphiregulin TGFb, PDGF, IGF hepatocyte growth factor hepapoietin A, scatter factor) as well as their receptors, as well as thrombospondin. 1. These data were obtained by analyzing the whole transcriptome (Affymetrix) in several skin samples from a man exposed to TCDD compared to human controls. The effect of TCDD is still characterized by a very important accumulation of hyaluronate in the dermis, parallel to the hyper-expression of hyaluronate synthases.

An object of the invention is therefore a topically administrable pharmaceutical composition intended to treat and prevent skin healing disorders and delays particularly related to dermatoporosis, this composition comprising at least one compound interacting as an agonist with the receptor. AhR. The means for obtaining the desired effect without inducing other toxic effects of TCDD type consist in the use of compounds whose kinetics of interaction with the AhR receptor is favorable to this dissociation: the invention proposes in particular the production in from a precursor such as tryptophan (Trp) or a derivative of Trp, administered in a pharmaceutical vehicle for transcutaneous bioavailability, followed by exposure to UV radiation, or the application in a chronic wound of Trp in a suitable vehicle, allowing in situ generation by AhR ligand medium according to the example shown in FIGS. 7,9,14-17, the interaction with the AhR receptor being limited by the in situ metabolism by enzymes of Phase 1.

Another object of the invention is therefore a topically administrable pharmaceutical composition for treating and preventing skin atrophy known as dermatoporosis (Ref Dermatology 2007.215, 284-294), which is characterized by a depletion of hyaluronate leading to a loss of the viscoelasticity of the skin, this composition comprising at least one compound interacting as an agonist with the AhR receptor, or a precursor thereof, of the type described above.

With respect to estrogen deprivation, the beneficial effect of TCDD is manifested by activation of estrogen pathways and by overexpression of estrogen receptors. Estrogens are endogenous hormones very important in many cutaneous functions; the estrogen deprivation causes disorders of each of these functions. In particular, it is a factor involved in dermatoporosis. It is known, on the other hand, that the local administration of estrogens is a conventional, but not without risk, means for combating hyperseborrhoeic states and androgenetic alopecia. Exposure to TCDD induces in the skin a strong expression of estrogen receptor genes and aromatase enzyme (Fig. 10), as well as an increase in cutaneous estradiol (Fig. The immunohistochemistry of these specimens reveals a strong and highly focal expression of the receptors in the pilosebaceous structures. Thus the applicant has identified for the first time the local induction by TCDD of a state of hyperestrogenesis focused on a skin structure which is known to be hormone-dependent. It is very likely that this contributes to the decrease in the size of the sebaceous glands.

An object of the invention is therefore a topically administrable pharmaceutical composition for treating and preventing disorders of cutaneous functions related to estrogen deprivation, this composition comprising at least one compound interacting as an agonist with the AhR receptor. The means for obtaining the desired effect on the expression of estrogens without inducing other toxic effects of TCDD type consist in the use of compounds whose kinetics of interaction with the AhR receptor is favorable to this dissociation: In particular, the invention provides in situ production from a precursor such as tryptophan or a Trp derivative, administered in a pharmaceutical vehicle for transcutaneous penetration, followed by exposure to UV radiation, interaction with the receptor. AhR is limited by in situ metabolism by phase 1 enzymes.

With respect to the anti-infectious defense, the beneficial effect of the TCDD found by the applicant is manifested by a marked resistance to skin infections. This resistance coincides with a permanent stabilization of the surface flora in favor of non-pathogenic saprophytic species. This resistance results in the absence of superinfection by pathogenic germs. It is parallel and probably partly linked to an overexpression of defensins, which beta defensin. It is also parallel and probably linked at least in part to overexpression of filaggrin. Both observations with therapeutic implications are on the one hand the reduction of staphylococcal colonization, and on the other hand the reduction of colonization by P. acnes.

An object of the invention is therefore a topically administrable pharmaceutical composition for treating and preventing the effects of bacterial skin infections, especially in subjects with high susceptibility, as well as other situations of chronic carriage, this composition comprising at least one compound interacting as an agonist with the AhR receptor. The means for obtaining the desired effect on cutaneous infections without inducing other toxic effects of the TCDD type consist in the use of compounds whose kinetics of interaction with the AhR receptor is favorable to this dissociation: the invention proposes particularly the production in situ from a precursor such as tryptophan or a Trp derivative, administered in a pharmaceutical vehicle allowing transcutaneous penetration, followed by exposure to UV radiation, or the generation in situ by the medium of AhR ligand according to the example shown in FIGS. 7, 9, 14-17. It should be noted in particular that certain saprophytic and pathogenic strains of the cutaneous surface generate an AhR activation which is potentiated by the presence of tryptophan (FIG.

Among the compounds which bind to AhR and are capable of producing the effects according to the invention, mention may be made, by way of nonlimiting examples:

- Flavonoids

Flavanols with alkyl, alkenyl, alkoxyl or carboxyl substituents at the 4 to 6 and 3 'to 5' positions

Flavanones with alkyl, alkenyl, alkoxyl or carboxyl substituents in 5, 6 and 3 'to 5' positions

Flavones with alkyl, alkenyl, alkoxyl or carboxyl substituents at the 3, 5, 6 and 3 'to 5' positions

Flavonols with alkyl, alkenyl, alkoxyl or carboxyl substituents at the 5, 6 and

3 'to 5'

Isoflavones with alkyl, alkenyl, alkoxyl or carboxyl substituents at the 5, 6 and 3 'to 5' positions

Anthocyanidins with alkyl, alkenyl, alkoxyl or carboxyl substituents at the 4 to 6 and 3 'to 5' positions Epigallocatechin gallate; esterification at any of the 5, 7, 3 'to 5', 3 "to 5" positions with fatty acids or retinoic acid

Cis-Stilbene stilbenes with hydroxyl, alkyl, alkenyl, alkoxyl or carboxyl substituents in positions 1 to 5; and 5 'trans-stilbenes with hydroxyl, alkyl, alkenyl, alkoxyl or carboxyl substituents in positions 1 to 5 and 5 '

- Derivatives of tryptophan

Indole-3-carbinol; alkyl, alkenyl, alkoxyl or carboxyl derivatives on the hydroxyl group

tryptamine

Kynurenine and ester of the carboxylic group with retinol

Indole acetic acid and ester of the carboxylic group with retinol

Indole pyruvic acid and ester of the carboxylic group with retinol

6-Formylindolo [3,2-b] carbazole and alkyl or alkenyl derivatives of the formyl group

3,3'-diindolylmethane

Trythantrine

Malassézine

indirubin

Indigo

- Other ligands

ITE [2- (methyl h-indole-3'-carbonyl) -thiazole-4-carboxylate] (endogenous, see US 20020183524)

YH439 {(Isopropyl-2- (1,3-dithioethan-2-ylidene) -2- [N- (4-methylthiazol-2-yl) carbamoyl] acetate} naphthyroflavone b-Naphthoflavone

Hispidine

carvedilol

rutaecarpine β-Carboline ethyl 3-carboxylate

esomeprazole

Metabolites of fatty acids, especially arachidonate

The ligands are chosen in that they preferably fulfill four criteria:

1. An AhR receptor activation capacity, as shown in Figures 14, 15, and 16 on two cellular systems.

2. A modulation capacity of an AhR-regulated gene, for example CYP IA1 as shown in FIG. 17.

3. A half-life in the short human body preferably between 2 hours and 96 hours, and more specifically, and depending on the application, between 6 and 24 hours.

4. A measurable positive effect on a recognized criterion of sebaceous hyperactivity, wound healing and dermatoporosis, bacterial colonization of the skin, and / or estrogenic deprivation according to, but not exclusively, the examples presented here for each of these effects.

Among the preferred AhR ligands, mention may be made of:

• Flavonoids a. Catechin b. Apigenin c. 3-Gallate of epigallocatechin d. Quercetin e. silibinin

• Stilbenes and derivatives f. Resveratrol g. yangonin

• Tryptophan and tryptophan derivatives h. Indole-3-carbinol i. Tryptamine j. kynurenine k. 6-Formylindolo [3,2-b] carbazole (FICZ) shown in Figures 3-14 and alkyl or alkenyl derivatives of the formyl group

1. 3,3'-diindolylmethane m. Trythantrine n. Malassein o. Indirubine p. Indigo • Other ligands q. Methyl [2- (methyl h -indole-3'-carbonyl) -thiazole-4-carboxylate] (endogenous, see US 20020183524) r. YH439 {(Isopropyl-2- (1,3-dithioethan-2-ylidene) -2- [N- (4-methylthiazol-2-yl) carbamoyl] acetate} Hispidine, as illustrated in FIGS. -Naphthoflavone and beta-Naphthoflavone v. Ellipticin W. Carvedilol illustrated in Figures 14, 15, 17 x Triclabendazole and Rutaecarpine illustrated in Figures 3, 14,15,16,17 z. Β-Carboline ethyl 3-carboxylate (B-CCE) illustrated in FIG.

14.15, 16.17 aa. Esomeprazole shown in Figures 14, 15.

The methods for delivering the AhR receptor ligand in conditions optimal for the desired effect include a. The direct application of the active ligand in a suitable galenic vehicle such as lotion, gel, cream, ointment, foam, impregnated dressing. b. The topical application, in a suitable galenic vehicle, of a proligand, which will be metabolized in ligand by the epidermal enzymes. vs. The application of a proligand on the surface of the skin to be treated, which will be activated in ligand by the effect of a physical agent such as UV; for example Tryptophan and UVB or UVA possibly in combination with Riboflavin; or according to the indication, by the endogenous or exogenous agents present in the treated disease: for example, activation of AhR ligand in the fluid of a chronic wound treated with tryptophan, or by Propionebaterium acnes on the face Seborrhoeic / acne treated with tryptophan (Figures 7,9, 14, 15, & 16).

Example 1 Sebaceous Glands

The sebaceous glands in large numbers in normal human skin disappear on exposure to toxic doses of TCDD. Figure 1 shows the number of sebaceous glands per square centimeter on different areas of the human body, which ranges from 50 to 900 depending on the areas and subjects. Thus on the face of an adult, the number is between 400 and 900 / cm2.

Figure 1 shows that during exposure to TCDD the sebaceous glands disappear in all areas studied. For each site, 3 to 10 skin biopsies were analyzed after formalin fixation and haematin-eosin staining. At least 10 sections per block were analyzed. Out of a total of 62 biopsies, totaling 620 sections, no sebaceous glands could be observed, while the sweat glands and hairs were in number and preserved structure. These data were obtained on human skin specimens containing from 930 to 2900 ppt of TCDD measured by gas chromatography / mass spectrography.

The effect on the sebaceous glands can be reproduced in animals by the topical application of TCDD. A reduction of 95% in the number of sebaceous glands in C57 BL / 6 mice can be observed after 45 days of application of TCDD 2 μg / ml (FIG. 2). To reproduce the useful effect of TCDD, in a therapeutic reduction approach, the reduction of the surface of the sebaceous glands in the ears of C57BL / O mice treated once a day during 45 days by different ligands according to the invention can be studied. The surface of the sebaceous glands is measured on three serial sections per animal. A reduction greater than 40% is sought, and obtained here with a concentration, a duration of treatment and a frequency of applications considered minimal. Isotretinoin is shown by way of example of a product used for the topical and systemic treatment of acne. Applying an AhR ligand such as 6-Formylindolo [3,2-b] carbazole (FICZ) topically to a concentration of 100 mcg / ml makes it possible to obtain a significant decrease in sebaceous glands of the order of 40%. Other ligands according to the invention (see Figs. 14, 15 & 17) also cause a decrease in the surface area of the sebaceous glands. The data indicate that the optimal doses for a reduction greater than 40% may be beyond the concentrations, the duration and the frequency of application shown in Fig. 3 and illustrate the concept of the invention.

FIG. 16 shows the AhR activation of HEP G 2 cells by various extracts of common bacteria from human skin in the presence of tryptophan (trp) Fl: flora G + F2: G + PA flora: P. acnes SE: S. epidermidis. The activation value 25.00 on the ordinate corresponds to 1/6 of the AhR stimulation by TCDD 1OnM in the same system (157.00). It can be seen that in particular with P. acnes an increase in activation is observed with the addition of tryptophan (trp). These observations illustrate the mode of activation of AhR by tryptophan partly by interaction with the surface flora.

The effect shown in Figure 16, where it is seen that the Propionebacterium Acnes (PA) generates AhR activation, which is very accentuated by the addition of tryptophan, can be used in the context of this invention. Indeed, PA is one of the key players in sebaceous gland diseases, and it is present in large amounts in the skin of the face. It could therefore contribute to producing therapeutic amounts of AhR ligands in the presence of tryptophan, or also other aromatic amino acids such as tyrosine and phenylalanine. To explore the relevance of this approach, the applicant performed a trial by applying a 1% tryptophan gel on the face in two men with hyperseborrheic acid for 3 weeks and observed a decrease in seborrhea which could be evaluated at 40% of the starting value by "casual level" sebumetry. Figure 4 shows the significant reduction of messenger RNAs of several enzymes of sebaceous lipogenesis in human skin exposed to TCDD. This observation identifies a new target for drugs to modulate sebaceous function, a concept exemplified here by the activation of AhR.

Example 2: Healing and skin atrophy

The healing time of superficial epidermal dermal wounds (dermabrasion) is considerably reduced by TCDD. Figure 5 shows the percentage of healed area as a function of time of 4 dermabrasion in case of presence of TCDD in the skin (TCDD 1-4, 930 to 2900 ppt TCDD measured by gas chromato graphy / mass spectrometry) compared with 4 controls (Cl -C4). Figure 6 shows the average values calculated from the data of Figure 5. Thus the complete healing time is reduced from 9.3 to 5 days.

FIG. 7 shows, in a model for wound healing in vitro on culture of human keratinocytes, that the gap in the culture is repaired by TCDD and by active substances according to the invention in proportions similar to what is observed after application of factors growth. The wound is induced by an incision with a yellow micropipette tip (10-200 μl). Migration index = value obtained with the ligand / that of the control without ligand. These results indicate a strong stimulation of TCDD scarring at nM concentrations, and by FICZ and tryptophan at μM concentrations. This indicates an activity of the AhR activation pathway on keratinocyte migration that is found in vivo in humans (Figures 5, 6, 10).

At the highest concentrations there is a decrease in the effect, or even an inhibition of healing with TCDD and FICZ. Tryptophan (Trp) generates AhR binding and activation activity under the conditions of this culture (see also FIGS. 14 & 15 & 16).

Expression of the messenger RNAs of endothelial and vascular growth factors (VEGF) and their receptors is increased in skin exposed to TCDD. Figure 8 shows the ratio (RT PCR) of human skin fragments containing 1800 ppt of TCDD, as measured by gas chromato graphy / mass spectrometry, in comparison with the controls.

The contraction of chronic wounds and the development of epithelial scarring with keratinocyte tongues was observed after application of a tryptophan gel generating factors in the wound that could activate AhR (Figs 15-16). There is also thickening of dermatoporotic skin around the wound which, in the opinion of specialists, is less atrophic and younger. Figure 9 is representative of a series of 10 cases of chronic wounds on dermatoporoetic legs.

Example 3: Estrogenic Effect

The expression of the messenger RNAs of the different ESRR and ER estrogen receptors, the estrogen ligand responsive finger protein (EFP), and the production aromatase enzyme estrogens are increased in skin exposed to TCDD. FIG. 10a shows the RT PCR reports on human skin fragments containing 1800 ppt of TCDD measured by gas chromato graphy / mass spectrometry) in comparison with the controls: the exposure of a man to large doses of TCDD increases significantly. in the skin the expression of messenger RNAs of estrogenic receptors and associated proteins, as well as aromatase, thus corresponding to a state of hyperoestrogeny. Figure 10b shows that in these skin fragments a strong ectopic and focal expression of the ER alpha receptor protein occurs in the pilosebaceous structures indicating that the local TCDD induction of a hyperestrogenic state is focused on a skin structure that is known to be hormone-dependent. Immunohistochemistry reveals a strong and highly focal expression of the estrogen receptor alpha ERa at the level of pilosebaceous structures.

Figure 11 shows the increase in the concentration of estradiol (E2) in human skin exposed to TCDD, determined by Elisa on human skin fragments containing 1800 ppt of TCDD, (measured by gas chromatography / mass spectrography). comparison with controls (f <40 women under 40, f> 40 women over 40, males 50). These results demonstrate for the first time in humans the local induction by TCDD of a hyperestrogenic state focused on a cutaneous structure that is known to be hormone-dependent.

Example 4 Anti-Infection Defense

As shown in Figure 12, the colonization of human skin by staphylococcus sp (CFU number, colony forming units, per cm2) is greatly reduced in case of exposure to TCDD. This figure shows the values obtained on 4 samples at 1 month interval in a human subject expressing more than 900 ppt of TCDD in the skin, as measured by gas chromatography / mass spectrography. The same effect is observed on colonization by p-acnes, and illustrated by Figure 13.

EXAMPLE 5 In Vitro Methods for Identifying Ligands According to the Invention: AhR Activation Tests Figures 14,15 & 16 show the activation of AhR in HEP G2 cells and normal human keratinocytes (NHK) stably transfected with the TATA-Luc plox-XRE lentivector.

HepG2 cells are cultured in DMEM (Gibco) + 10% Fetal Bovine Serum + Penicillin + Streptomycin, NHK in SFM keratinocyte medium (Gibco) + Penicillin + Streptomycin. At 0, HepG2 cells are seeded in 12-well plates at approximately 30,000 cells / cm 2. NHKs are seeded in 6-well plates at approximately 15,000 cells / cm 2. After 24h the medium is replaced by fresh medium and the cells are transduced by the lentivector plox-XRE TATA-Luc. After 48h the cells are firstly subcultured and maintained in culture and secondly tested for their reactivity to TCDD.

The tests are performed using Promega's "luciferase reporter assay System" kit. At 0, the cells are inoculated at a density of about 60% confluency and then treated with the test substance diluted to different concentrations in the appropriate culture medium. At D2 the cells are lysed in CLB buffer, the lysate is clarified by centrifugation from 5 'to 10,000 x g. The luciferase activity is measured in 20 microliters of lysate as recommended by the supplier using the luminoskan luminometer (Thermo). After 24h treatment with 10 nM TCDD, a 100-fold induction of luciferase activity was observed in TATA-Luc HepG2-plox-XRE cells and 10-fold in TATA-Luc NHK-plox-XRE cells.

FIG. 14 shows AhR activation of HEP G 2 cells by different ligands according to the invention. The values represent the percentage of activation relative to that obtained with the TCDD O.O1μM, by the concentration in the most active μM in the range tested for each substance. It is found that ethyl β-carboline 3-carboxylate (B-CCE), tryptophan, rutaecarpine, hispidine and FICZ have the most important activities.

Figure 15 shows, with the same representation, the activity of the same ligands on normal human keratinocytes. We see similar results. - Potentiation test by bacterial flora

FIG. 16 shows the AhR activation of HEP G 2 cells by various extracts of common bacteria from human skin in the presence of tryptophan (trp) Fl: flora G + F2: G + PA flora: P. acnes SE: S. epidermidis. The activation value 25.00 on the ordinate corresponds to 1/6 of the AhR stimulation by TCDD 1OnM in the same system (157.00). It can be seen that, in particular with P. acnes, an increase in activation is observed with the addition of tryptophan. These observations illustrate the concept of AhR activation by tryptophan, in part by interaction with surface flora.

- Test of CYPlAl activity

The tests are performed using Promega's "P450-GLO assay" kit. At 0, the cells are inoculated at a density of about 60% confluency and then treated with diluted test substance at different concentrations in the appropriate culture medium. At D2 the Cyp IA1 activity is measured as recommended by the supplier using the non-lytic cell monolayer protocol. After 24h treatment with 10 nM TCDD, 250-fold induction of Cyp IA1 activity was observed in HepG2 cells and 20-fold in NHK cells, as shown in Figure 17. Figure 17 similarly shows the induction of this dependent AhR biological activity by ligands according to the invention, carvedilol, B-CCE and rutaecarpine.

As will be appreciated by those skilled in the art, the test results set out above illustrate a methodology for selecting candidate molecules for use in the manufacture of a composition according to the invention.

Claims

1. A composition for topical use, intended to induce transiently the activation of AhR receptors of cutaneous tissues, said composition comprising an active substance selected from AhR agonist ligands with short half-life and in situ precursors of said ligands .

2. Composition according to claim 1, characterized in that said active substance is chosen from endogenous ligands of AhR.

3. Composition according to claim 1, characterized in that said active substance is a precursor chosen from the metabolic proligands of said AhR ligands.

4. Composition according to claim 1, characterized in that said active substance is an activatable precursor ligand of AhR under the effect of a physical agent, in particular under the effect of UV radiation or an endogenous agent or exogenous present in the treated pathology.

5. Composition according to claim 1, characterized in that said active substance has:

An ability to activate the AhR receiver,

A capacity of modulation of an AhR regulated gene,

A half-life in the short human organism, in particular between 2 hours and 96 hours, and more specifically between 6 and 24 hours.

- A measurable positive effect on a recognized criterion of sebaceous hyperactivity, wound healing and dermatoporosis, bacterial colonization of the skin, and / or estrogen deprivation.

6. Composition according to any one of the preceding claims characterized in that said ligands have half-lives in the human body between 2h and 96h, and more particularly between 6h and 24h.

7. Composition according to one of claims 1-6, for the treatment and / or prevention of diseases involving hypertrophy of the sebaceous glands.

8. Composition according to one of claims 1-6 for the treatment and / or prevention of bacterial skin infections.

9. Composition according to one of claims 1-6, for the treatment and / or prevention of diseases involving cutaneous colonization by bacterial species, including Propionebacterium acnes and Staphylococcus aureus.

10. Composition according to one of claims 1-6 for the treatment and / or prevention of cutaneous function disorders related to estrogen deprivation.

11. Composition according to one of claims 1-6 for the treatment and healing of skin wounds.

12. Composition according to one of claims 1-6, for the treatment and / or prevention of cutaneous atrophy, particularly in the context of a dermatoporosis.

13. Composition according to any one of the preceding claims, characterized in that said active substance is chosen from the group consisting of:

catechin

3-Epigallocatechin gallate

quercetin

apigenin

silibinin

resveratrol

yangonin

tryptophan

Indole-3-arbinol

tryptamine kynurenine

6-Formylindolo [3,2-b] carbazole (FICZ) and alkyl or alkenyl derivatives of the formyl group

3,3'-diindolylmethane

Trythantrine

Malassézine

indirubin

Indigo

ITE [2- (methyl h-indole-3'-carbonyl) -thiazole-4-carboxylate]

YH439 {(Isopropyl-2- (1,3-dithioethan-2-ylidene) -2- [N- (4-methylthiazol-2-yl) carbamoyl] acetate}

Hispidine alpha-Naphthoflavone beta-Naphthoflavone

ellipticine

carvedilol

triclabendazole

Rutaecarpine β-Carboline ethyl 3-carboxylate

Esomeprazole.