EP2097075A1

EP2097075A1 - Use of adapalene for modulating the expression of cd1d or il-10

Info

Publication number: EP2097075A1
Application number: EP07858717A
Authority: EP
Inventors: Brigitte Dreno
Original assignee: Galderma Research and Development SNC
Current assignee: Galderma Research and Development SNC
Priority date: 2006-11-20
Filing date: 2007-11-19
Publication date: 2009-09-09
Also published as: US20100130613A1; WO2008062132A1

Abstract

The invention relates to the use of adapalene for modulating the expression of CD1 d ou IL-10 in an inflammatory acne lesion.

Description

Use of adapalene to modulate the expression of CD1d or IL-10.

The invention relates to the use of adapalene to modulate the expression of CD1d or IL-10, particularly in patients with acne.

Acne is a chronic disease related to the hormonally dependent inflammation of the pilosebaceous follicle, which involves three well-defined steps (Cunliffe WJ et al., Br J Dermatol 2000: 142: 1084-1091, Gollnick HP et al. Dermatol 1991: 18: 489-499 The first stage, which usually begins at puberty, corresponds to the stimulation of sebaceous gland production, inducing hyper seborrhea.The second stage corresponds to the formation of micro comedones considered as the first elemental lesion of acne caused by abnormalities of proliferation, adhesion and differentiation of keratinocytes in the lower part of the sebaceous duct of the hair follicle.

The third step is the formation of inflammatory acne lesions in which Propionibacterium acnes (P. acnes), an anaerobic bacterium, plays an essential role.

The treatments used against acne are intended to curb the development of bacteria with antiseptics such as benzoyl peroxide or antibiotics, or to reduce the secretion of sebum (retinoids). Used only in case of severe acne, the oral treatments break down into three categories: antibiotics (tetracycline and erythromycin), retinoids (especially represented by isotretinoin) and hormonal treatments combining estrogen and progestin.

The inventors have surprisingly demonstrated that adapalene behaves as a modulator of the cytokines of the skin and makes it possible in particular to increase the innate and acquired immune responses of the skin by its action on two particular cytokines, CD1d and IL-10.

Indeed, the inventors have further explored the properties of this active ingredient in order to improve its therapeutic efficacy and highlighted the mechanisms of action of adapalene in the treatment of acne. An object of the present invention thus relates to the use of Adapalene, or a pharmaceutically acceptable salt thereof, for preparing a medicament for increasing the expression of CD1 d at the level of an inflammatory skin lesion, by particularly at the level of an inflammatory acne lesion. In particular, the invention is directed to the use of adapalene in a patient whose inflammatory acne lesions under-express CD1d.

Another object of the present invention is the use of Adapalene, or a pharmaceutically acceptable salt thereof, for preparing a medicament for decreasing the decrease in IL-10 intraperatinocyte production.

In particular, the invention is directed to the use of adapalene in a patient whose acne epidermal cells over-produce IL-10.

The target patients are preferably patients whose inflammatory acne lesions under-express CD1d and / or patients whose acne epidermal cells, especially keratinocytes, over-produce IL-10.

Another object of the invention is the use of adapalene to prepare a medicament for modulating the immune response of the epidermis by increasing the expression of CD1 d and / or decreasing the expression of IL-1. 10 by the cells of the acne epidermis, in particular the keratinocytes

In the previously described uses, the drug is preferably applied topically and may be in the form of a gel, lotion or cream. The invention therefore relates to pharmaceutical compositions comprising in a physiologically acceptable medium adapalene or a pharmaceutically acceptable salt thereof.

The invention therefore also relates to a method for inducing an increase in the expression of IL-10 and / or decreasing the expression of CD1d by the epidermal cells of a patient in need of treatment, characterized in that said patient is administered Adapalene or a pharmaceutically acceptable salt thereof.

The details of the invention will be set forth in the detailed description which follows. Adapalene is a chemically stable derivative of naphthoic acid. The chemical formula of this derivative is 6 [3- (1-adamantyl) -4-methoxyphenyl] -2-naphthoic acid. A method for preparing this compound is described in patent EP358574. Adapalene is a second-generation topical retinoid (such as tazarotene) that selectively binds to RARγ subtypes (found mainly in the epidermis) and RARβ (found mainly in dermal fibroblasts) of the nuclear acid receptor. retinoic acid (RAR), activating the genes responsible for cell differentiation. On the other hand, it does not bind to the binding proteins of cellular retinoic acid. However, at the clinical level, adapalene also has activity on superficial inflammatory lesions (Millikan LE, Int J Dermatol 2000: 39: 784-788). The mechanisms of anti-inflammatory activity seem in particular related to the modulation of non-specific immunity. Thus, adapalene inhibits the oxidative metabolism of arachidonic acid via the lipoxygenase pathway, the chemotaxis of neutrophils and the production of free radicals. It also inhibits the production of leukotrienes by the lipoxygenase pathway.

The inventors furthermore identify, in the context of the present invention, two molecules, expressed by the cells of the acne epidermis, playing an essential role in the development of cutaneous inflammation: CD1d and IL-10.

CD1d:

CD1d belongs to a conserved family of non-polymorphic cell surface glycoproteins acting as antigen presenting molecules. These glycoproteins are structurally, and in terms of distance, linked to the proteins of the classical class I major histocompatibility complex. The CD1d molecules are capable of presenting lipid and glycolipid antigens to NKT cells. The expression CD1d is rapidly induced on the keratinocytes of the normal skin when the latter undergoes a physical trauma which destroys its barrier function (Aderem A. et al., Nature 2000: 406: 782-787). This shows that keratinocytes are able to function as unconventional antigen presenting cells and activate NKT cells by presenting lipid antigens using CD1 d. IL-10:

Human IL-10 is a 19 kDa lymphokine produced by various cell types such as B and T cells and monocytes. In normal human skin, this cytokine is not expressed in keratinocytes, but its expression can be induced by UVB irradiation or antigenic stimulation. In vitro, normal human keratinocytes express the mRNA encoding IL-10. The expression of IL-10 is increased after stimulation with alpha-MSH. This cytokine exerts immunosuppressive and anti-inflammatory effects, produced in particular via an inhibitory effect on the production of interferon-gamma (Suh DH et al., Eur J Dermatol

2002: 12: 139-144.).

In the context of the present invention, the inventors have shown that adapalene induces a decrease or significantly decreases the expression of CD1 d by the cells of the acne epidermis, in particular the keratinocytes, thus increasing their cell function. antigen presenters vis-à-vis NKT cells. The antigens in question are in particular the lipid, glycolipid and bacterial antigens present in the epidermis or the follicle, and among them the P. acnes antigens.

The inventors have also demonstrated that adapalene induces a decrease or significantly decreases the expression of IL-10 in the epidermis of normal skin and in acne lesions, which facilitates activation of T cells by increasing interactions between Langherans cells and T lymphocytes

These modulations, and in particular the decrease in expression, make it possible to increase the interactions between dendritic cells and T lymphocytes and reinforce the antimicrobial activity against P. acnes. By modulation is meant an effect on the expression or activity of IL-I gene ¹ 10 and / or CD1d or their respective promoter or alternatively on the expression of these genes occurs.

By expression product of the gene is meant proteins or any product resulting from the transcription and / or translation of said gene. By activity is meant biological activity. By promoter activity of the gene is meant the ability of the promoter to induce the transcription of the DNA sequence under the control of said promoter.

The invention thus relates to the use of adapalene to prepare a composition for modulating the immune response of the epidermis by increasing the expression of CD1d and / or decreasing the expression of IL-10 by the acne epidermis, in particular keratinocytes. Indeed, as mentioned in the examples, under the action of adapalene, the expression of CD1d is significantly increased in the cells of the epidermis and in particular in the keratinocytes, thus increasing their function as antigen-presenting cells. Similarly, I ¹ II 10 being an immunosuppressive cytokine having an inhibitory role in the activation and proliferation of T cells, the decrease of this cytokine induced adapalene increases immune responses.

A particular object of the invention relates to the use of Adapalene, or a pharmaceutically acceptable salt thereof, for preparing a pharmaceutical composition for increasing the expression of CD1d at the level of a cutaneous lesion, in particular at level of an inflammatory acne lesion. By increase, it is meant an increase, an over-expression, an increase in the level of expression of the CD1d gene or an overproduction of its expression product (mRNA, protein).

Another particular object of the invention relates to the use of Adapalene, or a pharmaceutically acceptable salt thereof, for preparing a pharmaceutical composition for decreasing IL-10 intraperatinocyte production.

By decrease is meant a decrease, a reduction, a retro-regulation, a inhibition or a suppression (total or partial) of the expression of the gene of IL-10 or its product of expression (mRNA, protein) . By partial inhibition, it can be understood a reduction of at least 25% of the activity and preferably 35% even more preferred of at least 50%.

Preferably, the pharmaceutical compositions used here are intended to be used, in order to treat acne, optimally, in patients whose lesions inflammatory acne-like sub-express CD1 d and / or in patients whose acne epidermal cells, especially keratinocytes, over-produce IL-10.

The pharmaceutical composition comprises adapalene or a pharmaceutically acceptable salt thereof in a physiologically acceptable medium.

To give an order of magnitude, the composition according to the invention advantageously comprises between 0.001 and 5% and advantageously between 0.01 and 1% by weight of adapalene relative to the total weight of the composition, preferably between 0.1 and 0.4% by weight. adapalene weight, more preferably 0.3% by weight of adapalene.

The composition may furthermore comprise any additive usually used in the cosmetic or pharmaceutical field, such as penetrating agents, wetting liquid surfactants, sequestering agents, antioxidants, sunscreens, preservatives, fillers, electrolytes, humectants. , dyes, bases or usual acids, mineral or organic, perfumes, essential oils, cosmetic active ingredients, moisturizers, vitamins, essential fatty acids, sphingolipids, self-tanning compounds such as DHA , soothing and protective agents for the skin such as allantoin. Of course those skilled in the art will take care to choose this or these optional additional compounds, and / or their amount, such that the advantageous properties of the composition according to the invention are not, or not substantially impaired.

These additives can be present in the composition in a proportion of 0 to 20% by weight relative to the total weight of the composition.

Such a composition may be administered by any known route, conventionally associated with the treatment (using adapalene) of a dermatological disorder, Le., Topically, enterally, parenterally or ocularly. Preferably, the treatment is administered topically. The pharmaceutical composition is preferably in the form of a gel, lotion or cream.

Conventionally the composition is in the form of an aqueous gel. By aqueous gel is meant a composition containing, in an aqueous phase, a viscoelastic mass formed from colloidal suspensions (gelling agent).

By way of non-limiting example, there may be mentioned gelling agents of the family of polyacrylamides such as the mixture Sodiumacryloyldimethyltaurate copolymer / isohexadecane / polysorbate 80 sold under the name Simulgel 600 by the company Seppic, the polyacrylamide / isoparaffin mixture C13-14 / laureth- 7, for example, that sold under the name Sepigel 305 by the company Seppic, the family of acrylic polymers coupled to hydrophobic chains such as the PEG-150 / decyl / SMDI copolymer sold under the name Aculyn 44 (polycondensate comprising at least as elements, a polyethylene glycol of 150 or 180 moles of ethylene oxide, decyl alcohol and methylene bis (4-cyclohexylisocyanate) (SMDI), at 35% by weight in a mixture of propylene glycol (39%) and water (26%)), the family of modified starches such as modified potato starch sold under the name Solanace Structure or mixtures thereof. The preferred gelling agents are derived from the family of polyacrylamides such as Simulgel 600 or Sepigel 305 or mixtures thereof.

The gelling agent as described above may be used at preferential concentrations ranging from 0.1 to 15% and, more preferably, from 0.5 to 5%.

The compositions are suitable for the treatment of inflammatory lesions of any type of acne, namely in particular vulgar, comedonal, polymorphic, rosacea acne, nodulocystic acne, acne conglobata, senile acne, secondary acne such as solar acne, medication, occupational or occupational acne.

LEGEND OF FIGURES Figure 1. Expression of CD1 d in explants of healthy human skin (a) and in acne skin explants (b) after 24 hours of incubation with 10 ^{~ 7} M and 10 ^'6 M of adapalene or control medium (n = 8 in each group). Bars and error bars represent mean and SEM. ^* p <0.05 [matched Wilcoxon test adapalene vs. witness].

Figure 2. Expression of IL-10 in explants of healthy human skin (a) and in acne skin explants (b) after 24 hours of incubation with 10 ^{~ 7} M and 10 ^'6 M of adapalene or control medium (n = 8 in each group). Bars and error bars represent mean and SEM. ^* p <0.05 [matched Wilcoxon test adapalene vs. witness].

EXAMPLE

Skin biopsies

Inflammatory lesions of acne: a skin biopsy (1 x 1.5 cm) was obtained under local anesthesia from a superficial inflammatory lesion (papule) of the back in 8 patients suffering from moderate acne and not undergoing no topical (for 15 days) or general treatment (for one month, or three months for isotretinoin). The collected fragments were immediately cultured. Healthy human skin: Healthy skin fragments from 8 healthy donors were obtained from breastplasty (plastic surgery) or foreskin (infant surgery).

Sampling technique

Fragments (4 mm in diameter) from inflammatory skin biopsies, considered an inflammatory model, and healthy skin, were incubated at 37 ° C. in a humid atmosphere and in the presence of 5% CO ₂ for 24 hours in medium. KGM culture without hydrocortisone (PromoCell GmbH, Heidelberg, Germany). The culture medium was supplemented with two different concentrations of adapalene (Galderma, Sophia Antipolis, France): 10 ^-7 M and 10 ^-6 M. The culture medium alone served as a control medium. After incubation, the explants were removed from the culture medium and then frozen in liquid nitrogen for immunohistochemical study. Culture supernatants were stored at -80 ° C for subsequent measurement of protein secretion by an ELISA assay technique.

Antibodies used for immunohistochemistry

The primary antibodies [an anti-human IL-10 monoclonal mouse IgGI (Diaclone, Besançon, France), a rabbit polyclonal anti-CD1d antibody (Santa Cruz Biotechnology Inc.)] were used, in immunohistochemistry, to 2 μg / ml (anti-IL-10 antibody) or 5 μg / ml (anti-CD1d antibody). Negative control mouse IgGI at 2 μg / ml (DAKO, Copenhagen, Denmark) or secondary antibody alone were used as controls.

Cutaneous Section Immunohistochemistry Samples of frozen specimens (5 μm thick) were cut with a cryostat and then fixed with acetone at 4 ° C for 10 minutes. After saturation of non-specific sites with TBS (Tris-buffered saline) / BSA (Bovine Serum Albumin) at 0.1%, for 30 minutes at room temperature, the slides were incubated for 30 minutes with the primary antibodies at room temperature, in a humid atmosphere. and in the dark. The sections were then washed again for 15 min in 0.1% TBS / BSA / 0.05% Tween 20 and then incubated with a biotinylated secondary antibody (DAKO) for 30 min, at room temperature and in a humid atmosphere, then washed and incubated. with streptavidin / peroxidase (DAKO) for 30 min at room temperature and in a humid atmosphere. After a final wash, the reaction products were revealed using the peroxidase AEC (hydrogen peroxide / aminoethylcarbazole) (DAKO) substrate for 5 min. The slides were then counter-stained with Mayer's haemalun and observed using a Leitz microscope. The labeling intensity was quantified on a semi-quantitative scale: zero (0), low (1), medium (2) and high (3). For each slide, the marking intensity was determined by an average of three fields. In addition, for each cytokine or receptor studied, an average was established on 8 healthy donors or 8 acne patients in each of the "control", "adapalene 10 ^{'and 7} M" groups. "Adapalene 10 ^'6 M". The reading was performed by two independent investigators.

Cytokine Assays IL-10 concentrations were measured in culture supernatants using a solid-phase Enzyme Linked-Immuno-Sorbent Assay (ELISA) sandwich kit obtained from BioSource (International Inc, Ca, USA).

The slides were read at 450 nm using an Emax reader (Molecular Devices, CA, USA). The sensitivity of the assay IL-10 was less than 1 pg / ml and the minimum detectable level of IL-I ¹ 10 was 7.8 pg / ml.

For each cytokine studied, an average was established on 8 healthy donors or 8 acne patients in each of the "control", "adapalene 10 ^{~ 7} M" and "adapalene 10 ^'6 M" groups.

Statistical analysis

All results are expressed as mean +/- SEM. The statistical significance of differences between the values (compared to the control medium) was determined using the matched Wilcoxon test. A difference between the values was considered statistically significant for p <0.05.

RESULTS

Expression of CD1d

Explorers of Healthy Human Skin: CD1d expression is low to moderate (1.7 +/- 0.4) in all epidermis of healthy human skin incubated in control medium. Adapalene significantly decreased the expression of CD1 d in the epidermis of healthy donors compared to the control medium (p = 0.023 with 10 ⁷ M adapalene, p = 0.035 with 10 ⁶ M of adapalene) (Fig. 1 a).

Acne skin explants: Significantly lower expression (0.9 +/- 0.2) of CD1d is detected in all epidermis of acne biopsies compared to healthy skin (p = 0.002). The expression of CD1 d is significantly increased with both concentrations of adapalene, ranging from 0.9 (+/- 0.2) in control medium to 1.5 (+/- 0.5) (p = 0.034), in the presence of 10 ^'7 and 10 ^{~ 6} M of adapalene (Fig. 1b).

Expression of IL-10

Healthy human skin explants: the average expression of the cytokine is low in the epidermis incubated with a control medium (1 .3 +/- 0.7). IL-10 is mainly expressed by the cytokines of the basal layer of the epidermis. The addition of adapalene significantly decreases the expression of IL-10 in a dose-dependent manner. The inhibitory effect of 10 ^-6 M of adapalene on the expression of IL-I ¹ 10 is significant compared to the control medium (p = 0.016) (Fig. 2a).

Acne skin explants: The expression of the cytokine is average (1.9 +/- 0.4) in the basal layer of the acne epidermis incubated with the control medium. It is slightly stronger in the acne epidermis (1 .9 +/- 0.4) than in the healthy epidermis (1.3 +/- 0.7). The expression of IL-10 decreases significantly and dose-dependent in the presence of adapalene. Thus, the expression intensity of IL-10 decreases from 1.9 (+/- 0.4) to 0.9 (+/- 0.2) with 10 ^'7 M adapalene (p = 0.024) and up to 0.6 ( +/- 0.2) with 10 ^{~ 6} M adapalene (p = 0.0199) (Fig 2b).

Secretion of IL-10 in culture supernatants:

To determine whether adapalene influences the secretion of IL-10, cytokine levels were measured in explant supernatants.

Human Healthy Skin: The mean level of IL-10 in healthy human skin explant supernatants incubated with control medium was 17.8 μg / ml (+/- 20.9). The addition of 10 ^-7 M adapalene increases the secretion of IL-10. No modulation is observed with 10 ^{~ 6} M adapalene.

Acne Skin Explants: The average level of IL-10 in skin explant supernatants from acne patients incubated with control medium is 5.7 μg / ml (+/- 7.1) less than that of healthy skin supernatants. There is no modulation of IL-10 secretion with adapalene. Indeed, the levels of IL-10 were 6.1 μg / ml (+/- 7.2) and 4.0 μg / ml with 10 ^{~ 7} M and 10 ^{~ 6} M adapalene respectively.

The invention confirms, on the one hand, that CD1d is exhaled by healthy human skin keratinocytes from a region of the body other than the scalp (Aderem A. et al., Nature 2000: 406: 782-787). The results presented in the present invention show a high level of expression in superficial keratinocytes located near the lipid-rich stratum corneum. The inventors have demonstrated expression of CD1d in the other layers of the epidermis including basal keratinocytes (Kawai K et al., J Dermatol Sci 2002: 30: 185-194). These results indicate that basal and supra-basal keratinocytes are presenting cells involved in the binding of glycolipid antigens to activate NKT cells (Chatelain R. et al., M. Arch Dermatol Res 1998: 290: 477-482. Expression of CD1 d is slightly decreased in inflammatory acne epidermis and adapalene induces a significant increase in CD1d expression by acne epidermal cells, particularly keratinocytes, thereby increasing their cell function However, the keratinocytes may function in the skin as non-occupational antigen-presenting cells (Koreck A et al., Dermatology) for antigen-presenting agents for lipid and glycolipid antigens vis-à-vis NKT cells. 2003: 206: 96-105) The presentation of a lipid antigen in the skin by CD1 d-expressing cells is of primary importance in the host defense against pathogens By modulating, preferably by increasing the expression of CD1d by acne epidermal cells, especially keratinocytes, adapalene stimulates their NK activity against lipid and / or bacterial antigens present in the epidermis or the follicle and among them P. acnes.

The level of IL-10 expression is not significantly different in the epidermis of healthy skin and acne lesions. The invention shows that adapalene significantly decreases the expression of IL-10 in the epidermis of normal skin and in acne lesions which facilitates the activation of T cells by increasing interactions between Langherans cells. and T lymphocytes. Indeed, IL-10 is an immunosuppressive cytokine that inhibits activation and proliferation of T cells via inhibition of antigen presenting cells including Langerhans cells (Moore KW et al., Annu Rev Immunol 2001: 19: 683-765). Inhibition of ICAM-1 and other accessory molecules by IL-10 appears to be an important mechanism inhibiting the antigen-presenting function of Langerhans cells (Chatelain R. et al., M. Arch Dermatol Res 1998: 290 : 477-482). Thus, on the basis of the known effects of IL-10 on inflammatory responses, the decrease of its expression with adapalene makes it possible to increase both innate and acquired immune responses.

Claims

1. Use of Adapalene, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for increasing the expression of

CD1d at the level of an inflammatory acne lesion.

2. Use according to claim 1, wherein said patient is an individual whose inflammatory acne lesions sub-express CD1d.

3. Use of Adapalene, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for decreasing IL-10 intrakeratinocyte production.

The use of claim 3, wherein said patient is an individual whose acne epidermal cells over-produce IL-10.

Use according to claim 1, wherein said patient is an individual whose inflammatory acne lesions under-express CD1d and / or whose acne epidermal cells over-produce IL-10.

6. Use according to any one of the preceding claims, characterized in that the acne inflammatory lesions selected from acne vulgaris, comedonal acne, polymorphic acne, nodulocystic acne, acne conglobata and acne. secondary acne.

7. Use of adapalene to prepare a drug to modulate epidermal immune response by increasing CD1 d expression and / or decreasing IL-10 expression by acne epidermal cells , in particular keratinocytes

8. Use according to any one of the preceding claims, characterized in that the medicament is applied topically.

9. Use according to claim 7, characterized in that the drug is in the form of a gel, a lotion or a cream.

10. Pharmaceutical compositions comprising in a physiologically acceptable medium adapalene or a pharmaceutically acceptable salt thereof.

A method for inducing an increase in IL-10 expression and / or decreasing expression of CD1 d by epidermal cells of a patient in need of treatment, characterized in that it is administered to said patient Adapalene or a pharmaceutically acceptable salt thereof.