New composition for the treatment and/or prevention of vitiligo

Technical Field

[0001] The present invention relates to composition for the treatment and/or prevention of vitiligo.

Background Art

[0001] Vitiligo is a skin condition characterized by the appearance of white spots on the hands, feet, face or any other part of the body. These spots are caused by depigmentation, which corresponds to the disappearance of melanocytes. This depigmentation can be more or less important and the white spots of variable sizes. In some cases, the hair or hair growing inside the depigmented areas may also be white.

[0002] Vitiligo is neither contagious nor painful, but it can cause significant psychological distress. Note that people with dark skin suffer particularly because this condition is even more visible in them. However, the impact on quality of life is significant for all affected individuals. Vitiligo is still insufficiently treated by doctors.

[0003] Vitiligo affects approximately 0.5 to 2% of the world's population, regardless of ethnicity or gender. It generally appears around the age of 10 to 30, half of those affected are affected before the age of 20. Vitiligo is therefore quite rare in young children. It affects both men and women equally.

[0004] There are several types of vitiligo:

[0005] 1) Segmental vitiligo, localized on only one side of the body, for example on part of the face, upper body, leg or arm. This form of vitiligo appears more often in children or adolescents. This form appears rapidly in a few months, then generally stops evolving. [0006] 2) Non-segmental vitiligo, which appears in the form of spots that are often more or less symmetrical, affecting both sides of the body. The evolution is unpredictable, the spots can remain small and localized or spread rapidly.

[0007] Usually, the disease progresses at an unpredictable rate and may stop or spread continuously or most often in flare-ups. Vitiligo can thus evolve in phases, the aggravations sometimes occurring after a psychological or physical triggering event. In very rare cases, the plaques go away on their own. These people are also more likely to suffer from other autoimmune diseases.

[0008] Vitiligo is a multifactorial polygenetic disease causing the loss of melanocytes. Although genetic studies have discovered over 50 vitiligo susceptibility loci, the pediatric to adult shift in vitiligo age-of-onset over the last 30 years emphasizes the key role of environmental triggers and their contribution to initiation and/or exacerbation of vitiligo. These triggers include UV radiation, trauma, or chemical stress as well as pollutants.

[0009] To date, vitiligo treatments often require many months and are mainly effective for attacks on the face. Failures remain numerous, especially for bony prominences and hands and feet. The reference treatment combines phototherapy (at best UV-B) with topical treatments with topical corticosteroids or calcineurin inhibitors. Very active forms can benefit from cortisone mini-pulses. Finally, localized and stable forms and segmental vitiligo are good indications for surgical treatment, in particular by epidermal cell suspension grafting.

[0010] There is therefore a strong demand for a therapy that is more effective, well tolerated and more affordable. Summary of the invention

[0011] House dust mites (HDM) are a component of normal skin microbiota.

[0012] Classically, HDM drives Th2 responses but can also stimulate innate immunity through TLR activation. HDM, ubiquitous and perennial indoor respiratory allergens, are the undisputed trigger of allergic reactions such as wheezing and respiratory diseases including allergic rhinitis and asthma. HDM contains potent allergens, proteases and components of both bacterial (LPS) and fungal (0-D-glucan) cell wall, all which are known allergic triggers and powerful immunomodulators.

[0013] Dermatophagoides pteronyssinus (Der p) is a major HDM allergen with potent protease activity.

[0014] The inventors now established that HDM caused a specific disruption of E- cadherin between primary keratinocytes. This effect was dose-dependent and was not observed for other tight-junction proteins. Moreover, the keratinocytes from vitiligo patients were be more sensitive to HDM compared to healthy keratinocytes, similar to what has previously been reported in response to oxidative stress, confirming the intrinsic fragility of vitiligo cells to environmental stimuli.

[0015] The inventors further demonstrated that HDM-induced changes were driven by the major HDM allergen, Der pl, through MMP-9 induction but not MMP-2. Evidence of HDM-induced flying melanocytes, prior loss of E-cadherin and preliminary increased MMP-9 activity following HDM stimulation were confirmed using a 3D skin model and ex-vivo human skin biopsies.

[0016] Using cysteine protease inhibitor E64 (inhibiting Der pl) and monoclonal antibody against MMP-9, the inventors significantly restored E-cadherin levels and inhibited melanocyte detachment. [0017] Together these results highlight contribution of ubiquitous HDM as a trigger of melanocyte loss in susceptible individuals with fragile and/or genetically susceptible skin and MMP-9 and Der p 1 inhibitors as potential promising new therapeutic targets in vitiligo treatment.

[0018] Accordingly, a first object of the invention is directed to a composition comprising at least one mite peptidase 1 or Matrix metallopeptidase 9 (MMP-9) antagonist, and optionally a pharmaceutically acceptable carrier for use for preventing and/or treating vitiligo in a subject.

[0019] In another aspect, the present invention provides a method for preventing and/or treating vitiligo in a subject in need thereof, comprising providing to the subject an effective amount of a composition comprising at least one mite peptidase 1 or Matrix metallopeptidase 9 (MMP-9) antagonist, and optionally an pharmaceutically acceptable carrier.

Detailed description of the invention

[0020] As used herein, the mite peptidase 1 (Enzyme entry EC 3.4.22.65), also known as endopeptidase 1 (mite), is an enzyme found in various species of mites. This enzyme exhibits cysteine protease activity with broad endopeptidase specificity.

[0021] The various forms of peptidase 1 pertaining to individual mite species comprise the group 1 mite allergens. Following the naming conventions of allergens, these peptidase 1 variants include Der p 1 of the European house dust mite Dermatophagoides pleronyssinus: Der f 1 of the American house dust mite Dermatophagoides farinae: Eur m 1 of the Mayne's house dust mite Euroglyphus mayney and Pso o 1 of the sheep scab mite Psoroptes ovis.

[0022] Advantageously, the mite peptidase 1 is Der p 1 or Der f 1, preferably Der p 1. [0023] As used herein, a mite peptidase 1 antagonist is a compound inhibiting mite peptidase 1 protease activity from more than 50%, preferably from more than 75%.

[0024] Such a mite peptidase 1 antagonist are known from the skilled person and includes, as an examples, E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4- guanidino )butane), iodoacetate or iodoacetamide, cystatin (e.g. chestnut cystatin (CsC), egg white cystatin (EWC), Kiwifruit cysteine proteinase inhibitor 1 (KCPI1), and the antagonists disclosed in the international patent applications W02012004554 or WO201 1089396.

[0025] In a preferred embodiment, the mite peptidase 1 antagonist is a cystatin.

[0026] According to a preferred option, said cystatin is egg white cystatin. Such egg white cystatin may be in the form of an egg white extract.

[0027] According to another preferred option, said cystatin is phytocystatin, such chestnut cystatin (CsC) or Kiwifruit cysteine proteinase inhibitor 1 (KCPI1). Such phytocystatin may be in the form of an extract, like a chestnut extract or a kiwi seed extract.

[0028] According to another preferred embodiment, the mite peptidase 1 antagonist is one of the antagonists disclosed in the international patent applications W02012004554 or WO2011089396.

[0029] According to a preferred option, said mite peptidase 1 antagonist is selected in the group of compounds having the formula (I)

[0030] Wherein the substituents are as follows:

[0031] Advantageously, said mite peptidase 1 antagonist is selected in the group consisting of the compounds 12, 15, 17, 18 and 19.

[0032] Still preferably, said mite peptidase 1 antagonist is the compound 12 or 18, and preferably the compound 12 . [0033] Matrix metallopeptidase 9 (MMP-9), also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), is a matrixin. This protein belongs to the zinc- metalloproteinases family involved in the degradation of the extracellular matrix. In humans the MMP9 gene encodes for a signal peptide, a propeptide, a catalytic domain with inserted three repeats of fibronectin type II domain followed by a C-terminal hemopexin-like domain.

[0034] As used herein, a “MMP-9 antagonist” is compound inhibiting the MMP-9 protease activity from more than 50%, preferably from more than 75%.

[0035] Such an MMP-9 antagonist may correspond to an anti -MMP-9 antibody, as the one commercialized by ABCAM (Ab 142180) or a compound from the group comprising Z-PDLDA-NHOH (Z-Pro-D-Leu-D-Ala-NHOH) (CALBIOCHEM, catalog number 234140), MMP Inhibitor II (N-Hydroxy-l,3-di-(4-methoxybenzenesulphonyl)-5, 5-dimethyl-[l,3]-pip-erazine-2-carboxamide) (CALBIOCHEM,, catalog number 444247); MMP Inhibitor IV(HONH-COCH~CH~CO— FA-NH-) (CALBIOCHEM, catalog number 444271); MMP-2/MMP-9 Inhibitor I ((2R)-2-[(4- Biphenylylsulfonyl)amino]-3-phenylpropionic Acid) (CALBIOCHEM, catalog number 44424 l);MMP-2/MMP-9 Inhibitor II ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hy- droxy-3-phenylpropionamide)(CALBIOCHEM, catalog number 444249); MMP- 2/MMP-9 Inhibitor IV (SB-3CT)(CALBIOCHEM, catalog number 444274); MMP-9/ MMP-13 Inhibitor I (N-Hydroxy-I-(4-methoxyphenyl)sul-fonyl-4-(4- biphenylcarbonyl)piperazine-2-carboxamide) (CALBIOCHEM, catalog number 444252); MMP-9/MMP-13 Inhibitor II(N-Hydroxy-I-(4-methoxyphenyl)sul-fonyl-4- benzyloxycarbonylpiperazine-2-carboxamide) (CALBIOCHEM, catalog number 444253); GM6001 MMP Inhibitor (CHEMICON International, Inc., catalog number CC1000); Batimastat (BB94) (VERNALIS PIC); Marimastat (BB 2516) (VERNALIS PIC); Neovastat (AE-941) (AETERNA ZENTARIS INC ); and Prinomastat AG3340 (Pfizer Inc,).

[0036] According to a preferred embodiment, the composition of the invention comprised at least mite peptidase 1 antagonist.

[0037] ] As used herein “vitiligo” can correspond just as well to segmental vitiligo or to non-segmental vitiligo.

[0038] By “subject” is meant an animal, preferably a mammal such as a feline, a canine or a primate. According to a preferred embodiment, the subject is a human.

[0039] The expression "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar undesirable reactions, such as gastric upset, dizziness and the like when administered to a human. Preferably, as used herein, the expression "pharmaceutically acceptable" means approvable by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0040] The term "carrier" refers to a solvent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.

[0041] Such pharmaceutically acceptable carriers that can be used in the composition according to the invention are well known to one of skill in the art (Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed. , Pharmaceutical Press[2000]).

[0042] When appropriate, compositions of the invention may comprise any ingredient conventionally used in the fields under consideration, and particularly in cosmetics and dermatology.

[0043] Specific examples of ingredient include, but is not limited to, moisturizers and hydration agents, penetration agents, emulsifiers, natural or synthetic oil solutions, chelating agents, solvents, surfactants, gelling agents, emollients, fragrances, darkening or lightening agents, glitter, humectants, fillers, thickeners, waxes, odor absorbers, dyestuffs, coloring agents, powders such as mica or minerals, viscosity-controlling agents and water, powders, amino acids, polyamino acids and a salt thereof, sugar alcohol and alkylene oxide adducts thereof, lower alcohols, animal and plant extracts, nucleic acids, vitamins, enzymes, anti-inflammatory agents, additional antimicrobial agents such as antibiotics, preservatives, antioxidants, moisturizer, thickener, viscosity modifier, UV absorbers, adiaphoretics, pigments, dyes, flavors, pH adjusters, pearly sheen agents, wetting agents and the like. Lists of these excipients can be found in the patent application US 2016/0354294 Al. These are mere examples and components other than these can be added.

[0044] The pharmaceutical composition according to the invention may be suitable for local or systemic administration, in particular for oral, sublingual, cutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, topical, intra-tracheal, intranasal, transdermal, rectal, intraocular or intra-auricular administration. Preferably, the pharmaceutical composition according to the invention is suitable for cutaneous, oral, topical, intramuscular, intravenous, transdermal or subcutaneous administration. According to a particular embodiment, the pharmaceutical composition according to the invention is suitable for topical administration. The pharmaceutical composition according to the invention may be in the form of tablets, capsules, soft capsules, granulates, suspensions, emulsions, solutions, gels, pastes, ointments, creams, plasters, potions, suppositories, enemas, injectables, implants, patches, sprays or aerosols.

[0045] Thus, the form of the composition of the present invention is not particularly limited, and may take any form such as lotion, serum, suspension, emulsion, paste, cream, foam, ointment, gel, solid, powder and the like, spray or aerosol, tablet.

[0046] Now and preferably, the compositions of the invention will be suitable for topical administration.

[0047] When formulated for topical delivery, the composition of the invention may be formulated as cream, foam, a gel, a lotion or an ointment. Topical delivery means may include transdermal delivery (e.g. via an adhesive patch, iontophoresis or ultrasound device).

[0048] By “an effective amount” is meant an amount of pharmaceutical composition comprising at least one Der p 1 or Matrix metallopeptidase 9 (MMP-9) antagonist, which is sufficient to induce the regression or the disappearance of symptoms related to a condition caused by vitiligo. The doses used for the administration can be adapted as a function of various parameters, in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment. Naturally, the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend on the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the preferred dose can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

[0049] Advantageously, the composition of the invention is administrated to the subject to a dose from about 0.01 to about 100 mg of at least one Der p 1 or Matrix metallopeptidase 9 (MMP-9) antagonist per subject kilogram, preferably from about 0.1 to 10 mg/kg of said antagonist.

[0050] The following examples are given solely by way of illustration of the subject matter of the present invention, and in no way constitute a limitation thereof.

EXAMPLES

[0051] 1. HDM is present in healthy and vitiligo skin

[0052] Five vitiligo patients (n=5) with stable disease were recruited from the Department of Dermatology, L’Archet Hospital, CHU Nice and were enrolled in the study after informed, written consent was obtained. From vitiligo patients we obtained 4-mm skin punch biopsies from both lesional (L) and non-lesional (NL) sites.

[0053] Five healthy controls (n=5) were recruited from the same clinic. From healthy patients we obtained 4-mm skin punch biopsies from skin.

[0054] The two groups were matched for gender, age, location, and disease duration. The study was approved by the National ethics committee.

[0055] Section slides were fixed with 4% paraformaldehyde, permeabilized with Trisbuffered saline (PBS) in presence of 0.3% TRITON and blocked for 1 hour with PBS 5% BSA with 10% goat serum. Sections were then incubated overnight at 4°C with rabbit anti -/ Av pl (1/200, RAYBIOTECH). After three PBS washes, the skin was incubated with Alexa 594 goat anti-rabbit IgG (H+L). Sections were then mounted with Prolong Gold antifade reagent containing DAPI and images acquired using Nikon confocal AIR. [0056] Unexpectedly, the results established the presence of Der /?7-immunoreactive cells in healthy and vitiligo skin, at both non-lesional (NL) and lesional (L) sites.

[0057] 2. HDM induces biomarkers strongly associated with vitiligo in keratinocytes

[0058] The bodies of house dust mites contain many bio-reactive molecules which can penetrate the skin. However, little is known about how these molecules should modify skin function.

[0059] Accordingly, at the in vitro immune response of primary keratinocytes to Dermatophagoides pteronyssinus HDM was investigated.

[0060] Primary human keratinocytes were obtained from surgical samples of healthy abdominal skin (Department of Dermatology, L’Archet Hospital, CHU Nice) and cultivated in keratinocyte serum-free medium supplemented with bovine pituitary extract (30pg/mL), transferrin (lOpg/ml), insulin (5pg/ml), hydrocortisone (0.3pg/ml), epinephrine (0.39 pg/ml), CaCl (0.5mM) and epidermal growth factor (0.125 ng/ mL). Cells were maintained in a humidified atmosphere 5% CO2 at 37°C.

[0061] For the experiments, keratinocytes were stimulated with different concentrations of house dust mite (HDM) (1 pg/ml, 200pg/ml, and 400 pg/ml) for 24h.

[0062] Cell-free supernatants were harvested for measuring their cytokines and chemokines secretion in culture supernatants using PROTEOMEPROFILER™ multiplex assay, QPCR and ELISA. [0063] The figures 1A and IB show the measures of cytokines (IFNy), chemokines

(CXCL10, CXCL11, CXCL16), Serpin El and CXCR3B at mRNA or protein level respectively in keratinocytes stimulated in vitro for 24hr with 100 pg/ml HDM.

[0064] The experiments have demonstrated a HDM-induced, dose-dependent increase in secretion of innate chemokines (CXCL1, CXCL5, CXCL9, CXCL10, CXCL12), chemokine ligands (CCL3, CCL7, CCL17, CCL20) and pro- inflammatory/Thl/Th2/Thl7 cytokines (IL-la, IL-8, IFNy, MIF, GM-CSF, IL-5, IL-10, IL-24 and IL- 17 A) as well as proteases (Emmprin, MMP-9), protease inhibitors (Cy statin C, Serpin El), inhibitors of the Wnt pathway (DKK-1) and biomarkers of inflammation (uPAR).

[0065] The results established that the markers associated with vitiligo were strongly increased IFNy and IFNy-inducible chemokines (CXCL10, CXCL11), chemokine receptor CXCR3B and keratinocyte stress marker CXCL16 following stimulation of normal human keratinocytes (NHK) with 100 pg/ml HDM in vitro (Figure 1A for qPCR and Figure IB for ELISA assays)

[0066] These results suggest that HDM could be associated with vitiligo.

[0067] 3. HDM induces biomarkers strongly associated with vitiligo in ex vivo biopsies

[0068] Following these results on keratinocytes, the effect of HDM was investigated on ex-vivo using skin explants from healthy patients.

[0069] Skin from plastic surgery waste was used for the development of the ex vivo skin culture model. After subcutaneous fat was removed, we obtained 4-mm biopsies which was composed of dermis and epidermis. The biopsies were rapidly placed into a 0.4-pm TRANSWELL chamber maintained under semi-liquid culture conditions in skin long- term culture medium at 37°C in a 5% CO2 atmosphere. The biopsies were treated with of HDM lOOpg/ml.

[0070] Twenty-four hours later, the biopsy was used for RNA extraction and QPCR analysis.

[0071] The figure 1C shows the measures of chemokines (CXCL10, CXCL11, CXCL16), Serpin El and CXCR3B at mRNA level and protein level (CXCL16) in the human ex vivo biopsies stimulated with HDM.

[0072] The results showed a significant increase in CXCL10 mRNA and CXCL11 mRNA (Figure 1C). Although no increased of CXCL16 mRNA was detected at a significant increase of CXCL16 protein secretion in the explant supernatant was demonstrated (Figure 1C).

[0073] Together, these results indicate that the HDM can modulate not only NHK immune response in vitro but can also induce cutaneous production of ‘vitiligo-like’ mediators ex vivo in explanted skin.

[0074] 4. HDM induces disruption of tight junctions between keratinocytes

[0075] Presence of HDM has been detected in the skin of atopic and eczematous dermatitis however its link with cutaneous tight junction proteins remains to be explored. [0076] Accordingly, primary human keratinocytes were cultured as previously described before being stimulated with different concentrations of house dust mite (HDM) (50 pg/ml, pg/ml, and 100 pg/ml) for 24h.

[0077] Part of the cultures were then fixed with 4% paraformaldehyde, permeabilized with Tris-buffered saline (PBS) in presence of 0.3% TRITON and blocked for 1 hour with PBS 5% BSA with 10% goat serum. Sections were then incubated overnight at 4°C with mouse anti-E-cadherin antibody (1/200, BECTON DICKINSON). After three PBS washes, the skin was incubated with secondary antibodies, Alexa 488 goat anti-mouse IgG (H+L). Sections were then mounted with Prolong Gold antifade reagent containing DAPI and images acquired using NIKON confocal AIR.

[0078] The results have shown a dose-dependent disruption of E-cadherin protein in keratinocytes stimulated with HDM (50pg/ml or 100 pg/ml) for 24h, whereas E-cadherin mRNA expression remained unchanged.

[0079] Similar experiments were done on ex-vivo in human skin explant biopsies.

[0080] Part of the explant were fixed on section slides and incubated with mouse anti-E- cadherin antibody as previously described.

[0081] The figure 2 A shows the mean signal intensity quantitated for E-cadherin immunostaining in skin biopsies stimulated with HDM and in control.

[0082] Another part of the cultures was lysed in RIPA cell lysis buffer supplemented with protease inhibitors. Protein concentrations were measured using a BCA Protein Assay Kit. Protein lysates (30 pg) were separated by 10% SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. E-cadherin, ZO-1, occludin, Filaggrin, Claudin-1, P- cadherin, N-cadherin, actin and HSP90 were detected using mouse anti-E-cadherin antibody (1/1000, BECTON DICKINSON) or anti-E-cadherin (1/1000, SANTA CRUZ), rabbit anti-ZO-1 (INVITROGEN), anti-occludin, anti-Filaggrin, anti-Claudin-1, mouse anti-P-cadherin (SANTA CRUZ), mouse N-cadherin (SANTA CRUZ), mouse anti- HSP90 (SANTA CRUZ) and mouse anti-actin (CELL SIGNALING); all used at 1/1000, peroxidase-conjugated goat anti-mouse antibody (1/2000, DAKO) and peroxidase- conjugated goat anti-rabbit antibody (1/2000, DAKO). Detection was carried out using the ECL detection system (BIO-RAD) and a chemiluminescent image analyzer (LAS- 3000, FUJIFILM) and results normalized to HSP90 or actin. [0083] The Figure 2B shows the effect of HDM on other tight-junction proteins in keratinocytes of ex vivo explants.

[0084] The Figure 2C shows the effect of HDM on -cadherin isoforms, P-cadherin, and N-cadherin in keratinocytes of ex vivo explants.

[0085] The results confirmed the disruption of E-cadherin protein in keratinocytes stimulated with HDM (Fig. 2A). Now, this HDM-induced disruption seems to be specific for E-cadherin as HDM had no effect on other tight junction proteins such as ZO-1, occludin, Filaggrin or Claudin-1 (Fig. 2B) nor its two other isoforms, P-cadherin or N- cadherin (Fig. 2C).

[0086] 5. _ Cutaneous e ffect of HDM is driven by Per pl

[0087] Since Der pl is the major HDM protease, The question was then if HDM-induced destruction of E-cadherin is driven or not by Der pl.

[0088] According, primary human keratinocytes cultured as previously described were incubated with different doses (100 pg/ml, 1 pg/ml, 3 pg/ml, and 10 pg/ml) of recombinant Der pl protein (R&D) for 24hrs and measured E-cadherin expression by western blot.

[0089] The figure 3A shows the effect of Der pl on E-cadherin expression at 24hr. Images are representative of at least 3 different experiments. Results are shown as mean ± SEM. *P<0.05 versus control.

[0090] The results showed Der pl to cause a dose-dependent decrease in E-cadherin expression in NHK starting at 3 pg/ml with complete loss at 10 pg/ml (Fig. 3 A).

[0091] The hypothesis of a potential binding between Der pl and E-cadherin was tested by immunoprecipitation. [0092] For these immunoprecipitation assays, 1 mg of protein lysate were incubated with 2 pg of our protein of interest antibody E-cadherin (BD) or control protein IgG (cellsignaling) bound to PAG magnetic beads (ADEMTECH) overnight at 4°C. After three washes in lysis buffer (0.1% TRITON X-100), bead-bound proteins were subjected to western blot analysis using anti-E-cadherin (SANTACRUZ) or anti-Derp-1 (RAYBIOTECH) antibodies.

[0093] The figure 3B shows the immunoprecipitation test between E-cadherin and Der pl. Images are representative of at least 3 different experiments. Results are shown as mean ± SEM. *P<0.05 versus control.

[0094] The results of immunoprecipitation demonstrate no direct binding between E- cadherin and Der pl (Fig. 3B).

[0095] Together, these results suggest that HDM-induced decrease of E-cadherin is likely to be driven by Der pl. However, this decrease does not result from any direct interaction between E-cadherin and Der pl.

[0096] 6. _ Vitiligo keratinocytes are more sensitive to HDM than healthy keratinocyte

[0097] For testing keratinocytes sensitivity to HDM, ex vivo skin cultures of skin biopsies of vitiligo and healthy patients were cultured as described previously and treated or not with different doses (1 pg/ml, 5 pg/ml, 10 pg/ml, 50 pg/ml, and 100 pg/ml) of HDM. Twenty-four hours later, half of the biopsy was frozen in OCT medium for subsequent immunofluorescence staining on 7 pm tissue sections and the second half was used for RNA extraction (for QPCR analysis) and for protein analysis (for western blot).

[0098] The figure 4 A shows the measures of cytokines (IFNy, TNFa), chemokines

(CXCL10, CXCL11, CXCL16), Serpin El and CXCR3B at mRNA level in ex vivo skin cultures of vitiligo patients stimulated in vitro for 24hr with 100 pg/ml HDM (HDM) or not (C). The figure 4C shows the measures of cytokines (IFNy, TNFa), and chemokines (CXCL10, CXCL16) at mRNA level respectively in ex vivo skin cultures of vitiligo and healthy patients both stimulated in vitro for 24hr with 100 pg/ml HDM (HDM). All the results are normalized to healthy controls.

[0099] The figure 4B shows the E-cadherin protein expression in vitiligo keratinocytes stimulated in vitro for 24hrs with different doses (1 pg/ml, 5 pg/ml, 10 pg/ml, 50 pg/ml, and 100 pg/ml) of HDM.

[0100] The figure 4D shows the intensity of E-cadherin immunodetection in vitiligo non- lesional (NL) and lesional (L) skin compared to healthy skin and with or without HDM exposure for 48hr. The intensity of staining was quantified in the 3 groups at baseline. Images are representative of at least 3 different experiments. Results are shown as mean ± SEM. *P<0.05 and **P<0.01 versus control.

[0101] The results show that the keratinocytes isolated from vitiligo patients stimulated with lOOpg/ml of HDM showed increased IFNy, CXCL10, and CXCL11 mRNA, as well as decreased expression of serine protease inhibitor, Serpin El mRNA (Fig. 4A). There was a trend for increased CXCL16 (P=0.09) and TNFa (p=0.06) with HDM however these changes were not significant (Fig. 4A). E-cadherin expression was also decreased dose-dependently, the effect evident from 5 pg/ml of HDM (Fig. 4B).

[0102] When comparing keratinocyte responses from vitiligo patients exposed to HDM to healthy keratinocytes exposed to HDM, an increased production of all cytokines (IFNy and TNFa) and chemokines tested (CXCL10 and CXCL16) was observed for the vitiligo patients (Fig. 4C). This result suggests an increased sensitivity of vitiligo cells to HDM compared to normal cells. [0103] At baseline, we detected significantly reduced mean-intensity of E-cadherin- immunostaining in both non-lesional and lesional vitiligo skin compared to healthy skin (Fig. 4D). Now, the results have shown that lesional vitiligo skin (V-L) was significantly more sensitive to HDM-induced E-cadherin destruction than non-lesional vitiligo skin (V-NL) or healthy skin.

[0104] Together, these results suggest that HDM can induce immune responses and tissue disruption in vitiligo skin and that this ensuing effect is greater than the effect seen in healthy skin given the same stimuli.

[0105] 7. HDM can induce fl ‘ ying melanocytes ’ in vitiligo skin

[0106] The concept of flying melanocytes' refers to melanocyte detachment from the epidermal basal cell layer in the skin. This concept has been described as a mechanism contributing to loss of pigment-producing melanocytes leading to depigmentation in vitiligo (JIN et al., J. Invest. Dermatol., vol.140 (e4), p:241-243, 2020; and DAI et al, Biochimica et Biophy sica Acta (BBA) - Molecular Basis ofDisease, vol.1866, p: 165719, 2020). The proposed mechanisms suggest that melanocyte anchorage to the epidermis is dependent on the adhesion protein E-cadherin and this protein to be unevenly distributed in the epidermis before the lesions appear. More recently, BOUKHEDOUNI et al. (JCI Insight., vol.5(11), p:el33772, 2020) have demonstrated that keratinocytes stimulated with proinflammatory Thl cytokines IFNy and TNFa induce production of matrix metalloproteinase (MMP)-9. The produced MMP-9 thus cleaves E-cadherin to release its soluble form, resulting in disruption of extracellular matrix and subsequent detachment of melanocytes from their basal layer.

[0107] Accordingly, the question was whether HDM may contribute to melanocyte detachment (or flying') through its Der pl protease activity, inducing skin depigmentation and contributing to vitiligo flares in susceptible individuals.

[0108] For testing keratinocytes sensitivity to HDM, ex vivo skin cultures of skin biopsies of vitiligo and healthy patients were cultured as described previously and treated or not with 100 pg/ml of HDM. Twenty -four hours later, the biopsies were frozen in OCT medium for subsequent immunofluorescence staining on 7 mm tissue sections.

[0109] Section slides were fixed with 4% paraformaldehyde, permeabilized with Trisbuffered saline (PBS) in presence of 0.3% TRITON, blocked for 1 hour with PBS 5% BSA with 10% goat serum. Sections were then incubated overnight at 4°C with mouse anti-E-cadherin antibody (1/200, BECTON DICKINSON), rabbit anti-melanA (1/200, ABCAM), and mouse anti-collagen IV (1/200, ABCAM). After three PBS washes, the skin was incubated with secondary antibodies, Alexa 488 goat anti-mouse IgG (H+L) and Alexa 594 goat anti-rabbit IgG (H+L), respectively. Sections were then mounted with Prolong Gold antifade reagent containing DAPI and images acquired using NIKON confocal AIR.

[0110] The figure 5 A shows the number of fl ‘ ying melanocytes ’ (per epidermal length) at baseline (that is, before HDM stimulation), in healthy (H) or vitiligo (V) skin biopsies stimulated or not by HDM.

[0111] The figure 5B shows the mean intensity of E-cadherin immunodetection healthy (H) or vitiligo (V) skin biopsies stimulated or not by HDM. The intensity of staining was quantified in the 3 groups at baseline.

[0112] The results have shown that the exposure of normal skin to HDM induce an increased number of supra-basal melanocytes. Now, this induction is much more pronounced in vitiligo tissue. Assessing the number of flying melanocytes ’ (per epidermal length) at baseline (that is, before HDM stimulation), the results established an increased numbers in vitiligo compared to healthy skin (Fig. 5A). In both healthy and vitiligo skin, HDM increased the number of flying melanocytes however this effect was greater in vitiligo skin compared to healthy skin (P=0.059) (Fig. 5A). These changes in melanocyte positions or evidence of fl ‘ ying melanocytes ’ were associated with proportional loss of adherent protein E-cadherin in the same tissues (Fig. 5B). Although HDM caused decreased expression of E-cadherin in both healthy and vitiligo skin, this effect was significantly potentiated in vitiligo compared to the effect seen in the healthy skin (P<0.05) (Fig. 5B).

[0113] 8. HDM increases soluble E-cadherin in explant supernatants: Increased levels in vitiligo serum

[0114] Soluble E-cadherin was measured by ELISA (PEPRO TECH) in the serum of healthy (n=5) and vitiligo (n=5) patients and in cultured primary keratinocytes with or without stimulation with 100 pg/ml HDM for 24hr.

[0115] The results have shown that soluble E-cadherin (sE-cadherin) levels were significantly higher in the sera of vitiligo compared to healthy controls. Moreover, the results also shown that ex vivo exposure of healthy skin to lOOpg/ml HDM for 24hrs can induce similar increase in sE-cadherin production in cultured supernatants.

[0116] In order to determine the evolution of E-cadherin expression and localization, a Western blot analysis of extracellular and cytoplasmic E-cadherin expression in primary keratinocytes 24hrs after HDM stimulation was made and is presented in Figure 6. Results are representative of at least 3 different experiments. Results are shown as mean ± SEM. *P<0.05 versus respective.

[0117] The results show that while HDM caused a dose-dependent decrease in extracellular E-cadherin (at 130 kDa) whilst it increased cytoplasmic E-cadherin (37 kDa) (Fig. 6) suggesting that HDM cleaves the extracellular part of the protein which is then located and measured in the supernatant in its soluble form.

[0118] 9. HDM decreases E-cadherin by activating MMP-9

[0119] HDM is known to contain potent proteases, particularly cysteines proteases.

[0120] Simultaneously, E-cadherins are known to be proteolytically cleaved by several proteases including metalloproteases.

[0121] Since MMP-9 has previously been implicated in vitiligo (JIN et al., abovementioned, 2020) and its expression was increased in screening assay (data not shown), MMP9 effect was determined in keratinocytes following HDM exposure.

[0122] Primary keratinocytes were cultured as described previously and treated or not with different doses (1 pg/ml, 5 pg/ml, 10 pg/ml, 20 pg/ml, 50 pg/ml, and 100 pg/ml) of HDM. Twenty-four hours later, half of the biopsy was frozen in OCT medium for subsequent immunofluorescence staining on 7 pm tissue sections and the second half was used for RNA extraction (for QPCR analysis) and for protein analysis (for western blot). [0123] The culture supernatants were then collected.

[0124] Half of the samples were analyzed by zymography. For this, the proteins were separated in 7.5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) and 1 mg/ml gelatin. After electrophoresis, the gels were washed two times for 30 min with 2.5% TRITON X100 to remove the SDS and incubated overnight in 50 mmol/L Tris-HCl pH 7.5, 5 mmol/L CaC12 at 37°C for the development of zymolytic bands.

[0125] The figure 7A shows the obtained gel, wherein the protease bands were detected by absence of Coomassie Brilliant Blue staining and appear as white bands at 90 kDa (Pro MMP-9), 82 kDa (active MMP-9) and at 72 kDa (Pro MMP-2) against a dark blue background. [0126] The Figure 7B shows the analysis of the other part of the culture supernatants samples by western blot as described previously. MMP-9, actin and HSP90 were detected using mouse anti-MMP-9 (SANTA CRUZ) or rabbit anti-MMP-9 (ABCAM), and mouse anti-HSP90 (SANTA CRUZ); all used at 1/1000.

[0127] The zymography results showed increased Pro and active MMP-9 (but not MMP- 2) expression in NHK supernatants (Figure 7A). This increase in Pro and active MMP-9 is confirmed in keratinocytes lysates following in vitro exposure to lOOpg/ml HDM (Figure 7B).

[0128] For further testing MMP-9 activity, ex vivo skin cultures of skin biopsies of vitiligo and healthy patients were cultured as described previously and treated or not with 100 pg/ml of HDM in the presence or not of Abl42180 (5-50 pM), which is a specific MMP-9 inhibitor.

[0129] Twenty-four hours later, the biopsies were frozen in OCT medium for subsequent immunofluorescence staining on 7 mm tissue sections as previously. Simultaneously, the concentration of active MMP-9 in culture supernatants was determined by ELISA [0130] The figure 7C shows the concentration of active MMP-9 activity in supernatant of skin explants stimulated (HDM) or not (Control) by 100 pg/ml of HDM in the presence or not of Abl42180 (5-50 pM).

[0131] The results have shown an increased immunoreactivity of total MMP-9 protein in skin explants as in the primary keratinocytes. Moreover, this increased MMP-9 immunoreactivity was also observed in both NL and L vitiligo skin compared to healthy skin

[0132] Now, the results further established that the MMP-9 activity was increased following HDM stimulation, which increase was completely abolished by incubation of skin explants with 10 pM selective MMP-9 inhibitor, Ab 142180, simultaneously with

HDM (Fig. 7C).

[0133] 10. MMP-9 activation is related to Per p 1

[0134] In order to confirm that MMP-9 increased activity is related to Der p 1 activity, primary keratinocytes were cultured as described previously and treated or not with different doses (1 pg/ml, 5 pg/ml, 10 mg/ml, 50 mg/ml, and 100 mg/ml) of HDM together or not with different concentrations (1 mM, 5 mM, and 50 mM) of selective MMP-9 inhibitor Abl42180 or E64 (cysteine protease inhibitor). Twenty-four hours later, the keratinocytes were lysed and, MMP-9 and E-cadherin expression was determined by western blot as described previously.

[0135] The figure 8 A shows the MMP-9 protein expression in vitiligo keratinocytes stimulated in vitro for 24hrs with different doses (1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, and 100 mg/ml) of HDM.

[0136] The figure 8B shows the E-cadherin protein expression in vitiligo keratinocytes stimulated in vitro for 24hrs with 100 mg/ml of HDM with or without different concentrations (1 mM, 5 mM, and 50 mM) of selective MMP-9 inhibitor Abl42180.

[0137] The figure 8C shows the E-cadherin protein expression in vitiligo keratinocytes stimulated in vitro for 24hrs with 100 mg/ml of HDM with or without different concentrations (1 mM, 5 mM, and 50 mM) of E64 (cysteine protease inhibitor) inhibiting Der p 1.

[0138] The results confirmed a strong MMP-9 activity in vitiligo keratinocytes post HDM stimulation, both Pro and the active form, even at 50 mg/ml HDM (Fig. 8A).

[0139] As observed previously, the inhibition of MMP-9 activity with Abl42180 partially restored the HDM-induced disruption in E-cadherin (Fig. 8B). [0140] Now, the inhibition of Der p 1 activity with cysteine protease inhibitor E64 (5-50 mM) completely restored the HDM-induced disruption in E-cadherin (Fig. 8C).

[0141] These results suggest that HDM activates MMP-9 in human skin though its Der p 1 activity and inhibiting its Der p 1 activity can abolish the detrimental effect of HDM on E-cadherin which is integral in holding melanocytes at basement membrane.

[0142] 11. HDM stimulated keratinocyte medium disturbs melanocytes morphology and function

[0143] Primary human keratinocytes were cultured as previously described and incubated with or without 100 pg/ml HDM for 24hrs. After several washing steps, the keratinocytes were cultured for a new 24 hours in a new culture medium without HDM. The culture mediums (from stimulated and non-stimulated keratinocytes) were then collected and conditioned.

[0144] Primary melanocytes were then exposed for 24hours to the previously disclosed keratinocyte conditioned mediums.

[0145] The Figure 9 shows the cell death (p53, p21) and apoptosis (total and cleaved Caspase-3) markers expression examined by western blot analysis in primary human melanocytes stimulated with keratinocyte conditioned media (CM) from keratinocytes stimulated or not by HDM. Melanocyte’s morphology was then observed, and melanocytes were lysed in RIP A as described previously. Protein lysates were analyzed by western blot as described previously using rabbit anti P-53, anti-P21, and anti-caspase 3 and mouse anti HSP (SANTA CRUZ) all used at 1/1000.

[0146] The results have shown that morphology’s changes were observed in primary human melanocytes exposed to keratinocyte conditioned media (CM) (that is, supernatant collected from keratinocytes exposed to HDM for 24hr). The results also show that this exposure results in an increased expression of cell death markers (P53, P21) together with caspase-3 cleavage induction, thus resulting in apoptosis in melanocytes (Fig. 9).

[0147] Accordingly, it was that keratinocytes HDM stimulation induced MMP-9 activation and melanocyte detachment. Now, this keratinocytes HDM stimulation also induce melanocytes apoptosis.

[0148] 12. Mimicking vitiligo immuno-histopathology in 3D reconstructed skin exposed to HDM

[0149] The reconstructed human pigmented epidermis (RHPE) model was used as a proof of principle to examine whether HDM can reproduce human vitiligo features in human skin. In vitro RHPE containing both melanocytes and keratinocytes were generated from surgical samples of pediatric foreskins on polycarbonate culture inserts in duplicate. RHPE were cultured at the air-liquid interphase for 10 days in a humidified atmosphere 5% CO2 at 37°C and then treated for 24 hours with or without TNF-a /IFN-y (as positive control, 10 ng/mL) or HDM (1, 50 or 100 pg/ml).

[0150] RHPE was frozen in OCT for histological analysis (examination of E-cadherin disruption and proportion of supra-basal melanocytes in the epidermis) and supernatant collected to examine the impact of HDM on the regulation of the inflammatory response in the epidermis measuring cytokine and chemokine markers, soluble E-cadherin and MMP-9 by ELISA and multiplex assays.

[0151] The figure 10A shows the cytokines (CXCL9, CXCL10), MIP3a/CCL20, and

MMP-9 expression as determined by ELISA in supernatant of 3D reconstructed human pigmented epidermis (RHPE) skin exposed to HDM (1, 50 and lOOpg/ml) (TNF-a /IFN- y or PBS control). [0152] The figure 10B shows the soluble E-cadherin expression as determined by ELISA in supernatant of 3D reconstructed human pigmented epidermis (RHPE) skin exposed to HDM (lOOpg/ml) (TNF-a /IFN-y or PBS control).

[0153] The figure 10C shows the % flying melanocytes with or without HDM (1, 50 and lOOpg/ml) exposure as assessed from OCT frozen RHPE sections counting Mel A- positive supra basal melanocytes. IFNy/TNFa was an internal positive control.

[0154] The results show a significantly increased CXCL9, CXCL10, and MIP-3a (CCL20) production in the supernatant after 100 pg/ml HDM exposure and responses were similar, although less pronounced, to our positive control exposure to IFNy/TNFa for the same period (Fig. 10A). As determined, the levels of active MMP-9 were increased in cultured supernatants compared to non-stimulated control (Fig. 10A). [0155] In addition, the results have shown that HDM caused a dose-dependent reduction in E-cadherin immunoreactivity. This reduction is correlated with a significantly increased levels of soluble E-cadherin in the cultured supernatant (Fig. 10B). In parallel, HDM caused a dose-dependent increase in the number of detached supra-basal melanocytes (Fig. 10C), similar to what was detected in IFNy/TNFa condition.