WO2024028417A1 - Nouvelle composition pour traitement et/ou prévention du vitiligo - Google Patents

Nouvelle composition pour traitement et/ou prévention du vitiligo Download PDF

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WO2024028417A1
WO2024028417A1 PCT/EP2023/071476 EP2023071476W WO2024028417A1 WO 2024028417 A1 WO2024028417 A1 WO 2024028417A1 EP 2023071476 W EP2023071476 W EP 2023071476W WO 2024028417 A1 WO2024028417 A1 WO 2024028417A1
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hdm
vitiligo
composition
mmp
cadherin
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Thierry Passeron
Florence NADAL
Mélic TURI
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Institut National de la Santé et de la Recherche Médicale
Isispharma France
Universite Cote D'azur
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4425—Pyridinium derivatives, e.g. pralidoxime, pyridostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/56—Protease inhibitors from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the present invention relates to composition for the treatment and/or prevention of vitiligo.
  • 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.
  • 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.
  • 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.
  • 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.
  • Vitiligo is a multifactorial polygenetic disease causing the loss of melanocytes.
  • 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.
  • 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.
  • House dust mites are a component of normal skin microbiota.
  • 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.
  • Dermatophagoides pteronyssinus (Der p) is a major HDM allergen with potent protease activity.
  • 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.
  • 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.
  • 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.
  • MMP-9 Matrix metallopeptidase 9
  • 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.
  • a composition comprising at least one mite peptidase 1 or Matrix metallopeptidase 9 (MMP-9) antagonist, and optionally an pharmaceutically acceptable carrier.
  • mite peptidase 1 Enzyme entry EC 3.4.22.65
  • mite also known as endopeptidase 1 (mite)
  • mite is an enzyme found in various species of mites. This enzyme exhibits cysteine protease activity with broad endopeptidase specificity.
  • 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.
  • the mite peptidase 1 is Der p 1 or Der f 1, preferably Der p 1.
  • a mite peptidase 1 antagonist is a compound inhibiting mite peptidase 1 protease activity from more than 50%, preferably from more than 75%.
  • Such a mite peptidase 1 antagonist include, 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.
  • E-64 L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4- guanidino )butane
  • cystatin e.g. chestnut cystatin (CsC), egg white cystatin (EWC), Kiwifruit cysteine proteinase inhibitor 1 (KCPI1)
  • KCPI1 Kiwifruit cysteine proteina
  • the mite peptidase 1 antagonist is a cystatin.
  • said cystatin is egg white cystatin.
  • Such egg white cystatin may be in the form of an egg white extract.
  • said cystatin is phytocystatin, such chestnut cystatin (CsC) or Kiwifruit cysteine proteinase inhibitor 1 (KCPI1).
  • CsC chestnut cystatin
  • KCPI1 Kiwifruit cysteine proteinase inhibitor 1
  • Such phytocystatin may be in the form of an extract, like a chestnut extract or a kiwi seed extract.
  • the mite peptidase 1 antagonist is one of the antagonists disclosed in the international patent applications W02012004554 or WO2011089396.
  • said mite peptidase 1 antagonist is selected in the group of compounds having the formula (I)
  • said mite peptidase 1 antagonist is selected in the group consisting of the compounds 12, 15, 17, 18 and 19.
  • said mite peptidase 1 antagonist is the compound 12 or 18, and preferably the compound 12 .
  • Matrix metallopeptidase 9 also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB)
  • MMP-9 Matrix metallopeptidase 9
  • GELB gelatinase B
  • MMP9 matrix metallopeptidase 9
  • This protein belongs to the zinc- metalloproteinases family involved in the degradation of the extracellular matrix.
  • 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.
  • a “MMP-9 antagonist” is compound inhibiting the MMP-9 protease activity from more than 50%, preferably from more than 75%.
  • 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
  • composition of the invention comprised at least mite peptidase 1 antagonist.
  • vitiligo can correspond just as well to segmental vitiligo or to non-segmental vitiligo.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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]).
  • compositions of the invention may comprise any ingredient conventionally used in the fields under consideration, and particularly in cosmetics and dermatology.
  • 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
  • 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.
  • the pharmaceutical composition according to the invention is suitable for cutaneous, oral, topical, intramuscular, intravenous, transdermal or subcutaneous administration.
  • 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.
  • 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.
  • compositions of the invention will be suitable for topical administration.
  • 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).
  • 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.
  • 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.
  • 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.
  • MMP-9 Matrix metallopeptidase 9
  • HDM is present in healthy and vitiligo skin
  • 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.
  • PBS Trisbuffered saline
  • HDM induces biomarkers strongly associated with vitiligo in keratinocytes
  • keratinocytes were stimulated with different concentrations of house dust mite (HDM) (1 pg/ml, 200pg/ml, and 400 pg/ml) for 24h.
  • HDM house dust mite
  • 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.
  • HDM induces biomarkers strongly associated with vitiligo in ex vivo biopsies
  • 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.
  • 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.
  • HDM induces disruption of tight junctions between keratinocytes
  • HDM house dust mite
  • the figure 2 A shows the mean signal intensity quantitated for E-cadherin immunostaining in skin biopsies stimulated with HDM and in control.
  • 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.
  • ECL detection system BIO-RAD
  • LAS- 3000, FUJIFILM chemiluminescent image analyzer
  • the Figure 2C shows the effect of HDM on -cadherin isoforms, P-cadherin, and N-cadherin in keratinocytes of ex vivo explants.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HDM can induce fl ‘ ying melanocytes ’ in vitiligo skin
  • 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).
  • 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.
  • BOUKHEDOUNI et al. JCI Insight., vol.5(11), p:el33772, 2020
  • keratinocytes stimulated with proinflammatory Thl cytokines IFNy and TNFa induce production of matrix metalloproteinase (MMP)-9.
  • MMP-9 matrix metalloproteinase
  • 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.
  • 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.
  • 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.
  • PBS Trisbuffered saline
  • 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.
  • 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.
  • HDM increases soluble E-cadherin in explant supernatants: Increased levels in vitiligo serum
  • soluble E-cadherin sE-cadherin
  • results have shown that soluble E-cadherin (sE-cadherin) levels were significantly higher in the sera of vitiligo compared to healthy controls.
  • 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.
  • HDM decreases E-cadherin by activating MMP-9
  • HDM is known to contain potent proteases, particularly cysteines proteases.
  • E-cadherins are known to be proteolytically cleaved by several proteases including metalloproteases.
  • 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.
  • 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.
  • 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.
  • 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).
  • MMP-9 activation is related to Per p 1
  • 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.
  • 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.
  • 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.
  • 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.
  • HDM stimulated keratinocyte medium disturbs melanocytes morphology and function
  • 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.
  • CM keratinocyte conditioned media
  • CM keratinocyte conditioned media
  • keratinocytes HDM stimulation induced MMP-9 activation and melanocyte detachment. Now, this keratinocytes HDM stimulation also induce melanocytes apoptosis.
  • RHPE human pigmented epidermis
  • 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.
  • 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).
  • 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).
  • 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.

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Abstract

La présente invention concerne une composition comprenant au moins un antagoniste de la peptidase 1 ou de la métallopeptidase matricielle 9 (MMP-9) de l'acarien et, éventuellement un support pharmaceutiquement acceptable, destinés à être utilisés pour prévenir et/ou traiter le vitiligo chez un patient.
PCT/EP2023/071476 2022-08-03 2023-08-02 Nouvelle composition pour traitement et/ou prévention du vitiligo WO2024028417A1 (fr)

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