Compounds which restore the activity of p63 for treating a subject suffering from junctional epidermolysis bullosa (JEB)

Abstract

Inventors tested keratinocytes isolated from a patient with severe LM332-associated JEB carrying a homozygous variant (c.183delCA) in the LAMB3 gene (Vailly et al., 1998). Pathogenic variants in the LAMB3 gene account for 80% of severe LM332-associated JEB (Lincoln et al., 2018). They confirmed altered cell adhesion in these keratinocytes but then 10 found that treatment with PRIMA-1MET rescued cell adhesion in a dose-dependent manner. To further validate the rescuing effect of PRIMA-1MET, two additional epidermal cell lines derived from two patients with JEB carrying pathogenic variants in the ITGB4 gene were tested under the same conditions. These two JEB cells displayed similar altered cell adhesion, which is also partially 15 rescued by PRIMA-1MET.Asfor the LAMB3-associated JEB cells, PRIMA-1MET treatment increased matrix metal-loproteinase 1 gene MMP1 and ADAM8 gene expression in these cells (Figure 2b). Altogether, our results validate the effect of PRIMA-1MET on three different JEB lines, suggesting that it could benefit patients with JEB.

Description

[0001] METHODS AND COMPOSITIONS FOR TREATING A SUBJECT SUFFERING FROM JUNCTIONAE EPIDERMOEYSIS BUEEOSA (JEB)

[0002] FIEED OF THE INVENTION:

[0003] The present invention is in the field of dermatology, more particularly, the invention relates to methods and composition for promoting would healing in a subject suffering from junctional epidermolysis bullosa (JEB).

[0004] BACKGROUND OF THE INVENTION:

[0005] Wound healing is a process which starts immediately after an injury and may continue for months or years, and is essentially the same for all types of wounds. Variations in wound healing are the result of differences in location, severity of the wound, and the extent of injury to the tissues. Other factors affecting wound healing are the age, nutritional status and general state of health of a subject and its body reserves and resources for the regeneration of tissue. The wound healing process is divided into 3 or 4 distinct phases: (i) coagulation and haemostasis; (ii) inflammation; (iii) proliferation; and (iv) wound remodelling with scar tissue formation.

[0006] There are many diseases cause wound in the structure of an organ or tissue, including epithelium, connective tissue, and muscle tissue in the subject such as inflammatory skin disease; bacterial skin infection; fungal and yeast infection; viral infection; and infection caused by small macro organisms such as mites; diabetes or aging process.

[0007] For some skin diseases, more particularly rare diseases such as junctional epidermolysis bullosa (JEB). JEB is a group of rare, severe autosomal recessive forms of the hered-itary heterogeneous blistering disease epidermolysis bullosa (Has et al., 2020). JEB is caused by the absence or functional loss of heterotrimer LM332, integrins (a6, b4, and a3), or type XVII collagen. LM332 is secreted by keratinocytes and provides epi- dermaledermal adhesion by linking hemidesmosome-composing proteins such as integrin a6b4 and type XVII collagen to the anchoring fibril protein, type VII collagen. In severe LM332-associated JEB, LM332 is either absent or markedly reduced, leading to disrup-tion of hemidesmosomes with impaired epithelial adhesion and mucoepithelial erosions (Keith et al., 2020). In interme-diate or localized JEB, the patients pre-sent with a chronic disease that displays skin and mucosal erosions, inflamma-tion, and other comorbidities. Apart from the cell / gene therapy approach demonstrated by De Luca’s group (Kueckelhaus et al., 2021), there are currently no effective treatments available for blisters / erosions in patients with JEB.

[0008] Accordingly, there is a need to find new tool to treat this kind of pathologies.

[0009] SUMMARY OF THE INVENTION:

[0010] The present invention relates to a method to a method for treating junctional epidermolysis bullosa (JEB) in a subject need thereof comprising a step of administering to said subject a therapeutically effective amount of a compound which restores the activity of p63.

[0011] In particular, the invention is defined by claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Inventors tested keratinocytes isolated from a patient with severe LM332-associated JEB carrying a homozygous variant (c,183delCA) in the LAMB3 gene (Vailly et al., 1998). Pathogenic variants in the LAMB3 gene account for 80% of severe LM332-associated JEB (Lincoln et al., 2018). They confirmed altered cell adhesion in these keratinocytes but then found that treatment with PRIMA-1MET rescued cell adhesion in a dose-dependent manner.

[0014] To further validate the rescuing effect of PRIMA-1MET, two additional epidermal cell lines derived from two patients with JEB carrying pathogenic variants in the ITGB4 gene were tested under the same conditions.

[0015] These two JEB cells displayed similar altered cell adhesion, which is also partially rescued by PRIMA-lMET.Asfor the LAMB3 -associated JEB cells, PRIMA-1MET treatment increased matrix metal-loproteinase 1 gene MMP1 and ADAM8 gene expression in these cells (Figure 2b). Altogether, our results validate the effect of PRIMA-1MET on three different JEB lines, suggesting that it could benefit patients with JEB.

[0016] Accordingly, in a first aspect, the present invention relates to a method for treating junctional epidermolysis bullosa (JEB) in a subject need thereof comprising a step of administering to said subject a therapeutically effective amount of a compound which restores the activity of p63.

[0017] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0018] As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with junctional epidermolysis bullosa (JEB). More particularly, the subject is afflicted with or susceptible to be afflicted by JEB syndromes.

[0019] In another embodiment, the subject is afflicted with or susceptible to be afflicted by JEB syndromes including but not limited to the following syndromes: Fragile skin that blisters easily, especially on the palms and feet, nails that are thick or unformed., blisters inside the mouth and throat, scalp blistering and hair loss (scarring alopecia), skin that looks thin, tiny pimple-like bumps (milia), dental problems, such as tooth decay, difficulty swallowing. As used herein, the term “junctional epidermolysis bullosa (JEB)” is a group of rare, severe autosomal recessive forms of the hered-itary heterogeneous blistering disease epidermolysis bullosa (Has et al., 2020). JEB is caused by the absence or functional loss of heterotrimer LM332, integrins (a6, b4, and a3), or type XVII collagen. LM332 is secreted by keratinocytes and provides dermal-epidermal adhesion by linking hemidesmosome-composing proteins such as integrin a6b4 and type XVII collagen to the anchoring fibril protein, type VII collagen. In severe LM332-associated JEB, LM332 is either absent or markedly reduced, leading to disrup-tion of hemidesmosomes with impaired epithelial adhesion and mucoepithelial erosions (Keith et al., 2020). In interme-diate or localized JEB, the patients present with a chronic disease that displays skin and mucosal erosions, inflamma-tion, and other comorbidities.

[0020] As used herein, the term “a compound which restores the activity of p63” refers to a compound which allows to obtain the properties of the p63 wild type when p63 is mutated. p63 also known as tumor protein p63 (TP63) is a member of the p53 family of transcription factors. p63 encodes for two main isoforms by alternative promoters (TAp63 and ANp63). ANp63 is involved in multiple functions during skin development and in adult stem / progenitor cell regulation. Whereas TAp63 has been mostly restricted to its apoptotic function. p63 mutations associated with the ED syndrome are point mutations in the DNA-binding domain affecting all isoforms. In the context of the invention, the compound restores the proliferation and development of epithelial cells and epidermal cells. In a particular embodiment, the compound is STIMA-1 compound or PRIMA-lMetcompound.

[0021] In particular embodiment, the compound which restores the activity of p63 rescue epidermal cell differentiation. As used herein, the term “PRIMA-lMet” also known as APR-246 is a small molecule characterized by the following formula in the art: It is a methylated derivative and structural analog of PRIMA-1 which is a p53 reactivator and inductor of massive apoptosis. PRIMA-1Met, also known as APR-246 and 2- (hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one (C10H17NO3) has the following CAS number: 5291-32-7.

[0022] As used herein, the term “STIMA-1”, also known as 2-vinyl-4H-quinazolinol (C10H8N2O), refers to the p53 activator VII and is referenced under CAS 91634-12-7. This small molecule modulates the biological activity of p53 and has the following structure in the art:

[0023] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g.,PRIMA-lMetand / or STIMA-1) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0024] In a particular embodiment, the subject is administered subcutaneously, systemically, ocularly, intradermally or topically with a compound which restores the activity of p63 (e.g., PRIMA-1 Met and / or STIMA-1).

[0025] In a further embodiment, the method according to the invention, wherein STIMA-1 compound or PRIMA-1 Met compound are administered to the subject in need thereof simultaneously, separately or sequentially.

[0026] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

[0027] In a particular embodiment, a i) PRIMA-lMetcompound, and ii) STIMA-1 compound, as a combined preparation for simultaneous, separate or sequential use in the method for promoting wound healing in a subject suffering from JEB.

[0028] The method according to the invention, wherein the compound is formulated as a cream, an ointment, or gel.

[0029] The method according to the invention, wherein the compound is formulated as an ophthalmic drop or an ophthalmic ointment.

[0030] In another particular embodiment, a i) PRIMA-lMetcompound, and ii) STIMA-1 compound, as a combined preparation for simultaneous, separate or sequential for use in the method for treating JEB in a subject suffering in need thereof.

[0031] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient (PRIMA-lMetand / or STIMA-1) for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. In a particular embodiment, the subject is administered with 5mg / day of PRIMA-1METand 0,7mg / day of STIMA-1 simultaneously, sequentially or separately.

[0032] In a particular embodiment, the invention relates to a method for promoting wound healing in a subject suffering from junctional epidermolysis bullosa (JEB) in need thereof comprising a step of administering subcutaneously, intradermally or topically to said subject a therapeutically effective amount of a compound which restores the activity of p63.

[0033] As used, herein, the term “wound” refers to a break or discontinuity in the structure of an organ or tissue, including epithelium, connective tissue, and muscle tissue, caused by an external agent. Examples of wounds include, but are not limited to, skin wounds, bruises, ulcerations, bedsores, grazes, tears, cuts, punctures, psoriasis wounds, tympanic membrane perforations, and bums. Wound healing is a dynamic, interactive process involving soluble mediators, blood cells, extracellular matrix, and parenchymal cells. Wound healing has 3 phases that overlap in time: vascular phase and inflammation, new tissue formation including reepithelialization, and tissue remodelling. Wounds are currently treated by applying an emergency treatment to a wounded site and waiting for the wounds to spontaneously heal via the biological recovering power of their own.

[0034] As used herein, the term “promote” refers to “improve” or “enhance”, the wound healing generally means increasing the speed by which the wound or perforation heals or reducing the extent of residual scar or necrotic tissue during or after healing of the wound or perforation. In a particular embodiment, the wound healing process is increased in a subject suffering from JEB.

[0035] In a second aspect, the invention relates to a pharmaceutical composition comprising a compound which restores the activity of p63.

[0036] The pharmaceutical composition according the invention, wherein the compound is i) STIMA-1 compound or ii) PRIMA-lMetcompound. The compound which restores the activity of p63 as defined above and the pharmaceutical combination according to the invention, may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0037] The pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0038] In a particular embodiment, the pharmaceutical composition (PRIMA-1METand / or STIMA-1) is formulated as a cream, an ointment, or gel.

[0039] In another embodiment, the pharmaceutical composition (PRIMA-1METand / or STIMA- 1) is formulated as an ophthalmic drop or an ophthalmic ointment.

[0040] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being administered to the subject by intradermally subcutaneously or topically. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound which restores the activity of p63 (PRIMA_1METand / or STIMA-1) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0041] The pharmaceutical composition according to the invention, further is formulated with Seraqua™ or Versatile™ Rich cream. Typically, the pharmaceutical composition (PRIMA- 1METand / or STIMA-1) is solubilized in the water and Seraqua™ is added to the composition to obtain a homogenized pharmaceutical composition. Seraqua™ is composed by the following ingredients: purified water, glycerin, glycerol, propanediol, Oil Helianthus annuus seeds, prolyglyceryl-3-diidsostearate, tocopheryl acetate, polyacrylate-13, dimethicone, phenoxyethanol (0.5%) polyisobuten, gomme xanthane, acide benzoic and polysorbate 20.

[0042] In a further embodiment, the pharmaceutical composition is solubilized in the water and Versatile™ Rich cream. Versatile™ Rich cream is composed of the following ingredients: Aqua purificata, olus oil, cetearyl alcohol, glyceryl stearate, dicaprylyl carbonate, polysorbate 60, isohexadecane, isododecane, lecithin, PEG-75 stearate, polyacrylate-13, tocopheryl acetate, glycerin, polyisobutene, C13-C15 alkane, alcohol, disodium EDTA, simethicone, benzoic acid, sorbic acid, polysorbate 20, sodium hydroxide.

[0043] For a composition by therapeutic unity (e.g for cream tube 30g), the pharmaceutical composition can be performed by adding 10 to 20 mg of PRIMA-1MET, 0,1 to 5 ml of water purified and Seraqua™ uitity to obtain a total mass of 30g.

[0044] In particular embodiment, for a composition by therapeutic unity (e.g. for cream tube 30g), the pharmaceutical composition is performed by adding 18mg of PRIMA-1MET, 0,5ml of water purified and Seraqua™ unity to obtain a total mass of 30g.

[0045] The pharmaceutical composition as described above does not present any inflammations on the application site.

[0046] The pharmaceutical composition according to the invention, for use in the therapy.

[0047] The pharmaceutical composition according to the invention, for use in the treatment of junctional epidermolysis bullosa (JEB).

[0048] The pharmaceutical composition according to the invention, for use in the promoting wound healing in a subject suffering from junctional epidermolysis bullosa (JEB).

[0049] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0050] FIGURES:

[0051] Figure 1. PRIMA-1MET rescues cell adhesion of epidermal cells carrying a homozygous pathogenic variant (c.!83delCA) in the LAMB3 gene, (a) Detachment assay. A total of 3 xlO5keratinocytes from patients with JEB were seeded in six-well plates and treated for 10 hours with PRIMA-1MET (2 and 10 iiM) or its buffer DMSO. Then, cells were incubated in StemPro Accutase Cell Dissociation Reagent at RT, and detached cells were counted after indicated times (2.5, 5, 10, and 15 min). Viability was tested with trypan blue, n % 5. NHK served as control, (b) Total number of cells detached after 15 min. Statistical significance was determined by two-way ANOVA. *P < 0.01 and **P < 0.001 as compared with DMSO-treated controls, (c) RNA-seq analyses of triplicate samples of JEB cells treated or not treated with PRIMA. Gene ontology enrichment analysis of the 10 top molecular functions (GOrilla). Significance of term enrichment by FDR values is represented by log 10 bar values, (d) Validation of selected gene expression by RT-qPCR. n % 2. A.U., arbitrary unit; FDR, false discovery rate; JEB, junctional epidermolysis bullosa; min, minute; NHK, normal human keratinocyte; RNA-seq, RNA sequencing; RT, room temperature.

[0052] Figure 2. PRIMA-1METrescues cell adhesion of epidermal cells derived from additional EB patients, (a) Detachment assay. 3x105JEB patient keratinocytes carrying were seeded in 6-well plates and treated for 10 hours with PRIMA-1MET( I OuM) or its buffer DMSO. Cell adhesion assay was performed as described in legend to Fig. 1. n=5. ITGB4-C11 and ITGB4-C12 are JEB patient epidermal cells. NHK served as control, (b) Modulation of gene expression by PRIMA-1METwere tested by RT-qPCR on ITGB4-C11 and ITGB4-C12 cells. n=2.

[0053] EXAMPLE :

[0054] Inventors tested keratinocytes isolated from a patient with severe LM332-JEB patient carrying a homozygous variant (c,183delCA) in the LAMBS gene (Vailly et al., 1998). Pathogenic variants in the LAMBS gene account for 80% of severe LM332-JEB (Lincoln et al., 2018). They confirmed altered cell adhesion in these keratinocytes (Fig. la-b) but then found that treatment with PRIMA- 1METrescued cell adhesion in a dose-dependent manner. To clarify its mechanism of action (MOA), they performed a comparative RNA-seq analysis of the JEB cells treated (or not treated) with PRIMA- 1MET. These data have been assigned to the BioStudies accession number S-BSST1020. Remarkably, GO analysis mainly highlighted cell adhesion coupled to extracellular matrix (ECM) structure and organization as well as focal adhesion (Fig. 1c), strongly suggesting that the MOA of PRIMA-1METis directly related to ECM rearrangement. The analysis included 249 genes whose expression was modulated by the compound (79 up and 170 down). Among them, they identified integrins ITGA2, ITGB1, ITGA3, ITGA6), laminins (LAMA3, LAMB3, LAMC2), MMP1, ADAM8, SMOC2 and FBN2. The activation of selected genes by PRIMA-1METwas validated by RT-qPCR (Fig. Id). To further validate the rescuing effect of PRIMA-1MET, two additional epidermal cell lines derived from two JEB patients carrying pathogenic variants in the ITGB4 gene were tested under the same conditions. The first, named ITGB4-C11, is a compound heterozygote for the variants c.3750_3801del (51-bp deletion) and c.H50delG, producing a truncated 04 peptide (Vidal et al., 1995) while the second, named ITGB4-C12, is a compound heterozygote for the variants c.3802+lG>A and c.3986-19T>A, producing low level of 04 (Chavanas et al., 1999). As illustrated in Fig. 2a, these two JEB cells displayed similar altered cell adhesion, which is also partially rescued by PRIMA-1MET. AS for the LAMB3-SEQ cells, PRIMA-1METtreatment increased MMP1 and ADAM8 gene expression in these cells (Fig. 2b). Altogether, our results validate the effect of PRIMA-1METon three different JEB lines, suggesting that it could benefit JEB patients. From the promoter / reporter assay, PRIMA-1METdoes not act directly on p63 (not shown), as suggested previously (Aberdam et al., 2020).

[0055] Apart from the cell / gene therapy approach demonstrated by De Luca's group (Kueckelhaus et al., 2021), there are currently no effective treatments available for blisters / erosions in JEB patients. Since systemic administration of PRIMA-1METis already validated in phase III clinical trials for cancer patients without any toxicity (Sallman et al., 2021) and in daily topical application for some AEC patient skin erosions with proven efficacy (Aberdam et al., 2020), repurposing of the molecule either topically or systemically for JEB treatment could be translated rapidly to patients.

[0056] REFERENCES:

[0057] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0058] Aberdam E, Roux LN, Secretan PH, Boralevi F, Schlatter J, Morice-Picard F. et al. Improvement of epidermal covering on AEC patients with severe skin erosions by PRIMA- 1MET / APR-246. Cell Death Dis. 2020; 11 :30.

[0059] Chavanas S, Gache Y, Vailly J, Kanitakis J, Pulkkinen L, Uitto J. et al. Splicing modulation of integrin beta4 pre-mRNA carrying a branch point mutation underlies epidermolysis bullosa with pyloric atresia undergoing spontaneous amelioration with ageing. Hum Mol Genet. 1999;8:2097-105.

[0060] Has C, Bauer JW, Bodemer C, Bolling MC, Bruckner-Tuderman L, Diem A et al. Consensus reclassification of inherited epidermolysis bullosa and other disorders with skin fragility. Br J Dermatol. 2020;183:614-27.

[0061] Keith AR, Twaroski K, Ebens CL, Tolar J. Leading edge: emerging drug, cell, and gene therapies for junctional epidermolysis bullosa. Expert Opin Biol Ther. 2020;20:911-23.

[0062] Kueckelhaus M, Rothoeft T, De Rosa L, Yeni B, Ohmann T, Maier C. et al. Transgenic epidermal cultures for junctional epidermolysis bullosa - 5-year outcomes. N Engl J Med. 2021;385:2264-70.

[0063] Lincoln V, Cogan J, Hou Y, Hirsch M, Hai M, Alexeev V et al. Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa. Proc Natl Acad Sci USA. 2018; 115: E6536-E45.

[0064] Pecorari R, Bemassola F, Melino G, Candi E. Distinct interactors define the p63 transcriptional signature in epithelial development or cancer. Biochem J. 2022;479:1375-92.

[0065] Peschel N, Wright JT, Koster MI, Clarke AJ, Tadini G, Fete M, et al. Molecular pathway-based classification of ectodermal dysplasias: first five-yearly update. Genes 2022;13:2327.

[0066] Sallman DA, DeZem AE, Garcia-Manero G, Steensma DP, Roboz GJ, Sekeres MA. et al. Eprenetapopt (APR-246) and Azacitidine in TP53-mutant myelodysplastic syndromes. J Clin Oncol. 2021;39:1584-94.

[0067] Sen GL, Boxer LD, Webster DE, Bussat RT, Qu K, Zamegar BJ. et al. ZNF750 is a p63 target gene that induces KLF4 to drive terminal epidermal differentiation. Dev Cell. 2012;22:669-77.

[0068] Shalom-Feuerstein R, Serror L, Aberdam E, Muller FJ, van Bokhoven H, Wiman KG. et al. Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246 / PRIMA-1MET. Proc Natl Acad Sci U S A. 2013; 110:2152-6.

[0069] Soares E, Xu Q, Li Q, Qu J, Zheng Y, Raeven HHM. et al. Single-cell RNA-seq identifies a reversible mesodermal activation in abnormally specified epithelia of p63 EEC syndrome. Proc Natl Acad Sci U S A. 2019;116:17361-70.

[0070] Vailly J, Gagnoux-Palacios L, DelfAmbra E, Romero C, Pinola M, Zambruno G. et al. Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia. Gene Ther. 1998;5:1322-32. Vidal F, Aberdam D, Miquel C, Christiano AM, Pulkkinen L, Uitto J. et al. Integrin beta mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nat Genet. 1995;10:229-34.

Claims

1. CLAIMS:

1. A method for treating junctional epidermolysis bullosa (JEB) in a subject need thereof comprising a step of administering to said subject a therapeutically effective amount of a compound which restores the activity of p63.

2. A method for promoting wound healing in a subject suffering from junctional epidermolysis bullosa (JEB) in need thereof comprising a step of administering subcutaneously, intradermally or topically to said subject a therapeutically effective amount of a compound which restores the activity of p63.

3. The method according to claim 1 or 2, wherein the compound is STIMA-1 compound or PRIMA-lMetcompound.

4. The method according to claims 1 to 3, wherein STIMA-1 compound or PRIMA-lMetcompound are administered to the subject in need thereof simultaneously, separately or sequentially.

5. The method according to claim 1 or 4, wherein the compound is formulated as a cream, an ointment, or gel.

6. The method according to claim 1 or 4, wherein the compound is formulated as an ophthalmic drop or an ophthalmic ointment.

7. A pharmaceutical composition comprising a compound which restores the activity of p63.

8. The pharmaceutical composition according to claim 7, wherein the compound is i) STIMA-1 compound or ii) PRIMA-lMetcompound.

9. The pharmaceutical composition according to claim 7 or 8, further is formulated with Seraqua™ cream.

10. The pharmaceutical composition according to claim 7 or 8, further is formulated with Versatile™ Rich cream.

11. The pharmaceutical composition according to claims 8 to 11, for use in the therapy.

12. The pharmaceutical composition according to claim 7 to 11, for use in the promoting wound healing in a subject suffering from junctional epidermolysis bullosa (JEB).

13. The pharmaceutical composition according to claim 7 to 11, for use in the treatment of junctional epidermolysis bullosa (JEB).

Images