WO2022107179A1 - Selective activators of peroxisome proliferator‐activated receptors (ppars) for the treatment of vitiligo - Google Patents

Selective activators of peroxisome proliferator‐activated receptors (ppars) for the treatment of vitiligo Download PDF

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WO2022107179A1
WO2022107179A1 PCT/IT2021/050371 IT2021050371W WO2022107179A1 WO 2022107179 A1 WO2022107179 A1 WO 2022107179A1 IT 2021050371 W IT2021050371 W IT 2021050371W WO 2022107179 A1 WO2022107179 A1 WO 2022107179A1
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vitiligo
ppary activator
melanocytes
ppary
activator
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Mauro Michele Maria PICARDO
Barbara BELLEI
Daniela KOVACS
Federica PAPACCIO
Emanuela BASTONINI
Maria Lucia DELL'ANNA
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Picardo Mauro Michele Maria
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    • 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
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria

Definitions

  • PPARs Peroxisome proliferator-activated receptors
  • the present invention concerns specific activators of Peroxisome proliferator-activated receptors (PPARs), such as Pioglitazone, for the treatment of vitiligo.
  • PPARs Peroxisome proliferator-activated receptors
  • the present invention concerns specific and selective activators of Peroxisome proliferator-activated receptors y (PPARy), such as Pioglitazone, for the treatment of vitiligo, such as non- segmental vitiligo.
  • Vitiligo is an acquired chronic depigmenting disorder of the skin resulting from selective loss of melanocytes. According to international consensus, it is classified into three major forms, namely, non-segmental vitiligo (or simply, vitiligo), segmental vitiligo and mixed vitiligo [1 ,2], Non- segmental vitiligo, the commonest form of this unpredictable disease, is characterized by symmetrical and bilateral white patches. The lesions are typically distributed in an acrofacial pattern (hands and feet, periorificial facial involvement) or scattered symmetrically over the entire body. The hairs on the involved skin remain pigmented initially but after a prolonged time, leukotrichia (whiteness of the hair) might develop.
  • Vitiligo is quite common, with an estimated global prevalence of 0.5% to 2% but peaks of up to 8.8% have been reported in India, possibly relating to the inclusion of chemically induced depigmentation.
  • vitiligo The characteristic of vitiligo is the loss of functional melanocytes and, even if the mechanisms involved are not completely defined, oxidative stress, generation of inflammatory mediators, cell detachment and autoimmune responses have been considered pathogenetically relevant [4,5].
  • the pivotal role in melanocyte loss is played by CD8+ T cells which are more numerous in the skin of patients with active disease than in healthy controls and in patients with stable disease.
  • Keratinocytes which support melanocyte survival and melanin production via growth factors including bFGF (basic fibroblast growth factor) and SCF (stem cell factor) show apoptotic markers and reduced SCF production, in the lesional area [10].
  • bFGF basic fibroblast growth factor
  • SCF stem cell factor
  • vitiligo is not a “cosmetic disease’” so treatment should and can be offered to patients.
  • European guidelines for the diagnosis and management of vitiligo recommend narrow band (NB) ultraviolet (UV) B, tacrolimus and topical steroids but results are not always satisfactory [11],
  • Topical treatments are adopted when small areas are involved. Phototherapy in combination with topical ultrapotent or potent corticosteroids is preferred when >5-10% of the BSA is affected or when focal areas are unresponsive to steroids alone. For lesions on the face, neck, and intertriginous areas and lesions in children either mid-potency topical corticosteroids or calcineurin inhibitors are used. In published guidelines, the European Dermatology Forum group proposed twice daily topical calcineurin inhibitors for head and neck lesions as a first-line approach.
  • JAK inhibitors The newest options are represented by JAK inhibitors.
  • Apremilast is indicated in the treatment of rheumatoid arthritis and psoriasis and case reports have noted its use in the treatment of vitiligo.
  • Khemis et al. apremilast in combination with Narrow Band UVB (NB-UVB) did not demonstrate any significant improvement of the therapeutic effect obtained with NB-UVB alone [11].
  • an activator of Peroxisome proliferator-activated receptors PPARs
  • Pioglitazone is capable of activating melanocyte reservoir, mainly present in the hair follicles.
  • pioglidazone prevents or blocks the damage of melanocytes and presumably the induction of the immune response in vitiligo through a mechanism of action not previously identified.
  • PPARs are nuclear receptors, which have an important role in the mammalian physiological system.
  • the three PPAR isoforms, PPARa, [3 /5 and y share considerable sequence and structural homologies but exhibit different tissue distribution, selectivity and responsiveness to ligands, leading to regulation of distinct sets of genes by different receptors.
  • Activated PPARs heterodimerize with the retinoid X receptor (RXR) in the presence of activators and bind to a PPAR response element (PPRE) in the promoters of target genes.
  • PPARs also act through ligand-independent modes of action, such as trans-repression and constitutive activation of the receptors.
  • PPARy the most widely investigated subtype, is expressed predominantly in the adipose tissue but also, at lower levels, in the heart, colon, kidney, spleen, intestine, skeletal muscle, liver, macrophages and skin. It is involved in the regulation of the glucose and lipid metabolism. In the skin, it controls the expression of a network of genes involved in cell proliferation, differentiation and inflammatory responses [13].
  • PPARs are activated by a wide variety of ligands that are derived from the metabolism of fatty acids (FAs) with some degree of isoform specificity.
  • FAs fatty acids
  • PPARy ligands such as the thiazolidinediones (TZDs), some non-steroidal anti-inflammatory drugs (NSAIDs), tetrahydroisoquinoline etc.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • PPARy ligands have different therapeutic indications and side effects demonstrating that through the interaction with PPARy diverse intracellular pathways and targets are activated.
  • the type of PPARy ligands seems to determine the conformational modification of the receptor, the activation or inhibition of different clusters of genes and then the possible therapeutic indications.
  • PPARy selective activator are able to activate some gene clusters and possibly to inhibit the expression of others [14].
  • thiazolidindiones such as rosiglitazone, troglitazone, pioglitazone, all developed for the treatment of type 2 diabetes and metabolic diseases for their ability to improve glycemic control [3,15]
  • rosiglitazone and troglitazone have been removed from the market due to increased cardiovascular disease risk and increased hepatic toxicity, respectively.
  • Pioglitazone not presenting the same risk, is commercially available.
  • ciglitazone has been studied for its effects on melanocytes.
  • conflicting results are shown in literature.
  • Joong Sun Lee et al showed that ciglitazone stimulated the melanin content of cells and cultured skin [16]; however, the same authors showed also that ciglitazone caused inhibition of melanocyte growth in a dose-dependent manner, which seemed to occur through the induction of apoptosis. In fact, apoptosis increased after ciglitazone treatment [20].
  • selective PPARy activator NAC-GED is able to reduce the inflammatory process in human keratinocytes and sebocytes and treat an experimental mouse model of psoriasis in a micro-RNA assay has demonstrated a difference in the gene activation with respect to troglitazone [17],
  • PGZ at different concentrations was tested on melanocytes from apparently normal skin of vitiligo subjects, with the aim to evaluate whether PPARy activators were able to inhibit the inflammatory process in vitiligo.
  • Vitiligo melanocytes present several biological and metabolic differences from normal characterized by an impaired energetic metabolism, where the defective ATP production is tempted to be compensated by an increased activity of enzymes involved in glucose utilization [18,19]. These alterations lead to a lower replication rate, and a pre-senescent status.
  • Vitiligo can be defined as a mitochondrial disease associated, which could be an initial event of the inflammatory process.
  • a PPARy activator, or agonist for use in the treatment of vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption (or uptake), to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
  • PPARy activators suitable for use in the treatment of vitiligo are PPARy activators characterised by the fact that they provide specific metabolic improvement on vitiligo melanocytes, i.e. decreasing glucose consumption, increasing ATP production and inducing mtDNA synthesis.
  • a PPARy activator or agonist is intended as a drug able to bind and activate the nuclear receptor PPARy and through this mechanism activate some of the target genes involved in inflammatory, differentiation and mitogenesis process and possibly inhibit the expression of other genes. Therefore, PPARy activators of the present invention are specific PPARy activators that selectively modulate PPARy activity by activating some gene clusters and/or inhibiting others.
  • a person skilled in the art is able to measure the decrease of glucose consumption, the increase of mitochondrial DNA synthesis and the increase of ATP production by known methods, for example when said vitiligo melanocytes are cultured in vitro.
  • the glucose consumption can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of glucose in the culture medium of vitiligo melanocytes treated with the PPARy activator and in the culture medium of untreated vitiligo melanocytes.
  • the measure of concentration of glucose in the culture medium can be associated with the measuring the expression of the anaerobic glycolytic enzymes Hexo 2(Esochinase 2), PKM1 ,2 (Pyruvate kinase isozymes M1/M2), PDK4 (Pyruvate Dehydrogenase Kinase); an over-expression of these enzymes in vitiligo melanocytes treated with the PPARy activator with respect to their expression in untreated vitiligo melanocytes should be associated with a decrease of glucose consumption in melanocytes treated with the PPARy activator with respect to untreated vitiligo melanocytes.
  • the ATP production can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of ATP in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by using a mitochondria targeted luciferase assay.
  • the mitochondrial DNA synthesis can be measured by culturing vitiligo melanocytes in a culture medium and measuring the mitochondrial DNA in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by real-time PCR.
  • said PPARy activator can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ⁇ 5%.
  • the above-mentioned percentage can be calculated by comparing the concentration of glucose measured in the culture medium of untreated vitiligo melanocytes with the concentration of glucose measured in the culture medium of vitiligo melanocytes treated with the PPARy activator and by calculating the percentage decrease of glucose consumption.
  • the percentage can be calculated on the minimum decrease glucose consumption obtained treating vitiligo melanocytes with the PPARy activator, such as Pioglitazone, at micromolar concentrations.
  • said PPARy activator can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ⁇ 5%.
  • the above-mentioned percentage can be calculated by comparing the concentration of ATP measured in untreated vitiligo melanocytes with the concentration of ATP measured in vitiligo melanocytes treated with the PPARy activator and by calculating the percentage increase of ATP production.
  • said PPARy activator can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ⁇ 15%.
  • the above-mentioned percentage can be calculated by comparing the concentration of mtDNA measured in untreated vitiligo melanocytes with the concentration of mtDNA measured in vitiligo melanocytes treated with the PPARy activator and by calculating the percentage increase of mtDNA synthesis.
  • said PPARy activator can also be able to increase the mitochondrial membrane potential.
  • the mitochondrial membrane potential can be measured, for example by using a cytofluorimetric method, for example using JC-1 dye.
  • said PPARy activator is able to increase mitochondrial membrane potential of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 40% ⁇ 10%.
  • the vitiligo can be non- segmental vitiligo.
  • the PPARy activator or agonist can be pioglitazone.
  • said PPARy activator such as pioglitazone
  • said PPARy activator can be administered before, simultaneously or after a phototherapy treatment.
  • said PPARy activator can be administered by oral administration or by topic administration.
  • the present invention concerns also a pharmaceutical composition
  • a pharmaceutical composition comprising or consisting of a PPARy activator or agonist in combination with one or more excipients and/or adjuvants, for use in the treatment of vitiligo, such as nonsegmental vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
  • said PPARy activator can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ⁇ 5%.
  • said PPARy activator can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ⁇ 5%.
  • said PPARy activator can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80 % ⁇ 15%.
  • said PPARy activator can also be able to increase the mitochondrial membrane potential.
  • the mitochondrial membrane potential can be measured, for example by using a cytofluorimetric method, for example using JC-1 dye.
  • said PPARy activator is able to increase mitochondria membrane potential of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 40%.
  • the PPARy activator or agonist to be used in the pharmaceutical composition can be pioglitazone.
  • the pharmaceutical composition can be administered before, simultaneously or after a phototherapy treatment.
  • said pharmaceutical composition can be in a form for oral administration or in a form for topic administration.
  • the pharmaceutical composition can further comprise a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, and an immunosuppressive drug.
  • the present invention concerns a combination of a PPARy activator or agonist with a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, or an immunosuppressive drug for separate or sequential use in the treatment of vitiligo, such as non-segmental vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
  • “separate use” is understood as meaning the administration, at the same time, of the compounds of the combination according to the invention in distinct pharmaceutical forms. “Sequential use” is understood as meaning the successive administration of the compounds of the combination according to the invention, each in a distinct pharmaceutical form.
  • the PPARy activator of the combination can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ⁇ 5%. According to the present invention, the PPARy activator of the combination can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ⁇ 5%.
  • the PPARy activator of the combination can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ⁇ 15%.
  • said PPARy activator or agonist of the combination can be pioglitazone.
  • said PPARy activator such as pioglitazone
  • said PPARy activator can administered by oral administration or by topic administration.
  • the present invention concerns also a method for screening a PPARy activator suitable for the treatment of vitiligo, said method comprising measuring in vitro the following parameters a) the glucose intake of vitiligo melanocytes, b) the production of ATP in vitiligo melanocytes and c) the mitochondrial DNA synthesis in vitiligo melanocytes, said parameters being measured in vitiligo melanocytes treated with a PPARy activator and in untreated vitiligo melanocytes, wherein when the glucose consumption of vitiligo melanocytes treated with the PPARy activator is lower than the glucose consumption of untreated vitiligo melanocytes, when the production of ATP of vitiligo melanocytes treated with the PPARy activator is higher than the production of ATP of untreated vitiligo melanocytes and when the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator is higher than the
  • the method for screening a PPARy activator suitable for the treatment of vitiligo is intended as a method for screening a PPARy activator which is effective in the treatment of vitiligo.
  • the glucose consumption can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of glucose in the culture medium of vitiligo melanocytes treated with the PPARy activator and in the culture medium of untreated vitiligo melanocytes.
  • the ATP production can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of ATP in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by using a mitochondria targeted luciferase assay.
  • the mitochondrial DNA synthesis can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of mitochondrial DNA in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by real-time PCR.
  • the glucose consumption of vitiligo melanocytes treated with the PPARy activator can be lower than the glucose consumption of untreated vitiligo melanocytes of at least 20% ⁇ 5%
  • the production of ATP of vitiligo melanocytes treated with the PPARy activator can be higher than the production of ATP of untreated vitiligo melanocytes of at least 25% ⁇ 5%
  • the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator can be higher than the mitochondrial DNA synthesis of untreated vitiligo melanocytes of at least 80% ⁇ 15%.
  • FIG. 1 Melanocyte’s proliferation rate. Treatment with PGZ 2pM increased the proliferation rate of vitiligo melanocytes (VHM) at 48hrs evaluated by automatic cell counter, whereas no modification in normal human primary epidermal melanocytes (NHM) proliferation rate was observed.
  • VHM vitiligo melanocytes
  • NHS human primary epidermal melanocytes
  • Pioglitazone improves bioenergetic in non lesional vitiligo melanocytes. Since the ATP content has been previously demonstrated to be reduced in vitiligo melanocyte (VHM), using a mitochondria targeted luciferase assay, the ATP level was measured in cells treated and untreated with Pioglitazone 2pM. An increase in ATP content was detected in vitiligo melanocytes treated for 10 days whereas control melanocytes showed no significant modification. The mitochondrial dysfunction leads to a decrease in mitochondrial membrane potential. After 10 days, PGZ treatment restores mitochondrial membrane potential in VHM.
  • VHM vitiligo melanocyte
  • JC-1 5,5,6,6’-tetrachloro-1 , 1 ’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide
  • JC-1 tetraethylbenzimi-dazoylcarbocyanine iodide
  • Rosaiglitazone reduced ATP and JC1 levels in VHM.
  • FIG. 3 Pioglitazone improves glucose metabolisms.
  • Figure shows that Pioglitazone improves glucose metabolisms in vitiligo melanocytes (VHM) in unexpected way.
  • VHM vitiligo melanocytes
  • PGZ 2 pM produced a decrease in glucose consumption, evaluated on the base of the concentration in the cultural medium, associated with an over-expression of the anaerobic glycolytic enzymes Hexo 2(Esochinase 2), PKM1 ,2 (Pyruvate kinase isozymes M1/M2), PDK4 (Pyruvate Dehydrogenase Kinase) at 6 hrs.
  • NHM PGZ 2 pM treatment induces different alterations in the expression of the same anaerobic glycolytic enzymes without concomitant modification of glucose consumption.
  • PGZ increase mitochondrial DNA (mtDNA) copy number in Vitiligo Melanocytes.
  • PGZ increases the mitochondrial DNA (mtDNA) integrity and copy number in Vitiligo Melanocytes (VHM) that was lower than in NHM.
  • Mitochondrial DNA (mtDNA) copy number which regulates the capacity for mitochondrial energy production, was measured using a target sequence on nuclear genomic DNA as reference for data normalization. To execute a comparison, cells isolated from sun- unexposed areas of age-matched healthy donors were used. Significantly lower levels of mtDNA were observed in vitiligo melanocytes with respect to normal melanocytes. Treatment with PGZ at 2pM for 10 days significantly increased the mtDNA copy number. mtDNA copy number evaluation was performed using nuclear DNA as internal reference.
  • FIG. 5 PGZ modifies expression of senescence associated markers in VHM.
  • Figure indicates that Pioglitazone modulates the expression of senescence-associated markers insulin-like growth factor-binding protein 3(IGFBP3) and cycloxygenase 2 (COX2) gene in VHM (Vitiligo human melanocytes) and NHM (Normal human melanocytes).
  • IGFBP3 insulin-like growth factor-binding protein 3
  • COX2 cycloxygenase 2
  • Relative IGFBP3 and Cox2 mRNA expression were evaluated after 6 hrs of treatment with PGZ (2 pM) and measured by qRT-PCR upon normalization to a reference gene (ACTIN).
  • MITF Molethmia-associated transcription factor
  • NHM and VHM Semi-quantitative real-time PCR was used to measure MITF mRNA expression in NHM and VHM.
  • Vitiligo melanocytes show a higher Mitf expression level indicating a high level of differentiation, that was reverted by Pioglitazone treatment.
  • Figure 6 PGZ downregulates IL-6 expression in VHM.
  • Figure shows the results of IL-6 quantification by ELISA in VHM and NHM after 48 hours of treatment. Baseline levels of IL-6 are higher in VHM than in NHM and PGZ treatment induces a reduction in IL-6 expression. Contrarily, PGZ induces IL-6 expression in NHM.
  • EXAMPLE 1 Study on the capability of PPARy activation by PGZ of improving the biological characteristics of vitiligo melanocytes
  • vitiligo and 10 normal subjects age and sex matched with non- segmental vitiligo subtype were included in the study. Vitiligo subjects were classified according to the VETF criteria.
  • the control samples normal human primary epidermal melanocytes, NHM
  • NHM normal human primary epidermal melanocytes
  • VHM Primary epidermal melanocytes from vitiligo subjects
  • Isolated NHM and VHM were cultured in 254 Medium (Cascade Biologies, TermoFisher) supplemented with specific Growth Factors cocktail (Cascade Biologies) and penicillin/streptomycin (Gibco). All of the analyses were performed between 3 and 7 culture passage.
  • the intracellular level of ATP was measured using a commercial fluorimetric kit (ThermoFisher) according to the manufacturer’s instructions. The results were reported as pM mean value ⁇ SD.
  • RT-PCR Semiquantitative real-time polymerase chain reaction
  • Pioglitazone (PGZ) treatment PGZ (Sigma) was dissolved in dimethyl sulfoxide (DMSO) to a stock solution of 20 mM and added to the cell growth medium at the final concentrations of 2pM. In untreated control cells an equal volume of DMSO was added.
  • mtDNA quantification Total DNA was prepared from melanocytes using DNeasy Blood and Tissue (Qiagen) according to the manufacturer’s recommendations and stored at -20 °C. mtDNA content was measured by real-time PCR using an iQ5 real-time PCR (BioRad). Amplification conditions were as follows: 5min at 95 °C, then 45 cycles of 15s at 95 °C and 1 min at 58 °C.
  • a dissociation curve was also calculated for each sample to ensure presence of a single PCR product.
  • the experiment was performed in triplicate.
  • the relative quantification of mitochondrial DNA (mtDNA) over nuclear DNA (nuDNA) levels was determined using the difference in the threshold cycle values of nuclear TATA-box-binding protein region on chromosome 6 and the mitochondrial non-coding control region D-loop (ACt, namely, CtmtDNA-CtnuDNA).
  • the relative abundance of the mitochondrial genome was reported as 2-ACt.
  • the primers used were the following: mtDNA forward, GATTTGGGTACCACCACCCAAGTATTG (SEQ ID NO:15); reverse, GTACAATATTCATGGTGGCTGGCA (SEQ ID NO:16); and nuDNA forward, TTCCACCCAAGTATTG (SEQ ID NO:17); reverse, TGTTCCATGCAGGGGAAAACAAGC (SEQ ID NO:18)
  • Absolute mtDNA copy number analyzed by using the Absolute Human Telomere Length and Mitochondrial DNA Copy Number Dual Quantification qPCR Assay Kit according to the manufacturer’s instructions (ScienCell Research Laboratories, Sun Diego, CA).
  • IL-6 determination in the supernatants of control and vitiligo melanocytes were quantified by ELISA assay (IL-6 from 4ABiotech) according to the manufacturer’s protocol. The results were normalized for the number of cells contained in each sample and were expressed as picograms per 1x10 6 cells. The measurement was performed in duplicate for each sample and the experiments were repeated twice.
  • VHM melanocytes from apparently normal skin of vitiligo patients
  • ROS Reactive Oxygen Species
  • differentiation markers such as Microphthalmia-associated transcription factor (MITF);
  • IL-6 insulin growth factor binding protein 3
  • IGFBP3 insulin growth factor binding protein 3
  • Vitiligo melanocytes result to be characterized by an impaired energetic metabolism, where the defective ATP production is tempted to be compensated by an increased activity of enzymes involved in glucose utilization [18,19].
  • Peroxisome proliferator-activated receptors-c activator ciglitazone
  • ciglitazone inhibits human melanocyte growth through induction of apoptosis.

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Abstract

The present invention concerns a specific and of selective activators of Peroxisome proliferator-activated receptors γ (PPARγ), such as Pioglitazone, for the treatment of vitiligo, such as non-segmental vitiligo.

Description

Selective Activators of
Peroxisome proliferator-activated receptors (PPARs) for the treatment of vitiligo
The present invention concerns specific activators of Peroxisome proliferator-activated receptors (PPARs), such as Pioglitazone, for the treatment of vitiligo. In particular, the present invention concerns specific and selective activators of Peroxisome proliferator-activated receptors y (PPARy), such as Pioglitazone, for the treatment of vitiligo, such as non- segmental vitiligo.
Vitiligo is an acquired chronic depigmenting disorder of the skin resulting from selective loss of melanocytes. According to international consensus, it is classified into three major forms, namely, non-segmental vitiligo (or simply, vitiligo), segmental vitiligo and mixed vitiligo [1 ,2], Non- segmental vitiligo, the commonest form of this unpredictable disease, is characterized by symmetrical and bilateral white patches. The lesions are typically distributed in an acrofacial pattern (hands and feet, periorificial facial involvement) or scattered symmetrically over the entire body. The hairs on the involved skin remain pigmented initially but after a prolonged time, leukotrichia (whiteness of the hair) might develop. Different clinical subtypes have been described, including generalized, acrofacial, and universalis types, all with a bilateral distribution. Overall, progressive patchy loss of pigmentation from the skin, overlying hair, and sometimes mucosa remains the basis of the diagnosis. The involvement of non- cutaneous melanocytes such as ocular and cochlear melanocytes is controversial [1 ,3].
Vitiligo is quite common, with an estimated global prevalence of 0.5% to 2% but peaks of up to 8.8% have been reported in India, possibly relating to the inclusion of chemically induced depigmentation.
The characteristic of vitiligo is the loss of functional melanocytes and, even if the mechanisms involved are not completely defined, oxidative stress, generation of inflammatory mediators, cell detachment and autoimmune responses have been considered pathogenetically relevant [4,5].The pivotal role in melanocyte loss is played by CD8+ T cells which are more numerous in the skin of patients with active disease than in healthy controls and in patients with stable disease.
Melanocytes from apparently normal skin of vitiligo patients show several metabolic defects that lead to alteration in the free radicals scavenging activity that make harder for the cell to adapt to external stress. An increased level of Reactive Oxygen Species (ROS) has been demonstrated in vivo, on lesional and non-lesional areas and in vitro in melanocytes obtained from pigmented skin, associated with alterations of the anti-oxidant mechanisms [1 ,6]. The imbalance in the pro and anti- oxidative state is responsible for the increased susceptibility to external stressful stimuli and possibly of the premature senescence of the skin characterized by the production of different proteins of the so-called Senescence-associated Secretory Phenotype (SASP)[6]. Furthermore, the increased intracellular oxidative stress is also responsible for the accumulation of immature proteins, due to damage to the endoplasmic reticulum, with activation of the so-called "unfolded protein response" (UPR). Recent works have shown that some of the alterations are not exclusive to melanocytes and have also been described in other skin cells obtained from non lesional areas, suggesting that vitiligo leads to generalized degeneration [7], Fibroblasts exhibit oxidative stress, phosphorylation of p38, over-expression of p53 and a senescent phenotype. These changes are the basis for altered secretion of soluble growth factors supporting melanocyte survival and homeostasis [4,8,9]. Keratinocytes which support melanocyte survival and melanin production via growth factors, including bFGF (basic fibroblast growth factor) and SCF (stem cell factor) show apoptotic markers and reduced SCF production, in the lesional area [10]. The alteration of the dermal-epidermal network is probably the basis for melanocyte detachment [7],
It is important to note that vitiligo is not a “cosmetic disease’” so treatment should and can be offered to patients. European guidelines for the diagnosis and management of vitiligo recommend narrow band (NB) ultraviolet (UV) B, tacrolimus and topical steroids but results are not always satisfactory [11],
At present, no medical treatment for repigmenting vitiligo has been approved by the US Food and Drug Administration (FDA) and European Medicine Agency (EMA) and, therefore, treatments are used off-label.
Topical treatments are adopted when small areas are involved. Phototherapy in combination with topical ultrapotent or potent corticosteroids is preferred when >5-10% of the BSA is affected or when focal areas are unresponsive to steroids alone. For lesions on the face, neck, and intertriginous areas and lesions in children either mid-potency topical corticosteroids or calcineurin inhibitors are used. In published guidelines, the European Dermatology Forum group proposed twice daily topical calcineurin inhibitors for head and neck lesions as a first-line approach.
In addition, surgical treatments can be offered to some patients with limited and stable lesions.
The newest options are represented by JAK inhibitors. For example, Apremilast is indicated in the treatment of rheumatoid arthritis and psoriasis and case reports have noted its use in the treatment of vitiligo. However, in the randomized control trial by Khemis et al., apremilast in combination with Narrow Band UVB (NB-UVB) did not demonstrate any significant improvement of the therapeutic effect obtained with NB-UVB alone [11].
The major limitation of the currently available therapies, both topical and systemic, is the high percentage of recurrences (30 to 50% on average). The phenomenon is due to the fact that these treatments reduce the production or the release of inflammatory mediators and decrease the immune infiltrate but they do not interfere with the initial mechanisms responsible for the activation of the autoimmune response. Therefore, any further stressful event can produce a damage of the melanocytes with the subsequent release of DAMPS and PAMPS and reactivation of resident memory T Cells responsible for disease relapse [12],
Therefore, new therapies for vitiligo are needed, able to overcome the disadvantages of current ones and capable of activating melanocyte reservoir, mainly present in the hair follicles.
According to the present invention, the Applicant has now found that, an activator of Peroxisome proliferator-activated receptors (PPARs), Pioglitazone, is capable of activating melanocyte reservoir, mainly present in the hair follicles. In addition, according to the present invention, it has been found that pioglidazone prevents or blocks the damage of melanocytes and presumably the induction of the immune response in vitiligo through a mechanism of action not previously identified.
PPARs are nuclear receptors, which have an important role in the mammalian physiological system. The three PPAR isoforms, PPARa, [3 /5 and y share considerable sequence and structural homologies but exhibit different tissue distribution, selectivity and responsiveness to ligands, leading to regulation of distinct sets of genes by different receptors. Activated PPARs heterodimerize with the retinoid X receptor (RXR) in the presence of activators and bind to a PPAR response element (PPRE) in the promoters of target genes. PPARs also act through ligand-independent modes of action, such as trans-repression and constitutive activation of the receptors. PPARy, the most widely investigated subtype, is expressed predominantly in the adipose tissue but also, at lower levels, in the heart, colon, kidney, spleen, intestine, skeletal muscle, liver, macrophages and skin. It is involved in the regulation of the glucose and lipid metabolism. In the skin, it controls the expression of a network of genes involved in cell proliferation, differentiation and inflammatory responses [13]. PPARs are activated by a wide variety of ligands that are derived from the metabolism of fatty acids (FAs) with some degree of isoform specificity. Several families of compounds of different nature have been classified as PPARy ligands such as the thiazolidinediones (TZDs), some non-steroidal anti-inflammatory drugs (NSAIDs), tetrahydroisoquinoline etc. However, the different families of compounds have different therapeutic indications and side effects demonstrating that through the interaction with PPARy diverse intracellular pathways and targets are activated. The type of PPARy ligands seems to determine the conformational modification of the receptor, the activation or inhibition of different clusters of genes and then the possible therapeutic indications. PPARy selective activator are able to activate some gene clusters and possibly to inhibit the expression of others [14], Among the thiazolidindiones, such as rosiglitazone, troglitazone, pioglitazone, all developed for the treatment of type 2 diabetes and metabolic diseases for their ability to improve glycemic control [3,15], rosiglitazone and troglitazone have been removed from the market due to increased cardiovascular disease risk and increased hepatic toxicity, respectively. Pioglitazone, not presenting the same risk, is commercially available.
Among the thiazolidindiones, ciglitazone has been studied for its effects on melanocytes. However, conflicting results are shown in literature. In particular, Joong Sun Lee et al showed that ciglitazone stimulated the melanin content of cells and cultured skin [16]; however, the same authors showed also that ciglitazone caused inhibition of melanocyte growth in a dose-dependent manner, which seemed to occur through the induction of apoptosis. In fact, apoptosis increased after ciglitazone treatment [20].
Recently, the inventors of the present invention have demonstrated that selective PPARy activator NAC-GED is able to reduce the inflammatory process in human keratinocytes and sebocytes and treat an experimental mouse model of psoriasis in a micro-RNA assay has demonstrated a difference in the gene activation with respect to troglitazone [17],
According to the present invention, PGZ at different concentrations was tested on melanocytes from apparently normal skin of vitiligo subjects, with the aim to evaluate whether PPARy activators were able to inhibit the inflammatory process in vitiligo.
According to the present invention the biological and metabolic characteristics of melanocytes from skin of vitiligo patients have been studied. The results show that Vitiligo melanocytes present several biological and metabolic differences from normal characterized by an impaired energetic metabolism, where the defective ATP production is tempted to be compensated by an increased activity of enzymes involved in glucose utilization [18,19]. These alterations lead to a lower replication rate, and a pre-senescent status.
According to the results of the above-mentioned studies, it was found that Vitiligo can be defined as a mitochondrial disease associated, which could be an initial event of the inflammatory process.
In addition, according to the present invention the effects of PGZ on vitiligo melanocytes, were studied.
The unexpected results of the treatment with PGZ were that vitiligo melanocytes showed a different behaviour with respect to normal melanocytes. This makes it plausible that PGZ can be successfully used in the treatment of vitiligo as new therapeutic approach through the improvement of the energetic metabolism of the cells. Of note, most of the effects obtained by the treatment with PGZ were not previously described, such as:
• reduction of the glucose intake associated with the mitochondria genesis and increase of ATP production;
• reverting of the senescence phenotype in all skin cells;
• decreases the production of inflammatory mediators.
It is therefore a specific object of the present invention a PPARy activator, or agonist, for use in the treatment of vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption (or uptake), to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
Therefore, according to the present invention, PPARy activators suitable for use in the treatment of vitiligo are PPARy activators characterised by the fact that they provide specific metabolic improvement on vitiligo melanocytes, i.e. decreasing glucose consumption, increasing ATP production and inducing mtDNA synthesis.
A PPARy activator or agonist is intended as a drug able to bind and activate the nuclear receptor PPARy and through this mechanism activate some of the target genes involved in inflammatory, differentiation and mitogenesis process and possibly inhibit the expression of other genes. Therefore, PPARy activators of the present invention are specific PPARy activators that selectively modulate PPARy activity by activating some gene clusters and/or inhibiting others. A person skilled in the art is able to measure the decrease of glucose consumption, the increase of mitochondrial DNA synthesis and the increase of ATP production by known methods, for example when said vitiligo melanocytes are cultured in vitro.
In particular, the glucose consumption can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of glucose in the culture medium of vitiligo melanocytes treated with the PPARy activator and in the culture medium of untreated vitiligo melanocytes. The measure of concentration of glucose in the culture medium can be associated with the measuring the expression of the anaerobic glycolytic enzymes Hexo 2(Esochinase 2), PKM1 ,2 (Pyruvate kinase isozymes M1/M2), PDK4 (Pyruvate Dehydrogenase Kinase); an over-expression of these enzymes in vitiligo melanocytes treated with the PPARy activator with respect to their expression in untreated vitiligo melanocytes should be associated with a decrease of glucose consumption in melanocytes treated with the PPARy activator with respect to untreated vitiligo melanocytes.
The ATP production can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of ATP in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by using a mitochondria targeted luciferase assay.
The mitochondrial DNA synthesis can be measured by culturing vitiligo melanocytes in a culture medium and measuring the mitochondrial DNA in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by real-time PCR.
According to the present invention, said PPARy activator can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%. The above-mentioned percentage can be calculated by comparing the concentration of glucose measured in the culture medium of untreated vitiligo melanocytes with the concentration of glucose measured in the culture medium of vitiligo melanocytes treated with the PPARy activator and by calculating the percentage decrease of glucose consumption. For example, the percentage can be calculated on the minimum decrease glucose consumption obtained treating vitiligo melanocytes with the PPARy activator, such as Pioglitazone, at micromolar concentrations.
According to the present invention, said PPARy activator can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ± 5%.
The above-mentioned percentage can be calculated by comparing the concentration of ATP measured in untreated vitiligo melanocytes with the concentration of ATP measured in vitiligo melanocytes treated with the PPARy activator and by calculating the percentage increase of ATP production.
According to the present invention, said PPARy activator can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ± 15%.
The above-mentioned percentage can be calculated by comparing the concentration of mtDNA measured in untreated vitiligo melanocytes with the concentration of mtDNA measured in vitiligo melanocytes treated with the PPARy activator and by calculating the percentage increase of mtDNA synthesis.
According to the present invention, said PPARy activator can also be able to increase the mitochondrial membrane potential. The mitochondrial membrane potential can be measured, for example by using a cytofluorimetric method, for example using JC-1 dye. In particular, said PPARy activator is able to increase mitochondrial membrane potential of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 40% ± 10%. According to the present invention, the vitiligo can be non- segmental vitiligo.
According to an embodiment of the present invention, the PPARy activator or agonist can be pioglitazone.
According to the present invention, said PPARy activator, such as pioglitazone, can be administered before, simultaneously or after a phototherapy treatment.
Moreover, according to the present invention, said PPARy activator can be administered by oral administration or by topic administration.
The present invention concerns also a pharmaceutical composition comprising or consisting of a PPARy activator or agonist in combination with one or more excipients and/or adjuvants, for use in the treatment of vitiligo, such as nonsegmental vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
As mentioned above, said PPARy activator can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%.
According to the invention, said PPARy activator can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ± 5%.
According to the invention, said PPARy activator can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80 % ± 15%.
As stated above, according to the present invention, said PPARy activator can also be able to increase the mitochondrial membrane potential. The mitochondrial membrane potential can be measured, for example by using a cytofluorimetric method, for example using JC-1 dye. In particular, said PPARy activator is able to increase mitochondria membrane potential of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 40%.
As mentioned above, the PPARy activator or agonist to be used in the pharmaceutical composition can be pioglitazone.
According to an embodiment of the present invention, the pharmaceutical composition can be administered before, simultaneously or after a phototherapy treatment.
According to the present invention, said pharmaceutical composition can be in a form for oral administration or in a form for topic administration.
The pharmaceutical composition can further comprise a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, and an immunosuppressive drug.
In addition, the present invention concerns a combination of a PPARy activator or agonist with a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, or an immunosuppressive drug for separate or sequential use in the treatment of vitiligo, such as non-segmental vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
According to the present invention, “separate use” is understood as meaning the administration, at the same time, of the compounds of the combination according to the invention in distinct pharmaceutical forms. “Sequential use” is understood as meaning the successive administration of the compounds of the combination according to the invention, each in a distinct pharmaceutical form.
According to the present invention, the PPARy activator of the combination can be able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%. According to the present invention, the PPARy activator of the combination can be able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ± 5%.
According to the present invention, the PPARy activator of the combination can be able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ± 15%.
As mentioned above, said PPARy activator or agonist of the combination can be pioglitazone.
According to the combination of present invention, said PPARy activator, such as pioglitazone, can administered by oral administration or by topic administration.
The present invention concerns also a method for screening a PPARy activator suitable for the treatment of vitiligo, said method comprising measuring in vitro the following parameters a) the glucose intake of vitiligo melanocytes, b) the production of ATP in vitiligo melanocytes and c) the mitochondrial DNA synthesis in vitiligo melanocytes, said parameters being measured in vitiligo melanocytes treated with a PPARy activator and in untreated vitiligo melanocytes, wherein when the glucose consumption of vitiligo melanocytes treated with the PPARy activator is lower than the glucose consumption of untreated vitiligo melanocytes, when the production of ATP of vitiligo melanocytes treated with the PPARy activator is higher than the production of ATP of untreated vitiligo melanocytes and when the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator is higher than the mitochondrial DNA synthesis of untreated vitiligo melanocytes, the PPARy activator is suitable for the treatment of vitiligo.
According to the present invention, the method for screening a PPARy activator suitable for the treatment of vitiligo is intended as a method for screening a PPARy activator which is effective in the treatment of vitiligo.
According to the method of the invention, the glucose consumption can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of glucose in the culture medium of vitiligo melanocytes treated with the PPARy activator and in the culture medium of untreated vitiligo melanocytes.
According to the method of the invention, the ATP production can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of ATP in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by using a mitochondria targeted luciferase assay.
According to the method of the invention, the mitochondrial DNA synthesis can be measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of mitochondrial DNA in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes, for example by real-time PCR.
According to the present invention, when the PPARy activator is suitable for the treatment of vitiligo, the glucose consumption of vitiligo melanocytes treated with the PPARy activator can be lower than the glucose consumption of untreated vitiligo melanocytes of at least 20% ± 5%, the production of ATP of vitiligo melanocytes treated with the PPARy activator can be higher than the production of ATP of untreated vitiligo melanocytes of at least 25% ± 5% and the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator can be higher than the mitochondrial DNA synthesis of untreated vitiligo melanocytes of at least 80% ± 15%.
The present invention now will be described by an illustrative, but not limitative way, according to preferred embodiments thereof, with particular reference to the examples and the enclosed drawings, wherein:
Figure 1. Melanocyte’s proliferation rate. Treatment with PGZ 2pM increased the proliferation rate of vitiligo melanocytes (VHM) at 48hrs evaluated by automatic cell counter, whereas no modification in normal human primary epidermal melanocytes (NHM) proliferation rate was observed. K = Control; P = PGZ.
Figure 2. Pioglitazone improves bioenergetic in non lesional vitiligo melanocytes. Since the ATP content has been previously demonstrated to be reduced in vitiligo melanocyte (VHM), using a mitochondria targeted luciferase assay, the ATP level was measured in cells treated and untreated with Pioglitazone 2pM. An increase in ATP content was detected in vitiligo melanocytes treated for 10 days whereas control melanocytes showed no significant modification. The mitochondrial dysfunction leads to a decrease in mitochondrial membrane potential. After 10 days, PGZ treatment restores mitochondrial membrane potential in VHM. The technique involved using 5,5,6,6’-tetrachloro-1 , 1 ’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) that is a lipophilic, cationic dye which is able to enter into the mitochondria where it accumulates and (in a concentration-dependent manner) starts forming reversible complexes called J aggregates. Contrarily, other thiazolidindiones such as Rosaiglitazone reduced ATP and JC1 levels in VHM.
Figure 3. Pioglitazone improves glucose metabolisms. Figure shows that Pioglitazone improves glucose metabolisms in vitiligo melanocytes (VHM) in unexpected way. In VHM, PGZ 2 pM produced a decrease in glucose consumption, evaluated on the base of the concentration in the cultural medium, associated with an over-expression of the anaerobic glycolytic enzymes Hexo 2(Esochinase 2), PKM1 ,2 (Pyruvate kinase isozymes M1/M2), PDK4 (Pyruvate Dehydrogenase Kinase) at 6 hrs. In NHM, PGZ 2 pM treatment induces different alterations in the expression of the same anaerobic glycolytic enzymes without concomitant modification of glucose consumption.
Figure 4. PGZ increase mitochondrial DNA (mtDNA) copy number in Vitiligo Melanocytes. PGZ increases the mitochondrial DNA (mtDNA) integrity and copy number in Vitiligo Melanocytes (VHM) that was lower than in NHM. Mitochondrial DNA (mtDNA) copy number, which regulates the capacity for mitochondrial energy production, was measured using a target sequence on nuclear genomic DNA as reference for data normalization. To execute a comparison, cells isolated from sun- unexposed areas of age-matched healthy donors were used. Significantly lower levels of mtDNA were observed in vitiligo melanocytes with respect to normal melanocytes. Treatment with PGZ at 2pM for 10 days significantly increased the mtDNA copy number. mtDNA copy number evaluation was performed using nuclear DNA as internal reference.
Figure 5. PGZ modifies expression of senescence associated markers in VHM. Figure indicates that Pioglitazone modulates the expression of senescence-associated markers insulin-like growth factor-binding protein 3(IGFBP3) and cycloxygenase 2 (COX2) gene in VHM (Vitiligo human melanocytes) and NHM (Normal human melanocytes). However, while in VHM senescence markers decreased after treatment, NHM showed an inverse trend. Relative IGFBP3 and Cox2 mRNA expression were evaluated after 6 hrs of treatment with PGZ (2 pM) and measured by qRT-PCR upon normalization to a reference gene (ACTIN). MITF (Microphtalmia-associated transcription factor) is a specific melanocyte marker and a master regulator of melanocyte development. Semi-quantitative real-time PCR was used to measure MITF mRNA expression in NHM and VHM. Vitiligo melanocytes show a higher Mitf expression level indicating a high level of differentiation, that was reverted by Pioglitazone treatment.
Figure 6. PGZ downregulates IL-6 expression in VHM. Figure shows the results of IL-6 quantification by ELISA in VHM and NHM after 48 hours of treatment. Baseline levels of IL-6 are higher in VHM than in NHM and PGZ treatment induces a reduction in IL-6 expression. Contrarily, PGZ induces IL-6 expression in NHM.
EXAMPLE 1 : Study on the capability of PPARy activation by PGZ of improving the biological characteristics of vitiligo melanocytes
Materials and methods
Skin biopsies were collected after obtaining a written informed consent for collection and use of such biopsies from all patients, on the basis of current legislation.
In order to evaluate whether PPARy activation by PGZ improves the biological characteristics of vitiligo melanocytes, normal and vitiligo melanocytes were treated with Pioglitazone at concentrations ranging from 2 to 10 pM for periods from 6h to 10 days according to the type of parameter evaluated.
10 vitiligo and 10 normal subjects age and sex matched with non- segmental vitiligo subtype were included in the study. Vitiligo subjects were classified according to the VETF criteria. The control samples (normal human primary epidermal melanocytes, NHM) were obtained from subjects who underwent plastic surgery for diseases unrelated to pigmentation disorders. Primary epidermal melanocytes from vitiligo subjects (VHM) were isolated from 1 cm2 skin biopsy in non-lesional skin after informed consent. Isolated NHM and VHM were cultured in 254 Medium (Cascade Biologies, TermoFisher) supplemented with specific Growth Factors cocktail (Cascade Biologies) and penicillin/streptomycin (Gibco). All of the analyses were performed between 3 and 7 culture passage.
A TP Determination
The intracellular level of ATP was measured using a commercial fluorimetric kit (ThermoFisher) according to the manufacturer’s instructions. The results were reported as pM mean value ±SD.
Semiquantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted using the Aurum Total mini kit (BioRad, Milan Italy). cDNA was synthesized from 1 pg total RNA using the FirstAid kit (Fermentas, ThermoFisher Scientific, Waltham, MA, USA) and amplified in a reaction mixture containing SsoAdvanced Universal Syber Green Supermix (BioRad) and 25 pmol forward and reverse primers using a CXF96 Touch Cycler (BioRad). All samples were run in triplicate. Amplification of the p-actin (Pact) from each sample was included as the internal control. For each gene, the assessment of quality was performed by examining PCR melt curves after quantitative (q)RT-PCR to ensure product specificity. Table 1 shows the oligonucleotide sequences used to detect expression of reported target genes.
Table 1
Figure imgf000018_0001
Pioglitazone (PGZ) treatment PGZ (Sigma) was dissolved in dimethyl sulfoxide (DMSO) to a stock solution of 20 mM and added to the cell growth medium at the final concentrations of 2pM. In untreated control cells an equal volume of DMSO was added. mtDNA quantification Total DNA was prepared from melanocytes using DNeasy Blood and Tissue (Qiagen) according to the manufacturer’s recommendations and stored at -20 °C. mtDNA content was measured by real-time PCR using an iQ5 real-time PCR (BioRad). Amplification conditions were as follows: 5min at 95 °C, then 45 cycles of 15s at 95 °C and 1 min at 58 °C. A dissociation curve was also calculated for each sample to ensure presence of a single PCR product. The experiment was performed in triplicate. The relative quantification of mitochondrial DNA (mtDNA) over nuclear DNA (nuDNA) levels was determined using the difference in the threshold cycle values of nuclear TATA-box-binding protein region on chromosome 6 and the mitochondrial non-coding control region D-loop (ACt, namely, CtmtDNA-CtnuDNA). The relative abundance of the mitochondrial genome was reported as 2-ACt. The primers used were the following: mtDNA forward, GATTTGGGTACCACCCAAGTATTG (SEQ ID NO:15); reverse, GTACAATATTCATGGTGGCTGGCA (SEQ ID NO:16); and nuDNA forward, TTCCACCCAAGTATTG (SEQ ID NO:17); reverse, TGTTCCATGCAGGGGAAAACAAGC (SEQ ID NO:18)
Absolute mtDNA copy number analyzed by using the Absolute Human Telomere Length and Mitochondrial DNA Copy Number Dual Quantification qPCR Assay Kit according to the manufacturer’s instructions (ScienCell Research Laboratories, Sun Diego, CA).
Protein determination by sandwich enzyme-linked immunosorbent assay
IL-6 determination in the supernatants of control and vitiligo melanocytes were quantified by ELISA assay (IL-6 from 4ABiotech) according to the manufacturer’s protocol. The results were normalized for the number of cells contained in each sample and were expressed as picograms per 1x106 cells. The measurement was performed in duplicate for each sample and the experiments were repeated twice.
Results
Biological characteristics of skin cells from apparently normal skin of vitiligo patients.
Melanocytes
It was demonstrated that melanocytes from apparently normal skin of vitiligo patients (VHM) present, respect to normal melanocytes, some structural and functional alterations with:
• a significant slower replication rate;
• an increased susceptibility to mild stress;
• higher Reactive Oxygen Species (ROS)generation;
• decreased of the mitochondrial Transmembrane potential (AMJm) and of the expression of some Electron Transport Chain (ETC) proteins.
Moreover, they present higher expression of:
• p53;
• differentiation markers such as Microphthalmia-associated transcription factor (MITF);
• senescent associated secreted phenotype including interleukin 6
(IL-6) and insulin growth factor binding protein 3 (IGFBP3);
• an enhanced cellular uptake of glucose from the culture medium;
• Lower ATP level;
• Mitochondrial mass, as detected by Differential Light Scatter (DLS), as compensatory mechanism to obtain a sufficient energetic level in steady-state condition;
• increased expression of some mitochondrial genes (ND2, ND5, ND6 of Cxi, and Cxi, Cxll, Cxlll of CxIV), without an increase in mitochondrial DNA content.
Therefore, Vitiligo melanocytes result to be characterized by an impaired energetic metabolism, where the defective ATP production is tempted to be compensated by an increased activity of enzymes involved in glucose utilization [18,19].
Pioglitazone (PGZ) treatment
PGZ at the concentrations tested (up to 10 pM) did not modified cell viability in both normal and vitiligo melanocytes. On the contrary treatment with 2 pM PGZ increased in vitiligo melanocytes the duplication rate according to the period of culture (2 days). Considering that 2 pM was the lowest concentration able to induce an increase of cell proliferation rate, this concentration was used in the further experiments (Fig 1 ).
PGZ treatment On vitiligo melanocytes:
• improves mitochondria bioenergetic status, statistically significant increased ATP concentration by 27.8% after 10 days (Fig 2);
• Increases mitochondria membrane potential, evaluated by flow citometry, (Fig 2);
• Increases the expression of anaerobic glucose metabolism. Unexpected, however the glucose consumption was decreased by 14.3% (Fig 3);
• increases mitochondrial biogenesis as demonstrated by the significant increase of the mtDNA copies (Fig 4);
• produces a decrease expression of the differentiation marker MITF and of senescent marker IGFBP3 (Fig 5)
REFERENCES
[1] Picardo, M.; Dell'Anna, M.L.; Ezzedine, K.; Hamzavi, L; Harris, J.E.; Parsad, D.; Taieb, A. Vitiligo. Nat. Rev. Dis. Primers 2015, 1, 15011.
[2] Ezzedine, K.; Eleftheriadou, V.; Whitton, M.; van Geel, N. Vitiligo. Lancet 2015, 386, 74-84.
[3] Dumasia, R.; Eagle, K.A.; Kline-Rogers, E.; May, N.; Cho, L.; Mukherjee, D. Role of PPAR- Gamma Agonist Thiazolidinediones in Treatment of Pre-Diabetic and Diabetic Individuals: A Cardiovascular Perspective. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005, 5, 377-386.
[4] Boukhedouni, N.; Martins, C.; Darrigade, A.S.; Drullion, C.; Rambert, J.; Barrault, C.; Garnier, J.; Jacquemin, C.; Thiolat, D.; Lucchese, F. et al. Type-1 Cytokines Regulate MMP-9 Production and E- Cadherin Disruption to Promote Melanocyte Loss in Vitiligo. JCI Insight 2020, 5, e133772. doi: 10.1172/jci.insight.133772.
[5] Schafer, P.H.; Parton, A.; Gandhi, A.K.; Capone, L.; Adams, M.; Wu, L.; Bartlett, J.B.; Loveland, M.A.; Gilhar, A.; Cheung, Y.F. et al. Apremilast, a cAMP Phosphodiesterase-4 Inhibitor, Demonstrates Anti- Inflammatory Activity in Vitro and in a Model of Psoriasis. Br. J. Pharmacol. 2010, 159, 842-855. [6] Bellei, B.; Pitisci, A.; Ottaviani, M.; Ludovici, M.; Cota, C.; Luzi, F.; Dell'Anna, M.L.; Picardo, M. Vitiligo: A Possible Model of Degenerative Diseases. PLoS One 2013, 8, e59782.
[7] Bastonini, E.; Bellei, B.; Filoni, A.; Kovacs, D.; lacovelli, P.; Picardo, M. Involvement of Non-Melanocytic Skin Cells in Vitiligo. Exp. Dermatol. 2019, 28, 667-673.
[8] Cario-Andre, M.; Pain, C.; Gauthier, Y.; Casoli, V.; Taieb, A. In Vivo and in Vitro Evidence of Dermal Fibroblasts Influence on Human Epidermal Pigmentation. Pigment Cell Res. 2006, 19, 434-442.
[9] Khemis, A.; Fontas, E.; Moulin, S.; Montaudie, H.; Lacour, J.P.; Passeron, T. Apremilast in Combination with Narrowband UVB in the Treatment of Vitiligo: A 52-Week Monocentric Prospective Randomized Placebo-Controlled Study. J. Invest. Dermatol. 2020, 140, 1533-1537. e2.
[10] Moretti, S.; Fabbri, P.; Baroni, G.; Berti, S.; Bani, D.; Berti, E.; Nassini, R.; Lotti, T.; Massi, D. Keratinocyte Dysfunction in Vitiligo Epidermis: Cytokine Microenvironment and Correlation to Keratinocyte Apoptosis. Histol. Histopathol. 2009, 24, 849-857.
[11] Passeron, T. First Step in a New Era for Treatment of Patients with Vitiligo. Lancet 2020, 396, 74-75.
[12] Boniface, K.; Jacquemin, C.; Darrigade, A.S.; Dessarthe, B.; Martins, C.; Boukhedouni, N.; Vernisse, C.; Grasseau, A.; Thiolat, D.; Rambert, J. et al. Vitiligo Skin is Imprinted with Resident Memory CD8 T Cells Expressing CXCR3. J. Invest. Dermatol. 2018, 138, 355-364.
[13] Scheen, A.J.; Paquot, N. PPAR-Gamma Receptors, New Therapeutic Target in Metabolic and Cardiovascular Diseases. Rev. Med. Liege 2005, 60, 89-95.
[14] Pirat, C.; Farce, A.; Lebegue, N.; Renault, N.; Furman, C.; Millet, R.; Yous, S.; Speca, S.; Berthelot, P.; Desreumaux, P. et al. Targeting Peroxisome Proliferator-Activated Receptors (PPARs): Development of Modulators. J. Med. Chem. 2012, 55, 4027-4061.
[15] Wang, Y.; Li, S.; Li, C. Perspectives of New Advances in the Pathogenesis of Vitiligo: From Oxidative Stress to Autoimmunity. Med. Sci. Monit. 2019, 25, 1017-1023. [16] Lee, J.S.; Choi, Y.M.; Kang, H.Y. PPAR-Gamma Agonist, Ciglitazone, Increases Pigmentation and Migration of Human Melanocytes. Exp. Dermatol. 2007, 16, 118-123.
[17] Mastrofrancesco, A.; Ottaviani, M.; Cardinali, G.; Fiori, E.; Briganti, S.; Ludovici, M.; Zouboulis, C.C.; Lora, V.; Camera, E.; Picardo, M. Pharmacological PPARy Modulation Regulates Sebogenesis and Inflammation in SZ95 Human Sebocytes. Biochem. Pharmacol. 2017, 138, 96-106.
[18] Dell'Anna, M.L.; Ottaviani, M.; Albanesi, V.; Vidolin, A.P.; Leone, G.; Ferraro, C.; Cossarizza, A.; Rossi, L.; Picardo, M. Membrane Lipid Alterations as a Possible Basis for Melanocyte Degeneration in Vitiligo. J. Invest. Dermatol. 2007, 127, 1226-1233.
[19] Dell'Anna, M.L.; Ottaviani, M.; Kovacs, D.; Mirabilii, S.; Brown, D.A.; Cota, C.; Migliano, E.; Bastonini, E.; Bellei, B.; Cardinali, G. et al. Energetic Mitochondrial Failing in Vitiligo and Possible Rescue by Cardiolipin. Sci. Rep. 2017, 7, 13663-017-13961 -5.
[20] Hee Young Kang, Ji Yeoun Lee, Joong Sun Lee, You Mi Choi,
Peroxisome proliferator-activated receptors-c activator, ciglitazone, inhibits human melanocyte growth through induction of apoptosis. Arch Dermatol Res, 2006, 297: 472-476, DOI 10.1007/s00403-006-0646-4.

Claims

22
1 ) PPARy activator for use in the treatment of vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
2) PPARy activator according to claim 1 , for use according to claim 1 , wherein said PPARy activator is able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%.
3) PPARy activator according to any one of claims 1 -2, for use according to anyone of claims 1 -2, wherein said PPARy activator is able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ± 5%.
4) PPARy activator according to any one of claims 1 -3, for use according to anyone of claims 1 -3, wherein said PPARy activator is able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ± 15%.
5) PPARy activator according to any one of claims 1 -4, for use according to anyone of claims 1 -4, wherein the vitiligo is non segmental vitiligo.
6) PPARy activator according to any one of claims 1 -5, for use according to any one of claim 1 -5, wherein said PPARy activator is pioglitazone.
7) PPARy activator according to any one of claims 1 -6, for use according to any one of claims 1 -6, wherein said PPARy activator is administered before, simultaneously or after a phototherapy treatment.
8) PPARy activator according to any one of claims 1 -7, for use according to any one of claims 1 -7, wherein said PPARy activator is administered by oral administration or by topic administration. 9) Pharmaceutical composition comprising or consisting of a PPARy activator in combination with one or more excipients and/or adjuvants, for use in the treatment of vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
10) Pharmaceutical composition according to claim 9, for use according to claim 9, wherein said PPARy activator is able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%.
11 ) Pharmaceutical composition according any one of claim 9-10, for use according to any one of claim 9-10, wherein said PPARy activator is able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25% ± 5%.
12) Pharmaceutical composition according any one of claim 9-11 , for use according to any one of claim 9-11 , wherein said PPARy activator is able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80 % ± 15%.
13) Pharmaceutical composition according to any one of claim 9-12, for use according to any one of claim 9-12, wherein the vitiligo is non segmental vitiligo.
14) Pharmaceutical composition according to any one of claims 9-
13, for use according to any one of claim 9-13, wherein said PPARy activator is pioglitazone.
15) Pharmaceutical composition according to any one of claims 9-
14, for use according to any one of claims 9-14, wherein said pharmaceutical composition is administered before, simultaneously or after a phototherapy treatment.
16) Pharmaceutical composition according to any one of claims 9- 15, for use according to any one of claims 9-15, wherein said pharmaceutical composition is in a form for oral administration or in a form for topic administration.
17) Pharmaceutical composition according to anyone of claims 9-
16, for use according to any one of claims 9-16, said pharmaceutical composition further comprising a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, and an immunosuppressive drug.
18) Combination of a PPARy activator with a drug chosen from the group consisting of an anti-inflammatory drug, such a steroid, or an immunosuppressive drug for separate or sequential use in the treatment of vitiligo, wherein said PPARy activator is a PPARy activator that is able to decrease glucose consumption, to increase mitochondrial DNA synthesis and to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes.
19) Combination according to claim 18, for use according to claim 18, wherein said PPARy activator is able to decrease glucose consumption of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 20% ± 5%.
20) Combination according to any one of claims 18-19, for use according to any one of claims 18-19, wherein said PPARy activator is able to increase the ATP production of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 25%± 5%.
21 ) Combination according to any one of claims 18-20, for use according to any one of claims 18-20, wherein said PPARy activator is able to increase the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator in comparison to untreated vitiligo melanocytes of at least 80% ± 15%.
22) Combination according to any one of claims 18-21 , for use according to any one of claims 18-21 , wherein said PPARy activator is pioglitazone.
23) Method for screening a PPARy activator suitable for the 25 treatment of vitiligo, said method comprising measuring in vitro the following parameters a) the glucose intake, b) the production of ATP and c) the mitochondrial DNA synthesis, said parameters being measured in vitiligo melanocytes treated with a PPARy activator and in untreated vitiligo melanocytes, wherein when the glucose consumption of vitiligo melanocytes treated with the PPARy activator is lower than the glucose consumption of untreated vitiligo melanocytes, when the production of ATP of vitiligo melanocytes treated with the PPARy activator is higher than the production of ATP of untreated vitiligo melanocytes and when the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator is higher than the mitochondrial DNA synthesis of untreated vitiligo melanocytes, the PPARy activator is suitable for the treatment of vitiligo.
24) Method according to claim 23, wherein the glucose consumption is measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of glucose in the culture medium of vitiligo melanocytes treated with the PPARy activator and in the culture medium of untreated vitiligo melanocytes.
25) Method according to any one of claims 23-24, wherein the ATP production is measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of ATP in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes.
26) Method according to any one of claims 23-25, wherein the mitochondrial DNA synthesis is measured by culturing vitiligo melanocytes in a culture medium and measuring the concentration of mitochondrial DNA in vitiligo melanocytes treated with the PPARy activator and in untreated vitiligo melanocytes.
27) Method according to any one of claims 23-26, wherein, when 26 the PPARy activator is suitable for the treatment of vitiligo, the glucose consumption of vitiligo melanocytes treated with the PPARy activator is lower than the glucose consumption of untreated vitiligo melanocytes of at least 20% ± 5%, the production of ATP of vitiligo melanocytes treated with the PPARy activator is higher than the production of ATP of untreated vitiligo melanocytes of at least 25% ± 5% and the mitochondrial DNA synthesis of vitiligo melanocytes treated with the PPARy activator is higher than the mitochondrial DNA synthesis of untreated vitiligo melanocytes of at least 80% ± 15%.
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