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WO2021105219A1 - Inhibiteurs de mort cellulaire, parthanatos, destinés à être utilisés dans le traitement de psoriasis - Google Patents

Inhibiteurs de mort cellulaire, parthanatos, destinés à être utilisés dans le traitement de psoriasis Download PDF

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Publication number
WO2021105219A1
WO2021105219A1 PCT/EP2020/083380 EP2020083380W WO2021105219A1 WO 2021105219 A1 WO2021105219 A1 WO 2021105219A1 EP 2020083380 W EP2020083380 W EP 2020083380W WO 2021105219 A1 WO2021105219 A1 WO 2021105219A1
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Prior art keywords
parp
psoriasis
treatment
skin
par
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Ceased
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PCT/EP2020/083380
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Inventor
Victoriano Francisco MULERO MÉNDEZ
Ana Belén PÉREZ OLIVA
Diana GARCÍA MORENO
Francisco Javier MARTÍNEZ MORCILLO
Francisco Juan MARTÍNEZ NAVARRO
Joaquin CANTÓN SANDOVAL
María Luisa CAYUELA FUENTES
Teresa MARTÍNEZ MENCHÓN
Antonio Raúl CORBALÁN VÉLEZ
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Fundacion para la Formacion e Investigacion Sanitarias de la Region de Murcia
Universidad de Murcia
Original Assignee
Fundacion para la Formacion e Investigacion Sanitarias de la Region de Murcia
Universidad de Murcia
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Priority to EP20820342.2A priority Critical patent/EP4065097A1/fr
Publication of WO2021105219A1 publication Critical patent/WO2021105219A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
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    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
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    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
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    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
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    • G—PHYSICS
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    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis

Definitions

  • the present invention refers to the medical field. Particularly, the present invention refers to the use of a parthanatos cell death inhibitor selected from the group comprising: NAMPT inhibitor, PARP inhibitor and/or AIFM1 inhibitor in the treatment of skin inflammatory diseases.
  • a parthanatos cell death inhibitor selected from the group comprising: NAMPT inhibitor, PARP inhibitor and/or AIFM1 inhibitor in the treatment of skin inflammatory diseases.
  • Parthanatos is a form of programmed cell death that is distinct from other cell death processes such as necrosis and apoptosis. While necrosis is caused by acute cell injury resulting in traumatic cell death and apoptosis is a highly controlled process signalled by apoptotic intracellular signals, parthanatos is caused by the accumulation of poly (ADP) ribose (PAR) and the nuclear translocation of apoptosis-inducing factor 1 (AIFM1) from mitochondria. Parthanatos is also known as PAR polymerase 1 (PARPl) dependent cell death. PARPl mediates parthanatos when it is over-activated in response to extreme genomic stress and synthesizes PAR which causes nuclear translocation of AIFM1.
  • PARPl PAR polymerase 1
  • PARPl Over activation of PARPl is involved in diseases that afflict hundreds of millions of people worldwide.
  • Well known diseases involving PARPl overactivation include Parkinson's disease, stroke, heart attack, and diabetes. It also has potential use as a treatment for ameliorating disease and various medical conditions such as diabetes and obesity.
  • NAMPT, PARP and AIFM1 are key upstream regulators for the activation of parthanatos.
  • NAMPT gene encodes for a protein which is the rate-limiting enzyme in the Nicotinamide adenine dinucleotide (NAD+) salvage pathway that converts nicotinamide to nicotinamide mononucleotide (NMN) in mammals to enable NAD+ biosynthesis.
  • NAMPT has also been reported to be a cytokine (PBEF) that promotes B cell maturation and inhibits neutrophil apoptosis.
  • PBEF cytokine
  • PARPl has been described to play a relevant part in some acute and chronic inflammatory diseases such as neurological diseases (Parkinson's disease), sepsis, arthritis, colitis, diabetes and myocardial infarction.
  • DNA damage provoked by cellular metabolism is successfully handled by PARP1.
  • alkylating DNA damage, oxidative stress, hypoxia, hypoglycemia or activation of inflammatory pathways can trigger PARP1 hyperactivation.
  • Excessive PARylation deplete cellular NAD+ and ATP, although it does not necessarily imply cell death.
  • psoriasis and atopic dermatitis are two non-contagious skin chronic inflammatory diseases, which global prevalence is 0.1-3 % and 2-20 %, respectively.
  • prevalence seems to be increasing over time. While atopic dermatitis incidence is common in infancy, ameliorated in adolescence and reappears during the thirties, the prevalence of psoriasis increases after the age of forty.
  • both diseases have a genetic predisposition, environmental factors act as triggers of the pathology.
  • cytokines and chemokines produced in the lesion reach the blood and consequently, these patients suffer from comorbidities.
  • comorbidities In psoriasis, most common comorbidities caused by systemic inflammation (specially IL-Ib and TNFa) are cardiovascular complications, metabolic syndrome (such as obesity, dyslipidemia, atherosclerosis and type 2 diabetes mellitus) and autoimmune diseases (psoriasis complication develops psoriatic arthritis). Comorbidities development is progressive, being a process known as the “inflammatory skin march”.
  • Atopic dermatitis is characterized by redness eczematous skin lesions that provoke intense pruritus (itch, because of histamine release) and consequently induce epidermal barrier disruption due to scratching. Indeed, skin lesions vicinity shows hyperinnervation and increasing itchy feeling. Additionally, in these regions microbiota is altered (dysbiosis), with a principal colonization by Staphylococcus aureus. At the site of inflammation, there is an important presence of Langerhans cells (skin dendritic cells) and T helper cells (helper T lymphocyte CD4+). Predominantly, TH2 (release cytokines IL4, IL10 and IL13) and TH12 (produce IL22) immune responses contribute to the pathology.
  • Psoriasis vulgaris is the most widespread psoriasis form, affecting the skin and joints with a very relevant genetic susceptibility. Histological manifestation is keratinocyte hyperproliferation and increased tissue thickness (acanthosis), characterize by the development of silvered-covered erythematous scaly plaques. These lesions provoke pain, burning and itching sensation, being usually occurring on the scalp, trunk and extensor surfaces of extremities. Both innate and adaptive immune systems contribute to the pathology in the dermis and epidermis. Remarkably, keratinocytes, neutrophils, dendritic cell and T cells (TH17 immune responses mediated by IL17 and IL23) play a crucial role.
  • the present invention demonstrates that skin inflammatory diseases, for example human psoriasis and atopic dermatitis, showed differential mRNA levels of key genes which act as regulators of parthanatos cell death. Particularly, it is herein reported that patients with human psoriasis and atopic dermatitis, showed differential mRNA levels of genes encoding key enzymes of NAD+ metabolism. Genetic (with CRISPR-Cas9) and pharmacological inhibition of NAMPT with, for example, Daporinab (FK866, AP0866) effectively decreased hydrogen peroxide production, skin neutrophil infiltration, NFKB activation, and keratinocyte DNA damage, hyperproliferation and cell death in zebrafish models of skin chronic inflammation.
  • Daporinab FK866, AP0866
  • the present invention presents for the first time the inhibition of parthanatos cell death as a key strategy for treating skin inflammatory disorders, such as psoriasis and atopic dermatitis.
  • the inhibition of parthanatos cell death can be achieved by using inhibitors directed to any of the genes which participates in the regulation of parthanatos cell death, such as, for example, NAMPT, PARPl and/or AIFM1.
  • the special technical feature or contribution over the prior art which offers the present invention is the inhibition of parthanatos cell death for treating skin inflammatory disorders.
  • any of the proteins which participates in the regulation of parthanatos cell death can be used as therapeutic targets.
  • NAMPT nuclear-activated protein
  • PARPl and/or AIFM1 specific inhibitors directed to NAMPT, PARPl and/or AIFM1 are used in the present invention as proof of concept
  • any chemical or biological molecule able to inhibit NAMPT, PARP and/or AIFM1 could be used in the present invention to inhibit parthanatos cell death and, consequently, treat patients suffering from skin inflammatory disorders such as psoriasis and atopic dermatitis.
  • the first embodiment of the present invention refers to parthanatos cell death inhibitors selected from the group comprising: NAMPT inhibitors, PARP inhibitors and/or AIFM1 inhibitors, for use in the treatment of skin inflammatory diseases.
  • the present invention refers to a method for treating patients suffering from skin inflammatory diseases which comprises the administration of a therapeutically effective amount of a parthanatos cell death inhibitors selected from the group comprising: NAMPT inhibitor, PARP inhibitor and/or AIFM1 inhibitor.
  • the second embodiment of the present invention refers to a pharmaceutical composition
  • a pharmaceutical composition comprising a parthanatos cell death inhibitor selected from the group comprising: NAMPT inhibitor, PARP inhibitor or AIFM1 inhibitor and, optionally, pharmaceutically acceptable excipients and/or carriers, for use in the treatment of skin inflammatory diseases.
  • the third embodiment of the present invention refers to an in vitro method for identifying and producing candidate compounds useful in the treatment of skin inflammatory diseases which comprises: (a) Determining whether parthanatos cell death has been inhibited by measuring at least the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, nicotinamide (NAM), NAM mononucleotide (NMN), S-adenosylmethinonine (SAM), S- adenosylhomocysteine (SAH) and/or PAR; and/or the proteolysis of AIFM1 in a biological sample isolated from the patient after the administration of the candidate compound; (b) wherein if after the administration of the candidate compound it is determined that parthanatos cell death has been inhibited because the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and/or PAR; and/or the proteolysis of AIFM
  • the fourth embodiment of the present invention refers to an in vitro method for determining the efficacy or response to a treatment of skin inflammatory diseases which comprises: (a) Determining whether parthanatos cell death has been inhibited by measuring at least the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and/or PAR; and/or the proteolysis of AIFM1 in a biological sample isolated from the patient after the administration of the treatment; (b) wherein if after the administration of the treatment it is determined that parthanatos cell death has been inhibited because the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and/or PAR; and/or the proteolysis of AIFM1 are statistically lower as compared with the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and
  • the fifth embodiment of the present invention refers to an in vitro method for the diagnosis of skin inflammatory diseases and/or for selecting patients suffering from skin inflammatory diseases for a treatment with parthanatos cell death inhibitors which comprises: (a) Determining whether parthanatos cell death has been activated by measuring at least the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and/or PAR; and/or the proteolysis of AIFM1 in a biological sample isolated from the patient; (b) wherein if it is determined that parthanatos cell death has been activated because the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH and/or PAR; and/or the proteolysis of AIFM1 are statistically higher as compared with the enzymatic activity of NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, S
  • the sixth embodiment of the present invention refers to a kit adapted for identifying and producing candidate compounds useful in the treatment of skin inflammatory diseases, for determining the efficacy or response to a treatment of skin inflammatory diseases or for the diagnosis of skin inflammatory diseases which comprises: a) Tools for obtaining a biological sample from the patient, and b) tools or reagents for determining whether parthanatos cell death is activated or inhibited, by means of measuring at least the enzymatic activity NAMPT and PARP; the amount of NADH, NAD+, NAM, NMN, SAM, SAH, and/or PAR; and/or the proteolysis of AIFM1.
  • the skin inflammatory disease is psoriasis or atopic dermatitis.
  • the inhibitors are administered by means of a local or systemic administration, preferably topical administration.
  • the NAMPT inhibitor is selected from the list comprising: FK- 866 (AP0866 or daporinad), STF-118804, KPT-9274, GMX1778 (CHS828) and/or nicotinamide (NAM);
  • the PARP inhibitor is selected from the list comprising: olaparib (AZD2281, KU0059436), AZD2461, talazoparib (BMN 673), veliparib (ABT-288), niraparib, iniparib (INN, BSI 201), rucaparib (AG-014699,PF-01367338), CEP-9722, CEP- 8983, INO-1001 (3-Aminobenzamide), PJ34, A966492 and/or AG-14361; and the AIFM1 inhibitor is N-fenilmaleimida and p-chloromercuryphenylsulfonic acid (PCMBS).
  • PCMBS N-fen
  • the pharmaceutical is formulated as a cream, foam, gel, lotion, ointment, or is included in transdermal patches.
  • “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • terapéuticaally effective dose or amount an amount that, when administered as described herein, brings about a positive therapeutic response in a subject having a skin inflammatory disease.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, mode of administration, and the like.
  • An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.
  • reference value refers to the values used a “control”.
  • a “reference value” can be a threshold value or a cut-off value. Typically, a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value must be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Preferably, the person skilled in the art may compare the biomarker levels (or scores) obtained according to the method of the invention with a defined threshold value.
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is good.
  • NAD + treatment alters neutrophil distribution pattern. Schematic representation of a 3 dpf zebrafish larvae. Red dots represent neutrophils mostly positioned at CHT, highlighted with a green box (A). Quantification of the percentage of neutrophils out of the
  • Representative merge images (brightfield and red channels) of lyz:dsRED zebrafish larvae of every group are shown (B') The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p ⁇ 0.05, ****p ⁇ 0.0001.
  • NAD + and their precursors induce skin oxidative stress.
  • H2O2 imaging embryos of 72 hpf treated 48 hours with NAD + metabolites were incubated in 50 mM of acetyl-pentafluorobenzene sulphonyl fluorescein solution for 1 hour.
  • Quantification of fluorescence intensity for NAD + -mediated (A) and NAM-/NMN-mediated (B) induction of H2O2 in the zebrafish skin Representative merge images (brightfield and red channels) of lyz:dsKED zebrafish larvae of every group are shown (A' & B') The mean ⁇ S.E.M. for each group is shown.
  • P values were calculated using one-way ANOVA and Tukey multiple range test ****p ⁇ 0.0001.
  • FIG. 3 High concentrations of FK-866 triggers muscle inflammation. Quantification of fluorescence intensity in 3 dpf embryos treated 48 hours with increasing doses of FK-866 (1, 10 and 100 pM) (A). Representative images (green channel) of NF-kB:eGFP zebrafish larvae of every group are shown (A'). The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p ⁇ 0.0001.
  • NAD + reverses the induction of NFKB in the muscle by high concentrations of FK- 866. Quantification of fluorescence intensity in 72 hpf embryos treated 48 hours with 1 mM NAD + in the presence or absence of 100 pM FK-866 (A). Representative merge images (brightfield and green channel) of NF-kB:eGFP zebrafish larvae of every group are shown (A'). The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ****p ⁇ 0.0001.
  • FK-866 reduces oxidative stress in the skin.
  • Neutrophil distribution in zebrafish embryos of 3 dpf treated 48 hours with FK-866 (A), quantification of skin H2O2 production (B) and representative merge images (green and red channel) of lyz:dsRED zebrafish larvae of every group are shown (C).
  • P values were calculated using one-way ANOVA and Tukey multiple range test *p ⁇ 0.05, ****p ⁇ 0.0001.
  • FIG. 6 Pharmacological inhibition of Nampt diminishes oxidative stress and skin inflammation and restores epithelial integrity in spintla mutant.
  • Neutrophil distribution of zebrafish embryos of 3 dpf treated 48 hours with FK-866 (10, 50 and 100 pM) (A), quantification of skin H2O2 production (B) and representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (C). The mean ⁇ S.E.M. for each group is shown.
  • P values were calculated using one-way ANOVA and Tukey multiple range test **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • NAD + intensifies spintla mutant phenotype and interferes with FK-866 beneficial effects in the skin.
  • P values were calculated using one-way ANOVA and Tukey multiple range test ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG. 8 Total NAD + and NADH levels were efficiently modulated by treatments with FK- 866 and NAD + .
  • FIG. 9 FK-866 rescues enhanced skin NFKB transcriptional activity on spintla mutants. Quantification of fluorescence intensity of wild type and spintla mutant embryos of 72 hpf treated for 2 days with 10 pM FK-866 (A). Representative images (green channel) of NF- kB:eGFP zebrafish larvae of every group are shown (A') The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test
  • FIG. 10 Parpl pharmacological inhibitors reduce neutrophil scattering and skin inflammation and reestablish epithelial integrity. Wild type and spintla mutant embryos of 72 hpf treated for 2 days with olaparib (A & B), veliparib or talazoparib (C). Quantification of neutrophil dispersion out of the CHT (A & C) and NFKB transcriptional activity in the skin (B). Representative images (brightfield and red channel in A' & C'; green channel in B') of lyz:dsRED and NF-kB:eGFP zebrafish larvae of every group are shown. The mean ⁇ S.E.M. for each group is also shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p ⁇ 0.05, ****p ⁇ 0.0001.
  • Figure 11 The high levels of PAR in the skin of spintla mutant are counteracted by FK-866 and olaparib.
  • FIG. 13 DNA damage analysis reveals increased susceptibility and a higher number of lesions in the skin of spintla mutant larvae. Quantification of rgH2Ac positive cells from 48 hpf wild type and spintla mutant zebrafish embryos treated for 24 hours with 10 pM FK-866 or 100 pM olaparib (A). Similarly, around 60 fish fin folds were amputated and disaggregated into cells for comet assay analysis in alkaline conditions (B). Representative merge images of maximum intensity projection of an apotome Z stack from zebrafish larvae of every group are shown (A').
  • P values were calculated using one-way ANOVA and Tukey multiple range test (A) and Kruskal- Wallis test and Dunn's multiple comparisons test (B) *p ⁇ 0.05, **p ⁇ 0.01, ****p ⁇ 0.0001.
  • FIG. 14 Pharmacological inhibition of Parpl or Nampt does not induce apoptosis. Quantification of cleaved caspase 3 positive cells from 48 hpf wild type and spintla mutant zebrafish embryos treated for 24 hours with 10 pM FK-866 or 100 pM Olaparib (A). Representative merge images of maximum intensity projection of an apotome Z stack from zebrafish larvae of every group are shown (AQ. WIHC with anti-cleaved casp3 (green), anti- p63 (basal keratinocyte marker, red) were counterstained with DAPI (blue) (A') The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test.
  • FIG. 15 Olaparib blocks cell death in the skin of spintla mutants. Quantification of TUNEL+ cells from 48 hpf wild type and spintla mutant zebrafish embryos treated for 24 hours with 100 pM Olaparib (A). Representative images of zebrafish larvae of every group are shown (A'). The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test **p ⁇ 0.01, ***p ⁇ 0.001. Figure 16. The antioxidants NAC, mito-TEMPO and tempol rescue skin neutrophil recruitment and skin morphology of spintla mutant larvae.
  • NMMA does not alter skin neutrophil infiltration in spintla mutant larvae. Measurement of neutrophil distribution of 3 dpf zebrafish wild type and spintla mutant embryos treated 48 hours with 1 mM NMMA (A). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (A') The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test ns, not significant, ****p ⁇ 0.0001.
  • FIG. 18 Pharmacological inhibition of Aifml translocation reduces skin neutrophil infiltration and improves skin morphology. Measurement of neutrophil distribution of 3 dpf zebrafish embryos treated 48 hours with 10 nM N-phenylmaleimide (NP) (A). Representative merge images (brightfield and red channel) of lyz:dsRED zebrafish larvae of every group are shown (A'). The mean ⁇ S.E.M. for each group is shown. P values were calculated using one way ANOVA and Tukey multiple range test. ****p ⁇ 0.0001.
  • FK-866 and olaparib improves skin epithelial integrity in psoriasis mutants. Determination of the skin phenotype of 2.5 dpf zebrafish embryos treated 1.5 days with 50 mM FK-866 or 100 pM olaparib (A). Representative bright field images of zebrafish larvae of every group are shown (A'). The mean ⁇ S.E.M. for each group is shown. P values were calculated using Chi-square and Fisher's exact test *p ⁇ 0.05, ****p ⁇ 0.0001.
  • FIG. 20 FK-866, olaparib, N-phenylmaleimide (NP) and apocynin ameliorate inflammation in human psoriasis three-dimensional epidermal models.
  • A Thickness cell layer and transcriopt levels of the psoriasis marker DEFB4 were determined in human epidermal explants pre-treated with 30 ng/ml IL-17a and IL -22 in the presence of vehicles (ETOH and DMSO) or the indicated inhibitors.
  • A’ Representative bright field images of epidermis stained with hematoxylin and eosin.
  • A' The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test.
  • FIG 22 The expression of genes encoding key NAD + metabolic enzymes correlate with those of specific inflammatory gene markers of psoriasis and atopic dermatitis.
  • Transcriptomic data in lesioned skin from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database were employed to study their correlation with inflammatory marker genes expression. Linear regression for each group is shown. P values were calculated using Pearson's correlation coefficient. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG 23 Differential gene expression profile of parthanatos components and PAR hydrolases in psoriasis and atopic dermatitis.
  • GEO Gene Expression Omnibus
  • C Venn diagram showing common and specific gene expression in lesional skin. The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test (A) and t-Test (B). ns, not significant. *p£0.05, ****p ⁇ 0.0001.
  • Hypercholesterolemia Protein ARH1 ; Ectonucleotide Pyrophosphatase/Phosphodiesterase 1, ENPP; Mono-ADP Ribosylhydrolase 1, MACRODP, Mono-ADP Ribosylhydrolase 2, MACROD2; Nudix Hydrolase 16, NUDT16; Terminal ADP-Ribose Protein Gly cohydrolase 1, TARGL
  • FIG. 24 Correlation of the expression of genes involved in parthanatos and PAR metabolism with specific inflammatory gene markers of psoriasis and atopic dermatitis.
  • Transcriptomic data in lesioned skin from human psoriasis (GDS4602) (A) and atopic dermatitis (GSE57225) (B) samples from the Gene Expression Omnibus (GEO) database were employed to study their correlation with inflammatory gene marker expression levels. Linear regression for each group is shown. P values were calculated using Pearson's correlation coefficient. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Figure 25 Recovery of reduced levels of NAD + and SAM in serum of psoriasis patients treated with PUVA Measurement by HPLC-MS of serum metabolites of psoriasis patients before and after receiving a phototherapy treatment. The mean ⁇ S.E.M. for each group is shown. P values were calculated using one-way ANOVA and Tukey multiple range test *p ⁇ 0.05, **p ⁇ 0.01.
  • NAMPT (A) and PAR (B) are overexpressed in human psoriatic lesions.
  • the transgenic zebrafish line Tg(lyz:dsRED2) contains the genomic regulatory regions of the zebrafish lysozyme C gene, neutrophil-specific promoter, that controls the expression of the red fluorescent protein DsRED2, and Tg(NFicB-RE:eGFP) were provided by Profs. Phil Crosier and Stephen A. Renshaw, respectively.
  • Example 1.2 Chemical treatments.
  • Zebrafish embryos were manually dechorionated at 24 hpf. Larvae were treated from 24 hpf to 48 hpf or 72 hpf by chemical bath immersion at 28 °C. Incubation was carried out in 6-well plate containing 20-25 larvae/well in egg water (including 60 pg/mL sea salts in distilled water) supplemented with 1% dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Example 1.3 Imaging of zebrafish larvae.
  • Live imaging of 72 hpf larvae was obtained employing buffered tricaine (200 pg/mL) dissolved in egg water. Images were capture with an epifluorescence LEICA MZ16FA stereomicroscope set up with green and red fluorescent filters. All images were acquired with the integrated camera on the stereomicroscope and were analyzed to determine number of leukocytes (lyz:dsRED) and their distribution in the larvae. The transcriptional activity of NF-KB was visualized and measured with the zebrafish line NFicB-RE:eGFP.
  • H2O2 imaging was quantified employing the live cell fluorogenic substrate acetyl- pentafluorobenzene sulphonyl fluorescein (Cayman Chemical). Briefly, about 20 embryos of 72 hpf were rinse with egg water and collected in a well of a 24-well plate with 50 pM of the substrate in 1% DMSO for 1 hour.
  • ImageJ software was employed to determine mean intensity fluorescence of a common region of interest (ROI) placed in the dorsal skin for H2O2 production quantification. Similarly, a ROI located in muscle or skin was used to obtain mean intensity fluorescence of nfkb:egfp transgenic line.
  • ROI region of interest
  • Example 1.4 Whole-mount immunohistochemistry.
  • BrdU incorporation assay was used to determine cell proliferation. Embryos of 48 hpf were incubated in 10 mM of BrdU dissolved in egg water for 3 hours at 28 °C followed by a one- hour wash out with egg water and fixation in 4% PFA overnight at 4°C or 2 hours at room temperature (RT). For the rest of immunofluorescence techniques, embryos/larvae were directly fixed in 4% paraformaldehyde (PFA), as indicated above. Embryos/larvae were then washed with phosphate buffer saline (PBS) with 0.1% tween-20 (PBST) 3 times for 5 minutes.
  • PBS phosphate buffer saline
  • PBST 0.1% tween-20
  • larvae were wash 6 times for 5 minutes. Incubation in secondary antibody in blocking solution was performed for 2-3 hours in dark. From then on, the protocol followed in darkness. In order to remove unbound secondary antibody, embryos were washed 3 times for 10 minutes with PBT. In this step, the sample was ready for 2-(4-Amidinophenyl)-6- indolecarbamidine (DAPI) staining, a DAPI solution (1:1000) in PBT for 20 minutes followed by a wash out step of 3 times for 10 minutes with PBT. Finally, embryos were transferred to 80% glycerol/20% PBST and stored in dark at 4 °C until imaging.
  • DAPI 2-(4-Amidinophenyl)-6- indolecarbamidine
  • the following primary antibodies were used: rabbit anti-BrdU (Abeam, ab 152095, 1:200), mouse anti-p63 (Santa Cruz Biotechnology, sc-7255, 1:200), rabbit Anti- Active Caspase-3 (Bd Bioscience, #559565, 1:250) and rabbit anti-H2AX.XS139ph (phospho Serl39) (GeneTex, GTX127342, 1:200). Secondary antibodies were goat anti-rabbit Alexa Fluor-488 (Molecular probes, CAT#A11008, 1:1000) and goat anti-mouse Cyanine 3 (Life technologies, A10521, 1:1000). Images for BrdU staining were taken using a Zeiss Confocal (LSM710 META), the other stains were acquired by ZEISS Apotome.2. All images were processed using ImageJ software.
  • Emrbyo s/larvae were fixed and dehydrate as described above. Afterwards, embryos/larvae were rinsed with pre-cooled (-20 °C) 100% acetone and then incubated 100% acetone at -20 °C for 10 minutes. Samples were then washed 3 times for 10 minutes with PBST and incubated in a solution of 0.1% TritonX-100 and 0.1% sodium citrate (10%) in PBS for 15 minutes to further permeabilize the embryos/larvae. Next step consisted on rinse specimens 2 times for 5 minutes in PBST.
  • TUNEL reaction mixture composed by 5 pL of enzyme solution mixed with 45 pL of labeling solution (In Situ Cell Death Detection kit, POD, ROCHE, version 15.0) for 1 hour at 37 °C, followed by 5 wash with PBST for 5 minutes. Blocking step was carried out for at least 1 hours at RT with blocking buffer.
  • blocking buffer was removed and added 50 pL Converted-POD (anti-fluorescein antibody conjugated to peroxidase) for 1 hour at room temperature or overnight at 4 °C on rocker.
  • Embryo were rinsed 4 times for 30 minutes in PBST and incubated in 1 mL of 3,3'- Diaminobenzidine (DAB) solution for 30 minutes in the dark and transferred to a 24 well- plate. From then on, the protocol followed in darkness. Two pL of a fresh 0.3% H2O2 solution was added to initiate peroxidase reaction that was monitored 10-20 minutes followed by rinsing and a wash out step of 2 times for 5 minutes with PBST. Finally, embryos were transferred to 80% glycerol/20% PBST and stored in dark at 4 °C until imaging. Images were acquired by ZEISS Apotome.2 and processed using ImageJ software.
  • DAB 3,3'- Diaminobenzidine
  • Zebrafish embryos at 48 hpf were anesthetized in tricaine (200 pg/mL) dissolved in egg water and the end of the fin fold was amputated with a scalpel. Tissues collected from around 60 embryos were pooled, then spin and resuspended in 1 mL PBS. Liberase at l:65/volume of PBS (Roche, cat # 05401119001) was added and tissues were incubated at 28 °C for 35 minutes, pipetting up and down every 5 minutes. To stop the reaction, FBS was added to a final concentration of 5% in PBS. From now on, samples were kept on ice.
  • Disaggregated fin folds were filtered through a 40 pM filter and washed using PBS + 5% FBS.
  • Cell suspension was centrifuged at 650xg for 5 minutes and resuspended in 50 pL of PBS + 5% FBS.
  • Trypan Blue-treated cell suspension was applied to Neubauer chamber and cell were counted in an inverted microscope. Around 15.000 cells were employed to perform the Alkaline Comet Assay according to the manufacturer's protocol (Trevigen). Briefly, cells were added in low melting point agarose at 37 °C at a ratio of 1:10 (v/v) and then were placed onto microscope slides.
  • DNA was stained with SYBRTM Green I Nucleic Acid Gel Stain IO,OOOC (Invitrogen) and images were taken using a Nikon Eclipse TS2 microscope with lOx objective lens. Quantitative analysis of the tail moment (product of the tail length and percent tail DNA) was obtained using CASPLAB software. More than 100 randomly selected cells were quantified per sample. Values were represented as the median of the tail moment of treated cells relative to the median of the tail moment of untreated cells.
  • Example 1.7 Western blot.
  • Zebrafish embryos at 72 hpf were anesthetized in tricaine (200 pg/mL) dissolved in egg water and the end of the fin fold was amputated with a scalpel. Tissues collected from around 120 embryos were pooled, then spin and resuspended in 80 pL of 10 mM Tris pH 7.4 + 1% Sodium Dodecyl Sulfate (SDS). Samples were then incubated at 95 °C for 5 min with 1400 rpm agitation, followed by maximum speed centrifugation for 5 min. Supernatants were frozen at - 20 °C until proceeding. BCA kit was employed to quantify protein using BSA as a standard.
  • Fin lysates (10 pg) in SDS sample buffer were subjected to electrophoresis on a polyacrylamide gel and transferred to PVDF membranes.
  • the membranes were incubated for 1 h 30 min with TTBS containing 5% (w/v) skimmed dry milk powder and immunoblotted in the same buffer 16 h at 4 °C with the mouse monoclonal antibody to human poly(ADP- ribose) (1/400, ALX-804-220, Enzo).
  • the blot was then washed with TTBS and incubated for 1 h at room temperature with secondary HRP-conjugated antibody diluted 2500-fold in 5% (w/v) skimmed milk in TTBS. After repeated washes, the signal was detected with the enhanced chemiluminescence reagent and ChemiDoc XRS Biorad.
  • Example 1.8 Total NAD + & NADH determination.
  • Zebrafish embryos at 72 hpf were anesthetized in tricaine (200 pg/mL) dissolved in cold PBS in order to amputate the tail at the end of the yolk sac extension with a scalpel.
  • Tissues from around 120 embryos were pooled and collected in lysis buffer provided by the kit (Total NAD and NADH Assay Kit, ab 186032) according to the manufacturer's protocol (Abeam). Tissues were homogenized and centrifuged at 1400 rpm for 5 minutes at 4 °C. Supernatants were collected and centrifuged at maximum speed for 10 minutes at 4 °C. Supernatants were employed to protein quantification with BCA kit using BSA as a standard. To proceed with Total NAD and NADH determination, 50 pg of protein were employed.
  • Example 1.9 Gene Expression Omnibus (GEO) database.
  • GEO Gene Expression Omnibus
  • Example 1.10 Immunohistochemistry in human skin samples.
  • Inert transwells (Sigma-Aldrich MCHT12H48) were seeded with 10 5 human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 pL CnT-PR medium (CellnTec) in a 12 well format. After 48 hours, cultures were switched to CnT-PR-3D medium (CELLnTEC) for 24 hours and then cultured at the air-liquid interface for 12 days. From day 7 to 12 of the air-liquid interphase culture, the Thl7 cytokines IL-17 (30 ng/mL) and IL-22 (30 ng/mL) were added.
  • Pharmacological treatment was applied from day 10 to 12 and consisted of 100 mM apocynin, 100 nM FK-866 and 1 mM olaparib. Culture medium was refreshed every two days. At day 12, the tissues were harvested for gene expression analysis and immunohistochemistry.
  • Example 2.1 NAD + and its precursors contribute to skin inflammation.
  • CHT caudal hematopoietic tissue
  • H2O2 Given the role of H2O2 in driving neutrophil mobilization to acute and chronic insults, we used a H2O2 specific fluorescent probe to know if this molecule was implicated in the observed phenotype. Fluorescent probe indicated that NAD + treatment was able to enhance H2O2 production by skin keratinocytes in a dose-dependent manner, compared to the control group. Similar results were obtained with NAM, a well-known NAD + booster. However, while NMN precursor was unable to increase skin oxidative stress by itself, NAM and NMN combination synergistically induce the same effect as NAM alone (Figure 2B). Nevertheless, no differences in neutrophil redistribution were observed (Figure 2A' & B') These results might suggest that NAD + levels could regulate oxidative stress in the skin.
  • Example 2.3 Pharmacological inhibition of Nampt diminishes oxidative stress and skin inflammation and restores epithelial integrity in a zebrafish psoriasis model.
  • various zebrafish mutants in key genes that encode or regulate interconnecting anchoring structures that mediates stable association of dermis and epidermis have been described. These mutants with defective skin epithelial integrity show persistent leukocyte skin infiltration, a hallmark of psoriasis and other skin inflammatory diseases.
  • NAD + metabolism remarkably influences the spintla mutant phenotype. While NAD + supplementation exacerbated mutant phenotype, FK- 866-mediated NAD + depletion counteracts increased oxidative stress and NFKB transcriptional activity in the skin and neutrophil infiltration, characteristic of the spintla mutant. More importantly, FK-866 treatment dramatically improves skin epithelial integrity.
  • Example 2.4 Parpl activity inhibition rescues skin inflammation of spintla mutant larvae.
  • talazoparib and olaparib display similar enzymatic activity inhibition potency
  • talazoparib is close to 100 times more effective at trapping PARP1 with DNA.
  • this inhibitor is the less potent in both activities. This observation could be the rational for the different concentrations able to rescue skin inflammation in spintla mutant larvae: 500 pM veliparib > 100 pM olaparib >1 pM talazoparib.
  • Example 2.5 FK-866 and olaparib block keratinocyte hyperproliferation in spintla mutant larvae.
  • Example 2.6 Spintla mutants displays higher DNA damage, which is induced by Parpl inhibition while reduced by Nampt inhibition.
  • the main PARP1 biological function is to orchestrate spatio-temporal reparation of DNA damage, being indispensable for SSBR and participating in HR.
  • PARP inhibitors entrapping and accumulating inactive PARP1 on DNA trigger dsBs formation during replication.
  • PARP inhibitors Independently of synthetic lethality phenomena caused by PARP inhibitors when HR pathway is hampered, PARP inhibitors has also been described to induce apoptosis in proliferating cells. Apoptotic factors regulated by p53 trigger cytochrome c release from mitochondria and subsequent caspases cascade activation, ultimately cleaving caspase 3, among other targets.
  • Initial studies of spintla mutants although they found keratinocyte cell death, they were unable to block it targeting caspases or pro-apoptotic factors, suggesting an unidentified programmed cell death.
  • cleaved caspase 3 staining in 2 dpf spintla mutant embryos or wild type larvae was negative ( Figure 14A & A').
  • spintla mutants treated with FK-866 or olaparib did not show keratinocyte apoptosis ( Figure 14A & A'), even at 3 dpf upon 48 hours of treatment (data not shown).
  • TUNEL was a second approach to study cell death; this technique consists on the labelling of exposed 3 ⁇ H ends of DNA fragments. Therefore, TUNEL labels ssBs and dsBs.
  • Example 2.9 ROS scavenging molecules rescue skin inflammation of spintla mutant larvae.
  • Duoxl-derived H2O2 In first place, we decided to inhibit Duoxl-derived H2O2. The inhibition of this enzyme by the NADPH oxidase inhibitor dibenziodolium chloride (DPI) successfully diminishes skin oxidative stress and rescues a zebrafish skin chronic inflammatory model. However, upon different concentrations and experimental settings tested for this inhibitor, we concluded that Duoxl does not contribute to the spintla mutant skin phenotype. Similarly, genetic inactivation of Duoxl in skin keratinocytes by overexpressing a dominant negative form of Duoxl, also failed to rescue skin inflammation on spintla mutants.
  • DPI NADPH oxidase inhibitor dibenziodolium chloride
  • NAC N- acetylcysteine
  • GSH glutathione
  • Example 2.10 Nitric oxide synthesis inhibition does not affect skin inflammation of spintla mutant larvae.
  • NO reactive nitrogen specie
  • N G -monomethyl-L-arginine NMMA is an inhibitor of the 3 NOS isoforms.
  • zebrafish spintla mutant phenotype is characterized by skin oxidative stress (involving H2O2), skin inflammation, keratinocyte aggregation, hyperproliferation and subsequent cell death (unidentified) and DNA damage.
  • Parpl inhibition also improved mutant phenotype in relation to epithelial integrity, skin NFKB transcriptional activity, neutrophil infiltration, keratinocyte proliferation and, more importantly, reduced PAR activity and cell death.
  • Parthanatos is a PARP1 -dependent cell death in which extensive DNA damage induces PARPl overactivation. Accumulation of PAR polymers and PARylated proteins reach the mitochondria causing depolarization of the membrane potential and apoptosis-inducing factor (AIFM1) release.
  • AIFM1 apoptosis-inducing factor
  • AIFM1 is released into the cytosol, where it recruits macrophage migration inhibitory factor (MIF) to the nucleus.
  • MIF macrophage migration inhibitory factor
  • AIFMl-MIF nuclease activity execute a large-scale DNA fragmentation resulting in cell death.
  • N- phenylmaleimide N- phenylmaleimide
  • Example 2.13 Pharmacological inhibition of NADPH oxidases, Parpl or Nampt rescues inflammation in three-dimensional human psoriasis epidermal models.
  • Example 2.14 Altered expression profile of genes encoding key NAD + metabolic enzymes in atopic dermatitis and psoriasis.
  • Psoriasis and atopic dermatitis, or eczema are two non-contagious skin chronic inflammatory diseases.
  • environmental factors act as triggers of the pathologies
  • psoriasis and atopic dermatitis have a strong genetic component affecting individual susceptibility.
  • NAD + metabolism is affected in psoriasis and atopic dermatitis, probably paying a role in the pathologies, we analyzed transcriptomic data of enzymes implicated in NAD + metabolism.
  • NAD + salvage pathway (carried out by NAMPT and NMNAT1-3 ), generally used to keep intracellular NAD + levels in a great variety of tissues. This pathway is fueled by Preiss-Handler pathway (participating NAPRT and NADSYN ), de novo pathway (mediated by IDOl , TD02 and QPRT , among others) and nicotinamide riboside (NR) conversion (catalyzed by PNP and NRK1/2).
  • Preiss-Handler pathway participating NAPRT and NADSYN
  • de novo pathway mediated by IDOl , TD02 and QPRT , among others
  • NR nicotinamide riboside
  • NNM ⁇ N-methyltransferase
  • SAM S-adenosylmethionine
  • transcriptomic data obtained from human psoriasis where skin from healthy subjects and psoriasis patients (non-lesional and lesional skin) were compared (Figure 21A).
  • transcript levels were upregulated compared with non-lesional psoriatic skin and healthy samples.
  • the mRNA levels of the gene encoding NAMPT was increased, in contrast with the reduction in NMNAT3 levels.
  • NRK2 transcript levels also augmented in lesional skin, at the same level than non-lesional skin, compared with control group.
  • NAD + salvage pathway with a common reduction of NMNAT3 expression, which is in charge of controlling NAD + levels in the mitochondria.
  • de novo biosynthesis of NAD + could be also enhanced in both diseases, in those tissues in which it is active.
  • Preiss-Handler pathway is only altered in psoriasis, while no differences were found in atopic dermatitis analysis.
  • NNMT expression profile showed enhanced induction specifically in atopic dermatitis and both shared high levels of CD38 transcript levels (Figure 21C).
  • Example 2.15 The expression profile of genes encoding NAD + metabolic enzymes correlate with inflammatory gene markers.
  • cytokines related to T H 2 IL4 , IL10 and 11 A 3
  • T H 12 IL22
  • NAMPT and PNP robustly correlated its expression with IL1B and IL17A , similarly to IDOl and TD02 expression, but with lower significance ( Figure 22A).
  • NRK1 expression slightly correlated with IL23A and importantly, NAPRT showed an inverse correlation with the expression of this cytokine ( Figure 22A).
  • NAMPT correlated with IL4 and IL6, IDOl and TD02 were also linked to IL6 transcript levels and other cytokines (Figure 22B).
  • NNMT and CD38 mRNA levels showed very important association with those of genes encoding several cytokines, what could mean relevant correlation with disease severity.
  • NMNAT3 transcript levels displayed an inverse correlation with numerous genes coding for inflammatory cytokines, probably pointing out NMNAT3 expression as a protective factor (Figure 22B).
  • Example 2.16 Altered expression profile of genes encoding enzymes related to parthanatos in atopic dermatitis and psoriasis.
  • Psoriasis transcriptomic data revealed strong increased mRNA levels of PARP1, AIFM1 and MIF in lesional tissue compared with healthy skin and non-lesional psoriatic skin ( Figure 23 A). In addition, it was observed slight decreased and increased AIFM1 and MIF levels, respectively, in non-lesional skin from psoriasis patients ( Figure 23A).
  • Example 2.17 The expression of genes encoding key components involved in parthanatos and PAR metabolism correlate with inflammatory gene markers of psoriasis and atopic dermatitis
  • MACROD1 transcript levels showed robust inverse association with those of several cytokine genes (Figure 24B). Therefore, in both diseases parthanatos components and other genes related to PAR metabolism showed associated expression with those of genes encoding cytokines specific of every disease. Oppositely, MACROD1 and MACROD2 displayed an inverse correlation with inflammatory marker genes, what probably could indicate a protective role against the pathologies.
  • Example 2.18 Human serum levels of NAD + and SAM are altered in psoriasis patients
  • NAD + metabolism play a fundamental role in the regulation of oxidative stress and skin inflammation in a zebrafish preclinical model of psoriasis.
  • transcriptomic data displayed altered expression profile of genes encoding key NAD + metabolic enzymes in atopic dermatitis and psoriasis. Therefore, we set out to investigate if serum levels of metabolites relative to NAD + metabolism could be altered in psoriasis patients.
  • HPLC-MS high-performance liquid chromatography-mass spectrometry
  • NAD + , NAD + /NADH and/or SAM might be potential serum biomarkers to predict the response of psoriasis patients to phototherapy.
  • Example 2.19 NAMPT and PAR are overexpressed in the nucleus of human keratinocytes from psoriatic lesions.

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Abstract

L'inhibiteur de mort cellulaire, parthanatos, est destiné à être utilisé dans le traitement de maladies inflammatoires de la peau. La présente invention concerne l'utilisation d'un inhibiteur de mort cellulaire, parthanatos, choisi dans le groupe comprenant : des inhibiteurs de NAMPT, des inhibiteurs de PARP ou des inhibiteurs d'AIFM1 dans le traitement de maladies inflammatoires de la peau.
PCT/EP2020/083380 2019-11-26 2020-11-25 Inhibiteurs de mort cellulaire, parthanatos, destinés à être utilisés dans le traitement de psoriasis Ceased WO2021105219A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116656801A (zh) * 2022-12-02 2023-08-29 湖南家辉生物技术有限公司 一种Cowchock综合征致病基因AIFM1突变位点的应用及其检测试剂和应用

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