WO2022253910A1 - Nouveau procédé pour traiter une maladie cutanée inflammatoire - Google Patents

Nouveau procédé pour traiter une maladie cutanée inflammatoire Download PDF

Info

Publication number
WO2022253910A1
WO2022253910A1 PCT/EP2022/064940 EP2022064940W WO2022253910A1 WO 2022253910 A1 WO2022253910 A1 WO 2022253910A1 EP 2022064940 W EP2022064940 W EP 2022064940W WO 2022253910 A1 WO2022253910 A1 WO 2022253910A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
inhibitor
thl7
psoriasis
skin
Prior art date
Application number
PCT/EP2022/064940
Other languages
English (en)
Inventor
Magali SAVIGNAC
Lucette Pelletier
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Université Toulouse Iii – Paul Sabatier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Centre National De La Recherche Scientifique (Cnrs), Université Toulouse Iii – Paul Sabatier filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Publication of WO2022253910A1 publication Critical patent/WO2022253910A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention is in the field of dermatology.
  • the present invention relates to a method to treat an inflammatory skin disease.
  • Psoriasis is a chronic inflammatory skin disease frequently associated with co morbidities, including arthritis, cardio-metabolic disease, and psychological troubles. It affects 2-3% of the world's population. Skin lesions present as erythematous scaly patches characterized by keratinocyte hyper-proliferation and inflammatory cell infiltrates in the epidermis and dermis 1 ’ 2 . It is assumed that psoriasis results from abnormal dialogue between innate and adaptive immune cells in one hand and resident skin cells in another one 3 . Skin injury causes cell death and release of self DNA-anti-microbial products (as LL37), which can activate plasmacytoid dendritic cells in the skin resulting in IFNa production and dendritic cell (DC) activation.
  • as LL37 self DNA-anti-microbial products
  • Activated DC cells would migrate into the draining lymph node cells where they polarize a population of self-reactive CD4 + T lymphocytes in IL17, IL22, IFNy and TNFa through the secretion of IL12 and mainly IL23 4 .
  • These effector T cells are recruited into the skin and produce inflammatory cytokines inducing keratinocyte proliferation and promoting a positive feedback loop between epithelial and immune cells resulting in sustained inflammation 3 ’ 5 .
  • targeting TNFa/IL-23/IL- 17 cytokine axis is effective in the treatment of psoriasis 6 9 .
  • Ca v l channels are encoded by a multi-protein complex composed by pore-forming al subunit encoded by 4 genes ( CACNA1S , CACNA1C, CACNA1D and CACNA1F encoding for Ca v l.l to Ca v 1.4 channels, respectively), and auxiliary subunits b and a2-d (each encoded by 4 genes).
  • Ca v l expression is plastic depending on their activation and differentiation status 14 16 .
  • Ca v 1.2 and Ca v 1.3 channels are selectively expressed in mouse and human Th2-cells 13 15 and Ca v 1.4 is the only Ca v l calcium channel expressed in Thl7 cells from peripheral blood of healthy donors (HD) 15 .
  • Nicardipine a Ca v l channel antagonist, decreased TCR-induced IL17 production by human Thl7 (CCR6 + ) cells 15 .
  • the inventors highlighted the presence of Ca v 1.4 calcium channel in RORyt + CD4 + T cells from lesional psoriatic (LP) skin biopsies. These channels appear to be a valuable target in psorasis treatment as Ca v l inhibition dampened inflammatory cytokine production by Thl7, whatever their origin (blood or LP). Ca v l channels acted very early during Thl7 activation on the elementary Ca 2+ events (ECE) preceding global Ca 2+ response. Finally, Ca v l blocker, nicardipine was beneficial in a 3D model of psoriasis-like reconstructed skin induced by infiltration of activated Thl7-cells. Thus, the present invention relates to a Ca v 1.4 inhibitor for use in the treatment of an inflammatory skin disease in a subject in need thereof. In particular, the invention is defined by the claims.
  • Ca v 1.4 is present in skin infiltrating RORyT + CD4 + cells.
  • Ca v l.4 calcium channel is required to cytokine production induced by TCR engagement in human Thl7 cells from peripheral blood of healthy or psoriasis patients as well as by T-cells infiltrating psoriatic skin and for the expression of keratinocyte genes characteristic of psoriasis inflammation in human skin equivalents populated with Thl7 cells.
  • a first aspect of the present invention relates to a Ca v 1.4 inhibitor for use in the treatment of an inflammatory skin disease in a subject in need thereof.
  • the inflammatory skin disease is induced by the cytokine production of Thl7 lymphocytes and pathogenic T cells.
  • the inflammatory skin disease is psoriasis.
  • the present invention relates to nicardipine for use in the treatment of psoriasis in a subject in need thereof.
  • Ca v 1.4 denotes a Ca v l voltage-gated calcium channel comprising an al pore-forming subunit encoded by CACNA1F (Entrez Gene: 778; mRNA sequences references RefSeq: NM_001256789.3; protein sequence reference RefSeq: NP 001243718.1; Uniprot: Q60840).
  • This al subunit is a protein associated with the auxiliary subunits b and a2d.
  • the auxiliary subunits allow the addressing to the plasma membrane of al and also the regulation of its electrophysiological properties. Without these auxiliary subunits, the subunit al is not functional (or poorly functional).
  • Ca v 1.4 i.e.
  • Ca v l voltage-gated calcium channel having an al pore-forming subunit encoded by CACNA1F is the only Ca v l calcium channel expressed in Thl7 cells from peripheral blood of healthy donors (Robert et al. 2014 JACI).
  • the “inflammatory skin disease” may be psoriasis, itching, acne, eczema with Thl7 markers, urticaria and more generally every inflammatory skin disease with a Thl7 component.
  • a skin disease with a Thl7 component is an inflammatory skin disease induced by the cytokine production of Thl7 lymphocytes and pathogenic T cells.
  • T-cells can be recovered from the lesional skin and tested for their ability to produce Thl7 cytokines (IL17A and/or IL17F and/or IL22) after TCR engagement and to express the Thl7 specific transcription factor (RORyt).
  • inflammatory skin disease is psoriasis.
  • Psoriasis is a chronic inflammatory skin disease resulting from abnormal dialogue between innate and adaptive immune cells and cells and resident skin cells.
  • psoriasis is associated with higher prevalence of cardiovascular pathology, including high blood pressure, relative to non-psoriatic patients 17 .
  • hypertension is associated with higher risk of psoriasis.
  • the effect of antihypertensive drugs has been checked and b-blockers are the only drugs found to be associated with higher risk of developing psoriasis 18 .
  • the Ca v 1.4 inhibitor for use in patients suffering of both psoriasis and hypertension diseases is particularly indicated.
  • the invention relates to a Ca v 1.4 inhibitor for use in the treatment of psoriasis and hypertension.
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • the subject according to the invention is a human.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • Ca v l .4 inhibitor denotes a molecule or compound, which can inhibit the activity of a Ca v 1.4 subunit (al, b and/or a2d), or can destabilize Ca v 1.4.
  • a Ca v 1.4 inhibitor can inhibit protein transcription or translation of a Ca v 1.4 subunit (al, b and/or a2d).
  • the Ca v 1.4 inhibitor is an antagonist of Ca v 1.4 calcium channel.
  • An antagonist of Ca v 1.4 calcium channel may be, for example, a calcium antagonist that prevents calcium ions to enter the cell through voltage-gated calcium channels.
  • the Ca v 1.4 inhibitor may be a selective or a non-selective inhibitor of Ca v 1.4.
  • Ca v 1.4 putative inhibitor can be tested on Ca v 1.4-transfected cells (such HEK293 cells) together with auxiliary subunits.
  • the inhibitor effect of putative Ca v l .4 antagonist can be quantified on the calcium current after application of a voltage ramp (electrophysiology) and/or on calcium signal (with Ca 2+ -sensitive fluorescence probe) after a range of KC1.
  • Ca v 1.4 inhibitor also denotes an inhibitor of the expression of the gene coding for Ca v 1.4 a ⁇ pore-forming subunit (e.g. inhibitor of CACNA1F ), in order to selectively inhibit Ca v 1.4.
  • the inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the Ca v 1.4 inhibitor is a calcium antagonist.
  • the calcium antagonist is a dihydropyridine, as described by D.J.
  • the Ca v 1.4 inhibitor is nicardipine.
  • Nicardipine is a potent calcium channel blocker of formula (I):
  • the Ca v 1.4 inhibitor according to the invention is an antibody against Ca v 1.4.
  • Antibodies directed against Ca v 1.4 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against Ca v l .4 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et ah, 1983); and the EBV- hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti- Ca v 1.4 single chain antibodies.
  • Anti-Ca v 1.4 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to Ca v 1.4.
  • Humanized anti-Ca v 1.4 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibody according to the invention is a single domain antibody against Ca v 1.4.
  • the term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals, which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHHs can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • the VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • the high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B- cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E.
  • coli see for example US 6,765,087 and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • moulds for example Aspergillus or Trichoderma
  • yeast for example Saccharomyces, Kluyveromyces, Hansenula or Pichia
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the Ca v 1.4 inhibitor is a polypeptide.
  • polypeptides referenced in Findeisen et al. (2017) prevent the interaction between Ca v l a and b subunits and inhibit the activity of Ca v l calcium channel.
  • the polypeptide of the invention may be linked to a “cell-penetrating peptide” to allow the penetration of the polypeptide in the cell.
  • cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • the polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is particularly generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others.
  • Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown.
  • a common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages.
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate.
  • the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the Ca v 1.4 inhibitor according to the invention is an inhibitor of Ca v 1.4 subunits gene expression.
  • the Ca v 1.4 inhibitor is an inhibitor of Ca v 1.4 al, b and/or a2d subunits gene expression.
  • the Ca v 1.4 inhibitor is an inhibitor of Ca v 1.4 al pore-forming subunit gene expression (e.g. CACNA1F as previously describe).
  • the inhibitor of Ca v 1.4 al pore forming subunit gene expression is a siRNA against CACNA1F.
  • the inhibitor of Ca v 1.4 al pore-forming subunit gene expression is a shRNA against CACNA1F.
  • the inhibitor of Ca v 1.4 subunits gene expression is a siRNA or a shRNA against CACNA1F.
  • Small inhibitory RNAs can also function as inhibitors of Ca v 1.4 expression for use in the present invention.
  • Ca v 1.4 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Ca v 1.4 gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g.
  • the Ca v 1.4 inhibitor is an antisense oligonucleotide.
  • the antisense oligonucleotide is directed against Ca v 1.4 al, b and/or a2d subunits.
  • Ribozymes can also function as inhibitors of Ca v 1.4 gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Ca v 1.4 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of Ca v 1.4 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector” is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and, in particular, to the cells expressing Ca v 1.4.
  • the vector is particularly able to facilitate the transfer of the oligonucleotide siRNA or ribozyme nucleic acid to Thl7 cells.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Other vectors include plasmid vectors.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et ah, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene- gun.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • Another object of the invention relates to a method to treat an inflammatory skin disease in a subject in need thereof with a therapeutic composition comprising a Ca v 1.4 inhibitor according to the invention.
  • the inflammatory skin disease is induced by the cytokine production of Thl7 lymphocytes and pathogenic T cells.
  • the inflammatory skin disease is psoriasis.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • “Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the form of the therapeutic compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.
  • the therapeutic composition of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the therapeutic composition of the invention is formulated for a topical administration on inflamed patches of skin, in order to prevent side effects.
  • the therapeutic composition of the invention can be formulated for a topical administration in the treatment of an inflammatory skin disease induced by the cytokine production of Thl7 lymphocytes and pathogenic T cells.
  • the inflammatory skin disease is psoriasis.
  • the therapeutic compositions may also contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected.
  • compositions of the present invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising a Ca v 1.4 inhibitor according to the invention and a further therapeutic active agent.
  • Anti-psoriasis agents may be added to the therapeutic composition as described below.
  • Anti-psoriasis agents may be methotrexate (Novatrex), ciclosporin (Neoral), acitretin (Soriatane) or infliximab (Remsima).
  • Others anti -psoriasis agents may be, for example, leflunomide, apremilast, adalimumab, certolizumab, infliximab, etanercept, brodalumab, guselkumab, ixekizumab, risankizumab, ustekinumab, tildrakizumab or aprelimast.
  • additional anti -psoriasis may be selected from, but are not limited to, one or a combination of the following class of agents: immunosuppressants, retinoids, TNF-a inhibitors, phosphodiesterase inhibitors, corticosteroids, topical corticosteroids, interleukin inhibitors and calciferol derivatives.
  • Additional anti-psoriasis agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Fig. 1 Ca v 1.4 calcium channels are detected in Thl7 cells infiltrating psoriasis skin.
  • Violin plots represent the expression (-delta Ct with housekeeping gene) of CACNA1F (encoding Ca v 1.4 a ⁇ pore-forming subunit) between the different skin biopsies. Each dot represents one individual, the horizontal line denotes the median and the dotted line denotes the interquartile range.
  • Fig. 2 T cells infiltrating the skin of psoriatic patients produce Thl7 inflammatory cytokines, which are inhibited by Ca v l channel blocker.
  • Cells were expanded for 14 days by stimulation with anti-CD3/CD28/CD2 coated beads in presence of IL2 and IL23. After expansion, T cells were stimulated with anti-CD3/CD28 coated beads in the presence or the absence of nicardipine (10pg/ml).
  • Fig. 3 Inhibitor effect of nicardipine, a Ca v l channel blocker on TCR-induced cytokine production and expression by expanded T cells from the lesional skin of psoriatic patients.
  • Fig. 4 Ca v l channel blocker inhibits cytokine at protein level in Thl7 cells from healthy donors and psoriatic patients.
  • Fig. 5 Ca v l channels contribute to TCR-induced [Ca 2+ ]i rise in Thl7 cells.
  • A-B HD Thl7 cells were loaded with Fura2-AM and fluorescence was recorded before (F0) and after (F) TCR stimulation ⁇ nicardipine (nicar).
  • A Mean ⁇ SEM from 50-100 cells from 3 HD.
  • B the area under the curve.
  • C-E Elementary calcium events are recorded by TIRF. Number of ECE (one symbol by cell; pool from 2 HD, C), relative ECE number (D) and opening frequency (one symbol by ECE; 5-7 cells from 2 donors, E). P values were determined by Student’s t-test. * p ⁇ 0.05; ** pO.Ol; ***p ⁇ 0.005; ****p ⁇ 0.0001.
  • Fig. 6 CACNA1F knockdown reduces IL17A and IL22 production by Thl7 cells.
  • Thl7 cells obtained from the blood of healthy donors were sorted and expanded for 14 days.
  • Cells were transduced with viral particles encoding for GFP reporter control shRNAs (shCtr) or GFP reporter shRNAs against CACNA1F ( shCACNAlF ).
  • shCtr GFP reporter control shRNAs
  • shCACNAlF GFP reporter shRNAs against CACNA1F
  • A qRT-PCR analysis of CACNA1F , CACNA1C and CACNA1D in sorted GFP + shCtr cells or in sorted GFP + shCACNAlF cells. The expression levels were normalized to the housekeeping gene.
  • B Sorted GFP + cells were stimulated for 24h with anti-CD3/anti-CD28 antibodies to determine the amount of cytokines in the supernatant by ELISA. Results from 2-3 biological replicates from 3 experiments. P values were determined by paired wilcoxon test. * p ⁇ 0.05;
  • Fig. 7 Ca v l channel blocker diminishes the production of Thl7 cytokines and gene expression characteristic of psoriasis in a model of Thl7-induced psoriatic alterations.
  • Skin equivalent model comprising multi-stratified keratinocyte layers contacting a pseudo dermis structure was used.
  • Activated Thl7 cells were placed underneath the “dermis” with or without nicardipine (nicar). Medium was replaced each day by fresh medium containing or not the drug.
  • As control, ciclosporin A (CsA) was added 2 days after the beginning of the culture.
  • A Four days after starting the experiment, samples were cut and sections were revealed with A555-labeled anti-CD3 antibodies. The number of cells/field was counted and the mean was represented for each sample.
  • IL17 and IL22 amounts were measured in the medium after 24 hours. Each point corresponds to one reconstructed skin.
  • Fig. 8 The inhibition of Ca v l calcium channels decreases psoriasis-like inflammation in mice.
  • Imiquimod cream IMQ 5%
  • Vaseline was applied to the skin of control mice.
  • Intra-peritoneal nicardipine nicar. was given once a day or not from day 0 to day 6.
  • A-B Representative hematoxylin eosin staining at day 6. Bars, 37 pm (A) and 75 pm in (B).
  • C Ear thickness was measured with a caliper. Back skin thickness (epithelium and dermis) was measured. Statistical comparisons were done by one way Anova followed by multiple comparison tests.
  • Thl7 cell sorting and expansion From PBMC, CD4 + CXCR3 T cells were enriched using EasySepTM Human Thl7 Cell Enrichment Kit (STEMCELL Technologies). Then, CD4 + CD45RA CCR6 + CXCR3 (Thl7) cells were isolated by cell sorted on the FACS Aria cell sorter (BD Biosciences). Cells (0.2 10 6 ) were expanded during 14 days by stimulation with ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator (STEMCELL) in Immunocullt-XF (STEMCELL) medium containing IL-2 (5 ng/mL), anti-IFNy antibody (5 pg/ml, B27, BD Biosciences) and Ethanercept (10pg/ml).
  • IL-17A SCPL1362, BD Biosciences
  • IL-22 22URTI, eBioscience
  • TNFa MAbl 1, BD Biosciences
  • IFNy B27, BD Biosciences
  • Cells were analyzed using a Fortessa (BD Biosciences), and FlowJo software (Tree Star, Ashland, Ore) was used for analysis.
  • ELISA ELISA.
  • Expanded cells were seeded (5 c 10 4 cells per well) into 96-well flat-bottom plates and stimulated with ImmunoCultTM Human CD3/CD28 T Cell Activator (STEMCELL) for 24 hours, and cytokine production was quantified by ELISA. Plates were coated with anti-IFNy (3 pg/mL, MD-1, Biolegends) or anti-IL17A (1 pg/mL, eBio64CAP17, eBioscience). Plates were washed and supernatants were added.
  • TIRF Total internal reflection fluorescence
  • the excitation light with a critical angle produced by an expended beam of an Argon ion laser (488 nm), is sent to the specimen, triggering a total internal reflection at the interface glass/medium.
  • This evanescent wave is generated over less than 100 nm from the interface, sufficient to excite fluorophores located in this region.
  • Emitted light was collected at 510nm and recorded with a cooled (-80°C) back illuminated EMCCD camera (Andor iXon).
  • the frame rate was 500 Hz.
  • F0 corresponds to the mean of fluorescence recorded on the 100 first images before stimulation.
  • the opening frequencies were determined over a period of 4 sec (2000 consecutive images) as soon as a channel opens (30 sec after stimulation), and results were expressed in events/second.
  • T cell recovery from psoriatic patient biopsies The 3 biopsies of lesional skins of PP were cut into small pieces.
  • the gentleMACS Dissociators (Miltenyi Biotec, « Skin Ol » program) was first used for tissues dissociation before incubation with Collagenase IV (2.4 mg/ml) and DNase I (0,2mg/ml) in a shaking water bath at 37 °C for 60 min.
  • the biopsies underwent a second step of mechanical dissociation.
  • Cells were passed through a 70pm cell strainer to remove cell clumps/tissue debris, washed, and expanded during 14 days by stimulation with ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator (STEMCELL) in Immunocullt-XF (STEMCELL) medium containing IL-2 (5 ng/ml) and IL-23 (50ng/ml).
  • RNAscope In situ hybridization (ISH) was performed according to the protocol of the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, Advanced Cell Diagnostics, Cat. No. 320293). 15 pm cryosections were cut from frozen biopsies of 15 patients, dried on slides for 10 min at 40°C and kept at -20°C.
  • ISH In situ hybridization
  • slides were incubated in boiling antigen retrieval solution (ACD, 322000) for 5 min, washed in water twice, dehydrated in 100% ethanol and finally treated with Protease III for 30 min at 40°C, then rinsed twice in PBS.
  • ACD boiling antigen retrieval solution
  • C2 ( CD4 ) and C3 ( CACNA1F) probes were diluted in Cl ( RORC ) probes solutions at a 1 :50 ratio and incubated for 2hrs at 40°C.
  • RNA extraction and qPCR Expanded Thl7 cells from peripheral blood of PP and HD and expanded T cells from LP skins were stimulated during 4 hours with ImmunoCultTM Human CD3/CD28 T Cell Activator (STEMCELL) in R10 medium. Grinding of biopsies was performed with TissueLyser (QIAGEN SA) by mixing the sample with a metal bead and RLT buffer (QIAGEN SA). Nucleic acids were isolated by using AllPrep DNA/RNA Mini Kit (QIAGEN SA,) according to a randomized design. A quality control was performed on H2O eluted total RNA on TapeStation instrument with RNA ScreenTape Kit (AGILENT Tech.).
  • a reverse transcription step was performed with Quantitect RT Kit (QIAGEN SA) for cDNA synthesis with integrated genomic DNA removal. Transcriptomic experimentations were performed in duplicate with TaqMan Low Density Array technology. Custom array containing Housekeeping genes were run on ViiA 7 Real-Time PCR System (THERMOFISHER Sc.). Gene assay references targeting cytokines, CACNA1 and housekeeping genes are given in Table 1. Ct values were normalized with the delta Ct method to obtain delta Ct values (difference in Ct values between the target and the average of four housekeeping genes). To account for systematic variation between LP, NLP and AD biopsies, differentially expressed genes were identified using a mixed linear model on delta Ct values.
  • the group factor with three levels was included as a fixed effect, duplicate and patient as random effects.
  • Mean difference between the ACt values of two groups was given by least-squares means. Fold change was computed as 2 Ct value. Two-tailed tests were used.
  • cytokine expression genes the data are presented as the ratio of 2 DDa between stimulated in presence or not of nicardipine and unstimulated group.
  • the R software v3.5.0 was used for statistical analyses.
  • Lentiviral production and Thl7 cell transduction Two lentiviral vector encoding an shRNA targeting the CACNA1F mRNA (encoding for Cavl.4 al) and containing a gene encoding TurboGFP, under the control of the CMV promoter was purchased from Sigma (MISSION TRCN0000454237 and TRCN0000043718). Lentiviral particles were prepared by standard transfection into HEK293T cells with VSV-G envelope expressing plasmid (pMD.2G) and Packaging plasmid (PsPax2). Three days later, the supernatant was harvested, centrifuged and passed through 0.45 pm filter. Viral particles are concentrated by centrifugation OVN at 3200rpm at 10°C.
  • Human skin equivalents were obtained from the department of Pierre Fabre DermatoCosmetic. Expanded Thl7 cells from healthy donor were activated during 5 hours with ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator (STEMCELL) in presence of not of nicardipine (lOpg/ml) before to introduction in the skin equivalents.
  • STEMCELL ImmunoCultTM Human CD3/CD28/CD2 T Cell Activator
  • RNA extraction After 4 days of culture, a part of each insert is proceeded for histology study and another part for RNA extraction. Immunofluorescence and immunohistochemistry staining with anti-CD3 antibodies followed with A555 -secondary antibodies or peroxidase-secondary antibody, respectively to quantify the migration of Thl7 cells in the equivalent skin.
  • inserts were lysed in RLT buffer containing b-mercaptoethanol in Lysing Matrix M Tubes (MP biomedicals) using FastPrep® instruments. RNA extraction is thus done with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with the SuperScriptlll Reverse Transcriptase (Invitrogen, Carlsbad, Calif).
  • Transcripts were measured by using real-time quantitative PCR with a LightCycler 480 Instrument and the LightCycler 480 SYBR Green I Master (Roche, Carlsbad, Calif). List of primers are given in Table 2. Amounts of mRNA were calculated as arbitrary units relative to 60S acidic ribosomal protein P0 (RPL0) as follows: (2
  • Ca v 1.4 calcium channel is detected in RORyt + CD4 + cells in lesional psoriasis biopsies.
  • Thl/Thl7/Th22 cytokines 1L17A , IL26, IL23A , IFNG, CSF2 , IL22, IL6
  • chemokines that recruit Thl, Thl7 lymphocytes and neutrophils CCL20 and CXCL8
  • skin barrier function elafin (PI3), P-defensin2 ( DEFB4B ), Late Cornified Envelop ( LCE3A ), psoriasin ( S100A7 ), MPR6 ( S100A8 ), mKi67, keratinl6 ( KRT16 ), involucrin ( IVL )
  • genes involved in type 2-cell function/recruitment IL13 , CCL26
  • CA2 Carbonic Anhydrase 2
  • NELL2 Neural EGFL Like 2
  • TCR-induced cytokine production by T cells infiltrating psoriatic lesions is dependent of Ca v l channels.
  • Ca v 1.4 on T cells infiltrating the skin of PP, we isolated cells from biopsies of lesional skin of PP and expanded T cells by stimulating them with anti-CD3/CD28/CD2 coated beads in the presence of IL2 and IL23 according to 22 . After 14 days of culture, all cells were CD3 + T lymphocytes dividing equally into CD4 + and CD8 + T cells (data not shown).
  • CD4 + CD45RA On the basis of chemokine receptor expression 23 , we analyzed among CD4 + CD45RA the frequency of CD4 + CXCR3 + CCR6 (Thl), of CD4 + CXCR3 + CCR6 (Thl/Thl7) and of CD4 + CCR6 + CXCR3 (Thl7) on expanded cells.
  • CD4 + T cells around 30% were CXCR3 CCR6 + (Thl7), 30% CXCR3 + CCR6 + (Thl/Thl7), and 20% CXCR3 + CCR6 (Thl), (data not shown).
  • CD4 + T cells Upon anti-CD3/CD28 stimulation, CD4 + T cells produced significantly IL17A, IL22, IFNy and TNFa (Fig. 2).
  • nicardipine a Ca v l channel inhibitor at a concentration (10pg/ml) that was shown to inhibit Ca v l.2 channel function in human Th2 cells 15 .
  • Nicardipine reduced significantly TCR-driven IL-17 (36%), IL-22 (35%), ⁇ FNy (27%) and TNF-a (53%) production (Fig. 2 and 3A).
  • Nicardipine acted at the transcriptional level since it inhibited TCR-induced transcription of IL17A (52%), IL22 (57%) and at lesser level IFNG (37%) and TNFA (27%) (Fig. 3B).
  • IL17F and CSF2 transcripts were also TCR-induced in infiltrating T-cells and their expressions were significantly inhibited by nicardipine (54 and 42% respectively, Fig. 3B).
  • nicardipine 54 and 42% respectively, Fig. 3B.
  • Thl7 cells The TCR-induced cytokine transcription and production by expanded HD and PP blood Thl7 cells is also partially controlled by Ca v l channel activation. Based on chemiokine receptors expression, we showed the only population that was significantly increased in PP compared to HD, was Thl7 cells (data not shown). To investigate the role of Ca v 1.4 in Thl7 cell functions from peripheral blood of PP and HD, we set up a protocol of amplification to get enough Thl7 cells. We sorted Thl7 cells (CD4 + CD45RA CCR6 + CXCR3 ) and expanded them by anti-CD3/CD28/CD2 stimulation for 14-21 days in the presence of IL2, anti-IFN-/ and anti- TNFa antibodies.
  • Thl7 cells retained their ability to produce IL17, IL22 and IFN-/ (although at a lower level for the two latter cytokines) when compared to ex vivo production (data not shown).
  • RORyt, a Thl7-specific transcription factor and CACNA1F expression remained detectable in expanded Thl7-cells (data not shown).
  • Thl7 cells from PP and HD produced IL17A, IL22, IFNy and TNFa after anti-CD3/CD28 stimulation (Fig. 4A and 4B).
  • Nicardipine decreased not only the frequency of cytokine producing cells (Fig. 4A and 4B) but also the intensity of secretion by cell (GMFI), the amount of cytokines in culture supernatants and their transcriptions (data not shown).
  • the inhibitory effect of nicardipine on cytokines was similar in HD and PP, regarding cytokine secretion, production or transcription (data not shown). Together, these results show that Ca v l Ca 2+ channels contributed to optimal cytokine production by TCR-activated Thl7 lymphocytes from HD and PP.
  • Ca v l calcium channels are involved in TCR-driven initial calcium events in Thl7 cells.
  • Ca v l calcium channels play a role in the global calcium response of Thl7 cells.
  • Nicardipine strongly reduced the TCR-induced rise of [Ca 2+ ]i as testified by the trace over several minutes and the significant decrease of area under the curve (Fig. 5A and 5B).
  • TIRFM imaging also called optical patch clamp
  • ECE spatiotemporal single elementary calcium events
  • Nicardipine decreased the number of spontaneous ECE in resting cells (Fig. 5C) and precluded the increase of ECE numbers and opening frequencies upon TCR stimulation (Fig. 5C-E). These results demonstrate Ca v l channels support early calcium influx at plasma membrane in both resting and TCR-activated Thl7 cells.
  • the knockdown of Ca v 1.4 decreases IL17 and IL22 production by Thl7 cells.
  • nicardipine is an inhibitor of all Ca v l calcium channels that include Ca v l.l to Ca v 1.4
  • we tested the specific role of Ca v 1.4 in Thl7 functions by knocking-down it with GFP reporter shRNAs.
  • the transduction of expanded Thl7 cells from peripheral blood of HD with these shRNAs against CACNA1F (encoding Ca v 1.4) diminished by around 50% its expression in GFP + sorted cells relative to cells transduced with control shRNA (Fig. 6A).
  • This decreased Ca v 1.4 expression was associated with reduced production of IL17, IL22 after TCR stimulation by about 55 and 59%, respectively (Fig. 6B).
  • Ca v l calcium channels are involved in the development of psoriasic inflammation in 3D skin reconstruction model.
  • 3D skin reconstruction model in which the expression of inflammatory markers characteristic of psoriasis by keratinocytes is induced, as previously described by populating the skin with activated Thl7-cells 25 .
  • Expanded Thl7 cells from peripheral blood of HD were stimulated with anti-CD3/CD28-coated beads before being placed between the transwell membrane and dermal side of the skin equivalents to favour their migration in the dermis 25 .
  • nicardipine was added to Thl7 cells before the stimulation and are present during all the time of culture.
  • Ciclosporin A CsA was used as an inhibitor of the psoriasiform inflammation as previously described 25 .
  • the activation of Thl7 cells was confirmed by CD69 expression and was not modified by nicardipine (data not shown).
  • Thl7 cells did not modify the epidermis morphology, in agreement with previous work 25 , whether nicardipine or CsA were present or not (data not shown).
  • cytokine production in the skin equivalent supernatant.
  • Nicardipine tended to reduce IL17 production and significantly decreased IL22 production (Fig. 7B).
  • the markers of epidermal inflammation were quantified by qPCR in the inserts after four days of culture.
  • the infiltration of Thl7 cells induced a significant increase of psoriasis-associated genes, such as KRT16, PI3, and DEFB4 (encoding for cytokeratin 16, elafin and b-defensin 2, respectively), (Fig.
  • Nicardipine diminished these gene expressions at the same level as CsA (Fig. 7C). These data show that nicardipine can affect the psoriasis inflammation in 3D skin reconstruction model probably by acting on Thl7-cells functions, in accordance with locally decreased IL17 and IL22 production.
  • Murine imiquimod-induced psoriasis like model Imiquimod is applied to the shaved back (31,25 mg Aldara® per day for 6 days) and on one ear (5mg per day for 6 days) of BALB/c mice that received or not intraperitoneal injections of nicardipine (lmg per day for 6 days). Hematoxylin and eosin staining is performed to analyze the morphology of ear and back skin.
  • nicardipine was investigated the role of nicardipine in local lesions in murine imiquimod-induced psoriasis-like model.
  • imiquimod- treated mice presented dermal inflammation and epidermal acanthosis compared with untreated mice, in treated-ear and treated-back-skin (Fig. 8A and 8B) and a significant increase in imiquimod-treated-ear and imiquimod-treated-back-skin thickness (Fig. 8C).
  • Nicardipine greatly reduced both dermal inflammation and epidermal acanthosis and decreased the ear and back skin thickness in this experimental model.
  • Nicardipine a Ca v l channel antagonist reduced both TCR-induced cytokine transcription and production by CD4 + T cells infiltrating PP skin.
  • CD4 + T cells were the main producers of IL17A and IL22 (CD8 + T cells produced very few IL17A and IL22, not shown), IFNy and TNFa were also produced by infiltrating CD8 + T cells and nicardipine inhibited their production (not shown), in accordance with Ca v 1.4 expression by CD8 + T cells 15 .
  • nicardipine The potential inhibitory action of nicardipine on all cytokines and in the majority of PP, is interesting since these cytokines have their own and synergic effect, but also amplify the inflammatory loop on psoriasis skin inflammation 26,27 .
  • Thl7 cells expressed CACNA1F and kept this expression as well as their pattern of TCR-induced cytokine expression after expansion showing they represent a valuable tool for studying Thl7 functions.
  • TCR stimulation of Thl7 cells from PP and HD induced similar amounts of cytokines (transcription and production) and nicardipine inhibited them with a similar effect.
  • cytokines transcription and production
  • nicardipine reduced cytokine production by Thl7 cells even in inflamed tissues.
  • That nicardipine decreased the transcription and production of all inflammatory cytokines tested suggests that Ca v l channels regulate common pathways, upstream of cytokine gene transcription.
  • Ca 2+ is a second messenger playing a crucial role in Thl 7 functions and their pathological potential 28 explaining why ciclosporin A, that inhibits the Ca 2+ -dependent phosphatase calcineurin, was used in the treatment of PP 29 .
  • inhibition of Orai channels that mediate store-operated Ca 2+ entry in most if not all T-cells effectively reduced Thl7 functions and Thl7-cell mediated pathology but these treatments are not selective of Thl7-cells and can cause adverse effects 28 .
  • Ca v 1.4 known to activate at relatively negative cell membrane potential, closer to resting cell membrane potential than the other Ca v l channels, could deliver Ca 2+ in a very fast and localized manner facilitating the transduction machinery assembly.
  • Ca v l channels activate fast, before for example ORAI channels that require first the depletion of intracellular calcium stores to get activated 33 .
  • Ca v l.4 was the only Ca v l channel expressed in Thl7 cells and transduction of these cells with specific shRNA reduced inflammatory cytokine production confirming the role of Ca v 1.4 in Thl7-cell functions.
  • cytokines produced by T cells appear to be the main factor responsible for the psoriatic inflammatory phenotype 25 and that nicardipine diminishes IL17 and IL22 cytokines in the skin equivalent infiltrated with Thl7 cells, we propose that Ca v l channel blockage lessens psoriatic gene expression through the inhibition of cytokine production by T cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Le psoriasis est une maladie chronique de la peau avec un risque accru de maladies cardiovasculaires et rhumatismales. L'inhibition de l'axe IL17/IL22/TNFα est bénéfique, favorisant un rôle des cellules Th17 dans cette pathologie. Dans la présente invention, les inventeurs ont étudié la présence et le rôle des canaux calciques Cav1.4 dans les cellules Th17 des patients psoriasiques. Ils ont démontré, pour la première fois, l'expression du Cav1.4 dans les cellules RORgT+ CD4+ infiltrant la peau psoriasique. La nicardipine, un inhibiteur de Cav1 largement utilisé dans les maladies cardiovasculaires, a nettement réduit la production de toutes leurs cytokines. L'inactivation de Cav1.4 par le biais du ARNsh a également altéré la production de cytokines Th17 induite par le TCR dans les cellules Th17. Enfin, la nicardipine a réduit les expressions des gènes kératinocytaires caractéristiques de l'inflammation psoriasique dans des équivalents de peau humaine peuplés de cellules Th17. Dans l'ensemble, ces données ont encouragé le développement de médicaments inhibant les canaux Cav1.4 dans le traitement du psoriasis.
PCT/EP2022/064940 2021-06-02 2022-06-01 Nouveau procédé pour traiter une maladie cutanée inflammatoire WO2022253910A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP21305740.9 2021-06-02
EP21305740 2021-06-02

Publications (1)

Publication Number Publication Date
WO2022253910A1 true WO2022253910A1 (fr) 2022-12-08

Family

ID=76355435

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2022/064940 WO2022253910A1 (fr) 2021-06-02 2022-06-01 Nouveau procédé pour traiter une maladie cutanée inflammatoire

Country Status (1)

Country Link
WO (1) WO2022253910A1 (fr)

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US541A (en) 1837-12-26 Daniel desmond
US5874A (en) 1848-10-24 Apparatus eob baking- water
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
EP0937460A2 (fr) * 1994-04-12 1999-08-25 Adolor Corporation Utilisation d'un antidiarrhéique pour la préparation d'un médicament pour le traitement des inflammations
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5874A (en) 1848-10-24 Apparatus eob baking- water
US541A (en) 1837-12-26 Daniel desmond
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6015695A (en) 1992-08-21 2000-01-18 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
EP0937460A2 (fr) * 1994-04-12 1999-08-25 Adolor Corporation Utilisation d'un antidiarrhéique pour la préparation d'un médicament pour le traitement des inflammations
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn

Non-Patent Citations (37)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. Q60840
ACOSTA-RODRIGUEZ, E. V. ET AL.: "Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells", NAT IMMUNOL, vol. 8, 2007, pages 639 - 646, XP002496162, DOI: 10.1038/ni1467
ARMSTRONG, A. W.READ, C.: "Pathophysiology, Clinical Presentation, and Treatment of Psoriasis: AReview", JAMA, vol. 323, 2020, pages 1945 - 1960
BALIWAG, J.BARNES, D. H.JOHNSTON, A.: "Cytokines in psoriasis", CYTOKINE, vol. 73, 2015, pages 342 - 350, XP029576735, DOI: 10.1016/j.cyto.2014.12.014
BOEHNCKE WOLF-HENNING ET AL: "Psoriasis", THE LANCET, 27 May 2015 (2015-05-27), pages 983 - 994, XP055860219, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0140673614619097> [retrieved on 20211111], DOI: https://doi.org/10.1016/S0140-6736(14)61909-7 *
BOEHNCKE, W. H.SCHON, M. P.: "Psoriasis", LANCET, vol. 386, 2015, pages 983 - 994, XP055860219, DOI: https://doi.org/10.1016/S0140-6736(14)61909-7
CABRAL, M. D. ET AL.: "Knocking down Cavl calcium channels implicated in Th2 cell activation prevents experimental asthma", AM J RESPIR CRIT CARE MED, vol. 181, 2010, pages 1310 - 1317
DEMURO, A.PARKER, I.: "Optical patch-clamping'': single-channel recording by imaging Ca2+ flux through individual muscle acetylcholine receptor channels", J GEN PHYSIOL, vol. 126, 2005, pages 179 - 192
EHST, B. ET AL.: "Synergistic induction of IL-23 by TNFa, IL-17A, and EGF in keratinocytes", CYTOKINE, 2020, pages 155357
FESKE, S.SKOLNIK, E. Y.PRAKRIYA, M.: "Ion channels and transporters in lymphocyte function and immunity", NAT REV IMMUNOL, vol. 12, 2012, pages 532 - 547
GOMES, B. ET AL.: "Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation", AM J RESPIR CRIT CARE MED, vol. 175, 2007, pages 1117 - 1124
HE, X.DE OLIVEIRA, V. L.KEIJSERS, R.JOOSTEN, I.KOENEN, H. J.: "Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture", JOURNAL OF VISUALIZED EXPERIMENTS : JOVE, 2016, pages e52564
JHA, M. K. ET AL.: "Defective survival of naive CD8+ T lymphocytes in the absence of the beta3 regulatory subunit of voltage-gated calcium channels", NAT IMMUNOL, vol. 10, 2009, pages 1275 - 1282, XP055697702, DOI: 10.1038/ni.1793
JULLIEN, D.PRINZ, J. C.NESTLE, F. O.: "Immunogenicity of biotherapy used in psoriasis: the science behind the scenes", J INVEST DERMATOL, vol. 135, 2015, pages 31 - 38
KAUFMANN, U. ET AL.: "Calcium Signaling Controls Pathogenic Th17 Cell-Mediated Inflammation by Regulating Mitochondrial Function", CELL METABOLISM, vol. 29, 2019, pages 1104 - 1118
KRUEGER, J. G. ET AL.: "IL-17A is essential for cell activation and inflammatory gene circuits in subjects with psoriasis", J ALLERGY CLIN IMMUNOL, vol. 130, 2012, pages 145 - 154
LEONARDI, C. ET AL.: "Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis", N ENGL J MED, vol. 366, 2012, pages 1190 - 1199, XP002681513, DOI: 10.1056/NEJMoa1109997
LOWES, M. A.SUAREZ-FARINAS, M.KRUEGER, J. G.: "Immunology of psoriasis", ANNU REV IMMUNOL, vol. 32, 2014, pages 227 - 255
MARS MARION ET AL: "Cav1.4 calcium channels control cytokine production by human peripheral TH17 cells and psoriatic skin-infiltrating T cells", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, 1 October 2021 (2021-10-01), AMSTERDAM, NL, XP055860078, ISSN: 0091-6749, DOI: 10.1016/j.jaci.2021.09.030 *
MENTER, A. ET AL.: "Joint American Academy of Dermatology-National Psoriasis Foundation guidelines of care for the management of psoriasis with systemic nonbiologic therapies", JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, vol. 82, 2020, pages 1445 - 1486
NASH, P.: "Inhibition of interleukins 17A and 17F in psoriatic arthritis", LANCET, vol. 395, 2020, pages 395 - 396, XP086027157, DOI: 10.1016/S0140-6736(20)30220-8
NAULI, S. M. ET AL.: "Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells", NAT GENET, vol. 33, 2003, pages 129 - 137
NESTLE, F. O.KAPLAN, D. H.BARKER, J.: "Psoriasis", N ENGL J MED, vol. 361, 2009, pages 496 - 509
OMILUSIK, K. ET AL.: "The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis", IMMUNITY, vol. 35, 2011, pages 349 - 360, XP028298661, DOI: 10.1016/j.immuni.2011.07.011
PAPP, K. A. ET AL.: "Anti-IL-17 receptor antibody AMG 827 leads to rapid clinical response in subjects with moderate to severe psoriasis: results from a phase I, randomized, placebo-controlled trial", J INVEST DERMATOL, vol. 132, 2012, pages 2466 - 2469, XP055254737, DOI: 10.1038/jid.2012.163
PAPP, K. A. ET AL.: "Brodalumab, an anti-interleukin-17-receptor antibody for psoriasis", N ENGL J MED, vol. 366, 2012, pages 1181 - 1189, XP002726554, DOI: 10.1056/NEJMoa1109017
ROBERT VIRGINIE ET AL: "Protein kinase C-dependent activation of CaV1.2 channels selectively controls human TH2-lymphocyte functions", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 133, no. 4, 1 April 2014 (2014-04-01), AMSTERDAM, NL, pages 1175 - 1183.e12, XP055860220, ISSN: 0091-6749, DOI: 10.1016/j.jaci.2013.10.038 *
ROBERT, V. ET AL.: "Protein kinase C-dependent activation of CaVl.2 channels selectively controls human TH2-lymphocyte functions", J ALLERGY CLIN IMMUNOL, vol. 133, 2014, pages 1175 - 1183
ROSA, N. ET AL.: "The beta and alpha2delta auxiliary subunits of voltage-gated calcium channel 1 (Cav1) are required for TH2 lymphocyte function and acute allergic airway inflammation", J ALLERGY CLIN IMMUNOL, vol. 142, 2018, pages 892 - 903
SCHON, M. P.BOEHNCKE, W. H.: "Psoriasis", N ENGL J MED, vol. 352, 2005, pages 1899 - 1912, XP055534815
SHI, X. ET AL.: "Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids", NATURE, vol. 493, 2013, pages 111 - 115
TAKESHITA, J. ET AL.: "Psoriasis and comorbid diseases: Epidemiology", JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY, vol. 76, 2017, pages 377 - 390, XP029920494, DOI: 10.1016/j.jaad.2016.07.064
VAN DEN BOGAARD, E. H. ET AL.: "Crosstalk between keratinocytes and T cells in a 3D microenvironment: a model to study inflammatory skin diseases", J INVEST DERMATOL, vol. 134, 2014, pages 719 - 727, XP055436202, DOI: 10.1038/jid.2013.417
WASILEWSKA, A.WINIARSKA, M.OLSZEWSKA, M.RUDNICKA, L.: "Interleukin-17 inhibitors. A new era in treatment of psoriasis and other skin diseases", POSTEPY DERMATOLOGII I ALERGOLOGII, vol. 33, 2016, pages 247 - 252
WILLIAMS, H. C.BURNEY, P. G.PEMBROKE, A. C.HAY, R. J.: "The U.K. Working Party's Diagnostic Criteria for Atopic Dermatitis. III. Independent hospital validation", BR J DERMATOL, vol. 131, 1994, pages 406 - 416
WU, S.HAN, J.LI, W. Q.QURESHI, A. A.: "Hypertension, antihypertensive medication use, and risk of psoriasis", JAMA DERMATOLOGY, vol. 150, 2014, pages 957 - 963
YI, J.BALAGOPALAN, L.NGUYEN, T.MCINTIRE, K. M.SAMELSON, L. E.: "TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps", NAT COMMUN, vol. 10, 2019, pages 277

Similar Documents

Publication Publication Date Title
JP6141919B2 (ja) 新規細胞上のＣｌｅｖｅｒ−１をモジュレートし得る医薬組成物
US8871205B2 (en) Methods and compositions for the treatment of immune disorders
KR20140019303A (ko) 루푸스의 치료 또는 예방을 위한 조성물 및 방법
WO2015120138A2 (fr) Agents modulant la signalisation rgmb-neogénine-bmp et leurs procédés d&#39;utilisation
CA3146344A1 (fr) Traitement et prevention de maladies metaboliques
WO2014013005A1 (fr) Procédés de prévention et de traitement de la maladie rénale chronique (mrc).
EP3430404B1 (fr) Procédé précoce et non invasif pour l&#39;évaluation de risque d&#39;un sujet ayant un adénocarcinome intracanalaire pancréatique et procédés de traitement de ces maladies
US20140093494A1 (en) Alpha synuclein toxicity
US20180298104A1 (en) Methods and pharmaceutical compositions for the treatment of th17 mediated diseases
WO2022253910A1 (fr) Nouveau procédé pour traiter une maladie cutanée inflammatoire
JP2007517498A (ja) 骨形態形成タンパク質（ｂｍｐ）２ａ及びその使用
WO2020229648A1 (fr) Méthode de traitement d&#39;une inflammation de type 2 ou d&#39;une maladie dépendant des mastocytes
EP3452512B1 (fr) Procédés et compositions pharmaceutiques pour le traitement de lésion de tissu
US20150073034A1 (en) Methods and pharmaceutical compositions for the treatment of hypertension
WO2014064192A1 (fr) Procédé et composition pharmaceutique pour l&#39;utilisation dans le traitement et la prédiction d&#39;un infarctus du myocarde
WO2024028476A1 (fr) Méthodes de traitement de maladies médiées par th2
US9573997B2 (en) Method for use in the treatment of aneurysm
US20200062855A1 (en) Compositions and methods of promoting wound healing
US10144772B2 (en) Method for treating melanoma
WO2020208082A1 (fr) Méthode de traitement des maladies liées au cytomégalovirus
JP2021001165A (ja) オンコスタチンｍ受容体シグナリング制御による尿路結石の予防と治療
WO2024003131A1 (fr) Applications thérapeutiques basées sur l&#39;inhibition du récepteur 182 couplé aux protéines (gpr182)
WO2019211369A1 (fr) Procédés et compositions pharmaceutiques pour le traitement du cancer
EP2852679A1 (fr) Procédés pour diagnostiquer et traiter la glomérulosclérose segmentaire focale
EP3321280A1 (fr) Modulateurs immunitaires de réduction d&#39;immuno-résistance dans un mélanome et d&#39;autres maladies à évolution chronique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22731601

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22731601

Country of ref document: EP

Kind code of ref document: A1