WO2018037103A1 - Procaïnamide pour le traitement de troubles immunitaires et pour la modulation de l'expression de l'ido - Google Patents

Procaïnamide pour le traitement de troubles immunitaires et pour la modulation de l'expression de l'ido Download PDF

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Publication number
WO2018037103A1
WO2018037103A1 PCT/EP2017/071404 EP2017071404W WO2018037103A1 WO 2018037103 A1 WO2018037103 A1 WO 2018037103A1 EP 2017071404 W EP2017071404 W EP 2017071404W WO 2018037103 A1 WO2018037103 A1 WO 2018037103A1
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Prior art keywords
cells
antigen presenting
procainamide
presenting cells
pharmaceutically acceptable
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PCT/EP2017/071404
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English (en)
Inventor
Peter Ericsson
Hans Olov SJÖGREN
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Idogen Ab
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Priority claimed from GBGB1614593.0A external-priority patent/GB201614593D0/en
Priority claimed from GBGB1614590.6A external-priority patent/GB201614590D0/en
Priority claimed from GBGB1614591.4A external-priority patent/GB201614591D0/en
Priority claimed from GBGB1614592.2A external-priority patent/GB201614592D0/en
Priority claimed from GBGB1709399.8A external-priority patent/GB201709399D0/en
Priority claimed from GBGB1709401.2A external-priority patent/GB201709401D0/en
Application filed by Idogen Ab filed Critical Idogen Ab
Publication of WO2018037103A1 publication Critical patent/WO2018037103A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • PROCAINAM IDE FOR TREATING I M MUNE DISORDERS AND FOR MODULATING IDO EXPRESSION
  • the invention relates to a compound and compositions comprising said compound for use in the treatment or prophylaxis of an immune related disease, disorder or condition, and to related methods.
  • ID01 indoleamine 2,3-dioxygenase 1
  • ID01 is a heme-containing enzyme catalyzing the initial, rate-limiting step in tryptophan degradation that is known to have an important role in inducing immune tolerance in both experimental animals and in humans by inhibiting deleterious immune reactions.
  • IDO gene expression is known to be induced in antigen presenting cells. When intracellularly expressed by antigen presenting cells such as dendritic cells, ID01 functions as a natural immunoregulator by suppressing T cell responses which results in immune tolerance. Therefore control of IDO gene expression presents a possible mechanism for treating a variety of diseases in which the immune system is involved. For example, during mammalian pregnancy ID01 plays a key role. ID01 levels are increased during pregnancy and in this way it is essential for induction of immune tolerance against the foetus. Further, experimental inhibition of ID01 in mice has been shown to result in rejection of foetuses expressing different MHC molecules than their mother.
  • Antigen presenting cells are cells that display foreign peptide antigens complexed with major histocompatibility complexes (MHCs) on their surfaces; they process antigens and present them to T cells.
  • APCs fall into two categories: professional and non-professional, the professional APC being those that express MHC class II molecules. There are four main types of professional APCs: dendritic cells, macrophages, certain B-cells and certain activated epithelial cells. Interaction of APCs with CD4+ T-cells occurs in the lymph nodes, to which APCs are directed through the effect of chemotaxis.
  • Dendritic cells can be isolated and matured from a number of sources, such as bone marrow (particularly hematopoietic bone marrow progenitor cells) and blood (particularly peripheral blood mononuclear cells, of which some are immature and are expressing CD34). Dendritic cells may be obtained by differentiation of other less mature cells for example CD34+ hematopoietic stem cells or CD14+ monocytes. Depending on the circumstances, (for example, level of expression of co- stimulators such as CD80/86, CD40, or ID01 , PD-L1 or PD-L2 with suppressive activity), the interaction of dendritic cells and T cells in the lymph nodes can lead to an immune or a tolerant response.
  • sources such as bone marrow (particularly hematopoietic bone marrow progenitor cells) and blood (particularly peripheral blood mononuclear cells, of which some are immature and are expressing CD34). Dendritic cells may be obtained by differentiation of other less mature cells
  • a key interaction involves DCs producing immunosuppressive cytokines (e.g. IL-10 and TGF- ⁇ ), which induces the T cells to adopt a regulatory phenotype.
  • immunosuppressive cytokines e.g. IL-10 and TGF- ⁇
  • the regulatory T cells in their turn do not only suppress the effector T cells or naive T cells in their microenvironment, but they also induce immature DCs to differentiate to tolerogenic rather than immunogenic DCs thereby creating a tolerogenic loop response.
  • Procainamide is an antiarrhythmic drug used to treat abnormal heart rhythms. Procainamide has a short half-life and must be administered every 3 to 4 hours. Prolonged use of procainamide increases the risk of developing procainamide-induced antinuclear antibodies which may lead to various serious side effects.
  • a common side effect of long term procainamide use is development of systemic lupus erythematosus (Zhou et al. 2008; Rubin et al. 1999). The side effects have been reported to be due to acetylation of procainamide in vivo (Lawson et al., 1977). Lee et al.
  • procainamide specifically inhibits hemimethylase activity of DNA methyltransferase 1 (DNMT1 ), the enzyme thought to be responsible for maintaining DNA methylation patterns. It was found that procainamide is a partial competitive inhibitor of DNMT1 and reduces its affinity for its two substrates: hemimethylated DNA and S-adenosyl-L-methionine. DNMT1 has been strongly linked with epigenetic alterations in carcinogesis, and therefore indicates that procainamide may be useful in the prevention of cancer.
  • DNMT1 DNA methyltransferase 1
  • WO2008/147283 discloses the use of zebularine for the manufacturing of a medicament for the treatment of an auto-immune disorder or disease or immune rejection of transplants or gene therapeutically modified cells, wherein the treatment induces I DO.
  • WO2012/087234 discloses a composition comprising at least two compounds each of which induce indolamine 2,3-dioxygenase (IDO), for the treatment of an autoimmune disorder or disease or suffering from immune rejection of organs, tissues, normal cells or gene therapeutically modified cells, wherein said IDO inducers have different mechanisms of action and give rise to a synergistic effect on the IDO level. It also discloses a method of inducing IDO in a cell culture comprising the steps of (a) providing isolated cells in a suitable medium, (b) adding such a composition which comprises at least two compounds each of which induce IDO, (c) incubating said isolated cells with the composition and (d) obtaining a cell culture in which IDO is induced.
  • IDO indolamine 2,3-dioxygenase
  • It also discloses a method of treating a mammal having an autoimmune disorder or disease or suffering from immune rejection of organs, tissues, normal cells or gene therapeutically modified cells, wherein the treatment induces IDO, comprising firstly a treatment ex vivo of cells derived from the treated mammal or from another mammal, with a therapeutically effective amount of such a composition in the presence of one or more antigens associated with a condition being treated, followed by the transfer of treated cells to the mammal being treated.
  • procainamide is a potent inducer of I DO in cells in which IDO is capable of being induced, for example THP-1 cells. This discovery makes possible important new treatment opportunities involving procainamide in circumstances where IDO induction would be beneficial, particularly immunotherapy.
  • the invention provides procainamide or a pharmaceutically acceptable salt thereof, for use in the prophylaxis or treatment of an immune related disease, disorder or condition.
  • the present invention further provides use of procainamide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of an immune related disease, disorder or condition.
  • the present invention also provides a method of treating an immune related disease, disorder or condition comprising administering to a subject in need thereof a therapeutically effective amount of procainamide or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method of inducing IDO expression in a culture of antigen presenting cells obtained or derived from a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • antigen presenting cells or other cells which are capable of being matured or differentiated into antigen presenting cells, said antigen presenting cells being capable of IDO expression, from blood or bone marrow of the subject;
  • aspects of the invention involve culturing of CD4+ T cells isolated from the subject.
  • the present invention further provides such a method comprising the steps of:
  • antigen presenting cells or other cells which are capable of being matured or differentiated into antigen presenting cells, said antigen presenting cells being capable of IDO expression, and CD4+ T-cells which are capable of being differentiated into Treg cells, from the blood or bone marrow of the subject;
  • the present invention further provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • antigen presenting cells or other cells which are capable of being matured or differentiated into antigen presenting cells, said antigen presenting cells being capable of IDO expression, from blood or bone marrow of the subject;
  • the present invention yet further provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • antigen presenting cells or other cells which are capable of being matured or differentiated into antigen presenting cells, said antigen presenting cells being capable of IDO expression, and CD4+ T-cells which are capable of being differentiated into Treg cells, from the blood or bone marrow of the subject;
  • Figure 1 Induction of ID01 expression in THP-1 cells by procainamide.
  • FIG. 1 Induction of ID01 expression in THP-1 cells by decitabine.
  • Figure 3 Induction of ID01 expression in THP-1 cells by azacytidine.
  • indolamine dioxygenase or "IDO” as used herein means ID01 (indoleamine 2,3- dioxygenase, EC 1 .13.1 1.52) or ID02 (indoleamine-pyrrole 2,3 dioxygenase-like 1 , EC 1 .13.1 1 .-) these being two different proteins that can catabolize tryptophan, and can be expressed by APCs.
  • Procainamide means 4-amino-/V-(2-diethylaminoethyl) benzamide i.e. the compound of formula (I) below:
  • Decitabine also known as deoxyazacytidine, means 4-Amino-1 -(2-deoxy-3-D- ribofuranosyl)-1 ,3,5-triazin-2(1 H)-one i.e. the compound of formula (II) below:
  • Azacytidine means 4-Amino-1-(3-D-ribofuranosyl)-1 ,3,5-triazin-2(1 H)-one i.e. the compound of formula (III) below:
  • Immunorelated disease, disorder or condition means any disease, disorder or condition in which a pathological immune response is observed.
  • Immunotolerant means the lack of response to antigens (self- or foreign-antigens) and includes natural tolerance or induced tolerance (i.e. deliberate manipulation of the immune system).
  • Self-antigen means any molecule or chemical group of an organism which acts as an antigen in inducing a T effector lymphocyte response or antibody formation in another organism but to which the healthy immune system of the parent organism is tolerant. Under certain circumstances, for example, when a subject is suffering from or is susceptible to an autoimmune disease, the parent organism is not tolerant to the self-antigen and a specific adaptive immune response is mounted against self-antigens.
  • Exogenous therapeutic agent means any therapeutic agent for treatment of a subject that originates from outside the subject.
  • co-culturing means culturing two (or more) cell types in the presence of each other.
  • procainamide may be employed in the form of a pharmaceutically acceptable salt.
  • other substances to be used in combination with procainamide in different embodiments as referred to herein may also be employed in the form of a pharmaceutically acceptable salt.
  • Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art.
  • Pharmaceutically acceptable salts include those described by Berge et al., 1977.
  • Such pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid and organic acids e.g.
  • the salt is hydrochloric acid salt of procainamide, hereinafter referred to as "procainamide HCI”.
  • procainamide HCI hydrochloric acid salt of procainamide
  • Procainamide or a pharmaceutically acceptable salt thereof, may be used as a medicament, in particular for the prophylaxis or treatment of an immune related disease, disorder or condition.
  • the immune related disease, disorder or condition is selected from the group consisting of autoimmune diseases and disorders; immune rejection of transplants; and immune reactions to an exogenous therapeutic agent.
  • the immune related disease, disorder or condition is an autoimmune disease or disorder.
  • the autoimmune diseases or disorders are selected from the group consisting of Achlorhydria, Acute haemorrhagic leukoencephalitis, Addison's Disease, Alopecia Areata, Anemia, Ankylosing Spondylitis, Anti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome, Aplastic Anemia, Atopic Allergy, Autoimmune Atrophic Gastritis, Autoimmune Hearing Loss, Autoimmune hemolytic anemia, Autoimmune Hepatitis, Autoimmune hypoparathyroidism, Autoimmune hypophysitis, Autoimmune Lymphoproliferative, Autoimmune Myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal-Dystrophy, Autoimmune Polyendocrinopathy, Autoimmune Syndrome Type II, Behcet Syndrome, Celiac Disease, Chagas
  • the autoimmune disease is selected from the group consisting of Diabetes Mellitus Type 1 , Rheumatoid Arthritis, Chronic lymphocytic thyroiditis, Multiple Sclerosis and Ulcerative Colitis.
  • the immune related disease, disorder or condition are arteriosclerosis, Parkinson's disease, and Alzheimer's disease.
  • the immune related disease, disorder or condition is immune rejection of transplants.
  • the transplant is an organ, tissue or cells (including normal cells or genetically modified cells).
  • Organ transplants include transplant of kidney, liver, heart, lung, pancreas, intestines and thymus (or part of said organ).
  • the transplant is an organ, such as kidney, liver, heart or lung.
  • Tissue transplants include transplant of bones, tendons (collectively, musculoskeletal grafts), cornea, skin, tendon, heart valves, nerves and veins.
  • the tissue is nerve tissue, for example fetal ventral mesencephalic tissue.
  • the transplant is an allograft. Alternatively it might be a xenograft.
  • Cell transplants include cells derived from the subject's own stem cells differentiated in vitro to express molecules such as insulin or cells derived from the subject's own cells which have been genetically manipulated to express molecules that the subject is unable to express e.g. due to error of metabolism or genetic defect.
  • the transplanted cells may be pancreatic insulin-producing beta-cells.
  • the cell is a nerve cell, for example a neuron.
  • the immune related disease, disorder or condition is an immune reaction to an exogenous therapeutic agent.
  • the exogenous therapeutic agent may be any drug capable of generating an immune response for example any biological drug e.g. a protein drug.
  • the immune reaction typically includes the raising of antibodies thereto.
  • Example protein drugs include blood factors (including FVIII or Factor IX), hormones (including insulin and EPO), growth factors (including EGF, IGF, KGF, HGF and FGF), cytokines (e.g. interleukins), enzymes and the like.
  • the drug is FVIII.
  • the drug is Factor IX.
  • Biological drugs may contain polysaccharide components.
  • biological drugs include engineered proteins (such as fusion and chimeric proteins) and recombinant proteins.
  • the drug may be an antibody.
  • the drug may, for example, be a monoclonal antibody, such as a humanized or fully human monoclonal antibody.
  • the drug may also be a protein construct comprising fragments of immunoglobulin.
  • the term antibody includes antibody-drug conjugates and antibody-nanoparticle conjugates as well as PEGylated analogues.
  • the antibody may be a domain antibody including a single light chain antibody or VHH.
  • the drug may consist of an intact light chain immunoglobulin, or a fragment thereof which comprises at least a variable domain and at least part of the light chain constant region or a ScFv.
  • Antibodies include anti-TNF-a monoclonal antibodies, for example, REMICADETM (infliximab), ENBRELTM (etanercept), HUMIRATM (adalimumab), CIMZIA ® (certolizumab pegol), SIMPONI ® (golimumab; CNTO 148) and anti-VEGF antibodies.
  • Methods of the invention are suitable for the treatment of subjects who develop an immune reaction to a drug including the raising of anti-drug antibodies in a number of drug treatment situations, such as bleeding disorders (haemophilia A and B; respectively deficiency of FVIII and Factor IX), growth factor deficiency (deficiency of EGF, IGF, KGF, HGF, FGF etc), hormone deficiency (deficiency of insulin, EPO), enzyme replacement therapy and inflammatory and auto-immune disorders (anti-TNF-a monoclonal antibodies).
  • bleeding disorders haemophilia A and B; respectively deficiency of FVIII and Factor IX
  • growth factor deficiency deficiency of EGF, IGF, KGF, HGF, FGF etc
  • hormone deficiency deficiency of insulin, EPO
  • enzyme replacement therapy enzyme replacement therapy and inflammatory and auto-immune disorders (anti-TNF-a monoclonal antibodies).
  • procainamide or a pharmaceutically acceptable salt is administered as the sole active ingredient.
  • procainamide may be administered in combination with one or more additional active ingredients.
  • the one or more additional active ingredients are selected from metabolites of tryptophan; immunosuppressive reagents; aldehyde oxidase inhibitors; methotrexate; rapamycin; cyclophosphamide; antimetabolites; immunophilin-binding drugs; inhibitors of nucleotide synthesis; FTY720; lymphocyte depleting antibodies; non-depleting antibodies; anti-TNF antibodies; natalizumab; anti-CD154 antibodies; soluble cytokine receptors; soluble TNF receptors; and anakinra.
  • the additional active ingredient is rapamycin. Rapamycin may be expected to have a role in expanding the population of Treg cells in vivo.
  • procainamide is administered with one or more other additional active ingredients which also induce IDO.
  • the one or more additional active ingredients are selected from the group consisting of cytidine analogues; histone deacetylase inhibitors; vitamin D3 analogues; interferons; tolllike receptor ligands; gonadotropine receptor signalling hormones; prostaglandine E2 analogues; IDO stabilizers; soluble CTLA4 conjugates; and glycocorticoids. More suitably, the one or more additional active ingredients is a cytidine analogue, for example, zebularine.
  • the one or more additional active ingredients which also induce IDO are selected from the group consisting of psammaplin A, decitabine, guadecitabine (also known as SGI-1 10) and azacytidine and pharmaceutically acceptable salts thereof.
  • an additional active ingredient which also induces IDO is azacytidine or a pharmaceutically acceptable salt thereof.
  • procainamide is administered in combination with azacytidine or a pharmaceutically acceptable salt thereof.
  • an additional active ingredient which also induces IDO is decitabine or a pharmaceutically acceptable salt thereof.
  • procainamide is administered in combination with decitabine or a pharmaceutically acceptable salt thereof.
  • one or more additional active ingredients are other than psammaplin A, decitabine, guadecitabine or azacytidine, or pharmaceutically acceptable salts thereof.
  • the one or more additional active ingredients induce IDO.
  • the additional active ingredient is an interferon, for example interferon gamma or interferon alpha.
  • the additional active ingredient is a cytidine analogue, for example zebularine.
  • procainamide may be administered with two additional active ingredients such as a cytidine analogue (for example zebularine) and an interferon such as interferon gamma or interferon alpha.
  • Procainamide may be administered to the subject in a pharmaceutical composition. Therefore in one embodiment of the invention, there is provided a pharmaceutical composition comprising procainamide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition comprises procainamide or a pharmaceutically acceptable salt thereof, one or more additional active ingredients and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition comprises procainamide or a pharmaceutically acceptable salt thereof, one or more additional active ingredients which also induces IDO and a pharmaceutically acceptable excipient, diluent or carrier.
  • the one or more additional active ingredients in the pharmaceutical composition which also induce IDO are selected from the group consisting of psammaplin A, decitabine, guadecitabine (also known as SGI-1 10) and azacytidine and pharmaceutically acceptable salts thereof.
  • an additional active ingredient in the pharmaceutical composition which also induces IDO is azacytidine or a pharmaceutically acceptable salt thereof.
  • procainamide is administered in combination with azacytidine or a pharmaceutically acceptable salt thereof in a pharmaceutical composition.
  • an additional active ingredient in the pharmaceutical composition which also induces IDO is decitabine or a pharmaceutically acceptable salt thereof.
  • procainamide is administered in combination with decitabine or a pharmaceutically acceptable salt thereof in a pharmaceutical composition.
  • one or more additional active ingredients are other than psammaplin A, decitabine, guadecitabine or azacytidine, or pharmaceutically acceptable salts thereof.
  • the one or more additional active ingredients induce IDO.
  • the additional active ingredient in the pharmaceutical composition is an interferon, for example interferon gamma or interferon alpha.
  • the additional active ingredient in the pharmaceutical composition is a cytidine analogue, for example zebularine.
  • a cytidine analogue for example zebularine
  • an interferon such as interferon gamma or interferon alpha.
  • the diluent or carrier may be an aqueous or non-aqueous solution with the purpose of diluting or carrying the medicament.
  • the diluent or carrier may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils.
  • the excipient may be one or more of carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, sucrose, mannitol, and cyclodextrines, which are added to the pharmaceutical composition, e.g., for facilitating lyophilisation.
  • polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the pharmaceutical composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the pharmaceutical composition for reasons similar to those for polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the pharmaceutical composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
  • a pharmaceutical composition comprising procainamide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier for use in the prophylaxis or treatment of an immune related disease, disorder or condition.
  • the immune related disease, disorder or condition is as defined above.
  • the pharmaceutical composition for use in the prophylaxis or treatment of an immune related disease, disorder or condition comprises procainamide or a pharmaceutically acceptable salt thereof and one or more additional active ingredients.
  • the one or more additional active ingredients induces IDO.
  • Example active ingredients which induce IDO are defined above.
  • a pharmaceutical composition comprising procainamide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prophylaxis or treatment of an immune related disease, disorder or condition.
  • the pharmaceutical composition may further comprise one or more additional active ingredients.
  • the one or more additional active ingredients induces IDO.
  • Example active ingredients which induce IDO are defined above.
  • a method of treating an immune related disease, disorder or condition comprising administering to a subject in need thereof a therapeutically effective amount of procainamide or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition may further comprise one or more additional active ingredients.
  • the one or more additional active ingredients induces IDO.
  • Example active ingredients which induce IDO are defined above.
  • Procainamide or a pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising procainamide or a pharmaceutically acceptable salt thereof may be administered in vivo via any suitable route including oral, sublingual, buccal, nasal, by inhalation, parenteral i.e.
  • injection including cardiac, intravenous, intra-articular, intraorgan, intramuscular, intraperitoneal, intradermal, lymphatic and subcutaneous
  • retrograde perfusion through cerebral venous system, via catheter into the brain parenchyma or ventricles, direct exposure or under pressure onto or through the brain or spinal tissue, or any of the cerebrospinal fluid ventricles, injections into the subarachnoid, brain cisternal, subdural or epidural spaces, via brain cisterns or lumbar puncture, intra and peri-ocular instillation including application by injection around the eye (within the eyeball and its structures and layers), the ear (including the Eustachian tube, mastoid air cells, external and internal auditory canals, tympanic membrane, middle ear, inner ear including the cochlear spiral ganglion and labyrinthine organs), as well as via enteral, bowel, rectal, vaginal, urethral or bladder cisternal.
  • the preferred route may vary depending on the condition of the patient.
  • the route of administration is parenteral e.g. by injection.
  • procainamide or a pharmaceutically acceptable salt thereof may be administrated at dose levels that will achieve concentrations in vivo, at the sites or locations of action, such that the concentration in vivo is for example 0.1 - 100 mM such as 0.1 - 50 mM e.g. 0.1 -10 mM or other lower or higher levels that are effective, depending on which disease or disorder is to be treated.
  • azacytidine or pharmaceutically acceptable salts thereof may be administrated at dose levels that will achieve concentrations in vivo, at the sites or locations of action, such that the concentration in vivo is for example 0.1 -100 mM such as 0.1 -50 mM e.g. 0.1 -10 mM or other lower or higher levels that are effective, depending on which disease or disorder is to be treated.
  • decitabine or pharmaceutically acceptable salts thereof When used as an additional active ingredient to induce IDO, decitabine or pharmaceutically acceptable salts thereof, may be administrated at dose levels that will achieve concentrations in vivo, at the sites or locations of action, such that the concentration in vivo is for example 0.1 -100 microM such as 0.1 -50 microM or 0.1 -30 microM e.g. 0.1 -10 microM or other lower or higher levels that are effective, depending on which disease or disorder is to be treated.
  • Procainamide or a pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising procainamide or a pharmaceutically acceptable salt thereof, may be employed in an ex vivo method of induction of IDO expression.
  • procainamide or a pharmaceutically acceptable salt thereof is employed as the sole active ingredient in an ex vivo method of induction of IDO expression.
  • the present invention also provides a method of inducing IDO expression in a culture of antigen presenting cells obtained or derived from a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • Suitable antigen presenting cells are disclosed elsewhere herein and include especially dendritic cells. Antigen presenting cells are most suitably derived from blood of the subject.
  • the present invention also provides a method of inducing IDO expression in a culture of antigen presenting cells obtained or derived from a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of: (a) isolating cells which are capable of being matured or differentiated into antigen presenting cells, said antigen presenting cells being capable of IDO expression, from blood or bone marrow of the subject;
  • Cells which are capable of being matured or differentiated into antigen presenting cells include especially CD14+ monocytes and CD34+ hematopoietic stem cells. Such cells are most suitably derived from blood (e.g. peripheral blood) of the subject.
  • the present invention provides a method of inducing IDO expression in a culture of antigen presenting cells obtained or derived from a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • the present invention provides a method of inducing IDO expression in a culture of antigen presenting cells obtained or derived from a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • antigen presenting cells such as dendritic cells and T cells can interact with each other and a key interaction involves DCs expressing IDO and producing immunosuppressive cytokines (e.g. IL-10 and TGF- ⁇ ) which induces the T cells to adopt a regulatory phenotype.
  • immunosuppressive cytokines e.g. IL-10 and TGF- ⁇
  • IDO especially ID01 induced in DCs by procainamide, further enhances the TGF-beta and the IDO expression of the dendritic cells.
  • the regulatory T cells in their turn can also induce immature DCs to differentiate to tolerogenic rather than immunogenic DCs thereby creating a tolerogenic loop response.
  • the co-culturing of antigen presenting cells or other cells which are capable of being matured or differentiated into antigen presenting cells together with CD4+ T-cells in the culture is expected to lead to beneficial expansion of the tolerogenic cell population in the cell culture.
  • the procainamide may have a direct effect on the T-cells by inducing differentiation to Treg cells via demethylation of FoxP3 and CTLA4 thus leading to activation of these two genes for Treg regulating function.
  • particular benefits are expected to arise from the presence in the culture of antigen presenting cells, CD4+ T-cells and procainamide.
  • the present invention provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • the present invention provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • the present invention provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • the present invention provides a method of inducing tolerance in a subject who is suffering from or susceptible to an immune related disease, disorder or condition comprising the steps of:
  • step (c) either the antigen presenting cells in which IDO expression has been induced or the Treg cells are transferred back to the subject.
  • step (c) both the antigen presenting cells in which IDO expression has been induced and the Treg cells are transferred back to the subject.
  • Antigen presenting cells APCs
  • APCs suitable for use in the present invention are APCs which are capable of expressing I DO, especially ID01.
  • APCs are cells capable of inducing a tolerogenic loop, for example by generating antigen specific regulatory T cells or B cells.
  • the APCs are dendritic cells (DCs), such as bone marrow-derived dendritic cells (BMDCs).
  • DCs dendritic cells
  • BMDCs bone marrow-derived dendritic cells
  • cells which are capable of being matured or differentiated into antigen presenting cells such as dendritic cells.
  • Such cells include stem cells such as CD34+ hematopoietic stem cells or CD14+ monocytes.
  • Antigen presenting cells and cells which are capable of being matured or differentiated into antigen presenting cells such as CD34+ hematopoietic stem cells and CD14+ monocytes are suitably derived from peripheral blood.
  • DCs that are capable of proliferating are preferred.
  • the hematopoietic stem cells are readily expandable, but it also appears possible to induce DCs derived from peripheral blood CD14+ monocytes-to proliferate by appropriate treatment (see e.g. Langstein et al., 1999).
  • Dendritic cells may be mature dendritic cells or immature dendritic cells.
  • the dendritic cells may be dendritic cells that have been co-cultured with mesenchymal stem cells.
  • one or more antigens may be (and suitably are) included in the culture.
  • An antigen is any substance that causes the immune system to react e.g. by generating T-cells recognizing peptides derived from protein substances, and B-cells producing antibodies against the substance.
  • the antigen will bear one or more epitopes.
  • the one or more antigens comprise self-antigens e.g. antigens derived from collagen, cartilage or other tissues of the subject.
  • the one or more antigens comprise antigens derived from the transplant.
  • the antigen preparation corresponding to the exogenous therapeutic agent may contain the exogenous therapeutic agent, for example a drug, in whole or part, said part comprising an epitope containing fragment thereof.
  • a purified antigen will be used.
  • the antigen preparation corresponding to the exogenous therapeutic agent, for example a drug resembles as closely as possible the exogenous therapeutic agent, for example a drug, which is being administered to the subject.
  • various recombinant drugs are available which are suitable for this purpose including the commercial products ReFacto AF, Helixate NexGen, Kogenate Bayer, Advate, NovoEight, Nuwiq, Beriate, Beriate P, Haemoctin, Hemofil, Monoclate - P, Octanate [LV], Optivate and Recombinate.
  • various recombinant drugs are available which are suitable for this purpose including the commercial products Immunine, Mononine, Alprolix, Rixubis and Benefix.
  • the immune related disease, disorder or condition may be any one described above.
  • the subject is suffering from or susceptible to an autoimmune disease or disorder (as described above) and the one or more antigens comprise self-antigens e.g. antigens derived from collagen, cartilage or other tissues of the subject.
  • the subject is suffering from or susceptible to rheumatoid arthritis and the one or more antigens comprise antigens derived from collagen, cartilage or other tissues of the subject.
  • the subject is suffering from or susceptible to immune rejection of a transplant and the one or more antigens comprise antigens derived from the transplant.
  • the transplant is transplant of an organ, tissue or cells (including normal cells or genetically modified cells).
  • the organ, tissue or cells are as described above.
  • the subject is suffering from or susceptible to immune reaction to an exogenous therapeutic agent and the one or more antigens comprise antigens from the exogenous therapeutic agent.
  • the exogenous therapeutic agent is as described above and thus may be a biological drug such as FVIII, Factor IX or an antibody and the immune reaction comprises the raising of antibodies thereto.
  • one or more additional active ingredients are employed in step (b) of the aforementioned ex vivo methods in addition to procainamide.
  • Such active ingredients may be selected from metabolites of tryptophan; immunosuppressive reagents; aldehyde oxidase inhibitors; methotrexate; rapamycin; cyclophosphamide; antimetabolites; immunophilin-binding drugs; inhibitors of nucleotide synthesis; FTY720; lymphocyte depleting antibodies; non-depleting antibodies; anti-TNF antibodies; natalizumab; anti-CD154 antibodies; soluble cytokine receptors; soluble TNF receptors; and anakinra.
  • the one or more additional active ingredient includes rapamycin.
  • one or more additional active ingredients which induce IDO are employed in step (b) and are selected from compounds such as cytidine analogues; histone deacetylase inhibitors; vitamin D3 analogues; interferons; toll-like receptor ligands; gonadotropine receptor signalling hormones; prostaglandine E2 analogues; IDO stabilizers; soluble CTLA4 conjugates; and glycocorticoids. More suitably, the one or more additional active ingredients is a cytidine analogue, for example, zebularine.
  • the one or more additional active ingredients which induce IDO and are employed in step (b) are selected from the group consisting of psammaplin A, decitabine, guadecitabine (also known as SGI-1 10) and azacytidine and pharmaceutically acceptable salts thereof.
  • an additional active ingredient which induces IDO is azacytidine or a pharmaceutically acceptable salt thereof.
  • azacytidine or a pharmaceutically acceptable salt thereof is employed in step (b) of the aforementioned ex vivo methods in addition to procainamide.
  • an additional active ingredient which induces IDO is decitabine or a pharmaceutically acceptable salt thereof.
  • decitabine or a pharmaceutically acceptable salt thereof is employed in step (b) of the aforementioned ex vivo methods in addition to procainamide.
  • one or more additional active ingredients are employed in step (b) and are other than psammaplin A, decitabine, guadecitabine or azacytidine, or pharmaceutically acceptable salts thereof.
  • the one or more additional active ingredients induce IDO.
  • the additional active ingredient is an interferon, for example interferon gamma or interferon alpha.
  • the additional active ingredient is a cytidine analogue, for example zebularine.
  • procainamide may be administered with two additional active ingredients such as a cytidine analogue (for example zebularine) and an interferon such as interferon gamma or interferon alpha.
  • a cytidine analogue for example zebularine
  • an interferon such as interferon gamma or interferon alpha.
  • step (b) of the above ex vivo methods further comprises stimulating the cultured antigen presenting cells with oligonucleotides.
  • Cell cultures may be prepared in which IDO is induced ex vivo.
  • antigen presenting cells in which IDO expression has been induced obtained or obtainable by the any of the methods of the present invention.
  • the cell culture in which IDO expression has been induced comprises:
  • the cell culture in which IDO expression has been induced further comprises Treg cells obtained by differentiation in the cell culture of CD4+ T-cells isolated from blood or bone marrow of the subject.
  • the cell culture in which IDO expression has been induced further comprises one or more antigens associated with the immune related disease, disorder or condition.
  • the immune related disease, disorder or condition is selected from the group consisting of autoimmune diseases and disorders; immune rejection of transplants; and immune reactions to an exogenous therapeutic agent and are as defined above.
  • antigen presenting cells obtained or obtainable according to methods of the invention or isolated from a cell culture according to the invention for use in a method of treating a subject suffering from or susceptible to an immune related disease, disorder or condition whereby according to any of the methods of the present invention said cells are transferred back to said subject thereby to establish immune tolerance to the immune related disease, disorder or condition.
  • Treg cells isolated from a cell culture according to the invention for use in a method of treating a subject suffering from or susceptible to an immune related disease, disorder or condition whereby according to said method said cells are transferred back to said subject thereby to establish immune tolerance to the immune related disease, disorder or condition.
  • antigen presenting cells obtained or obtainable according to methods of the invention or isolated from a cell culture according to the invention in combination with Treg cells isolated from a cell culture according to the invention for use in a method of treating a subject suffering from or susceptible to an immune related disease, disorder or condition whereby according to said method said cells in combination are transferred back to said subject thereby to establish immune tolerance to the immune related disease, disorder or condition.
  • APCs such as DCs are first obtained from a subject for example a mammal, e.g. a human.
  • a subject for example a mammal, e.g. a human.
  • One suitable method is to collect immature PBMCs by apheresis (optionally after mobilization of immature cells with standard doses of recombinant G-CSF).
  • Another suitable method is to use Nycodenz centrifugation to separate low density monocytes after lyzing of the red blood cells.
  • immature CD34 + hematopoietic stem cells may be purified from PBMC using magnetic beads linked to anti-CD34 MAb (Dynal Biotech, Oslo, Norway). CD34 + hematopoietic stem cell purity can be confirmed by flow cytometry.
  • Purified CD34 + hematopoietic stem cells are typically differentiated into DCs by treatment with GM-CSF, SCF and IL-4. Thus, typically they are cultured at 1 x10 5 cells/mL in tissue culture flasks (Nunc, Roskilde, Denmark) in SCGM serum-free medium, CellGro serum-free medium or RPMI medium containing 10% autologous plasma, 2 mM I- glutamine (JHR), 20-100 ng/ml GM-CSF, 20-100 ng/mL SCF, 20-100 ng/mL, IL-4 (R&D Systems, Minneapolis, MN, USA), optionally together with 100 ng/mL IL3, 5-20 ng/mL IL10 and 50 ng/ml Flt-3L (R&D Systems), 10 ng/mL TNF-alpha and/or 10 ng/mL IL1 -beta.
  • Cells may also be cultured with foetal bovine serum (FBS) or human serum AB. Cultures may be maintained at 37°C in humidified 5% CO2 atmosphere for 14 days. As cellular expansion occurs, cell culture medium and cytokines are replenished (e.g. approximately every 4 days).
  • FBS foetal bovine serum
  • cytokines are replenished (e.g. approximately every 4 days).
  • DC cell differentiation can be induced and cells expanded as described above for CD34 + hematopoietic stem cells.
  • CD14+ monocytes are differentiated into DCs using a similar method to that described above.
  • CD14 cells are purified using a similar method as described above, using anti-CD14 Mab and cultured in CellGro serum free or SCGM serum free for 2-5 days in GM-CSF and IL4. The cell culture is matured over two to three days with TNF-alpha, IL1 -beta, PGE2 and/or IL-10.
  • immature DCs may be exposed to procainamide, together with an antigen preparation e.g. corresponding to the tissue for transplant, self-antigen or exogenous therapeutic agent.
  • concentration of procainamide and the length of exposure can be selected for optimum IDO induction.
  • a concentration of 0.1 mM-100 mM such as 0.1 -50 mM e.g. 0.1-10 mM is exemplary.
  • a concentration of 0.1 -100 mM such as 0.1 -50 mM e.g. 0.1 -10 mM is exemplary.
  • a concentration of 0.1 -100 microM such as 0.1 -50 microM or 0.1 -30 microM e.g. 0.1 -10 microM is exemplary.
  • An exposure of 2-4 days for each compound is exemplary.
  • Procainamide or a pharmaceutically acceptable salt thereof and the antigen preparation can be added to the DCs together or separately.
  • Antigens and procainamide or a pharmaceutically acceptable salt thereof may be added at the same time as maturation of immature cells such as CD34 + hematopoietic stem cells and CD14 + monocytes into DCs.
  • cells are typically treated for approximately 24 h with prostaglandin E2 (at concentration 1-10 ⁇ ) to induce required chemotactic receptors.
  • DCs may be harvested, suspended in medium and stored frozen in liquid nitrogen until being released for use.
  • Quality control should include tests for viability, sterility and endotoxin and expression of ID01 . Additionally, expressions of PD-L1 , PD-L2, the chemotactic receptor CCR7, level of expression of the co-stimulators CD80/CD86, CD40, and MHC-II are to be considered. Expression levels of these substances is an indicator that the DCs are tolerogenic.
  • CD4+CD25- T-cells are isolated by magnetic separation and co-cultured with APCs, or cells being capable of being differentiated into APCs particularly DCs, in CellGro serum free or SCGM serum free containing tryptophan restricted to 1 -3 uM for 3-10 days in the presence of procainamide and IL-2 (2000 U/ml) and anti-CD3 beads to allow expansion of the generated CD4+ Treg cells.
  • Cells may be administered back to the mammal by various routes e.g. intravenously, subcutaneously or intracutaneously.
  • the medium containing the cells is suitably containing human albumin as a cell-protecting protein.
  • an amount of 3-10 x 10 6 APCs/dose and/or 3-30 x 10 6 Treg cells/dose in 1-10 doses at weekly to bi-weekly intervals is administered. The treatment can be extended until the desired tolerance is achieved.
  • the subject may receive treatment with one or more other pharmaceutically active agents concurrently with the treatment with cells.
  • the subject may receive treatment with rapamycin.
  • rapamycin may be expected to have a role in expanding the population of Treg cells in vivo
  • Transplants may be treated with procainamide or a pharmaceutically acceptable salt thereof prior to implantation of the transplant to a subject in need thereof, such that IDO is induced in the transplant prior to implantation thereby establishing immune tolerance in said subject post- implantation of the transplant.
  • a method of inducing IDO in a transplant comprising the steps of:
  • transplant for example, organ, tissue or cells
  • the effective amount will, for example, be a therapeutically effective amount.
  • the present invention further provides a method of inducing immune tolerance to a transplant in a subject who is to receive said transplant comprising the steps of:
  • transplant for example, organ, tissue or cells
  • the transplant is an organ, tissue, or cells (including normal cells or genetically modified cells). Organ, tissue or cells may be any one of those described above.
  • the transplant is cells, in particular, cells derived from the patients' own stem cells differentiated in vitro to express molecules such as insulin or cells derived from the patients' own cells which have been genetically manipulated to express key molecules, wherein a specific immune response may be observed against the cell transplant.
  • the specific immune response may be against antigens selective for beta-cells or insulin, proinsulin or preproinsulin.
  • the transplanted cells may be pancreatic insulin-producing beta-cells. Patients who receive cells which have been genetically manipulated to express key molecules may be unable to express the key molecules, for example, due to errors of metabolism.
  • the effective amount of procainamide or pharmaceutically acceptable salt will depend on the transplant.
  • the effective amount of procainamide required to induce I DO may be between 0.1 - 100 mM such as 0.1 -50 mM e.g. 0.1 -10 mM.
  • the effective amount of azacytidine required to induce IDO may be between 0.1 -100 mM such as 0.1 -50 mM e.g. 0.1 -10 mM.
  • the effective amount of decitabine required to induce IDO may be between 0.1 -100 microM such as 0.1 -50 microM or 0.1 -30 microM e.g. 0.1 -10 microM.
  • procainamide is employed in step (b) as the sole active ingredient.
  • one or more additional active ingredients are employed in step (b) and are selected from metabolites of tryptophan; immunosuppressive reagents; aldehyde oxidase inhibitors; methotrexate; rapamycin; cyclophosphamide; antimetabolites;
  • immunophilin-binding drugs inhibitors of nucleotide synthesis; FTY720; lymphocyte depleting antibodies; non-depleting antibodies; anti-TNF antibodies; natalizumab; anti- CD154 antibodies; soluble cytokine receptors; soluble TNF receptors; and anakinra.
  • one or more additional active ingredients which induce IDO are employed in step (b) and are selected from compounds such as cytidine analogues (e.g. zebularine or a pharmaceutically acceptable salt thereof); histone deacetylase inhibitors; vitamin D3 analogues; interferons (e.g. interferon gamma or interferon alpha or a
  • toll-like receptor ligands gonadotropine receptor signalling hormones
  • prostaglandine E2 analogues IDO stabilizers
  • soluble CTLA4 conjugates IDO stabilizers
  • glycocorticoids glycocorticoids
  • one or more additional active ingredients which induce IDO are employed in step (b) and are selected from the group consisting of psammaplin A, decitabine, guadecitabine and azacytidine and pharmaceutically acceptable salts thereof.
  • the additional active ingredient which induces IDO is azacytidine or a pharmaceutically acceptable salt thereof.
  • the additional active ingredient which induces IDO is decitabine or a pharmaceutically acceptable salt thereof.
  • one or more additional active ingredients are employed in step (b) and are other than psammaplin A, decitabine, guadecitabine or azacytidine, or pharmaceutically acceptable salts thereof.
  • procainamide may be employed in step (b) with two additional active ingredients such as a cytidine analogue (for example zebularine) and an interferon such as interferon gamma or interferon alpha.
  • a cytidine analogue for example zebularine
  • an interferon such as interferon gamma or interferon alpha.
  • Procainamide is a specific inhibitor of DNA methyltransferase 1 ; Lee et al. J. Biol.
  • CD137 induces proliferation and endomitosis in monocytes; Langstein et al. J. Blood, 1999, 94(9), 3161 -3168.
  • TGF-b TGF-beta
  • THP-1 cells were harvested from appropriate number of T75 flasks. Cells were counted in a Bijrker chamber, stained with 0.4% Trypan Blue. The appropriate number of cells for each experiment were transferred to a new tube and centrifuged at 1200 rpm, 5 min at room temperature (RT). The supernatant was discarded and fresh RPMI with 5% FBS was added to get a cell concentration of 2x10 5 cells/mL. The compound was added to the five separate wells in 6-well plates such that five different final concentrations were obtained after adding the cells. A control was added to one well in the 6-well plate to which cells were added. 1 x10 6 cells per well were seeded out to achieve a total volume of 5 mL per well. The cells were incubated at 37 °C in 5% CO2 for 96 hours. After 96 hours the assay was stopped and the cells were lysed for the isolation of RNA.
  • RNA extraction and quantification of gene expression by RT-qPCR Total RNA was extracted from THP-1 cells using the RNA RNeasy Mini extraction kit (Qiagen), according to the manufacturer's instructions. A Superscript® VILOTM RT-kit (Thermo Fisher) was used to reverse transcribe 1 ⁇ g of total RNA to cDNA and qPCR was performed.
  • the primers used for amplification of the human ID01 gene were 5 ' - TTGTTCTCATTTCGTGATGG-3 ' (forward; SEQ ID No. 1 ) and 5 ' - TACTTTGATTGCAGAAGCAG-3 ' (reverse; SEQ ID No. 2).
  • the primers used for amplification of the endogenous reference gene for human HPRT1 were 5 ' - CTAATTATG G ACAG GACTG AAC-3 ' (forward; SEQ ID No. 3) and 5 ' - AG C AAAG AATTTATAG C C C C-3 ' (reverse; SEQ ID No. 4).
  • RT-qPCR was performed in 20 ⁇ reaction, consisting 10 ⁇ PowerUp SYBR Green Master Mix, 0.5 ⁇ of each primer, 4 ⁇ diluted cDNAfor amplification of ID01 and 1 .5 ⁇ diluted cDNAfor amplification of HPRT1 .
  • An iCycler iQ real-time detection system (Stratagene, Mx3000P, La Jolla, CA, USA) with the following thermal profile: UDG incubation at 50 °C for 2 min, then denaturation at 95 °C for 5 min, followed by 45 cycles at 94 °C for 15s, 58 °C for 30 s, and 60 °C for 30 s, was used to perform thermocycling and real-time detection of PCR products. After amplification a melting curve analysis was performed. The RT-qPCR experiments were always run in triplicate. ID01 was normalised to the geometric means of two HPRT1 reference gene, using the ACt method. Within-group comparisons were normalized to one control sample, using the AACt method.
  • procainamide may be used to induce IDO in cells such as antigen presenting cells in vivo or ex vivo.
  • decitabine may be used to induce I DO in cells such as antigen presenting cells in vivo or ex vivo.
  • azacytidine may be used to induce IDO in cells such as antigen presenting cells in vivo or ex vivo.

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Abstract

La présente invention concerne un procaïnamide (I): (I) destiné à être utilisé dans le traitement ou la prophylaxie d'une maladie, d'un trouble ou d'une affection lié à l'immunité, et des procédés associés.
PCT/EP2017/071404 2016-08-26 2017-08-25 Procaïnamide pour le traitement de troubles immunitaires et pour la modulation de l'expression de l'ido WO2018037103A1 (fr)

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WO2020035555A3 (fr) * 2018-08-15 2020-04-23 Pharmacyl Ab Benzamides substitués et leur utilisation en thérapie
CN113811363A (zh) * 2019-03-29 2021-12-17 汉诺威医学院 用于HvG病的治疗的CAR

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
WO2020035554A1 (fr) * 2018-08-15 2020-02-20 Pharmacyl Ab Nouveau traitement médical contre l'inflammation pathologique
WO2020035555A3 (fr) * 2018-08-15 2020-04-23 Pharmacyl Ab Benzamides substitués et leur utilisation en thérapie
CN112703036A (zh) * 2018-08-15 2021-04-23 法玛西尔有限公司 取代的苯甲酰胺及其在疗法中的用途
CN112770736A (zh) * 2018-08-15 2021-05-07 法玛西尔有限公司 病理性炎症的新医学治疗
JP2022501315A (ja) * 2018-08-15 2022-01-06 ファルマシル・アクチボラグ 病的炎症の新しい医療
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CN113811363A (zh) * 2019-03-29 2021-12-17 汉诺威医学院 用于HvG病的治疗的CAR

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