JP5415724B2 - A kind of oligonucleotide and its application - Google Patents

Description

  The present invention relates to a kind of oligonucleotide and an application for treating immune-mediated diseases using the oligonucleotide. Immune-mediated diseases include autoimmune diseases, transplant rejection, hypersensitivity reactions, diseases caused by self-antigens and microorganisms over-stimulating the host immune system, and diseases mediated by Toll-like receptors (TLR).

  The present invention provides oligonucleotides having a 5′-cctcctcctcctcctcctcctcct-3 ′ (SEQ ID NO: 1) nucleotide sequence and applications for treating immune-mediated diseases using such oligonucleotides.

  The immune system can protect the body from bacterial, parasite, fungal, viral infections and tumor cell growth. However, some immune responses are sometimes undesired and can cause immune-mediated diseases. Immune-mediated diseases include autoimmune diseases, transplant rejection, hypersensitivity reactions, diseases caused by microorganisms over-stimulating the host immune system, and diseases mediated by Toll-like receptors (TLRs).

  Autoimmune diseases are caused by acquired and innate immune responses, or by endogenous and / or exogenous antigens. Foreign substances derived from bacteria, parasites, fungi, and viruses are similar to self-proteins and stimulate the individual's immune system to cause an immune response against self-cells and tissues, causing autoimmune diseases. Autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE) and rheumatoid arthritis. Transplant rejection is the result of transplantation of an organ or tissue caused by an immune response to the transplant organ or tissue of the recipient of the transplant (host). When a subject is transplanted with a transplant, such as a kidney, pancreas, heart, lung, bone marrow, cornea, and skin, the subject can cause an immune response (rejection) to the transplant. Hypersensitivity is an inappropriate immune response that has deleterious effects, leading to serious tissue damage and even death. Hypersensitivity reactions are divided into four types, for example, type I, II, III and IV. Diseases associated with over-response of the host immune system by microorganisms are caused by infection of viruses such as influenza viruses and other microorganisms. When infected with an influenza virus or Gram-negative bacterium, an over-immune response to an intruder can be a fatal factor for the patient. The response is characterized by cytokine overproduction. Studies of septic shock syndrome indicate that cytokine overproduction / abnormal production leads to rapid death due to cytokine-mediated lethal shock (Slifka MK, et al. J Mol Med 2000; 78 (2): 74-80). Septic shock following gram-negative infection is a major cause of rapid death in critically ill people. Overproduction of cytokines is known to contribute to sepsis characterized by cytokine-mediated lethal shock (Espat NJ, et al, J Surg Res. 1995 Jul; 59 (1): 153- 8). Multiple organ dysfunction syndromes (MODS) are the leading cause of morbidity and mortality in severe sepsis and shock. Cytokine-mediated lethal shock as a result of overproduction of host cytokines is thought to be the main mechanism leading to MODS (Wang H, et al. Am J Emerg Med 2008 Jul; 26 (6): 711-5). Toll-like receptor (TLR) -mediated disease is a disease caused by activation of Toll-like receptors (TLRs). TLRs are a group of receptors that recognize microbial-derived molecular structures (pathogen-associated molecular patterns, PAMPs). Immune cells that express TLRs are activated by binding to PAMPs. TLRs recognize and activate products from many pathogens. Bacterial lipopolysaccharide (LPS) is synthesized into TLR4, lipochochoic acid and diacyl lipopeptide into TLR2-TLR6 dimer, triacyl lipopeptide into TLR2-TLR1 dimer, CpG-containing oligonucleotide derived from bacteria or viruses (CpG ODN) to TLR9, bacterial flagellar fibers to TLR5, zymosan to TLR2-TLR6 dimer, F protein from respiratory multinuclear virus (RSV) to TLR4, virus-derived double-stranded RNA (dsRNA) and poly I: C, synthetic analog dsRNA is recognized by TLR3, viral DNA is recognized by TLR9, and single-stranded viral RNA (VSV and influenza virus) is recognized by TLR7 and TLR8 (Foo Y. Liew, et al. Nature Reviews Immunology. Vol.5, June 2005, 446-458). In recent years, there are TLR activations including sepsis, dilated cardiomyopathy, diabetes, experimental autoimmune encephalomyelitis, systemic lupus erythematosus, atherosclerosis, asthma, chronic obstructive pulmonary disease, organ failure It has been linked to the occurrence of species diseases (Foo Y Liew, et al. Nature Review Immunology, Vol 5, 2005 446-458). Activation to TLR9 by self-DNA is performed in SLE (Christense SR, et al. Immunity 2006; 25: 417-28) and rheumatoid arthritis (Leadbetter EA, et al. Nature 2002; 416: 603-7; Boule MW, et al. J Exp Med 2004; 199: 1631-40) and is said to play an important role in the development of autoimmune diseases. In addition, production of high levels of interferons (IFNs) as a result of TLR activation has been reported to contribute to the development of systemic lupus erythematosus (Barrat FI, et al. J Exp Med 2005; 202 : 1131-9; Wellmann U. et al. Proc Natl Acad Sci USA 2005; 102: 9258-63).

  In the present invention, an oligonucleotide having a nucleic acid sequence of 5′-cctcctcctcctcctcctcctcct-3 ′ is disclosed. This suppresses the proliferation of human peripheral blood mononuclear cells (PBMC) activated by TLR9 agonists, and interferons from hPBMC induced by sera of TLR9 agonists, HSV-1, influenza virus and SLE patients Suppresses production and rescues mice from cytokine-induced shock. Accordingly, such oligonucleotides are useful as therapeutic applications for immune-mediated diseases.

  The oligonucleotide of the present invention suppresses the activation of TLR9. There is a report that a TLR9 agonist activates innate immune response and adaptive immune response (Arthur M Krieg Nature Reviews Drug Discovery, Vol 5 June 2006, 471-484) . CpG-containing oligonucleotides (CpG ODN) are TLR9 agonists (D M. Klinman, Nat Rev, Immunol 4 (2004) 249-258). The oligonucleotide of the present invention suppresses the proliferation of hPBMC and the production of interferon stimulated by CpG ODN. This indicates that the oligonucleotide can be used in the treatment of diseases related to TLR9 activation. Activation of TLR9 is SLE (Barrat FJ, et al. Exp Med 2005; 202: 1131-9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102: 9258-63; Christensen SR, et al. Immunity 2006 25: 417-28) and the development of rheumatoid arthritis (Leadbetter EA, et al. Nature 2002; 416: 603-7; Boule MW, et al. J Exp Med 2004; 199: 1631-40) Therefore, the oligonucleotide of the present invention can be used as a therapeutic application for SLE and rheumatoid arthritis by suppressing the activation of TLR9.

  The oligonucleotides of the present invention inhibit interferon production from hPBMC induced by sera from TLR9 agonists, HSV-1, influenza virus and SLE patients (Barrat FJ, et al. J Exp Med 2005; 202: 1131- 9; Wellmann U. et al. Proc Natl Acad Sci USA 2005; 102: 9258-63), the oligonucleotide of the present invention can be used as a therapeutic application of SLE by suppressing the production of IFN.

  The oligonucleotide of the present invention suppresses the production of interferon from hPBMC induced by influenza virus. Since there is a report that influenza virus can activate TLR-7 and TLR-8 (Wang JP, et al. Blood 2008 Jun 10 [Epub ahead of print]), the oligonucleotide of the present invention is By suppressing TLR-7 and TLR-8, it can be used as a therapeutic application for Toll-like receptor (TLR) -mediated diseases.

  The oligonucleotide of the present invention suppresses the production of interferon from hPBMC induced by HSV-1. Since there is a report that HSV-1 can activate TLR9 (Hubertus Hochrein et al. PNAS, 101, 11416-11421), the oligonucleotide of the present invention can suppress TLR9 activation. It can be used as a therapeutic application for Toll-like receptor (TLR) mediated diseases, including but not limited to SLE.

  In order to examine the in vivo activity of the oligonucleotides of the present invention, a mouse model of cytokine-mediated lethal shock was used. This mouse model was created by injecting CpG ODN into mice pre-sensitized with D-galactosamine (D-Gal). After being made, the model mice died within 12-24 hours. Analysis of plasma cytokines revealed overproduction of tumor necrosis factor (TNF), interleukin-12 (IL-12) and gamma interferon (Marshall AJ, et al. Infect Immun 1998 Apr; 66 (4): 1325-33; Peter M, Bode K, et al. Immunology 2008 Jan: 123 (1): 118-28). Using this model, we have demonstrated that the oligonucleotides of the invention can save mice from cytokine-mediated lethal shock. Cytokine-mediated lethal shock is septic shock (Slifka MK, et al. J Mol Med 2000; 78 (2): 74-80; Espat NJ, et al. J Surg Res 1995 Jul; 59 (1): 153-8 ) And multiple organ dysfunction syndromes (MODS) (Wang H, et al. Am J Emerg Med 2008 Jul; 26 (6): 711-5). Nucleotides can be used as therapeutic applications for sepsis and MODS by saving the host from cytokine-mediated lethal shock.

(Detailed description of the invention)
Unless otherwise noted, all terms in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include multiple references unless the context indicates otherwise. Similarly, the term “or” includes “and” unless the context indicates otherwise. If a conflict occurs, it is controlled in the specification of the present application (including explanation of terms). In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. “Treat”, “treating” or “treatment” have the same meaning regardless of grammar. Similarly, “prevent”, “preventing” or “prevention” have the same meaning regardless of grammar.

  “Oligonucleotide”: An “oligonucleotide” is a molecule composed of a polynucleotide, ie, a sugar (eg, dioxyribose), a phosphite group, and an exchangeable organic base that is substituted. Pyrimidine (Py) (eg, cytosine (C), thymine (T)) or substituted purine (Pu) (eg, adenine (A) or guanine (G)). As used herein, the term “oligonucleotide” refers to oligodioxyribonucleotide (ODN). Oligonucleotides can be obtained from existing nucleic acid sources (eg, genomic or cDNA), but synthetic are preferred. The oligonucleotide of the present invention can be synthesized by various commercially available automatic nucleic acid synthesizers. These oligonucleotides refer to “synthetic oligonucleotides”.

  “Chemical modification”: The oligonucleotides disclosed in the present invention can be subjected to various chemical modifications compared to natural DNA. For example, phosphodiester internucleoside bridges between nucleosides, ribose units and / or natural nucleoside bases (adenine, guanine, cytosine, thymine). The modification can be performed either during or after oligonucleotide synthesis. During synthesis it takes place inside or at the end of the base to be modified and after synthesis it is an active group (via an amino modifier, via a 3 'or 5' hydroxyl group or via a phosphate group). Can be used. Those skilled in the art are aware of various chemical modifications. The oligonucleotides of the invention may be subjected to one or more chemical modifications. Compared to oligonucleotides of the same sequence that constitute natural DNA, the chemical modification of the oligonucleotide of the present invention is located at a local internucleoside phosphodiester bridge and / or at a local ribose unit and / or at a local nucleoside base Can do. Chemical modifications include backbone modifications of the oligonucleotides of the invention. Here, the modified backbone of the oligonucleotide of the present invention includes, but is not limited to, a phosphorothioate backbone. A phosphorothioate backbone refers to the stable sugar phosphate backbone of a nucleic acid molecule. In it, the oxygen of the non-bridged phosphate in the bond between at least one nucleotide is replaced with sulfur. In one example, the oxygen of the non-bridged phosphate in the linkage between each or all nucleotides is replaced with sulfur. Other backbone modifications include non-ionic DNA analogs such as alkyl phosphonates, allyl phosphonates (where the charged phosphonate oxygen is substituted with alkyl, allyl groups), phosphodiesters and Refers to modification with an alkyl phosphate triester, where the charged oxygen moiety is alkylated. In other examples, the oligonucleotide can be a phosphorothioate / phosphodiester chimera. Chemical modifications also include base substitutions of the oligonucleotides disclosed in the present invention. The substituted purines and pyrimidines can be C-5 propyne pyrimidine and 7-deaza-7 substituted purines. Substituted purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymine, and other naturally occurring and non-naturally occurring nucleobases. The chemical modification of the oligonucleotide of the present invention further includes base modification of the oligonucleotide. Modified bases are those that are chemically different from naturally occurring bases typically found in DNA (e.g., A, T, C, G), but are fundamental to these naturally occurring bases. Shares a common chemical structure. The oligonucleotide of the present invention can be modified using a cytidine derivative. The term “cytidine derivative” refers to a cytidine-like nucleoside (excluding cytidine). The term “thymidine nucleoside derivative” refers to a thymidine-like nucleotide (excluding thymidine). Furthermore, the oligonucleotide of the present invention can be chemically modified by linking a diol such as tetraethylene glycol or hexaethylene glycol to one or both ends of the oligonucleotide.

  “Immune-mediated disease”: An “immune-mediated disease” is a disease caused by an immune response that the subject does not want. Such diseases include autoimmune diseases, transplant rejection, hypersensitivity reactions, diseases associated with over-stimulation of the host's immune system by microorganisms and diseases associated with TLR activation. The oligonucleotides disclosed in the present invention can be used as therapeutic applications for immune-mediated diseases.

  “Immune response”: An “immune response” is a response to stimulation of cells of the immune system, such as B cells, T cells, NK cells, dendritic cells, neutrophils and macrophages. The response includes an intrinsic immune response and an acquired (specific) immune response. Acquired (specific) immune responses include humoral and cellular immune responses.

  “Prevent or treat immune-mediated diseases”: “Prevention” as used herein refers to preventing complete development of immune-mediated diseases in the subject, and “treatment” refers to therapeutic intervention in the subject. Refers to alleviating signs or symptoms of immune-mediated diseases, halting progression or eliminating pathological conditions.

  “Subject”: As used herein, “subject” refers to human and non-human vertebrates. Non-human vertebrates are non-human primates, livestock and pets. The oligonucleotide of the present invention can be administered for prevention or treatment of immune-mediated diseases in the subject.

“Autoimmune disease”: The term “autoimmune disease” refers to a disease caused by the disruption of self-resistance. It responds to acquired and innate immune systems against self-antigens and leads to cell and tissue damage. Autoimmune diseases are often characterized in the single organ, or single cell type, or multiple organs or tissues in which they are involved. Autoimmune disease also refers to “collagen”, “collagen-vascular”, or “connective tissue” disease. Autoimmune diseases are often associated with hypersensitivity reactions. The oligonucleotides of the invention are useful for the treatment and prevention of various types of autoimmune diseases. In particular, non-limiting examples of autoimmune diseases include systemic lupus erythematosus, insulin-dependent (type I) diabetes, inflammatory arthritis, rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, chronic active hepatitis, Autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune atrophic gastritis, autoimmune pernicious anemia, autoimmune encephalomyelitis, autoimmune testicularitis, acquired hemophilia, ankylosing spondylitis , Chronic inflammatory demyelinating polyneuritis, scarring pemphigus, antiphospholipid antibody syndrome, cardiomyopathy, cold agglutinin disease, discoid lupus erythematosus, sympathetic ophthalmitis, idiopathic mixed cryoglobulinemia , IgA nephropathy, juvenile arthritis, systemic sclerosis, polyarteritis nodosa, polychondritis, dermatomyositis, primary acquired agammaglobulinemia, primary biliary cirrhosis, hyperimmunoglobulin E disease, Progressive systemic sclerosis, psoriasis, lighter Syndrome, nodule, stiff man syndrome, uveitis, vasculitis, vulgaris vulgaris, Hashimoto's disease, Goopastute disease, pernicious anemia, Addison's disease, Sjogren's syndrome, myasthenia gravis, Grave's disease, allergic encephalomyelitis, thread Sphere nephritis (N Engl J Med, Vol 345, No. 5, August 2, p340-350) is included. DNA or RNA released from DNA or RNA containing microorganisms stimulates the production of specific autoantibodies against self DNA or RNA containing complexes and thus autoimmune diseases such as SLE (limited to this). Not be).

  “Hypersensitivity reaction”: “Hypersensitivity reaction” refers to a disease caused as a result of a humoral or cellular (immune) response to an exogenous antigen, which is divided into four types. Type I hypersensitivity reactions (often referred to as hypersensitivity, rapid response, atopic, and specific IgE-mediated hypersensitivity reactions or allergies) are usually exogenous antigens that are specific to IgE-sensitized basophils and mast cells. Due to pharmacologically active ingredients released after contact with histamine, such as histamine, delayed-reactive substance (SRS-A) of hypersensitivity reaction and eosinophil chemotactic factor (ECF). Type I hypersensitivity reactions include, but are not limited to, allergic exogenous asthma, seasonal allergic rhinitis and systemic anaphylaxis. Type II hypersensitivity reactions (also called cytotoxic, cytolytic, complement-dependent or cell-stimulated hypersensitivity reactions) are those in which the antibody recognizes its own cell or tissue antigen or is coupled to the cell or tissue. Or it is caused by recognizing a hapten. Type II hypersensitivity reactions include, but are not limited to, autoimmune hemolytic anemia, granulocytopenia, fetal erythroblastosis, and Goodpasture's syndrome. Type III hypersensitivity reactions (also called toxic complexes, soluble complexes, or immune complex type hypersensitivity reactions) are caused by precipitation in the blood vessels or tissues of circulating soluble antigen-antibody complexes, at the immune complex precipitation site. Accompanying a severe inflammatory reaction. Type III hypersensitivity reactions include, but are not limited to, Arthurs reaction, serum sickness, systemic lupus erythematosus and glomerulonephritis. Type IV hypersensitivity reactions (often referred to as cellular, cell-mediated, delayed or tuberculin type hypersensitivity reactions) are caused by sensitized T cells, which result from contact with specific antigens. Type IV hypersensitivity reactions include, but are not limited to, contact dermatitis and allograft rejection (Richard A Goldsby Thomas J Kindt Barbara A Osborne Janis Kuby Immunology, Fifth Edition, 2003, WHFREEMAN AND COMPANY).

  “Diseases associated with over-stimulation of the host's immune system by microorganisms”: When the invasion of microorganisms becomes severe, a systemic inflammatory reaction may occur mainly at one time, which is associated with over-stimulation of the host's immune system by microorganisms. Can cause illness. When the disease progresses, for example, when infected with influenza virus A (H5NI) or bacteria, fairly high levels of the following substances are found in the blood: TNF-α, IL-1, IL-6, IL- 12, IFN-α, IFN-β, IFN-γ, chemokine interferon-inducing protein 10, monocyte migration factor 1, IL-8, IL-1-β. Such a response can cause cytokine-mediated lethal shock in people with sepsis, acute respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A / H5. Avian Influenza A (H5NI) Infection in Humans. N Engl J Med 2005; 353: 1374-85). The fairly high levels of cytokines in the blood following a microbial infection are called cytokineemia or cytokine storms. The study advises that patients in contact with avian influenza (virus) or SARS (virus) need drugs that suppress immune responses in addition to requiring antiviral drugs. The oligonucleotides of the invention can be used to treat and / or prevent diseases associated with microbial over-stimulation of the host immune system. Microorganisms causing such diseases include, but are not limited to, pathogenic pathogens of viruses, bacteria, fungi, parasites and sponge encephalopathy. Viruses that cause diseases associated with over-stimulation of the host's immune system by microorganisms include: SARS virus, influenza virus, avian influenza virus, HTV-1, poliovirus, hepatitis A virus, enterovirus, human Cocksackie virus, rhinovirus, echovirus, equine encephalitis virus, rubella virus, dengue virus, encephalitis virus, yellow fever virus, coronavirus, vesicular stomatitis virus, rabies virus, ebola virus, parainfluenza virus, epidemic parotid gland Flame virus, measles virus, respiratory multinuclear virus, nephrogenic hemorrhagic fever virus, bunga virus, flavovirus, nairo virus, hemorrhagic fever virus, reovirus, orbivirus, rotavirus, Hepatitis B virus, parvovirus, papilloma virus, polyoma virus, adenovirus, herpes simplex virus (HSV) -1, HSV-2, varicella-zoster virus, cytomegalovirus, herpes zoster virus, variola virus, vaccinia virus , Poxvirus, African swine fever virus, pathogenic agent of sponge encephalopathy, hepatitis D virus, hepatitis C virus, foot-and-mouth disease virus. Bacteria causing disease associated with over-stimulation of the host's immune system by microorganisms include: Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium species (eg , M. tuberculosis, M. avium, M. E intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Group A Streptococcus, Group B Streptococcus, linkage Streptococcus, Streptococcus, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, Pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Anthrax, Diphtheria, Corynebacterium species, Swine erysipelas, Clostridium perfringens, tetanus, Enterobacter aerogenes, Neisseria pneumoniae, Pasteurella multocida, Bacteroid Genus, Fusobacterium nutaleum, Streptobacillus moniliformis, syphilis treponema, flambedia treponema, leptospira and Israel actinomycetes. Fungi that cause diseases associated with over-stimulation of the host's immune system by microorganisms include: Cryptococcus neoformans, Histoplasma capsulatsum, Coccidioides imimitis, Blastmyces, Chlamydia trachomatis, Candida・ Albicans. Parasites that cause diseases associated with over-stimulation of the host's immune system by microorganisms include the malignant malaria parasite, Toxoplasma.

  “Transplant rejection”: “Transplant rejection” is an immune-mediated disease caused by transplantation of an organ or tissue. Transplantation means transfer of the transplant from the donor to the recipient. A transplant is a living cell, tissue or organ that is transferred from a donor to a recipient. An autograft is a transplant from one site of one's own tissue to another site. Syngeneic genotype transplantation (isograft) is a transplant between identical twins. A homograft is a transplant performed between allogeneic genetically distinct ones. A xenograft is a transplant between different species. If the subject is an allograft or xenograft recipient, the organism can produce an immune response against the donor tissue. Under this circumstance, there is a clear need to suppress the immune response to avoid transplant rejection (Richard A. et al. Immunology, Fifth Edition, 2003 WH FREEMAN AND COMPANY). The oligonucleotides of the present invention are useful in preventing transplant rejection. Examples of the transplant include heart, kidney, liver, bone marrow, skin, cornea, lung, pancreas, small intestine, limb, muscle, nerve, duodenum, and Langerhans islet cells. In some cases, the recipient may be an animal defined as the subject of the present invention.

  “Toll-like receptor (TLR) -mediated disease” refers to immune-mediated activation of TLR family members. It refers to sex diseases. The disease is: sepsis associated with activation of TLR4 by lipopolysaccharide (LPS), dilated cardiomyopathy associated with activation of TLR2, 3, 4, 9 and activation of TLR2, 3, 4, 9 Diabetes mellitus, experimental autoimmune encephalomyelitis (EAE) associated with TLR3 activation, systemic lupus erythematosus associated with TLR9 activation, atherosclerosis associated with TLR4 activation, LPS Including, but not limited to, asthma associated with TLR4 activation, chronic obstructive pulmonary disease associated with TLR4 activation, EAE associated with TLR4 activation, and organ weakness associated with TLR4 activation (Foo Y. et al. Nature Review Immunology, Vol 5, 2005, 446-458). A CpG-containing DNA (TLR9 agonist) derived from a nucleic acid-containing infectious agent has been identified from SLE serum that induces a sufficient immune response governed by the secretory action of IFN-α that appears to contribute to the development of SE. The oligonucleotides of the invention can be administered to treat or prevent TOLL-like receptor (TLR) mediated diseases, including but not limited to SLE in the subject.

“CpG ODN”: There are reports that TLR9 agonists activate both intrinsic and acquired immune responses (Arthur M. Krieg. Nature Reviews Drug Discovery, Vol. 5 June 2006, 471-484). CpG-containing oligonucleotides (CpG ODN) are TLR9 agonists (DM Klinman, 428 Nat Rev, Immunol 4 (2004) 249-258). CpG ODNs are classified into three types according to functional characteristics (Tomoki Ito, et al. Blood, 15 March 2006, Vol 107, Num 6: 2423-2431). Type A CpG ODN activates human plasmacytoid dendritic cells (pDCs) to produce large amounts of type I interferon (IFN-α / β) and vigorously activate NK cells. B-type CpG ODN mainly activates B cells and stimulates B cell proliferation and antibody secretion. C-type CpG ODN has the functions of both A-type and B-type CpG ODNs. As a TLR9 agonist, CpG2216, CpG2006 or CpG C274 is taken up into the cell gap where it is exposed to and activates TLR9. In pDC, activation of TLR9 triggers a rapid intrinsic immune response. It is characterized by secretion of proinflammatory cytokines (IL-6, tumor necrosis factor-α (TNFα)), secretion of type I interferon (IFN-α) and secretion of IFN-induced chemokines. Innate immune cells, including NK cells, monocytes, and neutrophils are secondarily activated by pDC through two IFN-dependent and IFN-independent pathways. B cells activated via TLR9 have greatly increased sensitivity to antigen stimulation and are efficiently differentiated into antibody secreting cells. Thus, stimulation of TLR9 contributes to an acquired immune response, particularly a humoral immune response. PDC activated via TLR9 secretes IFNα, which causes it to aggregate into lymph nodes and other surrounding lymphoid tissues. At these sites, pDC activates unsensitized T cells, assists in cross-presentation of soluble antigen to CD8 ⁺ cytotoxic T lymphocytes (CTLs), and promotes THI-type CD4 and CD8 T cell responses To do. Based on the above findings, formulations that antagonize the activity of CpG ODN can be used to prevent and treat immune-mediated diseases by suppressing intrinsic and acquired immunity.

  “Pharmaceutically acceptable carrier”: “Pharmaceutically acceptable carrier” refers to one or more solid or liquid fillers, diluents or embedding substances, which carriers are defined in the present invention. It is suitable for application to the main body of the oligonucleotide. The carrier may be organic, inorganic, natural or synthetic. The carrier is a solution, diluent, solvent, dispersant, liposome, emulsion, coating, antibacterial and antifungal agent, tonicity agent and absorption delaying agent, and other carriers suitable for application of the oligonucleotide of the present invention. Their use is well known in the art. The selection of a pharmaceutically acceptable carrier is made according to the method of use related to the application of the oligonucleotide of the present invention. Parenteral preparations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, dextrose solutions, glycerin and the like as carriers. . For solid compositions (e.g. in the form of powders, pills, tablets, capsules), conventional non-toxic solid carriers such as mannitol, lactose, starch and magnesium stearate with pharmaceutical purity can be included. In addition to the biologically neutral carrier, the pharmaceutical composition to be applied can contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents, For example, sodium acetate or monolaurate can be included.

  “Therapeutically effective amount”: In order to treat or prevent an immune-mediated disease, a therapeutically effective amount of the oligonucleotide of the present invention is mainly administered. A “therapeutically effective amount” of an oligonucleotide refers to a sufficient amount in the main body of the oligonucleotide used to treat or prevent an immune-mediated disease to obtain a desired result. The oligonucleotides of the invention may be used in pure form or in a pharmaceutically acceptable carrier. Alternatively, the oligonucleotide may be applied as a pharmaceutical composition. The “amount” in the present invention is referred to as “dose”. This “dose” can be determined by standard techniques well known to those skilled in the art, and depends on factors including but not limited to the size of the subject and / or the overall health status or severity of the disease. change. Introduction of the oligonucleotides of the invention can be done as a single treatment or as a series of treatments. Each dose range of the oligonucleotide of the present invention is 1 μg to 100 μg. However, the dose for treatment of immune mediated diseases may be 10 to 1,000 times the above dose. More preferred dosages can be prepared by those skilled in the art to provide the best therapeutic effect, for example within the appropriate medical judgment of the attending physician.

  “Route of administration”: When used in medicine, the oligonucleotides of the invention can be administered alone or formulated into a pharmaceutical composition and administered by any suitable route effective to achieve the desired therapeutic result. . The route of administration of the oligonucleotides of the invention can be enteral, injection (parenteral) and topical administration or inhalation. Enteral routes of administration of the oligonucleotides of the invention include oral, stomach, intestinal and rectal. Injection routes include intravenous, peritoneal, intramuscular, intrathecal, subcutaneous, local injection, vaginal, topical, nasal mucosa and pulmonary administration. The topical route of administration of the oligonucleotide of the present invention means the administration of the oligonucleotide from the outside into the epidermis, oral cavity, and into the ear, eye and nose.

  “Pharmaceutical composition”: “Pharmaceutical composition” refers to a composition comprising a therapeutically effective amount of an oligonucleotide of the invention, whether or not it contains a pharmaceutically acceptable carrier. It is. The pharmaceutical composition can comprise one or more oligonucleotides of the invention. Pharmaceutical compositions include aqueous solutions, saline solutions, particles, aerosols, pellets, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops and various Other pharmaceutical compositions suitable for use in drug delivery systems are included. The pharmaceutical composition may be injected (parenteral), oral, rectally, vaginally, intraperitoneally, locally (dosage form: powder, ointment, gel, drop, transdermal patch), buccal or buccal or transdermal. It may be administered by nasal spray. In all cases, the pharmaceutical composition must be sterile and stable under the conditions of manufacture and storage and protected from microbial contamination. The injectable pharmaceutical composition of the present invention comprises a pharmaceutically acceptable sterile aqueous or non-aqueous solution, dispersion, suspending agent or emulsifier, and similarly, with a sterile injection solution or dispersion before injection. Contains powder to be reconstituted. The oligonucleotide of the present invention is an aqueous carrier, for example, an isotonic buffer solution having a pH of 3.0 to 8.0, preferably a pH range of pH 3.5 to pH 7.4, pH 3.5 to pH 6.0, pH 3.5. It can be suspended in an isotonic buffer at pH 5.0. Buffers include sodium citrate-citric acid, sodium phosphate-phosphoric acid, sodium acetate-acetic acid. Orally administered pharmaceutical compositions are prepared with edible carriers to form powder tablets, pills, dragees, capsules, liquids, gels, syrups, slurries or suspensions. Conventional non-toxic solid carriers for solid compositions can include pharmaceutical grades of mannitol, lactose, starch and magnesium stearate. The pharmaceutical composition for buccal administration may be a traditional form of a tablet or tablet. The pharmaceutical composition for inhalation is an aerosol spray, and those skilled in the art can select and use from pressure packs, nebulizers or dry powders. In some cases, in order to prolong the effect of the oligonucleotide of the present invention, the oligonucleotide of the present invention is also appropriately administered in a sustained release system. The oligonucleotides of the present invention may be used in liquid suspensions of crystalline or amorphous materials that are poorly soluble in water. Delayed release of the parenterally administered drug form of the oligonucleotide is accomplished by dissolving or suspending the oligonucleotide in a hydrophobic material (eg, an acceptable oily carrier). Injectable depot forms include embedding oligonucleotides in liposomes, microemulsions, or biodegradable semipermeable polymer matrices such as polylactide-polyglycolides, polyorthoesters, and polyanhydrides. It is made with.

  “Active ingredient”: The oligonucleotides of the invention can be used alone or in combination, utilizing a pharmaceutically acceptable carrier, in combination with one or more additional active ingredients. Administration of the oligonucleotides and their additional active ingredients of the present invention can be sequential or simultaneous. Active ingredients include non-steroidal anti-inflammatory agents, steroids, non-specific immunosuppressants, biological response modifiers, compounds, small molecules, nucleic acid molecules, TLR antagonists. Active ingredients refer to agents that suppress immune activation in the following manner: antagonize chemotactic factors; induce production of regulatory T cells (CD4 + CD25 + T cells) Complement, inhibit matrix metalloproteases and NO synthases; block costimulatory molecules; inhibit signal transduction of immune cells; Non-steroidal anti-inflammatory agents include, but are not limited to, diclofenac, diflunisal, indoleacetic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, piroxicam, sulindac, tohnetin, celecoxib, rofecoxib. Steroids include, but are not limited to, adrenocorticotropic hormone, dexamethasone, cortisol, methylprednisolone, dexamethasone, prednisone and triamcinolone. A non-specific immunosuppressive agent means an agent used to suppress the development of immune-mediated diseases. Non-specific immunosuppressive agents include, but are not limited to, cyclophosphamide, cyclosporine, methotrexate, steroids, FK506, tacrolimus, mycophenolic acid, sirolimus. Biological response modifiers include recombinant human interleukin-1 receptor antagonist (Kineret or anakima), soluble p75 TNF-α receptor-IgG1 fusion protein (etanercept or Enbrel), or anti-TNF-α monoclonal antibody (infliximab or RemicadeX), including but not limited to, interferon β-Ia, interleukin-10 and TGFβ.

  “Delivery carrier”: The oligonucleotides of the invention can be administered in / with a delivery carrier or in a form bound to a single entity. Such carriers include sterols (eg, cholesterol), complex compounds, emulsions, ISCOMs; lipids (eg, cationic lipids and anionic lipids), liposomes; (poly) ethylene glycol (PEG); living bacterial vectors ( For example, Salmonella, E. coli, Bacillus Calmette-Gurin, Shigella, lactic acid bacteria, live virus vectors (eg, vaccinia, adenovirus, herpes simplex virus), virus particles, virus-like particles, micro Includes, but is not limited to, spheres, nucleic acid vaccines, polymers (eg, carboxymethylcellulose, chitosan), polymer rings and targeting agents (which recognize target cells through specific receptors).

  “Polyethylene glycolation”: “Polyethylene glycolation” is the process of covalent attachment of polyethylene glycol to other molecules (generally drugs or therapeutic proteins) of the polymer chain. Polyethylene glycolation is always achieved by incubating a reactive derivative of PEG with the target agent. Polyethylene glycolating agents can mask the agent from the host's immune system and increase the hydrodynamic size of the agent to extend its circulation time. The oligonucleotide of the present invention can be polyethylene glycolated.

Brief introduction of the examples In the first example, the present invention provides an oligonucleotide having a 5'-cctcctcctcctcctcctcctcct-3 '(SEQ ID NO: 1) nucleotide sequence, wherein the oligonucleotide is of the formula (5' Follow CCT3 ') n.

  In the second embodiment, the present invention is an immunization using the oligonucleotide of the present invention. It provides applications for treating sexually transmitted diseases. Immune-mediated diseases include autoimmune diseases, transplant rejection, hypersensitivity reactions, diseases associated with over-stimulation of the host immune system by autoantigens and microorganisms, and Toll-like receptor (TLR) -mediated diseases.

  In a third example, the present invention uses the oligonucleotides of the present invention to suppress TLR activation and IFN production (which is induced by DNA viruses, RNA viruses, sera from SLE patients), It provides applications to rescue the subject from cytokine-mediated lethal shock and treat immune-mediated diseases.

  In a fourth example, the present invention provides an application for treating SLE, sepsis and multiple organ dysfunction syndrome in a subject using the oligonucleotide of the present invention.

  In a fifth embodiment, the present invention administers the oligonucleotide of the present invention to the subject via enteral, parenteral (injection), local administration, and inhalation routes, alone or with a pharmaceutically acceptable carrier. Thus, it provides applications for treating immune mediated diseases.

  In a sixth example, the present invention provides a composition comprising a therapeutically effective amount of an oligonucleotide of the invention for the treatment of immune mediated diseases.

  In other embodiments, the present invention provides applications for treating immune-mediated diseases by administering the oligonucleotides of the present invention alone or in combination with additional active ingredients.

  In other embodiments, the present invention provides applications for treating immune mediated diseases by administering the oligonucleotides of the present invention by a delivery carrier.

  In one aspect, the present invention provides a compound of formula (5′CCT3 ′) n (where 5′CCT3 ′ is a repeating unit, n is an integer of 2 to 50, preferably 5′-cctcctcctcctcctcctcctcct-3 ′ (SEQ ID NO 1) is provided).

  In preferred embodiments, the phosphate backbone of the oligonucleotide may or may not be partially or fully phosphorothioate modified.

  In other preferred embodiments, the oligonucleotides evolve into their derivatives by adding one or more nucleotides to each end of the oligonucleotide and by changing one or more bases of the oligonucleotide. ing.

  In other preferred embodiments, the oligonucleotide is part of another DNA molecule, plasmid or viral vector.

  In a further preferred embodiment, the oligonucleotide is chemically modified.

In another aspect, the present invention provides the use of the above oligonucleotides to prepare applications for treating immune-mediated diseases in a subject.
In preferred embodiments, the immune-mediated disease is an autoimmune disease, hypersensitivity reaction, transplant rejection, a disease associated with over-stimulation of the host immune system by a microorganism, or a Toll-like receptor (TLR) -mediated disease.

In a further preferred embodiment, the treatment of immune-mediated diseases suppresses the proliferation of immune cells activated by a Toll-like receptor 9 agonist, suppresses the activation of Toll-like receptor 9, and produces interferon production. To suppress and rescue the subject from cytokine-mediated lethal shock.
Preferably, immune-mediated diseases are treated by suppressing systemic lupus erythematosus (SLE), which inhibits TLR9 activation and interferon production induced by TLR9 agonists, viruses and sera of SLE patients ), Sepsis (which is treated by rescuing the subject from cytokine-mediated lethal shock), or multi-organ dysfunction syndrome (which is treated by rescuing the subject from cytokine-mediated lethal shock) Is).
In another aspect, the present invention provides an application for administering the oligonucleotide to a subject who is at risk of developing an immune-mediated disease or at risk.

Preferably, the remedy (application therefor) further comprises a pharmaceutically acceptable carrier and / or an additional active substance. More preferably, the remedy (application therefor) is in a form for administration via enteral, parenteral (injection), local administration and inhalation routes.
In a preferred embodiment, the oligonucleotide is polyethylene glycolated.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the inhibitory effect of SAT05f on the proliferation of human peripheral blood mononuclear cells (hPBMC) stimulated by CpG 2006 and CpG C274.
FIG. 2 is a graph showing the inhibitory effect of SAT05f on interferon production from hPBMC stimulated by CpG C274.
FIG. 3 shows the inhibitory effect of SAT05f on interferon production from hPBMC stimulated with type I herpes simplex virus and influenza virus. here,
(A) is a comparison of the antiviral activity of interferon (IFN) induced by inactivated HSV-1 and recombinant human alpha interferon (IFN-α). “IU” represents an international unit.
(B) shows the inhibitory effect of SAT05f on the production of interferon from human PBMC stimulated by inactivated HSV-1.
(C) shows the dose effect of the inhibitory action of SAT05f on interferon production from human PBMC stimulated by HSV-1.
(D) is a comparison of the antiviral activity of inactivated PR8-induced interferon (IFN) and recombinant human alpha interferon (IFN-α).
(E) shows the inhibitory effect of SAT05f on IFN production from hPBMC stimulated with inactivated PR8.
(F) Dose effect on the suppression of SAT05f on IFN production from hPBMC stimulated with inactivated PR8.
The figure represents the results of one test out of three repeatable tests. HSV-1 represents type I herpes simplex virus and PR8 represents influenza virus (HINI / PR8).
FIG. 4 is a graph showing that SAT05f suppresses interferon production from hPBMC stimulated with serum of lupus erythematosus (SLE) patients.
FIG. 5 is a graph showing that SAT05f rescues mice from cytokine-mediated lethal shock.

Examples In the following examples, the invention is described in more detail. However, the present invention is not limited to these examples. In these examples, unless otherwise stated, experiments using commercially available kits and drugs were performed according to the attached protocol. One skilled in the art will appreciate the applicability of the oligonucleotides of the present invention to the treatment of immune-mediated diseases. The invention is illustrated in the following non-limiting examples.

  All drugs used to handle oligonucleotides (ODNs) in the following examples are pyrogen-free. In the preparation of ODN, endotoxin was tested by the Limulus modified cytolysis method (Associates of Cape Cod. Inc). Human peripheral blood mononuclear cells (hPBMC) used in the following examples were obtained from the human blood (The Blood Center of Jilin Province, China) using density gradient centrifugation (PM Daftarian et al. (1996): Journal of Immunology, 157, 12-20). The cells used were IMDM medium containing 10% (v / v) heat-inactivated fetal bovine serum (FBS; GIBCO), antibiotics (100 IU penicillin / ml and 100 IU streptomycin / ml), 5% The cells were cultured at 37 ° C. in a CO 2 incubator. The trypto blue staining method confirmed that the survival rate of hPBMC was 95% to 99%.

Example 1 Effect of SATO5f on human peripheral blood mononuclear cell (hPBMCs) proliferation induced by CpG ODN Oligonucleotides used in this example were synthesized by Shanghai Biotechnology Co., Ltd. (Shanghai, China) These are CpG2006 (5'-tcgtcgttttgtcgttttgtcgtt-3 '), CpG274 (5'-tcgtcgaacgttcgagatgat3'), A151 (5'-ttagggttagggttagggttaggg3 ') (Hidekazu Shirota, et al. The Journal of Immunology, 579: 174: , SATO5f (5'-cctcctcctcctcctcctcctcct-3 ') and control ODN (5'-gttagagattaggca--3').

  CpG2006 (Dominique De Wit, et al. Blood, 2004, Vol 103, Num 3: 1030-103) is a prototype of B-type CpG ODN. CpG274 (Omar Duramad, et al. The Journal of Immunology, 2005, 174: 5193-5200) is a prototype of C-type CpG ODN.

In ^[3 H] thymidine incorporation method it was tested whether to suppress the growth of hPBMCs which SATO5f is stimulated CpG2006 and CpG274. HPBMCs at a density of 5 × 10 ⁵ / well are seeded on 96-well U-bottom microtiter plates (Costar) and CpG2006 (1 μg / ml) in the presence of SATO5f, A151 or control oligonucleotide (control ODN). ) Or CpG274 (1 μg / ml) for a total of 48 hours, then [ ³ H] thymidine (New England Nuclear, Boston, Mass.) Was added, and the cells were continuously cultured for 16 hours. The cells were harvested with a glass fiber filter and the results were examined with a scintillation counter. Cell proliferation in 3 wells is expressed as mean cpm ± SD.

  As shown in FIG. 1, SAT05f suppresses the proliferation of hPBMCs stimulated with CpG ODN 2006 or CpG ODN 274, and its inhibitory effect is stronger than that induced by A151. Control ODN showed no inhibitory effect. Since CpG ODN including CpG2006 or CpG274 is a TLR9 agonist [DMKlinman, Nat Rav, Immunol 4 (2004) 249-258], this data indicates that SAT05 suppresses TLR9 activation and is associated with TLR9 activation. And can be used to treat other TOLL-like receptor (TLR) mediated diseases.

Example 2 Effect of SAT05f on interferon production from hBMC induced by CpG ODN <Test method>
Use IMDM medium containing 10% (v / v) heat-inactivated fetal bovine serum (GIBCO), antibiotics (100 IU penicillin / ml and 100 IU streptomycin / ml) in a 5% CO ₂ incubator, 37 Vero E6 cells (African green monkey kidney cell line, American Type Culture Collection) were cultured at ℃.

To test whether SAT05f baths INF production from hPBMC stimulated with CpG C274, an interferon bioassay was performed using Vero E6 cells and VSV. Oligonucleotides containing CpG C274, A151, SAT05f and control ODN were synthesized by Shanghai Biotechnology Co., Ltd. (Shanghai, China). Their sequences are as shown in Example 1. CpG C274 is a prototype of C-type CpG ODN and has both A-type CpG ODN and B-type CpG ODN activities. Type A CpG ODN can activate human plasmacytoid dendritic cells (pDCs) to produce large amounts of type I interferon. Inoculate hPBMCs (5 × 10 ⁵ / well) on 96-well microtiter plates (Costar) and incubate with CpG C274 (1 μg / ml) for 48 hours in the presence of SATO5f, A151 or control ODN, then collect supernatant Then, the interferon activity in the supernatant was verified. Vero E6 cells (3 × 10 ⁴ / well) were inoculated into a 96-well flat bottom plate and cultured for 24 hours. Next, the cells were cultured with 100 μl of the supernatant for 18 hours, and further 10 × TCID50 (50% tissue culture infectious dose) of vesicular stomatitis virus (VSV) was added and cultured continuously for 48 hours. VSV was proliferating in Vero E6 cells. After culturing, it was stored in aliquots at -70 ° C and awaited use. After staining with crystal violet 0.5%, the cytopathic effect of the virus was measured with a multiwell microtiter plate detector and displayed as the OD value (A578nm).

<Test results>
As shown in FIG. 2, SATO5f suppresses INF production from hPBMCs stimulated with CpG C274. It has been reported that high levels of INFs production as a result of TLR-9 activation promote the development of systemic lupus erythematosus (SLE) (Barrat FJ, et al. J Exp Med 2005; 202: 1131- 9; Wellmann U, et al. Proc Natl Acad Sci USA 2005; 102; 9258-63), so this data is mediated by SLE and other Toll-like receptors (TLR) by suppressing the production of high levels of INF It shows that SAT05f can be used as an application to treat sexually transmitted diseases.

Example 3 Inhibitory effect of SAT05f on interferon production from hPBMCs stimulated with type I herpes simplex virus and influenza virus
<Experiment method>
Vero E6 cells were cultured as in Example 2. Type I herpes simplex virus (HSV-1) and influenza virus (HINI / PR8, PR8) are derived from Department of Immunology, Beilin Medical School, Jilin University. HSV-1 (MO1 = 200) was grown on Vero E6 cells and PR8 (MO1 = 3) was grown on MDCK cells (Madin-Darby Canine Kidney Cells, ATCC). MDCK cells were cultured in IMDM medium containing 10% (v / v) heat-inactivated fetal bovine serum (FBS; GIBCO) and antibiotics (100 IU penicillin / ml and 100 IU streptomycin / ml). HSV-1 in IMDM medium containing 2% (v / v) FBS was inactivated by heating in a 70 ° C. water bath for 10 minutes. PR8 in IMDM medium containing 2% (v / v) FBS was inactivated by heating in a 56 ° C. water bath for 30 minutes.

Oligonucleotides containing SAT05F and CTRLODN (shown as CTRL in FIG. 3) and having a 5′-aaaaataaaaataaaataaaat-3 ′ nucleotide sequence were synthesized by Takara (Dalian, China). The sequence of SAT05f is described in Example 1.
Using 96-well microtiter plates (Costar), in the presence or absence of SAT05f or CTRL, hPBMCs (5 × 10 ⁶ ml) were inactivated HSV-1 or inactivated PR8 In addition, after culturing at 37 ° C. for 48 hours, the supernatant was collected for verification. Vero E6 cells (3 × 10 ⁴ / well) were inoculated into a 96-well flat bottom plate and cultured for 24 hours. The culture was then incubated with 100 μl of the collected supernatant (HSV-1 induced supernatant dilution was 1:20 and PR8 induced supernatant dilution was 1:80) for 16 hours. 10 × TCID50 (50% tissue culture infectious dose) of VSV was added and cultured continuously for 48 hours. After staining with crystal violet 0.5%, the cytopathic effect was measured with a multiwell microtiter plate detector and displayed as the mean OD value ± SD (A578 nm). Single data from 3 replicates are shown.

<Test results>
As shown in Figure 3-A, inactivated HSV-1 virus is able to induce interferon, which causes Vero E6 cells to interact with VSV as did recombinant human alpha interferon (IFN-α). It shows protection from attacks. As shown in FIG. 3-B, SAT05f suppresses interferon production from hPBMCs stimulated by inactivated HSV-1 virus. A dose-response analysis revealed that there was a dose-dependent relationship in suppressing interferon production from hPBMCs stimulated with HSV-1 inactivated SAT05f. SAT05F can mediate such suppression at 1 μg / ml (FIG. 3-C). As shown in Figure 3-D, inactivated PR8 was able to induce interferon, which made Vero E6 cells from VSV attack, as did recombinant human alpha interferon (IFN-α). Indicates that it is protected. As shown in FIG. 3-E, SAT05f suppresses interferon (IFN) production from hPBMCs stimulated by inactivated influenza virus (PR8). From a dose-response analysis, a dose of 2 μg / ml is sufficient for SAT05f to suppress interferon production from inactivated PR8-stimulated hPBMC, and if SAT05f is a dose of 4 μg / ml, It was revealed that the suppression was maximal (Figure 3-F).

  Research shows that influenza virus recognizes and activates TLR7 (Wang JP, et al. Blood 2008 Jun 10 [Epub ahead of print]), and HSV-1 recognizes and activates TLR9 (Hubertus Hochrein et al. PNAS, 101, 11416-11421), and it has been elucidated to stimulate interferon production. Combined with the results of this example, the oligonucleotides of the present invention are useful for the treatment of Toll-like receptor (TLR) mediated diseases such as SLE by inhibiting TLR7 or TLR9 activation and INF production. It is clear that it can be used as an application.

Example 4 Inhibitory effect of SAT05f on interferon production from serum-stimulated hPBMBs in SLE patients
<Experiment method>
The anti-double-stranded DNA-positive serum of patients with SLE is from the Department of Rheumatology, Jilin University Chunichi Clinic.
ONDs containing SAT05F and CTRL-ODN (shown as CTRL in FIG. 4) and having the sequence 5′-aaaaataaaaataaaataaaat-3 ′ were synthesized by Takara (Dalian, China). These are the SAT05f and the control ODN (CTRL). The sequence of SAT05f is described in Example 1.

Using a 96-well microtiter plate (Costar), hPBMCs (5 x 10 ⁶ ml) in the presence or absence of SAT05f or CTRLODN, anti-double-stranded DNA-positive serum from SLE patients (1: 1 dilution) And cultured for 48 hours. hPBMCs were cultured with medium alone. At the same time, the cells were cultured with anti-double-stranded DNA-positive serum from healthy donor blood and used as a control. The supernatant was collected and the interferon activity in the supernatant was verified. Vero E6 cells were cultured with the collected supernatant (1: 5 dilution) for 16 hours, after which 10X TCID50 VSV was allowed to act for 48 hours. After staining with crystal violet 0.5%, the cytopathic effect was measured at A578 nm using a multiwell microtiter plate detector.

<Test results>
As shown in FIG. 4, serum from SLE patients stimulated interferon production from hPBMC, and SAT05f suppressed interferon production from hPBMCs stimulated with serum from SLE patients. Single data from 3 replicates is shown. It is well known that production of high levels of IFNs promotes the development of SLE (Barrat FI, et al. J Exp Med 2005; 202: 1131-9; Wellmann U. et al. Proc Natl Acad Sci USA 2005; 102: 9258-63). In addition, there is a substance that induces endogenous INF in the serum of SLE patients (Kwok SK, et al. Arthritis Res Ther 2008; 10 (2): R29), and SLE patients are circulating inducers of IFN production. SLE patients' sera often induce INF production in PBMC medium from blood of healthy donors (Vallin H, et al. Clin Exp Immunol 1999 Jan; 115 (1): 196-202), and that anti-double-stranded DNA antibodies or DNA-anti-DNA antibody complexes function as endogenous INF-α derivatives in SLE patients and are also pathogenic agents for SLE (Vallin H, et al J Immunol 1999 Dec 1; 115 (1): 163 (11): 6306-13) is reported in the report. Taken together, these data indicate that SAT05 can be used to treat SLE patients by suppressing IFN production.

Example 5 Effect of SAT05f on rescue of mice from cytokine-mediated lethal shock
<Experiment method>
In order to elucidate the function of SAT05F in the body, a model of cytokine-mediated lethal shock has been developed using conventional methods (Marshall AJ, et al. Infect Immun 1998 Apr; 66 (4): 1325-33; Peter M, Bode K, et al. Immunology 2008 Jan: 123 (1): 118-28).
Female BALB / C mice (weight 20 ± 1 g) were obtained from Experimental Animal Center, Jilin University School of Medicine. During the experiment, the mice had free access to food and water. Experiments were performed according to local methods.
SAT05F having the sequence 5'-cctcctcctcctcctcctcctcct-3-3, CTRL-ODN having the sequence 5'-aaaaataaaaataaaataaaat-3 ', CpG-ODN 1826 (1826) having the sequence 5'-tccatgacgttcctgacgtt-3-3 (Sanjai Oligonucleotides including Kumar, et al. Infection and Immunity, February 2004, p. 949-957, Vol 72, No 2) were synthesized by Takara (Dalian, China). .
D-galactosamine (D-(+)-galactosamine HCL, D-GALN) is from DeBioChem (Nanjing, China).

  BALB / C mice were divided into the following groups (5 mice / 1 group): D-GALN + 1826, D-GALN + 1826 + SAT05F, D-GALN + 1826 + CTRL-ODN. All mice were intraperitoneally injected with 500 μl of D-galactosamine 32 mg / ml in PBS), and 1.5 hours later, 1826 (10 μg / mouse) dissolved in PBS was further intraperitoneally injected. Subsequently, SAT05F (50 μg / mouse) dissolved in PBS was intraperitoneally injected into mice of the D-GALN + 1826 + SAT05F group. Mice in the D-GALN + 1826 + CTRL-ODN group were intraperitoneally injected with CTRL-ODN (50 μg / mouse) dissolved in PBS. Mice in the D-GALN + 1826 group were injected intraperitoneally with only D-galactosamine and 1826. Mice were observed and mouse deaths were recorded.

<Experimental result>
As shown in FIG. 5, within 24 hours after the injection of D-galactosamine, all the mice in the D-GALN + 1826 group or the D-GALN + 1826 + CTRL-ODN group (5 mice in each group) died. In comparison, 168 hours after D-galactosamine injection, all mice in the D-GALN + 1826 + SAT05F group are alive, and SAT05f can rescue mice that received D-galactosamine and 1826. It is shown that. It has been reported that mice administered with D-galactosamine can be an animal model of itokine-mediated lethal shock when injected with CpG OD (Peter M, et al. Immunology. 2008 Jan; 123 (1) : 118-28). As can be clearly seen from these results, the data indicate that SAT05F is repressive in the body and suppresses cytokine-mediated lethal shock. Data from primary experiments of two independent repeatable experiments are shown.

It is a graph showing the inhibitory effect of SAT05f on the proliferation of human peripheral blood mononuclear cells (hPBMC) stimulated by CpG 2006 and CpG C274. It is a graph showing the inhibitory effect of SAT05f on interferon production from hPBMC stimulated by CpG C274. It represents the inhibitory effect of SAT05f on interferon production from hPBMC stimulated with type I herpes simplex virus and influenza virus. Here, (A) is a comparison of the antiviral activity of interferon (IFN) induced by inactivated HSV-1 and recombinant human α interferon (IFN-α). “IU” represents an international unit. (B) shows the inhibitory effect of SAT05f on the production of interferon from human PBMC stimulated by inactivated HSV-1. (C) shows the dose effect of the inhibitory action of SAT05f on interferon production from human PBMC stimulated by HSV-1. (D) is a comparison of the antiviral activity of inactivated PR8-induced interferon (IFN) and recombinant human alpha interferon (IFN-α). (E) shows the inhibitory effect of SAT05f on IFN production from hPBMC stimulated with inactivated PR8. (F) Dose effect on the suppression of SAT05f on IFN production from hPBMC stimulated with inactivated PR8. The figure represents the results of one test out of three repeatable tests. HSV-1 represents type I herpes simplex virus and PR8 represents influenza virus (HINI / PR8). FIG. 4 is a graph showing that SAT05f suppresses interferon production from hPBMC stimulated with serum of patients with lupus erythematosus (SLE). 3 is a graph showing that SAT05f rescues mice from cytokine-mediated lethal shock.

Claims (14)

An oligonucleotide which is 5'-cctcctcctcctcctcctcctcct-3 '(SEQ ID NO: 1) comprising:
The oligonucleotide characterized in that the phosphate backbone of the oligonucleotide is not modified or partially phosphorothioate-modified . Use of an oligonucleotide having the sequence 5'-cctcctcctcctcctcctcctcct-3 '(SEQ ID NO: 1) in the preparation of a medicament for treating immune mediated diseases in an individual. The immune-mediated disease, autoimmune diseases, hypersensitivity reactions, transplant rejection, claim microorganism including diseases disease and Toll-like receptors caused to over-stimulate the immune system of the host (TLR) mediated 2 Use as described in. Use according to claim 2 , wherein the individual is a human or other vertebrate. Treatment of the immune-mediated disease inhibits the stimulatory effect of Toll-like receptor 9 agonist on immune cell proliferation, inhibits Toll-like receptor 9, inhibits interferon production, and is mediated by cytokine-mediated lethality 3. Use according to claim 2 , carried out by a mechanism selected from the group consisting of being saved from sexual shock. Systemic lupus erythematosus (SLE), wherein the immune-mediated disease is treated by inhibiting TLR9 (Toll-like receptor 9) activation and interferon production induced by sera of TLR9 agonists, viruses and SLE patients ), use of claim 2 wherein the multiple organ dysfunction syndrome to be treated by the patient is saved from sepsis being treated or cytokine mediated lethal shock by being saved from lethal shock. Use according to claim 2, wherein the oligonucleotide can be chemically modified. Use according to claim 2, wherein the phosphate backbone of the oligonucleotide can be unmodified, or wholly or partially phosphorothioate modified. A drug for administration to an individual who is immune-mediated or at risk of immune-mediated disease, comprising an oligonucleotide having the sequence 5'-cctcctcctcctcctcctcctcct-3 '(SEQ ID NO: 1) .   13. A medicament according to claim 12, comprising a pharmaceutically acceptable carrier and / or an additional active ingredient.   14. The agent according to claim 13, wherein the oligonucleotide can be subjected to polyethylene glycolation treatment.   15. The drug according to claim 14, which is a dosage form administered by a route including intestinal, extra-intestinal, topical application and inhalation when the oligonucleotide is administered to an individual. 10. The agent of claim 9, wherein the oligonucleotide can be chemically modified. 10. The agent according to claim 9, wherein the phosphate backbone of the oligonucleotide can be unmodified, or wholly or partially modified with phosphorothioate.

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