Classifications

• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• • 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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
• • 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
• • 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
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/17—Immunomodulatory nucleic acids
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
• • 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
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
• • 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
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/31—Combination therapy
• • 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

• the present invention relates to the oligonucleotides and remedies for treating immune-mediated disorders, using the oligonucleotides.
• the immune-mediated disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation of host's immune system by autoantigens, microbes and Toll-like receptor (TLR)-mediated disease.
• TLR Toll-like receptor
• the immune system protects human body from bacterial, parasitic, fungal, viral infections and from the growth of tumor cells.
• Immunity can be classified as innate immunity or as adaptive immunity. Innate immune responses typically occur immediately upon infection for providing of an early barrier to infectious disease whereas adaptive immune responses occur later with the generation of antigen-specific long term protective immunity.
• the immune response can sometimes be unwanted and cause immune-mediated disorder.
• the disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation of host's immune system by microbes and Toll-like receptor (TLR)-mediated disease.
• TLR Toll-like receptor
• the autoimmune diseases results from an adaptive immune response or innate immune response or both against endogenous and/or exogenous antigens.
• Foreign substances derived from bacteria, parasites, fungi or viruses, may mimic self-proteins and stimulate the immune system to launch responses to a self-cell and tissue, resulting in the diseases including but not limited to systemic lupus erythematosus ( SLE ) and rheumatoid arthritis.
• the graft rejection is a consequence of organ or tissue transplantation caused by the immune response in the transplant recipient (host) to the transplanted organ/tissue.
• grafts including kidney, pancreas, heart, lung, bone marrow, cornea and skin
• the subject can launch an immune response (rejection) against the grafts.
• Hypersensitivity is an inappropriate immune response that has deleterious effects, resulting in significant tissue damage or even death.
• the hypersensitivity is divided into four types (e.g. Types I, II, III and IV.
• Disease associated with the over-stimulation of host's immune system by microbes is triggered by the infection of viruses such as flu viruses and other microbes.
• TLR Toll-like receptor
• TLRs are a family of receptors that recognize microbe derived molecular structures (pathogen-associated molecular patterns or PAMPs). TLR expressing immune cells are activated upon binding of PAMPs. TLRs recognize a range of pathogen-derived products and activated.
• TLR activation has been connected to the pathogenesis of some of diseases including sepsis, dilated cardiomyopathy, diabetes, experimental autoimmune encephalomyelitis, systemic lupus erythematosus, atherosclerosis, asthma, chronic obstructive pulmonary disease and organ failure (Foo Y. Liew, et al. Nature Review Immunology, Vol 5, 2005, 446-458).
• Activation of TLR9 by self DNA play an important role in the development of autoimmune diseases such as psoriasis (Gilliet M, et al. Nat. Rev. Immunol. 2008, 594-606), SLE (Christensen SR, et al.
• TLR9 agonist activates both innate and adaptive immune response (Arthur M. Krieg. Nature Reviews Drug Discovery, Vol 5. June 2006, 471-484). It was documented an oligonucleotide with a nucleotide sequence of 5 '-cctcctcctcctcctcctcctcctcctcctcctcct-3' prevented proliferation of human peripheral blood mononuclear cells (PBMCs) and production of IFNs, which induced by TLR9 agonists (US8030289B2).
• PBMCs peripheral blood mononuclear cells
• the present invention provides an oligonucleotide that comprises an oligonuleotide with a formula of (CCT)nCm, wherein the n is an integer from 6 to 16, the m is 0, 1, or 2; with the proviso that when n is 8, m is 1 or 2.
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcc-3' (SEQ ID NO: 1).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctccctcccccccccccccccccccccccccccccccccccccccccccccccccccccc-3' (SEQ ID NO: 2).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcct-3' (SEQ ID NO: 3).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctctctctctctcctcctcctcctcctcctcctc-3' (SEQ ID NO: 4).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccc-3' (SEQ ID NO: 5).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcct-3' (SEQ ID NO: 6).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctc-3' (SEQ ID NO: 7).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctccctccctccccccccccccccccccccccccccccccccccccccccccccccccccccccccctccctcctctcctctccctcctctcctctctctctctctctctctctct-3' (SEQ ID NO: 9).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctctctctctctcctctcctctcctctc-3' (SEQ ID NO: 10).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcccctcccccccctccccccccccccccccccccccctcccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccc
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctctctctctctctctct-3' (SEQ ID NO: 12).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctcctctcctctcctcctcctcctcctcctctcctctctct-3' (SEQ ID NO: 13).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctcctctctcctctcctctcctcctctcctcctcctctcctctctctctctctct-3' (SEQ ID NO: 14).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctccctcctcct-3' (SEQ ID NO: 15).
• the present invention provides an oligonucleotide with a nucleotide sequence of 5'-cctcctcctcctcctcctccctccctcctcctcct-3' (SEQ ID NO: 16).
• the present invention provides a remedy for treating immune-mediated disorder using the oligonucleotides of the invention.
• the immune-mediated disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation by of host's immune system by autoantigens, microbes and Toll-like receptor (TLR)-mediated disease.
• TLR Toll-like receptor
• the present invention provides a remedy for treating immune-mediated disorder using the oligonucleotides of the invention by inhibiting the TLR activation and IFNs production induced by TLR antagonists such as DNA virus, RNA virus and the serum from SLE patients.
• the present invention provides a remedy for treating immune-mediated disorders using the oligonucleotides of the invention by inhibiting production of proinflammatory cytokines and by rescuing a subject from cytokine-mediated lethal shock.
• the present invention provides a remedy for treating immune-mediated disorders using the oligonucleotides of the invention by inhibiting NF- ⁇ activation induced by TLR stimulation.
• the present invention provides a remedy for treating SLE, sepsis and multiple organ dysfunction syndromes in a subject using the oligonucleotides of the invention.
• the present invention provides methods of regulating an immune response in an individual, comprising administering to an individual immunostimulatory compounds in an amount sufficient to regulate an immune response in said individual.
• Immunoregulation according to the methods of the invention may be practiced on individuals including those suffering from a disorder associated with an unwanted activation of immune response.
• the present invention provides methods of inhibiting a TLR9 dependent immune response in an individual, comprising administering to an individual immunostimulatory compounds in an amount sufficient to prevent TLR9 dependent cytokine production in said individual.
• the present invention provides methods of inhibiting a TLR7/8 dependent immune response in an individual, comprising administering to an individual immunostimulatory compounds in an amount sufficient to prevent TLR7/8 dependent cytokine production in said individual.
• the present invention provides methods of inhibiting a NF- ⁇ dependent immune response in an individual, comprising administering to an individual immunostimulatory compounds in an amount sufficient to prevent NF- ⁇ dependent cytokine production in said individual.
• the present invention provides a remedy for treating immune-mediated disorder by administering the oligonucleotides of the invention alone or with a pharmaceutically acceptable carrier to a subject through the route of enteral, parenteral and topical administration or inhalation.
• the present invention provides a composition comprising therapeutically effective amount of the oligonucleotides of the invention for the treatment of immune-mediated disorder.
• the present invention provides a remedy for the treatment of immune-mediated disorder by administering the oligonucleotides of the invention alone or in combination with additional active ingredients.
• the present invention provides a remedy for the treatment of immune-mediated disorder by administering the oligonucleotides of the invention in delivery vehicles.
• FIG. 1 shows suppression of NF- ⁇ activation by inhibitory-ODNs in human plasmacytoid DC cell line.
• A The CAL-1/NFKB-GFP cell line was designed for monitoring the activity of NF- ⁇ transcription factor in cell-based assays. Vector encoding the GFP reporter gene driven by the NF- ⁇ consensus transcriptional response element was transfected into human plasmacytoid DC cell line; CAL-1. GFP expression was induced by TLR9 agonist; CpG2395.
• B GFP expression induced by TLR9 stimulation was blocked by the addition of inhibitory-ODNs.
• Figure 2 shows a graph depicting the suppression ability of inhibitory-ODNs on NF- ⁇ activation by TLR9 stimulation in the CAL-1/NFKB-GFP cell line.
• Figure 3 shows graphs depicting the suppression ability of inhibitory-ODNs on IL-6 and TNFa production from CAL-1 cells stimulated with TLR9 agonist; CpG2395. Comparison of the inhibition activity between (cct)8, (cct)8c, (cct)8cc, (cct)9, (cct)10, (cct)ll and (cct)12.
• Figure 4 shows graphs depicting the suppression ability of inhibitory-ODNs on IL-6 and TNFa production from CAL-1 cells stimulated with TLR9 agonist; CpG2395. Comparison of the inhibition activity between (cct)8, (cct)9, (cct)10, (cct)ll, (cct)12, (cct)14 and (cct)16.
• Figure 5 shows graphs depicting the suppression ability of inhibitory-ODNs on IL-6 and TNFa production from mouse DC cell line; D2SC/1. D2SC/1 cells were stimulated with TLR9 agonist; CpG1826 in the presence of inhibitory-ODNs.
• A Comparison of the inhibition activity between (cct)8, (cct)8c, (cct)8cc and (cct)9.
• B Comparison of the inhibition activity between (cct)8, (cct)9, (cct)10, (cct)ll, (cct)12, (cct)14 and (cct)16.
• Figure 6 shows a graph depicting the suppression activity of inhibitory-ODNs on IFNa production from human PBMC stimulated with CpG2216. Comparison of the inhibition activity between (cct)8, (cct)9, (cct)10, (cct)ll, (cct)12, (cct)14 and (cct)16 on IFNa production which induced by TLR9 agonist; CpG2216.
• Figure 7 shows a graph depicting the suppression ability of inhibitory-ODNs on NF- ⁇ activation by TLR7/8 stimulation in the CAL-1/NFKB-GFP cell line.
• A Comparison of the inhibition activity between (cct)6, (cct)7 and (cct)8 for NFKB activation which induced by TLR7/8 agonist; Gardiquimod.
• B Comparison of the inhibition activity between (cct)8, (cct)9, (cct)10, (cct)ll, (cct)12, (cct)14 and (cct)16 for NF-KB activation which induced by TLR7 agonist; CL264. DETAILED DESCRIPTION OF THE INVENTION
• the oligonuleotides of the present invention strongly inhibits TLR9 activation.
• CpG containing oligonucleotides CpG ODN
• the oligonuleotides of the invention strongly inhibits the cytokines stimulated by CpG ODN, indicating that the oligonucleotides of the invention can be used as a remedy for the treatment of diseases related to TLR9 activation. Because TLR9 activation has been reported to contribute to the development of psoriasis (Gilliet M, et al. Nat. Rev. Immunol.
• the oligonucleotides of the invention can be used as a remedy for the treatment of psoriasis, SLE and rheumatoid arthritis by inhibiting the TLR9 activation.
• the oligonuleotides of the present invention strongly inhibits IFN production from human PBMC induced by TLR9 agonist. Because the elevated production of IFNs has been reported to contribute to the development of 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), the oligonucleotides of the invention can be used as a remedy for the treatment of SLE by inhibiting IFN production.
• the oligonuleotides of the present invention strongly inhibits the cytokines production which induced by TLR7/8 agonist.
• the oligonucleotides of the present invention can be used as a remedy for the treatment of Toll-like receptor (TLR)-mediated disease by inhibiting TLR7 or TLR8.
• TLR Toll-like receptor
• TLR9 agonist CpG ODN with the D-galactosamine (D-Gal) into mice induced hyper immune reactions.
• D-Gal D-galactosamine
• the model mice died within 12 to 24 h.
• Analyses of plasma cytokines revealed over-production of proinflammatory cytokines such as TNFa (Marshall AJ, et al. Infect Immun. 1998 Apr; 66(4): 1325-33; Peter M, Bode K,et al. Immunology. 2008 Jan;123(l): 118-28).
• the oligonucleotides of the present invention strongly inhibits the production of TNFa from mouse cells induced by TLR9 stimulation.
• the oligonucleotides of the present invention can be used as a remedy for the treatment of sepsis and
• NF- ⁇ is clearly one of the most important regulators of proinflammatory gene expression. Activation of the NF-KB plays a central role in inflammation through its ability to induce transcription of proinflammatory cytokines (Baldwin (Jr) AS, et al. Annu Rev Immunol. 1996, 649-683). It has been demonstrated that NF- ⁇ plays a role in constitutive IL-6 production in rheumatoid arthritis (RA) synovial fibroblasts (Miyazawa K, et al. Am J Pathol 1998, 793-803).
• RA rheumatoid arthritis
• NF- ⁇ is intimately involved in activation of inflammatory genes by IL-1 or TNFa in human monocytes (Schottelius AJ, et al. J Biol Chem 1999, 31868-31874). The number of NF- ⁇ positive cells correlates with the degree of gastritis. Similarly, there is evidence of NF- ⁇ activation in inflammatory bowel disease, where lamina intestinal macrophages display activated NF-KB (Neurath MF, et al. Ann NY Acad Sci 1998, 859: 149-159).
• TLRs The activation of TLRs by the ligands induces the activation of transcription factors such as NF-KB and interferon responsive factors (IRFs). Those activated transcription factors further induce the production of cytokines such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNFa) and the interferons (IFNs).
• IRFs interferon responsive factors
• the oligonuleotides of the invention strongly inhibits NF- ⁇ activation induced by TLR stimulation, indicating that the oligonucleotides of the invention can be used as a remedy for the treatment of diseases related to NF- ⁇ activation.
• NF- ⁇ activation has been reported to contribute to the development of rheumatoid arthritis, gastritis and inflammatory bowel disease
• the oligonucleotides of the invention can be used as a remedy for the treatment of rheumatoid arthritis, gastritis and inflammatory bowel disease by inhibiting the NF- ⁇ activation.
• oligonucleotide refers to oligodeoxyribonucleotide (ODN).
• ODN oligodeoxyribonucleotide
• the oligonucleotide can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic.
• the oligonucleotide of the invention can be synthesized by a variety of automated nucleic acid synthesizers available in the market. These oligonucleotides are referred to as synthetic oligonucleotides.
• the oligonucleotide disclosed in the invention can encompass various chemical modifications, in comparison to natural DNA, involving a phosphodiester internucleoside bridge, a ribose unit and/or a natural nucleoside base (adenine, guanine, cytosine, thymine).
• the modifications can occur either during or after synthesis of the oligonucleotide.
• modified bases can be incorporated internally or on its end.
• the modification can be carried out using the active groups (via an amino modifier, via the 3' or 5' hydroxyl groups, or via the phosphate group).
• the skilled person knows examples of chemical modifications.
• An oligonucleotide according to the invention may have one or more modifications, wherein each modification is located at a particular phosphodiester internucleoside bridge and/or at a particular ribose unit and/or at a particular natural nucleoside base position in comparison to an oligonucleotide of the same sequence, which is composed of natural DNA.
• the chemical modification includes "back bone modification" of the oligonucleotide of the invention.
• the modified back bone of the oligonucleotide of the invention includes, but not limited to the "phosphorothioate backbone” that refers to a stabilized sugar phosphate backbone of a nucleic acid molecule in which a non-bridging phosphate oxygen is replaced by sulfur at least one intemucleotide linkage.
• a non-bridging phosphate oxygen is replaced by sulfur at each and every intemucleotide linkage.
• oligonucleotide can be is a phosphorothioate/phosphodiester chimera.
• the chemical modification also includes the base substitutions of the oligonucleotide disclosed in the invention.
• the substituted purines and pyrimidines can be C-5 propyne pyrimidine and 7-deaza-7-substituted purine.
• the substituted purines and pyrimidines include but are not limited to adenine, cytosine, guanine, and thymine, and other naturally and non-naturally occurring nucleobases.
• the chemical modification of the oligonucleotide of the invention further includes the modification of the bases of the oligonucleotide.
• a modified base is any base which is chemically distinct from the naturally occurring bases typically found in DNA such as T, C, G and A, but which share basic chemical structures with these naturally occurring bases.
• the oligonucleotide of the invention can be modified by using cytidine derivatives.
• cytidine derivative refers to a cytidine-like nucleotide (excluding cytidine)
• thymidine derivative refers to a thymidine-like nucleotide (excluding thymidine).
• the oligonucleotides of the invention can be chemically modified by linking a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini of the oligonuleotide.
• Immuno-mediated disorder An immune-mediated disorder is a disease caused by an unwanted immune response in a subject.
• the disorder includes autoimmune disease, graft rejection, hypersensitivity, diseases associated with the over-stimulation of host's immune system by microbes and diseases associated with TLR activation.
• the oligonucleotide disclosed in the invention can be used as a remedy to treat the immune-mediated disorder.
• Immuno response A response of a cells of the immune system, such as a B cell, T cell, natural killer cell, dendritic cell, neutrophil and macrophage to a stimulus.
• the response includes innate immune response and adaptive (specific or acquired) immune response.
• the adaptive (specific or acquired) immune response includes humoral immune response and cellular immune response.
• prevent refers to prevent the full development of an immune-mediated disorder in a subject; treat refer a therapeutic intervene in a subject so as to ameliorate a sign or symptom of, halt the progression of, or eliminate pathological condition of the immune-mediated disorder.
• Subject As used herein, a subject refers to a human or non-human vertebrate. Non-human vertebrates are non-human primates, livestock animals and companion animals.
• the oligonucleotide of the invention can be administered to prevent or/and treat immune-mediated disorder in a subject.
• autoimmune diseases refers to a disease caused by a breakdown of self-tolerance such that the adaptive and innate immune system responds to self antigens and mediates cell and tissue damage. Autoimmune diseases are frequently characterized by means of their involvement of single organ or single cell-types or involvement of multiple organs or tissue systems. Autoimmune diseases have also been referred to as “collagen,” or “collagen-vascular” or “connective tissue” diseases. Autoimmune disorders are frequently associated with hypersensitivity reactions.
• the oligonucleotides of the invention can be useful for treating and/or preventing various types of autoimmune diseases.
• autoimmune disorders are systemic lupus erythematosus, insulin-dependent (type I) diabetes mellitus, inflammatory arthritis, rheumatoid arthritis, multiple sclerosis, autoimmune hepatitis, chronic aggressive hepatitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, acquired hemophilia, ankylosing spondylitis, antiphospholipid syndrome, Beh.cedilla.et's syndrome, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinin disease, polymyositisdermatomyositis, discoid lupus, sympathetic ophthalmia, essential mixed cryoglobulinemia, fibromyalgia, fibromyositis, Guilla
• DNA or RNA released from DNA- or RNA-containing microbes could stimulate the production of autoantibody specific to self RNA- or DNA-containing complexes and consequently led to an autoimmune disease, including but not limited to SLE.
• Hypersensitivity A hypersensitivity is referred to the disorders wherein tissue injury occurs as a result of a humoral or cell-mediated response to antigens of endogenous or exogenous origin and has been classified into four types.
• Type I hypersensitivity (frequently referred to as anaphylactic, immediate-type, atopic, reagenic, IgE-mediated hypersensitivity reactions or allergy) generally result from the release of pharmacologically active substances such as histamine, slow-reacting substance of anaphylaxis (SRS-A), and eosinophilic chemotactic factor (ECF) form IgE-sensitized basophils and mast cells after contact with a specific exogenous antigen.
• SRS-A slow-reacting substance of anaphylaxis
• ECF eosinophilic chemotactic factor
• Type I hypersensitivity includes, but not limited to, allergic extrinsic asthma, seasonal allergic rhinitis and systemic anaphylaxis.
• Type II hypersensitivity also referred to as cytotoxic, cytolytic complement-dependent or cell-stimulating hypersensitivity reaction
• Type II hypersensitivity results when antibody reacts with antigenic components of cells or tissue elements or with an antigen or hapten, which has become intimately coupled to cells or tissue.
• Type II hypersensitivity includes, but not limited to, autoimmune hemolytic anemia, erythroblastosis fetalis and Goodpasture's disease.
• Type III hypersensitivity results from the deposition of soluble circulating antigen-antibody complexes in vessels or in tissues, with accompanying acute inflammatory reactions at the site of immune complex deposition.
• Type III hypersensitivity includes, but not limited to, Arthurs reaction, serum sickness, systemic lupus erythematosis, and certain types of glomerulonephritis.
• Type IV hypersensitivivity (frequently called cellular, cell-mediated, delayed, or tuberculin-type hypersensitivity reactions) are caused by sensitized T-lymphocytes which result from contact with a specific antigen.
• Type IV hypersensitivity includes, but not limited to, contact dermatitis and allograft rejection (Richard A. et al. Immunology, Fifth Edition, 2003, W.H. FREEMAN AND COMPANY).
• Diseases associated with the over-stimulation of host's immune system by microbes Microbe invasion, if severe, sometimes can cause systemic inflammatory response in a subject, leading to diseases associated with the over-stimulation of host's immune system by microbes.
• the events in the development of the diseases include the significantly elevated blood levels of TNFa, interleukin-1 (IL-1), IL-6, IL-12, interferon alpha (IFN-a), interferon beta (IFN- ⁇ ), interferon gamma (IFN- ⁇ ), chemokines interferon-inducible protein 10, monocyte chemoattractant protein 1, interleukin-8, interleukin-1 ⁇ , and monocyte chemoattractant protein 1.
• the oligonucleotide of the invention can be used to treat and/or prevent the diseases associated with the stimulation of host's immune system by microbes in a subject.
• the microbes causing the diseases includes, but not limited to, viruses, bacteria, fungi, parasites and etiological agents of Spongiform encephalopathies.
• the virus that cause the diseases associated with the over-stimulation of host's immune system by microbes include: SARS CoV, influenza viruses, avian flu virus HIV-1, polio viruses, hepatitis A virus; enteroviruses, human Coxsackie's viruses, rhinoviruses, echoviruses, equine encephalitis viruses, rubella viruses, dengue viruses, encephalitis viruses, yellow fever viruses, corona viruses, vesicular stomatitis viruses, rabies viruses, Ebola viruses, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus, influenza viruses, Hantan viruses, bunga viruses, phleboviruses, Nairo viruses, hemorrhagic fever viruses; reoviruses, orbiviurses and rotaviruses, Hepatitis B virus, parvoviruses, papilloma viruses, polyoma viruses, adenoviruses
• the bacteria that can cause the diseases associated with the over-stimulation of host's immune system by microbes include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M. tuberculosis, M. avium, M. E intracellulare, M. kansaii, M.
• the fungi that can cause the diseases associated with the over-stimulation of host's immune system by microbes include, but not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.
• the parasites that can cause the diseases associated with the over-stimulation of host's immune system by microbes include: Plasmodium falciparum and Toxoplasma gondii.
• the graft rejection is an immune-mediated disorder caused by organ or tissue transplantation, Transplantation means the transfer of transplants (grafts) from a donor to a recipient. Grafts are the living cells, tissues, or organs transplanted from a donor to a recipient.
• An autograft is the a graft transferred of one's own tissue from one location to another; a syngeneic graft (isograft) is a graft between identical twins; an allogeneic graft (homograft) is a graft between genetically dissimilar members of the same species; and a xenogeneic graft (heterograft) is a transplant between members of different species.
• oligonucleotides of the present invention are useful when administered for the prevention of the graft rejection.
• grafts examples include heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum ***, limb, muscle, nervus, duodenum, small-bowel, pancreatic-islet-cell, and the like.
• the recipient may be an animals as defined in "subject" of the invention.
• TLR-mediated diseases A Toll-like receptor (TLR)-mediated disease means an immune mediated disorder related to the activation of members of the TLR family.
• the disease includes, but not limited to, the diseases include but not limited to, sepsis associated with the activation of TLR4 by lipopolysaccharide (LPS), dilated cardiomyopathy associated with the activation of TLR2, 3, 4, 9, diabetes associated with the activation of TLR2,3,4,9, experimental autoimmune encephalomyelitis associated with the activation of TLR3, systemic lupus erythematosus associated with the activation of TLR9, atherosclerosis associated with the activation of TLR4, asthma associated with the activation of TLR4 by LPS, chronic obstructive pulmonary disease associated with the activation of TLR4, EAE associated with the activation of TLR4 and organ failure associated with the activation of TLR4 (Foo Y.
• LPS lipopolysaccharide
• dilated cardiomyopathy associated
• CpG-containing DNA a TLR9 agonist
• a nucleic acid-containing infectious agent could be identified from SLE serum that induces an efficient immune response dominated by IFN-a secretion that is thought to contribute the development of SLE.
• the oligonucleotides of the present invention can be administered for treating and/or prevent the Toll-like receptor (TLR)-mediated diseases including but not limited to SLE in a subject.
• TLR Toll-like receptor
• CpG ODN It has been documented that TLR9 agonist activates both innate and adaptive immune response (Arthur M. Krieg. Nature Reviews Drug Discovery, Vol 5. June 2006, 471-484). CpG containing oligonucleotides (CpG ODN) is a TLR9 agonist [D.M. Klinman, Nat. Rev, Immunol. 4 (2004) 249- 258]. Based on the functional characteristics, CpG ODNs are divided into three types (Tomoki Ito, et al. Blood, 2006, Vol 107, Num 6: 2423-2431).
• A-type CpG ODN activates human plasmacytoid dendritic cells (pDCs) to produce large amount of type I interferon (IFN-a/ ⁇ ) and strongly activates natural killer cells (NK cells).
• B-type CpG ODN primarily activates B cells, resulting in their proliferation and antibody secretion.
• C-type CpG ODN shares the activities of both A- and B-type CpG ODN.
• CpG ODN such as CpG 2216 or CpG 2006 or CpG 2395 can be endocytosed into a cellular compartment where they are exposed to and activate TLR9.
• TLR9 activation initiate a rapid innate immune response that is characterized by the secretion of pro-inflammatory cytokines [IL-6, tumor-necrosis factor-a (TNFa)], the secretion of type I interferon (IFN) and the secretion of secretion of IFN-inducible chemokines.
• pro-inflammatory cytokines IL-6, tumor-necrosis factor-a (TNFa)
• IFN type I interferon
• IFN-inducible chemokines Through both IFN-dependent and IFN-independent pathways, innate immune cells including natural killer (NK) cells, monocytes and neutrophils are secondarily activated by the pDC.
• NK natural killer
• B cells activated through TLR9 have a greatly increased sensitivity to antigen stimulation and efficiently differentiate into antibody-secreting cells, and therefore contributing to the adaptive immune response, especially humoral immune response.
• pDC activated through TLR9 secrete IFNa, which drives the migration and clustering of pDC to lymph nodes and other secondary lymphoid tissues where the pDC activates naive and memory T cells, assists the cross-presentation of soluble protein antigens to CD8+ cytotoxic T lymphocyte (CTL) and promotes strong TH1 biased cellular CD4 and CD8 T-cell responses.
• CTL cytotoxic T lymphocyte
• a pharmaceutically acceptable carrier denotes one or more solid or liquid filler, diluents or encapsulating substances that are suitable for administering the oligonucleotide of the invention to a subject.
• the carrier can be organic, inorganic, natural or synthetic.
• the carrier includes any and all solutions, diluents, solvents, dispersion media, liposome, emulsions, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and any other carrier suitable for administering the oligonucleotide of the invention and their use is well known in the art.
• the pharmaceutically acceptable carriers are selected depending on the particular mode of administration of the oligonucleotide.
• the parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
• pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• a therapeutically effective amount of an oligonucleotide of the invention is administered to a subject.
• the "therapeutically effective amount” of one of the oligonucleotides means a sufficient amount of the oligonucleotide used to achieve a desired result of treating or preventing an immune-mediated disorder in a subject.
• the oligonucleotides of the present invention may be employed in pure form or in pharmaceutically acceptable carriers. Alternatively, the oligonucleotides may be administered as pharmaceutical compositions.
• the “amount” in the invention shall refer to a dose.
• the dose can be determined by standard techniques well known to those skilled in the art and can vary depending the factors including, but not limited to the size or/and overall health of the subject or the severity of the disease.
• Introduction of the oligonucleotide of the invention can be carried out as a single treatment or over a series of treatments.
• Subject doses of the oligonucleotide of the invention for the administration range from about 1 ⁇ g to 100 mg per administration.
• doses for the treatment of immune-mediated disorder may be used in a range of 10 to 1,000 times higher than the doses described above. The more preferable doses can be adjusted to provide the optimum therapeutic effect by those skilled in the art, for example, by the attending physician within the scope of sound medical judgment.
• the oligonucleotides of the invention can be administered alone or formulated in a pharmaceutical composition via any suitable route of administration that is effective to achieve the desired therapeutic result.
• the "route" of administering the oligonucleotide of the invention shall mean the enteral, parenteral and topical administration or inhalation.
• the enteral routes of administration of the oligonucleotide of the invention include oral, gastric, intestinal, and rectal.
• the parenteral route includes intravenous, intraperitoneal, intramuscular, intrathecal, subcutaneous, local injection, vaginal, topical, nasal, mucosal, and pulmonary administration.
• the topical route of administration of the oligonucleotide of the invention denotes the application of the oligonucleotide externally to the epidermis, to the buccal cavity and into the ear, eye and nose,
• a pharmaceutical composition shall mean the composition comprising an therapeutically effective amount of the oligonucleotide of the invention with or without a pharmaceutically acceptable carrier.
• the pharmaceutical compositions can comprise one or more oligonucleotides of the invention.
• the composition includes but not limited to aqueous or saline solutions, particles, aerosols, pellets, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, drops and other pharmaceutical compositions suitable for use in a variety of drug delivery systems.
• compositions may be administered parenterally, orally, rectally, intravaginally, intraperitoneally, topically (in a dosage form as powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.
• the composition must be sterile and stable under the conditions of manufacture and storage and preserved against the microbial contamination.
• Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically-acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• the oligonucleotide of the invention can be suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.
• the buffer solution includes sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate-acetic acid buffers.
• the composition will be formulated with edible carriers to form powders tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like.
• conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
• the composition will be tablets or lozenges in conventional manner.
• the composition will be an aerosol spray from pressurized packs or a nebulizer or a dry powder and can be selected by one of skill in the art.
• the oligonucleotides of the invention are also suitably administered by sustained-release systems.
• the oligonucleotide of the invention can be used in a liquid suspension of crystalline or amorphous material with poor water solubility to slow the releasing of the oligonucleotide.
• delayed releasing of a parenterally administered drug form of the oligonucleotide is accomplished by dissolving or suspending the oligonucleotide in hydrophobic materials (such as an acceptable oil vehicle).
• injectable depot forms are made by entrapping the oligonucleotide in liposomes or microemulsions or other biodegradable semi-permeable polymer matrices such as polylactide-polyglycolide, poly (orthoesters) and poly (anhydrides).
• the oligonucleotides of the invention can be used alone, in combination with themselves, in a pharmaceutically acceptable carrier, in combination with one or more additional active ingredients.
• the administration of the oligonucleotide of the invention and additional active ingredients can be sequential or simultaneous.
• the active ingredients include non-steroidal anti-inflammatory agents, steroids, nonspecific immunosuppressive agent, biological response modifier, chemical compound, small molecule, nucleic acid molecule and TLR antagonists.
• the active ingredients also denote the agents that suppress the immune activation by antagonizing chemochines, by inducing the generation of regulatory T cells (CD4+CD25+ T cells), by inhibiting a complement, matrix metalloproteases and nitric oxide synthase, by blocking costimulatory factors and by inhibiting the signaling cascades in the immune cells.
• the non-steroidal anti-inflammatory agents include, but unlimited to, diclofenac, diflunisal, etodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tohnetin, celecoxib and rofecoxib.
• the steroids include, but unlimited to, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone.
• a nonspecific immunosuppressive agent means the agent used to prevent the development of immune-mediated disorder.
• the nonspecific immunosuppressive agents include but not limited to cyclophosphamide, cyclosporine, methotrexate, steroids, FK506, tacrolimus, mycophenolic acid and sirolimus.
• the biological response modifier includes a recombinant interleukin-1 - receptor antagonist (Kineret or anakima), a soluble p75 TNFa receptor-IgGl fusion protein (etanercept or Enbrel), or a monoclonal antibody against TNFa (infliximab or RemicadeX).
• the agents also include Interferon beta- la, interleukin-10 and TGF .
• the oligonucleotides of the invention can be administered in/with a delivery vehicle or in a form linked with a vehicle.
• the vehicle includes, but not limited to, sterol (e.g., cholesterol), cochleates, emulsomes, ISCOMs; a lipid (e.g., a cationic lipid, anionic lipid), liposomes; ethylene glycol (PEG); live bacterial vectors (e.g., Salmonella, Escherichia coli, bacillus Calmette-Gurin, Shigella, Lactobacillus), live viral vectors (e.g., Vaccinia, adenovirus, Herpes simplex), virosomes, virus-like particles, microspheres, nucleic acid vaccines, polymers (e.g., carboxymethylcellulose, chitosan), polymer rings and a targeting agent that recognizes target cell by specific receptors.
• sterol e.g., cholesterol
• cochleates
• Pegylation is the process of covalent attachment of poly (ethylene glycol) polymer chains to another molecule, normally a drug or therapeutic protein. Pegylation is routinely achieved by incubation of a reactive derivative of PEG with the target agent.
• the pegylated agent can "mask” the agent from the host's immune systems, increase the hydrodynamic size of the agent which prolongs its circulatory time.
• the oligonucleotides of the invention can be pegylated.
• ODNs The all oligonucleotides (ODNs) used in the example were synthesized in Hokkaido System Science Co. Ltd (Sapporo, Japan).
• TLR9 stimulatory ODNs were CpG2395 (5'-tcgtcgttttcggcgcgcgccg-3', SEQ ID No: 17), CpG1826 (5'-tccatgacgttcctgacgtt-3', SEQ ID No: 18), CpG2216 (5'-gggggacgatcgtcgggggg-3', SEQ ID No: 19).
• CAL-1/NFKB-GFP cell line was established for monitoring the activity of NF- ⁇ transcription factor in cell-based assays.
• Vector encoding the GFP reporter gene driven by the NFKB consensus transcriptional response element was transfected into human plasmacytoid DC cell line; CAL-1 by electroporation. Transfected cells were further selected with zeocin.
• A GFP expression induced by TLR9 agonist; CpG2395 was evaluated. Briefly, CAL-1/NFKB-GFP cells (l x l0 5 /well) were plated in 96-well flat-bottomed plate (Costar) and cultured with or without CpG2395 ( ⁇ ).
• the cells were incubated at 37° C in a 5% C0 2 humidified incubator for 6 hours.
• GFP expression level in the cells was evaluated by flow cytometer (FACS Calibur, BD Bioscience Co.Ltd). The percentage of GFP positive cells was described in the figure.
• B CAL-1/NFKB-GFP cells (1 ⁇ 10 5 /well) were pre-incubated with (CCT)7, (CCT)8 and (CCT)9 (0.1 ⁇ , 0.3 ⁇ , 1.0 ⁇ ) for 2 hours. The cells were stimulated with CpG2395 ( ⁇ ) for 6 hours.
• GFP expression level in the cells was evaluated by flow cytometer (FACS Calibur, BD Bioscience Co.Ltd). The percentage of GFP positive cells in each condition was described in the figure.
• CAL-1/NFKB-GFP cells (l x lOTwell) were pre-incubated with various inhibitory-ODNs described above for 2 hours. The cells were stimulated with CpG2395 ( ⁇ ) for 6 hours. GFP expression level of the cells in each condition was evaluated by flow cytometer (FACS Calibur, BD Bioscience Co.Ltd). The percentage of GFP positive cells with CpG2395 alone was defined as 100% in the graph. The percentage of GFP positive in each condition was calculated from the number.
• (CCT)14 and (CCT)16 were as same as the activity of (CCT)12 (Fig.2B), suggesting that (CCT)12 as well as (CCT)14 and (CCT)16 may have maximum efficacy for the inhibition of NF-KB activity, which induced by TLR9 stimulation.
• the inhibitory activity of (CCT)8 at 1.0 ⁇ was almost same as those of (CCT)ll and (CCT)12 at 0.1 ⁇ . This data indicate that (CCT)ll and (CCT)12 have ten times higher efficacy for the inhibition of NF- ⁇ activation than (CCT)8 in human cells.
• (CCT)8C and (CCT)8CC exhibited better inhibitory activity than (CCT)8. It was also demonstrated that (CCT)IOC and (CCT)IOCC exhibited better inhibitory activity than (CCT)10.
• (CCT)llC and (CCT)llCC had better inhibitory activity than (CCT)ll, while the inhibitory activity of (CCT) 11 was already almost saturated.
• NF- ⁇ further induce the production of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFa).
• IL-6 interleukin-6
• TNFa tumor necrosis factor alpha
• ODNs oligonuleotides
• NF- ⁇ activation has been reported to contribute to the development of autoimmune diseases such as rheumatoid arthritis, gastritis and inflammatory bowel disease
• the ODNs we examined can be used as a remedy for the treatment of the diseases by inhibiting the NF- ⁇ activation.
• CAL-1 cells were cultured (l x l0 5 /well) were plated in 96-well flat-bottomed plate (Costar) and stimulated with CpG2395 (0.4 ⁇ ) in the presence of inhibitory-ODNs for 24 hours (concentrations of the inhibitory-ODNs are described in the figure). After 24 hours stimulation, cultured supernatants were recovered and proinflammatory cytokines production was evaluated. The level of IL-6 and TNFa production were measured by ELISA as described in manufacture's protocol (R&D systems Co. Ltd, Minneapolis, USA).
• (CCT)9 and (CCT)10 blocked TNFa production with much better efficacy than (CCT)8.
• (CCT)ll, (CCT)12, (CCT)14 and (CCT)16 exhibited strong efficacy for the inhibition of TNFa production at very low dose in human cells.
• IL-6 and TNFa play key roles for the development of the diseases such as rheumatoid arthritis, gastritis and inflammatory bowel disease
• the ODNs we examined can be used a remedy for the treatment of the diseases by the inhibition of IL-6 and TNFa.
• Mouse DC cell line D2SC/1 cells were cultured D2SC/l(l x l07well) were plated in 96-well flat-bottomed plate (Costar) and stimulated with CpG1826 (0.65 ⁇ ) in the presence of inhibitory-ODNs for 24 hours (concentrations of the inhibitory-ODNs are described in the figure). After 24 hours stimulation, cultured supernatants were recovered and proinflammatory cytokines production was evaluated. The level of IL-6 and TNFa production were measured by ELISA as described in manufacture's protocol (R&D systems Co. Ltd, Minneapolis, USA).
• mice with CpG ODN developed cytokine-mediated lethal shock because of the induction of hyper immune reactions (Peter M, et al. Immunology. 2008 Jan;123(l): 118-28).
• Analyses of plasma cytokines revealed over-production of proinflammatory cytokines such as TNFa (Marshall AJ, et al. Infect Immun. 1998 Apr; 66(4): 1325-33; Peter M, Bode K,et al. Immunology. 2008 Jan;123(l): 118-28).
• the ODNs we evaluated strongly inhibits the production of TNFa from mouse cells induced by TLR9 stimulation.
• the cytokine-mediated lethal shock contributes to the septic shock (Slifka MK,et al. J Mol Med. 2000;78(2):74-80; Espat NJ, et al. J Surg Res. 1995 Jul;59(l): 153-8) and multiple organ dysfunction syndromes (MODS) (Wang H, et al. Am J Emerg Med. 2008 Jul;26(6):711-5), the ODNs we evaluated can be used as a remedy for the treatment of sepsis and MOGS by rescuing the host from cytokine-mediated lethal shock.
• huPBMCs Human peripheral mononuclear cells
• huPBMCs Human peripheral mononuclear cells
• FCS v/v
• antibiotics 100 IU of penicillin/ml and 100 IU of streptomycin/ml
• huPBMCs (5 l0 6 /ml) were plated into 96 well flat-bottomed plate and stimulated with CpG2216 (1 ⁇ ) in the presence of inhibitory-ODNs (0.1 ⁇ ); (CCT)8, (CCT)9, (CCT)10, (CCT)ll, (CCT)12, (CCT)14 and (CCT)16.
• the culture supernatants were collected for measuring the level of IFNa production.
• the level of IFNa production was measured by ELISA as described in manufacture's protocol (R&D systems Co. Ltd, Minneapolis, USA).
• huPBMCs produced IFNa in response to TLR9 agonist; CpG2216.
• CCT blocked IFNa production induced by CpG2216.
• the suppression efficacy of (CCT)8 was not so strong.
• (CCT)9, (CCT)IO, (CCT)ll, (CCT)12, (CCT)14 and (CCT)16 exhibited better inhibitory activity for IFNa production by CpG2216 than (CCT)8.
• (CCT)ll, (CCT)12, (CCT)14 and (CCT)16 strongly inhibited IFNa production induced by CpG2216.
• CAL-1/NFKB-GFP cells (l l07well) were pre-incubated for 2 hours with inhibitory-ODNs described previously. The cells were stimulated with TLR7/8 agonist; Gardiquimod or CL264 (Invivogen, USA), for 4 hours. GFP expression level of the cells in each condition was evaluated by flow cytometer (FACS Calibur, BD Bioscience Co.Ltd).
• the percentage of GFP positive cells with Gardiquimod alone was defined as 100% in the graph.
• the percentage of GFP positive in each condition was calculated from the number.
• Fig.7 (B) CAL-1/NFKB-GFP cells were stimulated with TLR7/8 agonist; CL264 ( ⁇ g/ml) for 4 hours in the presence of (CCT)8, (CCT)9, (CCT)IO, (CCT)ll, (CCT)12, (CCT)14 and (CCT)16 (O.OluM, 0.03uM and O. luM). The percentage of GFP positive in each condition was calculated as described previously.
• (CCT)8 showed better inhibitory activity for TLR9 stimulation than (CCT)6 and (CCT)7. Thus, this suggests (CCT)6 and (CCT)7 have unique inhibitory activity for TLR7, but not TLR9, stimulation.
• (CCT)12 efficiently blocked GFP expression by CL264 stimulation and exhibited better inhibitory activity than (CCT)8.
• CCT inhibitory activity
• (CCT)14 and (CCT)16 for TLR7 stimulation was much worse than the activity of (CCT)12.
• (CCT)12 may have maximum efficacy for the inhibition of NF-KB activity, which induced by TLR7 stimulation.
• Our data provide the ODNs we examined can block TLR7 stimulation in human cells. It has been demonstrated that uncontrolled IFN production contributes to the development of SLE (Barrat FJ, et al. J Exp Med 2005; 202: 1131-9; Wellmann U, et al. Proc Natl Acad Sci
• the ODNs we examined can be used as a remedy for the treatment of TLR-mediated disease such as SLE by inhibiting TLR7 or TLR9 activation.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Genetics & Genomics (AREA)
• Immunology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Epidemiology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Rheumatology (AREA)
• Pain & Pain Management (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Transplantation (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (13)

Applications Claiming Priority (1)

Related Child Applications (2)

Publications (1)

Family

ID=50827048

Family Applications (1)

Country Status (10)

Cited By (4)

Families Citing this family (5)

Citations (4)

Family Cites Families (3)

• 2012
  • 2012-11-29 WO PCT/CN2012/085547 patent/WO2014082254A1/en not_active Ceased
  • 2012-11-29 US US14/442,799 patent/US9688992B2/en active Active
  • 2012-11-29 RU RU2015120365A patent/RU2015120365A/ru not_active Application Discontinuation
  • 2012-11-29 JP JP2015544288A patent/JP6106280B2/ja active Active
  • 2012-11-29 SG SG11201504142WA patent/SG11201504142WA/en unknown
  • 2012-11-29 AU AU2012395641A patent/AU2012395641B2/en not_active Ceased
  • 2012-11-29 CA CA2891906A patent/CA2891906A1/en not_active Abandoned
  • 2012-11-29 KR KR1020157014060A patent/KR102027023B1/ko not_active Expired - Fee Related
  • 2012-11-29 MX MX2015006703A patent/MX369618B/es active IP Right Grant
• 2015
  • 2015-05-27 PH PH12015501182A patent/PH12015501182B1/en unknown
• 2017
  • 2017-05-25 US US15/605,488 patent/US10190122B2/en active Active
• 2018
  • 2018-02-14 RU RU2018105485A patent/RU2018105485A/ru not_active Application Discontinuation

Patent Citations (4)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events