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WO1997010840A1 - Medicament antisens anti-inflammatoire - Google Patents

Medicament antisens anti-inflammatoire Download PDF

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Publication number
WO1997010840A1
WO1997010840A1 PCT/JP1996/002682 JP9602682W WO9710840A1 WO 1997010840 A1 WO1997010840 A1 WO 1997010840A1 JP 9602682 W JP9602682 W JP 9602682W WO 9710840 A1 WO9710840 A1 WO 9710840A1
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WIPO (PCT)
Prior art keywords
antisense
inflammatory
seq
oligonucleotide
antisense drug
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PCT/JP1996/002682
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English (en)
Japanese (ja)
Inventor
Shuji Sato
Takeshi Goto
Akira Wada
Yousuke Suzuki
Shinichi Kawai
Yutaka Mizushima
Original Assignee
Hisamitsu Pharmaceutical Co., Inc.
Ltt Institute, Co., Ltd.
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Publication date
Application filed by Hisamitsu Pharmaceutical Co., Inc., Ltt Institute, Co., Ltd. filed Critical Hisamitsu Pharmaceutical Co., Inc.
Priority to AU70004/96A priority Critical patent/AU7000496A/en
Publication of WO1997010840A1 publication Critical patent/WO1997010840A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61K47/59—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P37/04—Immunostimulants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to an anti-inflammatory antisense drug. More specifically, this invention relates to inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, septic shock, Clone disease and AIDS
  • the present invention relates to an antisense drug that specifically blocks gene expression of a physiologically active substance involved in a refractory liver disease or a pathological condition in liver transplantation.
  • an antisense method has been known as a method for controlling the functional expression of chromosomal DNA.
  • This antisense method uses a DNA or RNA that has a base sequence that is partially or wholly complementary to the mRNA (sense strand) transcribed from the chromosome DNA encoding specific protein synthesis information.
  • This method uses A (antisense strand) to block information on protein synthesis from mRNA by utilizing the fact that the sense strand and antisense strand bind to each other through complementarity.
  • antisense DNA is often used as a binding sequence to the sense strand in the examples so far.
  • a targeting site (1) a splicing site, (2) a cabling site. (3) A vicinity of an AUG initiation codon (initiation codon) site is often selected, and in particular, an AUG initiation site. A relatively high antisense effect has been obtained for the codon region.
  • the three-dimensional structure of the mRNA must also be considered, and it is generally thought that antisense DNA is likely to bind to a single-stranded region such as a loop structure or a bulge structure.
  • ⁇ Gene therapy is limited to mono-gene disease, cancer, AIDS, etc. due to problems such as biopsy.
  • antisense DNA can be captured as a compound similar to conventional synthetic drugs, and its effects in vivo as well as in vitro have been reported. Sex search is entering a new research phase.
  • D-oligo has a fatal drawback as a drug that is degraded in a short time by nucleases and the like. For this reason, attempts have been made to chemically modify DNA to increase its biological stability. Among them, phosphorothioate-type nucleotides (hereinafter abbreviated as S-oligo) are particularly stable. Has high biological activity. This type is currently in clinical trials.
  • S-oligo phosphorothioate-type nucleotides
  • DNA or RNA is quantitatively degraded by nucleases (hereinafter abbreviated as DNase and RNase, respectively) and the like, and the blood half-life is extremely short, within 1 minute.
  • DNase and RNase nucleases
  • the mainstream is methylphosphine, in which one of the oxygen atoms of the phosphoric acid group is substituted with S—, which is substituted with an S—oligo CH 3 group.
  • oligonucleotides such as DNA are taken up into cells, and are mainly taken up by endcytosis, and about 8 OkD.
  • the membrane protein in a is considered as the estimated receptor.
  • Modified oligonucleotides are also mainly obtained through end-site analysis. Although the details are unknown, there are still a lot of unknown details, and the transfer amount into cells is still small.
  • a delivery method has been devised to enhance low membrane permeability, but there are still points to be resolved including problems such as toxicity.
  • steroids and non-steroid anti-inflammatory drugs have been widely used in recent years.
  • Steroids significantly improve the symptoms of various inflammatory diseases, but their effects gradually decrease with administration, and side effects include coronary artery insufficiency, peptic ulcer, cataract, sepsis, and susceptibility to infectious diseases.
  • side effects include coronary artery insufficiency, peptic ulcer, cataract, sepsis, and susceptibility to infectious diseases.
  • Non-steroid anti-inflammatory drugs also temporarily suppress inflammatory symptoms, but do not fundamentally cure inflammatory diseases. Therefore, at present, there is a demand for the development of a therapeutic agent for inflammatory diseases which has high efficacy, sustains its therapeutic effect and is highly safe.
  • rheumatoid arthritis is an unexplained chronic inflammatory disease with the synovium as the main lesion.
  • the affected area sometimes does not stop at the joint synovium, and the inflammation that initially develops in the synovium causes the destruction of bones and bones, eventually leading to the destruction of the whole body.
  • Empirical factors are strongly involved in the treatment of osteoporosis, and non-steroid anti-inflammatory analgesics have been used as first-line drugs.
  • non Suteroi de anti-inflammatory agent in the treatment of a chronic rheumatoid Umachi is analgesic effect by c its administration in shrinking can be expected, antirheumatic effect on non-steroid anti-inflammatory drugs It has become common knowledge that there is no clinical benefit, and it has become clear that the side effects of non-steroid anti-inflammatory analgesics, such as gastrointestinal disorders and renal dysfunction, cannot be ignored clinically. It is.
  • TNF Tumor Necrosis Factor
  • TNF is now being understood as a site that is involved in biological defense reactions through inflammation, which was initially found as a substance that damages tumors.
  • Genes of TNF have been clearly identified in a wide range of mammals, including humans, bushes, porcupines, magpies, and mice, and their primary structures have also been determined. According to this, the amino acid sequence between each animal has a homology of around 80% conserved, suggesting that TNF is an extremely important physiologically active substance in living organisms.
  • the human TNF precursor has 233 amino acid residues, and is composed of 155 or 157 amino acids in the mature form. Although its molecular weight is 17 kDa, it forms a 45 kDa trimer in vivo. It is thought that sugar chains are present in mouse TNF ⁇ , which is not present in humans. In mouse TNF, sugar chains are not essential for the expression of their activities.
  • TNF is expressed by inoculating BCG-sensitized animals with lipopolysaccharide (hereinafter abbreviated as LPS).
  • LPS lipopolysaccharide
  • macrophages can be prepared for TNF production by various methods, and TNF peaks at around 2 hours by an appropriate triggering stimulus (eg, bacterial cells—cell components such as LPS). Can be produced.
  • human Bok macro Roff Aji system cells e.g., U937 strain, etc.
  • TNF acts on a wide variety of cells.
  • TNF produced from macrophages acts on neutrophils and vascular endothelial cells, leading to the development of inflammation from the outset. Then, by acting on fibroblasts and hepatocytes, the inflammation ends and goes toward repair. The inflammatory response progresses in a way that many cells and mediators interact with each other, and usually disappears at a constant rate.However, if there is a substance to be an antigen and its amount is more than a certain level, the information Is then passed on to the immune system. TNF is thus also involved in the transition from a non-specific host defense response to a specific defense response. In addition, TNF is known to act on, for example, osteoblasts, osteoclasts, adipocytes, epithelial cells, pituitary gland, and especially synovial cells.
  • the pathogenesis of inflammatory diseases such as rheumatoid arthritis includes various immune response systems-abnormalities of inflammatory reactions.
  • TNF- ⁇ other than TNF- ⁇ , one of TNF.
  • oncogenes such as c-fos, cytokins such as human interleukin-1 ⁇ (hereinafter abbreviated as IL-1 ⁇ ) and IL-16 are involved.
  • IL-1 ⁇ human interleukin-1 ⁇
  • IL-16 human interleukin-1 ⁇
  • PG prostaglandin
  • the role of prostaglandin (hereinafter abbreviated as PG) and its synthase has been attracting attention as one of the mediators of inflammatory diseases. That is, since non-steroidal anti-inflammatory analgesics such as aspirin were reported by Vane et al.
  • C0X cycloxygenase
  • COX-2 gene is different from COX-1 in that it is an enzyme newly produced by stimulation of IL-11.
  • C 0 X-1 in macrophages is invariable by LPS stimulation, and is detected at a constant concentration regardless of stimulation or inflammation.
  • 2 has almost no evidence of its gene, and its expression is increased by LPS stimulation, and its expression is completely suppressed by dexamethasone.
  • the cDNA of human C0X-2 has been cloned, which is elucidating the regularity of C0X-2 development in inflammatory cells.
  • the greatest effect can be expected when the etiology corresponds to the genes on a 1: 1 basis due to the characteristics of the antisense method, rather than multiple genes that cause the disease. Therefore, in order to apply the antisense method to the treatment of inflammatory diseases, it is essential to identify the causative gene as well as accurately understand the pathology. In addition, it is necessary to identify the expression mechanism of the gene and to select an appropriate site for shutting down the expression. Disclosure of the invention
  • the present invention has been made in view of the circumstances described above, and includes inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, and septicemia.
  • the purpose of the present invention is to provide a new anti-inflammatory drug containing antisense DNA as a main component that specifically blocks the expression of a physiologically active substance involved in shock, Crohn's disease, AIDS, and the like.
  • the present invention provides, as a first invention for solving the above-mentioned problems, a synthetic polyamino acid or a derivative thereof, and an mRNA encoding a physiologically active substance involved in a human inflammatory disease.
  • the present invention provides an anti-inflammatory antisense drug comprising a complex with an antisense 'oligonucleotide complementary to a part or the entire nucleotide sequence of the anti-inflammatory antisense drug.
  • the synthetic polyamino acid is a nucleic acid conjugate comprising a repeating sequence of a lysine residue and a serine residue, and derivatives thereof.
  • the preferred embodiment is a block modification of polyethylene glycol (hereinafter abbreviated as PEG) of synthetic polyamino acid.
  • the present invention provides, as a second invention, a synthetic polyamino acid or a derivative thereof, which is complementary to a part or the whole nucleotide sequence of mRNA encoding human IL-1 / 3.
  • An anti-inflammatory agent comprising a complex with a nucleic acid or an oligonucleotide.
  • an antisense oligonucleotide complementary to a part or the entire nucleotide sequence of mRNA encoding IL-1 / 3 is represented by SEQ ID NO: 2.
  • the preferred embodiment is an oligonucleotide having a part or the entire nucleotide sequence of 4.
  • a synthetic polyamino acid or a derivative thereof and an antisense oligonucleotide complementary to a part or the entire nucleotide sequence of human TNF-encoding mRNA comprising an anti-inflammatory antisense drug comprising a complex with leotide.
  • the human TNF is human TNF- ⁇ , and a part or all of the mRNA encoding the human TNF- ⁇ is complementary to the entire nucleotide sequence.
  • the antisense oligonucleotide is a polynucleotide having part or all of the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. This is the preferred mode.
  • the synthetic poly A Mi Roh acid or a derivative thereof, a part of a series of PGE 2 synthase co one sul mRNA A properly is complementary to the entire nucleotide sequence
  • An anti-inflammatory antisense drug comprising a complex with an antisense oligonucleotide is provided.
  • the gonionucleotide is an oligonucleotide having a part or the entire nucleotide sequence of SEQ ID NO: 12.
  • FIG. 1 shows the chemical formula (a) of PLS and the chemical formula (b) of PLSP that can be used in the present invention.
  • FIG. 2 is a graph showing the effect of suppressing the production of IL-1 / 3 by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and a specific example.
  • FIG. 3 is a graph showing the IL-13 production inhibitory effect of an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 4 and a comparative example.
  • Figure 4 shows anti-inflammatory antisense drugs using the DNA strand of SEQ ID NO: 2 and comparison It is a graph which shows the production inhibitory effect of IL-11 ⁇ by an example.
  • FIG. 5 is a graph showing the effect of suppressing the production of TNF- ⁇ by an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 6 and a comparative example.
  • FIG. 6 is a graph showing the effect of suppressing the production of TNF- ⁇ by an anti-inflammatory antisense drug using the D ⁇ chain of SEQ ID NO: 8 or 10, and a comparative example.
  • FIG. 7 is a graph showing the effect of suppressing the production of TNF- ⁇ by an anti-inflammatory antisense drug using the D ⁇ chain of SEQ ID NO: 6 and a comparative example.
  • FIG. 8 is a graph showing the amount of PGE 2 produced by an anti-inflammatory antisense drug using the D ⁇ chain of SEQ ID NO: 11 or 12 and a control.
  • FIG. 9 is a graph showing the uptake amount of an anti-inflammatory antisense drug into U933 cells using the DNA chain of SEQ ID NO: 11 or 12.
  • FIG. 10 is a graph showing the amount of an anti-inflammatory antisense drug incorporated into synovial cells using the DNA chain of SEQ ID NO: 11 or 12.
  • FIG. 11 is a graph showing the relationship between the dose of the anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 and the effect of suppressing lethality in an endotoxin-induced shock model by a comparative example.
  • FIG. 12 is a graph showing the relationship between the lethal inhibitory effect of an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 on an endotoxin-induced shock model and usage.
  • FIG. 13 is a graph showing the relationship between the lethal inhibitory effect of an anti-inflammatory antisense drug using the DNA chain of SEQ ID NO: 2 on an endotoxin-induced shock model and the administration route.
  • FIG. 14 is a graph showing the difference in the effect of the anti-inflammatory antisense drug using the DNA strands of SEQ ID NO: 2 and SEQ ID NO: 6 on the endotoxin-induced shock model against lethality. .
  • Nucleic acid composites of synthetic polyamino acids are composed of irregular or regular repetitions of water-soluble amino acid serine residues and cationic amino acid lysine residues.
  • Serine The molar ratio of the residue to the lysine residue is about 1: 1 and its molecular weight is about 3000-50,000.
  • Such a polyamino acid is, for example, a polylysine: serine (hereinafter, abbreviated as PLS) which forms a complex in a homogeneous system with an oligonucleotide.
  • PLS polylysine: serine
  • Patent WO95 / No. 0909 can be used.
  • the above-mentioned modified PLS PEG block (hereinafter abbreviated as PLSP) can be exemplified as a novel one.
  • PLSP modified PLS PEG block
  • the structures of PLS and PLSP can be exemplified as, for example, the chemical formulas in FIGS. 1 (a) and 1 (b).
  • antisense 'oligonucleotides which are complementary to mRNA encoding bioactive substances involved in inflammatory diseases, or synthetic polyamino acids such as PLS or the like.
  • Oligonucleotides modified with derivatives can be prepared by known methods (Rajendra, BR et al., Human Genetics. 55, 3633, 1980, Lira, F. and Sun, A., M. , Science, 210, 908, 1980).
  • Oligonucleotides of SEQ ID NOS: 1 to 15 in the sequence listing were synthesized using a DNA synthesizer (Applied Biosystems, Inc. type 380B). Oligonucleotide was synthesized based on the phosphoramidite method (Nucleic Acid Res., Vol. 17, 7059-7071, 1989) using a t-butylmethylsilyl group as the protecting group for the 2'-hydroxyl group. Purification of the compound was performed according to the method described in the literature (Nucleic Acid Res., Vol. 19, 5125-5130, 1991).
  • SEQ ID NO: 1 is a known 20-nucleotide oligonucleotide, including the initiation codon of IL-11 / 9 gene, one of the physiologically active substances of rheumatoid arthritis. It is a sense DNA strand corresponding to 0 bases, and SEQ ID NO: 2 is complementary to this. Typical antisense DNA strand.
  • SEQ ID NO: 3 is a sense DNA strand corresponding to 20 bases including the untranslated region of the same IL-11 gene, and SEQ ID NO: 4 is an antisense DNA strand complementary thereto (particularly, Kaihei 6 — 4 1 1 8 5).
  • SEQ ID NO: 5 is a sense D-chain corresponding to 20 bases including the initiation codon of the TNF-gene, which is also one of the physiologically active substances of rheumatoid arthritis
  • SEQ ID NO: 6 is The complementary antisense DNA sequence
  • SEQ ID NO: 7 has a nucleotide sequence corresponding to 20 nucleotides (the TNF- ⁇ gene sequence at positions 1624 to 1643) containing the TNF- ⁇ gene splicing site.
  • SEQ ID NO: 8 is a complementary antisense DNA strand
  • SEQ ID NO: 9 is 20 bases including the TNF- ⁇ gene splicing site (TNF- ⁇ gene sequence 2161- SEQ ID NO: 10 is the complementary antisense DNA strand.
  • SEQ ID NO: 11 is a sense DNA strand corresponding to a 20-base region containing the initiation codon of COX-2 involved in the immune mechanism of inflammation such as human rheumatoid arthritis.
  • SEQ ID NO: 12 is the complementary antisense DNA strand (also SEQ ID NO: 13 contains the initiation codon of the mouse ⁇ L-11 ⁇ gene.
  • SEQ ID NO: 14 is the sense strand corresponding to 0 bases, and SEQ ID NO: 14 is the complementary antisense D ⁇ C strand, and SEQ ID NO: 15 is the mouse TNF- ⁇ ⁇ gene initiator.
  • Example 1 An antisense strand corresponding to 20 bases including a don.
  • One-terminal methoxy One-terminal amino group polyethylene oxide (molecular weight: 5000, manufactured by NOF Corporation) 4.0 g was dissolved in 15 ml of chloroform, and the solution was dissolved in ⁇ -carbobenzoxylidine.
  • N-one N—Carbo Acid anhydride and benzyl serine-N-potassium sulfonic acid were added to the solution.
  • the reaction mixture was dropped into 330 ml of getyl ether, and the precipitated polymer was collected by filtration, washed with getyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), and then vacuumed.
  • Poly-lysine a copolymer of serine (PLS: Sigma) or PLSP synthesized in Example 1 and purified Z and either antisense 'oligonucleotide (SEQ ID NOS: 2 and 4) Were formed by a known method.
  • an ultrafiltration tube (Nippon Millipore Limited): Ultrafiltration C3-GC UFC3 TGC 00 filter with membrane fractionation ability through which the formed complex does not pass
  • a mixture of antisense oligonucleotide and PLS or PLSP was placed in a centrifuge tube) and centrifuged.
  • the oligonucleotides were added so that the concentrations were 1 nM, 100 nM, and 100 nM.
  • the oligonucleotide of SEQ ID NO: 4 was also prepared at a concentration of 10 M.
  • antisense-PLS oligonucleotide complex
  • PLSP oligonucleotide complex
  • antisense-PLSP oligonucleotide complex
  • the concentration of the oligonucleotide was determined from the absorbance at 260 nm. As a result, it was confirmed that the oligonucleotides in the formed complex had the above concentrations. Also, the concentration of each charge neutralization in the formation of the ionic complex depends on PLS or PLS. Was calculated from the number of charges obtained from the molecular weights of PLSP and oligonucleotides. In addition, it was confirmed that each complex of antisense-PLS and antisense-PLSP is uncharged by charge-coupled electrophoresis (multi-channel electrophoretic device: CAPI-3000, MCPD-3600 SPECT). O MULTI CHANNEL DETECTOR equipment, manufactured by Otsuka Electronics Co., Ltd.) As a result, the peak of the complex was the same as the peak of uncharged phenylalanine.
  • the inhibitory effect of the antisense-PLS and antisense-PLSP complex prepared in Example 2 on IL-1 / 3 production was examined in a cultured cell line.
  • Human macrophage U933 cells (Dainippon Pharmaceutical Co., Ltd.) were used as cells. Cell culture was performed using 10% FCS (Fetal Calf Serum: Sanko Junyaku), 100 unit Zml of penicillin (Life's Technology), and 1 OOug Zml. be sampled replica Bok My Thin using the RPMI medium (Nikken made by Institute for biomedical Research), including the (line-off ⁇ made Techno Russia di one company), under the conditions of 3 7 ° C, 5% C 0 2 went. The number of cultured U933 cells was visually counted after staining with trypable.
  • FCS Fetal Calf Serum: Sanko Junyaku
  • penicillin Life's Technology
  • 1 OOug Zml be sampled replica Bok My Thin using the RPMI medium (Nikken made by Institute for biomedical Research), including the (line-off ⁇ made Techno Russia di one company), under the conditions of 3 7 ° C, 5% C 0 2
  • the mixture was sufficiently frozen at 170 ° C., and then thaw-thawed three times, and 2501 of the supernatant was gently collected.
  • ELISA kit (1L-1 / S ELISA System: manufactured by Amersham), and then subjected to data analysis using a micro-mouth plate reader (manufactured by Bio-Rad).
  • the complex of the anti-inflammatory antisense drug of the present invention in particular, the antisense DNA (SEQ ID NO: 2) against the initiation codon of the IL-1 ⁇ gene and PLS or PLSP was obtained as shown in FIG.
  • the production of IL-1 / 3 was suppressed by 100%. That is, at an antisense DNA concentration of 100 nM, 100% of IL-1 production was inhibited, at a concentration of 100 nM, 50% of IL-1 ⁇ production was inhibited, and at a concentration of 1 ⁇ , 2%. 0% IL-11 production was suppressed.
  • Example 3 at a concentration of 10 M, IL- IL production was inhibited by about 40%.
  • Example 2 In the same manner as in Example 3, IL of the complex of antisense DNA (SEQ ID NO: 2) and ribofectin (manufactured by Gibco) (hereinafter, abbreviated as antisense-livofectin) was used. The effect of inhibiting 13 production was measured. The results are as shown in FIG. This lipofectin has been highly evaluated as a carrier for oligonucleotides, and antisense lipofectin has a considerably higher antisense concentration (50%). M), the effect of inhibiting ⁇ L-1 / 3 production was about 20%.
  • Antisense drugs against TNF- ⁇ were prepared in the same manner as in Example 2 except that the antisense 'oligonucleotides of SEQ ID NOs: 6, 8 and 10 were used. PLS).
  • Example 5 For these antisense drugs, turbidity and precipitate formation, the concentration of the oligonucleotide, the charge thereof, and the like were measured in the same manner as in Example 2.
  • Example 5 For these antisense drugs, turbidity and precipitate formation, the concentration of the oligonucleotide, the charge thereof, and the like were measured in the same manner as in Example 2.
  • TNF- ⁇ production inhibitory effect of the antisense-PLS and antisense-PLS S complex prepared in Example 4 was examined using the same method as in Example 3.
  • the complex of antisense DNA (SEQ ID NO: 6) to the initiation codon of the TNF- ⁇ gene and PLSP When the concentration of DNA was 100 OpM, the production of TNF- ⁇ was suppressed by 80%, and at a concentration of 10 ⁇ , it was suppressed by 60%, but at a lower concentration, TNF- ⁇ was suppressed. No production suppression was observed. Remarkably, within the same low concentration range, remarkable angiogenesis specificity was also observed in the antisense strand and the sense strand. As a synthetic polyamino acid forming a complex with an antisense 'oligonucleotide, PLSP showed a higher TNF- ⁇ production inhibitory effect than pLS.
  • Example 6 In the same manner as in Example 5, the effect of the complex of antisense DNA (SEQ ID NO: 6) and lipofectin (manufactured by Gibco) on the inhibition of TNF- ⁇ production was measured. The results are as shown in FIG. 7. Antisense-lipofectin had an inhibitory effect on TNF- ⁇ production of about 20% even though the antisense concentration was very high (50%). In addition, there was no difference in the binding specificity of the oligonucleotide c . Further, although not shown in FIG. 7, the S-antisense (SEQ ID NO: 6) was obtained in the same manner as in Example 5. ) was also measured for its inhibitory effect on TNF- ⁇ production. As a result, this S-antisense had an inhibitory effect on TNF- ⁇ production of about 20% even at a concentration of 10%. Example 6
  • An antisense to C0X-2-PLSP was prepared in the same manner as in Example 2 except that the antisense 'oligonucleotide of SEQ ID NO: 11 was used.
  • human synovial cells prepared from tissue resected from a knee joint of a rheumatoid patient were used, and the culture was performed under the same conditions as the U933 cells in Example 3. The number of cells was counted.
  • Antisense DNA strands and antisense—PLSPs are available in several concentrations from 1 nM to 10 M (final concentration). After 2 hours of incubation, collect the supernatants 301 and dilute if necessary, then use the PGE 2 ELISA kit (Nippon Perceptive Limited, No. 8-6801) Was measured.
  • a complex (hereinafter referred to as A) of the S-antisense and gene transfer primers of Comparative Example 1 (Wako Pure Chemical Industries, Ltd .: Code 074-03621) and an antisense DNA chain was used. (Abbreviated as gene transfer).
  • Genetransfer is a ribosome-based carrier, and has been highly evaluated as an oligonucleotide carrier.
  • antisense-PLSP the anti-inflammatory antisense drug of the present invention
  • the antisense DNA strand was labeled by the following method.
  • DNA (1.830 D26; unit, 5 pmol) 27.3 ⁇ 1, autoclaved distilled water 15.71, 10X kinase buffer ( 25 OmM tris) HCl buffer: p H 7. 6), l OO mM DTT, T 4 polynucleotidyl click Rare Ichize solution (1 0 0 unit / / 1), 7 one 32 P - after mixing the ATP 1 ⁇ 1, The reaction was performed at 37 ° C for 1 hour.
  • T 4 polynucleotide (Code No. 2030, manufactured by Takara Shuzo) was used for labeling with 32 P.
  • Human macrophage U933 cells similar to those in Example 2 and human synovial cells similar to those in Example 7 were used as cultured cells. Under the same conditions as in Example II, 1 ⁇ 10 ⁇ 7 ml of U933 cells were cultured in a glass tube (Falcon's 25058, 6 ml—12X75). For human synovial cells, add 50 1 of antisense-PLSP solution adjusted to the desired concentration, and incubate.
  • Example 7 3 ml of the same PBS solution as used in Example 7 was added to the cells adhered to the bottom of the tube, and the cells were washed twice, followed by 1% SDS (sodium dodecyl sulfate). The cells were lysed by adding 500 51. Finally, a 10 ml hyperflora solution was added to all of the lysates, and measurements were taken overnight at the liquid scintillator. Also, as a control, S-antisense and antisense-transfer were measured in the same manner as in Example 7.
  • SDS sodium dodecyl sulfate
  • Antisense-PLSP against mouse IL-1 / 3 and mouse TNF- ⁇ in the same manner as in Example 2 except that the oligonucleotides of SEQ ID NOS: 13, 14 and 15 were used.
  • Anti-sense-PLSP was prepared, and its lethal effect on endotoxin-induced shock was examined using model mice.
  • Antisense-PLS II solution 2001 prepared to the desired concentration with physiological saline, was injected into the tail vein of male BALBZc mice (Japan SLC: approx. 20 g) at the age of 8 to 10 weeks. did. Immediately afterwards, endotoxin (LPS, Lot No. 68692 W. E. col i055: B5. Difco manufactured by physiological saline solution corresponding to 20 mg / kg) Solution 2
  • mice 0 1 was administered intraperitoneally to mice, and the number of surviving individuals was counted over time.
  • antisense-PLSP to 1L-13 When antisense-PLSP to 1L-13 was used, the survival rate of the mouse increased depending on the concentration of the antisense drug administered. In particular, when antisense-P L SP was administered at a concentration of 10 O mg / kg, no deaths were observed for 40 hours.
  • antisense-PLSP at a concentration of 1 O mgZ kg was administered once and antisense-PLSP at a concentration of 5 mgkg was administered twice.
  • the effects of separate administration total of 10 mg / kg
  • the ability to administer the antisense drug of the present invention in two divided doses at 12-hour intervals was less than that of single administration. It turned out to be effective.
  • FIG. 13 shows the difference between the effects of administration of antisense-PLSP in the tail vein and intraperitoneal administration.
  • Antisense-PLSP did not show a lethal inhibitory effect when administered intraperitoneally, but showed an excellent lethal inhibitory effect when administered in the tail vein. It was recognized that it caused a difference in the effect.
  • an anti-inflammatory antisense drug which binds to sense RNA in a primary structure-specific manner and exerts a sufficient effect of suppressing a physiologically active substance in a very low concentration region.
  • inflammatory diseases such as rheumatoid arthritis, periodontitis, nephritis, ulcerative colitis, arteriosclerosis, psoriasis, diseases such as septic shock, Crohn's disease and AIDS, or intractable diseases
  • Effective treatment for hepatic disease and pathological conditions due to liver transplantation is possible.
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid Sequence type: Other nucleic acid Synthetic DNA
  • Human TNF Sequence containing the splicing site (positions 1624 to 1643) of the ⁇ gene
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Mouse TNF 20-base antisense chain containing the initiation codon for the ⁇ ⁇ gene

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Abstract

La présente invention concerne un médicament antisens anti-inflammatoire qui comprend un complexe d'un acide polyaminé synthétique constitué par des séquences répétées de restes de lysine et de sérine ou de modifications de ces substances par le polyéthylène glycol, et un oligonucléotide antisens complémentaire de la séquence de base partielle ou totale d'un ARNm codant des substances physiologiquement actives qui participent à des affections inflammatoires humaines, telles que l'interleukine-1β, le facteur de nécrose des tumeurs ou l'α-cyclooxygénase. Ce médicament permet un traitement efficace d'affections inflammatoires telles que l'arthrite rhumatoïde, la parodontite, la néphrite, la colite ulcéreuse, la sclérose artérielle et le psoriasis, le choc septique, la maladie de Crohn, le SIDA, les affections hépatiques réfractaires, les pathologies liées à la greffe du foie, etc.
PCT/JP1996/002682 1995-09-18 1996-09-18 Medicament antisens anti-inflammatoire WO1997010840A1 (fr)

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JP7/238583 1995-09-18
JP23858395 1995-09-18
JP7/278184 1995-10-25
JP27818495 1995-10-25
JP8/149598 1996-06-11
JP8149598A JPH09176038A (ja) 1995-09-18 1996-06-11 抗炎症性アンチセンス薬物

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
US6080580A (en) * 1998-10-05 2000-06-27 Isis Pharmaceuticals Inc. Antisense oligonucleotide modulation of tumor necrosis factor-α (TNF-α) expression
US6228642B1 (en) * 1998-10-05 2001-05-08 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of tumor necrosis factor-(α) (TNF-α) expression
WO2002053185A3 (fr) * 2001-01-05 2002-10-31 Intercell Biomedizinische Forschungs & Entwicklungs Gmbh Utilisations de composes polycationiques
US7172769B2 (en) 1999-12-08 2007-02-06 Pharmacia Corporation Cyclooxygenase-2 inhibitor compositions having rapid onset of therapeutic effect
US7244438B2 (en) 2001-01-05 2007-07-17 Intercell Ag Uses for polycationic compounds

Families Citing this family (3)

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Publication number Priority date Publication date Assignee Title
JPH11335269A (ja) 1998-05-19 1999-12-07 Hisamitsu Pharmaceut Co Inc 遺伝子関連医薬の経口投与固形製剤
EP1883427A4 (fr) * 2005-01-20 2010-04-21 Univ Rochester Compositions et methodes permettant d'etudier et de traiter des maladies et des troubles inflammatoires
TWI489982B (zh) * 2012-09-18 2015-07-01 Univ China Medical 苯醌類化合物應用於抑制動脈粥狀血管硬化之用途

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DE4341471A1 (de) * 1993-12-02 1995-06-08 Schering Ag Tumor-Nekrose-Faktor-alpha-inaktivierende CDR-Peptide
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6080580A (en) * 1998-10-05 2000-06-27 Isis Pharmaceuticals Inc. Antisense oligonucleotide modulation of tumor necrosis factor-α (TNF-α) expression
US6228642B1 (en) * 1998-10-05 2001-05-08 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of tumor necrosis factor-(α) (TNF-α) expression
US7172769B2 (en) 1999-12-08 2007-02-06 Pharmacia Corporation Cyclooxygenase-2 inhibitor compositions having rapid onset of therapeutic effect
WO2002053185A3 (fr) * 2001-01-05 2002-10-31 Intercell Biomedizinische Forschungs & Entwicklungs Gmbh Utilisations de composes polycationiques
US7244438B2 (en) 2001-01-05 2007-07-17 Intercell Ag Uses for polycationic compounds

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