WO2007123010A1 - Agent pour améliorer le métabolisme osseux - Google Patents

Agent pour améliorer le métabolisme osseux Download PDF

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Publication number
WO2007123010A1
WO2007123010A1 PCT/JP2007/057793 JP2007057793W WO2007123010A1 WO 2007123010 A1 WO2007123010 A1 WO 2007123010A1 JP 2007057793 W JP2007057793 W JP 2007057793W WO 2007123010 A1 WO2007123010 A1 WO 2007123010A1
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Prior art keywords
cthrcl
bone
mrna
joint disease
preventive
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PCT/JP2007/057793
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English (en)
Japanese (ja)
Inventor
Kyoji Ikeda
Sunao Takeshita
Hiroyuki Aburatani
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Japan Health Sciences Foundation
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Priority to JP2008512064A priority Critical patent/JPWO2007123010A1/ja
Publication of WO2007123010A1 publication Critical patent/WO2007123010A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders

Definitions

  • the present invention relates to a bone metabolism improving agent, a bone / joint disease prevention or treatment agent, a bone / joint disease diagnosis kit, a bone 'joint disease diagnosis method, and a bone' joint disease prevention or treatment screening kit.
  • the present invention relates to a method for screening a preventive or therapeutic agent for bone 'joint disease, or a method for preventing or treating bone' joint disease.
  • Osteoporosis bone associated with cancer, calcium metabolism abnormalities, rheumatoid arthritis, Paget's disease, etc. • Joint diseases have become a major problem in the aged society. These diseases include osteoclasts, It is thought that abnormal bone metabolism consisting of osteoblasts is involved.
  • osteoporosis is caused by an unbalance between excessive bone resorption and relatively unreachable bone formation.
  • osteoclast inhibitors have been used to treat osteoporosis.
  • the current treatment is mainly focused on the suppression of bone resorption by bisphosphonate drugs.
  • Bisphosphonates are effective in maintaining bone density by prolonging the bone mineralization (secondary mineralization) process.
  • osteoblasts bone metabolism is established by maintaining a turnover that repeats bone resorption by osteoclasts and bone formation by osteoblasts (osteoblasts).
  • Non-patent Document 1 Non-patent Document 1
  • the preceding bone resorption is indispensable. If resorption is suppressed by a bisphosphonate drug, bone formation cannot be promoted even if PTH is injected. It turned out that.
  • Coupling factors or substances that enhance their production or function, are expected to maintain good quality bone by stimulating physiological bone metabolism.
  • Cthrcl is a known protein that is mainly expressed in vascular smooth muscle, lung, and brain, but is also expressed in bone tissue and is involved in some form of bone growth. For example, it is shown in Patent Document 1 below!
  • Patent Document 1 US2005 / 0147602A1 (2005.7.7)
  • Non-Patent Document 1 N Engl J Med 349: 1207-15, 2003 [0011]
  • Cthrcl overexpression inhibits bone formation and reduces bone strength and density, and antibodies against Cthrcl in the treatment of osteoporosis. This suggests the possibility of applying Cthrcl inhibitors such as (see [0705] etc.)
  • Cthrcl works to promote bone regeneration and prevent destruction and vulnerability by promoting coupling to bone resorption formation. It is clear that the overexpression of selenium suppresses the differentiation of osteoclasts and acts in the direction of inhibiting bone resorption.
  • a group force consisting of Cthrcl, Cthrcl analogs, or genes encoding them, their mRNAs, antibodies to Cthrcl, genes encoding Cthrcl and polynucleotides capable of forming complementary pairs with Cthrcl mRNA is also selected.
  • An agent for improving bone metabolism comprising one or more.
  • a group force consisting of Cthrcl, a Cthrcl analog, or a gene encoding these, its mRNA, an antibody to Cthrcl, a gene encoding Cthrcl and a polynucleotide capable of forming a complementary pair with Cthrcl mRNA is also selected.
  • a preventive or therapeutic agent for bone and joint diseases characterized by comprising one or more.
  • a bone / joint disease diagnostic kit comprising the following (1) or (2):
  • Biomechanical force A diagnostic method for bone and joint diseases, characterized by using Cthrcl or its mRNA in a collected sample as an index.
  • a screening kit for a bone or joint disease preventive or therapeutic agent comprising at least one selected from the group consisting of osteoclasts, Cthrcl, a gene encoding Cthrcl, or an mRNA thereof.
  • a screening method for a preventive or therapeutic agent for bone / joint diseases characterized by using Cthrcl, a gene encoding Cthrcl, or mRNA thereof as an index.
  • Group 1 consisting of an antibody against 1, a gene encoding Cthrcl and a polynucleotide capable of forming a complementary pair with Cthrcl mRNA Prevention or treatment of bone and joint diseases characterized by using at least one or more selected Method.
  • the bone resorption ability promotes the coupling to bone formation, thereby promoting the physiological regeneration of the bone and maintaining the normal metabolism of the bone.
  • Agent, bone and joint disease prevention or treatment agent, bone / joint disease diagnostic kit using Cthrcl, bone / joint disease A method for diagnosing a disease, a screening kit for a preventive or therapeutic agent for bone / joint disease using Cthrcl, a screening method, and a method for preventing or treating bone / joint disease can be obtained.
  • the bone metabolism improving agent of the present invention the preventive or therapeutic agent for bone 'joint disease, and the preventive or therapeutic method for bone' joint disease>
  • the bone metabolism improving agent and the preventive or therapeutic agent for bone and joint diseases of the present invention are those that promote Cthrcl increase calorie and promote bone metabolism (Cthrcl, Cthrcl analogs, or genes encoding them) , Its mRNA, etc.), and those that suppress Cthrcl and suppress excessive bone metabolism (antibodies to Cthrcl, genes encoding Cthrcl, and polynucleotides that can form complementary pairs with Cthrcl mRNA).
  • Cthrcl (collagen triple helix repeat containing 1) used in the present invention is a known protein, and is also described in US2005 / 0147602A1 (2005.7.7) and the like.
  • the sequence of human Cthrcl is represented by SEQ ID NO: 1
  • the gene sequence encoding human Cthrcl is represented by SEQ ID NO: 2 (see Accession No. NM_138455 etc.).
  • the mouse Cthrcl is represented by SEQ ID NO: 3
  • the gene sequence encoding mouse Cthrcl is represented by SEQ ID NO: 4 (see Accession No. AK_003674 and the like).
  • it is preferable to use human Cthrcl or a gene encoding Cthrcl because it is less likely to cause side effects.
  • FIG. 5 compares the Cthrcl amino acid sequences of mouse (m) and human (h).
  • the arrow is the place where it undergoes degradation during secretion, and the N-terminal side shows a hydrophobic signal sequence.
  • the signal sequence is a sequence involved in protein localization. The remaining sequence after separation of this hydrophobic signal sequence functions as mature Cthrcl.
  • Cthrcl includes mature Cthrcl.
  • the Cthrcl used as a bone metabolism improving agent, bone / joint disease preventive or therapeutic agent of the present invention is also a mature Cthrcl. Is included.
  • the portion enclosed by a square indicates a portion where the amino acid differs between mouse and human in the mature type (from the arrow to the C-terminus).
  • the underline indicates the collagen area, and the dotted line in it Minute (GEKGEC) indicates the region expected to undergo sugar chain modification.
  • the inverted triangle indicates a cysteine residue.
  • the homology is 93.9% overall and 97.7% in mature form.
  • the Cthrcl analog-like compound used in the present invention is a protein in which a part of the amino acid sequence of Cthrcl is substituted, deleted, added, inserted, etc., and has the same activity as Cthrcl (osteoblasts).
  • Cthrcl modified protein that has a function of promoting osteogenesis by enhancing its differentiation or proliferation function
  • pharmaceutically acceptable salts of Cthrcl and Cthrcl modified proteins organic acid salts, inorganic acids Salts, metal salts, etc.
  • ethers and esters thereof are examples of Cthrcl and esters thereof.
  • a signal sequence region can be considered as a position where amino acid sequence substitutions “deletion / addition” can be inserted.
  • the signal sequence is also a force that functions as Cthrcl even though there are 10 differences between humans and mice.
  • substitution / deletion / addition / insertion, etc. may be possible for the same reason at different positions between human Cthrcl and human Cthrcl.
  • the collagen region may also play an important role in the Cthrcl protein, the substitution 'deletion' and 'addition' and insertions include amino acid sequence regions other than that region. It seems to be suitable, but it does not exclude collagen region modifications.
  • the number of substitutions, deletions, additions, insertions, and the like may be one or more, but it is preferable that the number is one or less because it is highly likely to exhibit the same function as Cthrcl. .
  • amino acid residues are substituted with an amino acid chemically or structurally similar to the amino acid because the possibility of exerting the function of Cthrcl is high.
  • amino acid substitutions that are chemically or structurally similar that is, highly conservative, specific embodiments of amino acid substitutions are widely known by those skilled in the art!
  • glycine is proline (Pro), alanine (Ala) and parin (Val), leucine (Leu) is isoleucine (lie), glutamic acid (Glu) is glutamine (Gin), and aspartic acid ( Asp) is asparagine (Asn), cystine (Cys) is threonine (Thr), Thr is serine (Ser) and Ala, and lysine (Lys) is arginine (Arg).
  • the present invention is not necessarily limited to this.
  • Cthrcl and Cthrcl-related compounds are related to bone metabolism abnormalities (bones) It can be used as an agent for improving (inhibition of formation) or as a prophylactic or therapeutic agent for bone and joint diseases involving abnormal bone metabolism (inhibition of bone formation).
  • the antibody against Cthrcl used in the present invention may be any of monoclonal, polyclonal, chimeric antibody and humanized antibody.
  • Cthrcl-decomposed subunits and antibodies to multimers such as dimers are also included. Examples of the subunit include those listed in SEQ ID NOS: 11 and 13 in Patent Document 1, for example.
  • These antibodies against Cthrcl can be used as a metabolic improving agent in the case of excessive Cthrcl, or as a prophylactic or therapeutic agent for bone / joint diseases caused by excessive Cthrcl.
  • the gene encoding Cthrcl or Cthrcl-related compound used in the present invention is a gene encoding Cthrc 1 or Cthrc 1 modified protein.
  • gene in the present invention includes not only DNA but also cDNA prepared from mRNA using reverse transcriptase V.
  • Cthrcl or Cthrcl analogs are produced in vivo, and these are abnormal bone metabolism (bone formation due to lack of Cthrcl). It functions as a preventive or therapeutic agent for bone and related diseases in which abnormal bone metabolism (inhibition of bone formation) is involved.
  • the polynucleotide that can form a complementary pair with Cthrcl encoding gene or Cthrcl mRNA means a polynucleotide that can form a complementary pair with Cthrcl DNA or mRNA to the extent that Cthrcl expression can be suppressed, Specific examples include the following.
  • a polynucleotide capable of forming a complementary pair with the gene (DNA, cDNA) encoding Cthrcl or the full length or a part of mRNA thereof which can be used as a so-called antisense drug.
  • About 70% or more of the target is preferably a polynucleotide that is the same as the full length or a part of the target Cthrcl coding gene. More preferably, it is 80% or more, more preferably 90% or more, particularly preferably 95% or more.
  • the number of substituted bases is 1 to several. Is.
  • RNA interference drug having 15 to 30 bases of RNA capable of forming a complementary pair with a part of mRNA of Cthrcl. If it is about 15 bases, it is preferably completely complementary, but if it is about 30 bases, 70% or more can be used if it is complementary, preferably 80% or more, more preferably 90%. % Or more.
  • a polynucleotide capable of forming a complementary pair with these targets can be used as an agent for improving the metabolism when Cthrcl is excessive, or as a prophylactic or therapeutic agent for bone and joint diseases caused by Cthrcl excess.
  • purine bases such as adenine (A) and guanine (G) and pyrimidines such as thymine (T), uracil (U) and cytosine (C) are used. It contains bases and their modified bases as constituent elements, and is either single-stranded or double-stranded DNA, double-stranded RNA containing single-stranded or hairpin structures, single-stranded DNA and single-stranded RNA It also includes a hybrid that consists of the above, and a chimera in which RNA and DNA are combined into a single strand.
  • A adenine
  • G guanine
  • T thymine
  • U uracil
  • C cytosine
  • Genes encoding Cthrcl or Cthrcl analogs, or genes encoding Cthrcl and polynucleotides capable of forming complementary pairs with mRNA of Cthrcl are used as bone metabolism improving agents, bone or joint disease prevention or treatment.
  • DNA, RNA, plasmid, viral vector, etc. can be used as a gene form when used as an agent, and it may be single-stranded or double-stranded.
  • bone quality can be improved, or bone or joint disease can be prevented or treated.
  • Bone metabolism improvement refers to Cthrcl or Cthrcl analogs, genes encoding these, or their mRNAs, through the function (i) below, or to Cthrcl antibodies, genes encoding Cthrcl, Through the function (ii) of a polynucleotide capable of forming a complementary pair with Cthrcl mRNA, (i) restore (increase), maintain, or activate bone metabolism consisting of bone resorption and bone formation to normal levels. It means to hesitate or, conversely, (ii) to suppress excessive bone metabolism and return it to a normal level. [0040] (i) Osteoclast power A function that promotes bone formation by acting on osteoblasts after being secreted and enhancing their differentiation or proliferation function
  • Examples of the bone / joint diseases that are the subject of prevention / treatment of the present invention include osteoporosis, Paget's disease, bone dyscalcia associated with cancer, rheumatoid arthritis, and the like.
  • Osteoporosis is a disease caused when bone formation cannot catch up due to excessive bone resorption.
  • bone resorption of osteoclasts causes (1) release of cell growth factors in the bone, causing cancer cells to grow, and (2) bone It is thought to be caused by the creation of a space for cancer cells to settle in the bone by destruction. In many cases, bone formation by osteoblasts is suppressed.
  • Paget disease of bone is a disease in which bone metabolism is increased (J Clin Invest 115: 200-208, 2005).
  • the prophylactic or therapeutic agent of the present invention comprising an antibody against Cthrcl, a Cthrcl-encoding gene, or a polynucleotide capable of forming a complementary pair with Cthrcl mRNA, as an active ingredient is effective. It is thought that.
  • the bone metabolism improving agent and bone 'joint disease preventing or treating agent of the present invention can be used in combination with other bone metabolism improving agents and bone' joint disease preventing or treating agents.
  • SERM Selective Estrogen Receptor Modulator: a selective estrogen receptor modulator, such as raloxifene, such as bisphosphonates used as a prophylactic or therapeutic agent for osteoporosis, estrogen used in hormone replacement therapy, etc.
  • bone resorption inhibitors such as human RANKL (Receptor Activator of NF- ⁇ B Ligand) antibody currently in clinical trials.
  • the bone metabolism improving agent and the bone or joint disease preventive or therapeutic agent of the present invention may contain other components as long as the improvement effect does not inhibit the preventive or therapeutic effect.
  • pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, diluents, surfactants, emulsifiers, solubilizers, absorption enhancers, moisturizers. It can be formulated by known methods using additives such as adsorbents, adsorbents, fillers, extenders, moisturizers, and preservatives.
  • examples of the excipient include organic excipients and inorganic excipients.
  • the administration route is intravenous administration such as oral administration and intravenous injection.
  • intravenous administration such as oral administration and intravenous injection.
  • Intravenous administration such as intravenous injection is preferable in that it is safe to keep the blood concentration constant.
  • the bone metabolism-improving agent or bone / joint disease preventive or therapeutic agent of the present invention forms a complementary pair with a gene encoding Cthrcl or an analog thereof, its mRNA, a gene encoding Cthrcl, or an mRNA of Cthrcl.
  • a possible polynucleotide it can be administered as follows.
  • a method of directly administering an expression plasmid into a muscle (DNA vaccine method), a ribosome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electopore position method, etc.
  • the ribosome method is preferred.
  • viruses used in the virus vector include DNA viruses or RNA viruses such as retroviruses, adenoviruses, adenovirus-related viruses, herpes viruses, vaccinia viruses, box viruses, polyviruses, and synbis viruses.
  • viruses retroviruses, adenoviruses, adeno-related viruses, vaccinia viruses, etc. are preferred, especially adenoviruses.
  • an appropriate route of administration can be selected according to the disease, symptom, and the like to be treated.
  • administration routes include vein, artery, subcutaneous, intradermal, intramuscular, intraosseous and the like.
  • the "in vivo method” When administered by the "in vivo method", for example, it can be in the form of a preparation such as a liquid. In general, it is possible to add various components that are commonly used in injections and the like as required by the form of injections containing genes.
  • Cthrcl or a similar compound is used by infiltrating the carrier (eg, idroxyapatite or polymer). There is.
  • ribosomes or membrane-fused ribosomes containing genes can be used as forms of ribosome preparations such as suspensions, freezing agents, centrifugal concentrated freezing agents, and the like.
  • HVJ Sendai virus
  • the dosage form of the bone metabolism-improving agent or bone / joint disease preventive or therapeutic agent of the present invention is, for example, in the form of tablets, force capsules, granules, powders, pills, troches, syrups, injections, etc. Are listed. Injections include ribosome preparations and the like.
  • the content of the active ingredient in the bone metabolism-improving agent or bone 'joint disease preventive or therapeutic agent of the present invention varies depending on the dosage form and cannot be generally limited, and various dosage forms are possible. In this range, it may be selected as appropriate in relation to the dose.
  • the bone metabolism-improving agent or the preventive or therapeutic agent for bone and joint diseases of the present invention is a liquid agent containing Cthrcl, a Cthrc 1-related compound, or an antibody against Cthrcl, 0.0001 to 10 (wZ ⁇ %) , Preferably ⁇ is 0.001 to 5 (w / v%), especially in the case of injection, 0.0002 to 0.2 (w / v%), preferably 0.001 to 0.1 (w / v %), 0.01 to 50 (wZw%) for solid agents,
  • the force that can be preferably prepared as 0.02 to 20 (wZw%) or the like is not necessarily limited to this range.
  • the bone metabolism improving agent or the bone or joint disease preventive or therapeutic agent of the present invention has a complementary pair with Cthrcl, a gene encoding a Cthrcl-related compound, its mRNA, a gene encoding Cthrcl, or an mRNA of Cthrcl.
  • a formable polynucleotide it can be prepared, for example, as 0.0001 to 50 (wZw%), but is not necessarily limited to this range.
  • the dose of the bone metabolism improving agent or bone / joint disease preventive or therapeutic agent of the present invention varies depending on the type of disease, administration route, symptoms, age, body weight, form of the preventive or therapeutic agent, etc. If the bone metabolism improving agent, bone or joint disease preventive or therapeutic agent is Cthrcl, a Cthrcl-related compound, or an antibody against Cthrcl, the amount of the active ingredient in the therapeutic agent is the weight of the subject requiring treatment. 0.005 to 500 mg per kg, preferably 0.1 to LOOmg, but for adults, the lower limit is 0. Olmg (preferably 0. lmg), the upper limit is 20 g (preferably 2000 mg, It is desirable to administer according to the symptom in a single dose or in several doses so that the dose is more preferably 500 mg, more preferably lOO mg.
  • the bone metabolism improving agent or bone / joint disease prophylactic or therapeutic agent of the present invention is complementary to a gene encoding Cthrcl, a Cthrcl-related compound, its mRNA, a gene encoding Cthrcl, or an mRNA of Cthrcl.
  • the amount of gene or polynucleotide is 0. OOOlmg to: LOOmg, preferably 0. OOlmg ⁇ : LOmg is preferably administered once every several days to several months.
  • the bone / joint disease diagnosis kit of the present invention comprises the following (1) or (2).
  • (1) includes reagents generally used for Western blotting and ELISA described in the diagnostic method of the present invention described later.
  • primary antibody against Cthrcl secondary antibody
  • horseradish peroxidase (HRP) horseradish peroxidase
  • AP alkaline phosphatase
  • Secondary antibodies substrate labeled acts
  • buffering agents such as T wee n20, such as bovine serum albumin (BSA)
  • BSA bovine serum albumin
  • an acrylamide gel, a nitrocellulose membrane or the like is used in addition to these, and these can be combined into a kit.
  • the antibody against Cthrcl of (1) includes antibodies against Cthrcl itself, as well as antibodies against multimers such as Cthrcl-degraded subunits and dimers.
  • Examples of the subject include those listed in SEQ ID NOS: 11 and 13 in Patent Document 1, for example.
  • the "polynucleotide capable of forming a complementary pair with the mRNA of a gene encoding Cthrcl" in (2) is a polynucleotide that is completely complementary to the mRNA of a gene encoding Cthrcl, or a partial sequence thereof ( (Including oligonucleotides), preferably 15 bases or more, more preferably 20 bases or more, can be used as a probe.
  • nucleotide sequences including substitutions, deletions, additions, insertions, etc. may be included.
  • Higher homology with Cthrcl mRNA is preferred.
  • the nucleotides are identical to the gene encoding Cthrcl, more preferably 80% or more, and even more preferred. Is 90% or more, particularly preferably 1 to several substituted bases, and in the case of 15 bases, it is preferably almost 100% identical.
  • polynucleotides are considered to be capable of forming a complementary pair even when there is an inherited or acquired abnormality in the gene encoding Cthrcl. , Can be detected.
  • DNA is suitable as a gene probe from the viewpoint of the stability of a nucleic acid molecule.
  • Chimeric probes are used.
  • the above-mentioned polynucleotide is prepared by a method of artificially synthesizing using a DNA synthesizer or the like according to a conventional method, a method of extracting a naturally occurring polynucleotide, a polynucleotide extracted from nature.
  • a label such as a fluorescent substance to an antibody, a gene probe, or the like, if necessary.
  • the label is not particularly limited as long as it is generally used for the detection of proteins and genes.
  • fluorescent protein to be bound to the antibody include GFP (Green Fluorescence Protein), YFP (Yellow Fluorescence Protein: yellow protein), CFP (Cyan Fluorescence Protein: cyan protein), red fluorescent protein derived from sputum and the like.
  • FAM (6-carboxyfluor Examples include fluorescent dyes such as fluorescein-based fluorescent dyes such as fluorescein, piotine digoxigenin (DIG), HRP (horseradish peroxidase), and AP (alkaline phosphatase).
  • the measurement target is a gene
  • a method in which a gene in a specimen is amplified in advance by a nucleic acid amplification method or the like there is a method in which a gene in a specimen is amplified in advance by a nucleic acid amplification method or the like.
  • nucleic acid amplification methods performed prior to gene detection include, for example, PCR method (see polynucleic acid amplification method, merase chain reaction method, Polymerase Chain Reaction, EP200362, etc.), NASBA method (see EP329822, etc.), TAS method (WO88) / 10315 etc.), isothermal gene amplification method (ICAN method: Isothermal and Chimeric primer—initiated amplification of Nucleic acias, Takaf Bio Inc.) and the like.
  • PCR method see polynucleic acid amplification method, merase chain reaction method, Polymerase Chain Reaction, EP200362, etc.
  • NASBA method see EP329822, etc.
  • TAS method WO88
  • ICAN method Isothermal and Chimeric primer—initiated amplification of Nucleic acias, Takaf Bio Inc.
  • A Real-time PCR method capable of tracking quantitative changes in genes
  • B Nested-PCR method in which genes are amplified by PCR and then PCR is performed with another primer pair
  • C nested -Nested-real-time PCR method combining PCR method and real-time PCR method (method of performing the second PCR by real-time PCR method), and reverse transcription step (Reverse RNA) that reversely transcribes RNA before PCR method Transcription: RT) has been developed.
  • the nested-PCR method is preferable in that only specific sequence DNA in the target gene can be amplified more specifically by amplification based on two-step PCR.
  • the kit using (2) further includes, as a material for performing nucleic acid amplification in advance, an enzyme having a thermostable polymerase activity, a buffer solution, etc.
  • an enzyme having a thermostable polymerase activity e.g., a reverse transcriptase used for PCR or the like can also be included as a kit material.
  • the amount of (1) or (2) in the kit may be appropriately selected according to a conventional method, but it is used as a probe.
  • the diagnosis of bone and joint diseases is performed using Cthrcl or Cthrcl mRNA in the specimen from which the vitality of the subject is also collected as an index.
  • “Using Cthrcl or Cthrcl mRNA as an indicator” means directly or indirectly measuring the amount or activity of Cthrcl or Cthrcl mRNA.
  • Examples of the method for indirect measurement include targeting Cthrcl subunits, dimers and other multimers, cDNA obtained by reverse transcription of mRNA of Cthrcl, and the like.
  • the above-described diagnostic kit can be used.
  • Specimens include body fluids such as urine, plasma, serum, whole blood, cerebrospinal fluid, semen, etc., but the subject's ability to easily extract and the state of bones throughout the body are reflected. Serum is preferred because it is possible to evaluate bone and joint diseases, which are systemic bone metabolizing diseases.
  • Protein measurement methods include (1) Western plot analysis and (2) enzyme immunoassay methods such as ELISA, (3) radioimmunoassay methods, and (4) sugar chains bound to proteins.
  • a general method used for protein detection and quantification, such as a method for detecting the protein can be used. From the viewpoint of measurement accuracy, the ELISA method is preferred.
  • ELISA is an acronym for enzyme-linked immunosorbent assay, and is also called EIA (Enzyme Immune Assay: Enzyme Immune Assay).
  • the measurement can be performed according to a conventional method using an antibody against the target Cthrcl protein and a gene probe for the gene of the target protein.
  • antibodies include polyclonal antibodies and monoclonal antibodies.
  • the antibody may be any of a mammal-derived antibody, a chimeric antibody, an anthropomorphic antibody, or a humanized antibody. As long as it is an antibody against a human protein, the detection antibody itself does not need to be humanized.
  • mRNA is susceptible to enzymatic degradation and is unstable, it is fragile if RT-PCR is used to perform PCR analysis after converting mRNA to cDNA using reverse transcriptase! / ⁇ RN
  • the mRNA expression level can be quantified by using a labeled gene probe.
  • the bone and joint disease diagnosis method of the present invention when the amount of Cthrcl protein or the mRNA of Cthrcl is less than normal, bone turnover is impaired, and bone associated with osteoporosis and cancer • Calcium If the amount of Cthrcl protein or Cthrcl mRNA is higher than normal, it may cause Paget's disease due to increased bone metabolism or Can be diagnosed as having a high probability of
  • the kit for screening a preventive or therapeutic agent for bone / joint diseases of the present invention comprises at least selected from osteoclasts, Cthr cl, a gene encoding Cthrcl, or a group force comprising the mRNA.
  • Including one or more Including one or more.
  • a substance that increases or decreases the amount of Cthrcl produced by adding a candidate substance to a culture medium of osteoclasts, a solution containing a gene encoding Cthrcl or its mRNA, and the like can be appropriately selected.
  • a candidate substance to Cthrcl one that enhances or inhibits the osteogenic function of Cthrcl itself can be appropriately selected.
  • the ability to increase Cthrcl, or the bone formation function of Cthrcl itself, will decrease bone metabolism such as osteoporosis, bone abnormal calcium metabolism associated with cancer, and rheumatoid arthritis.
  • the ability to reduce Cthrcl or Cthrcl mRNA, or the ability to inhibit Cthrcl functions such as osteogenic function can be used to prevent bone-joint diseases such as Paget's disease where bone metabolism is increased. Alternatively, it can be selected as a therapeutic agent.
  • the Cthrcl increase or decrease function or the Cthrcl function enhancement or inhibition effect of the selected substance can be confirmed by adding the substance to osteoblasts or the like.
  • the screening method using Cthrcl, a gene encoding Cthrcl of the present invention, or its mRNA as an index can be carried out by the following (1) or (2).
  • “As an index” means directly or indirectly measuring the amount or activity of C thrcl, a gene encoding Cthrcl, or its mRNA.
  • Cthrcl in cells at each osteoclast stage was analyzed by RT-PCR. 1-4 show the following cells, respectively.
  • RNA was prepared, converted to cDNA, PCR was performed using primers specific to each gene, and the expression level was analyzed by gel electrophoresis.
  • Cathebsin K, calcitonin receptor, carbonic anhydrase II, and TRAP (tartrate-resistant acid phosphatase) are all osteoclast differentiation markers, GAPDH is a positive control up to 4 and all RNA Is used uniformly. The results are shown in Figure 1.
  • Figure 1 shows that Cthrcl is specifically expressed in activated osteoclasts.
  • Cthrcl is expressed in mouse parietal bone-derived osteoblasts using retrovirus, cultured in osteogenesis induction medium containing ascorbic acid and ⁇ -glyceport phosphate for 4 days, and stained with alkaline phosphatase, a marker of bone formation ( (The intensity of blue represents the degree of bone formation)
  • Figure 2 shows the results. As a result, it was found that Cthrcl promotes bone formation not only in the case of induction with an osteogenic medium but also in a normal medium.
  • Cthrcl is expressed in mouse parietal bone-derived osteoblasts using a retrovirus, cultured in an osteogenesis medium containing ascorbic acid and ⁇ -glyceport phosphate for 10 days, and calcium, a marker of calcification, is expressed with alizarin red. Staining (red deposition) was performed. The results are shown in Figure 3. From Fig. 3, it was found that Cthrcl promotes osteoblast mineralization.
  • Cthrcl itself or the substance that enhances its action.
  • an antibody that inhibits Cthrcl an antisense drug that inhibits the expression of a gene that encodes Cthrcl and mRNA of Cthrcl, or RNAi pharmacology Cthrcl excessive action (excessive growth of Cthrcl, excessive function of Cthrcl itself, etc.),
  • TRAP-positive mononuclear pre-osteoclasts derived from mouse bone marrow were treated with ivory pieces (+) treated with a solution of bisphosphonate! /, On the ivory pieces (-), RANKL and M- Culturing in a medium containing CSF for 2 days induced mature osteoclasts.
  • Figure 4 shows the results of preparing RNA from each cell and examining the expression of Cthrcl and GAPDH as a control by RT-PCR. From Fig. 4, it was found that bisphosphonate suppressed Cthrcl expression in osteoclasts activated on ivory pieces.
  • Cthrcl (1 part by weight) or Cthrcl antibody (1 part by weight) is dissolved in distilled water for injection (1000 parts by weight), and this aqueous solution is filtered through a membrane filter. Dispense 1 ml of the filtrate into an ampoule purged with nitrogen and sterilize at 120 ° C for 15 minutes to obtain the injection of Example 1 or 2.
  • Example 3 (Bone and joint disease therapeutic agent Z injection)
  • a viral vector in which the gene encoding Cthrcl is incorporated into a retrovirus is prepared, and water is added to make an injection.
  • a bone metabolism improving agent a bone / joint disease preventive or therapeutic agent that can maintain or activate normal bone metabolism
  • a bone / joint disease diagnostic kit using Cthrcl a bone
  • Methods for diagnosing joint diseases screening kits for agents for preventing or treating bone / joint diseases using Cthrcl, screening methods, methods for preventing or treating bone / joint diseases.
  • FIG. 1 A drawing showing the results of RT-PCR analysis of Cthrcl expression in cells in each osteoclast division stage.
  • FIG. 2 is a diagram showing bone formation by Cthrcl.
  • FIG. 3 shows osteoblast mineralization by Cthrcl.
  • FIG. 4 is a graph showing a decrease in Cthrcl production by bisphosphonate.
  • FIG. 5 shows the homology between the amino acid sequences of mouse Cthrcl and human Cthrcl.

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Abstract

L'invention concerne un agent pour améliorer le métabolisme osseux grâce auquel le métabolisme osseux normal peut être maintenu et activé ; un agent préventif ou un remède pour une maladie osseuse ou articulaire ; une trousse pour diagnostiquer une maladie osseuse ou articulaire qui utilise Cthrc1 ; une méthode de diagnostic de maladie osseuse ou articulaire ; une trousse de dépistage d'un agent préventif ou remède pour une maladie osseuse ou articulaire qui utilise Cthrc1 ; une méthode de dépistage ; et une méthode pour prévenir ou traiter une maladie osseuse ou articulaire. L'invention concerne donc un agent pour améliorer le métabolisme osseux caractérisé en ce qu'il contient au moins un élément sélectionné parmi Cthrc1, un composé analogue à Cthrc1, un gène qui encode Cthrc1, un ARNm de Cthrc1, un anticorps anti-Cthrc1 et un polynucléotide capable de former une paire complémentaire avec un gène qui encode Cthrc1 ou l'ARNm de Cthrc1 ; et un agent préventif ou un remède pour une maladie osseuse ou articulaire qui le comprend.
PCT/JP2007/057793 2006-04-10 2007-04-06 Agent pour améliorer le métabolisme osseux WO2007123010A1 (fr)

Priority Applications (1)

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JP2008512064A JPWO2007123010A1 (ja) 2006-04-10 2007-04-06 骨代謝改善剤

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JP2006-107460 2006-04-10

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050147602A1 (en) * 2000-10-19 2005-07-07 Maine Medical Center Research Institute Compositions, methods and kits relating to CTHRC1, a novel modulator of collagen matrix

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050147602A1 (en) * 2000-10-19 2005-07-07 Maine Medical Center Research Institute Compositions, methods and kits relating to CTHRC1, a novel modulator of collagen matrix

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DURMUS T. ET AL.: "Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)", GENE EXPRESSION PATTERNS, vol. 6, no. 8, 2006, pages 935 - 940, XP005636230 *
LINDNER V. ET AL.: "The novel collagen triple helix repeat containing gene (Cthrc1) indicates a role in cartilage and bone formation via regulation of collagen matrix production", JOURNAL OF BONE AND MINERAL RESEARCH, vol. 19, no. SUPPL. 1, 2004, pages S34 - S35 + ABSTR. NO. 1132, XP003012939 *

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