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WO2003001906A1 - Procede de construction d'un modele animal de maladie des articulations - Google Patents

Procede de construction d'un modele animal de maladie des articulations Download PDF

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Publication number
WO2003001906A1
WO2003001906A1 PCT/JP2002/006421 JP0206421W WO03001906A1 WO 2003001906 A1 WO2003001906 A1 WO 2003001906A1 JP 0206421 W JP0206421 W JP 0206421W WO 03001906 A1 WO03001906 A1 WO 03001906A1
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WIPO (PCT)
Prior art keywords
joint
gene
human mammal
disease
function
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English (en)
Japanese (ja)
Inventor
Atsushi Nishimura
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Publication of WO2003001906A1 publication Critical patent/WO2003001906A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates

Definitions

  • the present invention relates to functional analysis of a disease-specific variable gene. More specifically, drugs and genes used to analyze the effects of joint diseases and joint disease-specific variable genes on joints, joint tissues and cells constituting joints involving osteoarthritis, rheumatoid arthritis or osteoporosis, etc. Or a model animal to which the gene product is administered. Background art
  • Bone diseases include non-metabolic bone diseases such as fractures, bone deformation, and osteogenesis imperfecta, and metabolic bone diseases such as osteoporosis and osteomalacia.
  • Cartilage diseases include chronic diseases such as osteoarthritis and rheumatoid arthritis.
  • Arthritis includes inflammatory diseases such as periarthritis of the shoulder, tendon, tendon sheath and peritenitis.
  • the joint disease that often affects patients refers to a disease in which degeneration of articular cartilage is the main lesion (eg, rheumatoid arthritis or osteoarthritis).
  • the extracellular matrix composed of collagen and proteodalican depends on various factors for proteodalican, then collagenase-3 in the order of collagen. Is degraded by an extracellular streptolytic enzyme called matrix metaoral protease. This causes the cartilage that covers the surfaces of the joints such as knees, jaws, elbows, hips, fingers and jaws to harden and break down, deforming the joints and leading to dysfunction in severe cases. It is known that the incidence of osteoarthritis increases with age, and it is expected that the number of patients will increase in aging societies including Europe and the United States.
  • the main clinical symptoms are inadequate knee expansion and contraction of the jaw due to joint pain, and when the knee joint or hip joint develops, walking may be difficult.
  • cartilage tissue has poor regeneration ability, so once cartilage destruction progresses, it takes time to regenerate cartilage. If severe pain is involved, total joint replacement or osteotomy is performed. A method of preserving the knot function is taken.
  • palliative treatments such as hyaluronic acid preparations and anti-inflammatory drugs have been used to prevent and treat osteoarthritis for the purpose of reducing pain. It has not been developed and there is no fundamental treatment. Disclosure of the invention
  • the present invention is intended for the manufacture of a model animal to which a drug, a gene or a gene product thereof is administered in relation to the development of a therapeutic agent from the viewpoint of creating a joint destruction inhibitor and a therapeutic agent for joint repair, and to analyze the function of the search target.
  • the present invention provides a method for producing a model animal to which a drug, a gene or a gene product thereof is efficiently and specifically administered to a joint disease for the purpose of evaluating the efficacy of the drug.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a conventional method of cutting a ligament of a knee joint to artificially induce a joint disease (osteolite squirt cartage, ninth ed. Volume, p. 308, 2001), the method of artificially inducing joint disease by adding meniscal resection of the knee joint to the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament amputation is unexpected. It was found that even more severe cartilage destruction could be induced. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.
  • a meniscal ligament resection model non-human mammal containing the gene characterized in that the gene is administered to the non-human mammal obtained by the production method according to (1), a joint thereof, and a joint thereof.
  • a non-human mammal capable of producing the gene product characterized in that the gene is administered to the non-human mammal obtained by the production method according to (1), a joint thereof, and a joint tissue thereof. Or a method for producing cells constituting the joint, (4) The method according to any of (1) to (3) above, wherein the non-human mammal is an animal from which the drug effect or gene function of a drug, gene or gene product can be inferred.
  • a method for elucidating the function of the gene or its gene product which comprises administering a gene of unknown function to a non-human mammal obtained by the production method according to (1),
  • a non-human mammal having the ability to produce a gene product obtained by the production method described in (3), a joint thereof, a joint tissue thereof, or a cell constituting the joint, wherein the gene or A kit for elucidating the function of the gene product, (13) The function is elucidated using the method according to any one of (7) to (9) or the kit according to any one of (10) to (12).
  • a medicament comprising the genetically modified gene,
  • a pharmaceutical comprising
  • a screening method for a joint disease modulator characterized by the following: (19) the joint disease in the case where the test compound is added to or not to the non-human mammal, the joint, the joint tissue, or the cells constituting the joint; The screening method according to the above (17) or (18), wherein the amount of mRNA corresponding to the specific variable gene is measured.
  • test compound When the test compound is added to the non-human mammal, its joint, its joint tissue or cells constituting the joint, and when the test compound is not added, the non-human mammal, its joint, its joint tissue or The screening method according to the above (17) or (18), wherein the function of a cell constituting the joint is searched for,
  • a kit for screening for a joint disease modulator (23) A non-human mammal having the ability to produce a joint-related disease gene product obtained by the production method according to (3), a joint thereof, a joint tissue thereof, or a cell constituting the joint. Screening kit for a joint disease modulator
  • a medicament comprising a compound whose efficacy has been evaluated using the method according to any of (28) to (31) above, (33)
  • I will provide a.
  • the method for producing a meniscal ligament resection model non-human mammal (hereinafter abbreviated as a model animal) of the present invention substantially comprises the medial collateral ligament, anterior cruciate ligament and posterior cruciate ligament of the knee joint of a non-human mammal. It is characterized by artificially inducing joint disease by applying medial meniscectomy to the cutting of the three ligaments of the ligament, thereby inducing significant cartilage destruction.
  • the model animals obtained by this production method are targeted for joint drug-specific and efficient drug, gene or gene product administration for the purpose of functional analysis of drug discovery and drug efficacy evaluation.
  • non-human mammals include, for example, guinea pigs, rats, mice, egrets, bushes, higgins, magpies, monkeys, and the like.Small animals are preferred, and rodents such as rats and mice are particularly preferred. Rats are particularly preferred.
  • Meniscectomy of the knee joint refers to opening the knee joint and removing part or all of the cartilage meniscus intervening between the bones that form the upper and lower joint cavities. As a result, the mobility of the joint is impaired, and the upper and lower bones come into contact with each other, thereby destroying the cartilage existing at the epiphysis. Meniscectomy can be performed by a method known per se, for example, a method for producing an arthritis model animal (Toxicolological Pathology 1, Vol. 27, p. 134, 1999) or a method analogous thereto.
  • Drugs genes, gene products (eg, proteins, peptides, etc.) are used as substances to be administered to meniscal ligament resection model animals.
  • the drug is not particularly limited as long as it has a therapeutic / preventive effect on various diseases such as inflammation as well as joint diseases. Specifically, drugs listed in the Japanese Pharmacopoeia are used.
  • test compounds for example, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds (including peptides), microbial cultures, cell extracts, plant extracts, and animal tissue extracts are used. These compounds may be new compounds or known compounds.
  • Genes can be either genes whose function is unknown or genes whose function is elucidated. And it may be either genomic DNA or cDNA. Further, the gene may be either sense DNA or antisense DNA.
  • the DNA can be administered alone, but it may be administered in the form of ribosome or a viral vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector; It can be used by inserting it into a vector.
  • genes may encode a full-length protein or peptide, or may be gene fragments encoding a part of the protein or peptide.
  • the gene product may be any of a protein and a peptide.
  • a gene product whose function is unknown or a gene product whose function is elucidated can be used.
  • the order of the surgical procedures is not particularly limited, and specifically, usually, incision of the skin, cutting of the medial collateral ligament for exposing the joint cavity, cutting of the anterior cruciate ligament, A method of cutting the posterior cruciate ligament, followed by medial meniscectomy, and suturing to close the joint cavity is used.
  • the method of ablation and the like can be appropriately changed. That is, surgery on a non-human mammal is usually composed of about 1 to 4, preferably about 15 to 30, individual procedures. Can be replaced with the acquired technique. These procedures can be obtained from known books or literatures. Therefore, the method for producing a model animal of the present invention can be performed using substantially the same method as the above-described specific method, and performing the three ligament cuts and medial meniscectomy shown above for the target non-human mammal. It can be modified as appropriate.
  • the degree of modification is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more identical to the above-mentioned production method. Range.
  • the method includes the step of resecting the inner meniscus with the three ligaments of the knee joint of a non-human mammal cut, and as a whole about 70% or more, preferably about 80% or more, more preferably about 9% or more. 0% or more, most preferably about 95% or more identity Is preferred.
  • a joint disease model animal can be produced specifically and efficiently.
  • OA osteoarthritis
  • joints, joint tissues or cells constituting the joints can be extracted by conventional means.
  • the cells constituting the joint include bone cells, osteoblasts, osteoclasts, chondrocytes, joint synovial cells, fibroblasts and the like.
  • the above-described gene may be contained in the above-described model animal of the present invention using a virus vector or the like.
  • a model animal containing the gene or a model animal capable of producing the gene product can be produced.
  • a joint containing the gene, a cell constituting the joint tissue or a cell constituting the joint, or a joint capable of producing the gene product, a joint tissue or a cell constituting the joint is isolated from these model animals by a conventional means. be able to.
  • the method for producing the cells constituting the joint containing the above-mentioned gene is substantially performed by cutting three ligaments of the medial collateral ligament, the anterior cruciate ligament and the posterior cruciate ligament in a non-human mammal knee joint.
  • the method is characterized by removing the inner meniscus.
  • the specific operation may be such that the above-described gene is contained in the above-described model animal of the present invention using a viral vector or the like.
  • the method for isolating the cells constituting the joint from the joint is known per se, for example, the method described in Osteolites cultivation (Vol. 9, pp. 73-84, 2001) or a method similar thereto. A method is used.
  • the method for elucidating the function of a gene of the present invention is to elucidate the function of the gene by administering a gene whose function is unknown to the model animal of the present invention.
  • the gene whose function is unknown may be genomic DNA or cDNA. Further, the gene may encode a full-length protein or peptide, or may be a gene fragment encoding a part of the protein or peptide.
  • the function includes, for example, a function relating to the mobility of a joint, and more specifically, a joint destruction inhibitory action, a joint repair action, and the like.
  • the search for the function of the joint (which may be any of assay, detection, or measurement, but preferably measurement) is performed by, for example, Arthritis Rheum, Vol. 44, 107, 1081 , 2001).
  • the search for the function of the cells constituting the joint is performed, for example, by the method of Arthritis Rheum, Vol. 1071-1081, 2001).
  • Culture of the cells constituting the joint is performed by a method known per se, for example, The method can be carried out using the method described in Arthritis Rheum, Vol. 44, pp. 1071-11081, 2001.
  • the functions of the cells constituting the joint can be elucidated more efficiently.
  • the pressurized condition refers to a condition as if the bone were pressurized by applying a centrifugal operation or the like in the process of culturing the cells constituting the joint, for example, osteoblasts. Under pressurized conditions, osteoblasts are subjected to pressurized stress, and the cells organize, transmit, and provide a means for stress.
  • Fatal conditions include serum removal and administration of highly toxic anticancer drugs (eg, adriamycin) to the cells that make up the joint.
  • highly toxic anticancer drugs eg, adriamycin
  • the kit for elucidating the function of the present invention comprises a model animal containing the gene, a joint thereof, a joint tissue thereof, or a cell constituting the joint, or a model animal capable of producing the gene product, a joint thereof, a joint tissue thereof. Or it contains the cells that make up the joint.
  • Examples of the function elucidating kit of the present invention include the following.
  • the solution may be sterilized by filtration through a 0.45 m filter, and stored in, or may be prepared at use.
  • the cells constituting these joints 1 2-well plates and passaged 5 X 1 0 5 or holes, 3 7, 5% C 0 2, 9 with 5% air 2 days followed by culturing.
  • the function of a joint containing a gene whose function is unknown is compared with that of a joint that does not contain a gene whose function is unknown.
  • about 20% or more, preferably about 30% or more Preferably, when it is increased by about 50% or more, it can be determined that the gene has a joint function promoting (or improving or enhancing) effect.
  • the function of the joint when it does, and the function of the cells that make up the joint are about 20% or more, preferably about 30% or more.
  • the gene is reduced by about 50% or more, it can be determined that the gene has a joint function lowering action.
  • joint function regulating genes Genes elucidated to have a joint function promoting effect or genes elucidated to have a joint function lowering effect are referred to as joint function regulating genes, respectively, as pharmaceuticals such as joint function regulators (preferably drugs for treating joint diseases).
  • pharmaceuticals such as joint function regulators (preferably drugs for treating joint diseases).
  • Useful as a composition In particular, genes that have been found to have joint function-promoting effects are useful, for example, as agents for preventing and treating bone or joint diseases.
  • bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone petiet's disease, ankylosing myelitis; representatives of cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.
  • non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery
  • bone defects osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone petiet'
  • genes that have been shown to have joint function-promoting effects can be used as bone tissue repair agents after surgery for multiple myeloma, lung cancer, breast cancer, etc.
  • treatment of periodontal disease, periodontal disease It can be used to repair periodontal tissue defects in disease, stabilize artificial dental roots, repair ridge formation and cleft palate.
  • the gene When the gene whose function has been elucidated is used as the above-mentioned pharmaceutical composition, the gene may be used alone or after being inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. It can be carried out according to conventional means.
  • the gene can be administered as is or with an auxiliary to promote uptake, such as with a gene gun or a catheter such as Hyde-mouth gel catheter.
  • the gene can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, they can be used parenterally in the form of injections such as suspensions.
  • the gene It can be manufactured by admixing it with known approved carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. it can.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), non-ionic surfactant (eg, Polysorbate 80 TM, HCO-50) May be.
  • oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • solubilizers such as benzyl benzoate and benzyl alcohol.
  • phosphate buffer, sodium acetate buffer for example, phosphate buffer, sodium acetate buffer
  • soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizer eg, human serum albumin, polyethylene dalicol, etc.
  • preservative eg, benzyl) Alcohol, phenol, etc.
  • antioxidants e.g, benzyl benzoate and benzyl alcohol.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • mammals eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.
  • the dosage of the gene varies depending on the administration target, target organ, symptoms, administration method, etc.
  • oral administration in general, for example, in patients with osteoarthritis (60 kg), about 0.1 to 100 mg, preferably about 1.0 to 5 Omg, more preferably Is about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • an injection it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg, preferably about 0 .: to about 2 Omg, and more preferably about 0.1 to 1 Omg by intravenous injection. It is convenient.
  • the dose can be administered in terms of 6 O kg.
  • the drug candidate compound can be screened using the following method.
  • the present invention is a.
  • a method for screening for a drug for regulating a joint function which comprises using cells constituting a joint of a model animal of the present invention containing a gene, and
  • the gene examples include a gene involved in joint function, a joint disease-specific variable gene such as a joint function regulating gene, and the like. More specifically, Connective Tissue Res. Research (Connect Tissue Res 41, 175-184, 2000) and the like are used.
  • This screening method is, for example, as described above,
  • the cells constituting the joint can be cultured under lethal conditions.
  • Fatal conditions include serum removal and high toxicity to joint cells.
  • Administration of anticancer drugs eg, adriamycin.
  • this screening method includes, for example,
  • test compound for example, a non-peptide compound derived from a living body (such as a carbohydrate or a lipid), a synthetic compound (including a peptide), a microorganism culture, a cell extract, a plant extract, or an animal tissue extract can be used. These compounds may be new compounds or known compounds.
  • the expression level of mRNA can be determined by a method known per se, for example, Northern blotting or reverse transcription-polymerase chain reaction (RT-PCR)- ⁇ TaqMan polymerase chain reaction or the like. It can be measured according to the method.
  • RT-PCR reverse transcription-polymerase chain reaction
  • the production amount of the gene product can be measured by a method known per se, for example, a method using an antibody, a method using chromatography, and the like.
  • This screening kit contains cells constituting the joint, and the same kit as the above-described kit for elucidating the function of a gene is used.
  • the amount of mRNA and gene product when administered is about 20% or more, preferably about 30% or more, compared to the case where no test compound is administered.
  • the test compound has a joint function promoting action.
  • the amount of mRNA and the amount of gene product when administered are reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case where no test compound is administered.
  • the test compound has a joint function lowering effect.
  • Each of the test compounds having a joint function promoting action or a joint function reducing action is useful as a pharmaceutical composition such as a joint function regulator (preferably a therapeutic agent for joint disease).
  • a test compound having a joint function promoting effect is useful, for example, as an agent for preventing or treating bone or joint diseases.
  • bone or joint disease include Non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, dysplasia, and scoliosis in the field of plastic surgery; bone defects, osteoporosis, osteomalacia, rickets, and lines Metabolic bone diseases such as fibrous osteomyelitis, renal osteodystrophy, bone Petiet's disease, and stiff myelitis; and joint diseases represented by cartilage diseases such as osteoarthritis and rheumatoid arthritis.
  • Non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, dysplasia, and scoliosis in the field of plastic surgery
  • Metabolic bone diseases such as fibrous osteomyelitis, renal osteodys
  • test compounds having a joint function-promoting effect can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, for treatment of periodontal disease and dental treatment for periodontal disease. It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.
  • the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method.
  • the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions.
  • the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in generally accepted unit dosage forms required for the formulation. By doing so, it can be manufactured.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cellulose.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • Aqueous liquids for injection include, for example, saline, dextrose and others
  • An isotonic solution containing an auxiliary agent eg, D-sorbitol, D-mannitol, sodium chloride, etc.
  • a suitable solubilizing agent for example, alcohol (eg, ethanol), polyalcohol (eg, propylene) Glycol, polyethylene glycol) and nonionic surfactants (eg, Polysorbate 80 TM, HCO-50).
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer eg, human serum
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, monkeys, etc.). Can be administered.
  • mammals for example, humans, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, monkeys, etc.
  • the dose of the compound varies depending on the administration target, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a patient with osteoarthritis (60 kg), one day About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in patients with osteoarthritis (6 O kg )
  • the dose can be administered in terms of 60 kg.
  • a model animal of the present invention having the ability to produce a gene product or a joint thereof
  • a method for screening for a joint function modulator which is characterized in that it is used.
  • As the model or its joint the same one as described above is used.
  • test compound for example, a non-peptide compound derived from a living body (such as a carbohydrate or a lipid), a synthetic compound (including a peptide), a microorganism culture, a cell extract, a plant extract, or an animal tissue extract can be used. These compounds may be new compounds or known compounds.
  • Examples of the gene include a gene involved in joint function, a joint disease-specific variable gene such as a joint function regulating gene, and the like. More specifically, Connective Tissue Resume (Vol. 41, pp. 175-184, 2000) and the like are used.
  • the joint function of the model animal or its joints is searched for (either assay, detection or measurement, preferably measurement), with or without the test compound added to the model animal. It can be implemented by doing The measurement of the joint function can be performed in the same manner as described above.
  • the joint function when administered was increased by about 20% or more, preferably about 30% or more, and more preferably about 50% or more, compared to the case where no test compound was administered. In this case, it can be determined that the test compound has a joint function promoting effect.
  • the joint function and the function of the cells constituting the joint are reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more as compared with the case where the test compound is not administered. In this case, it can be determined that the test compound has a joint function lowering effect.
  • test compound having a joint function promoting action or a joint function lowering action is useful as a pharmaceutical composition such as a joint function modulator (preferably a therapeutic agent for joint disease).
  • a test compound having a joint function promoting action is useful, for example, as a prophylactic / therapeutic agent for bone or joint disease.
  • bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone petietosis, ankylosing myelitis; Joint diseases represented by cartilage diseases such as arthritis and rheumatoid arthritis.
  • test compound having a joint function-promoting effect is used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, treatment of periodontal disease, dental treatment for periodontal disease It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.
  • the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method.
  • the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions.
  • the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. By doing so, it can be manufactured.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Excipients that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cell mouth, corn starch, gelatin, Swelling agents such as alginic acid, lubricating agents such as stear magnesium phosphate, sweetening agents such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry.
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cell mouth
  • corn starch gelatin
  • Swelling agents such as alginic acid
  • lubricating agents such as stear magnesium phosphate
  • sweetening agents such as sucrose, lactose or saccharine
  • flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule
  • the above type of material can further contain a liquid carrier
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, polysorbate 80 TM, HCO-50) Any combination may be used.
  • oily liquid for example, sesame oil, soybean oil, and the like are used.
  • the oily liquid may be used in combination with a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • a solubilizing agent such as benzyl benzoate or benzyl alcohol.
  • phosphate buffer, sodium acetate buffer for example, phosphate buffer, sodium acetate buffer
  • soothing agent for example, benzalkonium chloride, pro-hydrochloride, etc.
  • stabilizer for example, human serum albumin, polyethylene glycol, etc.
  • preservative for example, For example, benzyl alcohol, phenol, etc.
  • antioxidants for example, For example, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule. Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats
  • the dose of the compound varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, in osteoarthritis patients (as 6 O kg)
  • It is about 0.1 mg to 100 mg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • an injection it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg per day, preferably about 0 :!
  • the dose can be administered in terms of 60 kg.
  • the joint function of a mammal can be regulated by administering to the mammal an effective amount of a joint function-regulating gene whose function has been clarified by using the method for elucidating the function of a gene of the present invention.
  • the dose of the gene to a mammal varies depending on the administration subject, symptoms, and the like. Usually, for example, in a patient with osteoarthritis (as 6 O kg), about 0.01 to 3 O mg, preferably about It is advantageous to administer about 0.1-20 mg, more preferably about 0.1-1 Omg, directly into the joint cavity. In the case of other animals, the dose can be administered in terms of 60 kg. (Compound evaluation method)
  • the present invention is a.
  • a method for evaluating the efficacy of a compound of the present invention which comprises administering a test compound to a model animal, a joint thereof, a joint tissue thereof, or a cell constituting the joint, is provided.
  • Test compounds other than genes for example, gene products, non-peptide compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds (including peptides), microbial cultures, cell extracts, and plant extracts An animal tissue extract or the like is used, and these compounds may be novel compounds or known compounds.
  • gene products for example, gene products, non-peptide compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds (including peptides), microbial cultures, cell extracts, and plant extracts
  • synthetic compounds including peptides
  • microbial cultures such as carbohydrates and lipids
  • cell extracts such as carbohydrates and lipids
  • plant extracts An animal tissue extract or the like is used, and these compounds may be novel compounds or known compounds.
  • the administration method of the test compound is the same as the administration method of the present invention described above.
  • Fatal conditions include serum removal and administration of highly toxic anticancer drugs (eg, adriamycin) to the cells that make up the joint.
  • highly toxic anticancer drugs eg, adriamycin
  • This drug efficacy evaluation method can be carried out in the same manner as the above-described method for elucidating the function of the gene of the present invention and the method for screening a drug candidate compound.
  • the joint function or the function of the cells constituting the joint when administered is about 20% or more, preferably about 30% or more as compared with the case where the test compound is not administered. , More preferably about 50% or more, It can be determined that the test compound has a joint function promoting action.
  • the joint function or the function of the cells constituting the joint when administered is about 20% or more, preferably about 30% or more, and more preferably about 50% or more, compared to when the test compound is not administered. When it decreases, it can be determined that the test compound has a joint function lowering effect.
  • test compound having a joint function promoting action or a joint function lowering action is useful as a pharmaceutical composition such as a joint function modulator (preferably a therapeutic agent for joint disease).
  • a test compound having a joint function promoting action is useful, for example, as a prophylactic / therapeutic agent for bone or joint disease.
  • bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery;
  • non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery
  • metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis
  • cartilage diseases such as osteoarthritis and rheumatoid arthritis Representative joint diseases are mentioned.
  • test compounds having a joint function-promoting effect can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, for treatment of periodontal disease and dental treatment for periodontal disease. It can be used for repair of peri-tissue defects, stabilization of artificial dental roots, ridge formation and repair of cleft palate.
  • the compound obtained by using the above-described screening method or screening kit is used as the above-mentioned pharmaceutical composition, it can be prepared into a pharmaceutical preparation by using a conventional method.
  • the compound can be administered as a sugar-coated tablet, capsule, elixir, microcapsule, orally, or aseptic solution with water or other pharmaceutically acceptable liquid, as appropriate, Or parenteral use in the form of injections such as suspensions.
  • the compound can be mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc. in the unit dosage form generally required for the practice of formulations. By doing so, it can be manufactured.
  • the amount of active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • additives that can be mixed with tablets, capsules, etc.
  • binders such as gelatin, corn starch, tragacanth, gum arabic, and crystalline cells.
  • Excipients such as loin, leavening agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, peppermint, coconut oil or cherry.
  • sweeteners such as sucrose, lactose or saccharine, peppermint, coconut oil or cherry.
  • sweeteners such as sucrose, lactose or saccharine, peppermint, coconut oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) Is also good.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer eg, human serum
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • mammals eg, humans, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.
  • the dose of the compound varies depending on the administration target, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a patient with osteoarthritis (60 kg), one day About 0.1 mg to 100 mg, preferably about 1.0-5 mg, more preferably about 1.0-2 mg.
  • parenteral administration the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc.
  • an injection it is usually used, for example, in patients with osteoarthritis (6 O kg ), About 0.01 to 3 Omg per day, preferably about 0:! To 20 mg
  • the drug by intravenous injection, for example, about 0.1 to 1 Omg.
  • the dose can be administered in terms of 6 O kg.
  • the present invention provides an antibody against a gene product, a partial peptide or a salt thereof, whose function or efficacy has been clarified by using the method for elucidating the function or the efficacy evaluation method of the invention described above.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody as long as it can recognize the gene product, its partial peptide, or a salt thereof.
  • Antibodies against the gene product, a partial peptide thereof, or a salt thereof can be produced by using the gene product as an antigen in accordance with a known method for producing an antibody or antiserum. it can.
  • the gene product is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier or diluent.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and mice and rats are preferably used.
  • a monoclonal antibody-producing hybridoma When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer from a mouse, and collect the spleen or lymph node 2 to 5 days after the final immunization Then, a monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained therein with myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled gene product described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation is performed by known methods, for example, the method of Koehler and Milstein [Ne Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
  • PEG polyethylene glycol
  • Sendai virus Preferably, PEG is used.
  • myeloma cells examples include NS-1, P3U1, and 3 to 20, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably, PEG 1000 to PEG6000) is about 10 to 80%.
  • the cell fusion can be carried out efficiently by incubating at about 20 to 40: preferably about 30 to 37 for about 1 to 10 minutes.
  • the hybridoma culture supernatant is applied to a solid phase (eg, a microplate) on which the antigen of the gene product is directly or adsorbed together with a carrier. Then, add an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A, labeled with a radioactive substance or an enzyme.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse
  • protein A labeled with a radioactive substance or an enzyme.
  • the selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.)
  • a serum-free culture medium for doma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
  • SFM-101 Nissui Pharmaceutical Co., Ltd.
  • the culturing temperature is usually 20 to 40, preferably about 37.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or specific antibody that is collected by using an active adsorbent such as protein A or protein G to dissociate the bond and obtain the antibody. Purification method].
  • the polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (the gene product) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the antibody and separating and purifying the antibody.
  • the type of the carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier.
  • any material can be cross-linked at any ratio.
  • ⁇ serum albumin, ⁇ thyroglobulin, keyhole 'limpet, hemocyanin, etc. are converted into hapten 1 by weight ratio.
  • a method of coupling at a ratio of about 0.1 to 20, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing antibodies.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above.
  • Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody. Since the antibody of the present invention can specifically recognize the gene product, it can be used for quantification of the gene product in a test solution, particularly for quantification by sandwich immunoassay. That is, the present invention provides, for example,
  • one antibody is an antibody that recognizes the N-terminal of the gene product and the other antibody is an antibody that reacts with the C-terminal of the gene product.
  • the gene product can be measured using a monoclonal antibody against the gene product (hereinafter, sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the measurement method using an antibody against the gene product is not particularly limited, and the amount of the antibody, the antigen, or the antibody-antigen complex corresponding to the amount of the antigen in the test solution (for example, the amount of the gene product) is determined. Any measurement method may be used as long as it is detected by chemical or physical means and is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ I4 C] and the like are used.
  • the above enzyme a stable enzyme having a large specific activity is preferable.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system may be used for binding the antibody or antigen to the labeling agent.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and the labeled monoclonal antibody of the present invention is further reacted (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the gene product binds. That is, the antibody used in the primary reaction and the secondary reaction is preferably, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • competition method the antigen in the test solution and the labeled antigen were allowed to react competitively with the antibody.
  • the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody (BZF separation), and the amount of B or F label is measured, and the amount of antigen in the test wave is determined. Quantify.
  • a soluble antibody is used as the antibody
  • BZF separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the above antibody or a solid phase antibody is used as the first antibody.
  • An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.
  • an antigen in a test wave and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated.
  • the antigen is allowed to react with an excess amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated.
  • the amount of label in either phase is measured to determine the amount of antigen in the test wave.
  • nephelometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test wave is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser scattering is preferably used.
  • a measurement system for the gene product may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
  • a measurement system for the gene product may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method.
  • reviews and compendiums for example, Hiroshi Irie “Radio Noatssey” (Kodansha, published in 1974), Irie III ” “Continuing Radio Imnoatssey” (Kodansha, issued in 1979), "Enzyme Immunoassay” edited by Eiji Ishikawa et al.
  • the gene product can be quantified with high sensitivity by using the antibody of the present invention.
  • the antibody of the present invention can detect a gene product whose function has been elucidated, it is useful as a diagnostic agent for a disease involving the gene product.
  • the antibody of the present invention is useful as a diagnostic agent for joint function, more specifically as a diagnostic agent for bone or joint disease.
  • bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibroostitis, renal osteodystrophy, bone Petiet's disease, ankylosing myelitis; cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.
  • non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery
  • bone defects osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibroostitis, renal osteodystrophy, bone Petiet's disease
  • the antibody of the present invention can be used for specifically detecting the gene product present in a subject such as a body fluid or a tissue. It is also used for preparing an antibody column used for purifying the gene product, detecting the gene product present in each fraction during purification, and analyzing the behavior of the gene product in test cells. Can be
  • the antibody of the present invention has low toxicity and can regulate the function of the gene product in vivo. Therefore, for example, a pharmaceutical composition such as a mammalian joint function regulator (preferably a therapeutic agent for joint disease) It is useful as an agent for preventing and treating bone or joint diseases.
  • a mammalian joint function regulator preferably a therapeutic agent for joint disease
  • bone or joint diseases include non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis; typical cartilage diseases such as osteoarthritis and chronic rheumatoid arthritis Joint disease.
  • non-metabolic bone diseases such as bone fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfecta, and scoliosis in the field of orthopedic surgery
  • Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease,
  • the antibody of the present invention can be used for skeletons after surgical operations such as multiple myeloma, lung cancer, and breast cancer.
  • tissue restorative and in the dental field, it can be used for treatment of periodontal disease, repair of periodontal tissue defects in periodontal disease, stabilization of artificial dental roots, repair of ridges and cleft palate.
  • the antibody of the present invention when used as the above-mentioned pharmaceutical composition, it can be prepared by a conventional method. For example, it can be produced and used in the same manner as a pharmaceutical composition containing a compound having a joint function promoting action or a joint function reducing action described above.
  • the present invention provides an antisense nucleic acid for a gene whose function has been elucidated using the function elucidation method of the present invention.
  • DNA encoding a gene product that has been cloned or determined from an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a gene whose function has been elucidated (hereinafter abbreviated as the gene).
  • an antisense polynucleotide capable of inhibiting the replication or expression of a gene whose function has been elucidated (hereinafter abbreviated as the gene).
  • nucleotide nucleic acid
  • Such a polynucleotide (nucleic acid) can hybridize to the RNA of the gene, inhibit the synthesis or function of the RNA, or interact with the RNA related to the gene product. It can regulate and control gene expression.
  • corresponding means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • the “correspondence” between a nucleotide, a nucleotide sequence or a nucleic acid and a peptide (protein) means that the amino acid of the peptide (protein) specified by the sequence derived from the nucleotide (nucleic acid) sequence or its complement is usually used. pointing.
  • 5 'end hairpin loop of the gene 5' end 6—base pair 'repeat, 5' end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3 'end untranslated region, 3 'End palindrome region, and 3' end to aviation
  • the loop may be selected as a preferred target region, but any region within the gene may be selected as a target.
  • Antisense polynucleotides may be 2-deoxy-D-ribose-containing polydeoxynucleotides, D-report-containing polydeoxynucleotides, N-glycosides of purine or pyrimidine bases, and others.
  • polymers having a non-nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds provided that the polymer is Pairing of bases as found in DNA and RNA (contains nucleotides having a configuration permitting base attachment)).
  • They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can further comprise unmodified polynucleotides (or unmodified oligonucleotides).
  • Nucleotides as well as those with known modifications, e.g., those with labels, capped, methylated, or one or more naturally occurring nucleotides with analogs, as known in the art Modified, intramolecularly nucleotide-modified, such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, Those having phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nucleases' inhibitors, toxins) Antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., which have side-chain groups, or interacting compounds (for example, acridine, psoralen, etc.), chelates Those containing compounds (eg, metals, radioactive metals, boro
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. These modifications are It may contain methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.
  • the antisense nucleic acid of the present invention may contain a modified or modified sugar, base, or bond, provided in a special form such as ribosome, microsphere, or applied by gene therapy. Or can be given in additional form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake.
  • polycations such as polylysine, which act to neutralize the charge on the phosphate backbone
  • lipids which enhance interaction with cell membranes or increase nucleic acid uptake.
  • crude water-based substances such as phospholipids and cholesterol are exemplified.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond.
  • Other groups include 3 'or 5' ends of nucleic acids.
  • Specific capping groups for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the antisense nucleic acid of the present invention has low toxicity and can regulate the function of the gene or the gene product in a living body, it can be used, for example, in regulating a joint function of a mammal (preferably a drug for treating a joint disease). It is useful as a pharmaceutical composition, as an agent for preventing or treating bone or joint diseases.
  • bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteomyelitis, osteosarcoma, myeloma, osteogenesis incomplete, scoliosis in the orthopedic field; Metabolic bone diseases such as osteoporosis, osteomalacia, rickets, fibrous ostitis, renal osteodystrophy, bone Behcet's disease, stiff myelitis; and cartilage diseases such as osteoarthritis and rheumatoid arthritis Representative joint diseases are mentioned.
  • the antisense nucleic acid of the present invention can be used as a bone tissue repair agent after surgery for multiple myeloma, lung cancer, breast cancer, etc., and in the dental field, treatment of periodontal disease, periodontal tissue in periodontal disease It can be used to repair defects, stabilize artificial dental roots, repair ridge formation and cleft palate.
  • the antisense nucleic acid of the present invention is used as the above pharmaceutical composition
  • the antisense nucleic acid is used alone or in an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. After insertion, it can be performed according to conventional means.
  • the antisense nucleic acid can be administered as it is or together with an auxiliary for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • the antisense nucleic acid can be sterilized orally as a sugar-coated tablet, capsule, elixir, or micron-capsule, etc., as necessary, or with water or other pharmaceutically acceptable liquids.
  • the antisense nucleic acid can be combined with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of the formulation. Can be manufactured by mixing You. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • additives that can be mixed with tablets, capsules, etc.
  • binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc.
  • Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • aqueous liquid for injection examples include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like.
  • Agents for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), non-ionic surfactant (eg, Polysorbate 80 TM, HCO-50) May be.
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • the above-mentioned pharmaceutical composition includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer eg, human serum It may be combined with preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • the pharmaceutical composition thus obtained is safe and has low toxicity, it can be used, for example, in mammals (for example, humans, rats, mice, rabbits, sheep, bush, horses, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the antisense nucleic acid varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in an osteoarthritis patient (as 60 kg), About 0.1 mg to 100 mg per day, preferably about It is 1.0-5 O mg, more preferably about 1.0-2 O mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method and the like. 60 kg) per day, about 0.01 to 3 Omg per day, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg by intravenous injection. It is convenient to administer. For other animals, the equivalent dose per 60 kg can be administered.
  • the antisense nucleic acid can also be used as a diagnostic oligonucleotide probe for examining the presence of DNA developed in tissues or cells using the method of the present invention and the state of expression thereof.
  • the antisense nucleic acid of the present invention can detect a gene whose function has been elucidated, it is useful as a diagnostic agent for a disease involving the gene.
  • the antisense nucleic acid of the present invention is useful as a diagnostic agent for joint function and as a diagnostic agent for bone or joint disease.
  • bone or joint diseases include non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery; bone defects, osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, ankylosing myelitis; represented by cartilage diseases such as osteoarthritis and rheumatoid arthritis Joint disease.
  • non-metabolic bone diseases such as fractures, refractures, bone deformities and osteoporosis, osteosarcoma, myeloma, osteogenesis imperfections, and scoliosis in the field of orthopedic surgery
  • bone defects osteoporosis Metabolic bone diseases such as osteomalacia, rickets, fibrous osteomyelitis, renal osteodystrophy, bone Behcet's disease, an
  • Genes whose functions have been elucidated using the method for elucidating gene functions of the present invention can be used, for example, as probes to obtain human or warm-blooded animals (for example, rats, mice, guinea pigs, (Eg heron, bird, sheep, widow, bush, horse, dog, dog, cat, monkey, chimpanzee, etc.) in the gene (abnormality), such as damage, mutation or reduced expression of the gene, It can be used as a gene diagnostic agent because it can detect an increase in the gene or overexpression of the gene.
  • human or warm-blooded animals for example, rats, mice, guinea pigs, (Eg heron, bird, sheep, widow, bush, horse, dog, dog, cat, monkey, chimpanzee, etc.
  • abnormality such as damage, mutation or reduced expression of the gene
  • the above-described genetic diagnosis can be performed by, for example, the known Northern hybridization or PCR-SSCP method.
  • the disease is a disease such as joint disease accompanied by decreased joint function. It can be diagnosed as high.
  • the present invention is a.
  • a method for screening a compound that regulates the promoter activity which comprises measuring the promoter activity when a test compound is not administered.
  • a primary cell or H9c2 cell line that constitutes a joint or a primary cell or H9c2 cell line that constitutes a joint into which a gene produced using the meniscal ligament resection model animal of the present invention has been introduced, is used as a host cell.
  • the repo overnight gene accession can be performed by a method known per se, for example, The Journal of Biological Chemistry (JB), Vol. 272, pp. 22800-22808, 1997, Development, Vol. 124, 793- 804, 1997) can be used.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds derived from living organisms (such as carbohydrates and lipids), synthetic compounds, microorganism cultures, cell extracts, plant extracts, and animal tissue extracts. However, these compounds may be novel compounds or known compounds.
  • Promoter activity can be measured by examining the expression level of a reporter gene.
  • the amount of reporter gene expression is measured according to a method such as Northern blotting or Reverse transcrip tion-polymerase chain reaction (RT-PCR)- ⁇ TaqMan polymerase chain reaction or a method analogous thereto. be able to.
  • RT-PCR Reverse transcrip tion-polymerase chain reaction
  • the expression level of the reporter gene when the test compound is administered is about 20% higher than that when the test compound is not administered, preferably 30% or more, More preferably, a compound that enhances about 50% or more can be selected as a compound that enhances the activity of a gene whose function has been elucidated or a gene product thereof.
  • the expression level of the reporter gene when the test compound is administered is about 20% or more, preferably 30% or more, and more preferably about 50% or more as compared with the case where the test compound is not administered.
  • the compound that inhibits can be selected as a compound that inhibits the activity of the gene whose function has been elucidated or the gene product thereof.
  • a compound that enhances the activity of a gene whose function has been elucidated or its gene product is useful as a drug such as a function enhancer for the gene or its gene product.
  • a compound that inhibits the activity of a gene whose function has been elucidated or its gene product is useful as a drug such as a function inhibitor of the gene or its gene product.
  • These medicaments can be produced and used in the same manner as the above-mentioned pharmaceutical composition.
  • the medial collateral ligament is hit during the medial incision.
  • the medial collateral ligament was cut, taking care not to damage the joint surface.
  • To open the joint cavity the femur and tibia were cut down to the rear of the knee.
  • ophthalmic scissors were inserted from the front of the knee joint, and the anterior cruciate ligament and the posterior cruciate ligament connecting the femur and tibia while expanding the joint cavity were confirmed and cut in order. At this time, care should be taken not to damage the blood vessels in the branches of the femoral artery and vein existing around the joint.
  • Example 2 three knee ligaments, the medial collateral ligament, the anterior cruciate ligament, and the posterior cruciate ligament, are cut, whereby the knee joint can be greatly opened laterally and posteriorly.
  • the knee joint was greatly opened posteriorly and posteriorly, and it was confirmed that the meniscus was connected to the tibia by connective tissue.
  • the medial side of the meniscus was excised with ophthalmic scissors.
  • the meniscus is formed symmetrically from the center of the joint, but this resection is limited to the meniscus inside the center.
  • the outer meniscus will remain on the proximal tibia articular surface, connected to the connective tissue.
  • Example 4 Knee joint closure and suture
  • the drug was administered to the joint space of the right limb from which the meniscus was removed.
  • Aru used a dissolved formulation of 10 mg Zm1.
  • Ro-32-355-55 was dissolved in DMSO and ethanol, and the final drug concentration was set to 20 nmo1.
  • the solution was used in a state of being sufficiently mixed with the waltz (10 mg Zm1).
  • the administration method was 1001 twice a week for 2 weeks, 2 times a week for each joint.
  • the administration drug was prepared at the time of use.
  • the rats were sacrificed by exsanguination under ether anesthesia, and the knee joints were collected.
  • the knee joint was fixed with 4% paraformaldehyde at 4 ° C.
  • the knee joint was demineralized with decalcifying solution B (Wako Pure Chemical Industries) for 2 weeks.
  • paraffin embedding was performed in accordance with the usual method (Histology Research Method, Yutaka Sano, Nanzando, 1985). Micro made paraffin block! And sliced to a thickness of 5 m. After slicing, the sections were stretched in a warm bath, mounted on a slide glass, and dried at 37 with a dryer.
  • the prepared sections were stained with hematoxylin-eosin and safranin O according to standard methods, and mounted (Histology Research, Yutaka Sano, Nanzando, 1985). The joint site to which the drug was administered was observed histologically in bright field and compared with the control group.
  • the production method of the present invention efficiently produces a joint disease model animal such as osteoarthritis, and the meniscal ligament resection model non-human mammal obtained by the production method of the present invention has a function. It has been shown that it can be used for methods for elucidating the functions of unknown genes, screening methods for drug candidate compounds, and methods for evaluating drug efficacy.
  • the manufacturing method of the meniscal ligament resection model non-human mammal of the present invention can efficiently produce a joint disease model animal such as osteoarthritis.
  • the meniscal ligament resection model non-human mammal obtained by the production method of the present invention can be used for a method for elucidating the function of a gene whose function is not known, a method for screening a drug candidate compound, a method for evaluating the efficacy, and the like.

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé de construction d'un modèle mammifère non humain de méniscectomie mis en oeuvre comme modèle animal d'une maladie des articulations, telle que la polyarthrite rhumatoïde. Ce procédé est caractérisé en ce qu'il consiste à couper le ligament latéral interne du genou, le ligament croisé antérieur du genou et le ligament croisé postérieur du genou d'un mammifère non humain (notamment, un rat) et puis à couper le ménisque interne de la même jambe, induisant ainsi sensiblement une arthrite mutilante.
PCT/JP2002/006421 2001-06-27 2002-06-26 Procede de construction d'un modele animal de maladie des articulations Ceased WO2003001906A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2001195335 2001-06-27
JP2001-195335 2001-06-27
JP2002-22431 2002-01-30
JP2002022431 2002-01-30

Publications (1)

Publication Number Publication Date
WO2003001906A1 true WO2003001906A1 (fr) 2003-01-09

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PCT/JP2002/006421 Ceased WO2003001906A1 (fr) 2001-06-27 2002-06-26 Procede de construction d'un modele animal de maladie des articulations

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WO (1) WO2003001906A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000000161A2 (fr) * 1998-06-30 2000-01-06 The Procter & Gamble Company Procedes pour l'inhibition d'activite tef-3

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000000161A2 (fr) * 1998-06-30 2000-01-06 The Procter & Gamble Company Procedes pour l'inhibition d'activite tef-3

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BENODE A. ET AL., TOXICOLOGIC PATHOLOGY, vol. 27, no. 1, 1999, pages 134 - 142, XP002965861 *
KARAHAN S. ET AL., COMPARATIVE MEDICINE, vol. 51, no. 6, 2001, MENPHIS, pages 504 - 512, XP002965886 *
MESSNER K., CLINICAL BIOMECHANICS, vol. 9, no. 1, 1994, pages 37 - 43, XP000418364 *

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