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WO2007000884A1 - Agent prophylactique/therapeutique pour les maladies osseuses /articulaires et procede de criblage de l’agent - Google Patents

Agent prophylactique/therapeutique pour les maladies osseuses /articulaires et procede de criblage de l’agent Download PDF

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Publication number
WO2007000884A1
WO2007000884A1 PCT/JP2006/311467 JP2006311467W WO2007000884A1 WO 2007000884 A1 WO2007000884 A1 WO 2007000884A1 JP 2006311467 W JP2006311467 W JP 2006311467W WO 2007000884 A1 WO2007000884 A1 WO 2007000884A1
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Prior art keywords
group
optionally substituted
serine
bone
expression
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Japanese (ja)
Inventor
Yukio Yoneda
Eiichi Hinoi
Takeshi Takarada
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Kanazawa University NUC
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Kanazawa University NUC
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a cell differentiation regulator, a bone / joint disease prevention / therapeutic agent, a substance capable of regulating cell differentiation, or a screening method for a substance capable of preventing / treating bone / joint disease, and a method that causes changes in cell differentiation.
  • the present invention provides a method for identifying a mutation such as a type, a method for determining an animal's biological state or cell differentiation efficiency with respect to cell differentiation, and a bone / chondrogenic cell with regulated gene expression.
  • Serine (2-amino-3-hydroxypropionic acid) is one of the major amino acids that constitute proteins in the body.
  • L-serine is synthesized from glycolytic intermediate 3-phosphoglycerate (3 PG) through three reactions.
  • 3-phosphohydroxypyruvic acid is produced from 3 PG by 3-phosphoglycerate dehydrogenase (3 PGDH), which is the rate-limiting enzyme in a series of L-serine synthesis reactions.
  • O-phosphoserine is produced from 3-phosphohydroxypyruvate by phosphoserine aminotransferase (PSAT), and finally L-serine is synthesized by phosphoserine phosphatase (PSPH).
  • PSAT phosphoserine aminotransferase
  • PSPH phosphoserine phosphatase
  • D-serine is present in the mammalian brain and activates the glutamate ZN-methinolol D-aspartate (NMDA) receptor responsible for neurotransmission. Therefore, D-serine is thought to play an important role in the brain.
  • NMDA glutamate ZN-methinolol D-aspartate
  • SR serine racemase
  • SR is a pyridoxal-15, monophosphate (PLP) dependent enzyme such as brain, liver, etc. Expression in tissues has been confirmed. Therefore, SR is thought to be responsible for the production of D-serine in the brain, and its importance is recognized in neurotransmission.
  • the present invention provides a drug or reagent having a new mechanism of action against various diseases based on the knowledge obtained by the functional analysis of a series of serine synthases, and development of the drug or reagent.
  • the purpose is to provide useful means.
  • SR can negatively regulate the differentiation of cells having the ability to form bone and cartilage.
  • 3 PGDH unlike SR, can positively regulate the differentiation of cells having the ability to form bone and cartilage.
  • the present inventors have further found that this action of 3 PGDH can be brought about by L-serine synthesized thereby. Therefore, by regulating the expression or function of selenium synthase, or by using L-serine or its natural precursor, differentiation of osteoblasts, chondrocytes and other cells that have bone / cartilage-forming ability, Alternatively, it may be possible to regulate bone and cartilage formation.
  • screening for substances that regulate the expression or function of serine synthase has the ability to regulate the differentiation of cells that have bone and / or chondrogenic capacity, and / or drugs and research for diseases caused by abnormal bone and / or chondrogenesis. It is thought to be useful for the development of chemical reagents.
  • the present invention is as follows:
  • R ls R 2 each independently represents a hydrogen atom, an optionally substituted Cis alkyl group, an optionally substituted C 2 8 alkenyl group, or an optionally substituted C 2 _ 8 Rukiniru group, an optionally substituted C 3 _ 8 cycloalkyl group, optionally substituted ⁇ 3 - 8 Shikuroaruke - group, optionally substituted C 8 Ashiru group, may be substituted C 6 10 aryl group, which may be substituted.
  • R 3 is a hydrogen atom, an optionally substituted hydroxyl group, an optionally substituted amino group, an optionally substituted 8 alkyl group, an optionally substituted C 2 8 alkenyl group, a substituted which may be C 2 8 alkynyl group, an optionally substituted C 3 8 cycloalkyl group, a substituted C 3 _ 8 optionally Shikuroaruke two group, an optionally substituted C 6 10 Ariru group, a substituted are unprotected C 7 _ 10 Ararukiru group., a heterocyclic group, or a halogen atom, a substituted 4-7 may membered,
  • R 4 is a hydrogen atom, an optionally substituted hydroxyl group, an optionally substituted phosphate group, an optionally substituted amino group, or an optionally substituted thiol group.
  • an osteoblastic mako is a promoter of chondrocyte differentiation.
  • An agent for promoting osteoblast or chondrocyte differentiation comprising L-serine or a natural precursor thereof, a prodrug thereof, or a salt thereof.
  • the food material is a food material having a bone / cartilage forming action, [6] or
  • a preventive or therapeutic agent for bone / joint diseases comprising the compound represented by the above formula (I) or a salt thereof.
  • a preventive or therapeutic agent for bone / joint diseases comprising L-serine, a natural precursor thereof, a prodrug thereof, or a salt thereof.
  • bone and joint disease is a disease selected from the group consisting of osteoporosis, rheumatoid arthritis, arthritis, synovitis, and osteoarthritis.
  • An agent for regulating osteoblast or chondrocyte differentiation comprising a substance that regulates the expression or function of serine synthase.
  • Substances that regulate serine synthase expression or function include antisense nucleic acids, ribozymes, RNA i-inducible nucleic acids, decoy nucleic acids, targeting vectors, one, antibodies, dominant negative mutants, and their expression vectors.
  • antisense nucleic acids ribozymes, RNA i-inducible nucleic acids, decoy nucleic acids, targeting vectors, one, antibodies, dominant negative mutants, and their expression vectors.
  • a preventive or therapeutic agent for bone / joint diseases comprising a substance that regulates the expression or function of serine synthase.
  • a method comprising using a test substance and a cell capable of measuring the expression of serine synthase, and evaluating whether the test substance can regulate the expression of serine synthase in the cell.
  • [20] including evaluating whether the test substance can regulate the function of serine synthase in the reconstitution system using a reconstitution system capable of measuring the function of the test substance and serine synthase
  • a screening method for a substance capable of preventing or treating a bone / joint disease comprising evaluating whether or not a test substance is capable of regulating the expression or function of serine synthase.
  • the bone / joint disease is selected from the group consisting of bone or cartilage cancer, dysplasia of bone or cartilage, osteoporosis, rheumatoid arthritis, arthritis, synovitis, osteoarthritis and osteopetrosis
  • a method for identifying a serine synthase mutation that causes a change in osteoblast or chondrocyte differentiation comprising analyzing an effect of a specific mutation of serine synthase on osteoblast or chondrocyte differentiation.
  • a diagnostic agent for the biological state of an animal with respect to osteoblast or chondrocyte differentiation comprising a reagent for measuring the expression level of serine synthase.
  • a diagnostic agent for the biological state of an animal against osteoblast or chondrocyte differentiation comprising a reagent for measuring the amount of L-serine or its natural precursor.
  • a diagnostic agent for the biological state of an animal against osteoblast or chondrocyte differentiation comprising a reagent for measuring a polymorphism of serine synthase.
  • a diagnostic agent for osteoblast or chondrocyte differentiation efficiency comprising at least one component selected from the group consisting of: (i) to (i i i):
  • Kit including (a) and (b) below:
  • osteoblast or chondrocyte is a serine synthase stable expression osteoblast cell line or soft bone cell line.
  • [3 8] A method for preventing or treating bone / joint diseases, comprising administering an effective amount of L-serine, a natural precursor thereof, a prodrug thereof, or a salt thereof to a subject.
  • a method for preventing or treating bone / joint diseases comprising administering to a subject an effective amount of a substance that regulates the expression or function of serine synthase.
  • the modulator of the present invention may be useful for regulating differentiation of cells having the ability to form bone and cartilage, and for preventing and treating bone and joint diseases.
  • the screening method of the present invention is useful for development of an agent for regulating differentiation of cells having the ability to form bone and cartilage, and a prophylactic and therapeutic agent for bone and joint diseases.
  • the diagnostic agent of the present invention enables the evaluation of the biological state of an animal with respect to the differentiation of cells having the ability to form bone and cartilage, or in the treatment of patients with bone and joint diseases, the expression of serine synthase or This is useful because it enables prediction of the therapeutic effect of the substance that modulates the function in the patient concerned.
  • Fig. 1 shows the results of confirmation of serine racemase (SR) expression in the tibia of 1-day-old Wistar rats by in situ hybridization.
  • Figure 2 shows the results of in situ high lysis confirming the expression of serine racemase (SR) in osteoblasts found in the tibia of 1-day-old Wistar rats. Arrows indicate osteoblasts.
  • Fig. 3 shows the results of confirmed SR expression in the established SR stably expressing ATDC 5 cell line by Northern blotting.
  • FIG. 7 shows the results of confirming the expression of 3PGDH in the prepared 3PGDH stable expression MC 3 T3 cell line by Northern plotting.
  • FIG. 8 shows the results of observation of nodule formation in MC 3 T 3-3 PGDH cells using a phase contrast microscope. Arrows indicate nodules.
  • ALP activity was measured on day 14 after culture, and Ca 2+ accumulation was measured on day 28 after culture. ** P ⁇ 0. 0 1 Best mode for carrying out the invention
  • the present invention includes a substance that regulates the expression or function of serine synthase, and controls differentiation of cells having the ability to form bone and cartilage. Agents, modulators of SOX 9 expression, or preventive and therapeutic agents for bone and joint diseases. If necessary, the present invention also provides a substance itself that regulates the expression or function of serine synthase.
  • Serine synthase refers to a protein involved in the biosynthesis of serine (L-serine or D-serine).
  • serine synthase 3-phosphoglycerate (3 PG) to '3-phosphohydroxypyruvate 3-phosphodalysate dehydrogenase (3 PG DH) with catalytic activity to form phosphoserine aminotransferase (P SAT) with catalytic activity to produce O-phosphoserine from 3-phosphohydroxypyruvic acid, O-phosphoserine Phosphoserine phosphatase (PS PH) having catalytic activity to produce L-serine from selenium, and serine racemase (SR) having catalytic activity to convert L-serine to D-serine.
  • 3-phosphoglycerate 3-phosphodalysate dehydrogenase (3 PG DH) with catalytic activity to form phosphoserine aminotransferase (P SAT) with catalytic activity to produce O-phosphoser
  • L-serine synthase among the above-mentioned serine synthases, is 3-phosphodalylate dehydrogenase (3 PGDH) involved in L-serine biosynthesis, phosphoserine Aminotransferase (PSAT) and phosphoserine phosphatase (PS PH) are generically meant, but serine racemase (SR) is excluded.
  • PGDH 3-phosphodalylate dehydrogenase
  • PSAT phosphoserine Aminotransferase
  • PS PH phosphoserine phosphatase
  • 3—Phosphoglycerate dehydrogenation ⁇ “1se (3 PGDH) includes human 3 PGDH (eg, GenBank accession number: orchid — 006623) or orthologs thereof, or variants thereof (including SNPs, haplotypes) 3
  • the ortholog of PGD H is not particularly limited, for example, mammals (eg, urchin, hidge, pig, goat, sanore, usagi, rat, hamster, mo / lemot, mouse), birds (eg, As long as it can regulate (eg, promote) differentiation of bone and cartilage-forming cells, it can be used for the prevention or treatment of bone and joint diseases.
  • the amino acid sequence encoded by the amino acid sequence may have catalytic activity to produce 3-phosphohydroxypyruvic acid from 3-phosphoglycerate (3 PG) 1 or more (for example, 1 to 10) in the amino acid sequence encoded by the amino acid sequence (eg, the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 2)
  • the amino acid may have a mutation (eg, deletion, substitution, addition, insertion).
  • the phosphoserine aminotransferase can be human P SAT (eg, GenBank accession number: Re_058179) or its ortholog, some of which can be variants (including SNPs, haplotypes).
  • P SAT Ol The solog is not particularly limited, and is derived from animals such as mammals (eg, urushi, hidge, pig, goat, sal, usagi, rat, hamster, guinea pig, mouse), birds (eg, turkey), etc. Can be.
  • PSAT can regulate the differentiation of cells with bone / cartilage-forming ability, or as long as it can maintain its function (eg, nodule-forming ability) enough to prevent or treat bone / joint diseases, or As long as it has the catalytic activity to produce O-phosphoserine from 3-phosphohydroxypyruvic acid, mutations of one or more amino acids in the encoded amino acid sequence (eg, 1 to: LO)
  • the phosphoserine phosphatase can be human P S PH (eg, GenBank accession number: ⁇ —004577) or an ortholog thereof, or a variant thereof (including SNP, haplotype).
  • the orthologue of PS PH is not particularly limited. For example, mammals (eg, rabbits, hidges, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice), birds (eg, chickens), etc. May be derived from other animals.
  • PS PH can regulate the differentiation of cells with bone / cartilage formation ability, or as long as it can maintain its function (eg, nodule formation ability) enough to prevent or treat bone / joint diseases, Alternatively, as long as it has catalytic activity to produce L-serine from O-phosphoserine, one or more (eg, 1 to: 10) amino acid mutations (eg, deletion, substitution, addition, (Insert) may be included.
  • Serine racemase can be human SR (eg, GenBank accession number: see ⁇ -021947) or its orthologs, or mutants thereof (including SNP, no-protype).
  • the ortholog of SR is not particularly limited. For example, it can be applied to animals such as mammals (eg, rabbits, hidges, pigs, goats, monkeys, rabbits, rats, hamsters, guinea pigs, mice) and birds (eg, chickens). It can be derived.
  • SR can regulate (eg, suppress) differentiation of cells that have the ability to form bone and cartilage, or as long as it can maintain its function to a degree sufficient to prevent or treat bone or joint disease
  • factors that can play a role eg, SOX 9
  • the encoded amino acid In an acid sequence (eg, an amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 3 or an amino acid sequence represented by SEQ ID NO: 4), one or more (eg, 1 to 10) amino acids It may have a mutation (eg, deletion, substitution, addition, insertion).
  • the substance that regulates the expression or function of serine synthase can be a substance that promotes the expression of serine synthase.
  • serine synthase means that a translation product (ie protein) from the serine synthase gene is produced and functionally localized at the site of action. Therefore, a substance that promotes the expression of serine synthase acts at any stage, such as serine synthase transcription, post-transcriptional regulation, translation, post-translational modification, and localized opiprotein folding. It may be a thing. As used herein, the promotion of serine synthase expression includes the supplementation of serine synthase (protein) itself.
  • serine synthase protein
  • expression vector containing nucleic acid encoding serine synthase protein
  • lipopolysaccharide Wu et al. , Ann NY Acad Sci. 1035: 133-46 (2004)
  • amyloid J3 peptide Wu et al., J Neuroinflararaation. 1 (1): 2 (2004).
  • the serine synthase can be a natural protein or a recombinant protein.
  • Serine synthase can be prepared by a method known per se, for example, a) Serine synthase can be recovered from a biological sample containing serine synthase (eg, bone, cartilage, liver, brain) ⁇ :, b) A transformant is prepared by introducing a serine synthase expression vector (described later) into host cells (eg, Escherichia, Bacillus, yeast, insect cells, insects, animal cells), Serine synthase produced by the transformant may be recovered.
  • host cells eg, Escherichia, Bacillus, yeast, insect cells, insects, animal cells
  • Serine synthase produced by the transformant may be recovered.
  • C) Use rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc.
  • Serine synthase uses solubility methods such as salting out and solvent precipitation; dialysis, ultrafiltration, gel filtration, and SDS_polyacrylamide gel electrophoresis. ; Method utilizing difference in charge such as ion exchange chromatography; Method utilizing specific affinity such as affinity chromatography and use of serine synthase antibody; Hydrophobic such as reverse phase high performance liquid chromatography It is appropriately purified by a method using a difference in sex; a method using a difference in isoelectric point such as isoelectric focusing; a method combining these, etc.
  • the substance that regulates the expression or function of serine synthase can be a substance that suppresses the expression of serine synthase.
  • the substance that suppresses the expression of serine synthase may act at any stage such as transcription of serine synthase, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding.
  • substances that suppress the expression of serine synthase are serine synthase transcripts, specifically mRNAs or antisense nucleic acids for early transcripts.
  • An antisense nucleic acid consists of a base sequence that can hybridize with the target mRNA (initial transcript) under physiological conditions of a cell that expresses the target mRNA (initial transcript), and the target mRNA in the hybridized state.
  • the type of antisense nucleic acid may be DNA, RNA, or DNA / RNA NA.
  • the length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the transcript of serine synthase, and is short. It may be about 15 bases in length, and may be long and include a sequence complementary to the entire mRNA (initial transcription product) sequence.
  • an oligonucleotide consisting of about 15 bases or more, preferably about 15 to about 30 bases, more preferably about 18 bases to about 30 bases is exemplified.
  • antisense nucleic acids not only hybridize with serine synthase transcripts to inhibit translation, but also bind to double-stranded DNA to form a triplex and transcribe into mRNA. It may be capable of inhibiting.
  • a serine synthase transcript specifically mRNA or an initial transcript, specific within the coding region (including the intron in the case of the initial transcript)
  • a ribozyme that can be cleaved Ribozyme is an RNA having an enzyme activity that cleaves nucleic acids.
  • oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleavage activity. In the present invention, as long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA.
  • RNAs found in infectious RNAs such as viroids and virsoids
  • hammerheads and hairpins are known.
  • a ribozyme is used in the form of an expression vector containing a DNA encoding the ribozyme, a hybrid liposome in which a tRNA-modified sequence is further linked in order to promote translocation to the cytoplasm. [Nucleic Acids Res. 29 (13): 2780-2788 (2001)].
  • RNA i-inducible nucleic acid refers to a polynucleotide that can induce the RNA i effect when introduced into a cell, and is preferably RNA.
  • the RNA i effect is a phenomenon in which a double-stranded RNA containing the same nucleotide sequence (or a partial sequence thereof) as mRNA suppresses the expression of the mRNA.
  • the double-stranded structure may be composed of different strands, or may be a double-stranded structure provided by a single RNA system loop structure.
  • the RNA i-inducible nucleic acid include siRNA, st RNA, miRNA and the like.
  • Another example of a substance that suppresses the expression of serine synthase is a decoy nucleic acid.
  • Decoy nucleic acid refers to a polynucleotide that mimics the region to which a transcriptional regulatory factor binds. Decoy nucleic acid as a substance that suppresses the expression of serine synthase is a region to which a transcriptional activator for serine synthase binds Can be included. The decoy nucleic acid is not limited as long as it includes a region to which a transcriptional activator for serine synthase binds.
  • a transcriptional activator for 3PGDH S1 binding site to which Sp1 binds may contain an AP 1 binding site to which the activating factor AP 1 binds, or contain about 2 kbp upstream of these genes.
  • the decoy nucleic acid for example, an oligonucleotide (S-oligo) having a thiophosphoric diester bond in which the oxygen atom of the phosphoric diester bond portion is substituted with a sulfur atom, or a methyl phosphate group having an uncharged phosphodiester bond.
  • S-oligo oligonucleotide having a thiophosphoric diester bond in which the oxygen atom of the phosphoric diester bond portion is substituted with a sulfur atom, or a methyl phosphate group having an uncharged phosphodiester bond.
  • substituted oligonucleotides and the like which are modified to make the oligonucleotide less susceptible to degradation in vivo.
  • the decoy nucleic acid may completely coincide with the region to which the transcriptional activator binds, but it is sufficient that the decoy nucleic acid retains the identity that allows the transcriptional activator to bind to the serine synthase.
  • the length of the deconucleic acid is not particularly limited as long as the transcriptional activator binds.
  • the decoy nucleic acid may include the same region repeatedly.
  • the targeting vector used in the present invention comprises a first polynucleotide and a second polynucleotide homologous to a serine synthase gene capable of inducing homologous recombination of a serine synthase gene, and a selection marker as necessary.
  • the first and second polynucleotides are polynucleotides having sufficient sequence identity and length to cause homologous recombination with genomic DNA containing serine synthase.
  • the first and second polynucleotides are inherited from serine synthase When the genomic DNA partial region that exists between two regions similar to the first and second polynucleotides is deleted in the genomic DNA containing the pup, the functional defect of the serine synthase gene is lost. Selected to be brought.
  • Selectable markers include positive selectable markers (eg, neomycin resistance gene, hygromycin B phosphotransferase (BPH) gene, blasticidin S deaminase gene, puromycin resistance gene), negative selectable markers (eg, herpes simplex) Pesz virus (HSV) thymidine ⁇ "oneze (tk) gene, diphtheria toxin A fragment (DTA) gene” etc.
  • Targeting vector One is either a positive selection marker or a negative selection marker
  • a targeting vector can also contain two or more recombinase target sequences (eg, 1 o XP sequence used in the Cre / lox P system from Batateriophage P 1, F from yeast F RT sequence used in LPZFRT system) Good.
  • the substance that regulates the expression or function of serine synthase can be a substance that suppresses the function of serine synthase.
  • the substance that suppresses the function of serine synthase is not particularly limited as long as it can interfere with the action of serine synthase, but serine synthase inhibitor protein (eg, serine synthase dominant negative mutant, serine synthesis) Enzyme antibodies), and expression vectors containing nucleic acids encoding them 9
  • the antibody against serine synthase may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique.
  • the antibody may be an antibody fragment (eg, Fab, F (ab ′) 2 ) or a recombinant antibody (eg, single chain antibody). .
  • a polyclonal antibody can be obtained by using a serine synthase or a fragment thereof (which can be a complex cross-linked to a carrier protein such as ushiserum anolevmin, KLH (Keyhole Limpet Hemocyanin) as an antigen.
  • a carrier protein such as ushiserum anolevmin, KLH (Keyhole Limpet Hemocyanin)
  • an adjuvant eg, complete or incomplete Freudite adjuvant
  • 2 to 4 times subcutaneously or intraperitoneally every 2 to 3 weeks measure the antibody titer of the partially collected serum by a known antigen-antibody reaction and confirm its rise
  • animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.
  • Monoclonal antibodies can also be produced by cell fusion methods (eg, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal antibodies and cancer one basic and clinical one”, 2-14 Page, Science Forum Publishing, 1985).
  • the factor is administered to a mouse subcutaneously or intraperitoneally 2-4 times with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected.
  • This leukocyte and myeloma cells are fused to obtain a hybridoma that produces a monoclonal antibody against the factor.
  • Cell fusion is based on the PEG method [J. Immunol. Methods, 81 (2): 223-228
  • hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method.
  • Hypridoma producing monoclonal antibodies can be cultured in vitro or in vivo in mice or rats, preferably mouse ascites: You can get it.
  • the antibodies of the present invention may be chimeric antibodies, humanized or humanized antibodies.
  • Chimeric antibodies are described in, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies are described in Japanese Patent Publication No. 4-506458, According to Kokai 62-296890, etc., human antibodies such as "Nature Genetics, Vol. 15, p. 146-156, 1997", “Nature Genetics, Vol. 7, p. 13-21, 1994” Table 4-504365 Publication, International Application Publication No. TO94 / 25585 Publication, “Nikkei Science, June Issue: Pages 40-50, 1995” “Nature, Vol. 368, p. 856-859, 1994”, JP-T 6-500233, etc. can be used for reference.
  • a dominant negative mutant of serine synthase is one whose activity has been reduced by introducing a mutation into serine synthase.
  • the dominant negative mutant can indirectly inhibit its activity by competing with natural serine synthase.
  • the dominant negative mutant can be prepared by introducing a mutation into a nucleic acid encoding serine synthase. Examples of the mutation include an amino acid mutation (for example, deletion of one or more amino acids, substitution, addition, insertion) that causes a decrease in the function of the functional site.
  • the dominant negative mutant can be prepared by a method known per se using PCR or a known kit.
  • the functional site of SR is pyridoxal-1-5-phosphate binding region (see, for example, Wolosker et al., Proc. Natl. Acad. Sci. USA 96 (23): 13409-13414 (1999)). Can be mentioned.
  • the agent of the present invention has an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. You can also.
  • the oligonucleotide or polynucleotide encoding the above-mentioned nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in mammalian cells to be administered.
  • the promoter used is not particularly limited as long as it can function in the mammal to be administered, for example, SV 40-derived early promoter, cytomegalovirus LTR, rous sarcoma virus LTR, Mo Mu LV-derived Examples include viral promoters such as LTR and adenovirus-derived early promoters, and mammalian constituent protein gene promoters such as the / 3-actin gene promoter, PGK genetic promoter, and transferrin gene promoter.
  • a promoter specific for cells having an ability to form bone and soft bone eg, osteoblasts, chondrocytes
  • Such promoters are specific to cells that have the ability to form bone.
  • the osteoblast specific promoter is type I collagen
  • the osteocalcin gene-derived promoter is type II collagen
  • a promoter derived from an X-type collagen gene can be used.
  • the present invention also provides an expression vector having such a promoter.
  • the expression vector preferably contains a transcription termination signal, that is, a terminator region downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule.
  • a transcription termination signal that is, a terminator region downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule.
  • selectable marker genes for transforming cells such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, genes that complement auxotrophic mutations, etc. It can also be included.
  • the basic backbone vector used as the expression vector may be a plasmid or a viral vector, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes. Examples include virus vectors such as virus, vaccinia virus, box virus, polio virus, Sindbis virus, sendai virus, and Epstein-Barr virus.
  • the agent of the present invention may contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that regulates the expression or function of serine synthase.
  • pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, and other excipients, senorelose, methinorescenellose, hydroxy Propinoresenorelose, polypropinolepyrrolidone, gelatin, gum arabic, polyethylene glycolol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium glycol glycol starch, sodium bicarbonate, calcium phosphate , Disintegrants such as calcium citrate, lubricants such as magnesium stearate, oil jelly, talc, sodium lauryl sulfate, citrate, menthol, Glycyllysine 'Ammonium salt, fragrance such as g
  • Preparations suitable for oral administration include solutions prepared by dissolving effective amounts of substances in diluents such as water and physiological saline, capsules, sachets or tablets containing effective amounts of substances as solids or granules.
  • diluents such as water and physiological saline
  • capsules, sachets or tablets containing effective amounts of substances as solids or granules.
  • a suspension in which an effective amount of a substance is suspended in an appropriate dispersion medium an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed and emulsified in an appropriate dispersion medium, or a powder, granule, etc. is there.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which are antioxidants Agents, buffers, antibacterial agents, tonicity agents and the like may be included.
  • Aqueous and non-aqueous sterile suspensions can also be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the preparation can be enclosed in a container such as an ampoule or a vial at a unit dose or multiple doses.
  • the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and dissolved or suspended in a suitable sterile vehicle immediately before use, and stored in a state.
  • the dosage of the agent of the present invention includes the activity and type of the active ingredient, the mode of administration (eg, oral, parenteral), the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, although it varies depending on the age, etc., it cannot be generally stated, but it is usually about 0.01 mg to about 2.0 g as an active ingredient amount per day for an adult.
  • the agent of the present invention is useful, for example, as a pharmaceutical or research reagent.
  • this agent can be used to prevent or treat bone / joint diseases or to induce bone / joint disease models.
  • Bone joint disease examples thereof include bone or cartilage cancer, bone or cartilage dysplasia, osteoporosis, rheumatoid arthritis, arthritis, synovitis, osteoarthritis, and osteomarl disease.
  • the agent of the present invention is a substance that suppresses the expression or function of L-serine synthase (ie, 3 PGDH, PSAT, PS PH) as a substance that regulates the expression or function of serine synthase.
  • differentiation of cells having the ability to form bone and cartilage eg, osteoblasts, chondrocytes
  • 3PGDH and L-serine produced thereby are factors that can promote the differentiation of cells having the ability to form bone and cartilage.
  • a substance that suppresses the expression or function of 3 PGDH and any of P SAT and PSPH involved in L-serine synthesis releases the differentiation promotion of cells capable of forming bone and cartilage by L-serine (ie, It is considered that the differentiation of cells having the ability to form bone / cartilage can be suppressed), and thus the number of differentiated cells can be decreased, or the formation of bone / cartilage can be suppressed.
  • SR is a factor capable of suppressing the differentiation of cells having the ability to form bone and cartilage.
  • the agent of the present invention can be used for the prevention or treatment of diseases in which inhibition of bone / cartilage formation is desired, or the preparation of a disease model (eg, cell, animal) in which bone / cartilage formation is inhibited (in this specification Medium: ⁇ Below, abbreviated as “preventing the treatment of diseases where inhibition of bone formation” is desired.
  • diseases for which suppression of bone / cartilage formation is desired include bone or cartilage cancer, bone or cartilage dysplasia, and bone marble disease.
  • the disease in which bone / soft bone formation is suppressed in the disease model in which bone / cartilage formation is suppressed can be the same as the later-described disease in which promotion of bone / cartilage formation is desired.
  • the agent of the present invention contains a substance that regulates the expression or function of serine synthase, for example, a substance that promotes the expression or function of L-serine synthase, or a substance that suppresses the expression or function of SR
  • a substance that regulates the expression or function of serine synthase for example, a substance that promotes the expression or function of L-serine synthase, or a substance that suppresses the expression or function of SR
  • Differentiation of bone and cartilage-forming cells Can promote.
  • 3PGDH and L-serine produced thereby are factors that can promote the differentiation of cells having the ability to form bone and cartilage. Therefore, a substance that promotes the expression or function of 3 PGDH and any of PSAT and PSPH involved in L-serine synthesis promotes the differentiation of cells with the ability to form bone and cartilage, thereby increasing the number of differentiated cells.
  • SR is a factor that can suppress the differentiation of cells having the ability to form bone and cartilage. Therefore, a substance that suppresses the expression or function of SR cancels the inhibition of differentiation of cells having the ability to form bone / cartilage by SR (ie, promotes the differentiation of cells having the ability to form bone / cartilage), and thus It is thought that it may cause an increase in the number of differentiated cells or bone 'chondrogenesis.
  • the agent of the present invention can be used for the prevention or treatment of diseases where bone / cartilage formation is desired, or the preparation of disease models (eg, cells, animals) in which bone / cartilage formation is promoted
  • it may be used as “prophylactic or therapeutic treatment of a disease for which promotion of bone / cartilage formation is desired” if necessary.
  • the disease for which promotion of bone / cartilage formation is desired include osteoporosis, rheumatoid arthritis, arthritis, synovitis, and osteoarthritis.
  • the disease in which bone / cartilage formation is promoted in the disease model in which bone / cartilage formation is promoted may be the same as the above-mentioned disease in which suppression of bone / cartilage formation is desired.
  • the agent of the present invention contains the above-mentioned substance that regulates the expression or function of serine synthase as an active ingredient
  • the agent is used in combination of two or more of these substances or the substance and other active ingredients. It can be.
  • Other effective components when the agent of the present invention is a concomitant agent include, for example, substances that can regulate the differentiation of cells having the ability to form bone and cartilage, and preventive and therapeutic agents for bone and joint diseases.
  • substances capable of regulating the differentiation of cells having bone 1 cartilage formation 13 ⁇ 4 as other active ingredients include, for example, substances that can promote osteoblast differentiation (eg, BMP 2 ) Substances that can inhibit osteoblast differentiation (eg, noggin), etc. Substances that can regulate osteoblast differentiation, and substances that can promote chondrocyte differentiation (eg, TGF-iS), suppress chondrocyte differentiation Substances that can (eg PTH) chondrocyte differentiation Substances that can be adjusted are mentioned.
  • preventive / therapeutic agents for bone / joint diseases include, for example, preventive-therapeutic treatments for diseases in which suppression of bone / cartilage formation such as anticancer agents is desired Drugs, as well as osteoporosis prophylaxis (eg, estrogen preparations, bisphosphonate preparations, vitamin D preparations, vitamins (eg, K2) preparations), anti-rheumatic drugs (eg, DMARD (Disease Modifying Ant i-Rheumatic Drug), Examples thereof include prophylactic / therapeutic agents for diseases in which bone / cartilage formation is desired, such as gold compounds), steroidal anti-inflammatory drugs, and non-steroidal anti-inflammatory drugs (eg, selective inhibitor of COX2).
  • preventive-therapeutic treatments for diseases in which suppression of bone / cartilage formation such as anticancer agents is desired Drugs
  • osteoporosis prophylaxis eg, estrogen preparations, bisphosphonate preparations, vitamin D preparations, vitamins (eg, K2) preparations
  • anti-rheumatic drugs
  • the present invention provides an agent for regulating differentiation of cells having the ability to form bone and soft bone, or a preventive / therapeutic agent for bone / joint diseases, comprising L-serine or an analog thereof or a salt thereof.
  • L ′ log is represented by the following formula (I)
  • R 1 R 2 are each independently a hydrogen atom, an optionally substituted C 8 alkyl group, an optionally substituted C 2 8 alkenyl group, a substituted C 2 _ 8 optionally ⁇ Rukieru group , optionally substituted c 3 _ 8 cycloalkyl group, an optionally substituted C 3 8 cycloalkenyl group, optionally substituted Ashiru group, an optionally substituted C 6 10 Ariru group, An optionally substituted C 7 10 aralkyl group or an optionally substituted force that is a 4-7 membered heterocyclic group, or R 2 together with the adjacent nitrogen atom may be substituted Good 4-7 membered heterocycle Form a group,
  • R 3 is a hydrogen atom, an optionally substituted hydroxyl group, an optionally substituted amino group, an optionally substituted alkyl group, an optionally substituted C 2 _ 8 alkenyl group, substituted which may be C 2 - 8 alkyl group, an optionally substituted C 3 - 8 cycloalkyl group, an optionally substituted C 3 - 8 Shikuroaruke two group, optionally substituted c 6 - A 10 0 aryl group, an optionally substituted c 7 — 1 0 aralkyl group, an optionally substituted 4 to 7-membered heterocyclic group, or a halogen atom,
  • R 4 is a hydrogen atom, an optionally substituted hydroxyl group, an optionally substituted phosphate group, an optionally substituted amino group, or an optionally substituted thiol group.
  • Optionally substituted C 8 alkyl group” - 8 alkyl group is a straight chain or branched chain, preferably 1 to 6 carbon atoms, more preferably 1-4 carbon atoms.
  • ⁇ ⁇ 8 Alkyl groups include, for example, methyl, ethyl, propyl, isopropinole, petitnore, isobutinole, sec-petitinole, tert — puchinole, pentinole, issopentinole, neopentinole, 1-ethylpropyl, hexyl, heptyl, octyl It is done.
  • C 2 - 8 alkenyl group is a straight or branched chain, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms is there.
  • the C 2 _ 8 alkenyl group for example, Eparu, 1 one propenyl,
  • Examples include 3 -butenore, 3 -methyl 1 -butur, 1 -pentyl, 2 -pentur, 3 -pentur, 4 1 pentur, hexenyl, heptul, and otatur.
  • C 2 _ 8 alkynyl group of the "optionally substituted C 2 _ 8 alkynyl group” is a straight or branched chain, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms is there.
  • the C 2 _ 8 alkynyl group for example crucible, E Ji - le, 1 one Purobyuru, 2 1 Propienore, 1 1 Petit 2 Nore, 2 1 Petit 2 Nore, 3-Buttininore, 1 1 Pentininore, 2 Pentinore, 3 1 Pentinyl, 4 Pentininore, Hexnore Nore and octyl.
  • “Substituted have may be C 3 - 8 cycloalkyl group", "C 3 _ 8 cycloalkyl group” is preferably 3 to 6 carbon atoms, more preferably from 3 to 4 carbon atoms.
  • C 3 - The 8 cycloalkyl group for example, cyclopropyl, cyclobutyl, cyclopentyl pentyl, hexyl consequent opening, the consequent opening heptyl include Shikurookuchinore.
  • C 3 - 8 Shikuroaruke group is preferably 3 to 6 carbon atoms, more preferably from 3 to 4 carbon atoms.
  • the C 3 _ 8 cycloalkenyl group such as 2-cyclopentene one 1 one I le, 3 over cyclopentene one 1 Inore, 2-cyclopropyl hexene one 1 Inore, 3-cyclohexylene key Sen one 1- Inore, 3- Examples include cycloheptene 1-1-inole and 4-cyclootaten 1-1-yl.
  • the “di- 8- acyl group” in the “optionally substituted 8- acyl group” is linear or branched, preferably having 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms.
  • Examples of the C- 8- acyl group include formyl, acetyl, propionyl, propylyl, valeryl, hexanol, heptanoyl and octanol.
  • Substituted ⁇ may 6 - 10 Ariru group" as the “C 6 one 10 Ariru group", for example, 7 Eniru, naphthyl (e.g., 1 one-naphthyl, 2-naphthyl) is exemplified al are.
  • Ci-s alkyl group C 2 _ 8 alkenyl, C 2 _ 8 alkynyl group, C 3 one 8 Shikuroarukinore group, C 3 - 8 cycloalkenyl group, Ji 8 Ashiru group, C 6 - 10 Ari group, And the same.
  • the aralkyl group may have 1 to 3 substituents at substitutable positions.
  • substituents include halogen atoms (eg, fluorine Element, chlorine, bromine, iodine), carboxyl group, hydroxyl group, amino group, amidino group, thiol group, sulfo group, cyano group, azide group, nitro group, nitroso group, oxo group, phosphoric acid group (or more, substituted) group a), and may have one to three substituent a at substitutable positions, respectively, ⁇ Bok 6 alkyl group, C 2 - 6 alkenyl group, C 2 - 6 Arukieru group, C 3 _ 6 cycloalkyl group, C 3 _ 6 cycloalkenyl group, C!
  • halogen atoms eg, fluorine Element, chlorine, bromine, iodine
  • carboxyl group e.g, hydroxyl group, amino group, amidino group, thiol group, sulfo group, cyano group, azide group
  • 1-6 acyl group C s — i. Ariel group,.
  • Ararukiru group a monocyclic aromatic and non-aromatic Hajime Tamaki 5-6 membered (as described later), ⁇ bets 6 alkoxy group (e.g., 6 ⁇ Rukiruokishi groups, C 2 _ 6 alkenyl - Ruokishi groups, C Bok 6 alkynyloxy groups).
  • the “4- to 7-membered heterocyclic group” in the “optionally substituted 4- to 7-membered heterocyclic group” is selected from oxygen atoms, sulfur atoms, and nitrogen atoms in addition to carbon atoms as ring-constituting atoms.
  • Examples of such monocyclic aromatic heterocyclic groups include furi / le, cheninole, pyridinole, pyrimigenole, pyridazinole, pyrazinole, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl. It is done.
  • Such monocyclic non-aromatic heterocyclic groups include, for example, pyrrolidinyl, piperidyl, morpholinyl, thiomorpholinyl, piperazil, hexamethyleneiminyl, oxazolidinyl, thiazoliduryl, imidazolidinyl, dixolyl, dioxolanyl, tetrahydrobila Nyl and tetrahydrothiopyranyl.
  • the “4- to 7-membered heterocyclic group” described above may have 1 to 3 substituents at substitutable positions.
  • substituents for example, above-mentioned alkyl groups, C 2 _ 8 alkenyl, C 2 _ 8 alkynyl group, C 3 - 8 cycloalkyl group, C 3 - 8 consequent Roarukeniru group, - 8 Ashiru group, Examples thereof include those similar to the substituents that the C 6 — 10 aryl group and the C 7 — 10 alkyl group may have.
  • two or more substituents of a 4- to 7-membered bicyclic ring group can be formed together, and may be substituted 4 It may represent a ⁇ 13 membered fused aromatic or non-aromatic heterocycle.
  • fused aromatic heterocyclic groups include quinolyl, isoquinolyl, quinazolyl, quinoki salinole, phthalajunore, benzofurinole, benzochenore, benzoxazolinole, benzothiazolinole, benzimidazolinore, indolinole
  • the Examples of such a condensed non-aromatic heterocyclic group include dihydroquinolinyl, tetrahydroquinolinyl, dihydroisoquinolinyl, tetrahydroisoquinolinole, dihydrobenzofuraninole, dihydrobenzodioxyninole, tetrahydroben Examples include Zofrael and dihydr
  • Such 4-13 membered substituent which may be optionally has Chijimigofuku ring, for example, above-mentioned alkyl groups, C 2 - 8 alkenyl group, C 2 - 8 alkynyl group, C 3 - 8 cycloalkyl alkyl group, C 3 _ 8 consequent Roarukeniru group, d-8 Ashiru group, C 6 - 10 Ariru groups, and C 7 - 10 Araruki Le group are the same as those of the substituent which may have.
  • Examples thereof include a aralkyl group, a hydroxyl group optionally substituted with a substituent selected from 4 to 7-membered heterocyclic groups, and a thiol group.
  • amino group may be substituted respectively, CI- 8 alkyl, C 2 - 8 alkenyl group, C 3 - 8 cycloalkyl group, C 3 _ 8 Shikuroaruke - Le group, C 6 _ 10 Ariru group, C 7 _ 10 Ararukiru group, and amino group which may optionally be mono- or di-substituted with a substituent selected from the heterocyclic group of 4 to 7 membered. ,
  • the "which may phosphate group even if substituted" may be substituted respectively, - 8 alkyl group, C 2 one 8 alkenyl group, C 3 - 8 cycloalkyl group,
  • C 8 alkyl group in “optionally substituted hydroxyl group”, “optionally substituted thiol group”, “optionally substituted amino group” and “optionally substituted phosphate group” C 2 - 8 alkenyl group, C 3 one 8 cycloalkyl group, C 3 - 8 cycloalkenyl group, C 6 - 10 Ariru group Contact Yopi C 7 - the 10 Ararukiru groups include those respectively described above.
  • C 8 alkyl group in “optionally substituted hydroxyl group”, “optionally substituted thiol group”, “optionally substituted amino group” and “optionally substituted phosphate group” C 2 - 8 Arukeeru group, C 3 _ 8 cycloalkyl group, C 3 - 8 cycloalkenyl group, C 6 - 10 Ariru group, C 7 - 10 Ararukiru groups and 4-7 membered heterocyclic group, It may have 1 to 3 substituents at substitutable positions.
  • substituents for example, above-mentioned alkyl groups, C 2 _ 8 ⁇ alkenyl group, C 2 _ 8 alkynyl group, C 3 - 8 cycloalkyl group, C 3 - 8 cycloalk Kenyir group, Ce-i. Ariru groups, and C 7 _ 10 Ararukiru groups include those similar to the substituent which may have.
  • R 2 is is as described above, preferably a hydrogen atom, may be substitution CI- 6 alkyl group, optionally substituted C 2 _ 6 alkenyl group, an optionally substituted C 2 _ 6 alkynyl group, a substituted ⁇ may 3 _ 6 cyclo alkyl group, optionally substituted C 3 _ 6 cycloalkenyl group, optionally substituted 6- acyl group, optionally substituted.
  • Aromatic or aromatic complex A ring group, or an optionally substituted 5- to 1-membered fused non-aromatic or aromatic complex ring group), or R and R 2 together with the adjacent nitrogen atom are substituted
  • a 5- to 6-membered heterocyclic group eg, an optionally substituted 5- to 6-membered monocyclic non-aromatic or aromatic heterocyclic group, or an optionally substituted 5- to 11-membered A condensed non-aromatic or aromatic heterocyclic group
  • preferably a hydrogen atom preferably a hydrogen atom.
  • R 3 is as described above, and among them, a hydrogen atom, an optionally substituted hydroxyl group, and an optionally substituted amino group are preferable, and an optionally substituted hydroxyl group. Groups are more preferred.
  • the optionally substituted hydroxy group is as described above, but may be substituted — 6 alkyl group, optionally substituted c 2 _6 alkenyl group, optionally substituted. Good c 3 _
  • Ji may be substituted 3 _ 6 cycloalkenyl group, an optionally substituted C 64.
  • An aryl group, a hydroxyl group substituted with a substituent selected from an optionally substituted C 7 10 aralkyl group, or an unsubstituted hydroxyl group is preferred.
  • R 4 is as described above, and among them, an optionally substituted hydroxyl group or an optionally substituted phosphate group is preferable.
  • the hydroxyl group which may be substituted is as described above.
  • the alkyl group which may be substituted the C 2 6 alkenyl group which may be substituted, or the C 3 6 which may be substituted.
  • Aryl group may be substituted
  • a hydroxyl group substituted with a substituent selected from an aralkyl group or an unsubstituted hydroxyl group is preferred.
  • good phosphate group substituted is as described above, among others, it may be substituted - 6 alkyl group, optionally substituted C 2 _ 6 alkenyl group, optionally substituted C 3 6 cycloalkyl group, an optionally substituted C 3 _ 6 Shikuroarukeyuru group, may be substituted.
  • aryl group optionally substituted C 7 .
  • a substituent selected from alkyl groups Substituted phosphate groups or unsubstituted phosphate groups are preferred, and unsubstituted phosphate groups (eg, monophosphate groups, diphosphate groups, and triphosphate groups) are preferred.
  • the “X group” in the “optionally substituted X group” in R x , R 2 , R 3 and R 4 may have 1 to 3 substituents at substitutable positions. Examples of such a substituent include those described above.
  • the substituent A, and an alkyl group which may have one or two substituents A at each substitutable position C 2 - 4 alkenyl group, C 2 - 4 alkyl - le group, C 2 - 4 alkoxy group (e.g., C 2 - 4 Arukiruokishi group) may be.
  • the compound represented by the formula (I) may be L-serine or O-phosphoserine.
  • L-serine or an analog thereof can be L-serine or a natural precursor thereof or a prodrug thereof.
  • the natural precursor of L-serine can be an L-serine precursor in the L-serine biosynthetic pathway, for example, 3-phosphodaricerate, 3-phosphohydroxypyruvic acid, O-phosphoserine, Pyruvate and O-phosphoserine are preferred.
  • Prodrugs of L-serine or its natural precursor can be converted into L-serine or its natural precursor by reactions under physiological conditions in vivo (eg, enzymatic oxidation, reduction or hydrolysis reaction, or gastric acid hydrolysis reaction).
  • a compound that can be converted into a body eg, enzymatic oxidation, reduction or hydrolysis reaction, or gastric acid hydrolysis reaction.
  • a prodrug of L-serine or its natural precursor for example, a compound in which the amino group of L-serine or its natural precursor is acylated or alkylated (for example, the amino group of L-serine or its natural precursor is The above-described optionally substituted Ci-sacyl group, a compound substituted with an optionally substituted Ci-8 alkyl group), L-serine or the carboxyl group of its natural precursor is esterified or Amylated compounds (eg, compounds in which the carboxyl group of L-serine or its natural precursor is substituted with the above-mentioned optionally substituted hydroxyl group or optionally substituted amino group), or L —Serine or its heaven
  • a compound in which the hydroxyl group of the precursor is acylated or alkylated (for example, the hydroxyl group of L-serine or its natural precursor may be substituted as described above, or may be substituted C i — Compounds substituted with 8 alkyl
  • the salt of L-serine or an analog thereof is not particularly limited, but is preferably a pharmaceutically or food-acceptable salt.
  • an inorganic base eg, alkali metal such as sodium or strength, alkaline earth such as calcium or magnesium
  • organic bases eg, trimethylamine, tritisylamine, pyridine, picoline, ethanolamine, diethananolamine, triethanolamine, dicyclohexylamine, ⁇ , ⁇ -dibenzyl Ethylenediamine
  • inorganic acids eg, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid
  • organic acids eg, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, queeze Acid, succinic acid, malic acid, methanesulfonic acid, benzene Sulfonic acid, .r
  • toluene sulfonic acid basic amino acids (eg, arginine, lysine, Ol two Chin) or acidic amino acids (e.g., Asuparagin acid, glutamic acid) and salts with the cited et be.
  • basic amino acids eg, arginine, lysine, Ol two Chin
  • acidic amino acids e.g., Asuparagin acid, glutamic acid
  • L-serine or an analog thereof or a salt thereof can be produced by a per se known organic chemical method or biochemical method (eg, enzymatic reaction).
  • the agent of the present invention may contain an optional carrier such as a pharmaceutically or food acceptable carrier in addition to L-serine or an analog thereof or a salt thereof.
  • a pharmaceutically or food acceptable carrier can be the same as the pharmaceutically acceptable carrier described above.
  • the agent of the present invention can also be a preparation suitable for the above-mentioned oral administration or parenteral administration.
  • the dosage or intake of the agent of the present invention can be determined by the mode used (eg, pharmaceuticals, foods such as drinking water), the activity and type of the active ingredient, the mode of administration (eg, oral, parenteral), the severity of the disease, the administration It depends on the target animal species, the drug acceptability of the administration target, body weight, age, etc., but it cannot be generally stated, but it is usually about 0.1 111 ⁇ to about 5 ⁇ as the amount of active ingredient per day for adults. .
  • the agent of the present invention is useful, for example, as a pharmaceutical, food, or research reagent. When the agent of the present invention is used as a medicine or a research reagent, it can be used, for example, as a prophylactic / therapeutic agent for the above-mentioned bone-related diseases or an inducer of the disease model.
  • examples of the food include general food (eg, bread, dairy products (milk, yogurt), confectionery, candy, rice cake, chocolate, cake, pudding, jelly, soft drink Water, foods), health foods, dietary supplements, and foods for specified health use and functional nutritional foods as defined in the Health Functional Food System of the Ministry of Health, Labor and Welfare.
  • the agent of the present invention when the agent of the present invention contains L-serine, an analog thereof, or a salt thereof, it can promote the differentiation of bone and chondrogenic cells (eg, osteoblasts, chondrocytes).
  • the present inventors have found that L-serine is a factor that can promote differentiation of cells having the ability to form bone and cartilage. Therefore, L-serine or its analog may promote the differentiation of cells having the ability to form bone and cartilage, thereby increasing the number of differentiated cells or promoting the formation of bone and cartilage.
  • the agent of the present invention can be used for the prevention / treatment of the above-mentioned diseases for which promotion of bone / cartilage formation is desired.
  • agent of the present invention When the agent of the present invention eats L-serine or an analog thereof or a salt thereof as an active ingredient, two or more of these compounds, or a combination drug containing these compounds and other active ingredients It can be.
  • Other active ingredients when the agent of the present invention is a concomitant drug include, for example, the other active ingredients described above, as well as amino acids or peptides, carbohydrates (eg, monosaccharides, disaccharides, oligosaccharides), Examples include lipids (eg, unsaturated fatty acids), vitamins, inorganic salts or ions (eg, Ca salts, Ca 2 + ), natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the agent of the present invention may be another substance as an active ingredient (eg, a substance having the ability to regulate differentiation of cells having a bone / chondrogenic ability, bone / joint disease) Substances that have preventive and / or therapeutic effects) In different forms (eg, stored as a mixture in the same container) or isolated from each other (eg, stored in different containers).
  • the agent of the present invention can be in any form such as liquid (water-soluble, insoluble), solid, powder, granule and the like.
  • the agent of the present invention when it is in the form of solid, powder, granule or the like, it can be used by dissolving in water or an aqueous solution.
  • the agent of the present invention may contain a solubilizer.
  • the agent of the present invention for example food, can also be characterized in terms of amino acid composition.
  • the amino acid means a natural L-amino acid that constitutes a natural protein.
  • the agent (food product) of the present invention may contain only L-serine (or an analog or a salt thereof) as an amino acid, or may have the highest amount of L-serine as an amino acid.
  • the amount of L-serine is the highest as an amino acid
  • the amount of L-serine is, for example, 1.5 times or more, preferably 2 times or more, more preferably 3 times by weight with respect to the amount of other specific amino acids.
  • the amount of L-serine in the total amount of amino acids is, for example, about 30% or more by weight, preferably May be about 40% or more, more preferably about 50% or more, even more preferably about 60% or more, and most preferably about 90% or more, or about 95% or more.
  • the present invention also provides a method for producing the agent of the present invention, for example, a food.
  • a method for producing the agent of the present invention for example, a food.
  • adding or enhancing bone / cartilage formation including adding L-serine or analogues or salts thereof to food materials to obtain foods with added or enhanced bone / cartilage formation.
  • a method for producing a processed food product is provided.
  • food material refers to L-serine or analogs thereof or their salts prior to addition ( ⁇ any edible material.
  • Food material refers to L-serine or analogs thereof or
  • the food material may also be a food material that has a bone / cartilage forming action, for example, the formation of bone and cartilage.
  • the food product of the present invention may also be a food product to which L-serine or an analog thereof, or a salt thereof is added or increased in this manner.
  • the present invention also provides a method for regulating differentiation of cells having the ability to form bone and cartilage, which may be a form of in vitro use of the agent of the present invention.
  • This method uses, for example, a substance that regulates the expression or function of a serine synthase and / or serine or its analog or a salt thereof to express serine synthase in a cell having the ability to form bone or cartilage. Or culturing to regulate function.
  • Bone and cartilage-forming cells can be derived from any animal, such as birds, mammals (eg, rabbits, hidges, pigs, goats, sanores, humans, rabbits, rats, hamsters, monoremots, mice) It can be a cell.
  • the cell may be a cell induced to differentiate from a stem cell such as an embryonic stem cell or a somatic stem cell.
  • cells suitable for allogeneic transplantation can be obtained by using cells derived from the same species as the animal intended for transplantation, and transplantation is intended. By using cells derived from individual individuals, cells suitable for allogeneic transplantation can be obtained.
  • the cells can be obtained by a method known per se (eg, F A C S) using a marker molecule (described later).
  • the medium used for cell culture is not particularly limited as long as it is suitable for the differentiation of cells having the ability to form bone and cartilage, and is appropriately selected.
  • minimal essential medium MEM
  • minimal Dulbecco modification Essential medium DMEM
  • F 12 medium F 12 medium or RPMI 16 40 medium
  • additives to the culture medium include various amino acids, various inorganic salts, various vitamins, various antibiotics, and buffering agents.
  • the culture conditions are also appropriately determined.
  • the pH of the culture medium is about 6 to about 8, and the culture temperature is usually about 30 to about 40 ° C.
  • the medium is Serum may or may not be included, but when cell transplantation is intended, a serum-free medium is preferable from the viewpoint of preventing the introduction of unidentified components and reducing the risk of infection.
  • the cells obtained by the method of the present invention can be used for cell therapy (transplantation).
  • the substances and materials used in the culture are the same as those of the subject receiving cell therapy from the viewpoint of allogeneic transplantation. It is preferable to unify substances and materials derived from the same species.
  • the regulation method of the present invention can further comprise isolating a cell whose differentiation is regulated (eg, a cell whose differentiation is promoted or suppressed) from a cell having the ability to form bone and cartilage. Isolation of cells can be performed by a method known per se using a cell marker.
  • the regulation method of the present invention can also include further regulating the differentiation of cells induced to differentiate from cells having the ability to form bone and cartilage.
  • a substance that regulates the expression or function of serine synthase, and / or L A method of culturing cells in the presence of serine or an analog thereof or a salt thereof and another differentiation regulator (eg, a substance capable of regulating the differentiation of cells having the ability to form bone / cartilage described above), serine synthase And / or methods of using other differentiation regulators after culturing cells with the ability to form bone and chondrocytes in the presence of L-serine or its analogs or their salts. . ,
  • the regulation method of the present invention is capable of, for example, regulating the differentiation of cells having the ability to form bone and cartilage, or when differentiating cells other than cells having the ability to form bone and cartilage more specifically from stem cells.
  • it is useful because the differentiation efficiency can be increased by inhibiting the differentiation into cells having the ability to form bone and cartilage. .
  • the present invention regulates SOX 9 expression, a substance capable of regulating the differentiation of cells having the ability to form bone and chondrogenesis, comprising evaluating whether a test substance can regulate the expression or function of serine synthase.
  • substances that can be obtained, or that can prevent or treat bones or related diseases Provided are a screening method, a substance obtained by the screening method, and a differentiation regulator or preventive / therapeutic agent containing the substance.
  • the test substance to be subjected to the screening method may be any known compound or a novel compound, such as a nucleic acid (eg, nucleoside, oligonucleotide, polynucleotide), carbohydrate (eg, monosaccharide, disaccharide). Sugars, oligosaccharides, polysaccharides), lipids (eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides), small organic compounds Compound libraries created using combinatorial chemistry technology, random peptide libraries produced by solid-phase synthesis, or natural components derived from microorganisms, animals and plants, marine organisms, etc. It is done.
  • a nucleic acid eg, nucleoside, oligonucleotide, polynucleotide
  • carbohydrate eg, monosaccharide, disaccharide
  • the screening method of the present invention can be carried out in any form as long as it can be evaluated whether or not a test substance can regulate the expression or function of serine synthase.
  • the screening method of the present invention comprises 1) measurement of serine synthase expression using a cell capable of measuring serine synthase expression, and 2) a reconstitution system capable of measuring the function of serine synthase (non- Measurement of serine synthase function using cultured cell systems), 3) measurement of serine synthase function using serine synthase expressing cells, 4) measurement of serine synthase expression or function using non-human animals Or the like.
  • the screening method using cells capable of measuring the expression of serine synthase can include, for example, the following steps (a) to (c):
  • step (a) of the above method the test substance is placed in contact with a cell capable of measuring the expression of serine synthase.
  • Cell coverage of serine synthase Contact of the test substance can be carried out in the culture medium.
  • a cell capable of measuring the expression of serine synthase is a cell capable of directly or indirectly evaluating the expression level of a serine synthase product (eg, transcription product, translation product).
  • a cell that can directly evaluate the expression level of a serine synthase product can be a serine synthase-expressing cell, while a cell that can indirectly evaluate the expression level of a serine synthase product can It may be a cell that allows reporter assay for the transcriptional regulatory region of the enzyme gene.
  • the cell capable of measuring the expression of serine synthase can be an animal cell, for example, a mammalian cell such as mouse, rat, hamster, monoremot, usagi, inu, sanore, human.
  • Serine synthase-expressing cells are not particularly limited as long as they potentially express serine synthase. Such cells can be easily identified by those skilled in the art, and primary cultured cells, cell lines derived from the primary cultured cells, commercially available cell lines, cell lines available from cell banks, and the like can be used. In addition, it is also preferable to use cells having the ability to form bone and cartilage (eg, osteoblasts, chondrocytes) as serine synthase-expressing cells. Further, cells derived from a bone / joint disease model animal (described later) may be used.
  • bone and cartilage eg, osteoblasts, chondrocytes
  • a cell that enables reporter assembly for the transcriptional regulatory region of the serine synthase gene is a cell that includes a transcriptional regulatory region of the serine synthase gene and a reporter gene operably linked to the region.
  • the transcriptional regulatory region of the serine synthase gene, the reporter gene can be inserted into an expression vector.
  • the transcriptional regulatory region of the serine synthase gene is not particularly limited as long as it can control the expression of serine synthase. For example, the region from the transcription start point of each serine synthase gene to about 2 kbp upstream.
  • the reporter gene can be any gene that encodes a detectable protein or an enzyme that produces a detectable substance, such as GFP (green firefly). Photoprotein) gene, GUS (j3-darc mouth nidase) gene, LUC (luciferase) gene, CAT (chloramphenicol acetyltransferase) gene, and the like.
  • a cell into which a transcriptional regulatory region of a serine synthase gene and a reporter gene operably linked to the region are introduced, as long as the transcriptional regulatory function of the serine synthase gene can be evaluated, that is, the expression level of the reporter gene Is not particularly limited as long as it can be quantitatively analyzed. However, since it expresses a physiological transcriptional regulatory factor for serine synthase and is considered to be more appropriate by evaluating the regulation of serine synthase expression, the introduced cell may have serine synthase expression. Cells are preferred.
  • the medium in which the test substance is contacted with the cells capable of measuring the expression of serine synthase is appropriately selected according to the type of cells used. For example, about 5 to 20% urine fetal serum is used. Including minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DM EM), RPMI 1640 medium, 1999 medium, and the like.
  • the culture conditions are also appropriately determined according to the type of cells used, but for example, the pH of the medium is about 6 to about 8, the culture temperature is usually about 30 to about 40 ° C, and the culture time Is about 12 to about 72 hours.
  • step (b) of the above method first, the expression level of serine synthase in the cell contacted with the test substance is measured.
  • the expression level can be measured by a method known per se in consideration of the type of cells used. For example, when serine synthase-expressing cells are used as cells capable of measuring serine synthase expression, the expression level is determined by a method known per se for serine synthase products, such as transcripts or translation products. More measurable.
  • the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like.
  • the expression level of the translation product can be measured by preparing an extract from the cells and immunologically. Immunological methods include radioisotope immunoassay (RIA method), EL ISA method
  • a fluorescent antibody method can be used.
  • serine synthase transcription can be controlled by measuring serine synthase expression. When cells that allow reporter assembly for a region are used, the expression level can be measured based on the signal intensity of the reporter.
  • the expression level of serine synthase in the cell contacted with the test substance is compared with the expression level of serine synthase in the control cell not contacted with the test substance.
  • the comparison of the expression level is preferably performed based on the presence or absence of a significant difference.
  • the expression level of serine synthase in the control cells that were not contacted with the test substance was measured at the same time, even if the expression level was measured in advance, compared to the expression level of serine synthase in the cells contacted with the test substance.
  • the expression level measured at the same time is preferable from the viewpoint of the accuracy and reproducibility of the experiment.
  • a test substance that regulates the expression level of serine synthase is selected. For example, reduce the expression level of L-serine synthase (ie, 3 PGDH, PSAT, PS PH) (suppress expression) or increase the expression level of SR (promote expression).
  • L-serine synthase ie, 3 PGDH, PSAT, PS PH
  • SR promoter expression
  • a test substance that increases the expression level of L-serine synthase or decreases the expression level of SR can be used for the prophylaxis or treatment of diseases in which the promotion of bone and cartilage formation is desired.
  • the reconstitution system that can measure the function of serine synthase is a non-cultured cell that can evaluate the ability of the test substance to regulate the function of serine synthase, including serine synthase (protein) and other factors.
  • the screening method of the present invention using a reconstitution system can include, for example, the following steps (a) to (c):
  • step (c) A step of selecting a test substance that regulates the function of serine synthase based on the comparison result of (b) above.
  • step (a) of the above method the test substance, serine synthase, and its substrate are contacted in an Atsy system that can convert a substrate to a product by serine synthase.
  • New substances eg, substances labeled with radioactive isotopes, etc.
  • This Atsy system is also configured to contain a capture factor (which may be labeled with a radioisotope or the like as necessary) necessary for the catalytic activity of serine synthase.
  • a capture factor which may be labeled with a radioisotope or the like as necessary
  • cofactors include pyridoxal 15 and monophosphate (SR).
  • cell homogenates containing serine synthase (and cofactor) etc. eg, homogenates of cells transfected with serine synthase expression vectors
  • step (b) of the above method first, the amount of product when the test substance is contacted is measured.
  • the amount of product can be measured by a method known per se, for example, HPLC, M S, or by an immunological technique using an anti-product antibody, or based on detection of radioactivity using a liquid scintillation counter or the like.
  • the amount of product can also be assessed indirectly by measuring the amount of substrate reduction or cofactor consumption. Such a methodology is also included in this step (b).
  • the amount of product when the test substance is contacted is compared with the amount of product when the test substance is not contacted.
  • the comparison of product amounts is preferably performed based on the presence or absence of significant differences.
  • the amount of product when the test substance is not contacted may be the amount of product measured in advance or the amount of product measured at the same time as the measurement of the amount of product when the test substance is contacted.
  • the amount of product measured at the same time is preferable from the viewpoint of experimental accuracy and reproducibility.
  • the reconstitution system capable of measuring the function of SR as a serine synthase see, for example, Wolosker et al., Proc. Natl.
  • step (C) of the above method a test substance that adjusts the amount of product is selected.
  • 3 PGDH produces 3-phosphohydroxypyruvate from 3-phosphodallylate, O-phosphoserine from 3-phosphohydroxypyruvate by PSAT, or L-serine from O-phosphoserine.
  • production by L-serine synthase if necessary
  • test substance that promotes the production of L-serine synthase or suppresses the production of D-serine by SR is used for the prevention / treatment of diseases in which bone / cartilage formation is desired to be promoted. obtain.
  • the screening method for measuring the functional level of serine synthase using serine synthase-expressing cells can include, for example, the following steps (a) to (c):
  • step (a) of the above method the test substance is brought into contact with serine synthase-expressing cells.
  • Contact of the test substance with serine synthase-expressing cells can be performed in a medium.
  • the serine synthase-expressing cell used here may be a cell capable of expressing serine synthase to such an extent that serine synthase can be assayed at the protein level.
  • Preferable examples of such serine synthase-expressing cells include cells in which a serine synthase expression vector is transformed.
  • Contact of the test substance with the serine synthase-expressing cell can be performed in a medium. Substrates and cofactors similar to those in 2) above can be used. 11467
  • step (b) of the above method first, the functional level of serine synthase in the cell contacted with the test substance is measured.
  • the functional level of serine synthase can be measured and evaluated using, for example, the amount of product from the substrate by serine synthase, the amount of SOX9 expression or its promoter activity as an index. These measurements can be performed by a method known per se.
  • the amount of product produced by serine synthase can be measured by the same method as in step b) of the above method 2) or by measuring the amount of product in the culture supernatant.
  • anti-D-serine antibodies Shell et al., Proc. Natl. Acad. Sci.
  • a test substance that regulates the functional level is selected.
  • a test substance that suppresses the production of L-serine synthase, promotes the production of D-serine from L-serine by SR, or reduces the expression level of SOX 9 or its promoter activity is It can be used for the prevention or treatment of diseases where suppression of cartilage formation is desired.
  • test substances having these opposite effects can be used for the prevention / treatment of diseases in which the promotion of bone / cartilage formation is desired.
  • the screening method of the present invention using a non-human animal can include, for example, the following steps (a) to (c):
  • animals such as non-human mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys, and birds such as chickens are used as animals.
  • bone / joint disease model animals eg, ovariectomized mice (postmenopausal osteoporosis model mice), SAMP 6 (senile osteoporosis model mice), collagen-induced arthritis model rats
  • Administration of a test substance to an animal can be performed by a method known per se.
  • step (b) of the above method the amount of serine synthase generated or the level of function can be measured by a method known per se.
  • the expression level or the functional level of serine synthase in osteoblasts or chondrocytes or bones or cartilage cells or tissues isolated or collected from animals is determined by the steps (b) and (b) of the method of 1) to 3) above. It can be measured by a similar methodology.
  • the comparison of the expression level in this step (b) and the step (c) of the above method can also be carried out in the same manner as the methodologies of the above 1) to 3).
  • the screening method of the present invention makes it possible to screen for substances that can regulate the differentiation of cells having the ability to form bone and cartilage, substances that can prevent or treat bone and joint diseases, or substances that can regulate SOX 9 expression. To do. Therefore, the screening method of the present invention is useful for medical rate or development of research reagents.
  • the present invention comprises analyzing the effect of a specific mutation of serine synthase on the differentiation of cells having the ability to form bone / cartilage or the risk of developing bone / joint disease.
  • a method for identifying a mutation in a serine synthase gene that causes a change in the differentiation of a cell having a bone or a change in the risk of developing a bone-related disease, and a protein containing a serine synthase gene mutation identified by the method Provide nucleic acid molecules.
  • Serine synthase gene mutation is any mutation in serine synthase, Includes polymorphisms of serine synthase and artificial mutations of serine synthase.
  • a polymorphism of serine synthase means a nucleotide sequence mutation found at a certain frequency in genomic DNA containing a serine synthase gene in a certain population, and is one or more in a genomic DNA containing a serine synthase gene.
  • DNA substitutions, deletions, additions, insertions eg, SNPs, haplotypes
  • repeats, inversions, translocations, etc. of partial regions in the genomic DNA.
  • the type of serine synthase polymorphism identified by the method of the present invention is a nucleotide sequence that can change the differentiation efficiency of cells having the ability to form bone and cartilage among all types of polymorphisms in serine synthase. Or a nucleotide sequence variation that differs in frequency between animals affected by a given disease (eg, bone / joint disease) and animals not affected.
  • the animal to be analyzed for serine synthase polymorphism is not particularly limited as described above, but human is preferred.
  • the analysis can be performed by a method known per se. For example, if there is a significant difference in the frequency of occurrence of a specific polymorphism depending on the frequency of occurrence of the disease (eg, bone / joint disease) and severity as a result of an analysis method such as linkage analysis, that type of polymorphism Can be determined to be a polymorphism that changes the differentiation of cells having the ability to form bone and cartilage, or changes the risk of developing bone and joint diseases. Analysis can also be performed in vitro.
  • cells that have bone and cartilage forming ability introduced with a serine synthase expression vector containing a specific mutation are cultured, and their differentiation efficiency is determined as a control cyst with a control serine synthase expression vector. Mutations can be analyzed by comparing the efficiency with a specific mutation (eg, polymorphism)
  • a DNA sample prepared from a biological sample derived from a mammal is subjected to sequencing, and a new type of serine synthase polymorphism is determined, or by introducing a nucleotide mutation, serine is introduced.
  • a step of artificially producing a mutant of the synthase gene can also be included.
  • Biological trials can use not only tissues that express selenium synthase or cells or other samples (eg, blood) but also any tissue containing genomic DNA such as hair, nails, skin, and mucous membranes.
  • the biological sample is preferably hair, nails, skin, mucous membrane, blood, plasma, serum, saliva, or the like.
  • Polymorph determination is applied to biological samples with different origins. This can be done by analyzing a large number of nucleotide sequences of the contained genome or transcript and identifying mutations found at a certain frequency in the determined nucleotide sequence.
  • the identification method of the present invention includes, for example, identification of a polymorphism that can affect the risk of developing a predetermined disease, or a variant of a serine synthase gene (eg, a mutation in which differentiation regulating ability or specific activity is enhanced or suppressed). Body).
  • the present invention determines the biological state of an animal with respect to differentiation of cells having the ability to form bone and cartilage (eg, bone and joint diseases) based on the measurement of the expression level of serine synthase or the amount of L-serine or its natural precursor. Provide a method.
  • the determination method of the present invention includes the following steps (a) and (b): (a) expression level of serine synthase or L-serine or a natural precursor thereof in a biological sample collected from an animal Measuring the amount of
  • step (a) of the above method the expression level of serine synthase or L-serine or its natural precursor is measured in a biological sample collected from an animal.
  • the animal is not particularly limited as described above, but is preferably human.
  • the biological sample is not particularly limited as long as it is a sample that can measure the expression level of serine synthase or the amount of L-serine or its natural precursor, and examples thereof include bone and cartilage (or osteoblasts and chondrocytes). .
  • the expression level of serine synthase or the amount of L-serine or a natural precursor thereof can be measured in the same manner as the measurement in the screening method of the present invention.
  • step (b.) the biological state of the animal with respect to the differentiation of cells having the ability to form bone and cartilage can be evaluated based on the expression level of serine synthase or the amount of L-serine or its natural precursor. .
  • first, 'emergence of the measured serine synthase The present amount or the amount of L-serine or its natural precursor is compared with the expression amount of serine synthase in an animal without normal differentiation or in a normal animal.
  • the comparison of the expression levels is preferably performed based on the presence or absence of a significant difference.
  • the expression level of serine synthase or the amount of L-serine or its natural precursor in an animal without normal differentiation or in a normal animal can be determined by a method known per se.
  • the differentiation of cells capable of forming bone and chondrogenesis is further suppressed, while when the amount of L-serine or its natural precursor is high, the bone It is considered that the differentiation of cells having the ability to form cartilage can be further promoted. Furthermore, it is known that a change in the expression level of a specific gene or a change in the amount of a substance in a living body is often observed before and after the onset of a specific disease. Therefore, by analyzing the expression level of serine synthase or the amount of L-serine or its natural precursor, abnormal differentiation of cells with the ability to form bone and cartilage, as well as the onset or onset risk of Z or a given disease are determined. It is considered possible to do.
  • the present invention also provides a diagnostic agent capable of the above determination, which contains a reagent for measuring the expression level of serine synthase or the amount of L-serine or its natural precursor.
  • the reagent for measuring the expression level of serine synthase is not particularly limited as long as the expression of serine synthase can be quantified.
  • the above-mentioned antibody against serine synthase, a nucleic acid probe for serine synthase transcript, or serine synthesis may contain a plurality of primers capable of amplifying the enzyme transcript.
  • the diagnostic agent of the present invention can further contain the labeling substance.
  • labeling substances include fluorescent substances such as FITC and FAM, luminescent substances such as noreminol, luciferin and lucigenin, 3 H, 14 C, 3 2 P, 3 5 S, 1 2 3 I and the like.
  • fluorescent substances such as FITC and FAM
  • luminescent substances such as noreminol, luciferin and lucigenin
  • 3 H, 14 C, 3 2 P, 3 5 S, 1 2 3 I and the like examples include radioisotopes, affinity substances such as piotin and streptavidin.
  • the nucleic acid probe for the serine synthase transcript may be either DNA or RNA, but DNA is preferred in consideration of stability and the like.
  • the probe may be either single-stranded or double-stranded.
  • the size of the probe is not particularly limited as long as the transcript of the serine synthase can be detected. For example, it is about 15 to 100 O bp, preferably about 50 to 500 bp.
  • the probe may be provided in a form fixed on a substrate like a microarray.
  • a plurality of primers capable of amplifying serine synthase are selected such that a detectable size nucleotide fragment is amplified.
  • the detectable size nucleotide fragment is not particularly limited, and may have a length of, for example, about 100 bp or more, preferably about 200 bp or more, more preferably about 400 bp or more.
  • the size of the primer is not particularly limited as long as serine synthase can be amplified, but it is preferably about 15 to 100 bp, more preferably about 18 to 50 bp, and even more preferably about 20 to 30. Can be bp.
  • the diagnostic agent of the present invention can further contain reverse transcriptase.
  • the reagent for measuring the amount of L-serine or its natural precursor is not particularly limited as long as L-serine or its natural precursor can be quantified.
  • an antibody against L-serine eg, available from Cosmo Bio
  • it may contain an antibody against the natural precursor of L-serine.
  • This reagent also contains serine antibodies (antibodies that recognize L and D forms in the same manner) and Z or D-serine antibodies (Schell et al., Proc.
  • the diagnostic agent of the present invention may further contain the labeling substance.
  • the labeling substance include those described above.
  • the determination method and diagnostic agent of the present invention can determine, for example, abnormal differentiation of cells having the ability to form bone and soft bone, the onset or risk of developing bone / joint disease in a predetermined subject, or a predetermined It is determined that the abnormality or disease in the subject is caused by suppression or promotion of serine synthase expression, and that the result is a decrease or increase in the amount of no or L-serine or its natural precursor. Therefore, it is useful for determining a treatment guideline for a predetermined disease in the subject or for improving lifestyle habits for the purpose of preventing a predetermined disease or the like.
  • the present invention provides a method for determining the biological state of an animal with respect to differentiation of cells having the ability to form bone and cartilage (eg, bone and joint diseases) based on measurement of polymorphism of serine synthase.
  • the determination method of the present invention includes the following steps (a) and (b):
  • step (a) of the above method polymorphism of serine synthase is measured in a biological sample collected from an animal.
  • the animals are not particularly limited as described above, but humans are preferred.
  • the biological sample can be the same as described above in the identification method of the present invention.
  • the polymorphic type can be measured by a method known per se.
  • TaqM an PCR method, Invader method, RFLP (Restriction enzyme fragment length polymorphism) method, PCR-S SCP (—Single-stranded DNA higher order structure polymorphism analysis) method, A S O (Allele
  • oligonucleotide method direct sequence method
  • ARMS Amplification Refracting Mutation System
  • step (b) of the above method based on the serine synthase polymorphism, The biological state of the animal relative to the differentiation of cells having can be assessed. Specifically, it is determined whether an animal is likely to have abnormal cell differentiation (eg, excessive promotion or suppression), or whether it is more or less likely to suffer from a given disease in the future. obtain.
  • abnormal cell differentiation eg, excessive promotion or suppression
  • L-serine synthase contains a polymorphism that reduces its function
  • NO or SR contains a polymorphism that enhances its function
  • differentiation of cells with bone / cartilage formation ability when L-serine synthase contains a polymorphism that enhances its function, and Z or SR contains a polymorphism that reduces its function, bone and cartilage formation It is considered that the differentiation of cells having ability can be promoted. It is also known that animals that are likely to develop a specific disease often have a specific type of polymorphism in the gene associated with the disease.
  • an animal containing a polymorphism that enhances or decreases the function of serine synthase has a relatively high possibility of developing a bone / joint disease. Therefore, it is considered possible to determine the possibility of the development of a given disease by analyzing the polymorphism. For example,
  • polymorphisms that can be measured can be such polymorphisms.
  • the polymorphism that can be measured can be obtained, for example, by the identification method of the present invention.
  • the present invention also provides a diagnostic agent capable of the above determination, which comprises a reagent for measuring a polymorphism of serine synthase.
  • the reagent for measuring polymorphism of serine synthase is not particularly limited as long as the type of polymorphism can be determined, and has a specific antibody against a partial peptide containing a polymorphic site and a specific type of polymorphism. It can be a nucleic acid probe capable of specifically measuring serine synthase or a plurality of primers capable of specifically amplifying serine synthase having a specific type of polymorphism.
  • the nucleic acid probe or primer may be for genomic DNA containing a serine synthase or a serine synthase transcript.
  • the reagent may be labeled with a labeling substance. 'The reagent is labeled with a labeling substance. If not recognized, the diagnostic agent of the present invention can further contain the labeling substance.
  • the diagnostic agent of the present invention may also contain a nucleic acid probe, a primer, or a reagent for extracting a transcription product or genomic DNA.
  • the specific antibody against the partial peptide containing the polymorphic site is not particularly limited as long as it recognizes the serine synthase containing the polymorphism more selectively than the serine synthase not containing the polymorphism. .
  • Production of a specific antibody against a partial peptide containing a polymorphic site can be performed by appropriately selecting a partial peptide to be used as an antigen.
  • a partial peptide having a shorter size including the polymorphic site is preferably used in order to enhance recognition of a specific polymorphism.
  • the size of the partial peptide is not particularly limited as long as it has immunogenicity. For example, it may be a peptide composed of 8, 10 or 12 or more consecutive amino acids.
  • the nucleic acid probe capable of specifically measuring a serine synthase having a specific type of polymorphism is not particularly limited as long as a serine synthase having a specific type of polymorphism can be selected.
  • the probe may be either DNA or RNA, but DNA is preferred in consideration of stability and the like.
  • the probe may be either single-stranded or double-stranded.
  • the polymorphism is SNP
  • the size of the probe should be as short as possible so that a serine synthase having a predetermined SNP can be selected.
  • the size of the probe is about 15 to 30 bp. obtain.
  • the probe enables, for example, an ASO (Allele Specific Oligonucleotide) hybridization method.
  • a plurality of primers capable of specifically amplifying a serine synthase having a particular type of polymorphism are selected such that measurable nucleotide fragments are amplified.
  • a plurality of primers is designed to include a polymorphic site at the 3 ′ end of one of the primers, for example.
  • the measurable size of the nucleotide fragment, the size of the primer can be the same as described above.
  • Seri In the case where the means capable of measuring polymorphism of the synthase is a plurality of primers for the serine synthase transcript, the diagnostic agent of the present invention can further contain a reverse transcriptase.
  • the serine synthase polymorphism measuring reagent include those containing a restriction enzyme that recognizes a specific polymorphic site. Such a reagent enables polymorphism analysis by RFLP.
  • the determination method and diagnostic agent of the present invention can determine, for example, abnormal differentiation of cells having bone-soft bone formation ability, risk of developing bone / joint disease, etc. in a predetermined subject, or predetermined subject It is possible to determine that the abnormality or disease is caused by suppression or promotion of serine synthase expression. Therefore, the purpose is to determine treatment guidelines for a given disease in the subject or to prevent a predetermined disease, etc. It is useful for improving lifestyle habits.
  • the present invention regulates the expression or function of serine synthase
  • a method for determining the differentiation efficiency of cells having the ability to form bone and cartilage using a substance, or L-serine or an analog thereof or a salt thereof.
  • the determination method of the present invention includes the following steps (a) and (b):
  • step (b) A step of evaluating the differentiation efficiency of the cells based on the characteristics of the cultured cells.
  • a substance that regulates the expression or function of serine synthase, or L-serine or an analog thereof, or a salt thereof hereinafter referred to as “serine synthase expression or Abbreviated as “substances that regulate function”
  • spinal cord cells eg, bone or cartilage-derived cells such as osteoblasts and chondrocytes
  • Substances that regulate the expression or function of serine synthase can be as described above. L-serine or its natural precursor, serine synthase expression vector is preferred.
  • the animal may also be as described above, but preferably, a subject for which it is desired to determine the differentiation efficiency of cells capable of forming bone and cartilage (eg, a substance that regulates the expression or function of serine synthase, etc.) (Subjects for whom treatment with) is being considered.) Cells collected from force can be used.
  • a subject for which it is desired to determine the differentiation efficiency of cells capable of forming bone and cartilage eg, a substance that regulates the expression or function of serine synthase, etc.
  • Cells collected from force can be used.
  • step (b) of the above method differentiation efficiency can be evaluated based on the analysis result of the characteristics of the cells cultured as described above.
  • the analysis of cell traits is not particularly limited as long as it can characterize cells with the ability to form bone and cartilage.
  • cell-specific marker molecules eg, type I collagen, osteotensive noresin
  • chondrocyte-specific molecules eg, aggrecan, type II collagen, type X collagen
  • differentiation efficiency is evaluated from the viewpoint of whether or not it is sufficient to affirm the usefulness of prevention / treatment of a given disease using a substance that regulates the expression or function of serine synthase. Also good. Furthermore, if necessary, the differentiation efficiency of cells cultured using a substance that regulates the expression or function of serine synthase is different from the differentiation efficiency of control cells (eg, normal cells) cultured using the substance. May be compared. The differentiation efficiency is compared based on, for example, the presence or absence of a significant difference in parameters for the trait. The differentiation efficiency of the control cells may be the efficiency measured in advance or the efficiency measured at the same time with respect to the measurement of the differentiation efficiency of the cells collected from the animal and cultured using the substance.
  • the present invention also provides a diagnostic agent that enables the determination, including a substance that regulates the expression or function of serine synthase. .
  • the determination method and diagnostic agent of the present invention can determine, for example, abnormal differentiation of cells having bone-soft bone formation ability in a predetermined subject, or expression or function of serine synthase in a predetermined subject. Prophylaxis using a substance that regulates the disease ⁇ Because it can predict the effect of treatment, it is useful for determining treatment guidelines for a given disease in the subject. It is for.
  • kits comprising components such as any of the substances, reagents, etc. mentioned herein.
  • the kit of the present invention comprises the following (a), (b):
  • Substance that regulates the expression or function of serine synthase, L-serine or an analog thereof, or a salt thereof, a reagent for measuring the expression level or polymorphism of serine synthase, and the amount of L-serine or its natural precursor At least one selected from the group consisting of:
  • a substance that regulates the expression or function of serine synthase, L-serine or an analog thereof, or a salt thereof a reagent for measuring the expression level or polymorphism of serine synthase, and L
  • the reagent for measuring the amount of -serine or its natural precursor is as described above.
  • the reagent for analyzing cells having the ability to form bone and cartilage is not particularly limited as long as it contains a component that enables analysis of the cell-specific character.
  • Bone ⁇ Reagent for staining cells that have cartilage-forming ability eg, Alizarin Red stain, Alcian blue stain, deposited reagent that contains components that allow quantitative determination of Ca2 +
  • bone 'cartilage-forming ability Reagents for measuring cell-specific activity eg, alfa phosphatase activity
  • bone / cartilage-forming cells eg, osteoblast or chondrocyte-specific marker molecule as described above
  • a reagent comprising a nucleic acid probe or a plurality of primers capable of detecting or quantifying the marker molecule eg, alfa phosphatase activity
  • the reagent for regulating differentiation of cells having the ability to form bone / cartilage is a substance that can regulate the differentiation of cells having the ability to form bone / cartilage (for example, the substance that can regulate the differentiation of osteoblasts or chondrocytes described above). May be included.
  • the kit of the present invention is useful because the preparation of the agent of the present invention and the determination method of the present invention can be carried out easily.
  • the present invention provides a cell having the ability to form bone and cartilage in which the expression of serine synthase (eg, SR, 3 PGDH) is regulated.
  • serine synthase eg, SR, 3 PGDH
  • the cell of the present invention can be any cell having the ability to form bone and cartilage, and examples thereof include osteoblasts and chondrocytes.
  • the cell of the present invention can also be a cell in which the expression of serine synthase is introduced (for example, promoted or suppressed) into which a substance that regulates the expression or function of serine synthase has been introduced.
  • Serine synthase expression cells into which serine synthase expression vectors have been introduced may be used.
  • the cells of the present invention further include, for example, mice such as mice, rats, hamsters, guinea pigs, usagis, inu, cats, mice, horses, hidges, goats, pigs, monkeys, humans and other mammals, birds such as chickens, etc.
  • the cell of the present invention can also be, for example, a primary cultured cell or a cell line (eg, a cell line derived from a primary cultured cell, a commercially available cell line, a cell line available from a cell bank), preferably a cell line. is there.
  • a primary cultured cell or a cell line eg, a cell line derived from a primary cultured cell, a commercially available cell line, a cell line available from a cell bank
  • a cell line eg, a cell line derived from a primary cultured cell, a commercially available cell line, a cell line available from a cell bank
  • an osteoblast cell line and a chondrocyte cell line are preferable as the cells having the ability to form bone and cartilage.
  • osteoblast cell lines include MC3T3-E1 cells, SV-HFO cells, TE-8.5 cells, and U20S cells. Among these, MC3T3-E1 cells are preferred.
  • MC3T3-E1 cells are preferred.
  • the chondrocyte cell line into which the gene is introduced include ATD C5 cells, £ 103-3 / 8 cells, and C20ZA4 cells. Among them, ATDC5 cells are preferable.
  • the cell of the present invention is, for example, a cell that can transiently or stably express the above-mentioned gene, but may preferably be a cell that can stably express.
  • “Stable expression” means that the expression of the target gene is not transient. Specifically, after the cell is cultured (for example, passaged), and after cryopreservation of Z or the cell, the target gene is expressed. Means that the activity level is maintained.
  • the cells of the present invention can be those whose differentiation has been suppressed or promoted, and exhibit various specific traits.
  • differentiation into osteoblasts can be suppressed or promoted, and the amount of acid mucopolysaccharide, the amount of deposited Ca 2+ , alkaline phosphatase activity, and the expression level of the marker molecule described above are decreased or increased.
  • differentiation into chondrocytes can be suppressed or promoted in the cells of the present invention, and the amount of acidic mucopolysaccharide, alkaline phosphatase activity, and the expression level of the above-described marker molecule can be decreased or increased.
  • the cell of the present invention can be produced by a method known per se, for example, the above-described culture method.
  • the cells of the present invention are useful, for example, for screening for substances that can regulate the differentiation of cells having the ability to form bone and cartilage, or substances that can prevent or treat bone / joint diseases (described above).
  • the cells of the present invention can also be used for screening of bone and joint disease pathological marker genes, screening of bone and Z or chondrocyte marker genes, analysis of pathological mechanisms of bone-related diseases, and differentiation of bone and / or chondrocytes. This is useful for analyzing the mechanism. These can be performed by expression profile analysis using, for example, a microarray, a protein chip (eg, an antibody chip or a non-antibody chip such as a chip manufactured by Cipher Jessie) or the like.
  • Example 1 Expression of serine racemase (SR) mRNA in cartilage tissue
  • SR mRNA expression was observed in the entire cartilage and woven tissue, particularly in the hypertrophic layer (Fig. 1). Similarly, SR mRNA was also expressed in osteoblasts. ( Figure 2) .
  • a stable expression strain of serine racemase (w t SR), a D_Ser synthase, was prepared in ATDC 5 cells, which are pre-chondrocytes.
  • Pc DNA—wt SR (a vector for overexpression of wt SR) was introduced into ATDC 5 cells using Lipofectamine 2000, and then G 4 18 resistant cells (ATDC 5— wt SR # 2,3,5,6,7,10,1 1,1 3,14,1 5,1 7,1 9,20,2 1,23,28 (total 16 types) were collected. Further, as the cells of these experiments control, in place of p c DNA- wt SR: the c DNA was introduced into ATDC 5 cells, as well as G4 1 8 cells resistant (ATDC 5- pc DNA # 3, 4 in total 2 types) were collected.
  • Example 3 Analysis of the effect of SR on chondrocyte differentiation process
  • Alcianpur is a substance that exhibits a blue color in response to the acidic polysaccharide that is the extracellular matrix of cartilage tissue, and it is known that the staining of Alcian blue increases as the differentiation stage of chondrocytes progresses. Yes.
  • ATDC 5-pc DNA # 4 ATDC 5—wt SR # l 1 and AT DC 5% wt SR # 28 at a density of 2X 10 4 (cell cm 2 ) on culture plate, 5% FBS and hormone mix
  • DMEMZF 1 2 containing 10 ⁇ gZmL insulin, 10./i gZm L transferrin, 30 nM sodium selenate, and perform Alcian blue staining on day 7 and day 14 of culture. It was.
  • the stainability of Alcian blue staining was quantified using image analysis software. The results are shown in Fig. 4.
  • ATDC 5-wt SR # 11 the staining of Alcian blue on day 7 and 14B of culture was significantly reduced compared to ATDC 5-pc DNA # 4.
  • ATDC 5—wt SR # 28 the staining color of cyan blue on day 7 of culture was not significantly different from that of ATDC 5_pcDNA # 4. was significantly reduced.
  • Example 4 Analysis of the effect of SR on cell proliferation
  • the MTT assay is an analysis method that quantifies the number of living cells by utilizing MTT as a substrate for intracellular mitochondrial dehydrogenase.
  • Br dU is an analog of thymidine and is taken up specifically in the S phase of the cell cycle, so the ability to take up Br dU is an indicator of cell growth rate.
  • ATDC 5-c DNA # 4 and ATDC 5— wt SR # ll are plated on culture plates at a density of 2X 10 4 (cells / cm 2 ) and the next day 5% FBS and hormone mix (10 ⁇ g / The culture was started in DMEM / F12 containing mL insulin, 10 ⁇ g / L transferrin, and 30 nM sodium selenate), and MTT assembly was performed 24 hours after the culture. As a result, there was no significant change in the MTT reduction rate in ATDC 5—p cDNA # 4 and ATDC 5_w t SR # 11 (FIG. 5).
  • ATDC 5 -pc DNA # 4 and ATDC 5—wt SR # ll are seeded in culture plates at a density of 0.25 x 10 4 (cells / cm 2 ) and the next day DMEM / F 12 containing 5% FS The cells were labeled with Br dU for 2 hours, and the ability of these cells to take up Br dU was measured by ELISA. As a result, no significant changes were observed in Br dU uptake ability in ATDC 5—pc DN A # 4 and ATDC 5—wt SR # 11 (Fig. 5).
  • SOX 9 is a transcription factor known to play an important role in the differentiation of cartilage progenitor cells from mesenchymal stem cells and subsequent differentiation into chondrocytes.
  • COS 7 cells were seeded on a culture plate at a density of 3.8 X 10 4 (cells Zcm 2 ), and the following day, using Lipofectamine 2000, 4 X 48— ⁇ 8 9—Luc vector (collagen II)
  • Lipofectamine 2000 4 X 48— ⁇ 8 9—Luc vector (collagen II)
  • PRK_w t SR vector overexpressing wild-type SR
  • PRK—SR K5 6G
  • Example 6 Preparation of a stable expression strain of 3-phosphodalysate dehydrogenase (3 PGDH).
  • MC-3T3-E1 cells an osteoblast cell line, a stable expression line of 3PGDH, an L-Ser synthase, was prepared.
  • pcDNA—wt SR vector for overexpression of wt SR
  • G4 1 8 cells were used.
  • MC 3 T 3-3 PGDH G4 18 resistant cells
  • MC 3 T 3-3 P GDH cells which are 3 PGDH stable expression strains, were cultured and then observed with a phase contrast microscope. Incubate the cells at a density of 1 X 10 4 cells / cm 2 , and then add the next 3 medium with 5 Omg / mL ascorbic acid 5 mM b-glycephosphate 10% FB S— ⁇ was replaced, and this time was set as day 0 of culture for 28 days.
  • Example 8 Analysis of the effect of L-Ser on chondrocyte differentiation process
  • Example 6 and 3 PGDH are known to be rate-limiting enzymes for L_Ser synthesis, suggesting that L-Ser synthesized by 3 PGDH may promote bone and cartilage formation. It was done. Therefore, for the purpose of analyzing the effect of L-Ser on the differentiation ability of cultured chondrocytes, L_Ser was applied to primary cultured chondrocytes at a concentration of 0.6 (mM) from day 0 of culture. Exposure was continued until day 28 and day 28, and Al force phosphatase activity was measured on day 14 of culture and Ca 2+ accumulation was measured on day 283 of culture.
  • mM concentration of 0.6
  • the primary cultured chondrocytes were prepared and cultured as follows.
  • the costal cartilage was removed from a Wistar female rat and immersed in GPBS. Take out the bones and weaves one by one in the clean bench, kill the attached meat pieces, and look slightly transparent when exposed to light. Cut out the growing cartilage tissue site that is close to the cartilage-one bone transition site, GPB S Recovered inside. The tissue was minced with dissecting iron, stirred at 37 ° C for 10 min in 0.1 mL of 0.1% EDTA solution, decalcified, and then the supernatant was removed.
  • the cartilage tissue Enzyme solution (DMEM containing 0.3% collagenase) 1 After stirring at 37 ° C for 3 hours in 01111 ⁇ , the cell suspension fraction was collected in 10% FBS-DMEM. The collected fraction is filtered with a cell filtration filter. The filtrate is centrifuged at 250 g for 5 minutes, and the sediment is suspended in 10% FB S—DMEM, each at a density of 1 ⁇ 10 4 cells / cm 2. Seeded on plate. On the next day, the medium was changed, and this time was designated as day 0 of culture.
  • the regulator of the present invention makes it possible to regulate the differentiation of cells having the ability to form bone and cartilage, and to prevent and treat bone-related diseases.
  • the screening method of the present invention makes it possible to develop a differentiation regulator for cells having the ability to form bone and soft bone, and a preventive / therapeutic agent for bone / joint diseases.
  • the diagnostic agent of the present invention makes it possible to evaluate the biological state of an animal with respect to differentiation of bone and cartilage-forming cells, and regulates the expression or function of serine synthase in the treatment of patients with bone and joint diseases. This makes it possible to predict the therapeutic effect of the substance on the patient.

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Abstract

La présente invention concerne un régulateur de la différenciation d’un ostéoblaste ou d’un chondrocyte ou un agent prophylactique ou thérapeutique pour une maladie osseuse/articulaire présentant un mécanisme d’action nouveau. Plus particulièrement, l’invention décrit les éléments suivants : un régulateur de la différenciation d’un ostéoblaste ou d’un chondrocyte ou un agent prophylactique ou thérapeutique comprenant une substance capable de réguler l’expression ou la fonction d’une sérine synthétase ou d’une sérine ou d’un analogue, ou d’un sel de la substance, de la sérine ou de l’analogue; un procédé de criblage d’une substance capable de réguler la différenciation d’un ostéoblaste ou d’un chondrocyte ou d’une substance capable de prévenir ou de traiter une maladie osseuse/articulaire, le procédé consistant à déterminer si une substance à tester peut réguler l’expression ou la fonction d’une sérine synthétase; un procédé d’identification d’une mutation dans un gène de sérine synthétase, susceptible de provoquer une quelconque modification dans la différenciation d’un ostéoblaste ou d’un chondrocyte; un agent pour le diagnostic d’un état biologique d’un animal quant à la différenciation d’un ostéoblaste ou d’un chondrocyte; un agent diagnostique relatif à l’efficacité de la différenciation d’un ostéoblaste ou d’un chondrocyte; un ostéoblaste ou un chondrocyte dans lesquels l’expression d’une sérine synthétase est régulée, etc.
PCT/JP2006/311467 2005-06-29 2006-06-01 Agent prophylactique/therapeutique pour les maladies osseuses /articulaires et procede de criblage de l’agent Ceased WO2007000884A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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WO2009081854A1 (fr) * 2007-12-21 2009-07-02 University Of Toyama Biomarqueur pour une maladie allergique et utilisation de celui-ci
WO2013121828A1 (fr) * 2012-02-13 2013-08-22 国立大学法人東京工業大学 Procédé pour le criblage pour une substance thérapeutique pour une anomalie osseuse, du cartilage ou dentaire
WO2015166845A1 (fr) * 2014-05-01 2015-11-05 株式会社島津製作所 Procédé pour l'évaluation de l'état de différenciation de cellules
CN116139119A (zh) * 2021-11-19 2023-05-23 北京大学口腔医学院 用于防治骨质疏松的丝氨酸制剂及其应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009081854A1 (fr) * 2007-12-21 2009-07-02 University Of Toyama Biomarqueur pour une maladie allergique et utilisation de celui-ci
WO2013121828A1 (fr) * 2012-02-13 2013-08-22 国立大学法人東京工業大学 Procédé pour le criblage pour une substance thérapeutique pour une anomalie osseuse, du cartilage ou dentaire
WO2015166845A1 (fr) * 2014-05-01 2015-11-05 株式会社島津製作所 Procédé pour l'évaluation de l'état de différenciation de cellules
CN116139119A (zh) * 2021-11-19 2023-05-23 北京大学口腔医学院 用于防治骨质疏松的丝氨酸制剂及其应用

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