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WO2017030394A1 - Composition pharmaceutique pour le traitement ou la prévention de l'obésité - Google Patents

Composition pharmaceutique pour le traitement ou la prévention de l'obésité Download PDF

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Publication number
WO2017030394A1
WO2017030394A1 PCT/KR2016/009118 KR2016009118W WO2017030394A1 WO 2017030394 A1 WO2017030394 A1 WO 2017030394A1 KR 2016009118 W KR2016009118 W KR 2016009118W WO 2017030394 A1 WO2017030394 A1 WO 2017030394A1
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Prior art keywords
cluh
bmper
protein
obesity
agent
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Korean (ko)
Inventor
이기호
신현진
정원희
송지영
이은주
함용호
김성섭
홍성희
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Korea Institute of Radiological and Medical Sciences
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Korea Institute of Radiological and Medical Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity

Definitions

  • the present invention relates to a pharmaceutical composition for treating or preventing obesity, comprising an agent for inhibiting the expression or activity of at least one protein of CLUH (Clustered mitochondrial homolog) and BMPER (BMP binding endothelial regulator) protein, or a gene encoding the at least one protein.
  • a pharmaceutical composition for inhibiting or promoting differentiation into adipocytes and a method of treating obesity comprising administering the pharmaceutical composition.
  • screening of an anti-obesity agent or an agent for controlling differentiation into adipocytes comprising the step of measuring the expression or activity of the protein or gene and a composition for diagnosis of obesity comprising an agent for measuring the expression or activity of the protein or gene
  • the present invention relates to a method for diagnosing obesity.
  • the present invention relates to a kit for screening an obesity agent or a differentiation modulator comprising an agent for measuring the expression level of the protein or gene and a kit for assaying the efficacy of an obesity agent or a differentiator. Furthermore, the present invention relates to compositions and kits for detecting markers for the diagnosis of obesity. Furthermore, the present invention relates to a composition and kit for detecting markers capable of measuring differentiation into adipocytes.
  • Obesity is a biological phenomenon caused by the interaction of genetic, metabolic, environmental and behavioral complex factors and is generally recognized as overweight.
  • BMI body mass index
  • Fat stored in fat cells is used as an important energy source in the body.
  • adipocytes increase numerically but also large amounts of triglyceride synthesis by the differentiation of excessive adipocytes are accompanied by morphological changes including the increase of adipocyte size and various gene expression changes.
  • Increasing the size of fat cells is induced by synthesizing and storing surplus energy in the form of triglycerides.
  • the increase in the size of fat cells can be increased by about 20 times in diameter, and as a result, the cell volume is known to increase by several thousand times.
  • the size of the adipocytes is generally controlled by diet, but the process of differentiating new progenitor cells into adipocytes is not effective as dietary control. It is important to adjust.
  • CLUH Clustered mitochondrial homolog
  • mitochondrial protein mRNA to regulate the in vivo synthesis of mitochondria. Only some are known about the involvement of in vivo synthesis (Aurelia De Pauw et al., Am J Pathol. 2009, 175 (3): 927-939, 2009.09). The fact that CLUH is involved in the process of adipocyte differentiation is unknown.
  • BMPER bone morphogenetic protein
  • BMP bone morphogenetic protein
  • the BMPER is known to be involved in tumor growth (J. Heinke et al., Oncogene, 2012, 31, 2919.2930, 2011.10.24) and is known to have a protective effect against atherosclerosis (Xinchun Pi et. al., Arterioscler Thromb Vasc Biol. 2012, 32 (9): 2214.2222, July 5, 2012), the fact that it is involved in the process of adipocyte differentiation is unknown.
  • the present inventors have tried to find a pharmaceutical composition that can prevent or treat obesity, and as a result, it is possible to prevent or treat obesity by inhibiting the expression of CLUH or BMPER, and to determine the expression level of the anti-obesity drug can be screened.
  • the present invention was completed.
  • One object of the present invention is to treat obesity comprising an agent that inhibits the expression or activity of one or more proteins of a clustered mitochondrial homolog (CLUH) and a BMP binding endothelial regulator (BMPER), or a gene encoding the one or more proteins, or It provides a preventive pharmaceutical composition, and a pharmaceutical composition for controlling differentiation into adipocytes.
  • CLUH clustered mitochondrial homolog
  • BMPER BMP binding endothelial regulator
  • Another object of the present invention is a composition for diagnosing obesity, comprising an agent for measuring the expression level of a clustered mitochondrial homolog (CLUH) or a BMP binding endothelial regulator (BMPER) or a gene encoding the protein, and a diagnostic kit and a fat tax It is to provide a composition capable of measuring differentiation of captivity, and a measurement kit.
  • CLUH clustered mitochondrial homolog
  • BMPER BMP binding endothelial regulator
  • Still another object of the present invention is to provide a diagnostic marker for obesity of CLUH or BMPER protein or gene, and a marker for determining differentiation into adipocytes.
  • Yet another object of the present invention is to provide a method for screening an agent for treating obesity or an agent for differentiation into adipocytes, which comprises measuring the expression level or activity of a CLUH or BMPER protein or a gene encoding the protein.
  • Yet another object of the present invention is to provide a kit for screening an agent for treating obesity or controlling differentiation into adipocytes, the method comprising measuring the expression level of a CLUH or BMPER protein or a gene encoding the protein.
  • the pharmaceutical composition of the present invention can inhibit the differentiation and accumulation of fat into adipocytes, thereby preventing or treating obesity.
  • CLUH Clustered mitochondrial homolog
  • BMPER BMP binding endothelial regulator
  • 1 is a diagram showing the increase in the expression of CLUH during the accumulation of fat and adipocyte differentiation.
  • FIG. 2 is a diagram showing the reduction of fat accumulation and adipocyte differentiation by inhibiting CLUH using CLUH siRNA.
  • FIG. 3 is a diagram showing the increase in the expression of BMPER in the process of fat accumulation and adipocyte differentiation.
  • FIG. 4 is a diagram showing the reduction of fat accumulation and adipocyte differentiation by inhibiting BMPER expression using BMPER siRNA.
  • an aspect of the present invention is an agent that inhibits the expression or activity of at least one protein of CLUH (Clustered mitochondrial homolog) and BMPER (BMP binding endothelial regulator) protein, or the gene encoding the at least one protein It provides a pharmaceutical composition for treating or preventing obesity.
  • the composition may include CLUH, BMPER, or an agent that inhibits the expression or activity of CLUH and BMPER proteins or genes encoding the proteins.
  • CLUH Clustered mitochondrial homolog
  • CLUH Clustered mitochondrial homolog
  • Specific sequencing and protein information of the gene encoding the CLUH can be obtained from a known database such as GeneBank of NCBI (for example, NM_001081158.2, NP_001074627.1.)
  • the CLUH As long as it exhibits the effect of inducing differentiation of fat cells and inducing fat accumulation, homologous proteins or variant proteins thereof may also be included in the scope of CLUH provided by the present invention.
  • the specific base sequence of the gene encoding the CLUH may be SEQ ID NO: 5, but is not limited thereto.
  • BMPER bone morphogenetic protein
  • BMP bone morphogenetic protein
  • the protein is known to inhibit BMP2- and BMP4-dependent osteoblast differentiation and BMP-dependent chondrogenic cell differentiation. Mutations in the gene encoding the protein are known to be associated with severe skeletal disease. Specific base sequence and protein information of the gene encoding the BMPER can be obtained from a known database such as GeneBank of NCBI (for example, NM_028472.2, NP_082748.1).
  • homologous proteins or variant proteins thereof may also be included in the scope of the BMPER provided by the present invention, as long as they exhibit the effect of inducing differentiation of fat cells and inducing fat accumulation.
  • the specific base sequence of the gene encoding the BMPER may be SEQ ID NO: 10, but is not limited thereto.
  • the term "homology" is intended to indicate a degree of similarity to the amino acid sequence of a wild type protein or a base sequence encoding the same, and the amino acid sequence or base sequence of the present invention and Includes sequences having more than one percent identical sequence. Such homology may be determined by visually comparing two sequences, but may be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. Homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA).
  • Automated alignment algorithms in this module include Needleman & Wunsch and Pearson & Lipman and Smith & Waterman sequence alignment algorithms. Algorithms and homology determinations for other useful arrays are automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.
  • the term "agent capable of inhibiting expression or activity” refers to a substance capable of inhibiting the production of a transcript or protein expressed and produced in a gene.
  • the agent includes a transcription factor that binds to a gene and inhibits it at the transcription level; Interfering RNA such as miRNA, siRNA, shRNA, etc., which bind to the transcript that is transcribed and synthesized to degrade the transcript; Compounds which inhibit the expression or inhibit the activity of the CLUH or BMPER, such as low molecular weight compounds; It may be an antibody, aptamer, antagonist, etc. which can bind to the expressed protein, but is not limited thereto.
  • the term "short interfering RNA” refers to double-stranded RNA capable of inducing RNAi that inhibits the activity of a gene.
  • the interference RNA may be miRNA, siRNA, shRNA, etc., which can inhibit the expression of CLUH or BMPER, but the interference RNA may be in any form as long as it inhibits the expression or activity of the CLUH or BMPER gene. It is possible.
  • siRNA obtained by chemical synthesis or biochemical synthesis or in vivo synthesis, or 10 base band or more double stranded RNA in which about 40 bases or more of double stranded RNA is degraded in the body can be used.
  • siRNA that inhibits the expression of CLUH may be siRNA of SEQ ID NO: 3, or siRNA of SEQ ID NO: 4, but is not limited thereto.
  • siRNA that inhibits the expression of BMPER may be siRNA of SEQ ID NO: 8, or siRNA of SEQ ID NO: 9, but is not limited thereto.
  • the interfering RNA is not limited to some of the genes encoding the respective proteins, but specifically about 70%, 75%, 80%, 85%, 90%, 95% or 100% It may be composed of homologous sequences, and RNA or modified variants thereof including double stranded portions may be used.
  • the term “low molecular weight compound” is not limited, and may be included in the present invention as long as it can suppress the expression or inhibit the activity of CLUH or BMPER, and may be a substance derived from nature or a synthesized substance. Specifically, it may be an organic synthetic material or a natural material.
  • the term "antibody” refers to a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody may be prepared by cloning each gene into an expression vector according to a conventional method. The protein encoded by the marker gene can be obtained and prepared by conventional methods from the obtained protein.
  • the form of the antibody is not particularly limited and, if it is a polyclonal antibody, a monoclonal antibody or antigen-binding, a part thereof may be included in the antibody of the present invention and may include all immunoglobulin antibodies, as well as humanized antibodies. It may also contain a special antibody of.
  • the antibodies include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule means a fragment having at least antigen binding function and may be Fab, F (ab '), F (ab') 2 and Fv.
  • the antibody may be an antibody that can specifically bind to CLUH or BMPER protein.
  • aptamer refers to a nucleic acid molecule having binding activity to a given target molecule.
  • the aptamers may be RNA, DNA, modified nucleic acids, or mixtures thereof, and may be linear or cyclic.
  • the shorter the sequence of nucleotides constituting the aptamer the more chemical synthesis and mass production. It is known to be easier, cost-effective, easy to modify chemical formula, excellent in vivo stability and low toxicity.
  • the aptamer may be interpreted as a means capable of inhibiting the activity of the protein by binding to the CLUH or BMPER protein.
  • the term "antagonist” refers to a molecule capable of directly or indirectly reducing a biological activity of a receptor, and when used with a ligand of a receptor, a molecule capable of reducing the action of the ligand is used. Including but not limited to.
  • the antagonist includes a molecule which inhibits the activity of CLUH or BMEPR protein without limitation, and in particular, the antagonist means a molecule that inhibits activity by binding to CLUH or BMPER protein, but is not limited thereto.
  • the term "obesity" means a condition or disease with excess body fat due to energy imbalance.
  • prevention of the present invention means any action that inhibits or delays the development of obesity by administration of the pharmaceutical composition according to the present invention.
  • the expression of CLUH or BMPER is increased during the differentiation and adipocyte accumulation process to adipocytes It was confirmed that (Figs. 1 and 3).
  • the present inventors can prevent or treat obesity by controlling the expression of CLUH or BMPER into adipocytes and accumulating fat, and furthermore, by measuring its expression level, diagnosing obesity or screening for obesity treatment I could see that.
  • the pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for treating or preventing obesity, further comprising a suitable carrier, excipient, or diluent commonly used in the manufacture of the pharmaceutical composition, wherein the carrier is an unnatural carrier. (non-naturally occuring carrier).
  • the pharmaceutical composition may be formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be.
  • carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form forms at least one excipient such as starch, calcium carbonate, sucrose or lactose. (lactose), gelatin, etc. are mixed and prepared.
  • lubricants such as magnesium styrate and talc are also used.
  • Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the content of the formulation included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight based on the total weight of the final composition.
  • the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount
  • the term "pharmaceutically effective amount" of the present invention is to treat or prevent the disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention
  • Sufficient amount means an effective dose level means the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of excretion
  • the duration of treatment, factors including drugs used in combination or coincidental with the composition of the invention used, and other factors well known in the medical art can be determined.
  • the pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects.
  • the dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the age, weight, sex, history, or type of substance used as an active ingredient.
  • the pharmaceutical composition of the present invention may be administered to a mammal, including humans, at 10 to 100 mg / kg, more preferably 10 to 30 mg / kg, per day, and the frequency of administration of the composition of the present invention may be
  • the present invention is not limited thereto, but may be administered once to three times a day or several times in divided doses.
  • the present invention provides a method for preventing or treating obesity, comprising administering a pharmaceutical composition for treating or preventing obesity to an individual at risk of developing obesity or obesity.
  • the composition may include CLUH, BMPER, or an agent that inhibits the expression or activity of CLUH and BMPER proteins or genes encoding the proteins.
  • prevention of the present invention is as described above.
  • treatment of the present invention means any action that improves or advantageously changes the condition of obesity by administering the pharmaceutical composition of the present invention.
  • a method for preventing or treating obesity through the administration of a pharmaceutical composition for treating or preventing obesity which may improve or benefit the symptoms of obesity in an obese individual, or suspect an onset of obesity or a risk of developing obesity Obesity can be prevented from developing.
  • the present invention provides a composition for diagnosing obesity, comprising an agent for measuring the expression level of one or more proteins of a clustered mitochondrial homolog (CLUH) and a BMP binding endothelial regulator (BMPER), or a gene encoding the one or more proteins.
  • CLUH clustered mitochondrial homolog
  • BMPER BMP binding endothelial regulator
  • the composition may include a CLUH, BMPER, or an agent for measuring the expression level of the CLUH and BMPER protein or the gene encoding the protein.
  • CLUH Clustered mitochondrial homolog
  • BMPER BMP binding endothelial regulatr
  • the term “agent capable of measuring the level of a protein or expression level of a gene encoding the protein” specifically refers to an antisense oligonucleotide, primer pair that specifically binds to mRNA of the CLUH or BMPER gene. , But may be selected from the group consisting of probes or antibodies, aptamers, antagonists, and combinations thereof that specifically bind to the CLUH or BMPER protein, but is not limited thereto.
  • the antibodies, aptamers, and antagonists are the same as described above.
  • primer refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths can be modified based on what is known in the art.
  • the primers can be modified, for example methylation, encapsulation, substitution of nucleotides or modifications between nucleotides, for example uncharged linkages such as methyl phosphonate, phosphoester, phosphoroamidate , Carbamate, etc.) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).
  • uncharged linkages such as methyl phosphonate, phosphoester, phosphoroamidate , Carbamate, etc.
  • charged linkers eg, phosphorothioate, phosphorodithioate, etc.
  • the 5 'or 3' end of the primer may be labeled with a fluorescent material or the like.
  • the fluorescent material used is not particularly limited, but FAM (6-carboxyfluorescein), HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), NED TM Etc. can be used.
  • primer pairs of the present invention include SEQ ID NOs: 1 and 2; Or it may be a primer having a sequence of SEQ ID NO: 6 and 7, but is not limited thereto.
  • probe refers to a nucleic acid fragment such as RNA or DNA, which may correspond to a short base to several hundred bases, which may achieve specific binding with a gene or mRNA, and includes an oligonucleotide probe, It may be prepared in the form of single stranded DNA probe, double stranded DNA probe, RNA probe, or the like, and may be labeled for easier detection.
  • diagnosis of the present invention refers to confirming the presence or characteristics of a pathological state, and for the purposes of the present invention, may be to confirm whether obesity exists.
  • the CLUH or BMPER protein or a gene encoding the protein of a sample isolated from a normal person and a subject suspected of obesity is measured to determine the CLUH or BMPER protein of the subject suspected of obesity or the same.
  • Obesity can be determined when the expression level of the encoding gene is increased than normal.
  • the normal person means an individual who is not obese.
  • the present invention provides a kit for diagnosing obesity, comprising the composition for diagnosing obesity.
  • the diagnostic composition for obesity may include a CLUH, BMPER, or an agent for measuring the expression level of the CLUH and BMPER protein or the gene encoding the protein.
  • the "kit” of the present invention may further include instructions for describing optimal reaction performance conditions. Instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet.
  • the kit may be an RT-PCR kit or a DNA analysis (eg, DNA chip) kit, or a protein chip kit.
  • the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR.
  • RT-PCR kits in addition to each primer pair specific for CLUH or BMPER, RT-PCR kits can be used in test tubes or other appropriate containers, reaction buffers (variable in pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq- Enzymes such as polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as a quantitative control.
  • DNA chip kits are those in which a nucleic acid species is attached in a gridded array to a glass surface that is generally no larger than a flat solid support plate, typically a microscope slide.
  • hybridization between the nucleic acid of the phase and the complementary nucleic acid contained in the solution treated on the surface of the chip (hybridization) is a tool that allows massively parallel analysis.
  • the protein chip kit may be a kit in which one or more antibodies against a marker are arranged at a predetermined position on a substrate and immobilized at a high density.
  • the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex, which can be read to confirm the presence or expression level of the protein.
  • Obesity can be determined using the obesity diagnostic kit of the present invention by measuring the expression level of CLUH or BMPER protein or a gene encoding the protein of an obese subject in question.
  • the present invention provides a composition for inhibiting adipocyte differentiation, which comprises an agent for inhibiting the expression or activity of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the composition may include CLUH, BMPER, or an agent that inhibits the expression or activity of CLUH and BMPER proteins or genes encoding the proteins.
  • an agent that inhibits the expression or activity of a CLUH or BMPER protein or a gene encoding said protein is as described above.
  • composition for inhibiting adipocyte differentiation of the present invention can suppress differentiation into adipocytes by inhibiting the expression or activity of CLUH or BMPER.
  • the present invention provides a method for diagnosing obesity, comprising measuring the expression level of one or more proteins of the CLUH and BMPER proteins, or a gene encoding the one or more proteins.
  • the diagnostic method of obesity may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER protein or gene encoding the protein.
  • the "diagnosis method of obesity" of the present invention CLUH or BMPER of the subjects suspected of obesity by measuring the expression level of CLUH or BMPER protein or the gene encoding the protein, respectively, from samples isolated from normal and suspected obesity Obesity can be determined when the expression level of the protein or gene encoding it is increased than normal.
  • the normal person means an individual who is not obese.
  • Determination of the expression level of a CLUH or BMPER protein or a gene encoding the protein can be performed by antisense oligonucleotides, primer pairs, probes that specifically bind to the mRNA of the CLUH or BMPER gene, or specifically binding to the CLUH or BMPER protein. It may be measured using an antibody, aptamer, antagonist, but is not limited thereto.
  • the term "individual" of the present invention means all animals, including humans, who are likely to develop or develop such obesity.
  • isolated sample of the present invention which is isolated from the individual, including the mRNA of the CLUH or BMPER protein or the gene encoding the protein, in the present invention can measure the expression level of the protein or gene Meaning a sample, the sample is not particularly limited, but may be, for example, isolated tissue or separated cells.
  • a method for treating a CLUH or BMPER gene which comprises: (a) treating a candidate agent for treating obesity; (b) measuring the expression level or activity of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein, in cells treated with the anti-obesity agent candidate in step (a); And (c) when the expression amount or activity measured in step (b) is lower than that of the control cells that did not treat the obesity candidate drug substance, judging the candidate agent for obesity treatment as an obesity agent. It provides a screening method.
  • the screening method of the anti-obesity agent may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER protein or gene encoding the protein.
  • the term “therapeutic agent for obesity” refers to a substance that induces inhibition of adipocyte differentiation in an obese individual and has effects such as body fat reduction, fat accumulation inhibition, weight loss, and the like.
  • Nucleic acids, proteins, other extracts or natural products, or compounds, and the like, and specifically, may be, but not limited to, low molecular weight compounds, organic synthetic materials, natural materials, microRNA, siRNA, shRNA, antibodies, aptamers, etc. .
  • the term “substance candidate for obesity” refers to a substance capable of becoming an anti-obesity agent as described above, and is estimated to have a possibility of treating obesity or randomly selected according to a conventional selection method.
  • Individual nucleic acids, proteins, other extracts or natural products, or compounds, and the like, and specifically, may be low molecular weight compounds, organic compounds, natural materials, microRNAs, siRNAs, shRNAs, antibodies, aptamers, and the like. It doesn't work.
  • the measurement of the expression level or activity of the protein or the gene encoding the protein may be to measure the expression level of the CLUH or BMPER protein or the mRNA of the gene.
  • measuring the mRNA expression level of the CLUH or BMPER gene may use antisense oligonucleotides, primer pairs, probes or a combination thereof that specifically binds to the mRNA of the gene, which may include reverse transcriptase polymerase reaction, Competitive reverse transcriptase polymerase reaction, real time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting and DNA microarray chip can be performed using an assay selected from the group consisting of, but not limited to.
  • measuring the expression level of the CLUH or BMPER protein may use an antibody, aptamer, antagonist or a combination thereof that specifically binds to the protein, which may be Western blotting, ELISA, radioimmunoassay, radioimmunoassay.
  • control refers to a cell or tissue expressing CLUH or BMPER that has not been treated with an anti-obesity candidate, and that is in a parallel relationship with the group treated with the candidate.
  • screening method of the obesity treatment agent by identifying the expression of CLUH and BMPER involved in the process of differentiation into adipocytes, the activity and expression of the CLUH and BMPER and the control cells not treated with the candidate drug substance It is designed in a way of comparison. Measuring the expression level of the gene of the present invention or the level of the protein encoded by the gene in a cell in the absence of a candidate agent capable of preventing or treating obesity, and also in the presence of the candidate agent After comparing the two by measuring the expression level or the level of the protein encoded by the gene, the expression level of the gene of the present invention when the candidate substance is present or the level of the protein encoded by the gene is absent of the candidate substance. Substances that decrease below levels can be predicted as agents for the prevention or treatment of obesity.
  • Substances obtained by this screening method will act as a leading compound in the future development of a prophylactic or therapeutic agent for obesity, and its structure may be modified so that the leading substance can exhibit an inhibitory effect on CLUH or BMPER or a gene encoding the same.
  • new obesity prophylaxis or treatments can be developed, and these substances can have a partial or complete inhibitory effect on CLUH or BMPER or genes encoding them, thereby preventing or treating obesity-related diseases.
  • the present invention provides a therapeutic agent for obesity comprising an agent for measuring the expression level of one or more proteins of the clustered mitochondrial homolog (CLUH) and the BMP binding endothelial regulator (BMPER), or a gene encoding the one or more proteins.
  • kits for screening comprising an agent for measuring the expression level of one or more proteins of the clustered mitochondrial homolog (CLUH) and the BMP binding endothelial regulator (BMPER), or a gene encoding the one or more proteins.
  • the kit for screening an obesity agent may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the protein.
  • an obesity therapeutic agent can be selected from an obesity therapeutic candidate.
  • the agent for measuring the expression level of the CLUH or BMPER protein or the gene encoding the protein and the kit are as described above.
  • the present invention provides a kit for assaying efficacy of an obesity therapeutic agent comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the kit for assaying efficacy of the anti-obesity agent may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the protein.
  • Agents and kits for measuring the expression level of the CLUH or BMPER protein or the gene encoding the protein in the present invention are as described above.
  • Effectiveacy assay kit for the treatment of obesity agent includes an agent for measuring the expression level of the protein or the gene encoding the protein, CLUH or BMPER protein in cells expressing CLUH or BMPER treated with the anti-obesity agent Or it may be a kit capable of measuring the expression level of the gene. The lower the expression level of CLUH or BMPER measured above can be assayed as an effective obesity treatment.
  • “Efficacy Assay Kit for Obesity Therapeutics” of the present invention includes an agent for measuring the expression level of the protein or the gene encoding the protein, and the CLUH or BMPER protein in cells expressing CLUH or BMPER treated with the anti-obesity agent Or it may be a kit capable of measuring the expression level of the gene. The lower the expression level of the CLUH or BMPER can be assayed as an effective obesity treatment.
  • the present invention provides a method for treating a cell comprising: (a) treating a candidate adipocyte differentiation candidate with a cell expressing a CLUH or BMPER gene; (b) measuring the expression level or activity of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein in cells treated with the adipocyte differentiation promoter candidate in step (a); And (c) determining the adipocyte differentiation candidate as an adipocyte differentiation promoter when the amount of expression or activity measured in step (b) is lower than that of the control cells not treated with the adipocyte differentiation candidate. It provides a method for screening adipocyte differentiation promoter comprising a.
  • the screening method of the adipocyte differentiation promoter may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • the term “fat cell differentiation regulator” refers to any substance capable of promoting or inhibiting differentiation into adipocytes (nucleic acid, protein, other extract or natural product, or compound, etc.), and specifically, the differentiation regulator is MLPH. Induced over-expression or inhibiting expression can promote or inhibit differentiation into adipocytes, or any substance that can cause a significant change in the amount of MLPH expression in the process of promoting or inhibiting differentiation into adipocytes. do. "Fat cell differentiation promoter” and “fat cell differentiation inhibitor” are included in “fat cell differentiation regulator”.
  • the term "dipose cell differentiation promoter candidate” is a single nucleic acid, protein, other extract or natural product presumed to have the potential to promote adipocyte differentiation according to a conventional selection method or randomly selected; Or a compound, and the like, and specifically, may be a low molecular compound, an organic synthetic material, a natural material, a microRNA, siRNA, shRNA, an antibody, aptamer, and the like, but is not limited thereto.
  • screening method of adipocyte differentiation promoter confirms that expression of CLUH or BMPER is involved in the process of differentiating into adipocytes, thereby preventing the activity and expression of CLUH or BMPER to treat adipocyte differentiation candidate candidates. It was designed in a way that compares with control cells.
  • Measuring the expression level of the gene of the present invention or the level of the protein encoded by the gene in the cell in the absence of a candidate agent capable of promoting the differentiation of adipocytes, and also in the presence of the candidate agent After comparing the two by measuring the expression level of the gene or the level of the protein encoded by the gene, the expression level of the gene of the present invention when the candidate substance is present or the level of the protein encoded by the gene is determined as the candidate substance. Substances that increase above the level in the absence of can be predicted as adipocyte differentiation promoters.
  • the present invention provides a method for treating a cell comprising: (a) treating an adipocyte differentiation inhibitor candidate to a cell expressing a CLUH or BMPER gene; (b) measuring the expression level or activity of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein in cells treated with the adipocyte differentiation inhibitor candidate in step (a); And (c) determining the adipocyte differentiation inhibitor candidate as an adipocyte differentiation inhibitor when the amount of expression or activity measured in step (b) is lower than that of the control cells not treated with the adipocyte differentiation inhibitor candidate. It provides a method for screening an adipocyte differentiation inhibitor comprising a.
  • the screening method of the adipocyte differentiation inhibitor may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER protein or gene encoding the protein.
  • the term “dipotent differentiation inhibitor candidate” refers to individual nucleic acids, proteins, other extracts or natural products presumed to have the possibility of inhibiting adipocyte differentiation according to a conventional selection method or randomly selected; Or a compound, and the like, and specifically, may be a low molecular compound, an organic synthetic material, a natural material, a microRNA, siRNA, shRNA, an antibody, aptamer, and the like, but is not limited thereto.
  • screening method of adipocyte differentiation inhibitor confirms that expression of CLUH or BMPER is involved in adipocyte differentiation, thereby not treating the adipocyte differentiation inhibitor candidates with the activity and expression of CLUH or BMPER. It was designed in a way that compares with control cells.
  • Measuring the expression level of the gene of the present invention or the level of the protein encoded by the gene in the cell in the absence of a candidate substance capable of inhibiting the differentiation of adipocytes, and also in the presence of the candidate substance After comparing the two by measuring the expression level of the gene or the level of the protein encoded by the gene, the expression level of the gene of the present invention when the candidate substance is present or the level of the protein encoded by the gene is determined as the candidate substance. Substances that decrease below levels in the absence of can be predicted as adipocyte differentiation inhibitors.
  • the material obtained by this screening method can be used as an inhibitor of adipocyte differentiation, and especially can be used for the prevention or treatment of obesity.
  • the present invention provides a kit for screening an adipocyte differentiation promoter comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the kit for screening the adipocyte differentiation promoter may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • the fat cell differentiation promoter can be selected from the adipocyte candidates for adipocyte differentiation using the kit for screening the adipocyte differentiation promoter of this invention.
  • the "agent for measuring the expression level of the CLUH or BMPER protein or the gene encoding the protein” and the “kit” are as described above.
  • the present invention provides a kit for screening an adipocyte differentiation inhibitor comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the kit for screening the adipocyte differentiation inhibitor may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • the kit for screening an adipocyte differentiation inhibitor of the present invention can be used to select an adipocyte differentiation inhibitor from a candidate adipocyte differentiation inhibitor.
  • the "agent for measuring the expression level of the CLUH or BMPER protein or the gene encoding the protein” and the “kit” are as described above.
  • the present invention provides a kit for assaying the efficacy of an adipocyte differentiation promoter comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the kit for assaying efficacy of the adipocyte differentiation promoter may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • Effectiveacy assay kit for adipocyte differentiation promoter includes an agent for measuring the expression level of the protein or the gene encoding the protein, in the cells expressing CLUH or BMPER treated adipocyte differentiation promoter It may be a kit capable of measuring the expression level of the CLUH or BMPER protein or the gene. The higher the expression level of CLUH or BMPER measured can be assayed as an effective adipocyte differentiation promoter. Such an efficacy assay kit can be effectively used for verifying the titer of adipocyte differentiation promoter in a general adipocyte differentiation process.
  • the present invention provides a kit for assaying the efficacy of an adipocyte differentiation inhibitor comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the kit for assaying efficacy of the adipocyte differentiation inhibitor may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • Effectiveacy assay kit for adipocyte differentiation inhibitor includes an agent for measuring the expression level of the protein or the gene encoding the protein, in the cells expressing CLUH or BMPER treated adipocyte differentiation inhibitor It may be a kit capable of measuring the expression level of the CLUH or BMPER protein or the gene. The lower the expression level of CLUH or BMPER measured above can be assayed as an effective adipocyte differentiation inhibitor. Such an efficacy assay kit can be effectively used for verifying the titer of adipocyte differentiation inhibitor in general adipocyte differentiation process.
  • the present invention provides a composition for detecting a marker for diagnosing obesity, comprising an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the marker detection composition for diagnosing obesity may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER protein or gene encoding the protein.
  • the agent for measuring the expression level of the CLUH or BMPER protein or the gene encoding the protein is as described above.
  • the term “marker for diagnosing obesity” may refer to an organic biomolecule that exhibits a significant difference in expression in normal individuals and obese individuals.
  • the marker detection composition for diagnosing obesity of the present invention the level of expression equal to or higher than that of CLUH or BMPER, and for the diagnosis of obesity, the expression level in the non-obese and obese individuals shows a significant difference Markers can be detected.
  • the present invention provides a marker detection kit for diagnosing obesity, comprising a composition for detecting markers for diagnosing obesity.
  • the marker detection composition for diagnosing obesity may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER protein or gene encoding the protein.
  • the term “agent for measuring the expression level of a CLUH or BMPER protein or a gene encoding the protein”, “a marker for diagnosing obesity”, “a composition for detecting a marker for diagnosing obesity”, and a kit are described above. As shown.
  • the marker detection kit for diagnosing obesity of the present invention it is possible to detect a marker for diagnosing obesity, which shows a significant difference in expression level than in a control sample.
  • the present invention provides a use of the pharmaceutical composition in the manufacture of a medicament for preventing or treating obesity.
  • the present invention provides the use of a diagnostic marker for obesity of a CLUH or BMPER protein or gene.
  • the present invention provides a composition for promoting adipocyte differentiation comprising an agent for overexpressing one or more proteins of CLUH and BMPER proteins, or a gene encoding the one or more proteins.
  • an agent that overexpresses one or more proteins of CLUH and BMPER protein, or a gene encoding the one or more proteins increases the expression level of CLUH, BMPER or both CLUH and BMPER, thereby promoting differentiation into adipocytes.
  • Any material that can be promoted is not limited, and may be nucleic acids, proteins, other extracts or natural products, or compounds, and specifically, may be low molecular weight compounds, organic synthetic materials, natural materials, and the like. It doesn't work.
  • composition for promoting adipocyte differentiation of the present invention may promote differentiation into adipocytes through overexpression of CLUH or BMPER.
  • the present invention provides a composition for detecting a diagnostic marker for differentiation into adipocytes, which comprises an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • composition for detecting a diagnostic marker for differentiation into adipocytes may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • agent for measuring the expression level of one or more proteins of CLUH and BMPER proteins, or genes encoding said one or more proteins are as described above.
  • differentiation diagnostic marker to adipocytes refers to an organic biomolecule showing a significant difference in expression according to the process of differentiation of adipocytes.
  • composition for detecting a diagnostic marker for differentiation into adipocytes of the present invention can detect organic biomolecules having a difference in expression level at a level equivalent to or higher than that of CLUH or BMPER during the differentiation into adipocytes.
  • the present invention provides a kit for diagnosing differentiation into adipocytes, which comprises an agent for measuring the expression level of at least one of CLUH and BMPER proteins, or a gene encoding the at least one protein.
  • the diagnostic kit for differentiation into adipocytes may be to measure the expression level of CLUH, BMPER, or CLUH and BMPER proteins or genes encoding the proteins.
  • agent for measuring the expression level of one or more proteins of CLUH and BMPER proteins, or genes encoding said one or more proteins are as described above.
  • the diagnostic kit for differentiation into adipocytes of the present invention can measure and diagnose the process of differentiation into adipocytes by measuring the amount of CLUH or BMPER expression.
  • Example 1 Confirmation of the relationship between the clustered mitochondrial homolog (CLUH) expression and adipocyte differentiation
  • 3T3-L1 is a mouse embryonic fibroblast cell line and is mainly used for metabolic disease and obesity research models.
  • Cell lines purchased from ATCC were used by culturing in DMEM (Dulbecco's modified Eagle's medium) including Bovine Calf serum (BCS).
  • DMEM Dulbecco's modified Eagle's medium
  • BCS Bovine Calf serum
  • 3T3-L1 0.8 ⁇ 10 5 cells were seeded in a 35 mm dish to make day 0 when 5 days had passed.
  • differentiation cocktail IBMX 0.5 mM, insulin 10 ug / mL, dexamethson 1uM
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • Complementary DNA cDNA was also synthesized using the iScript TM cDNA Synthesis Kit (Biorad, USA), and reverse transcriptase-polymerase chain reaction (RT-PCR) using Maxime PCR PreMix kit (i-StarTaq; Intron Biotechnology). was performed. Primers used for RT-PCR are as follows.
  • the cells were fixed with 3.7% formaldehyde and then incubated with 60% isopropanol for 5 minutes. Thereafter, cell suction was performed and the dish was completely dried. Oil Red and water were diluted 3: 2 and stained with cells. After 1 hour, the cells were washed 2-3 times with water.
  • the 3T3-L1 cell line was used to induce differentiation into adipocytes, and it was confirmed that expression of the CLUH gene of the present invention was increased in the course of differentiation into adipocytes. As disclosed in A of FIG. 1, it was confirmed that the expression of CLUH was increased from the second day of differentiation.
  • siRNA was produced.
  • siCLUH # 1 GCTTCAATCCTGACATCTT SEQ ID NO: 3
  • siCLUH # 3 GGGCATCATTGGCAATGAT SEQ ID NO: 4
  • transfection 100 nM siRNA using Lipofectamine RNAi MAX reagent (Invitrogen, CA) and Opti-MEM (Invitrogen, CA) was performed. transfection) was performed, and from the next day, 3T3-L1 cells were induced to differentiate into adipocytes using a differentiation cocktail (IBMX 0.5 mM, insulin 10 ug / mL, dexamethasone) 1 uM. Insulin treatment every 2 days.
  • a differentiation cocktail IBMX 0.5 mM, insulin 10 ug / mL, dexamethasone
  • the anti-obesity agent can be screened by inhibiting the expression of the CLUH gene to prevent or treat obesity, or by measuring its expression level.
  • BMPER BMP binding endothelial regulator
  • 3T3-L1 is a mouse embryonic fibroblast cell line and is mainly used for metabolic disease and obesity research models.
  • Cell lines purchased from ATCC were used by culturing in DMEM (Dulbecco's modified Eagle's medium) including Bovine Calf serum (BCS).
  • DMEM Dulbecco's modified Eagle's medium
  • BCS Bovine Calf serum
  • 3T3-L1 0.8 ⁇ 10 5 cells were seeded in a 35 mm dish to make day 0 when 5 days had passed.
  • differentiation cocktail IBMX 0.5 mM, insulin 10 ug / mL, dexamethson 1uM
  • DMEM Dulbecco's modified Eagle's medium
  • FBS Fetal bovine serum
  • Complementary DNA cDNA was also synthesized using the iScript TM cDNA Synthesis Kit (Biorad, USA), and reverse transcriptase-polymerase chain reaction (RT-PCR) using Maxime PCR PreMix kit (i-StarTaq; Intron Biotechnology). was performed. Primers used for RT-PCR are as follows.
  • the cells were fixed with 3.7% formaldehyde and then incubated with 60% isopropanol for 5 minutes. Thereafter, cell suction was performed and the dish was completely dried. Oil Red and water were diluted 3: 2 and stained with cells. After 1 hour, the cells were washed 2-3 times with water.
  • the 3T3-L1 cell line was used to induce differentiation into adipocytes, and it was confirmed that expression of the BMPER gene of the present invention was increased in the course of differentiation into adipocytes. As disclosed in FIG. 3A, it was confirmed that the expression of BMPER was increased from the second day of differentiation.
  • BMPER is involved in the process of differentiating into adipocytes, and furthermore, to determine whether or not it can suppress the production of fat by inhibiting the BMPER.
  • siRNA was produced.
  • SiBMPER # 1 SEQ ID NO: 8
  • siBMPER # 3 SEQ ID NO: 9
  • siRNAs of BMPER were made to order and control siRNA (si-Con, SEQ ID NO: 19) was purchased and used (Mbiotech, South Korea, Seoul). .
  • siBMPER # 1 GCATAATGTGTGTGTGTTT SEQ ID NO: 8 siBMPER # 3 GCACAGTATGCACTTGCAA SEQ ID NO: 9
  • transfection 100 nM siRNA using Lipofectamine RNAi MAX reagent (Invitrogen, CA) and Opti-MEM (Invitrogen, CA) was performed. transfection) and the following day was induced to differentiate 3T3-L1 cells into adipocytes using a differentiation cocktail (IBMX 0.5 mM, insulin 10 ug / mL, dexamethasone (1uM)). . Insulin treatment every 2 days.
  • a differentiation cocktail IBMX 0.5 mM, insulin 10 ug / mL, dexamethasone (1uM)
  • BMPER Gene expression of BMPER was measured by RT-PCR during differentiation of 3T3-L1 cells treated with siBMPER # 1 and siBMPER # 3, si-Con, si-Con, siRNA of BMPER. As a result, it was confirmed that the expression of BMPER was inhibited on the third day of differentiation, and the expression of PPAR gamma, a positive marker of differentiation, was also reduced. In addition, it was confirmed that the expression of Id-1, a downstream molecule of BMPER, was also reduced (FIG. 4A).
  • the anti-obesity agent can be screened by inhibiting the expression of the BMPER gene to prevent or treat obesity, or by measuring its expression level.
  • the present inventors have found that CLUH and BMPER are involved in fat accumulation and differentiation into adipocytes from the above examples, and tried to determine whether human cells perform the same function.
  • CLUH and BMPER were confirmed by real time PCR by inducing adipose differentiation in various cells (3T3L1, C3H10T1 / 2, Messenchymal stem cells), including 3T3-L1 cells used in the above examples.
  • Primer sequences of CLUH and BMPER were as follows.
  • RNA sequence is as follows. mRPL13A (Forward 5'-CCTGCTGCTCTCAAGGTTGTT-3 ', SEQ ID NO: 15; Reverse 5'-CGATAGTGCATCTTGGCCTTT-3', SEQ ID NO: 16), ⁇ -actin (Forward 5'-GCACCACACCTTCTACAATGA-3 ', SEQ ID NO: 17; Reverse 5'- TAGCACAGCCTGGATAGCAAC-3 ', SEQ ID NO: 18).

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Abstract

La présente invention concerne un homologue mitochondrial aggloméré (CLUH, clustered mitochondrial homolog) ou un régulateur de l'endothélium se liant à la BMP (BMPER) qui est impliqué dans la différenciation en adipocytes et l'accumulation de graisse. Ainsi, en inhibant le CLUH ou le BMPER, la différenciation en adipocytes peut être inhibée et l'obésité peut être traitée ou prévenue. En outre, en mesurant le niveau d'expression du CLUH ou du BMPER, l'obésité peut être diagnostiquée et le criblage d'un agent de traitement de l'obésité ou d'un agent de régulation de la différenciation en adipocytes est possible.
PCT/KR2016/009118 2015-08-18 2016-08-18 Composition pharmaceutique pour le traitement ou la prévention de l'obésité Ceased WO2017030394A1 (fr)

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