WO2000001405A1 - Promoteurs de differentiation des cellules de cartilage - Google Patents

Promoteurs de differentiation des cellules de cartilage Download PDF

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Publication number
WO2000001405A1
WO2000001405A1 PCT/JP1999/003577 JP9903577W WO0001405A1 WO 2000001405 A1 WO2000001405 A1 WO 2000001405A1 JP 9903577 W JP9903577 W JP 9903577W WO 0001405 A1 WO0001405 A1 WO 0001405A1
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Prior art keywords
cells
cartilage
agent
gene
active ingredient
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PCT/JP1999/003577
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English (en)
Japanese (ja)
Inventor
Yasuyuki Ishiduka
Reiko Mochizuki
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Sumitomo Pharmaceuticals Company, Limited
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Publication date
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Publication of WO2000001405A1 publication Critical patent/WO2000001405A1/fr

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel agent for promoting chondrocyte differentiation and a therapeutic agent for cartilage disorders. More specifically, C16N, a protein also known as "neurochondrin," C16N-1 and C16N-2, isoforms of the C16N, or the proteins of these proteins. A similar protein (hereinafter referred to as C16N, C16N-1, C16N-2, or a similar protein of these proteins is collectively referred to as a C16N-related protein), or encodes the C16N-related protein
  • the present invention relates to an agent for promoting chondrocyte differentiation or a therapeutic agent for cartilage disorders, comprising a gene for cartilage damage as an active ingredient.
  • C16N is a proteinaceous factor found by the expression cloning method in BW5147 cells, which are cells prone to bone metastasis.
  • the present inventors recently have a C 1 N amino acid sequence having the entire amino acid sequence of C 16 N, and further having a C 1 N-terminal longer by 150 amino acids or 133 amino acids.
  • An object of the present invention is to provide a novel agent for promoting chondrocyte differentiation and a therapeutic agent for cartilage disorders. That is, an object of the present invention is to provide an agent for promoting differentiation of chondrocytes or a therapeutic agent for cartilage disorders, which contains a C16N-related protein or a gene encoding these C16N-related proteins as an active ingredient.
  • the present inventors have studied the known effects of C16N-related proteins. Activity to induce differentiation into cells with hydroxyapatite degradability, (2) survival-maintaining activity of neurons, (3) osteoblast proliferation inhibitory activity, and (4) promotion of type I collagen expression in osteoblasts As a result of intensive studies on the effects of the C16N-related protein on the effects other than the activity, it was revealed that these C16N-related proteins have the effect of promoting the differentiation of undifferentiated chondrocytes into mature chondrocytes. did. That is, the gist of the present invention is:
  • An agent for promoting chondrocyte differentiation comprising as an active ingredient C16N, C16N-1, C16N-2, or a similar protein thereof.
  • a chondrocyte differentiation promoting agent comprising as an active ingredient a gene encoding any of the following proteins:
  • a therapeutic agent for cartilage disorders comprising C16N, C16N-1, C16N-2, or a similar protein thereof as an active ingredient; 4) C16N, C16 An agent for treating cartilage disorders, characterized by containing as an active ingredient a gene encoding any of N-1, C16N-2, or a similar protein thereof, and
  • Cartilage disorders include osteoarthritis, cartilage dysplasia, loss of cartilage in fractures, damage to articular cartilage or disc due to trauma, acute suppurative arthritis, tuberculous arthritis, syphilitic arthritis, rheumatoid arthritis, rheumatism
  • the therapeutic agent for cartilage disorders according to the above 3) or 4) which is any one of fever, systemic lupus erythematosus, osteoarthritis, and herniated disc.
  • C16N and “gene encoding C16N” used in the present invention refer to C16N and its gene described in International Publication W097 / 40150. As described in Biochem. Biophys. Acta., May 6, 1450 (1): 92-98 (1999), the C 16N is also called “neurochondrin”.
  • the gene encoding C16N can be cloned by the method described in the international publication, and the C16N can also be prepared by the method described in the international publication. Note that C Both the base sequence and amino acid sequence of 16N are described in the sequence listing of the international publication.
  • C 16 N — 1 and C 16 N—2 and “genes encoding C 16 N—1 and 16 N—2” used in the present invention are described in Japanese Patent Application No. 10-134440. C 16N-1 and C 16N-2 and their genes. These C 1
  • C16N-1 and C16N-2 are isoforms of C16N as described in Biochem. Biophys. Acta., May 6, 1450 (1): 92-98 (1999).
  • the gene encoding CI 6N-1 or C 16 N-2 can be obtained, for example, by using a part of C 16 N DNA described in WO 97/40150 as a probe or a PCR primer, for example, mouse or human. It can be cloned by screening a cDNA library of the brain, hippocampus, testis or the like. The cloning can be easily performed by those skilled in the art according to, for example, Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989). More specifically, C16N can be obtained by performing the 5'-RACE method (Marathon-Ready cDNA Amplification Kit (manufactured by CL0NTECH)) using an appropriate portion of C16N DNA as a PCR primer.
  • 5'-RACE method Marathon-Ready cDNA Amplification Kit (manufactured by CL0NTECH)
  • C16N-1 or C16N-2 can be obtained by ligating the cloned C16N-1 or C16N-2 gene to a known expression vector such as pBK-CMV. And expression and production.
  • Either prokaryotic or eukaryotic cells can be used as a host.
  • Escherichia coli strains and animal cell strains are already widely spread and readily available, unless otherwise specified.
  • animal cell hosts include COS-1, COS-7, and CHO cells.
  • a known method such as the LIP0FECTIN method (Feigner P., et al, Proc. Natl. Acad. Sci.
  • the C16N-1 or C16N-2 of the present invention can be purified by a conventional method from the cell membrane fraction prepared by the method described above.
  • the nucleotide sequence of C 16 N-1 obtained as described above is represented by SEQ ID NO: 1 (mouse) and SEQ ID NO: 5 (human), and the amino acid sequence of C 16 N-1 is represented by SEQ ID NO: 2
  • the aforementioned C16N, C16N-1 or C16N-2 analogous protein, and its gene can also be used.
  • the term "similar protein” means, for example, 1) a DNA that hybridizes under stringent conditions to a gene encoding C16N, C16N-1 or C16N-2.
  • proteins having a chondrocyte differentiation promoting action which is a feature of the present invention.
  • Such a similar protein and its gene can be prepared by the method described in International Publication WO97 / 41050.
  • the agent for promoting chondrocyte differentiation and the agent for treating cartilage disorders according to the present invention comprises the above-mentioned C 16 N-related protein or its gene as an active ingredient, and differentiates undifferentiated chondrocytes into mature chondrocytes. By promoting it, it can be used effectively for treatment and prevention of various diseases involving cartilage disorders.
  • the diseases include, for example, osteoarthritis, chondrodysplasia, loss of cartilage in fracture, damage to articular cartilage or disc due to trauma, acute suppurative arthritis, tuberculous arthritis, syphilitic arthritis, rheumatoid arthritis, Rheumatic fever, systemic lupus erythematosus, osteomyelitis, or herniated disc.
  • chondrocyte differentiation promoting agent and the cartilage disorder treating agent of the present invention When a protein is used as an active ingredient, the following administration methods, administration forms and dosages can be used.
  • administration by intravenous injection is preferable as a method for administering the agent for promoting chondrocyte differentiation and the agent for treating cartilage disorders of the present invention to a patient.
  • Oral administration, administration as a suppository, subcutaneous injection, intramuscular injection, local injection, peritoneal cavity Internal administration and the like can be performed.
  • the agent for promoting chondrocyte differentiation and the agent for treating cartilage disorders of the present invention can be administered in various unit dosage forms depending on the administration method described above.
  • the protein of the present invention can be used by dissolving or suspending it in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • aqueous carrier for example, water, buffered water, 0.4% physiological saline, and the like can be used.
  • the aqueous solution thus prepared can be packaged as it is or lyophilized, and the lyophilized preparation may be used by dissolving it in a sterile aqueous solution before administration.
  • the above preparations may contain pharmaceutically acceptable auxiliaries, such as ph regulators or buffering agents, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, sodium chloride It may contain sodium, potassium chloride, calcium salt, sodium sorbitan monolaurate, triethanolamine or the like.
  • auxiliaries such as ph regulators or buffering agents, tonicity regulators, infiltrants, and more specifically, for example, sodium acetate, sodium lactate, sodium chloride It may contain sodium, potassium chloride, calcium salt, sodium sorbitan monolaurate, triethanolamine or the like.
  • the proteins of the invention can be used in unit dosage forms of powders, tablets, pills, capsules, and syrups.
  • the protein of the present invention can be used by dissolving or suspending it in an aqueous or oily carrier. Alternatively, it may be administered as a sustained-release preparation using a biocompatible material such as collagen.
  • the daily dose of the above-mentioned agent for promoting chondrocyte differentiation and the agent for treating cartilage disorders can be administered at a daily dose of 0.0001 to 100 mg, preferably about 0.001 to 10 mg, until the symptoms are improved. .
  • chondrocyte differentiation promoting agent and the cartilage disorder treating agent of the present invention when a gene encoding a C16N-related protein is used as an active ingredient and used as a gene therapeutic agent, the following gene transfer method is used.
  • the mode and amount of introduction can be used.
  • the gene is directly there are two methods: an in vivo method in which cells are introduced into cells in the body, and an ex vivo method in which certain cells (such as bone marrow cells) are removed from a human, a gene is introduced into the cells outside the body, and the cells are returned to the body (Nikkei Science, April 1994, pp. 20-45, Special Issue on Experimental Medicine, 12 (15) (1994)).
  • a method using a recombinant virus for example, a method of incorporating the DNA of the present invention into a DNA virus such as an adenovirus, a herpes virus, a vaccinia virus, a box virus, a polio virus, a simbis virus, or an RNA virus and introducing the DNA is used. No. Among them, a method using adenovirus or vaccinia virus is particularly preferable.
  • DNA vaccine method a method in which the expression plasmid is directly administered intramuscularly
  • liposomal method a lipofectin method, and the like, with the ribosome method being particularly preferred.
  • Examples of the ex vivo method include a microinjection method, a canopy phosphate method, and an electroporation method.
  • the method of administration to a patient can be by an appropriate administration route depending on the disease, symptom and the like for the purpose of treatment.
  • it can be administered intravenously, arterially, subcutaneously, sarcastically, intramuscularly, and the like.
  • a conventional carrier may be added.
  • liposomal preparations such as suspensions, freezers, and centrifugal concentrated freezers can be prepared.
  • the content of the gene in the preparation can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, etc., but is usually 0.0001 to 100 mg, preferably 0.000 to 10 mg .
  • Fig. 1 shows the number of ATDC 5 cells transfected with C16N-1 or empty vector over time.
  • FIG. 2 is a photomicrograph of ATDC5 cells transfected with an empty vector cultured in a confluent state for 10 days.
  • FIG. 3 is a micrograph of ATDC5 cells into which the C16N-1 gene has been introduced, cultured for 10 days in a confluent state.
  • human C 16N-1 and C 16N-2 DNAs were obtained as follows.
  • the human and mouse C16N nucleotide sequences have extremely high homology, they can be used even when the primer used for PCR of mouse DNA is human. Therefore, in order to examine the presence of a gene encoding a human C16N-like protein, the mouse C1 sequence described in SEQ ID NO: 3 in the sequence listing of WO 97/40150 was used.
  • the antisense DNA (R24) (5'-CGAGAGCCAGCCGTACCTCC-3 ') at positions 732 to 752 of the 6N nucleotide sequence, the antisense DNA (R9) at positions 686 to 705, and Nucleotide sequence of mouse C 16 N as described in SEQ ID NO: 3 in SEQ ID NO: 3 of the sense DNA (F7) (5′-AGGAGCTGCTACAACTGCTA-3 ′) at positions 1779 to 1798 and WO97 / 40150 2078th to 2097th Sense DN A
  • the first PCR was performed with the F7 and AP2 primer sets, the PCR product was used as a template, and the second PCR was performed with the F14 and AP1 primer sets. From the above experiment, it was examined whether a cDNA clone longer in the 5 ′ direction or 3 ′ direction than C16N was obtained. As a result, a cDNA clone longer in the 3 'direction was not obtained, but two clones longer in the 5' direction than CI 6N were obtained.
  • Each of these two clones is subcloned into a TA cloning vector (pGEM-T Easy vector), and after confirming the nucleotide sequence, cut with restriction enzymes Sa1I and Bg1II, and human C
  • a plasmid having 16N cDNA (SEQ ID NO: 5 in the sequence listing of WO 97/40150) was ligated to a plasmid cleaved with the same restriction enzyme to obtain a novel human.
  • pBK / hC16N-1 having the cDNA of C16N-1
  • pBKZhC16N-2 having the cDNA of the new human protein CI6N-2 were obtained.
  • the nucleotide sequences of mouse and human CI6N-1 and C16N-2 were determined using an Auto Read Sequencing kit (manufactured by Pharmacia Biotech) according to the instructions attached to the ALF redDNA automatic sequencer. This was performed using Insertion of the plasmids pBK / mC16N-1, pBK / mC16N-2, pBK / hC16N-1 and pBK / hC16N-2, nucleotide sequence of cDNA It was determined.
  • the nucleotide sequence of mouse C16N-1 is shown in SEQ ID NO: 1, its amino acid sequence is shown in SEQ ID NO: 2, and the nucleotide sequence of mouse C16N-2 is shown in SEQ ID NO: 3.
  • the amino acid sequence is set forth in SEQ ID NO: 4.
  • the nucleotide sequence of human C 16N-1 is shown in SEQ ID NO: 5
  • the three amino acid sequences are shown in SEQ ID NO: 6
  • the nucleotide sequence of human CI 6N-2 is shown in SEQ ID NO: 7.
  • the amino acid sequence is set forth in SEQ ID NO: 8.
  • the mouse C 16 N-1 and C 16N-2 were compared with the mouse C 16 N described in SEQ ID NO: 4 in the sequence listing of WO 97/40 150, respectively, at the N-terminal at the amino acid level.
  • the side was 150 amino acids and 133 amino acids longer.
  • human C 16 N-1 and C 16 N-2 were also compared with human C 16 N described in SEQ ID NO: 6 in the sequence list of WO97 / 40150. At the amino acid level, the N-terminal side was 150 amino acids and 133 amino acids longer.
  • C16N, C16N-1 and C16N-2 cDNAs were added to mouse ATDC5 cells (cell development bank), which are undifferentiated cells capable of differentiating into chondrocytes, using pB
  • Plasmids integrated into K-CMV were introduced by the lipofection method, and transfected cells were selected using the drug marker (neomycin) as an index. The following three experiments were performed using the transfected cells.
  • transfected cells The growth rates of C16N, C16N-1 and C16N-2 gene transfected cells and cells transfected with vectors that do not incorporate these genes (hereinafter abbreviated as transfected cells) The comparison was made by measuring the number of cells.
  • Figure 1 shows the results for C 16 N-1. In the figure, the horizontal axis represents time (days), and the vertical axis represents the number of cells.
  • the C16N-1 transfected cells did not change until the third day after the start of culture, and the number of cells increased only about 10% of the empty vector transfected cells on the seventh day.
  • the growth rates of C 16 N-2 and C 16 N-introduced cells also decreased, these C 16N, C 16 N-1 and C 16 N-2 suppressed the growth of AT DC 5 cells. It was suggested that this promoted differentiation.
  • Fig. 2 shows the results of the cells transfected with the vector
  • Fig. 3 shows the results of the cells transfected with the C16N-1 gene.
  • primer sequences for the amplification of type II collagen gene 5'-ATTGAAGAGGAGAGGGCTT-3 'and 5'-CCAGTTCAGGTCTCTTAGAA-3 were used as 5' primers and 3 'primers. The DNA size amplified with these primer combinations is 463 base pairs.
  • the DNA size amplified by these primer combinations is 941 base pairs.
  • GAPDH glycosydehyde-3-phosphate dehydrogenase
  • 5'-CACCATGGAGAAGGCCGGGG-3 ' was used as a 5' primer
  • 5'-GACGGACACATTGGGGG TAG-3 ' was used as a 3 primer.
  • the amplified DNA size is 418 base pairs.
  • the reaction composition was performed using a DNA thermal cycler according to standard procedures, and heat annealing at 94 ° C for 1 minute 60. . For 1 minute and chain extension reaction 72 for 2 minutes for 30 cycles. One-tenth of the reaction mixture was electrophoresed on 1% agarose to confirm the band.
  • undifferentiated ATDC5 cells did not express type II collagen, but AT16 transfected with either C16N or C16N_1,12 gene.
  • the type II collagen gene was clearly detected in DC5 cells. Also, there was no difference in the expression of type I collagen and GAPDH gene in all ATDC5 cells.
  • C 16N, C 16 N—1 and C 16 N—2 were obtained from ATDC 5 It has been clarified that it has a differentiation promoting effect on cells.
  • a C16N-related protein which is also called a neurochondrin, or a chondrocyte differentiation promoting agent or a cartilage containing a gene encoding the C16N-related protein as an active ingredient
  • An agent for treating a disorder can be provided.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
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  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne des promoteurs de différentiation des cellules de cartilage ou des médicaments administrés pour remédier à une insuffisance de cartilage. Leur principe actif est une protéine C16N, également appelée neurochondrine, C16N-1 et C16N-2 qui sont des isoformes de la protéine C16N, leurs protéines analogues ou les gènes codant pour ces protéines liées à C16N.
PCT/JP1999/003577 1998-07-06 1999-07-02 Promoteurs de differentiation des cellules de cartilage WO2000001405A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP19088998 1998-07-06
JP10/190889 1998-07-06

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WO2000001405A1 true WO2000001405A1 (fr) 2000-01-13

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040150A1 (fr) * 1996-04-23 1997-10-30 Sumitomo Pharmaceuticals Company, Limited Proteines c16 et c16n ou genes codant pour ces proteines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997040150A1 (fr) * 1996-04-23 1997-10-30 Sumitomo Pharmaceuticals Company, Limited Proteines c16 et c16n ou genes codant pour ces proteines

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