WO2005084702A1

WO2005084702A1 - Agent for preventing and treating organ fibrosis

Info

Publication number: WO2005084702A1
Application number: PCT/JP2004/002570
Authority: WO
Inventors: Yasuo Kokai
Original assignee: Hokkaido Technology Licensing Office Co., Ltd.
Priority date: 2004-03-02
Filing date: 2004-03-02
Publication date: 2005-09-15
Also published as: WO2005082402A1

Abstract

An agent for preventing and treating organ fibrosis that is effective in prevention of fibroses and prevention/treatment of organ fibrosis. In particular, an agent for preventing and treating organ fibrosis, comprising a granulocyte colony stimulating factor, which agent can exert anti-fibrosis effect and exhibits efficacy in the prevention and treatment of organ fibrosis for which no effective therapeutic method has been available.

Description

Description Organ fibrosis prevention and treatment agent Technical field

The present invention relates to an agent for preventing and treating organ fibrosis, which is capable of preventing and treating organ fibrosis. Background art

Organ fibrosis such as pulmonary fibrosis, skin sclerosis, and cirrhosis is considered to be a disease caused by the deposition of extracellular matrix in tissues. Excessive extracellular matrix causes irreversible tissue changes, leading to poor prognosis leading to organ failure.

For example, pulmonary fibrosis is a disease in which chronic inflammation of the alveolar wall and an increase in collagen fibrils cause destruction of the alveolar structure, eventually leading to respiratory failure.

The liver is also known as a very regenerative organ.However, in chronic liver disease, fibrosis occurs during the regenerative process following sustained hepatocyte necrosis, preventing normal hepatocyte regeneration. It is thought to happen by.

Skin sclerosis is characterized by hardening of the skin and is a chronic disease. In the systemic case, the visceral sclerosis hardens and hard fibers accumulate around the myocardium, causing constrained cardiomyopathy.

Pharmaceuticals for preventing and suppressing organ fibrosis include, for example, Japanese Patent Application Laid-Open No. 8-268096, a pulmonary fibrosis preventive agent containing a liver growth factor as an active ingredient. Japanese Patent Application Laid-Open No. 2007-076 discloses an antifibrotic agent containing a keratan sulfate oligo as an active ingredient, and Japanese Patent Application Laid-Open No. 2002-37010 discloses a lung containing a thrombomodulin-like protein as an active ingredient. Prevention of fibrosis · An antiprogression agent is disclosed.

It has been difficult to completely prevent and treat organ fibrosis with some prophylactic agents disclosed in the above publication. Therefore, an object of the present invention is to provide an agent for preventing and treating organ fibrosis which can prevent fibrosis and prevent and treat organ fibrosis. Disclosure of the invention

The present inventors have conducted intensive studies to achieve the above object. As a result, the granulocyte colony-stimulating factor, which is a site power-in that selectively acts on hematopoietic cells and promotes neutrophil differentiation and proliferation. Have found that they have an anti-fibrotic effect, and have found that granulocyte colony stimulating factor can prevent and treat organ fibrosis, thus completing the present invention.

The present invention has been made based on the above findings, and provides an agent for preventing and treating organ fibrosis, which comprises a granulocyte colony stimulating factor.

The granulocyte colony stimulating factor contained in the agent for preventing or treating organ fibrosis of the present invention may be a recombinant granulocyte colony stimulating factor.

The agent for preventing or treating organ fibrosis of the present invention is preferably a sustained-release preparation.

The sustained-release preparation is preferably formulated so as to release granulocyte colony-stimulating factor for at least 7 days, and may be a medical pump.

The present invention also provides an agent for preventing and treating visceral fibrosis, which comprises a granulocyte colony stimulating factor gene.

The present invention also provides a method for preventing and treating organ fibrosis, which comprises using the above agent for preventing and treating organ fibrosis. Brief Description of Drawings

FIG. 1 is a graph showing the results of measuring the thickness of collagen fibers in mice.

FIG. 2 shows the results of Western blot analysis of the expression of decorin and TGF-J3 in mouse organs.

FIG. 3 is an electron micrograph of a mouse collagen fiber.

FIG. 4 is a graph showing the results of measuring the thickness of collagen fibers in mice. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the agent for preventing and treating organ fibrosis of the present invention will be described first.

The preventive / therapeutic agent for organ fibrosis of the present invention contains granulocyte colony stimulating factor (hereinafter, also referred to as “G-CSF” in the present specification). G-CSF contained in the agent for preventing and treating organ fibrosis of the present invention is defined as a cytokine that selectively acts on hematopoietic cells and promotes neutrophil differentiation and proliferation. Any polypeptide that has G-CSF activity may be included. For example, a polypeptide extracted, separated, and purified from a natural product (such as a human biological sample), or a G-CSF-producing cell is cultured and cultured. G-CSF-producing hybridomas formed using cell fusion method and those obtained from these cells. Transformation obtained by transforming a host such as Escherichia coli or animal cells by genetic recombination. Any of those produced from the body, isolated and purified, or those obtained by chemically modifying the same can be used.

In the present invention, any one having at least a certain degree of homology with any of the aforementioned G-CSFs can be used. The homology is preferably at least 30%, more preferably at least 50%.

In the present invention, a recombinant G-CSF may be used. When preparing G-CSF using a gene recombination technique, an appropriate DNA portion is used as a PCR primer based on the information of the gene encoding G-CSF, for example, the base sequence represented by SEQ ID NO: 1. For example, cloning can be performed by performing an RT-PCR primer reaction. The cloning, for example, Molecular Cloning;. A Laboratory Manual 2 nd Ed, Cold accordance with the present specification, such as Spring Harbor Labroratory Press (1989), can be easily implemented by those skilled in the art. Further, the G-CSF gene used in the present invention is not limited to the one represented by SEQ ID NO: 1, and may have a modification or the like that does not impair the activity.

For example, in the amino acid sequence of (i) a DNA that hybridizes with the DNA represented by SEQ ID NO: 1 under stringent conditions, or (ii) a protein obtained by expressing the DNA represented by SEQ ID NO: 1 A DNA encoding a protein consisting of an amino acid sequence in which some amino acids have been deleted, substituted or added, In addition, any DNA that can exhibit G-CSF activity when expressed can be used in the present invention. The DNA of the above (i) can be obtained by an ordinary hybridization method, and the DNA of the above (ii) can be obtained by introducing a mutation into the DNA of the above (i). As a method for introducing a mutation into DNA, for example, a known method such as the Kunkel method or the Gapped duple method or a method analogous thereto can be adopted. For example, mutations can be introduced using a mutagenesis kit (Mutant-K (manufactured by TAKARA) or Mutant-G (manufactured by TAKARA)) utilizing site-directed mutagenesis. Examples of the “stringent conditions” include the conditions for hybridization described in Molecular Cloning described above, and specifically, DIG DNA Labeling (manufactured by Kuchi-Shu Diagnostics). When the probe was labeled, it was hybridized in a 32 ° C DIG Easy Hyb solution (Roche's Diagnostics), and the solution was added at 40 ° C in a 0 lxSSC solution (0.1 l ° / .w). / v] SDS) (incl. SDS) is a condition that hybridizes to the above DNA probe by Southern blot hybridization under the conditions for washing the membrane (lxSSC is 0.15 M NaCl, 0.015 M sodium citrate). The G-CSF used in the present invention is prepared by preparing a recombinant vector containing the above DNA by a method known in the art, transforming the obtained recombinant vector into a host cell, It can be produced by culturing a transformant, producing and accumulating G-CSF, and collecting the protein. The fact that the protein is accumulated after culturing means not only the culture supernatant, but also any of cultured cells or cultured cells or crushed cells or cells. In the present invention, the method for culturing the transformant is not particularly limited, and may be a usual method used in culturing a host.

The vector to be used is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. This is performed by cutting out a DNA fragment containing the DNA represented by SEQ ID NO: 1 and ligating the DNA fragment downstream of the promoter in an appropriate expression vector. Plasmids derived from Escherichia coli (eg, pBR322, pBR325, pUC18, pUC19, pUC118, or pBluescript), and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 or pC194), yeast-derived plasmid (eg, pSH19, pS H15, YEp13 or YCp50), phage phage such as λ phage, and animal viruses such as retinovirus, vaccinia virus or baculovirus can be used. As the promoter used in the present invention, any promoter may be used as long as it is appropriate for the host used for gene expression. For example, when the host is Escherichia coli, trp promoter, lac promoter, rec A promoter, LPL promoter, 1 pp promoter, T7 promoter, T3 promoter, _araBAD promoter, etc., and the host is Bacillus sp. If the host is Bacillus subtilis, the SPOl promoter, penP promoter, XYL promoter, HWP promoter, CWP promoter, etc. , PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When animal cells are used as hosts, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned. When an insect cell is used as a host, a polyhedrin promoter, an OplE2 promoter and the like are preferable.

In addition to the above, expression vectors include, if desired, enhancers, splicing signals, poly A addition signals, selection markers, SV40 replication origins (hereinafter sometimes abbreviated as SV40 ori), which are known in the art. Can be added. Further, if necessary, the protein encoded by the DNA of the present invention can be expressed as a fusion protein with another protein (for example, daltathione S transferase and protein A). Such a fusion protein can be cleaved using a site-specific protease and separated into respective proteins.

As the host cell, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used. Specific examples of the genus Escherichia include Escherichia coli K12 · DH1 (Proc. Natl. Acad. Sci. USA, 60, 16 (1968)), JM 1 03 (Nucleic Acids Research, Vol. 9, 309 (1 981)), JA 221 (Journal of Molecular Biology, Vol. 120, 5 17 (1 9 78)), HB 101 (Journal of Molecular Biology, 41, 45 9 (1969)), DH5α and JM109 are used. Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 (Gene, 24 volumes, 255 (1 983)), 207-21 (Journal of Biochemistry, 95 volumes) And Bacillus' brevis, etc. Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD. — 5 D, 20 B—12, Schizosaccaromyces pombe NCYC 19 13, NCYC 203, Pichia pastoris, Hansenula polymorpha, etc. As animal cells, for example, monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter CHO (dhfr- ) Cells, mouse L cells, mouse AtT—20, mouse smiero And GH3, human FL cells and HEK293 cells.

Transformation of the host cells described above can be performed according to methods known in the art. For example, the following literature describes a method for transforming a host cell. Natl. Acad. Sci. USA, 69, 2 11 (1 972); Gene, 17, 1 07 (1 98 2); Molecular & General Genetics, 16 8 Vol. 1 1 1 (1 9 7 9); Methods in Enzymology, Vol. 1 94, 18 2—1 8 7 (1 9 9 1); Proc. Natl. Acad. Sci. US A), Vol. 7 5, 1 9 2 9 (1 9 7 8); Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 26 3—26 7 (1 995) (published by Shujunsha); and Virology, 52, 4 5 6 (1 9 7 3).

The method for introducing the recombinant vector into bacteria such as Escherichia coli is not particularly limited as long as it can introduce DNA into bacteria. For example, a method using calcium ions (Cohen, SN et al. Natl. Acad. Sci., USA, 69: 2110 (1972), and the election port method.

When a yeast is used as a host, the method of introducing the replacement vector into the yeast is not particularly limited as long as it can introduce DNA into the yeast, and examples thereof include an electroporation method, a flow-through plastic method, and an acetate method. The lithium method and the like can be mentioned. When an animal cell is used as a host, the method of introducing the recombinant vector into the animal cell is not particularly limited as long as the method can introduce DNA into the animal cell, and examples thereof include an electoral poration method and a calcium phosphate method. Lipofection method and the like. When an insect cell is used as a host, the method for introducing the recombinant vector into the insect cell is not particularly limited as long as it is capable of introducing DNA into the insect cell. For example, a phosphate method, a lipofection method, and the like. And the electoral portation method. As a method for confirming whether or not the gene has been integrated into the host, for example, a PCR method, a Southern hybridization method, a Northern hybridization method, or the like can be used. For example, DNA is prepared from a transformant, a DNA-specific primer is designed, and PCR is performed. PCR is performed under the same conditions as those used for preparing the plasmid. Next, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, and stained with bromide tube, SYBR Green solution, and the like. Transformation can be confirmed. PCR may be performed using primers previously labeled with a fluorescent dye or the like to detect amplification products. Further, a method may be used in which the amplified product is bound to a solid phase such as microbrate or the like, and then the amplified product is confirmed using fluorescence or an enzyme reaction.

G-CSF used in the preventive / therapeutic agent for organ fibrosis of the present invention can be produced by culturing the transformant, producing and accumulating G-CSF, and collecting the protein. The accumulation of G_CSF means not only the culture supernatant, but also the cultured cells or cultured cells, or the crushed cells or cells. In the present invention, the method of culturing the transformant in the present invention is not particularly limited, and may be a usual method used in culturing a host.

For example, when the host is a microorganism such as Escherichia coli or yeast, the culture medium for culturing the transformant contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism to efficiently culture the transformant. As long as the medium can be used, either a natural medium or a synthetic medium can be used. Examples of the carbon source include carbohydrates such as glucose, lactose, sucrose, and starch; organic acids such as acetic acid and propionic acid; and alcohols such as ethanol and propanol. Call. Nitrogen sources include, for example, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, and corn steep. Rica and the like. Examples of the inorganic substance include potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually performed under aerobic conditions such as infiltration culture or aeration and stirring culture. When the host is Escherichia coli, the culture is performed at a temperature of about 15 to 43 ° C for about 12 to 48 hours. When the host is a bacterium belonging to the genus Bacillus, the reaction is performed at a temperature of about 30 to 40 ° C for about 12 to 100 hours. When the host is yeast, the reaction is carried out at a temperature of about 20 to 35 ° C. for about 24 to 100 hours. In addition, ventilation and stirring can be added as necessary. If it is necessary to adjust pH, use an inorganic or organic acid, alkaline solution, or the like.

When culturing a transformant transformed with an expression vector using an inducible promoter as a promoter, add an inducer to the medium, if necessary, before culturing.

When culturing a transformant obtained using animal cells as a host, examples of the medium to be used include commonly used RPMI1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums. . The cultivation is preferably performed in the presence of about 5% carbon dioxide at a temperature of about 37 ° C for 1 to 30 days.

If G_CSF is produced in cells or cells after culturing, cells or cells are collected by a known method, suspended in an appropriate buffer, and sonicated, lysozyme or After disrupting cells or cells by freeze-thawing or the like, a method of obtaining a crude protein extract by centrifugation or filtration may be used. The buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark). When G-CSF is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected. The protein contained in the culture supernatant or the extract obtained in this manner can be purified by appropriately combining known separation and purification methods. That is, for example, ammonium sulfate precipitation, The target protein can be produced by using single chromatography, ion exchange chromatography, affinity mouth chromatography, etc. alone or in an appropriate combination.

The G-CSF thus obtained can be converted to a salt by a known method or a method analogous thereto. Conversely, when the G-CSF is obtained as a salt, the free form can be converted by a known method or a method analogous thereto. Or it can be converted to other salts. Further, the protein produced by the recombinant can be arbitrarily fragmented before or after purification by the action of an appropriate protein-modifying enzyme such as trypsin and chymotrypsin. In addition, the protein can be arbitrarily modified by the action of a protein-modifying enzyme such as a kinase. The presence of G—CSF can be measured by various binding assays and enzyme immunoassays using specific antibodies.

G_CSF used as a therapeutic agent for the prevention of organ fibrosis of the present invention may be used as it is, or may be used, if necessary, with known pharmacologically acceptable carriers, excipients, etc. It can be mixed orally or parenterally as a pharmaceutical composition.

Examples of the dosage form for oral administration include tablets, pills, capsules, granules, syrups, emulsions, suspensions and the like. Such a dosage form can be produced by a method known per se and contains a carrier or an excipient usually used in the field of formulation. Examples of the carrier include carriers for tablets, and examples of the excipient include ratatose, maltose, saccharose, starch, and magnesium stearate.

Dosage forms for parenteral administration include, for example, ointments, injections, compresses, liniments, suppositories, nasal absorbents, pulmonary absorbents, transdermal absorbents, topical sustained release agents, etc. Can be The solution preparation is a method known per se, for example, G-CSF is usually used after dissolving in a sterile aqueous solution used for injection or suspending in an extract, emulsifying and embedding in ribosomes. obtain.

Solid preparations can be prepared as freeze-dried products by a method known per se, for example, by adding mannitol, trezulose, sorbitol, lactose, glucose and the like to G_CSF as excipients. Furthermore, this lyophilized product is powdered and used. May be. In addition, the powdered freeze-dried product is mixed with polylactic acid / glycolic acid, etc.

May be. It may be used after gelation.

The agent for preventing or treating organ fibrosis of the present invention is preferably used as a sustained-release preparation. Sustained-release preparations mean that the release of the active ingredient is not delayed immediately after administration, but rather delayed by some period of time. This release can be done at one time, and the release can be done gradually and continuously. It is preferable to use a sustained-release preparation formulated so as to release G—CSF for at least 7 days. Further, it is preferable to use a product which is formulated to release G-CSF for 14 days. Examples of such sustained-release preparations include medical pumps (osmotic pumps). G-CSF is continuously released by using an osmotic pump.

The dose of the agent for preventing or treating organ fibrosis of the present invention is preferably such that the amount of G-CSF is preferably 0.1 to 1 µg Zkg / day. The G-CSF content in the preventive and therapeutic agent for organ fibrosis of the present invention is not particularly limited, and may be contained so as to have the above-mentioned dose. Next, a prophylactic / therapeutic agent for organ fibrosis containing the granulocyte colony stimulating factor gene of the present invention (hereinafter, also referred to as “gene-containing organ fibrosis preventive / therapeutic agent” in the present specification) will be described. The gene used in the agent for preventing and treating organ fibrosis containing the G-CSF gene of the present invention uses an appropriate DNA portion as a PCR primer based on information on the nucleotide sequence represented by SEQ ID NO: 1, for example, RT- Cloning can be performed by performing a PCR primer reaction. As the above gene, those used in the above-mentioned agent for preventing and treating organ fiber fatigue can be used.

The method of administering the agent for preventing or treating organ fibrosis containing the G-CSF gene of the present invention includes a case where a non-viral vector is used and a case where a viral vector is used. Such administration methods are described in, for example, Separate Volume Experimental Medicine, Basic Techniques for Gene Therapy, Yodosha, 1996, Separate Volume Experimental Medicine, Gene Transfer & Expression Analysis Experiments, Yodosha, 1997, Japan Society for Gene Therapy It is described in detail in experiment guides such as the Handbook of Research and Development of Gene Therapy, NTS, 1999. These methods are briefly described below. As a method of administration using a non-viral vector, a G-CSF gene is added to a cell or tissue by the following method using a recombinant expression vector in which the G-CSF gene is incorporated into a conventional gene expression vector. Can be introduced.

Examples of a method for introducing a gene into cells include a coprecipitation method with calcium phosphate; a direct injection method of DNA using a micro glass tube, and the like.

Examples of the gene transfer method into tissues include, for example, a gene transfer method using liposome (internal type liposome), a gene transfer method using electrostatic liposome (electrostatic type liposome), an HVJ-ribosome method, and an improved HVJ-ribosome. Method (HVJ-AVE ribosome method), receptor-mediated gene transfer method, method of transferring DNA molecules into cells with a carrier (metal particles) using a particle gun, direct transfer method of naked-DNA, transfer method using positively charged polymer And the like. By such a method, it is possible to incorporate the recombinant expression vector into cells.

Examples of the expression vector used in the above method include pCAGGS (Gene 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, Zeo SV (Stratagene, Invitrogen). Company).

When a virus vector is used, examples of the virus vector include a recombinant adenovirus and a retrovirus. More specifically, detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, poliovirus, symbisporenos, Sendai virus, SV40, immunity The G-CSFR gene can be introduced into a cell by introducing the PGIS gene of the present invention into a DNA virus or RNA virus such as deficiency virus (HIV) and infecting the cell with the recombinant virus.

It is known that, among the above-mentioned virus vectors, the infection efficiency of adenovirus is much higher than when other virus vectors are used. Therefore, it is preferable to use an adenovirus vector system.

Methods for introducing the agent for preventing or treating gene-containing organ fibrosis of the present invention into a patient include the in ViVo method in which the agent for preventing and treating gene-containing organ fibrosis is directly introduced into the body, and human. Remove seed cells and prevent gene-containing organ fibrosis outside the body There is an exvivo method that introduces and returns the cells to the body (for example, Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48, 1994; Experimental Medicine, 12 (15), 1994, see Gene Therapy Development Research Handbook, edited by the Japanese Society of Gene Therapy, N.T.S, 1999). In the present invention, it is preferable to use the in vivo method, since the effect of preventing or treating organ fibrosis is induced in cells into which the agent for preventing and treating gene-containing organ fibrosis has been introduced.

When administering an agent for preventing or treating gene-containing organ fibrosis by the invivo method, it can be administered by an appropriate administration route according to the target cell, tissue, target organ, and the like. For example, it may be administered intravenously, intravenously, subcutaneously, intradermally, intramuscularly, or the like, or may be directly administered locally directly to an organ itself expected to have a prophylactic-therapeutic effect on organ fibrosis.

The preparation may be in the form of a solution or the like suitable for each of the above-mentioned administration forms. For example, in the case of an injection containing the DNA of the active ingredient, the injection can be prepared by a conventional method. For example, it is prepared by dissolving in an appropriate solvent (buffer such as PBS, physiological saline, sterile water, etc.), filtering and sterilizing with a filter if necessary, and filling in an aseptic container. be able to. The injections may be exempted from commonly used carriers and the like, if necessary.

In order to facilitate the presence of the gene around the diseased site, it is possible to prepare a sustained-release preparation (mini-pellet preparation, etc.) and implant it near the affected part, or to use an osmotic pump to continuously connect the affected part to the affected part. It is also possible to administer gradually.

The content of the G-CSF gene in the preparation can be appropriately adjusted depending on the disease to be treated, the age and weight of the patient, and the like. For example, the amount of the DNA of the active ingredient is preferably 0.0001 to 100 mg, and more preferably 0.001 to 10] 31 §. The agent for preventing or treating organ fibrosis according to the present invention has an anti-fibrotic effect, and includes pulmonary fibrosis, skin sclerosis, cirrhosis, arteriosclerosis, interstitial myocarditis, interstitial cystitis, and thread. Grade nephritis, vasculitis, diabetic nephropathy, hypertensive renal sclerosis, HIV nephropathy, IgA nephropathy, lupus nephropathy, interstitial nephritis, a / Letzheimer's disease, liver fibrosis, Crohn's disease, fibrosis It can be used for the prevention and treatment of cystitis, chronic bronchitis, radioactive fibrosis, cystic fibrosis, etc. In addition, the agent for preventing or treating organ fibrosis of the present invention can be used for mammals (for example, It is applicable to the prevention and treatment of the above diseases in horses, pigs, sheep, dogs, cats, etc.

The method for preventing and treating organ fibrosis of the present invention uses the aforementioned agent for preventing and treating organ fibrosis of the present invention. Hereinafter, the present invention will be described in more detail by way of an actual example, but the present invention is not limited thereto.

Example 1

A transgenic mask expressing human granulocyte colony-stimulating factor (hG-CSF) was prepared. To generate transgenic mice expressing hG-CSF, the SRa promoter that induces hG_CSF was used. The SRct promoter is constructed from the human T cell leukemia virus RU5 sequence and the SV40 early promoter. The full length cDNA of hG—CSF was inserted into the EcoRI site of pSR296 plasmid, and the transgene expression unit was cut with Sa1I to obtain pSRahG—CSF. pSR ahG—2.3 kilobase S a1 I fragment of CSF was purified using glass powder (DNA PREP, manufactured by Asahi Glass Co., Ltd.), and 1 OmM Tris—HC1, 0.1 mM EDTA (pH In step 7.5), the cells were dissolved to 10 g / m1 for microinjection. Mouse fertilized eggs were collected from the cumulus of the fallopian tubes of superovulated (C57B LZ6 XDB A 2) BDF1 female mice mated with BDFIosos mice. The DNA fragment was injected into the most accessible pronucleus of fertilized eggs (l-5f1). 0.5 days later, the embryo into which the DNA was injected was transplanted into the oviduct of a pseudopregnant MCH-ICR mouse and allowed to give birth to obtain a transgenic mouse expressing hG-CSF.

Expression of hG-CSF in the transgenic mice obtained as described above was confirmed by quantification of hG-CSF protein and measurement of hG-CSF biological activity. The hG-CSF protein was quantified by ELISA, and the biological activity of hG-CSF was measured by an assay using the hG_CSF-dependent mouse promyelocytic leukemia cell line (NSF60). The antifibrotic effect of the transgenic mice obtained as described above was examined for 5-week-old females, females, 12-week-old females, and 32-week-old females. The antifibrotic effect was assayed by measuring the thickness of collagen fibers in mice as follows.

Mouse skin was harvested from the back, stretch-fixed, and left overnight in 2.5% Daltar aldehyde. Next, electron microscope data was prepared in accordance with a standard method. That is, the fixed tissue was minced to a size of 1 m, post-fixed with osmic acid, and dehydrated with alcohol. Thereafter, the tissue embedded in ebon was sliced with a glass knife, stained with toluidine blue, and the site observed with an electron microscope was determined. Ultrathin sections of the determined site were prepared, stained with acetic acid acetate, and observed with an electron microscope. The thickness of collagen fibers was measured for 1,000 fibers.

Similarly, for the MCH-ICR mouse as a control, the thickness of collagen fibers was similarly measured for 5-week-old females, females, 12-week-old females, and 32-week-old females.

The results are shown in Figure 1. FIG. 1 is a graph showing the results of measuring the thickness of collagen fibers of hG_CSF-producing transgenic mice and normal mice. Figure 1 (b) shows the measurement results for transgenic mice, and Figure 1 (a) shows the measurement results for normal mice. In the graphs of FIGS. 1 (a) and (b), the horizontal axis is the thickness of collagen fibers, and the vertical axis is the number of collagen fibers.

As shown in Fig. 1 (a), the size of collagen fibers in mice that did not produce hG-CSF were 5 weeks old (L 5), female (L 5 early), and 12 weeks old. It can be seen that there is a peak at 60-70 nm or 70-80 nm in females (L12 early) and 32-week-old females (L32 early). On the other hand, as shown in Fig. 1 (b), the transgenic mice showed that the 5-week-old osseous collagen fiber had a peak thickness of 70-80 nm, while the 5-week-old female The thickness peak was 60-70 nm for 12-week-old females and 40-50 nm for 32-week-old females.

As is clear from these results, in transgenic mice that produce G_CSF, collagen! : Polymerization is suppressed, which has the effect of suppressing organ fibrosis Force s force δ. Example 2

The expression of decorin and TGF-J3 in the skin of transgenic mice expressing hG-CSF obtained in Example 1 was measured by Western blotting.試料 Samples used for the estamplot method were prepared as follows.

That is, the dermis was removed from the back of the mouse, an extract containing 1% NP-40 was prepared, and SDS-PAGE was performed. After SDS-PAGE, the proteins were electrically transferred to PVDF membrane, and TGF_b and decorin were identified on the membrane.

In addition, the measurement was performed on a normal mouse as a control / re. TGF- is known to promote organ fibrosis, and decorin is known to suppress organ fibrosis (Cell, Vol. 35, No. 4, 2003 Molecular Pathology of Extracellular Matrix).

The result is shown in figure 2. FIG. 2 shows the results of Western blot analysis of the expression of decorin and TGF- in organs of hG-CSF-producing transgenic mice and normal mice. FIG. 2 (a) shows the results for decorin, and FIG. 2 (b) shows the results for TGF_. In Figs. 2 (a) and (b), lane 1 is a normal mouse of 5 weeks, lane 2 is a normal mouse of 12 weeks, lane 3 is a transgenic mouse of 5 weeks, and lane 4 is 1 The results are for a 2 week old transgenic mouse. As is evident from FIG. 2 (a), decorin production was higher in transgenic mice than in normal mice. In contrast, as shown in FIG. 2 (b), transgenic mice produced less TGF-) 3 than normal mice. Based on the above results, the amount of TGF-, which promotes organ fibrosis, and the amount of decorin, which suppresses organ fibrosis, are lower in organs in transgenic mice that favor hG-CSF than in normal mice. This indicates that hGC S-F has a function of suppressing organ fibrosis. Example 3

110_. Transgenic mouse bone marrow cells expressing Was examined for the effect of suppressing organ fibrosis when administered to a mouse that does not express the protein. From the femur of the transgenic mouse expressing hG-CSF obtained in Example 1, the bone marrow cells are collected by flushing with a 23 G needle, and pipetting with a 27 G needle. In this way, a suspension of individual cells was obtained. Cellular debris and tissue debris were removed by passing through a nylon mesh. Then, the cells were washed twice with Du1 becco's phosphate buffered saline to obtain bone marrow cells for bone marrow transplantation.

C57BL6 mice were administered acidic water and neomycin 7 days before irradiation. Lethal dose of radiation (9 0 0.0 ₇ lines, 2 5 0 c G y / min) within 3 hours by irradiating, 5 0 0 1 bone marrow cells (5, 0 0 0, 0 0 0 / ml) was injected into the tail vein to implant bone marrow cells. At 12 weeks and 32 weeks after bone marrow cell transplantation, the antifibrotic effect was examined in the same manner as in Example 1.

Collagen fibers were collected in the same manner as in Example 1, and an electron micrograph of the collagen fibers was taken. Figure 3 shows the photograph taken. Fig. 3 (a) is a photograph of collagen fibers of a mouse 12 weeks after transplantation of bone marrow cells, and Fig. 3 (b) is a collagen fiber of a mouse 32 weeks after injection of bone marrow cells. It is a photograph of a fiber. As is clear from FIGS. 3 (a) and (b), the collagen cells of the mice 32 weeks after the transplantation of the bone marrow cells were thinner than those of the mice 12 weeks after the transplantation.

Next, in the same manner as in Example 1, the collagen fibers were observed with an electron microscope, and the thickness of the collagen fibers was measured. Figure 4 shows the results of the U determination. FIG. 4 is a graph showing the results of measuring the thickness of collagen fibers in mice to which bone marrow cells of transgenic mice that produce hG—CSF are transplanted. In FIG. 4, the horizontal axis is the thickness of collagen fibers, and the vertical axis is the number of collagen fibers. In FIG. 4, the dashed line shows the results for mice 12 weeks after transplantation of bone marrow cells, and the solid line shows the results for mice 32 weeks after transplantation of bone marrow cells.

As shown in FIG. 4, it can be seen that the collagen fiber thickness of the mouse 12 weeks after bone marrow cell transplantation has a peak at 50 to 70 nm. In contrast, it can be seen that the collagen fiber thickness of the mouse 32 weeks after transplantation of bone marrow cells has a peak at 50 nm. As is evident from these results, transgenic mice producing G-CSF In mice transplanted with bone marrow cells, the collagen fibers become thinner with time.

Therefore, it is clear that in mice transplanted with bone marrow cells of G-CSF-producing transgenic mice, the polymerization of collagen fibers is suppressed and the effect of suppressing organ fibrosis is exhibited. As described in detail above, it has been found that granulocyte colony stimulating factor has an antifibrotic effect. The agent for preventing and treating organ fibrosis of the present invention has an anti-fibrotic effect, and exhibits an effective effect for preventing and treating the organ fibrosis, which has been conventionally used as a treatment method.

Further, the method for preventing and treating organ fibrosis of the present invention is effective for the prevention and treatment of organ fibrosis, which has not been conventionally treated.

Claims

The scope of the claims

1. An agent for preventing or treating organ fibrosis, which comprises granulocyte colony stimulating factor.

2. The preventive / therapeutic agent for organ fibrosis according to claim 1, wherein the granulocyte colony stimulating factor is a recombinant granulocyte colony stimulating factor.

3. The preventive or therapeutic agent for organ fibrosis according to claim 1 or 2, which is a sustained-release preparation.

4. The preventive / therapeutic agent for organ fibrosis according to claim 3, wherein the sustained-release preparation is formulated to release granulocyte colony-stimulating factor for at least 7 days.

5. The preventive or therapeutic agent for organ fibrosis according to claim 3, wherein the sustained-release preparation is a medical pump.

6. Prevention of organ fibrosis, characterized by containing a granulocyte colony stimulating factor gene.

7. Organ fibrosis, pulmonary fibrosis, cutaneous sclerosis, liver cirrhosis, arteriosclerosis, interstitial myocarditis, interstitial cystitis, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive kidney Sclerosis, HIV nephropathy, IgA nephropathy, Lupus nephropathy, interstitial nephritis, Alzheimer's disease, hepatic fibrosis, Crohn's disease, fibrogenic splenitis, chronic bronchitis, radiofibrosis or 7. The preventive / therapeutic agent for organ fibrosis according to claim 1, wherein the agent is cystic fibrosis.

8. A method for preventing and treating organ fibrosis, comprising using the agent for preventing and treating organ fibrosis according to any one of claims 1 to 5.