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    • 专利摘要:
    The present invention relates to a medicament containing the HGF gene. The agent of the present invention can be applied locally to the target organ so that the effect can be selectively selected, thereby minimizing the side effects of HGF.
    公开号:KR19990044279A
    申请号:KR1019980701515
    申请日:1996-08-22
    公开日:1999-06-25
    发明作者:류이찌 모리시따；도시오 오기하라；도시까즈 나까무라；데쓰야 도미따；다까히로 오찌
    申请人:다께우찌 마사야쓰；스미또모 세이야꾸 가부시키가이샤；
    IPC主号:
    专利说明:

    Drugs containing HGF gene
    HGF is a bioactive peptide that exhibits various pharmacological activities. The pharmacological activity of HGF is described, for example, in JIKKEN-IGAKU (Experimental Medicine), Vol. 10, No. 3 (extra issue), 330-339 (1992). In terms of its pharmacological activity, HGF is a drug for treating cirrhosis or kidney disease; Endothelial cell growth promoters; Anticancer agents; Anti-side effect agents during cancer therapy; Drugs for treating pulmonary disease, gastrointestinal injury or head nerve disease; Immunosuppressive side effects inhibitors; Collagen degradation promoters; Agents for treating cartilage disease, arterial disease, pulmonary fibrosis, liver disease, blood coagulation disorders, plasma hypoproteinemia or trauma; Neurological disorder improvers; Hematopoietic stem cell synergistic factor; And it is expected to be useful as a hair growth promoter [Japanese Patent Laid-Open Nos. 4-18029 and 4-49246, EP 492614, Japanese Patent Laid-Open Nos. 6-25010, WO 93/8821, and Japanese Patent Laid-Open No. 6- 172207, 7-89869 and 6-40934, WO 94/2165, Japanese Patent Laid-Open Nos. 6-40935, 6-56692 and 7-41429, WO 93/3061, Japanese Patent Official Publication No. 5-213721, etc.].
    As gene therapy, there is currently extensive research and investment worldwide around adenosine deaminase deficiency, AIDS, cancer, purulent fibrosis or hemophilia.
    However, gene therapy using the HGF gene has not been known. It is still unclear whether the gene therapy can be used effectively.
    Problem to be solved by the present invention
    HGF is known as one of the drugs with short half-life in the blood. Naturally, HGF is only being sustained topical administration.
    In terms of the various pharmacological activities of HGF, HGF is expected to be developed as a drug having a wide range of uses against various diseases. On the other hand, when HGF is administered systematically, side effects may be caused by various pharmacological activities of HGF. In addition, when HGF itself is administered intravenously, HGF faces the drawback that a significant amount of HGF remains in the liver resulting in a decrease in the amount of HGF reaching the target organ.
    Problem solving
    The present invention seeks to solve the preceding problem. In summary, the present invention relates to:
    (1) a medicament containing the HGF gene;
    (2) liposomes containing the HGF gene;
    (3) the liposome according to (2), which is a membrane fused liposome fused with Sendai virus;
    (4) a medicament containing the liposome according to (2) or (3);
    (5) the agent according to (1) or (4), which is used for treating arterial disease;
    (6) The agent according to (1) or (4), which is used for treating cartilage injury.
    The present invention relates to a medicament for use in gene therapy and the like. More particularly, the invention relates to medicaments containing liposomes containing the HGF gene as well as the hepatocyte growth factor (HGF) gene.
    Figure 1 shows the expression of HGF in rat coronary endothelial cells sensitized with Hemagglutinating virus of Japan (HVJ) -liposomal-DNA in Test Example 1.
    In FIG. 2, the (line) graph shows the cell growth rate in the presence or absence of HGF from HVJ-liposome-cont-sensitized endothelial cells in Test Example 2, where "DSF" represents a group of endothelial cells sensitized with HVJ-liposome-cont "HGF" refers to a group cultured in the presence of a predetermined concentration of recombinant human HGF. In FIG. 2, bar represents the cell growth rate of HVJ-liposome-DNA-sensitized endothelial cells in Test Example 2, where "DSF" represents an endothelial cell group sensitized with HVJ-liposome-cont, and "HGF vector" Endothelial cell population sensitized with HVJ-liposome-DNA.
    FIG. 3 shows the cell growth rate of endothelial cells sensitized with HVJ-liposome-DNA in the presence or absence of anti-HGF antibody in Test Example 2, wherein the “control” was HVJ-liposome-cont- cultured in the presence of IgG control. Sensitized endothelial cell populations; "HGF" refers to a group of HVJ-liposome-DNA-sensitized endothelial cells cultured in the presence of IgG control; And "HGFab" refers to a group of HVJ-liposomal-DNA-sensitized endothelial cells cultured in the presence of rabbit anti-human HGF antibodies. Cell growth rate (%) is expressed as relative% when the growth rate of the control is 100.
    FIG. 4 is a graph showing the effect of cell growth on rat coronary endothelial cells of culture supernatants from HVJ-liposomal-DNA-sensitized rat vascular smooth muscle cells (hereinafter abbreviated as VSMCs) in Test Example 3. FIG. , "Control" refers to the culture supernatant from HVJ-liposomal-cont-sensitized rat VSMCs, and "HGF" refers to the culture supernatant from HVJ-liposome-DNA-sensitized rat VSMCs. Group.
    5 is a graph showing the results in Test Example 3 in which the HGF concentration of supernatants from cultured HVJ-liposomal-DNA-sensitized rat VSMCs was determined using anti-human HGF antibodies. In Figure 5, "untreated group" refers to a group of culture supernatants of non-sensitized VSMCs; "Control" refers to a group of supernatants from cultured HVJ-liposomal-cont-sensitized rat VSMCs; And “HGF” refers to the group of supernatants from cultured HVJ-liposomal-DNA-sensitized rat VSMCs.
    FIG. 6 is a graph showing the results in Test Example 3 in which the HGF concentration of supernatant from cultured HVJ-liposomal-DNA-sensitized rat VSMCs was determined using anti-mouse HGF antibody. In Figure 6, "untreated group" refers to a group of culture supernatants of non-sensitized VSMCs; "Control" refers to a group of supernatants from cultured HVJ-liposomal-cont-sensitized rat VSMCs; And “HGF” refers to the group of supernatants from cultured HVJ-liposomal-DNA-sensitized rat VSMCs.
    7 is a graph showing the effect of cell growth on the rat coronary endothelial cells of the culture supernatant from HVJ-liposomal-DNA-sensitized rat coronary endothelial cells cultured in Test Example 4, where A, B and C were cultured, respectively. The supernatant of HVJ-liposome-DNA-sensitized rat coronary endothelial cells, the supernatant of cultured HVJ-liposome-cont-sensitized rat tubular endothelial cells, and the group of non-treated animals Indicates.
    FIG. 8 shows the cell growth effect of HVJ-liposomal-DNA-sensitized rat coronary endothelial cells on rat coronary endothelial cells in the presence of anti-HGF antibody in Test Example 4. FIG. In FIG. 8, A represents the group to which supernatant from cultured HVJ-liposomal-DNA-sensitized rat coronary endothelial cells was added; B represents the group to which supernatant from HVJ-liposomal-cont-sensitized rat coronary endothelial cells was added; C represents the group to which anti-HGF antibody was added to the supernatant from cultured HVJ-liposomal-DNA-sensitized rat coronary endothelial cells; D represents the group to which the control antibody was added to the supernatant of cultured HVJ-liposomal-DNA-sensitized rat coronary endothelial cells.
    9 shows cell growth of endothelial cells in Test Example 5 when HVJ-liposomal-DNA-sensitized human VSMCs were co-cultured with non-sensitized human endothelial cells. In Figure 9, "control" represents a group co-cultured with HVJ-liposomal-cont-sensitized VSMCs and "HGF" represents a group of supernatants from cultured HVJ-liposome-DNA-sensitized VSMCs. .
    10 shows cell growth of endothelial cells in Test Example 6 when HVJ-liposomal-DNA-sensitized rat VSMCs were co-cultured with unsensitized rat coronary endothelial cells. In FIG. 10, "control" represents a group co-cultured with HVJ-liposome-cont-sensitized VSMCs and "HGF" represents a group of culture supernatants of HVJ-liposome-DNA-sensitized VSMCs.
    11 shows the increase in the number of micro-vessels in the rat heart muscle directly injected with HVJ-liposomal-DNA in Test Example 8, "HGF" shows the number of the micro-vessels in the rat heart muscle injected directly with HVJ-liposomal-DNA, "Control" refers to the number of microvasculature in rat heart muscle directly injected with HVJ-liposome-cont.
    FIG. 12 shows the development of cartilage-like cells after 3 weeks of HVJ-liposome-DNA administration to the joints in Test Example 9, wherein the synthesis of double toluidine blue-stained proteoglycans is observed.
    FIG. 13 shows the development of cartilage-like cells 4 weeks after HVJ-liposome-DNA administration in the joints, and the synthesis of double toluidine blue-stained proteoglycans is observed.
    14 shows the development of cartilage-like cells by observing the synthesis of toluidine blue-stained proteoglycans even after 4 weeks of administering HVJ-liposome-DNA (TGF-β) prepared in Comparative Example 2 to the joint. Figure 9 shows no appearance.
    FIG. 15 shows that even after 4 weeks of administration of the HVJ-liposome-cont prepared in Comparative Example 1 to the joint, the development of the cartilage-like cells was not shown in Test 9 by observing the synthesis of toluidine blue-stained proteoglycans. Figure showing.
    Best mode for realizing the invention
    "HGF gene" used in the present invention refers to a gene capable of expressing HGF. Therefore, as long as the expressed polypeptide has substantially the same effect as that of HGF, the HGF gene can be partially deleted, substituted or inserted in the nucleotide sequence, or ligated with it at its 5'-end and / or 3'-end. Other nucleotide sequences. As typical examples of the HGF gene, Nature, 342 , 440 (1989), Japanese Patent Laid-Open No. 5-111383, Biochem. Biophys. Res. Commun., 163 , 967 (1989) and the like. The genes can be used in the present invention.
    The HGF gene is introduced into an appropriate vector and an HGF gene-containing vector is provided for use. For example, the HGF gene can be used in the form of a viral vector with the HGF gene described below, or in the form of a suitable expression vector with the HGF gene.
    "Pharmaceutical composition" as used herein refers to a medicament for the treatment or prevention of human diseases, which is due to the pharmacological activity of HGF. For example, agents for treating or preventing the diseases described above are exemplified.
    According to the present invention, the HGF gene is introduced into the cell, and HGF is expressed in the cell to exhibit pharmacological action. Therefore, the medicament of the present invention can be effectively used for diseases in which HGF itself is effective.
    When the HGF gene is introduced into a cell, for example, the growth of vascular endothelial cells is accelerated, as is demonstrated in later examples, while the undesirable growth of vascular smooth muscle is not accelerated. Furthermore, as demonstrated in later examples, angiogenesis is observed when the HGF gene is introduced into the heart in an in vivo animal test using mice. Therefore, the HGF gene is caused by an arterial disease, in particular a disorder mainly involving abnormal proliferation of vascular smooth muscle cells (e.g., restenosis after percutaneous endoscopic coronary angioplasty (PTCA), arteriosclerosis, peripheral circulatory insufficiency, etc.) It is effective in the treatment and prevention of various diseases, as well as in the treatment and prevention of diseases such as myocardial infarction, myocardium, peripheral vascular adhesion, heart failure. Since HGF promotes proliferation of vascular endothelial cells but does not promote vascular smooth muscle cell growth, HGF itself is also useful in the treatment and prevention of diseases as mentioned above. The pharmacological effect of the HGF gene is due to the pharmacological effect of HGF itself.
    As demonstrated in the Examples below, introduction of the HGF gene into the joint promotes proliferation of proteoglycan-synthetic cells by promoting healing of arterial chondrocytes. Therefore, the HGF gene is effective in the prevention and treatment of various cartilage injuries, such as osteoplastic abnormalities, degenerative arthritis, degenerative disc plaque, fracture healing and restorative insufficiency, trauma caused by exercise, key puncture disease and the like. Since HGF promotes the healing and growth of chondrocytes, HGF itself is useful for the treatment and prevention of the diseases described above. The effect of the HGF gene is based on the effect of HGF itself.
    "Liposomes" are suture carriers of a lipid bilayer that enclose an aqueous fraction therein. Lipid bilayer structures are known to be particularly similar to biological membranes. To prepare the liposomes of the present invention, phospholipids are used. Typical examples of phospholipids include phosphatidylcholine, such as lecithin, lyso lecithin, and the like; Acidic phospholipids such as phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl acid and the like; Or phospholipids obtained by replacing acyl groups of the acidic phospholipids with lauroyl, myristoyl, oleyl, and the like; And sphingophospholipids such as phosphatidylethanolamine, sphingomyelin and the like. Neutral lipids such as cholesterol may also be added to the phospholipids. Liposomes can be prepared by conventional methods from naturally occurring substances such as lipids in normal cell membranes. Liposomes containing the HGF gene of the present invention can be prepared, for example, by suspending a thin layer of purified phospholipids in a solution containing the HGF gene and then treating the suspension by conventional methods such as sonication.
    Liposomes containing the HGF gene of the present invention can be appropriately fused with viruses to form membrane fusion liposomes. In this case, it is preferable to inactivate the virus through, for example, ultraviolet irradiation. Particularly preferred examples of membrane fused liposomes are membrane fused liposomes fused with Sendai virus (Japanese hemagglutinin virus: HVJ). Membrane fusion liposomes are described in NIKKEI Science, April, 1994, p 32-28, J. Biol. Chem., 266 (6), 3361-3364 (1991), and the like. More specifically, HVJ-fused liposomes (HVJ-liposomes) are mixed with liposome suspensions containing HGF gene vectors, for example, purified HVJ inactivated by, for example, ultraviolet irradiation, the mixture is gently shaken and then sucrose It can be prepared by removing unbound HVJ by density gradient centrifugation. Liposomes can be bound to a substance having affinity for a target cell to enhance the efficiency of gene introduction into the target cell. Examples of materials having affinity for target cells include ligands such as antibodies, receptors, and the like.
    For the introduction of the HGF gene into cells, conventional methods are used, including introduction via viral vectors and other methods [NIKKEI Science, April, 1994, p 20-45; GEKKAN YAKUJI, 36 (1), 23-48 (1994) and references cited therein. Both methods are available for the manufacture of the medicaments of the present invention.
    Electronic methods using viral vectors consist of inserting the HGF gene into, for example, a retrovirus, adenovirus, adeno-associated virus, Hepes virus, Bexonia virus, poliovirus, Sindbis virus or other RNA virus. . Of these viruses, it is particularly preferred that retroviruses, adenoviruses and adeno-associated viruses are used for introduction.
    Examples of other methods include liposome methods, lipofectin methods, microinjection methods, calcium phosphate methods, electroporation methods. Of these methods, liposome methods are particularly preferred.
    For normal use of the HGF gene as a medicament, it is advantageous to inject HGF directly into the human body (in vivo methods). In contrast, any cells are harvested from humans and the HGF gene is then introduced into the cell ex vivo to return the HGF gene-induced cells back to the living body (ex vivo method). The methods are described in NIKKEI Science, April, 1994, p 20-45, GEKKAN-YAKUJI, 36 (1), 23-48 (1994) and the references cited therein. . Any of the above methods are appropriately selected depending on the disease, target organ, etc. to be treated and applied to the pharmaceutical composition of the present invention.
    In vivo methods are less expensive and less laborious and therefore more common than ex vivo methods, but the latter methods provide higher efficiency of HGF gene introduction into cells.
    When the medicament of the invention is administered by an in vivo method, the medicament may be administered via any route suitable for the disease, target organ, and the like to be treated. The agent may be administered intravenously, intraarterally, subcutaneously, intramuscularly or the like, or may be administered directly to the target organ of the disease, such as the kidneys, liver, lungs, brain, nerves and the like. Direct administration to the target site can selectively treat the target organ. For example, in gene therapy with genes for restenosis after PTCA, the composition can be administered intraarterally (JIKKEN-IGAKU, 12 (extra issue 15), 1298-1933 (1994)). Preferably, the medicament of the present invention is coated on the end of the instrument used in PTCA to rub the tip against blood vessels so that the medicament can be injected directly into vascular endothelial cells and vascular smooth muscle cells.
    When ex vivo methods are used to introduce HGF genes as described above, human cells (eg, lymphocytes or hematopoietic stem cells) are harvested by conventional methods and the harvested cells can be used as a medicament of the invention for gene introduction. Sensitize HGF-producing cells are then backinserted into humans.
    When the medicament is administered in vivo, the medicament may be prepared in various forms, including in liquid form. In general, the medicament may preferably be prepared as an injection solution containing the HGF gene as an active ingredient. If desired, conventional carriers may be added to the composition. Injectable solutions are dissolved in conventional methods, for example, by dissolving the HGF gene in a suitable solvent (e.g., sterile water, buffer, physiological saline, etc.), filtering through a filter or the like for sterilization, and filling the solution into a sterile container. Can be prepared. A medicament may be prepared using HGF gene-introduced viral vectors instead of the HGF gene itself. When liposomes (or HVJ-liposomes) containing the HGF gene embedded therein are used, the medicament may be in the form of liposome preparations such as suspensions, frozen preparations, centrifugally concentrated frozen preparations and the like.
    The HGF gene content in the medicament may vary as appropriate depending on the disease to be treated, the target organ, the age or weight of the patient, and the like. However, when calculated as the HGF gene, it is appropriate to administer at a dose of 0.0001 mg to 100 mg, preferably 0.001 mg to 10 mg. Dosages can be given in days or months.
    Hereinafter, the present invention will be described in more detail with reference to Examples, but is not limited thereto. The materials and methods used in the examples below are outlined below.
    Substances and Methods
    (1) HGF expression vector
    HGF expression vectors include human HGF cDNA [2,2 kb, Biochem. Biophys. Res. Commun., 172 , 321-327 (1990); Japanese Patent Laid-Open No. 5-111383] was prepared by inserting between the EcoRI and NotI positions of the pUC-SRα expression vector [FEBS, 333 , 61-66 (1993)]. In this plasmid vector, transcription of HGF cDNA is regulated by the SRa promoter (Nature, 342 , 440-443 (1989)).
    (2) cell culture
    Rat coronary endothelial cells are isolated from the enzymatically cleaved heart of 8 week old Sprague-Dawley (SD) mice by density gradient centrifugation [Transplantation, 57 , 1653-1660 (1994)]. Rat aortic vascular smooth muscle cells (VSMCs) are obtained by enzymatic treatment from 12 week old SD rats [J. Clin. Invest., 93 , 355-360 (1994)]. The cells are maintained in DMEM medium fed with 10% (vol / vol) fetal bovine serum, penicillin (100 U / ml) and streptomycin (100 μg / ml). Cells are incubated in a humidified 95% air-5% CO 2 atmosphere at 37 ° C. The culture medium is exchanged daily at two day intervals. Immunopathological and morphological observations reveal that the cells are endothelial cells and smooth muscle cells, respectively.
    Human aortic endothelial cells (5 pathways) and human VSMCs (5 pathways) are obtained from Kurabo Co. Endothelial cells similar to the above method in MCDB131 medium fed 5% fetal bovine serum, epidermal growth factor (10 ng / ml), basic fibroblast growth factor (2 ng / ml) and dexamethasone (1 μM) Incubate with.
    Endothelial cells at rest are described in J. Clin. Invest., 86 , 1690-1697 (1990), ibid., 94 , 824-829 (1994).
    (3) In Vitro Infection of HGF Gene with HVJ-Liposomes
    Endothelial cells or VSMCs are seeded and propagated for sensitization on 6-well plates having a cell number of 10 6 to reach an 80% population. Cells are washed three times with balanced salt solution (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6; then abbreviated as "BSS") fed with 2 mM calcium chloride. Thereafter, 1 ml of the solution of HVJ-liposome-DNA obtained from Example 1 (containing 2.5 mg of lipid and 10 mg of embedded DNA), or 1 ml of the solution of HVJ-liposome-cont obtained from Comparative Example 1, was then added to the cells. Add. The resulting mixture is incubated at 4 ° C. for 5 minutes and further incubated at 37 ° C. for 30 minutes. The cells are washed and maintained in fresh medium containing 10% bovine serum in a CO 2 incubator.
    (4) HGF concentration assay in endothelial cells and VSMCs
    The concentration of HGF produced from sensitized endothelial cells and VSMCs is assayed by ELISA. That is, rat or human endothelial cells or VSMCs are seeded on 6-well plates (Corning agent) having a cell density of 5 × 10 4 cells / cm 2 and then incubated for 24 hours. The medium is replenished 24 hours after sensitization and incubated for another 48 hours. To determine if HGF was released, sensitized cells (48 hours after sensitization) were washed and added to 1 ml of serum-free medium containing 5 × 10 −7 M insulin, 5 μg / ml transferrin and 0.2 mM ascorbate do. After 24 hours, the culture medium is harvested, centrifuged at 600 g for 10 minutes and then stored at -20 ° C.
    HGF concentration in the medium is measured by enzymatic immunoassay using anti-mouse HGF antibody or anti-human HGF antibody [Exp. Cell Res., 210 , 326-335 (1994); Jpn. J. Cancer Res., 83 , 1262-1266 (1992). Rabbit anti-rat or anti-human HGF IgG is coated on a 96-well plate (Corning agent) at 4 ° C. for 15 hours. After blocking with 3% bovine serum albumin-containing PBS (phosphate buffered saline), the culture medium is added to each well and the incubation is carried out at 25 ° C. for 2 hours. Each well was washed three times with PBS (PBS-Tween) containing 0.025% Tween, and then biotinylated rabbit anti-rat HGF IgG or anti-human HGF IgG was added to each well and incubated at 25 ° C. for 2 hours. Let's do it. After washing with PBS-Twin, each well is incubated with a hose radish peroxidase-linked streptoavidin-biotin complex (PBS-Twin solution). A substrate solution (containing 2.5 mM o-phenylenediamine, 100 mM sodium phosphate, 50 mM citrate and 0.015% hydrogen peroxide) is added to initiate the enzymatic reaction. The reaction is terminated by adding 1 M sulfuric acid to the system. Absorbance is measured at 490 nm. Anti-human HGF antibodies cross-react only with human HGF and not murine HGF. Anti-rat HGF antibodies cross-react only with rat HGF and do not respond to human HGF.
    (5) HGF
    The human and murine recombinant HGFs used are purified from cultures of CHO cells or C-127 cells infected with expression plasmids comprising human or murine HGF cDNA [Cell, 77 , 261-271 (1994); J. Clin. Invest., 93 , 355-360 (1994)].
    (6) statistical analysis
    Repeat all experiments three or more times. Measured data are expressed as mean ± standard error. Statistical analysis of the measured data is performed according to Duncan's test.
    (7) Hematoxylin-Esosine (HE) Staining and Azan Staining
    Ten days after transduction, HGF gene-induced mice are sacrificed by perfusion of heparinized saline. Then, it is fixed overnight with 4% paraformaldehyde PBS solution. After fixation, tissue is embedded in paraffin. Slides are prepared and stained with HE and azan in conventional manner. Examine the slide under a microscope to determine the number of capillaries.
    Example 1
    Preparation of HVJ-Liposomes Containing HGF Expression Vectors
    Phosphatidylserine, phosphatidylcholine and cholesterol are mixed with tetrahydrofuran in a weight ratio of 1: 4.8: 2. By distilling off tetrahydrofuran via a rotary evaporator, the lipid mixture (10 mg) is precipitated on the vessel wall. 96 μg of purified High Mobility (HMG) 1 nucleoprotein from bovine thymus is mixed with BBS (200 μl) solution of plasmid DNA (300 μg) at 20 ° C. for 1 hour, and the mixture is added to the obtained lipid mixture . The resulting liposome-DNA-HMG 1 complex suspension is circulated, sonicated for 3 seconds and shaken for 30 minutes.
    Purified HVJ (strain Z) is inactivated by UV irradiation (110 erg / mm 2 sec) for 3 minutes immediately before use. BSS is added and mixed with the liposome suspension (0.5 ml, containing 10 mg of lipid) and HVJ (20,000 erythrocyte aggregation units) obtained above to make the total volume to 4 ml. The mixture is incubated at 4 ° C. for 10 minutes and shaken more gently at 37 ° C. for 30 minutes. Unreacted HVJ is removed from HVJ-liposomes by sucrose density gradient centrifugation. That is, the upper layer in the sucrose density gradient is collected to obtain HVJ-liposomes containing HGF expression vector (containing 10 μg / ml of HGF expression vector). HVJ-liposomes containing HGF expression vectors are hereinafter referred to as HVJ-liposomes-DNA.
    Example 2
    Administration of HVJ-liposomes containing HGF Expression Vectors to Mice
    HVJ-liposomes containing HGF expression vectors are prepared by the method as described in the above examples using 64 μg of HMG 1 nucleoprotein and 200 μg of plasmid DNA. BSS is added and mixed with the liposome suspension (0.5 ml, containing 10 mg of lipid) and HVJ (35,000 erythrocyte aggregate units) to bring the total volume to 2 ml.
    SD rats (weight 400-500 g; sold by Japan Charles River) are anesthetized by parenteral administration of sodium pentobarbital (0.1 ml / 100 mg) and breathed in an automatic respirator with incubation. Thoracoscopic intubation is performed on the left side of the rat. HVJ-liposomal-DNA or HVJ-liposomal-cont (20 μl) is carefully injected directly over the heart using a 30 G syringe.
    Comparative Example 1
    Preparation of HVJ-Liposomes That Contain No HGF Expression Vector
    Vectors containing no HGF gene were treated in the same manner as described in Example 1 to prepare HVJ-liposomes containing no HGF expression vector. HGF expression vector-free HVJ-liposome followed by HVJ-liposome-cont.
    Comparative Example 2
    Preparation of HVJ-Liposomes Containing Human TGF-β Expression Vector
    HVJ-liposomes containing human TGF-β expression vectors were prepared in a similar manner to Example 1 except for using human TGF-β expression vectors.
    HVJ-liposomes containing human TGF-β expression vectors are hereinafter referred to as HVJ-liposomal-DNA (TGF-β).
    Test Example 1
    Expression of HGF in Rat Coronary Endothelial Cells Sensitized with HVJ-Liposome-DNA
    Rat coronary endothelial cells (cell number: 10 6 ) are sensitized with HVJ-liposome-DNA (concentration of HGF expression vector in liposomes: 10 μg). HGF production is measured by ELISA. For control, a similar test is performed using HVJ-liposome-cont. HGF production is also measured on unsensitized rat coronary endothelial cells (untreated group). The results are shown in FIG. 1 (n = 6), and “HGF” represents a group of rat coronary endothelial cells sensitized with HVJ-liposome-DNA.
    As shown in FIG. 1, rat coronary endothelial cells sensitized with HVJ-liposome-DNA produce and secrete high levels of HGF. In contrast, HGF production is not substantially observed in either the natural group or the group of rat coronary endothelial cells sensitized with HVJ-liposomal-cont.
    The cell numbers in the groups tested show that the HGF founders show significantly higher cell numbers.
    Test Example 2
    Effect of sensitized HGF expression vector on proliferation of endothelial cells
    Human endothelial cells are sensitized with HVJ-liposome-cont. The sensitized cells are cultured in the absence or presence of added exogenous recombinant human HGF (1, 10 and 100 ng / ml) and the cell growth rate (%) is measured. The results are shown in Figure 2 ((line) graph, n = 6), "DSF" represents a group of endothelial cells sensitized with HVJ-liposome-cont and "HGF" was cultured in the presence of recombinant human HGF at constant concentration. Group (for DSF, *: P <0.05, **: P <0.01).
    The (line) graph shown in FIG. 2 shows that the growth of endothelial cells is promoted by externally added HGF.
    Endothelial cells sensitized with HVJ-liposomal-DNA (concentration: 10 μg / ml) are similarly cultured and the increased cells are counted to determine cell growth rate (%). For control, endothelial cells sensitized with HVJ-liposome-cont are also cultured and the increased cells are counted to determine cell growth rate (%). The results are shown in FIG. 2 (n = 6), where "DSF" represents an endothelial cell group sensitized with HVJ-liposome-cont, and "HGF" represents an endothelial cell group sensitized with HVJ-liposome-DNA (to DSF **: P <0.01, # for HGF: P <0.05, 100 ng / ml).
    As can be seen from FIG. 2, the results show that the growth rate of HVJ-liposomal-DNA-sensitized endothelial cells is significantly higher than that of the control group, and even higher compared to the growth rate of externally added HGF.
    Endothelial cells sensitized with the aforementioned HVJ-liposomal-DNA are cultured in the presence or absence of rabbit anti-human HGF antibodies. Increased cells are counted to determine cell growth rate. For control, endothelial cells sensitized with HVJ-liposome-cont are cultured and the increased cells are counted in a similar manner to measure cell growth rate. Rabbit anti-human HGF antibody (10 μg / ml) was described in Jpn. J. Cancer Res., 83 , 1262-1266 (1992). The antibody may neutralize biological activity of 10 ng / ml at a concentration of 10 μg / ml. Anti-human HGF antibodies cross-react only with human HGF and not mouse HGF, while anti-rat HGF antibodies cross-react only with mouse HGF and do not react with human HGF. Usually rabbit serum IgG (10 μg / ml) is used as a control.
    The results are shown in FIG. 3 (n = 6), of which “control” refers to the group of HVJ-liposome-cont-sensitized endothelial cells cultured in the presence of IgG control; "HGF" refers to a group of HVJ-liposome-DNA-sensitized endothelial cells cultured in the presence of an IgG control; And “HGFab” refers to a group of HVJ-liposomal-DNA-sensitized endothelial cells cultured in the presence of rabbit anti-human HGF antibodies. The cell sperm percentage (%) is expressed as a relative percentage when the growth rate of the control group is 100 (*: P <0.01 for the control group, #: P <0.05 for the HGF). As shown in FIG. 3, the growth of HVJ-liposomal-DNA-sensitized endothelial cells is retarded in the presence of anti-human HGF antibody, so the cell growth rate is substantially the same as that of the control group. The results clearly demonstrate that HGF is a growth factor for endothelial cells.
    Test Example 3
    Effect of Cultured HVJ-Liposome-DNA-Sensitized Rat VSMCs Supernatant on Rat Coronary Endothelial Cells
    Supernatants from cultured HVJ-liposome-DNA-sensitized rat VSMCs are added to a stationary rat tubular endothelial cell culture system (cell number: 10 5 ). After incubation for 3 days, the increased number of endothelial cells is measured. As a control, supernatants of cultured HVJ-liposomal-cont-sensitized rat VSMCs are treated in a similar manner as described above and the increased endothelial cells are counted as described above. The results are shown in Figure 4 (n = 6), where "control" represents the supernatant from cultured HVJ-liposome-cont-sensitized rat VSMCs, and "HGF" represents the cultured HVJ- The supernatant of liposome-DNA-sensitized rat VSMCs is shown.
    As shown in FIG. 4, a significant increase in endothelial cell number is seen in the supernatant group of cultured HVJ-liposomal-DNA-sensitized rat VSMCs.
    The concentration of HGF in the culture supernatant of rat VSMCs sensitized with HVJ-liposomal-DNA or HVJ-liposomal-cont as described above is assayed by ELISA using anti-human HGF antibody and anti-mouse HGF antibody. HGF concentration in the culture supernatant of non-sensitized VSMCs is also assayed (untreated group).
    Results obtained using anti-human HGF antibody and anti-mouse HGF antibody are shown in FIGS. 5 and 6 (n = 6 in both tests), respectively. In the figure, "control" refers to the supernatant group from cultured HVJ-liposomal-cont-sensitized rat VSMCs; And “HGF” refers to the group of supernatants from cultured HVJ-liposomal-DNA-sensitized rat VSMCs.
    As shown in FIG. 5, HGF is detected in supernatants of HVJ-liposomal-DNA-sensitized rat VSMCs, and HGF concentration is significantly higher than the control.
    6 also shows that rat HGF is also detected in the supernatants of HVJ-liposomal-DNA-sensitized rat VSMCs, with HGF concentrations significantly higher than controls.
    As observed in FIGS. 5 and 6, in both the supernatant of the native group and the control group, HGF is not present in an amount detectable by ELISA.
    Test Example 4
    Effect of Cultured HVJ-Liposome-DNA-Sensitized Rat Coronary Endothelial Supernatant on Rat Coronary Endothelial Cells
    Supernatants of cultured HVJ-liposomal-DNA-sensitized rat tubular endothelial cells are added to a stationary rat tubular endothelial cell culture system (cell number: 10 5 ). After incubation for 3 days, the increased number of endothelial cells is measured. As a control, endothelial cells are cultured in a similar manner using culture supernatants of HVJ-liposome-cont-sensitized rat coronary endothelial cells and counted for increased endothelial cells. The results are shown in FIG. 7, in which A, B and C are the groups to which the culture supernatant was added to HVJ-liposomal-DNA-sensitized rat coronary endothelial cells (n = 8), HVJ-liposomal-cont-sensitized, respectively. Group supernatant (n = 8), and untreated group (n = 15).
    As shown in FIG. 7, a significant increase in endothelial cell number was seen in the group to which the culture supernatant was added to HVJ-liposomal-DNA-sensitized rat coronary endothelial cells, whereas in the control group, the cell number was nearly untreated. The same (control: 0.117 ± 0.002, group A: 0.148 ± 0.03, P <0.01).
    Next, anti-HGF antibody is added to the culture supernatant of HVJ-liposomal-DNA-sensitized rat coronary endothelial cells. The increased number of endothelial cells is investigated as described above. The results are shown in FIG. 8 (n = 8), where A represents the group to which the culture supernatant of HVJ-liposomal-DNA-sensitized rat coronary endothelial cells was added; B represents the group to which the culture supernatant of HVJ-liposomal-cont-sensitized rat coronary endothelial cells was added; C represents a group in which anti-HGF antibody was added to the culture supernatant of HVJ-liposomal-DNA-sensitized rat coronary endothelial cells; And D represents a group in which a control antibody was added to the supernatant from cultured HVJ-liposomal-DNA-sensitized rat coronary endothelial cells.
    As shown in Figures 8, A and C, cell growth promoting activity of the culture supernatant of HVJ-liposomal-DNA-sensitized rat coronary endothelial cells disappears due to the addition of anti-HGF antibody. The results indicate that the cell growth promoting activity of the culture supernatant of HVJ-liposomal-DNA-sensitized rat tubular endothelial cells was due to HGF.
    Test Example 5
    Effect of HVJ-Liposome-DNA-Sensitized Human VSMCs on Human Endothelial Cells
    Human VSMCs are seeded on cell culture inserts (Coaster, 0.45 μm pore diameter) and then grown in DMEM medium fed with 10% bovine serum. In contrast, human endothelial cells are seeded on 6-well plates and maintained in DMEM medium fed with 10% bovine serum. When VSMCs proliferated and reached 80% colony, VSMCs were incubated for 5 minutes at 4 ° C and incubated for 30 minutes at 37 ° C with HVJ-liposome-DNA (10 μg of DNA in liposomes) or HVJ-liposome-cont. Let's do it. After sensitization, an insert containing sensitized VSMCs is added to each well containing stationary human endothelial cells. VSMCs and endothelial cells are co-cultured for 3 days in DMEM medium fed with 0.5% bovine serum. Thereafter, the cell number is measured with a WST-cell counter kit (manufactured by Wako Co.). The results are shown in FIG. 9 (n = 6). In the figure, "control" represents a group co-cultured with HVJ-liposome-cont-sensitized VSMCs, and "HGF" represents a group of supernatants from cultured HVJ-liposome-DNA-sensitized VSMCs.
    The results shown in FIG. 9 show that human VSMCs sensitized with HVJ-liposome-DNA can significantly increase the growth of stationary non-sensitized human endothelial cells.
    Test Example 6
    Effect of HVJ-Liposome-DNA-Sensified Rat VSMCs on Coronary Endothelial Cells
    HVJ-liposomal-DNA-sensitized rat VSMCs (cell number: 10 6 ) are co-cultured on stationary phase with rat coronary endothelial cells (cell number: 10 5 ) for 3 days in the stationary phase. Thereafter, the increased number of endothelial cells is examined. For control, endothelial cells are co-cultured in a similar manner using HVJ-liposome-cont-sensitized rat VSMCs and the increased endothelial cells are measured. The results are shown in FIG. 10 (n = 6), of which “control” represents the group of rat VSMCs sensitized with HVJ-liposome-DNA, and “HGF” represents the group of rat VSMCs sensitized with HVJ-liposome-cont.
    As shown in FIG. 10, the growth of endothelial cells is stimulated by HGF released from HVJ-liposomal-DNA-sensitized rat VSMCs and increased cell numbers are observed (control: 0.126 ± 0.006, HGF group: 0.156 ±) 0.01, P <0.05).
    Test Example 7
    Growth of Rat VSMCs Sensitized with HVJ-Liposome-DNA
    Rat VSMCs sensitized with HVJ-liposome-DNA and rat VSMCs suddenly abbreviated with HVJ-liposome-cont were incubated respectively to compare the increased cell numbers. Sensitization with HVJ-liposomal-DNA does not affect cell growth at all. The results indicate that HGF does not have a cell growth promoting effect on VSMCs.
    Test Example 8
    Induction of Angiogenesis in Rat Cardiac Muscle Directly Injected with HVJ-Liposome-DNA
    Rat heart muscle injected directly with HVJ-liposome-DNA, rat heart muscle injected directly with HVJ-liposomal-cont, and untreated cardiac muscle of rats were stained with HE and azan, respectively, and examined under a microscope to determine the number of microvessels. Measure The results are shown in FIG. 11, of which "HGF" represents the number of microvessels in rat cardiac muscle directly injected with HVJ-liposome-DNA, and "control" in rat cardiac muscle directly injected with HVJ-liposome-cont. Indicates the number of microvessels.
    As can be seen from FIG. 11, the number of fine blood vessels was compared with the rat heart muscle injected directly with HVJ-liposomal-cont and the rat heart injected with HVJ-liposomal-DNA directly as compared to the rat untreated cardiac muscle. Increase significantly in muscle. The results show that HGF with endothelial cell growth activity shows angiogenic activity in vivo.
    Test Example 9
    Healing articular cartilage by injecting HVJ-liposomal-DNA directly into the joint
    Ten week old Fisher rats are injured between the femoral joints through the cartilage using Kirschner wire with a diameter of 1.8 mm. One week after surgery, HVJ-liposome-DNA prepared in Example 1 (100 μl / knee) is injected directly into the joint. For control, HVJ-liposome-cont prepared in Comparative Example 1 and HVJ-liposome-DNA (TGF-β) prepared in Comparative Example 2 are administered directly in equal amounts to the joints. 1, 3 and 4 weeks after injection of the gene, etc., the mice were sacrificed to observe the treated area histologically.
    As shown in FIG. 12, the results show that the synthesis of proteoglycans stained with toluidine blue was observed 3 weeks after administration of HVJ-liposome-DNA to the joint, indicating the development of cartilage-like cells. In addition, as shown in FIG. 13, 4 weeks after the HVJ-liposome-DNA was administered to the joint, a tendency to further expand the site where cartilage-like cells were developed was observed, where synthesis of proteoglycan was confirmed.
    As shown in FIG. 14, when HVJ-liposome-DNA (TGF-β) prepared in Comparative Example 2 was injected into a joint, development of the cartilage-like cells was not observed even after 4 weeks of administration. Furthermore, as shown in FIG. 15, when the HVJ-liposome-cont prepared in Comparative Example 1 was injected into the joint, the development of the cartilage-like cells was not observed even 4 weeks after administration.
    The medicament of the present invention, when compared to HGF itself, provides a lasting therapeutic effect. Moreover, the medicament of the present invention can be applied locally to the target organ so that the effect can be selectively shown, thereby minimizing the side effects of HGF.
    权利要求:
    Claims (6)
    [1" claim-type="Currently amended] Drugs containing the HGF gene
    [2" claim-type="Currently amended] Liposomes containing the HGF gene.
    [3" claim-type="Currently amended] The liposome of claim 2 which is a membrane fused liposome fused with Sendai virus.
    [4" claim-type="Currently amended] A medicament containing the liposome according to claim 2 or 3.
    [5" claim-type="Currently amended] The agent according to claim 1 or 4, which is used for treating arterial disease.
    [6" claim-type="Currently amended] The agent according to claim 1 or 4, which is used for the treatment of cartilage injury.
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    同族专利:
    公开号 | 公开日
    DE69634567D1|2005-05-12|
    ES2240999T3|2005-10-16|
    NZ315769A|1999-05-28|
    WO1997007824A1|1997-03-06|
    AU6754496A|1997-03-19|
    EP0847757A4|2000-04-12|
    US20040105882A1|2004-06-03|
    PT847757E|2005-06-30|
    EP0847757B1|2005-04-06|
    KR100765687B1|2007-10-11|
    JP3431633B2|2003-07-28|
    US7285540B2|2007-10-23|
    CN1198675A|1998-11-11|
    US20040220126A1|2004-11-04|
    KR100725199B1|2007-08-16|
    CA2230819A1|1997-03-06|
    DE69634567T2|2006-02-16|
    CA2230819C|2009-04-14|
    US20090004260A1|2009-01-01|
    AT292476T|2005-04-15|
    KR20040097246A|2004-11-17|
    CN100478033C|2009-04-15|
    EP0847757A1|1998-06-17|
    US6248722B1|2001-06-19|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1995-08-29|Priority to JP95-245475
    1995-08-29|Priority to JP24547595
    1996-02-20|Priority to JP5846796
    1996-02-20|Priority to JP96-58467
    1996-08-22|Application filed by 다께우찌 마사야쓰, 스미또모 세이야꾸 가부시키가이샤
    1999-06-25|Publication of KR19990044279A
    2007-08-16|Application granted
    2007-08-16|Publication of KR100725199B1
    优先权:
    申请号 | 申请日 | 专利标题
    JP95-245475|1995-08-29|
    JP24547595|1995-08-29|
    JP5846796|1996-02-20|
    JP96-58467|1996-02-20|
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