TWI236373B - Medicament composition comprising HGF gene - Google Patents

Abstract

The invention relates to a medicament composition comprising HGF gene. The medicament of the invention possesses better sustained release effects than HGF itself. Moreover, since such medicament has a selective local action which possibly can reduce the side-effects caused by HGF.

Description

1236373

The invention belongs to the same technical field. The present invention relates to medicines which can be used for gene therapy and the like. More specifically, it relates to medicine composed of HGF (hepatocyte growth factor) genes, and liposomes containing HGF genes. HGF is a physiologically active peptide that exhibits various pharmacological effects. The pharmacological effects of HGF are described in, for example, Experimental Medicine Vol.io, No. 3 (Supplement) 33〇 3 39 (1 992). HGF can be used as a therapeutic agent for liver cirrhosis, a therapeutic agent for renal diseases, an epithelial cell proliferation promoting agent, an anticancer agent, a side effect preventing agent for cancer therapy, a therapeutic agent for lung disorders, a therapeutic agent for stomach and duodenal damage, and a neurological disorder for its pharmacological effects Agent, immunosuppressive side effect preventive agent, collagen cleavage promoter, therapeutic agent for osteoporosis, therapeutic agent for arterial disease, therapeutic agent for pulmonary fibrosis, therapeutic agent for liver disease, therapeutic agent for abnormal blood coagulation, therapeutic agent for low blood plasma, trauma A therapeutic agent, an agent for improving a neurological disorder, an agent for increasing hematopoietic stem cells, a hair growth promoter, etc. (Japanese Patent Laid-Open No. 4_18〇28; Japanese Patent Laid-Open No. 4-49246; EP 4926 1; Japanese Patent Laid-Open No. 4-250; 1 Publication No. 0, WO 93/8 82 1, JP-A No. 1 72207, JP-A No. 7_89869, JP-A No. 6_4〇934, WO 9 4/2 1 65, JP-A No. 6-40 93 5, Japanese Unexamined Patent Publication No. 6- 5 6 692, Japanese Unexamined Patent Publication No. 7_4 1 429, 1 ^ 93/3061, Japanese Unexamined Patent Publication No. 5-2 1 3 7 2 1, etc.). Regarding gene therapy, currently active research and development on adenosine deaminase deficiency, AIDs gene therapy, oncogene therapy, cystic fibrosis gene therapy, and hemophilia gene therapy are underway internationally. -4- This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 mm). 5. Description of the invention (2. However, gene therapy using HGF gene is not known, and it is not possible for gene therapy to be possible. Understand. The solution of 诬 Α, which is one of the drugs with a short half-life in blood. Therefore, it is expected to be administered locally. Also, HGF has various pharmacological effects, so it is expected to develop various therapeutic agents. On the other hand, due to its various pharmacological effects 'in the systemic administration, side effects will also become a problem. In addition, if the GF itself is administered intravenously', a considerable amount of it will stay in the liver, so there is a problem of achieving the treatment goal. The disadvantage of reducing the amount of organs. Lead is the solution to the problem. The present invention is designed to solve the previous problems. The main points of the present invention are (1) medicine composed of HGF gene, ⑵ liposomes containing HGF gene, and ⑶ fusion with Sendai virus. The plastid described in the above description of the membrane fusion lipid f body, (4) a medicine composed of the liposome described in the above description or ⑶, ⑸ is a treatment agent for liver cirrhosis, a treatment agent for kidney diseases, and epithelium Cell proliferation promoter, anticancer agent, side effect preventing agent for cancer therapy, sodium for treating lung disorders, 3, treatment agent for duodenal injury, treatment agent for neurological disorder, preventing agent for immunosuppressive side effects, collagen degradation promoting agent ', two bones Disorder therapeutic agent, therapeutic agent for arterial disease, therapeutic agent for pulmonary fibrosis ,: therapeutic agent for visceral disease, therapeutic agent for abnormal blood coagulation, therapeutic agent for low blood content, therapeutic agent for trauma, neurological disorder improving agent, hematopoietic stem cell; 1236373 A7 '_____B7 V. Description of the invention (3) Additive or hair growth promoter medicine according to item 1 or 4 of the scope of patent application, (6) Medicine described in the treatment of arterial disease treatment, and (7) cartilage injury treatment Brief description of the medicine in the book. Figure 1 is a graph showing the expression of HGF in rat coronary artery endothelial cells sensitized with H v J · liposome-DNA in Test Example 1. Polyline in Figure 2 The figure shows the cell increase rate in the presence or absence of hgf of endothelial cells sensitized to η v J -liposome-cont in Test Example 2. In the figure, HGF indicates HVJ-liposome-cont Min The endothelial cell population, HGF represents a population cultured in the presence of a predetermined concentration of recombinant human HGF. The bar graph of FIG. 2 is shown in Test Example 2, which is an endothelial cell sensitized to η VJ-liposome_ D Ν Α The graph of the increase rate. In the figure, ds F represents the endothelial cell population sensitized by HVJ-liposome-cont, and HGF carrier represents the endothelial cell population sensitized by HVJ-liposome-DNA. Fig. 3 is shown in Test Example 2 Graph of cell growth rate of HVJ-liposome-DNA sensitized endothelial cells in the presence or absence of anti-HGF antibodies. In the figure, the comparison table shows η VJ-liposomes cultured in the presence of the I g G control group. -cont stimulated endothelial cell population; HGF represents HVJ-liposome-DNA stimulated endothelial cell population cultured in the presence of IgG control group; HGFab represents HVJ-liposome-DNA stimulated culture in the presence of rabbit anti-human HGF antibody Endothelial cell population. The increase rate is expressed as a relative% of the increase rate of the control. Figure 4 is shown in Test Example 3, HVJ-liposome-DNA sensitization is large-6- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1236373 A7 · ____ B7 Five invention descriptions (4 ~) ~ The effect of VSMC culture supernatant on the cell proliferation of rat coronary artery endothelial cells. The control group was HVJ-liposome a-sensitized rat VSMC culture supernatant, and HGF was hvj-lipid bone bean-DNA-sensitized rat vs μ c culture supernatant. Group. Fig. 5 is a graph showing the results of measuring the HGF concentration in the culture supernatant of HVJ_liposome-〇 ^^ eight-sensitized rat VSMC in Test Example 3 with an anti-human hgf antibody. In the figure, the non-sensitized VSMC culture supernatant group was used as a control, and the control group was a culture supernatant group of rats sensitized with Η VJ · liposome · c 0 nt; HGF is the HVJ- A population of culture supernatants of liposome-DNa-sensitized rat VSMCs. Fig. 6 is a graph showing the HGF concentration in the culture supernatant of rat VSMC sensitized with hvj_liposome_DNa in Test Example 3 and measured with an anti-rat HGF antibody. In the figure, the culture supernatant group without treatment was unsensitized VSMc; the control is the culture supernatant group with HVJ-liposome-cont unsensitized rat VSMC; HGF is the HVJ-liposome_DNA Non-sensitized rat VSMC culture supernatants. Fig. 7 is a graph showing the cell proliferation effect of rat coronary artery endothelial cells by the culture supernatant of rat coronary artery endothelial cells sensitized with HVJ-liposome-DNA in Test Example 4. In the figure, A is a group of culture supernatant of rat coronary artery endothelial cells sensitized with η VJ-liposome-D Ν Α; B is a group of rat coronary arteries with HVJ-liposome-cont sensitized Group of culture supernatant of endothelial cells; C is the untreated group. FIG. 8 shows the experimental supernatant of rat coronary artery endothelial cell culture sensitized with HVJ-liposome-DNA in the presence of anti-HGF antibody in Test Example 4. Chinese paper standard (CNS) A4 (210X) 297 directors) 1236373

Graph of cell proliferation effect of rat coronary artery endothelial cells. In the figure, A is a group of culture supernatant of rat coronary artery endothelial cells sensitized with HVJ-liposome_DNA; B is a group of rat coronary artery endothelial cells sensitized with HVJ-liposome 乂 ... Supernatant group; C is a group with HGF antibody added to the culture supernatant of rat distal artery endothelial cells sensitized to η Vj_liposome_ DNA; D is a sensitization of HVJ-liposome_DNA A control antibody was added to the culture supernatant of rat coronary endothelial cells. Fig. 9 is a graph showing an increase in the number of endothelial cells in Test Example 5 when HVJ liposome_DNA-sensitized human VSMCs were co-cultured with non-sensitized human endothelial cells. In the figure, the control is a co-culture group with HVJ_liposomal_cnt-sensitized VSMC; HGF is a culture supernatant group of HVJ-liposome-DNA-sensitized VSMC. Fig. 10 is a graph showing an increase in the number of endothelial cells in Test Example 6 when HVJ-liposome-DNA-sensitized rat VSMC was co-cultured with non-sensitized rat coronary artery endothelial cells. In the figure, the control is a co-culture group with H VJ-liposome · cont-sensitized VSMC; HGF is a culture supernatant group of HVJ-liposome-DNA-sensitized VSMC. FIG. 11 is a graph showing an increase in the number of microvessels in rat myocardium directly injected with HVJ-liposome-DNA in Test Example 8. FIG. In the figure, HGF is the number of microvessels in rat myocardium directly injected with HVJ-liposome-DNA, and the control is the number of microvessels in rat myocardium directly injected with ΗVJ-liposome · c ο n t. Fig. 12 is a diagram showing the appearance of cartilage-like cells that synthesize the stained protein polymerase by toluidine blue staining 3 weeks after H VJ-liposome · DNA was administered to the joint in Test Example 9. -8-This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1236373 A7 * ____B7 V. Description of the invention (6) Figure 13 is shown in Test Example 8 and 8 VJ was administered in the joint -Liposomal_ Toluidine blue staining 4 weeks after DNA confirmed the appearance of chondrocyte-like cells that synthesize the stained protein polymerase. Figure 14 shows toluidine blue staining in Test Example 9 after intra-articular administration of HVJ-liposome-DNA (TGF-p) prepared in Comparative Example 2 for 4 weeks, and it was not confirmed that the protein was identified as being synthesized and stained. Diagram of chondrocytes of polymerase. Fig. 15 shows toluidine blue staining in test example 9 after intra-articular administration of HVJ_liposome-cont prepared in comparative example 4 in 4 weeks, and cartilage that was identified as being synthesized by stained protein polymerase could not be confirmed Diagram of cell-like cells. The "HGF gene" used in the practice of the present invention to cover the fly can refer to a gene capable of expressing Hgf in the gene, and the expressed polypeptide as long as it is substantially synergistic with Hgf also includes the gene sequence Some are deleted or replaced by other bases, some are inserted into other test sequences, and factors that bind bases at the ends. Examples of such HGF genes include Nature, 342 · 440 (1 989), Japanese Patent Application Laid-Open No. 1 1 8.3, Biochem Bi () phys Res. Commun. J ^, 967 (1 989), special Kaiping 3_2 5 5096 and other 1 hgf motifs already contained, these genes can be used in the present invention. The HGF gene is incorporated into an appropriate vector. For example, it can be used as a viral vector in which the HGF gene is incorporated into the gene of the virus mentioned later, or an appropriate expression vector in which the HGF gene is incorporated. In the present invention, "medicine" refers to a therapeutic or preventive agent for human diseases based on the pharmacological effects of HGF, such as the therapeutic or preventive agent described above. According to the present invention, after the HCG gene is introduced into the cell, the HGF is in this fine size. -9- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1236373 A7 B7

Cell performance ' Its HGF shows a pharmacological effect. Therefore, the medicine of the present invention is effective for the same target diseases as those for HG.F. For example, the HGF gene was introduced into the cell #, as described in the examples, which promotes the proliferation of vascular endothelial cells, but does not promote vascular smooth muscle cells (proliferation. In addition, as described in the examples, in animal experiments using rats When the HGF gene is introduced into the heart of the body, angiogenesis can be seen. Therefore, the 'HGF gene is used for various diseases caused by obstacles that are mainly caused by arterial diseases, especially abnormal proliferation of vascular flat skeletal muscle cells (for example, blood expansion) (PTCA) re-narrowing, arteriosclerosis, peripheral circulatory insufficiency, etc.) treatment, prevention, myocardial infarction, cardiomyopathy, peripheral vascular occlusive disease, cardiac insufficiency and other diseases. Hgf itself It also promotes the proliferation of vascular endothelial cells, but does not promote the proliferation of vascular smooth muscle cells, and can be used as the same therapeutic agent and preventive agent. The effect of the hgf gene is based on the effect of HGF itself. In addition, as described in the examples, the The introduction of HGF gene into the joint promotes the repair of articular chondrocytes and the proliferation of cells that synthesize protein polymerase. Therefore, HGF For various cartilage injuries, such as disorders of bone formation, deformable joint disease, deformable intervertebral disc disease, repair of fractures, incomplete insults, injuries from sports, Chipacha (Feng-Yayoshi) disease, etc. It is effective for prevention and treatment. HGFM also promotes the repair and proliferation of chondrocytes. It can be used as the same therapeutic agent and preventive agent. The effect of hgf gene is based on the effect of HGF itself. The closed cell body formed by two layers of lipid, it is known that its lipid 2 molecular membrane structure is very similar to the biological membrane. As -10- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1236373 A7 j ------ ^ ___ B7 V. Description of the invention (8-t Phospholipids used in the present invention < liposomes, for example, _ phospholipids, soluble 'etc. Fermented serine acid, fermented glutamate: acidic phospholipids such as phosphoinositide, phosphoinositide, or fermented acid, or these fermented groups are replaced with phospholipids such as ten-, fourteen-, and oleyl groups Sphingomyelin, phospholipids, ethanolamine, sphingomyelin, etc. Further, cholesterol may also be added as ... shoulder blade from thin shell may be present in the typically with the lipids of the cell membrane of the day and the like;: 1: manufactured in a generally known methods of the present invention containing the HGF gene.

< The plastid can be produced by suspending a thin film of refined phospholipid in a solution containing the HGF gene, and applying ultrasonic treatment. In addition, the liposomes of the HGF gene of the present invention can also be used as appropriate membrane fusion liposomes for f-toxin fusion. In this case, it is better to inactivate the virus by, for example, ultraviolet rays. As a particularly good membrane fusion liposome, a membrane fusion liposome fused with Sendai disease mother (Japanese hemagglutinating virus: HVJ) can be mentioned. This membrane fusion liposome can be used in Nikkei Science, April 1944, pages 32-38, j.

Bwl Chem ·, 266 (6), 3 3 6 1 -3 3 64 (1 99 1) and other methods, for example, mixing inactivated purified HVJ with ultraviolet rays and the liposome suspension of the HGF gene carrier After slowly dropping, unbound HVJ is removed by oral sucrose density gradient centrifugation to prepare hvj fusion liposomes (HVJ-liposomes). Furthermore, binding the target cells with liposomes can increase the efficiency of gene introduction into human cells. Examples of the target having affinity include ligands such as antibodies and receptors. As a method for introducing HGF gene into human cells, it can be largely divided into those who use viral vectors and others (Yuejing Science, April, 1994, pp. 20-45, Monthly Pharmacy '36 (1) '23- 48 (1 994), and the cited documents-11-This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 male fluorescent) 1236373

Etc.) Any method can be applied to the medicine of the present invention. As a method of introduction by a viral vector, for example, a retrovirus, an adenovirus, an adeno-associated virus, a rash virus, a vaccinia virus, a polio virus, a Simbis virus, or other RNA viruses can be mentioned. Incorporate the method of H GF gene introduction. Among them, methods using retroviruses, adenoviruses, and adeno-associated viruses are particularly preferred. As the other introduction method, a liposome method, a liposin method, a microinjection method, a calcium phosphate method, an electrophoretic pulsation method, and the like are mentioned, and the liposome method is particularly preferable. In addition, in fact, the HGF gene is used as a medicine for the in vivo method of directly introducing the HGF gene in vivo, and a certain cell is taken from a human, the HGF gene is introduced into the cell in vitro, and the cell is returned to the body. In vitro method (Jingjing Science, April 1, 994, pp. 20-45, Monthly Pharmaceutical Affairs, M1U, 2 3 -48 (1 994), and references cited above). In the medicine of the present invention, any suitable method may be selected according to the disease to be treated, the target organ, and the like. Compared with the Ex Vivο method, the in vivo method is less expensive and less time consuming, and is simpler. In vitro method, H GF gene is efficiently introduced into cells. When the medicine of the present invention is administered by the In Vivo method, it can be administered according to an appropriate administration route for the disease to be treated, the target organ, and the like. For example, it can be administered to veins, arteries, subcutaneously, intramuscularly, etc., or it can be directly administered to the target part of diseases such as kidney, liver, lung, brain, nerve, etc. If directly administered to the diseased part, the organ can be selectively treated. For example, the use of gene therapy "for restenosis after rp T c A" can be performed by intra-arterial administration (Experimental Medicine '12 (15 Supplement) '1 298-1 993 (1 994)), the present invention is preferably- 12- This paper size is in accordance with Chinese National Standard (CNS) A4 (210X 297 mm) 1236373 A7 1 p --- —__ B7_ V.)--Apply the medicine to the ball end of p τ c A and apply Blood vessels can thus be introduced into vascular endothelial cells and vascular smooth muscle cells. Also, in the case of ex vivo methods, according to customary usage, human cells (such as lymphocytes, hematopoietic stem cells, etc.) are taken, sensitized with the medicine of the present invention, and after the gene is introduced, the HGF-producing cells are returned. Humanity. In the case of in vivo administration, various preparation forms (such as liquid preparations) can be taken, but injections containing H GF gene as an active ingredient are generally used. , Press ,,?, If necessary, you can also add the usual carrier. The injection can be prepared according to the custom. / For example, the HGF gene can be dissolved in a suitable solvent (for example, sterilized, buffered solution, physiological saline, etc.), then filtered and sterilized with a filter membrane, and then filled in It can be prepared in a sterile container and replaces the hgf gene.

A viral vector incorporating the HGF gene was formulated. In addition, liposomes (or H JV-liposomes) in which hgF genes are embedded can be made into the form of liposome preparations such as suspensions, refrigerants, and concentrated refrigerants by centrifugation. The content of the HGF gene for making money can be suitably prepared according to the disease to be treated, the target organ, the patient's age, weight, etc., but it is usually used as the hgf-based Q with 0.0001 ¾ ~ 100 mg, preferably 0001 亳 g ~ 10 mg, it is appropriate to administer this once a few days to several months. Example = Two, the present invention will be described in more detail with reference to the examples, but the present invention is not limited to the example of the temple. The experimental materials and methods used are outlined below. Materials and methods for inspection ① H GF gauge. Preparation of carrier GF manifestation 'By manifestation carrier-13- Chinese paper standard (CNS) for paper size-1236373 A7 B7 11 V. Description of invention 1JJ_, 6 1 -66 (1 993)) inserting human HGF cDNA (2.2 kb: Biochem. Biophys. Res Commun ·, 172 · 32 1 -327 (1 990) between Eco RI and Not I sites; Japanese Patent Laid-Open No. 5-1 1 1 3 8 3). In this plastid vector, the transcription of the HGF cDNA is controlled by the SRa promoter (Nature 342 · 4 40-4 4 3 (1 9 8 9)). ② Coronary artery endothelial cells from cell cultured rats were digested with enzymes from the hearts of Sprague-Dawley (SD) rats at 8 weeks of age and separated by density gradient centrifugation (Transplantation 5 7, 1653-1660 (1994) ). Arterial smooth muscle cells of rats (hereinafter referred to as VSMC) were obtained from SD rats treated with enzymes at 12 weeks of age (J. Clin. Invest., 9 3, 3 5 5 -3 60 (1 994)). These cells were maintained in DMEM medium containing 10% (vol / vol) bovine fetal serum, penicillin (100 units / ml), and streptavidin (100 micrograms / ml). The cells were cultured at 37 ° C, 95% air-5% (: 02) in warmed atmosphere, and the culture medium was exchanged every 2 days. These cells showed that they were endothelial cells and morphological observations, respectively. Smooth muscle cells. Human aortic endothelial cells (fifth generation) and human VSMC (fifth generation). Those purchased from Clapow (Ximajinshui) Company, using the method described above, containing 5% bovine fetal serum, epithelium Growth factor (10 ng / ml), bacterial fibroblast growth factor (2 ng / ml), and dexamethasone (1 μM) were cultured in MCDB131 medium. In addition, endothelial cells in the stationary phase were cultured in accordance with J. Clin Invest. 8 6, 1 690- 1 697 (1 990); same as above, 2_1, 8 2 4 · 8 2 9 (1 9 9 4) -14- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1236373 A7 B7 V. Description of the invention (12) Preparation. Gene-sensitized endothelial cells or VSMCs are introduced into the plastid camp, and 108 seeds are sown in 6 well selection dishes. Proliferate to 80% confluent culture. Cells are treated with a balanced salt solution containing 2 mM chloride chloride (137 mM NaCl, 5.4 mM K C1, 10 mM TriS_HCl, pH 7.6, hereinafter referred to as "BSS"), washed three times, and added the HVJ liposome-DNA (containing 2.5 g of lipid and 10 micrograms of embedded DNA) obtained in Example 1 1 ml of the solution or 1 ml of the HVJ-liposome_c nt solution obtained in Comparative Example 1 was cultured at 4 ° C, 5 minutes, and 37 ° C, 30 minutes. The cells were washed and contained 10% bovine serum The fresh medium was maintained in a C02 incubator. 浓 HGF concentration in endothelial cells and VSMCH The measurement of HGF concentration in sensitized endothelial cells and VSMCs was performed by ELIS A method. That is, rats or humans Endothelial cells or VSMCs were seeded in 6-well culture dishes (manufactured by Corning) at a cell density of 5 X 104 cells / cm 2 and cultured for 24 hours. After 24 hours after sensitization, the medium was exchanged and cultured again 48 hours. In order to review the release of HGF, the sensitized cells (after 48 hours of sensitization) were washed and added to the cells containing insulin (5 X 1 (Γ7 Μ), transferrin (5 μg / ml) and ascorbic acid ( 0.2 mM) in serum-free medium. After 24 hours, the medium was collected, centrifuged at 600 g for 10 minutes, and stored at -20 ° C. The concentration of HGF in the culture medium, using anti-rat HGF antibody or anti-human-15- [This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) ^ 1236373 A7 'B7 V. Description of the invention (13 ) Enzyme immunoassay for HGF-like antibodies (Exp. Cell. Res. 210, 3 26-3 3 5 (1 994); Jpn. J. Cancer Res. Mad, 1262-1266 (1992)). The anti-rat or anti-human HGF IgG was spread on a 96-well culture dish (manufactured by Corning) at 4 ° C for 15 hours. After blocking with PBS (phosphate buffered saline) containing 3% bovine serum albumin, add medium to each well and incubate at 25 ° C for 2 hours. The holes were washed 3 times with PBS (PBS-Thun) containing 0.05% Tween (Built-Torn), and then biotinylated anti-rat HGF IgG or anti-human HGF IgG was added. Incubate at 25 ° C for 2 hours. After washing with PBS-Thun, the holes were cultivated with Western camellia, peroxidase-binding streptavidin-biotin complex (p BS-Thun solution). Enzyme reactions were started by adding a matrix solution (containing 2.5 mM o-phenylenediamine, 100 mM sodium phosphate, 50 mM citric acid, 0.001% 5% hydrogen peroxide). The enzyme reaction was stopped by adding 1 M H2S04, and the absorbance at 490 nm was measured. Moreover, the anti-human HGF antibody only cross-reacts with human HGF and does not react with rat HGF, and the anti-rat HGF antibody only cross-reacts with rat HGF and does not react with human HGF.

⑤ Recombinant human and rat used for HGF H GF was purified from culture fluid containing CΗ 〇 cells or C-1 2 7 cells containing human or rat H GF cDN Α (Cell, 77_5 26 1 -27 1 (1 994); J. Clin. Invest, 9 3. 3 5 5 -3 60 (1994)) 0 ⑥ Statistical analysis of all experiments performed at least 3 times, the measured value is the mean ± standard error -16-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1236373 A7 '_______ B7 V. Description of the invention (14). Statistical analysis of measured values was performed by Duncan's test. Hematoxylin and eosin (HE) staining, HGF gene transduction in Azabuchi rats 10 days after gene introduction 'perfused with normal saline with heparin, slaughtered' and then prepared by p B s 4% polyacid is fixed overnight. After fixation, paraffin was embedded, and sections were made, and HE staining and Azhan staining were performed according to the usual method. Count the microvessels under the microscope. Example 1 HVJ-liposome containing HGF expression morphology In tetrahydrofuran, the phosphoric ester amidinoserine, phosphoryl amidylcholine, and cholesterol were mixed at a weight ratio of 1: 4 · 8: 2. Tetrahydrofuran was distilled off on a rotary evaporator to allow this lipid mixture (10 g) to precipitate out of the container wall. 96 micrograms of HMG 1 nucleoprotein (high fluidity group 1 nucleoprotein) purified from bovine thymus was mixed with a BSS (200 microliters) solution of plastid DNA (300 micrograms) at 200 for 1 hour, then added the above Of lipids. The lipid i complex suspension was mixed by vortexing, ultrasonically treated for 3 seconds, and stirred for 30 minutes. The refined ΗV J (Z strain) was inactivated by ultraviolet irradiation (丨 丨 〇 格 / square pen meter seconds) for 3 minutes before the salt was used. The liposome suspension (0.5 ml containing 10 mg of lipid) obtained above was mixed with HVJ (20,000 hemagglutination units) and BSS to make 4 ml 'of the whole liquid. The mixture was incubated at 4 for 10 minutes, and then stirred at 37 C for 30 minutes. Unfused η V J was removed from HVJ-liposomes by sucrose density gradient centrifugation. That is, η VJ-liposomes containing HGF expression carrier (including 丨 〇 克 mill Mill-17) were collected on the upper layer of the sucrose density gradient, and this paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) ) ''-1236373 A7 '___B7 V. Description of the invention (15) HGF expression carrier in liters). The HVJ-liposome containing the HGF expression vector is hereinafter referred to as HVJ-liposome-DNA. Example 2 Preparation of all the HVJ-liposomes of the vector in Table 1 for the preparation of HVJ-liposomes containing HGF expression vectors in rats, using 64 micrograms of HMG 1 nucleoprotein, 200 micrograms of plastid DNA, according to the examples The method described was performed. In addition, 0.5 ml of liposome suspension containing 10 mg of lipid) and HVJ (3 5,000 hemagglutination units) plus BSS were added to make a total liquid volume of 2 ml and mixed. For SD rats (400-500 g; purchased from the Japanese company Charles Riba (XXI)), pentobarbital sodium (0.1 ml / 10 mg) was administered intraperitoneally. , Keep warm, ensure breathing by automatic respirator. Rats underwent left thoracotomy with a 30G injection needle to inject HVJ-liposome_DNA or HVJ-liposome-cont (20 μl) directly and carefully into the apex. Comparative Example 1 (Production of HVJ-lipid body containing HGF expression vector) HVJ-liposome without HGF expression vector was produced in the same manner as described in Experimental Example 1 on a vector containing no HGF gene. Hereinafter, the HVJ-liposome containing no HGF expression vector will be referred to as HVJ_liposome_cont ° Comparative Example 2 Human TGF-β expression vector-containing VJ · liposome Preparation and implementation of human TGF-β expression vector Example i A HVJ-liposome containing a human TGF-β expression vector was produced in the same manner. -18- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1236373 A7 ^ B7 V. Description of the invention (16 ^ — HVJ-liposome containing human TGF-β expression carrier is hereinafter referred to as HVJ -Liposome-DNACTGF-β). Test Example 1 HVJ-liposome-DNA-sensitized rat coronal facial movement-ancient skin fine makeup> HGF bombardment HVJ-liposome-DNA (HGF expression carrier concentration in liposomes: 10 micrograms / Ml) was sensitized in rat coronary artery endothelial cells (cell number: 108), and the amount of HGF produced was measured by ELISA. As a control, the test described above was performed using HVJ_liposome-cont. HGF production was also measured in non-sensitized coronary endothelial cells (untreated group). The results are shown in Figure 1 (n = 6). In the figure, HGF is a coronary artery endothelial cell population of rats sensitized with HVJ-liposome_DNA. As shown in Figure 1, HVJ-liposome-DNA-sensitized rat endothelial cells produce high levels of H G F secretion. In contrast, the non-treatment group and the Η V J -liposome_ c ο n t sensitized coronary artery endothelial cell group have not been found to produce substantially HGF. Test Example 2 Effect of sensitized HGF expression carrier on endothelial sprint proliferation HVJ-liposome-cont was sensitized in human endothelial cells in the presence of exogenously added recombinant human HGF (1, 10, and 10) (0 nanograms / ml), or cultured in the absence, and the increase rate (%) of the number of cells was measured. The results are shown in FIG. 2 (line chart) (n = 6). In the figure, the DSF table expresses HVJ-liposome-cont-sensitized endothelial cell population, and the monarch sheep cultured in the presence of recombinant human η GF at the concentration specified in the HGF table. (* ·· Ρ < 0 · 05, **: Ρ < 〇 · 〇ΐ (for DSF)) 0 -19- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1236373

As shown in the line chart of Fig. 2, it is understood that exogenous addition of H G F promotes endothelial cell proliferation. On the one hand, endothelial cells sensitized with HVJ-liposome-DNA (concentration: 10 µg / mL) were cultured, and the increase in the number of cells was measured to determine the increase rate (%). As a control, endothelial cells sensitized with HVJ-liposome- 体 ... were cultured, and the increase in the number of cells was measured to determine the increase rate (%). The results are shown in Figure 2 (bar graph) (n = 6). In the figure, dsf indicates the HVJ-liposome-cont-sensitized endothelial cell population, and HGF vector indicates the HVJ-liposome-DNA-sensitized endothelial cell population (**: ρ < 0.01 (for DSF), #: ρ < 〇 · 〇5 (for HGF, 100 μg / ml). As shown in the bar graph of FIG. 2, it is clear that the increase rate of endothelial cells sensitized by HVJ-liposome_dnA is similar to that of the control group. The ratio is significantly higher, and the effect of exogenously added HGF is also intentionally higher. In addition, the endothelial cells sensitized by VJ-liposome-D Ν Α, in the presence of rabbit anti-human HGF antibody The measurement was performed in the presence or absence, and the increase in the number of cells was measured to determine the increase rate (%). As a control, HVJ_liposome_cont-sensitized endothelial cells were cultured, and the increase rate (%) of the number of cells was determined in the same way. In addition, rabbit anti-human HGF antibody was purified by the method described in the literature (Jpn · j · cancer Res, ϋ, 1 262-1 266 (1 992)). This antibody can be purified at a concentration of 10 μg / ml. 〇 Nanograms / ml of biological activity neutralization. In addition, anti-human HGF antibodies only cross-react with human η GF, and do not react with rat HGF 'anti-rat HGF The body only cross-reacts with rat HGF and does not react with human HGF. In addition, normal rabbit serum I g G (10 μg / ml) was used as a control group. -20- This paper standard applies to China National Standards (CNS) A4 specification (210X297 mm) 1236373 A7 B7 V. Description of the invention (18 The results are shown in Figure 3 (n = 6). In the figure, the comparison table shows HVJ-liposome-cont-sensitized endothelial cells cultured in the presence of IgG controls. Cell populations; HGF represents HVJ-liposome_DNa-sensitized endothelial cell populations cultured in the presence of IgG controls; HGFab represents jjvj-liposome-DNA-sensitized endothelial cell populations cultured in the presence of anti-human HGF antibodies. Also, The increase rate (%) is expressed as the relative% of the increase rate of the control 100 (*: P < 0. 〇1 (for the control), #: Ρ < 0 · 05 (for the HGF). As shown in FIG. 3 'Due to the presence of anti-human HGF antibodies, the proliferation of η ν j _ liposome-DN A-sensitized endothelial cells is inhibited, which is the same rate of cell increase as the control. It is clear that HGF is a proliferation factor of endothelial cells. Test Example 3 iiYJ-liposome-DNA sensitized rat VSMCs were cultured with slag. The HVJ-liposome-DNA-sensitized rat VSMC culture supernatant was added to the rat coronary artery endothelial cell culture line (cell number · 105) in a stationary phase, and cultured for 3 days. Increase in the number of endothelial cells. As a control ', an increase in the number of endothelial cells was examined similarly using a culture supernatant of rat Vs-MC sensitized with VJ-liposome-cont. The results are shown in Figure 4 (11 = 6). In the figure, the control is a group of culture supernatants of rat VSMC sensitized with HV plant liposome-^ ...; 11 (317 is a culture supernatant of rat VSMC with HVJ_liposome-DNA sensitization added As shown in Figure 4, in the group of culture supernatants of rat VSMCs sensitized with HVJ_liposome_DNa, the number of endothelial cells can be increased intentionally 0 -21-1236373 A7 '___ B7 _ V. Description of the invention (19) The HGF concentration of the culture supernatant of HVJ-liposome-DNA or HVJ-liposome-cont-sensitized rat VSMC, to use anti-human HGF antibodies and anti-rat HGF The antibody was measured by ELISA method. HGF concentration was also measured in the culture supernatant of non-sensitized VSMC (without treatment group). The measurement results using anti-human HGF antibodies are shown in Figure 5, and the results using anti-rat HGF antibodies Figure 6 (n = 6 for any one). In the figure, the control is a culture supernatant group of rat VSMC sensitized with HVJ-liposome-cont; HGF is sensitized with HVJ-liposome-DNA The culture supernatant group of rat VSMC. As shown in Fig. 5, HGF was detected in the culture supernatant of rat VSMC sensitized with HVJ-liposome-DNA, and its value phase It was intentionally high in the control. As shown in FIG. 6, rat HGF was also detected in the culture supernatant of HVJ-liposome-DNA-sensitized atmospheric VSMC, and its value was intentionally compared with the control. In addition, as shown in Fig. 5 and Fig. 6, in the untreated group and the control group, there was no HGF in the culture supernatant to the extent that it can be measured by the ELIS A method. Test Example 4 A sensitized rats were in the state Intra-arterial ancient fine_ > Effects of Zhengye solution on rat coronary artery endothelial cellsHVJ-liposome-dna-sensitized rat coronary artery endothelial cell culture supernatant was added to the rat coronary artery endothelium at rest In a cell culture line (105 cells), the increase in the number of endothelial cells was investigated after 3 days of culture. As a control, the culture of rat coronary artery endothelial cells sensitized with HVJ-liposome_cont was used. Serum, similarly investigated the increase in the number of endothelial cells -22- i Paper size applies Chinese National Takaoka Standard (CNS) 8 4 ^ (210 > < 297 mm)-1236373 A7 1 __B7 _ V. Invention Explanation (20) plus. The results are shown in Figure 7. In the figure, a is the addition of rats that sensitized HVJ-liposome_DNA. Group of culture supernatants of coronary endothelial cells (n = 8); B is a group of culture supernatants of rat coronary artery endothelial cells sensitized with HVJ-liposome_cont (n = 8); C It is a non-treatment group (n = 15). As shown in FIG. 7, the number of endothelial cells can be intentionally increased by adding a culture supernatant of coronary artery endothelial cells sensitized with HVJ-liposome-DNA. Compared with the control group, the number of cells was the same as that of the non-treated group. (Control group: 0 · 1 1 7 ± 0 · 〇〇2; group a: 0 · 1 4 8 ± 0 · 0 3, P < 0 · 01) ° Second, the HVJ-liposome_DNA was sensitized Anti-HGF antibody was added to the culture supernatant of rat coronary artery endothelial cells, and the increase in the number of endothelial cells was investigated in the same manner as described above. The result is not as shown in FIG. 8 (n == g). In the figure, A is a group added with culture supernatant of rat coronary artery endothelial cells sensitized by HVJ-liposome; B is a culture group added with rat coronary artery endothelial cells sensitized by HVJ-liposome-cont The group of serum; C is the group with anti-HGF antibody added to the culture supernatant of VJ-liposome_ DNA-sensitized rat coronary endothelial cells; D is the group of J-VJ-liposome-DNA sensitive A control antibody group was added to the culture supernatant of the rat's spinal artery endothelial cells. As shown in FIGS. 8A and 8C, the cell proliferation-promoting activity of the culture supernatant of rat arterial endothelial cells sensitized with HVJ-liposome-dna completely disappeared by adding anti-H G F antibody. From this, it is understood that the cell proliferation-promoting activity of the culture supernatant of rat coronary artery endothelial cells sensitized with η v j _ liposome-DN A was attributed to H G F. -23- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1236373 A7 1 B7 V. Description of the invention (21) Test example 5 Human VSMC sensitized with HVJ-liposome-DNA to human ancient The effect of 晌 晌 is seeded on a human VSMC cell culture insert (made by Kester (Yiyi), with a pore diameter of 0.45 micrometers), and 10% bovine serum in DMEM medium is added for proliferation. In one aspect, human endothelial cells were seeded in 6-well culture dishes and maintained in DMEM medium supplemented with 10% bovine serum. When VSMC reaches 80% content, incubate with HVJ-liposome-DNA (DNA content in liposome: 10 micrograms) or HVJ-liposome-cont for 5 minutes at 4 ° C, and then incubate at 37 ° C for 30 minutes. Minute. After sensitization, a plate containing sensitized VSMC was added to the pores of human endothelial cells containing stationary phase. VSMC and endothelial cells were co-cultured in DMEM medium containing 0.5% bovine serum for 3 days, and the cell number was measured using a WST-cell number measurement kit (manufactured by Wako Corporation). The results are shown in Fig. 9 (n = 6). In the figure, the control is a co-culture group with HVJ-liposome_cont-sensitized VSMC; HGF is a culture supernatant group of HVJ-liposome-DNA-sensitized VSMC. As shown in FIG. 9, it is understood that V J -liposome-DNA sensitized human V S M C intentionally increases the proliferation of non-sensitized human endothelial cells in the stationary phase. Test example 6 HVJ-liposome-DNA-sensitized rat VSMC # Coronary toughness of rats shows the effect of endothelial cells 旲 HVJ-liposome-DNA-sensitized rat VSMC (cell number: 108) and stationary phase Coronary Artery Endothelial Cells in Rats (Number of Cells: 105) and -24-This paper size applies to Chinese National Standard (CNS) A4 (210X 297 mm) " '— 1236373 A7' __ B7 V. Invention Note (22) The same culture was performed for 3 days, and the increase of the coronary endothelial cells was investigated. As a control, HVJ-liposome-cont-sensitized rat VSMC was used, and co-cultured in the same manner to investigate the increase in the number of endothelial cells. The results are shown in Fig. 10 (n = 6). In the figure, HGF is a VSMC group of rats sensitized with HVJ-liposome DNA, and a control is a VSMC group of rats sensitized with HVJ-liposome-cont. As shown in Figure 10, the proliferation of endothelial cells was stimulated by HGF released from HVJ-liposome-DNA-sensitized rat VSMCs, and an increase in the number of cells was confirmed (control group: 0.126 ± 0.06, HGF Group: 0.156 ± 0.01 (P < 0.05). Test Example 7 Proliferation of liposome_DNA-sensitized rat VSMC HVJ-liposome-DNA-sensitized rat VSMC and HVJ-liposome-cont-sensitized rat VSMC were cultured separately to review the increase in the number of cells However, the sensitization of HVJ-liposome-DNA had no effect on cell proliferation. Since then, it has been understood that HGF has no cell proliferation promoting activity against VSMC. Test Example 8: Neovascularization in rat myocardium directly injected with HVJ-liposome-DNA i Rat myocardium directly injected with HVJ-liposome-DNA, rat myocardium directly injected with VJ-liposome-c ont and Myocardium of untreated rats was stained with HE and Azhan, and the number of microvessels was counted with a microscope. The results are shown in Figure 11. The HGF in the picture is the rat myocardium directly injected with HVJ liposome-DNA-25. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1236373 A7 '_______ B7 V. The description of the invention (23) is small The number of blood vessels, the control is the number of microvessels of rat myocardium directly injected with HVJ-liposome_cont. As shown in Figure 11, HVJ_liposomal DNA-infused rat myocardium, and / or host HVJ-liposomal_cnt myocardium and non-treated rat myocardium are more thoughtful than 乂Increase the number of tiny blood vessels. This shows that HGF, which has an endothelial cell proliferation effect, has an angiogenesis effect in the living body. Test Example 9-Liposome-Γ) ΝA joint joint cheekbone repair hair 10 weeks old Fei (7 4 7 eve ten-) rat femoral interosseous region, using 1.8 mm Kirch (Feng Xiaoshi 21) Steel wire penetrates the subchondral bone to make injuries. HVJ-liposome-DNA (100 microliters / knee) prepared in Example 1 was directly introduced into the joint at a time point of 1 week after the operation. As a control, HVJ-liposome_cont produced in Comparative Example 1 and ΗV J-liposome-d N A (T G F-β) produced in Comparative Example 2 were administered to the joint in the same amount. One, three, and four weeks after the introduction of these genes, the rats were sacrificed and the repair site was observed histologically. The results are shown in Fig. 12. Three weeks after the administration of ηVJ-liposome-DNA into the joint, it was confirmed in a part of the repaired tissue that the cartilage-like cells identified as synthesizing protein polymerase stained with toluidine blue were synthesized. appear. As shown in FIG. 13, 4 weeks after the administration of η VJ_liposome-DN A into the joint, the tendency of the appearance of the chondrocyte-like cells identified as a protein synthetase to expand was confirmed. As shown in Figure 14, the HVJ-liposome produced in Comparative Example 2 -26- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1236373 A7 1 B7_ V. Description of the invention (24) When DNA (TGF-p) is administered into the joint, the appearance of such chondrocytes cannot be confirmed 4 weeks after the administration. As shown in FIG. 15, in the case where HV J-liposome-cont produced in Comparative Example i was administered into the joint, the appearance of such chondrocytes could not be confirmed 4 weeks after the administration. Test Example 10 The expression of HGF in rat coronary arterial endothelial cells sensitized with adenovirus containing rat HGF DNA Adenovirus containing rat HGF DNA (RIKEN GENE BANK: Adexl CA rat HGF) was infected with 293 cells from human fetal kidneys, and a high-valence virus solution was obtained 2 to 3 days later (Experimental Medicine, 1 804 to 1 8 1 0 (1 994)). The obtained virus solution 1 to 5 X 108 pfu was sensitized in rat coronary arterial endothelial cells (cell number: 108) with MO II to 5 and the amount of HGF produced was measured by ELISA method. As a result, rat coronary artery endothelial cells sensitized with adenovirus containing DNA of rat HGF produced high levels of H G F secretion. Industrial Applicability Compared with the administration of HGF itself, the medicine of the present invention has a sustained therapeutic effect, and can selectively act locally, thereby reducing the side effects of HGF. -27- This paper is suitable for standard paper ® Standards (CNS) Α4 size (21GX297 / ^ jy

Claims (1)

Patent No. 123 6373i〇4825 for Chinese Patent Application Replacement (October 1993) 8 8 8 8 ABCD Ιο ο > 々, Application Patent Scope 1. A pharmaceutical composition for treating arterial disease, which is A pharmaceutical composition for administration to a muscle using an expression vector containing an HGF gene as an active ingredient. This paper size applies to China National Standard (CNS) Α4 (210 X 297 mm)