HK1085948A - Combined use of g-csf with angiogenetic factor - Google Patents

Description

Combination of G-CSF with factors having a revascularization effect

Technical Field

The present invention relates to a therapeutic agent for ischemic diseases, i.e., a composition for treating ischemic diseases, which contains, as active ingredients, a granulocyte colony stimulating factor (G-CSF) and a factor having a revascularization activity.

Background

The present invention relates to an agent for treating ischemic diseases, and first, description will be given of arteriosclerosis obliterans, which is one of typical ischemic diseases.

Occlusive atherosclerosis is a disease in which atheromatous substances mainly composed of fat are deposited on the intima of arterial walls due to arteriosclerotic (atherogenic) lesions, and an occlusion or stenosis is produced in the limbs, particularly in the main arteries of the lower limbs, and the periphery thereof produces ischemic disorders. The clinical symptoms include cold or paralysis, intermittent claudication, pain at rest, ulcer and necrosis. About 10 ten thousand patients with occlusive arteriosclerosis have been reported in Japan (Yu Fu, therapeutics 31, 289 page 292; 1997), and it is expected that the population of the elderly will increase and the European beautification of food will increase in the future.

As a method for treating occlusive arteriosclerosis, exercise therapy, drug therapy, vascular repair, etc. can be performed depending on the symptoms, the state of the patient, and the like. In addition, at present, in order to avoid cutting of the critical ischemic limb, a revascularization therapy (gene therapy, autologous bone marrow cell transplantation, etc.) for promoting revascularization has been studied. These therapies have achieved certain results for the treatment of arteriosclerosis obliterans, but each of them has the following problems.

Exercise therapy is an example in which the effect of extending walking distance is found in mild cases, but the effect of this therapy is difficult to predict. In addition, it has been reported that even if patients having the effect of extending the walking distance are not satisfied and the vascular repair is desired, 30% (Taitian, Japanese medical news report, 3935, 25-29, 1990) of the cases, it cannot be said that the treatment is very effective.

As for the drug therapy, the antiplatelet agent as a main prescription can only reach the degree of preventing the worsening of the disease. Even the recently developed agents for improving microcirculation blood flow and improving oxygen transport ability are expected to be applied to only mild cases. The current situation is that no medicine for fundamentally treating arteriosclerosis obliterans is.

In contrast, vascular repair is currently the most effective treatment, and percutaneous angioplasty or bypass surgery is performed depending on the symptoms, lesion site or range of the patient. However, these treatments are large-scale treatments accompanied by surgery, and there are problems such as the failure to expect long-term survival in addition to complications and death accompanied by surgery.

Gene therapy using a revascularization factor is a therapeutic method for improving ischemia by allowing collateral blood vessels to develop. As the angiogenesis factor, Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor (FGF), and the like are known. Clinical studies using human HGF are being conducted in japan. Intramuscular injection of lower limb muscles using HGF plasmids has been studied for cases of critical limb ischemia, and its effect is expected. However, the method has not been widely used because the method has been limited to experimental treatment range and the evaluation of safety and effect has not been fixed.

In addition, intramuscular transplantation of autologous bone marrow cells, which has recently been attracting attention, is a method of treating a disease by transplanting bone marrow cells into muscles near a lesion and differentiating and forming blood vessels in vascular endothelial cells. The autologous bone marrow cell transplantation was confirmed to have no adverse effect on the immune system, and to have differentiation of bone marrow cells into endothelial cells and an increase in the number of blood vessels in an animal model. In the future, it is necessary to increase the number of cases and evaluate the effect, but the evaluation is expected as a future treatment method from the viewpoint of treating severe cases as well. However, since bone marrow is collected under general anesthesia in clinical practice, it is considered to be one of the problems associated with increased burden on patients and medical staff.

Recent studies have revealed that hematopoietic stem cells capable of differentiating into vascular endothelial cells exist not only in bone marrow but also in peripheral Blood (referred to as "endothelial cell precursor cells" from the viewpoint of the function of differentiating into endothelial cells, but since the cells are derived from hematopoietic stem cells, the term "hematopoietic stem cells" is used in the present specification in the concept of cell colonies capable of converting into endothelial cells), and this has been found to be involved in vascular regeneration (Qun Shiet al, Blood, Vol.92, 362-Alpha 367, 1998; Takayuki Asahara et al, Circulation Research, vol.85, 221-Alpha 228, 1999; Mario Peichev et al, Blood, vol.95, 952-958, 2000). Therefore, it is expected that occlusive arteriosclerosis can be treated by collecting hematopoietic stem cells in peripheral blood and transplanting them into muscles near the lesion. In this case, although there is an advantage that the burden on patients and medical staff in collecting peripheral blood stem cells is smaller than that in transplanting stem cells in bone marrow, the frequency of hematopoietic stem cells in peripheral blood is generally extremely low, and therefore there is a great question as to whether a sufficient amount of hematopoietic stem cells necessary for treating arteriosclerosis obliterans can be obtained.

Human G-CSF is a hematopoietic factor found as a differentiation growth factor of granulocyte-based hematopoietic precursor cells, and is clinically useful as a therapeutic agent for neutropenia after bone marrow transplantation or cancer chemotherapy because it promotes neutrophil hematopoiesis in the body. In addition to the above-mentioned effects, human G-CSF acts on hematopoietic stem cells to stimulate their proliferation and differentiation and mobilize hematopoietic stem cells in bone marrow into peripheral blood. In practice, peripheral blood stem cell transplantation, in which peripheral blood hematopoietic stem cells mobilized by human G-CSF are transplanted, is performed in clinical practice for the purpose of promoting hematopoietic recovery in cancer patients after intensive chemotherapy based on the latter effect. This mobilization of hematopoietic stem cells by G-CSF is much stronger than GM-CSF, which is also a granulocyte hematopoietic factor. Also, G-CSF is superior to GM-CSF in that it has fewer side effects.

HGF is a protein produced by various mesenchymal cells and targeting most epithelial cells, neurons, endothelial cells, and a part of mesenchymal cells. It is known that the cell growth-promoting activity and the cell motility-promoting activity and the epithelial morphogenesis (lumen structure, etc.) -inducing activity are possessed. From the viewpoint of the function as an organ regeneration factor for promoting the regeneration of the kidney, lung, and digestive tract, which are represented by the liver, in an adult, it is expected as a therapeutic agent for organ diseases.

Disclosure of Invention

Since the frequency of hematopoietic stem cells in bone marrow is expected to increase by administering G-CSF before a treatment for intramuscular transplantation of bone marrow cells is first performed on a patient with arteriosclerosis obliterans, the number of bone marrow punctures at the time of collecting bone marrow cells can be reduced, and the burden on the patient can be reduced. In this case, by obtaining transplanted hematopoietic stem cells from peripheral blood, the burden on the patient and the medical staff can be further reduced. Furthermore, it is presumed that the hematopoietic stem cells in peripheral blood contribute to angiogenesis, and that the administration of G-CSF increases the number of hematopoietic stem cells in peripheral blood to promote angiogenesis. Therefore, it is expected that G-CSF alone can be administered to a patient to treat arteriosclerosis obliterans. Treatment of occlusive arteriosclerosis by administration of G-CSF is clearly capable of greatly reducing the burden on patients and medical workers in terms of no need to collect or transplant hematopoietic stem cells.

Furthermore, the therapeutic effect is expected to be increased by gene therapy using a revascularization factor in combination. That is, it is predicted that the additive or synergistic effect of the angiogenesis action is obtained by acting G-CSF on hematopoietic stem cells to promote growth and differentiation thereof, mobilizing hematopoietic stem cells in bone marrow into peripheral blood to promote vascularization (vasculogenesis), and promoting angiogenesis (angiogenesis) by HGF or FGF, and by exerting different actions of both.

The treatment of arteriosclerosis obliterans using G-CSF can be expected to have an effect even in severe patients, and thus brings great benefit to patients, but when used in combination with a factor having an angiogenic effect such as Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor (FGF), or a gene therapy thereof, which promotes the differentiation and growth of vascular endothelial precursor cells, the therapeutic effect is expected to be increased. In this case, the above-mentioned factor or its gene may be administered to the patient, for example, in the vicinity of the lesion. Similarly, it is expected that the therapeutic effect of the present invention will be enhanced by using the present invention in combination with an antiplatelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, a hyperlipemia therapeutic agent, and the like, which are clinically used as a pharmacotherapy for arteriosclerosis obliterans.

As a result of the above studies, the present inventors have found that administration of HGF or FGF as a revascularization factor together with G-CSF can achieve a particularly high improvement effect in ischemic vessels, and have completed the present invention.

Accordingly, the present invention provides a therapeutic agent for ischemic diseases, which comprises G-CSF and HGF or FGF as active ingredients.

The therapeutic agent of the present invention is also applicable as a therapeutic agent for the following diseases, which are ischemic diseases. That is, the present invention provides a therapeutic agent for trauma, rejection during transplantation, ischemic cerebrovascular disorders (e.g., cerebral apoplexy and cerebral infarction), ischemic renal diseases, ischemic pulmonary diseases, ischemic diseases associated with infection, ischemic diseases in the limbs, ischemic cardiac diseases (e.g., ischemic myocardial diseases, myocardial infarction and ischemic heart failure) and the like, which contains G-CSF and HGF or FGF as active ingredients.

Further, the present invention provides a therapeutic agent for arteriosclerosis obliterans.

The present invention also provides an angiogenesis agent or a muscle regeneration agent.

Drawings

FIG. 1 is a graph showing the effect of administration of physiological saline (control), HGF, G-CSF and HGF + G-CSF, respectively, to mice with left foot ischemia on the weight ratio (%) of lower limb muscles of the mice.

FIG. 2 is a graph showing the effect of administration of physiological saline (control), HGF, G-CSF and HGF + G-CSF, respectively, to mice with left foot ischemia on the lower limb blood flow ratio (%) of the mice.

FIG. 3 is a schematic representation of the effect of administering physiological saline (control), HGF, G-CSF and HGF + G-CSF, respectively, to mice with left foot ischemia on the blood flow rate of the mice. The red portion is the highest speed portion, and the low speed portions are represented in the order of yellow, green, and blue.

In FIG. 4, A is a fluorescent micrograph showing the effect of administration of physiological saline (control: HGF-, G-CSF-), HGF plasmid (HGF +, G-CSF-), G-CSF (HGF-, G-CSF +), G-CSF + HGF plasmid (HGF +, G-CSF +) to mice with left foot ischemia, respectively, on the ratio of the number of GFP positive cells in the mice. Green indicates GFP positive cells and blue indicates nuclei. B is a graph in which the number of GFP-positive cells calculated in the micrograph was scored.

In FIG. 5, A is a fluorescent micrograph showing the effect of the administration of physiological saline (control: HGF-, G-CSF-), HGF plasmid (HGF +, G-CSF-), G-CSF (HGF-, G-CSF +), G-CSF + HGF plasmid (HGF +, G-CSF +) to the mouse vWF positive cell count ratio, respectively, to the mouse with left-foot ischemia. Red indicates vWF-positive cells and blue indicates nuclei. B is a graph in which the number of vWF-positive cells calculated in the micrograph was scored.

In fig. 6, a represents a fluorescence microscope photograph of GFP-positive cells (green), vascular endothelial cells (red), and nuclei (blue), and a photograph in which these cells are overlaid. B shows fluorescence micrographs of GFP positive cells (green), vascular smooth muscle cells (red) and nuclei (blue), and photographs obtained by overlaying these cells. Where, nucleoei represents "nucleus" and merged represents "overlap".

In fig. 7, a represents a fluorescence microscope photograph of GFP-positive cells (green), skeletal muscle cells (red), and nuclei (blue), and a photograph in which these cells are overlaid. B denotes overlapping photographs of consecutive slices. Wherein actin means "actinin", nucleolei means "nucleus" and aggregated means "overlap".

FIG. 8 is a graph showing the effect of administration of physiological saline (a: the day next to surgery, b: after 4 weeks of administration), HGF plasmid (c: after 4 weeks of administration), G-CSF (d: after 4 weeks of administration) and G-CSF + HGF plasmid (e: after 4 weeks of administration) on blood flow velocity in lower limbs of mice in mice with left-paw ischemia. "+" indicates p < 0.05(vs. saline group), "+" indicates p < 0.01(vs. saline group), "#" indicates p < 0.05(vs. hgf plasmid group), "§ indicates p < 0.05(vs. g-CSF group).

FIG. 9 is a graph showing differentiation of lower limb injury degree meters after 4 weeks of administration. White for no necrosis, grey for paw end necrosis and black for limb necrosis.

FIG. 10 is a graph showing the effect of administration of physiological saline, HGF plasmid, FGF, G-CSF + HGF plasmid, and G-CSF + FGF plasmid to mice with left foot ischemia on blood flow velocity in lower limbs of mice. "denotes p < 0.05(vs. saline group)," "denotes p < 0.01(vs. saline group)," # "denotes p < 0.05(vs. G-CSF group)," § denotes p < 0.05(vs. G-CSF group),p < 0.05(vs. FGF).

FIG. 11 is a graph showing differentiation of lower limb injury degree meters after 4 weeks of administration. White for no necrosis, grey for paw end necrosis and black for limb necrosis.

Detailed Description

The present invention relates to a therapeutic agent for ischemic diseases, which comprises G-CSF and HGF or FGF.

The term "ischemic disease" as used herein refers to a disease associated with ischemia caused by organic disorders of blood supply (e.g., arterial stenosis). Examples of ischemic diseases include trauma, rejection reaction at the time of transplantation, ischemic cerebrovascular disorders (cerebral apoplexy, cerebral infarction, etc.), ischemic renal diseases, ischemic pulmonary diseases, ischemic diseases associated with infection, ischemic diseases of limbs, and ischemic cardiac diseases (ischemic myocardial diseases, myocardial infarction, ischemic heart failure, etc.).

Furthermore, the present invention relates to an angiogenesis or muscle regeneration agent comprising G-CSF and HGF or FGF.

Human G-CSF is a well-known protein consisting of 174 amino acid residues.

When G-CSF, which is an active ingredient of the therapeutic agent for ischemic diseases of the present invention, is used, any G-CSF can be used, but highly purified G-CSF is preferable. Specific examples thereof include mammalian G-CSF, particularly human G-CSF, and G-CSF having substantially the same biological activity as human G-CSF. The source of G-CSF is not particularly limited, and natural G-CSF, G-CSF obtained by gene recombination, and the like can be used. G-CSF obtained by gene recombination may be the same as the amino acid sequence of natural G-CSF (for example, Japanese patent publication No. 2-5395, Japanese patent laid-open No. 62-236488, etc.), or G-CSF in which 1 or more amino acids are deleted, substituted and/or added in the amino acid sequence, but has the same biological activity as natural G-CSF, etc. For example, polypeptides having the same function as G-CSF can be prepared by introducing appropriate mutations into the amino acid sequence of G-CSF using site-directed mutagenesis (Gotoh, T.et al. (1995) Gene152, 271-275; Zoller, M.J. and Smith, M. (1983) Methods Enzymol.100, 468-500; Kramer, W.et al. (1984) nucleic acids Res.12, 9441-9456; Kramer, W.and Fritz H.J. (1987) Methods Enzymol.154, 350-367; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA, 82, 488-492; Kunkel (1988) Methods Enzymol.85, 2763-2766), etc. Mutations of amino acids can also occur in nature. Polypeptides having amino acid sequences modified by deletion, addition and/or substitution of 1 or more amino acid residues of an amino acid sequence are known to maintain their biological activity (Mark. D.F.et al, Proc. Natl.Acad.Sci.USA (1984)81, 5662-.

Therefore, a polypeptide having the same function as that of G-CSF, which is composed of an amino acid sequence in which 1 or more amino acids are mutated in the amino acid sequence of G-CSF, can also be used as a therapeutic agent for ischemic diseases of the present invention. The number of amino acid mutations in such a polypeptide is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids).

In substitution mutants, it is desirable to substitute other amino acids that retain the properties of the amino acid side chain. Examples of the amino acid capable of maintaining the property of an amino acid side chain include a hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), a hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl-containing side chain (S, T, Y), an amino acid having a sulfur atom-containing side chain (C, M), an amino acid having a carboxylic acid-or amide-containing side chain (D, N, E, Q), an amino acid having a base-containing side chain (R, K, H), and an amino acid having an aromatic side chain (H, F, Y, W). (the abbreviations in parentheses each represent a single letter of an amino acid)

The G-CSF-containing fusion polypeptide is included in the polypeptide in which a plurality of amino acid residues are added to the amino acid sequence of G-CSF. The fusion polypeptide is a substance in which G-CSF is fused to another polypeptide, and such a polypeptide can also be used in the present invention. When a fusion polypeptide is prepared, for example, a DNA encoding G-CSF and a DNA encoding another polypeptide are ligated so as to be in frame, introduced into an appropriate expression vector, and expressed by an appropriate host. Other polypeptides to be used in fusion with G-CSF are not particularly limited as long as the fusion polypeptide retains biological activity equivalent to that of G-CSF.

There have been many reports on G-CSF derivatives having a modified amino acid sequence of G-CSF, and therefore, these known G-CSF derivatives can also be used (for example, USP5581476, USP5214132, USP5362853, USP4904584, etc.).

Also, chemically modified G-CSF can be used. Examples of the chemically modified G-CSF include G-CSF in which a sugar chain is converted in structure, added, or deleted, and G-CSF to which a compound such as polyethylene glycol is bonded (for example, U.S. Pat. No. 5,5824778, U.S. Pat. No. 5,5824784, WO96/11953, WO95/21629, WO94/20069, U.S. Pat. No. 5,5218092, and JP-A-4-164098).

The G-CSF of the present invention can be produced by any method. For example, G-CSF can be isolated and purified by culturing a cell line of human tumor cells and extracting it by various methods, or mammalian cells such as Escherichia coli, yeast, Chinese hamster ovary cells (CHO cells), C127 cells, COS cells, myeloma cells, BHK cells, etc., using genetic engineering methods; G-CSF and the like produced by insect cells and purified by various methods (for example, Japanese patent publication (Kokoku) Nos. 1-44200, 2-5395, 62-129298, 62-132899, 62-236488 and 64-85098).

Such a method for producing G-CSF can be any method as long as it can produce the above-mentioned G-CSF, and specifically, it can be produced using a G-CSF-producing tumor, a G-CSF-producing hybridoma, or a transformed host to which a G-CSF-producing ability is imparted by gene recombination, and depending on the structure of the G-CSF to be produced, an appropriate modification operation or various modification operations can be carried out at an appropriate stage of the production process. In addition, the host used in the production by gene recombination is not limited, and a commonly used host such as Escherichia coli and animal cells can be used.

In the present invention, G-CSF can be administered as a protein or as a gene encoding G-CSF as in gene therapy.

HGF is a well-known heterodimeric protein consisting of an alpha chain of 69kDa and a beta chain of 34 kDa.

The method of administering HGF is not particularly limited, and HGF may be administered as a protein, but it is preferable to administer a gene encoding HGF as in gene therapy. The gene encoding HGF is usually administered as an expression vector or the like containing an expression cassette. The vector is not particularly limited, and a non-viral vector or a viral vector (Experimental medicine, Tokyo, 1997 (Experimental methods for Gene transfer and expression analysis, Tokyo, 1997; Experimental medicine, Tokyo, 1996, etc.) can be used as the vector, and a plasmid vector, a viral vector, a phage vector, a cosmid vector, a YAC vector, etc. can be mentioned.

The gene can be introduced by any method, for example, calcium phosphate coprecipitation method, lipofection method, method using lipid membrane, naked DNA method, receptor-mediated gene introduction method, method using a gene gun, DEAE-dextran method, method using a capillary, and the like. In the present invention, the gene may be introduced directly into the body, or the gene may be introduced into the taken-out cell and the cell may be returned to the body.

There have been many reports on HGF and HGF expression vectors (HGF expression plasmids), and thus those skilled in the art can appropriately select and administer them (Nakamura, T., Nishizawa, T., Hagiya, M.et al. Nature 1989, 342, 440-443; Hayashi, S., Morishita, R., Higaki, J.et al. biochem Biophys Res Commun 1996, 220, 539-545; Morishita, R., Sakaki, M., Yamamoto, K.et al. circulation, 2002, 105, 1491-1496, etc.). Furthermore, the administration of the gene encoding HGF may be carried out by a method known to those skilled in the art (e.g., WO01/32220, WO01/26694, WO97/07824, WO01/21214, etc.).

Further, when HGF is administered as a protein, any HGF may be used, but highly purified HGF is preferred. Specifically, there may be mentioned HGF of a mammal, particularly human HGF, or HGF having substantially the same biological activity as that of human HGF. The source of HGF is not particularly limited, and natural HGF, HGF obtained by gene recombination, and the like can be used. HGF obtained by gene recombination may have the same amino acid sequence as that of natural HGF (for example, GenBank Accession Number; M73239, M73240, M29145, L02931, M60718), or may have 1 or more amino acids deleted, substituted and/or added in the amino acid sequence, but HGF having the same biological activity as that of natural HGF may be used. For example, a polypeptide functionally equivalent to HGF can be prepared by introducing appropriate mutations into the amino acid sequence of HGF using site-directed mutagenesis (Gotoh, T.et al (1995) Gene152, 271-275; Zoller, M.J. and Smith, M. (1983) Methods Enzymol.100, 468-500; Kramer, W.et al (1984) Nucleic Acids Res.12, 9441-9456; Kramer, W.and Fritz H.J. (1987) Methods Enzymol.154, 350-367; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA, 82, 488-492; Kunkel (1998) Methods Enzymol.85, 2763-2766), etc. Amino acid mutations can also arise from nature. Polypeptides having amino acid sequences modified by deletion, addition and/or substitution of 1 or more amino acid residues of an amino acid sequence are known to maintain their biological activity (Mark. D.F.et. et. Proc.Natl.Acad.Sci.USA (1984)81, 5662-.

Therefore, among the amino acid sequences of HGF, a polypeptide consisting of an amino acid sequence in which 1 or more amino acids are mutated and having the same function as HGF can also be used as a therapeutic agent for ischemic diseases of the present invention. The number of amino acid mutations in such a polypeptide is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids).

In the substitution mutant of HGF, it is desired to substitute other amino acids capable of maintaining the properties of the amino acid side chain, as in G-CSF. A fusion polypeptide containing HGF is included in a polypeptide in which a plurality of amino acid residues are added to the amino acid sequence of HGF. The fusion polypeptide is a substance in which HGF is fused with other polypeptides, and such a polypeptide can also be used in the present invention. When a fusion polypeptide is prepared, for example, a DNA encoding HGF and a DNA encoding another polypeptide are ligated so as to be in frame, introduced into an appropriate expression vector, and expressed by an appropriate host. Other polypeptides to be fused with HGF are not particularly limited as long as the fusion polypeptide retains the same biological activity as HGF.

The HGF gene encoding the present invention also includes a gene encoding a polypeptide functionally equivalent to such HGF.

The FGF comprises acidic FGF (FGF1, 140 amino acids) and basic FGF (FGF2, 155-157 amino acids). Further, as functional analogs, FGF3(int-2), FGF4(hst-1), FGF5, FGF6(hst-2), FGF7 (KGF: keratinocyte growth factor, Keratinocytomegawatt factor), FGF8 (AIGF: androgen-induced growth factor), FGF9 (GAF: glial activating factor, Glia-activating factor), and the like can be given. The "FGF" in the present invention includes all of the above.

The method of administration of FGF is not particularly limited, and FGF may be administered as a protein, but it is preferable to administer a gene encoding FGF as in gene therapy. The gene encoding FGF can be usually administered using an expression vector or the like containing an expression cassette. The vector is not particularly limited, and a non-viral vector or a viral vector (Experimental medicine, Tokyo, 1997 (Experimental methods for Gene transfer and expression analysis, Tokyo, 1997; Experimental medicine, Tokyo, 1996, etc.) can be used as the vector, and a plasmid vector, a viral vector, a phage vector, a cosmid vector, a YAC vector, etc. can be mentioned.

The gene can be introduced by any method, for example, calcium phosphate coprecipitation method, lipofection method, method using lipid membrane, naked DNA method, receptor-mediated gene introduction method, method using a gene gun, DEAE-dextran method, method using a capillary, and the like. In the present invention, the gene may be introduced directly into the body, or the gene may be introduced into the taken-out cell and the cell may be returned to the body.

There have been many reports on FGF and FGF expression vector (FGF expression plasmid), and thus administration can be appropriately selected by those skilled in the art (Kurokawa, T et al, FEBS Lett.213, 189-194, (1987), GenBank Accession Number: J04513, M27968). Furthermore, the administration of the Gene encoding FGF can be carried out by methods known to those skilled in the art (e.g., Jejurikar S et al, Journal of scientific Research, 67(2), 137-146, (1997), Reynolds PN et al, Tumor Targeting, 3(3), 156-168, (1998), Ruffini F et al, Gene Therapy, 8(16), 1207-1213, (2001), etc.).

Further, when FGF is administered as a protein, any FGF may be used, but a highly purified FGF is preferred. Specifically, a mammalian FGF, particularly a human FGF, or an FGF having substantially the same biological activity as that of the FGF can be mentioned. The source of FGF is not particularly limited, and natural FGF, FGF obtained by gene recombination, and the like can be used. The FGF obtained by gene recombination may have the same amino acid sequence as that of a natural FGF (for example, FEBS Lett.213: 189-194, 1987.GenBank accession Number: J04513, M27968, etc.), or may have 1 or more amino acids deleted, substituted and/or added in the amino acid sequence, but may have the same biological activity as that of a natural FGF. For example, a polypeptide having the same function as FGF can be prepared by introducing appropriate mutations into the amino acid sequence of FGF using site-directed mutagenesis (Gotoh, T.et al. (1995) Gene152, 271-275; Zoller, M.J. and Smith, M. (1983) Methods Enzymol.100, 468-500; Kramer, W.et a1.(1984) Nucleic Acids Res.12, 944-9456; Kramer, W. and Fritz H.J. (1987) Methods Enzymol.154, 350-367; Kunkel, T.A. (1985) Proc.Natl.Acad.Sci.USA, 82, 488-492; Kunkel (1998) Methods Enzymol.85, 2763-2766), etc. Mutations of amino acids can also occur in nature. Polypeptides having amino acid sequences modified by deletion, addition and/or substitution of 1 or more amino acid residues of an amino acid sequence are known to maintain their biological activity (Mark. D.F.et al, Proc.Natl.Acad.Sci.USA 81, (1984), 5662-.

Therefore, a polypeptide having the same function as that of FGF consisting of an amino acid sequence in which 1 or more amino acids in the amino acid sequence of FGF are mutated can also be used as the therapeutic agent for ischemic diseases of the present invention. The number of amino acid mutations in such a polypeptide is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids).

In the substitution mutant of FGF, as in G-CSF, substitution with other amino acids capable of maintaining the properties of amino acid side chains is desired. The polypeptide in which a plurality of amino acid residues are added to the amino acid sequence of FGF includes a fusion polypeptide containing HGF. Fusion polypeptides are substances in which an FGF is fused to another polypeptide, and such polypeptides are also useful in the present invention. When preparing a fusion polypeptide, for example, a DNA encoding FGF and a DNA encoding the other polypeptide are ligated so as to be in frame, introduced into an appropriate expression vector, and expressed by an appropriate host. Other polypeptides to be fused with FGF are not particularly limited as long as the fusion polypeptide retains the same biological activity as HGF.

The gene encoding the FGF of the present invention also includes a gene encoding a polypeptide functionally equivalent to such FGF.

Furthermore, chemically modified HGF or FGF can also be used in the present invention. Examples of the chemically modified HGF or FGF include HGF subjected to sugar chain structure conversion, addition, or deletion, HGF to which a compound such as polyethylene glycol is bonded, and FGF.

The HGF or FGF of the present invention can be produced by any method. For example, HGF or FGF obtained by culturing a cell line of human tumor cells and extracting and isolating the cells therefrom by various methods, or mammalian cells derived from Escherichia coli, yeast, Chinese hamster ovary cells (CHO cells), C127 cells, COS cells, myeloma cells, BHK cells, etc.; HGF, FGF and the like produced by insect cells and the like and purified therefrom by various methods. Such a method for producing HGF or FGF may be any method as long as it can produce HGF or FGF as defined above, and may be produced using a transformed host to which HGF or FGF producing ability is imparted by gene recombination. In addition, the host used in the production by gene recombination is not limited, and a commonly used host such as Escherichia coli and animal cells can be used.

G-CSF, HGF and FGF are commercially available, and their commercially available products can be used.

The therapeutic agent for ischemic diseases of the present invention may contain a stabilizer or an anti-adsorption agent in addition to necessary pharmaceutical carriers and excipients in order to obtain a pharmaceutical preparation, and the dosage form may be selected from appropriate preparations such as injections (subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, and the like), long-acting preparations, nasal preparations, oral preparations (tablets, capsules, granules, liquid preparations, suspensions, and the like), pulmonary preparations, transdermal preparations, transmucosal preparations, and the like, and appropriate administration devices may be used as necessary.

The therapeutic agent for ischemic diseases of the present invention may be appropriately added with a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc., as required, depending on the administration method and the dosage form thereof.

Examples of the suspending agent include methylcellulose, tween80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, and polyoxyethylene sorbitan monolaurate.

As the solubilizer, polyoxyethylene solidified castor oil, Tween80, nicotinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, etc. can be mentioned.

Examples of the stabilizer include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metabisulfite and the like.

Examples of the isotonic agent include D-mannitol and sorbitol.

Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

Examples of the anti-adsorption agent include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hardened castor oil, and polyethylene glycol.

Examples of the sulfur-containing reducing agent include thiol-containing substances such as N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and C1-7 mercaptoalkanoic acids.

Examples of the antioxidant include a chelating agent such as erythorbic acid, dibutylhydroxytoluene, butylated hydroxyanisole, α -tocopherol, tocopherol acetate, L-ascorbic acid and a salt thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate or disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

In addition, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts such as sodium citrate, potassium citrate, sodium acetate, and the like.

The dose and frequency of administration of G-CSF contained in the therapeutic agent for ischemic diseases of the present invention can be determined depending on the symptoms of the subject patient, and the dose is usually 0.1 to 500. mu.g/kg/day, preferably 1 to 50. mu.g/kg/day per adult, and the frequency of administration is 1 to 7 days per week and 1 to 3 times per day. The administration method is preferably intravenous, subcutaneous, intramuscular, or the like.

When administered with the G-CSF gene, the amount of the G-CSF gene is selected from the range of 0.1. mu.g to 100mg, preferably 0.001 to 10mg per adult, and when taken in the form of liposome, the amount of the G-CSF gene is selected from the range of about 1. mu.g to about 4mg, preferably about 10. mu.g to about 400. mu.g per adult.

In the present invention, when the HGF gene is administered, an appropriate administration method and administration site are selected depending on the disease, symptom, and the like to be treated. The site of administration is preferably intramuscular. The method of administration is preferably non-oral.

The administration amount varies depending on the symptoms of a patient, and the HGF gene is 0.1. mu.g to 100mg, preferably 0.001 to 10mg, per adult. When taken in the form of liposomes, the amount to be administered is selected in the range of about 1. mu.g to about 4mg, preferably about 10. mu.g to about 400. mu.g, per adult human, based on the HGF gene. The number of administrations is appropriately selected depending on the symptoms of the patient. Administration 1 time over a period of days to weeks is suitable. Preferably 1 time a week, several times the administration. More preferably 8 times.

When HGF is administered as a protein, the dose and the frequency of administration are determined according to the symptoms of the subject patient, and the dose is usually 0.1 to 500. mu.g/kg/day, preferably 1 to 50. mu.g/kg/day per adult, and the frequency of administration is 1 to 7 days per week and 1 to 3 times per day. The administration method is preferably intravenous, subcutaneous, intramuscular administration.

In the present invention, when an FGF gene is administered, an appropriate administration method and administration site are selected according to the disease, symptom, and the like to be treated. The site of administration is preferably intramuscular. The method of administration is preferably non-oral.

The dose varies depending on the symptoms of the patient, and is 0.1. mu.g to 100mg, preferably 0.001 to 10mg, per adult in terms of FGF gene. When taken in the form of liposome, the dose is selected in the range of about 1. mu.g to about 4mg, preferably about 10. mu.g to about 400. mu.g, per adult human, in terms of FGF gene. The number of administrations is appropriately selected depending on the symptoms of the patient. Administration 1 time over a period of days to weeks is suitable. Preferably 1 time a week, several times the administration. More preferably 8 times.

When FGF is administered as a protein, the dose and the frequency of administration are determined according to the symptoms of the subject patient, and the dose is usually 0.1 to 500. mu.g/kg/day, preferably 1 to 50. mu.g/kg/day per adult, and the frequency of administration is 1 to 7 days per week and 1 to 3 times per day. The administration method is preferably intravenous, subcutaneous, intramuscular administration.

However, the present invention is not limited to the above amounts of G-CSF, HGF or FGF. In the present invention, G-CSF, HGF or FGF can be prepared and administered as 1 preparation, or can be prepared and administered separately.

The use of the therapeutic agent for ischemic diseases of the present invention can increase the number of hematopoietic stem cells, and by collecting bone marrow or peripheral blood and performing autologous bone marrow transplantation of a patient, angiogenesis in the peripheral blood can be promoted, and ischemic diseases can be treated. Furthermore, by administering the therapeutic agent of the present invention, hematopoietic stem cells are mobilized into peripheral blood, and ischemic diseases can be treated without collecting or transplanting hematopoietic stem cells.

The therapeutic agent for ischemic diseases of the present invention can be used in combination with conventionally used drugs effective against ischemic diseases, such as an antiplatelet agent, a vasodilator, a microcirculation improver, an anticoagulant, and a therapeutic agent for hyperlipidemia, and can also be used in combination with gene therapy.

The present invention will be described in more detail below by way of examples (pharmacological effects) and examples (formulation examples), but the present invention is not limited thereto.

Examples

EXAMPLE 1 (pharmacological Effect)

(1) A wild mouse (C57BL/6, 8-10 weeks old; CLEA, Tokyo, Japan) was irradiated with a single lethal dose of radiation (850cGy) over the whole body. Bone marrow cells (5X 10) were collected from GFP gene recombinant mice (C57BL/6, 10-12 weeks old) (Okabe et al (1997) FEBS. Lett.407, 313-doped 319)⁶One) and transplanted from the tail vein into wild mice. After 2 months of transplantation, the left femoral artery was ligated at 2 sites to prepare a model of ischemia in the lower limb. The administration was randomly divided into 4 groups of a normal saline administration group, a G-CSF administration group, an HGF plasmid administration group, and a G-CSF + HGF plasmid administration group (5 in each group). HGF plasmid (Nakamura, T., Nishizawa, T., Hagiya, M.et al. Nature 1989, 342, 440-443; Hayashi, S., Morrishita, R., Higaki, J.et al. biochem Biophys Res Commun 1)996, 220, 539-545; morishita, R., Sakaki, M., Yamamoto, K.et al.circulation, 2002, 105, 1491-1496) was prepared according to the manufacturer's laboratory manual using a plasmid purification kit (manufactured by QIAGEN Co., Ltd.). The physiological saline administration group and the G-CSF (recombinant human G-CSF (manufactured by Chinese and foreign pharmaceutical Co., Ltd.)) (300. mu.g/kg/day) administration group were subcutaneously administered for 10 days after ligating the cells for 24 hours, respectively. The HGF plasmid-administered group was administered at 500. mu.g/mouse by intramuscular injection 24 hours after ligation. The G-CSF + HGF plasmid administration group was administered with HGF (500. mu.g/mouse) intramuscularly 24 hours after ligation, followed immediately by G-CSF administration (300. mu.g/kg/day) for 10 days.

The lower limb muscle weight ratio (fig. 1), lower limb blood flow ratio (fig. 2), and representative blood flow rate (fig. 3) of each mouse after 4 weeks of administration are shown in the figure. In addition, the experimental data are shown in table 1.

TABLE 1

	Weight (g)		Lower limb muscle weight (g)		Left foot/right foot muscle weight ratio (%)	Blood flow ratio of left foot/right foot (%)
								Before the experiment	After the experiment	Right foot	Left foot
Physiological saline	20.28±1.52	19.08±1.18	0.96±0.05	0.70±0.08								72.10±5.68	87.80±2.92
					HGF plasmid	21.48±0.75	19.33±0.23	0.88±0.05	0.78±0.03	88.44±6.11	91.43±2.34
G-CSF	19.04±1.00	18.26±0.83	0.90±0.03	0.72±0.07								79.95±6.47	88.24±2.55
					HGF plasmid + G-CSF	20.98±0.45	19.52±0.55	0.92±0.04	0.88±0.07	95.27±4.29	94.56±1.64

Even though the group administered with the G-CSF and HGF plasmids alone showed improvement tendency compared with the group administered with physiological saline, the group administered with the G-CSF and HGF plasmids showed statistically significant recovery in the lower limb muscle weight ratio, lower limb blood flow ratio and blood flow rate, and the lower limb injury was reduced compared with the other groups.

In conclusion, by combining HGF and G-CSF, their therapeutic effects were shown to be increased compared to the respective individual treatments.

(2) For histological examination, wild mice treated in the same manner as described above were anesthetized with ketamine (30mg/kg) and xylazine (6mg/kg), and the lower limb vessels were perfused with PBS and fixed with 4% paraformaldehyde dissolved in PBS. The ischemic lower limb muscles were removed, embedded in OCT compound (Miles Scientific, Naperville, IL, USA), and then snap frozen with liquid nitrogen to make thin sections. Frozen sections (6 μm) were washed with PBS and stained with antibody overnight at 4 ℃. For the antibody, vascular endothelial cells were stained with an anti-vovWillebrand factor (vWF) antibody (clone F8/86; DAKO), vascular smooth muscle cells were stained with an alpha-smooth muscle actin antibody (clone 1A 4; Sigma Aldrich), skeletal muscle cells were stained with an anti-actinin antibody (clone EA; Sigma Aldrich). Then, the cells were washed 3 times with PBS, incubated at 4 ℃ for 4 hours (red) using TRITC (DAKO, Japan) in combination with 2 times the antibody, and the nuclei were stained with TOTO-3(Molecular Probes, Inc.) (blue). The immunostained sections were visualized using a laser confocal microscope (LSM510 META; Carl Zeiss; Jena, Germany) (FIGS. 4A and 5A).

The Image taken with the laser confocal microscope was input to a computer and analyzed with NIH Image. The number of GFP-positive cells (FIG. 4B) and the number of vWF-positive cells (FIG. 5B) were counted relative to the number of nucleated cells in each section of the saline administration group (HGF-, G-CSF-).

In addition, the overlap of the typical GFP-positive cell count and the photograph of vascular endothelial cells in the serial sections is shown in fig. 6A, the overlap of the GFP-positive cell count and the photograph of vascular smooth muscle cells is shown in fig. 6B, and the overlap of the GFP-positive cell count and the photograph of skeletal muscle cells is shown in fig. 7A.

It was found that GFP-positive cells derived from bone marrow cells were differentiated into vascular smooth muscle cells or vascular endothelial cells by immunostaining of lower limb muscles. Even though the HGF plasmid-administered group alone showed enhanced angiogenesis compared with the normal saline-administered group, the G-CSF + HGF plasmid-administered group showed a synergistic angiogenesis effect compared with the other groups. Furthermore, it was confirmed that bone marrow cells derived regenerated lower limb muscles. It is inferred that the combined therapeutic effect of G-CSF and HGF on lower limb injury is based on the effect on the regeneration of blood vessels and lower limb muscles in ischemic limbs.

EXAMPLE 2 (pharmacological Effect)

Ligation was performed at 2 of the left femoral artery in nude mice (BALB/cA) to create a model of lower limb ischemia. The administration was randomly divided into 4 groups of normal saline group (20), G-CSF group, HGF plasmid group, G-CSF + HGF plasmid group (10 in each group). In the same manner as in example 1, the physiological saline administration group and the G-CSF (300. mu.g/kg/day) administration group were subcutaneously administered for 10 days after 24 hours of ligation, respectively. The HGF plasmid-administered group was administered at 500. mu.g/mouse by intramuscular injection 24 hours after ligation. The G-CSF + HGF plasmid administration group was administered with HGF (500. mu.g/mouse) intramuscularly 24 hours after ligation, followed immediately by G-CSF administration (300. mu.g/kg/day) for 10 days. Representative blood flow rates of the groups of mice 4 weeks after administration are shown in fig. 8. The blood flow rate was expressed as the mean ± sEM, and the statistical difference between the means was calculated by ANOVA. The comparison was performed using the log-rank test or Fisher nonparametric multiplex test. "+" indicates p < 0.05(vs. saline group), "+" indicates p < 0.01(vs. saline group), "#" indicates p < 0.05(vs. hgf plasmid group), "§ indicates p < 0.05(vs. g-CSF group).

Differentiation of lower limb injury degree was evaluated 4 weeks after administration (fig. 9). White for no necrosis, grey for paw end necrosis and black for limb necrosis.

Even the group administered with the G-CSF and HGF plasmids alone showed statistically significant effects in improving blood flow in lower limbs as compared with the group administered with physiological saline, and the group administered with the G-CSF and HGF plasmids showed statistically significant effects in restoring blood flow in lower limbs as compared with the other groups. The effect was similar to that of the wild-type mouse (experimental example 1), but the effect was more significant in the nude mouse. In addition, lower limb injuries were also statistically improved.

EXAMPLE 3 (pharmacological Effect)

Ligation was performed at 2 of the left femoral artery in nude mice (BALB/cA) to create a model of lower limb ischemia. The administration was randomly divided into 6 groups of a normal saline administration group (20 individuals), a G-CSF administration group, an HGF plasmid administration group, an FGF administration group, a G-CSF + HGF plasmid administration group, and a G-CSF + FGF administration group (5 individuals per group). In the same manner as in example 1, the physiological saline administration group and the G-CSF (300. mu.g/kg/day) administration group were subcutaneously administered for 10 days after 24 hours of ligation, respectively. The HGF plasmid administration group was administered by intramuscular injection at 500. mu.g/mouse 24 hours after the operation. The FGF-administered group was injected with FGF at 500. mu.g/mouse intramuscularly 24 hours after ligation (Trafermin, scientific research pharmaceutical). The G-CSF + HGF plasmid administration group was administered with HGF (500. mu.g/mouse) intramuscularly 24 hours after ligation, followed immediately by G-CSF administration (300. mu.g/kg/day) for 10 days. The group administered with G-CSF + FGF was administered with FGF intramuscularly (500. mu.g/mouse) 24 hours after the operation and then immediately administered with G-CSF (300. mu.g/kg/day) for 10 days. Representative blood flow rates of the groups of mice 4 weeks after administration are shown in fig. 10. The blood flow rate was expressed as mean. + -. SEM, and the statistical difference between the means was calculated by ANOVA. The comparison was performed using the log-rank test or Fisher nonparametric multiplex test. "denotes p < 0.05(vs. saline group)," "denotes p < 0.01(vs. saline group)," # "denotes p < 0.05(vs. G-CSF group)," § denotes p < 0.05(vs. G-CSF group),p < 0.05(vs. FGF).

Differentiation of lower limb injury degree was evaluated 4 weeks after administration (fig. 11). White for no necrosis, grey for paw end necrosis and black for limb necrosis.

Even though the group administered with the G-CSF and HGF plasmids alone showed statistically significant effects in improving blood flow in lower limbs as compared with the group administered with physiological saline, the group administered with the G-CSF and HGF plasmids showed statistically significant effects in restoring blood flow in lower limbs as compared with the other groups. The effect was similar to that of the wild mouse (experimental example 1), but the effect was more significant in the nude mouse. In addition, lower limb injuries were also statistically improved.

Example 1 (formulation example)

Tween20 (Tween 20: polyoxyethylene sorbitan monolaurate) as a nonionic surfactant was added to 50. mu.g/mL of human G-CSF (10mM phosphate buffer, pH7.0) to a concentration of 0.1mg/mL, the osmotic pressure was adjusted to 1 with NaCl, and then filter sterilization was performed using a filter having a pore size of 0.22. mu.m. The resulting solution was filled into a sterilized ampoule, and the ampoule was filled with a rubber stopper which was sterilized in the same manner, and then sealed with an aluminum cap to prepare a solution preparation for injection. The injection can be stored in shade below 10 deg.C.

Example 2 (formulation example)

Tween80 (Tween 80: polyoxyethylene sorbitan monooleate) as a nonionic surfactant was added to 100. mu.g/mL of human G-CSF (10mM phosphate buffer, pH7.0) to a concentration of 0.1mg/mL, the osmotic pressure was adjusted to 1 with NaCl, and then filter sterilization was performed using a filter having a pore size of 0.22. mu.m. The resulting solution was filled into a sterilized ampoule, and the ampoule was filled with a rubber stopper which was sterilized in the same manner, and then sealed with an aluminum cap to prepare a solution preparation for injection. The injection can be stored in shade below 10 deg.C.

Example 3 (formulation example)

Tween20 (Tween 80: polyoxyethylene sorbitan monolaurate) as a nonionic surfactant was added to 50. mu.g/mL of human G-CSF (10mM phosphate buffer, pH7.0) to a concentration of 0.1mg/mL, HAS was added to 10mg/mL and mannitol was added to 50mg/mL for solubilization, followed by filtration sterilization using a filter having a pore size of 0.22. mu.m. The resulting solution was filled into a sterilized ampoule, half-stoppered with a sterilized rubber stopper, and freeze-dried to obtain a lyophilized preparation for injection. The lyophilized preparation for injection is stored at a temperature below room temperature. It can be dissolved in distilled water for injection.

Industrial applicability

The therapeutic agent for ischemic diseases containing G-CSF and HGF or FGF as active ingredients of the present invention is expected to have a therapeutic effect on a relatively severe case of arteriosclerosis obliterans, as shown in examples 1 to 3. The effects of G-CSF and HGF or FGF are presumed to be based on the promotion of angiogenesis, and therefore, therapeutic effects on other ischemic diseases, i.e., trauma, rejection at the time of transplantation, ischemic cerebrovascular disorders (cerebral apoplexy, cerebral infarction, etc.), ischemic renal diseases, ischemic pulmonary diseases, ischemic diseases associated with infection, ischemic diseases of limbs, and ischemic cardiac diseases (ischemic myocardial disorders, myocardial infarction, ischemic heart failure, etc.) are also expected. The treatment method of the present invention is simple, safe and effective as compared with conventional treatment methods.

Claims (35)

1. A therapeutic agent for ischemic diseases contains granulocyte colony stimulating factor and hepatocyte growth factor or fibroblast growth factor as active ingredients.

2. The therapeutic agent for ischemic diseases according to claim 1, wherein the ischemic diseases are trauma, rejection during transplantation, ischemic cerebrovascular disorders, ischemic renal diseases, ischemic lung diseases, ischemic diseases associated with infection, ischemic diseases in limbs, and ischemic heart diseases.

3. The therapeutic agent for ischemic diseases according to claim 1, wherein the ischemic diseases are cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, and arteriosclerosis obliterans.

4. The agent for treating ischemic diseases according to claim 1, wherein the ischemic diseases are arteriosclerosis obliterans.

5. The therapeutic agent for ischemic diseases according to claim 1, which is used for the treatment of ischemic diseases in which hematopoietic stem cells are administered to a patient, in order to obtain a sufficient amount of hematopoietic stem cells from bone marrow.

6. The therapeutic agent for ischemic diseases according to claim 2, which is used for obtaining a necessary and sufficient amount of hematopoietic stem cells from bone marrow in the treatment of trauma of hematopoietic stem cells, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease in limbs, or ischemic cardiomyopathy in a patient.

7. The therapeutic agent for ischemic diseases according to claim 3, which is used for obtaining hematopoietic stem cells in a sufficient amount necessary for cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, or arteriosclerosis obliterans, by administering hematopoietic stem cells to a patient.

8. The therapeutic agent for ischemic diseases according to claim 4, which is used for obtaining a sufficient amount of hematopoietic stem cells from bone marrow in the treatment of arteriosclerosis obliterans in which hematopoietic stem cells are administered to a patient.

9. The therapeutic agent for ischemic diseases according to claim 1, which is used for obtaining a sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of ischemic diseases in which hematopoietic stem cells are administered to a patient.

10. The therapeutic agent for ischemic diseases according to claim 2, which is used for obtaining a necessary and sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of trauma of hematopoietic stem cells, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease in limbs, or ischemic cardiomyopathy in a patient.

11. The therapeutic agent for ischemic diseases according to claim 3, which is used for obtaining a necessary and sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, or arteriosclerosis obliterans, in which the patient is administered with the hematopoietic stem cells.

12. The therapeutic agent for ischemic diseases according to claim 4, which is used for obtaining a sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of arteriosclerosis obliterans in which hematopoietic stem cells are administered to a patient.

13. The agent for treating an ischemic disease according to any one of claims 1 to 4, wherein hematopoietic stem cells increased in peripheral blood by administration contribute to vascularization in a patient.

14. A method for treating ischemic diseases, characterized by using a combination of a therapeutic agent for ischemic diseases, which is characterized by administering a factor having a revascularization activity or a gene thereof to a patient, and a therapeutic agent for ischemic diseases, which contains a granulocyte colony stimulating factor as an active ingredient.

15. The method for treating ischemic diseases according to claim 14, wherein the factor having an angiogenic effect is hepatocyte growth factor or fibroblast growth factor.

16. A method for treating arteriosclerosis obliterans, characterized by using a combination of a therapeutic agent for arteriosclerosis obliterans characterized by administering a factor having a revascularization activity near a lesion or a gene thereof and a therapeutic agent for ischemic diseases containing a granulocyte colony stimulating factor as an active ingredient.

17. The method for treating arteriosclerosis obliterans according to claim 16, wherein the factor having an angiogenic effect is hepatocyte growth factor or fibroblast growth factor.

18. A method for treating ischemic diseases, which comprises administering a pharmaceutical agent used as a clinical drug therapy for ischemic diseases, such as an antiplatelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, or a hyperlipemia therapeutic agent, in combination with the therapeutic agent for ischemic diseases according to any one of claims 1 to 3.

19. A method for treating arteriosclerosis obliterans, characterized by using a drug used as a clinical drug therapy for arteriosclerosis obliterans, such as an antiplatelet agent, a vasodilator, a microcirculation improver, an anticoagulant, or a hyperlipemia therapeutic agent, in combination with the therapeutic agent for ischemic diseases according to claim 4.

20. The application of granulocyte colony stimulating factor and hepatocyte growth factor or fibroblast growth factor in treating ischemic diseases.

21. The use according to claim 20, wherein the ischemic disease is trauma, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic cardiomyopathy.

22. The use as claimed in claim 20, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure or arteriosclerosis obliterans.

23. The use according to claim 20, wherein the ischemic disease is arteriosclerosis obliterans.

24. Use of a granulocyte colony stimulating factor and a hepatocyte growth factor or fibroblast growth factor for obtaining a necessary and sufficient amount of hematopoietic stem cells from bone marrow in the treatment of arteriosclerosis obliterans to which the patient is administered autologous hematopoietic stem cells.

25. The use according to claim 24, wherein the ischemic disease is trauma, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic cardiomyopathy.

26. The use according to claim 24, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, or arteriosclerosis obliterans.

27. The use according to claim 24, wherein the ischemic disease is arteriosclerosis obliterans.

28. Use of a granulocyte colony stimulating factor and a hepatocyte growth factor or fibroblast growth factor for obtaining a necessary and sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of arteriosclerosis obliterans in which hematopoietic stem cells are administered to a patient.

29. The use according to claim 28, wherein the ischemic disease is trauma, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic cardiomyopathy.

30. The use as claimed in claim 28, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure or arteriosclerosis obliterans.

31. The use of claim 28, wherein the ischemic disease is arteriosclerosis obliterans.

32. Use of granulocyte colony stimulating factor, and hepatocyte growth factor or fibroblast growth factor in the treatment of ischemic diseases characterized by administration of a factor having a revascularization effect or a gene thereof to a patient.

33. The use according to claim 32, wherein the ischemic disease is trauma, rejection in transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic cardiomyopathy.

34. The use as claimed in claim 32, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic heart failure or arteriosclerosis obliterans.

35. Use of granulocyte colony stimulating factor, and hepatocyte growth factor or fibroblast growth factor in the treatment of arteriosclerosis obliterans characterized by administration of a factor having a revascularization effect in the vicinity of a lesion or a gene thereof.