HK1059053A - Remedies for ischemic diseases - Google Patents

Description

Therapeutic agent for ischemic diseases

Technical Field

The present invention relates to a therapeutic agent for ischemic diseases, which comprises a human granulocyte colony stimulating factor (hereinafter, abbreviated as human G-CSF) as an active ingredient.

Background

The present invention relates to a therapeutic agent for ischemic diseases, and first, one of typical diseases of ischemic diseases, arteriosclerosis obliterans will be described.

Occlusive arteriosclerosis is a disease in which an aorta of a limb, particularly a lower limb is occluded or narrowed by arteriosclerotic lesions, and ischemic disorders occur at the end of the arteriosclerotic lesions, and is classified into cold feeling, paralysis, intermittent claudication, pain at rest, ulcer, and necrosis as clinical symptoms. About 10 ten thousand patients suffering from occlusive arteriosclerosis of our country (Yufu, therapeutics 31, 289 page 292; 1997) are presumed to increase in the future due to an increase in the population of elderly people or an European beautification of diet. In recent years, gene therapy, intramuscular transplantation of bone marrow cells, and the like have been attempted in addition to exercise therapy, drug therapy, and blood flow reconstruction depending on symptoms, patient conditions, and the like.

Although the above-described therapies have achieved certain effects on the treatment of arteriosclerosis obliterans at present, the respective therapies have the following problems. That is, in the present situation, although the effect of prolonging the walking distance is confirmed in the case of mild symptoms, the effect of the exercise therapy is difficult to predict, and even if the effect of prolonging the walking distance is obtained, the patient does not satisfy the effect, and 30% of reports are reported on the case where the blood flow reconstruction is desired (Taiguan: Japanese medical news report, 3935, 25-29 pages; 1999), and it cannot be said that the exercise therapy is a sufficiently effective treatment method.

In the present situation, an antiplatelet agent mainly formulated for drug therapy is expected to prevent the deterioration of the disease state, and even recently developed agents for improving microcirculation blood flow and improving oxygen transport ability are expected to be applied only to mild cases, and no fundamental therapeutic agents for arteriosclerosis obliterans are available for all drugs.

In contrast, blood flow reconstruction is currently the most effective treatment, and percutaneous angioplasty and bypass surgery are performed depending on the condition of the patient, the location and the extent of a lesion, but there are problems in that there are particular concerns about the surgery involved in these procedures, and long-term survival cannot be expected in addition to complications and death cases involved in the surgery.

In addition, gene therapy using factors having an angiogenic effect such as a vascular endothelial growth factor and an epithelial cell growth factor has been carried out in the field of gene therapy, but currently, evaluation of safety and effect outside the field of experimental therapy has not been fixed, and therefore, it has not been generally widespread.

Recently, the intramuscular transplantation of bone marrow cells, which has reported a therapeutic effect, is a therapy for treating a disease by transplanting bone marrow cells into muscles near a diseased site to differentiate and form blood vessels in vascular endothelial cells, and in the future, although it is necessary to increase the number of disease cases and evaluate the effect, serious disease cases can be treated, and thus, the intramuscular transplantation of bone marrow cells is expected as a future therapy. However, this therapy also has a problem that a burden on patients and medical staff is large due to bone marrow collection.

Recently, it has been found that hematopoietic stem cells capable of differentiating into vascular endothelial cells (called "endothelial cell precursor cells" from the viewpoint of the function of differentiating into endothelial cells) exist not only in bone marrow but also in peripheral blood (in the present specification, the term "hematopoietic stem cells" is used conceptually from a cell population capable of forming endothelial cells since the cells are originally derived from hematopoietic stem cells), and that they are involved in angiogenesis (Qun Shi et al. blood vol92, 362 vol 367; 1998, Takayuki Asahara et al. blood 95, 952 vol 958; 2000). Therefore, it is expected that occlusive arteriosclerosis will be treated by collecting hematopoietic stem cells in peripheral blood and transplanting them into muscles near the affected part. The advantages are that: in this case, the burden on the patient and medical staff involved in collecting peripheral blood stem cells is less than in the case of transplanting stem cells in bone marrow. However, since the frequency of hematopoietic stem cells in peripheral blood is usually quite low, it is questionable whether a sufficient amount of hematopoietic stem cells necessary for the treatment of arteriosclerosis obliterans can be obtained.

Disclosure of Invention

Human G-CSF is a hematopoietic factor found as a differentiation and proliferation factor of hematopoietic precursor cells of the granulocyte lineage, and is clinically used as a neutropenia therapeutic agent after bone marrow transplantation or cancer chemotherapy because it promotes the hematopoiesis of neutrophils in an organism. In addition to the above-mentioned effects, human G-CSF has an effect of stimulating the proliferation and differentiation of hematopoietic stem cells and an effect of mobilizing hematopoietic stem cells in bone marrow to peripheral blood, and in fact, based on the latter effect, peripheral blood stem cell transplantation, in which peripheral blood hematopoietic stem cells mobilized with human G-CSF are transplanted, is performed in clinical practice in order to promote hematopoietic recovery of cancer patients after chemotherapy. This hematopoietic stem cell mobilization of G-CSF is much stronger than the hematopoietic factor GM-CSF of the same granulocytic lineage. In addition, G-CSF is superior to GM-CSF in that it has less side effects.

Therefore, before treatment by intramuscular transplantation of bone marrow cells into a patient with arteriosclerosis obliterans, the frequency of hematopoietic stem cells in the bone marrow can be expected to increase by administration of human G-CSF, and therefore the number of bone marrow punctures at the time of bone marrow cell collection 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 medical staff can be further reduced. Furthermore, since hematopoietic stem cells in peripheral blood show angiogenesis, it is considered that administration of human G-CSF increases hematopoietic stem cells in peripheral blood to promote angiogenesis. Therefore, it is expected that the treatment of arteriosclerosis obliterans can be carried out by administering only human G-CSF to a patient. It is clear that the treatment of occlusive atherosclerosis by such administration of human G-CSF greatly reduces the burden on patients and medical staff in terms of not requiring hematopoietic cell collection or transplantation.

Brief description of the drawings

FIG. 1 is a graph showing the effect of administration of single peripheral blood cells (B) and administration of G-CSF (C) to rats on the density of capillary vessels in ischemic limbs, in mice to which G-CSF was administered. Capillary density of each individual was plotted against group B, group C, and control group (a).

The treatment of occlusive arteriosclerosis using human G-CSF of the above 3 types is expected to be effective even in severe patients and therefore is a great gospel for patients, but it is expected to further increase the therapeutic effect when used in combination with a factor having an angiogenic effect such as Vascular Endothelial Growth Factor (VEGF), epithelial cell growth factor (EGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor (FGF) or a gene thereof, which promotes the differentiation and proliferation of vascular endothelial precursor cells. At this time, these factors or their genes may be administered to the patient, for example, in the vicinity of the diseased part. Similarly, it is expected to increase the therapeutic effect of an anti-platelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, a therapeutic agent for hyperlipidemia, and the like, which are clinically used as a pharmacotherapy for arteriosclerosis obliterans.

Further, the G-CSF of the present invention can also be used as a therapeutic agent for the following diseases, which are the same ischemic diseases. That is, the present invention provides a therapeutic agent for treating trauma, graft rejection, ischemic cerebrovascular disorders (e.g., cerebral apoplexy and cerebral infarction), ischemic renal diseases, ischemic diseases of the extremities, ischemic pulmonary diseases, ischemic diseases associated with infection, ischemic cardiac diseases (e.g., ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, etc.), and the like, using G-CSF as an active ingredient.

The present invention achieves the above-described findings. That is, the present invention provides a therapeutic agent for ischemic diseases, which comprises human G-CSF as an active ingredient.

The present invention will be described in detail below.

Detailed Description

Human G-CSF is a protein having an amino acid sequence represented by the following formula 1, but human G-CSF used in the present invention may be a modified protein having a sequence obtained by substituting, adding or deleting 1 or more amino acids in the protein, or a protein represented by the following formula 1 or a modified protein, and may be one having any G-CSF activity. The term "various modifications" as used herein refers to structural changes, additions, deletions of sugar chains, or to inorganic or organic compounds such as polyethylene glycol and vitamin B12 bonded thereto.

Formula 1: amino acid sequence Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val 48Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys 64Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser 80Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser 96Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp 112Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro 128Ala Leu Gln Pro Thr Gln Gly of human G-CSF Ala Met Pro Ala Phe Ala Ser Ala Phe 144Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe 160Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 174

The method for producing human G-CSF can be any method as long as it is capable of producing the sequence defined in the above paragraph, and specifically, a human G-CSF-producing tumor or a human G-CSF-producing hybridoma is used, and the sequence defined in the above paragraph is further produced using a transformed host to which G-CSF-producing ability is imparted by genome conversion. In the case of production by gene conversion, a host generally used for the production of Escherichia coli, animal cells, or the like is used regardless of the type of the host.

The therapeutic agent for ischemic diseases of the present invention may contain a necessary carrier or excipient for the preparation, and may further contain a stabilizer or an absorptiveness preventing agent in order to obtain a form as a pharmaceutical preparation, and the dosage form may be selected from appropriate preparations such as an injection, a controlled release preparation, a nasal agent, an oral agent, a transpulmonary agent, a transdermal agent, and a transmucosal agent, and an appropriate device may be used as needed.

The dose and frequency of administration of human G-CSF containing the therapeutic agent for ischemic diseases of the present invention can be determined in consideration of the pathological condition of a subject patient, but the dose is usually 0.1 to 500. mu.g/kg/day, preferably 1 to 50. mu.g/kg/day per adult. In addition, the administration frequency can be 1 week to 7 days. The administration method is preferably intravenous, subcutaneous, intramuscular, or the like. However, the present invention is not limited to the amount of human G-CSF. In addition, the present invention can also be used in combination with a drug expected to have an effect on ischemic diseases, such as an antiplatelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, and a therapeutic agent for hypertension, and further gene therapy.

The present invention will be described in more detail below with reference to experimental examples (pharmacological effects) and examples (formulation examples), but the present invention is not limited thereto. EXAMPLE 1 (pharmacological Effect)

The left femoral artery and vein of a nude mouse (BALB/cAJcl-nu) was ligated and then excised to prepare a lower limb ischemia model. In the treatment-free group, 3 (60%) of 5 cases after 2 weeks of ischemia were found to have lower limb loss and 2 (40%) of the cases had lower limb necrosis. On the other hand, in the group to which a total of 5 subcutaneous administrations of G-CSF of 100. mu.g/kg/day were given from 3 days before the lower limb ischemia to 1 day after the operation, 1 (20%) of 5 cases had lower limb abscission, 3 (60%) had necrosis, and 1 (20%) had no damage, and the lower limb damage was reduced as compared with the non-treated group. Therefore, it is known that G-CSF may have an effect of ameliorating lower limb injury after ischemia by promoting angiogenesis. EXAMPLE 2 (pharmacological Effect)

Mice (BALB/cA) were subcutaneously administered G-CSF 100. mu.g/kg/day for 5 days,after that, blood was collected, and a monocyte fraction was fractionated by a density gradient method (d ═ 1.077). In addition, the left femoral artery and vein of a nude rat (F344/N Jcl-rnu) was excised and made into a lower limb ischemia model. After 1 day of ischemia, peripheral blood mononuclear cells derived from mice administered with G-CSF intramuscularly to nude rats (F344/N Jcl-rnu) with ischemic lower limbs were approximately 2X 10⁷cell/head (about 5mL equivalent to peripheral blood), transplanted. In the control group, phosphate buffer was administered intramuscularly. After 1 week of transplantation, a tissue specimen of the lower limb was prepared and stained with alkaline phosphatase to measure the capillary density. As a result, the peripheral blood mononuclear cell administration group tended to have a higher capillary density than the control group. (control group; 38.3. + -. 1.7, peripheral blood mononuclear cell administration group; 42.3. + -. 2.1 capillary number/5-fold of field, average. + -. standard deviation). The results are shown in A and B of FIG. 1.

These results suggest that G-CSF promotes the mobilization of endothelial precursor cells into peripheral blood of mice, and as a result, promotes angiogenesis in rats into which peripheral blood has been transplanted, suggesting that G-CSF is likely to be applied to the treatment of peripheral circulatory disorders. EXAMPLE 3 (pharmacological Effect)

The left femoral artery and vein of a nude rat (F344/N Jcl-rnu) were excised to prepare a lower limb ischemia model, and the tissue specimens of the lower limbs 1 week after ischemia were stained with alkaline phosphatase to measure the vascular density. The group administered with G-CSF of 100. mu.g/kg/day from 4 days before ischemia to 1 week after ischemia (G-CSF administered group) was compared with the control group. For the control group, phosphate buffer was administered intramuscularly. The results showed that the capillary density of the G-CSF group was higher than that of the control group (control group; 38.3. + -. 1.7, peripheral blood mononuclear cell administration group; 44.7. + -. 2.4 number of capillaries per 5 of each field of view, mean. + -. standard deviation). The results are shown in A and C of FIG. 1.

The results showed that G-CSF has a promoting effect on angiogenesis at the ischemic site, suggesting that G-CSF has a possibility of being applied to the treatment of peripheral circulatory disorders.

Example 1 (formulation example)

To 50. mu.g/ml of human G-CSF (10mM phosphate buffer pH7.0) was added polysorbate 20(Tween 20: polyoxyethylene sorbitan monolauryl) as a surfactant to give a concentration of 0.1mg/ml, and the mixture was adjusted to an osmotic pressure of 1 with NaCl and then sterilized by filtration through a membrane filter having a pore size of 0.22 mM. The resulting solution was filled into a sterilized ampoule, which was covered with a rubber stopper sterilized in the same manner, and then sealed with an aluminum cap to obtain a solution preparation for injection. The preparation for injection is stored in a cold dark place at a temperature of 10 deg.C or below.

Example 2 (formulation example)

To 100. mu.g/ml of human G-CSF (10mM phosphate buffer pH7.0) was added polysorbate 80(Tween 20: polyoxyethylene sorbitan monooleate) as a surfactant to give a concentration of 0.1mg/ml, the osmotic pressure was adjusted to 1 with NaCl, and the mixture was filtered and sterilized with a membrane filter having a pore size of 0.22 mM. The resulting solution was filled into a sterilized ampoule, which was covered with a rubber stopper sterilized in the same manner, and then sealed with an aluminum cap to obtain a solution preparation for injection. The preparation for injection is stored in a cold dark place at a temperature of 10 deg.C or below.

Example 3 (formulation example)

In 50. mu.g/ml of human G-CSF (10mM phosphate buffer pH7.0), 0.1mg/ml of polysorbate 20(Tween 20: polyoxyethylene sorbitan monolaurate), 10mg/ml of HAS, and 50mg/ml of mannitol were added and dissolved, followed by filtration sterilization using a membrane filter having a pore size of 0.22 mM. The resulting solution was filled into sterilized ampoules, half-capped with similarly sterilized rubber stoppers, and freeze-dried to obtain lyophilized agents for injection. The injection preparation is stored at a temperature below room temperature, and is dissolved in distilled water for injection before use.

Industrial applicability

The therapeutic agent for ischemic diseases containing G-CSF as an active ingredient of the present invention is expected to have a therapeutic effect on a relatively severe disease case among arteriosclerosis obliterans, as shown in experimental examples 1 to 3. Since the therapeutic effect of G-CSF is estimated to be based on the promotion of angiogenesis, it is expected to have a therapeutic effect on other ischemic diseases, i.e., trauma, rejection at the time of transplantation, ischemic cerebrovascular disorders (e.g., cerebral apoplexy and cerebral infarction), ischemic renal diseases, ischemic lung diseases, ischemic diseases associated with infection, ischemic diseases of limbs, and ischemic heart diseases (e.g., ischemic myocardial diseases, myocardial infarction, and ischemic cardiac insufficiency). The treatment according to the invention is simpler, safer and more effective than the existing treatments.

In <110> of the sequence table <120> ischemic disease therapeutic agent <130>002078<160>1<210>1<211>174<212> PRT <212> human <400>1Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 151015 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln

20 25 30Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val

35 40 45Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys

50 55 60Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser65 70 75 80Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser

85 90 95Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp

100 105 110Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro

115 120 125Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe

130 135 140Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe145 150 155 160Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro

165 170

Claims (33)

1. An agent for treating ischemic diseases, which comprises human granulocyte colony stimulating factor as an active ingredient.

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

3. The agent for treating ischemic diseases according to claim 1, wherein the ischemic diseases are cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, and arteriosclerosis obliterans.

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

5. The therapeutic agent for ischemic diseases according to claim 1, which is used for obtaining hematopoietic stem cells from bone marrow in an amount necessary and sufficient for treating ischemic diseases by administering hematopoietic stem cells of a patient per se.

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, graft rejection, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic heart disease by administration of hematopoietic stem cells to a patient.

7. 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 bone marrow in the treatment of cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency or arteriosclerosis obliterans by administering hematopoietic stem cells of a patient per se.

8. The therapeutic agent for ischemic diseases according to claim 4, which is used for obtaining a necessary and sufficient amount of hematopoietic stem cells from bone marrow in treating arteriosclerosis obliterans by administering hematopoietic stem cells of a patient per se.

9. The therapeutic agent for ischemic diseases according to claim 1, which is used for treating ischemic diseases by administering hematopoietic stem cells of a patient per se, and which is capable of obtaining a necessary and sufficient amount of hematopoietic stem cells from peripheral blood.

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 when administered to a patient's own hematopoietic stem cells for treating trauma, graft rejection, ischemic cerebrovascular disorders, ischemic renal diseases, ischemic pulmonary diseases, ischemic diseases associated with infections, ischemic diseases of limbs, or ischemic heart diseases.

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 cardiac insufficiency, or arteriosclerosis obliterans by administering the hematopoietic stem cells per se to a patient.

12. The therapeutic agent for ischemic diseases according to claim 4, which is used for treating arteriosclerosis obliterans by administering hematopoietic stem cells of a patient himself to obtain a necessary and sufficient amount of hematopoietic stem cells from peripheral blood.

13. The agent for treating ischemic disease according to claim 1 to 4, wherein hematopoietic stem cells increased in peripheral blood are imparted with angiogenesis at the diseased site by administration.

14. A method for treating ischemic diseases, which comprises administering a factor having an angiogenic effect or a gene thereof to a patient, and which comprises using the agent for treating ischemic diseases according to claims 1 to 3 in combination.

15. A method for treating arteriosclerosis obliterans, characterized by comprising administering a factor having an angiogenic activity or a gene thereof to the vicinity of a disease site, and using the agent for treating ischemic disease according to claim 4 in combination.

16. A method for treating ischemic diseases, which comprises using an anti-platelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, a therapeutic agent for hyperlipidemia, or the like, which is clinically used as a drug therapy for ischemic diseases, in combination with the therapeutic agent for ischemic diseases according to claims 1 to 3.

17. A therapeutic method for arteriosclerosis obliterans, characterized in that an anti-platelet agent, a vasodilator, a microcirculation improving agent, an anticoagulant, a therapeutic agent for hyperlipidemia, or the like, which is clinically used as a drug therapy for arteriosclerosis obliterans, is used in combination with the therapeutic agent for ischemic diseases according to claim 4.

18. Use of human granulocyte colony stimulating factor for the treatment of ischemic diseases.

19. The use of human granulocyte colony stimulating factor of claim 18, wherein the ischemic disease is trauma, rejection at transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic heart disease.

20. The use of human granulocyte colony stimulating factor of claim 18, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, or arteriosclerosis obliterans.

21. The use of human granulocyte colony-stimulating factor of claim 18, wherein the ischemic disease is arteriosclerosis obliterans.

22. Use of human granulocyte colony stimulating factor for obtaining hematopoietic stem cells in a sufficient amount from bone marrow in the treatment of ischemic diseases by administering hematopoietic stem cells to a patient.

23. The use of human granulocyte colony stimulating factor of claim 22, wherein the ischemic disease is trauma, rejection at transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic heart disease.

24. The use of human granulocyte colony stimulating factor of claim 22, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, or arteriosclerosis obliterans.

25. The use of human granulocyte colony-stimulating factor of claim 22, wherein the ischemic disease is arteriosclerosis obliterans.

26. Use of human granulocyte colony stimulating factor for obtaining a necessary and sufficient amount of hematopoietic stem cells from peripheral blood in the treatment of ischemic diseases by administering hematopoietic stem cells to a patient.

27. The use of human granulocyte colony stimulating factor of claim 26, wherein the ischemic disease is a therapeutic agent for trauma, transplant rejection, ischemic cerebrovascular disorder, ischemic renal disease, ischemic lung disease, ischemic disease associated with infection, ischemic disease of limbs, ischemic heart disease, or the like.

28. The use of human granulocyte colony stimulating factor of claim 26, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, or arteriosclerosis obliterans.

29. The use of human granulocyte colony-stimulating factor of claim 26, wherein the ischemic disease is arteriosclerosis obliterans.

30. Use of a human granulocyte colony stimulating factor for treating ischemic diseases, characterized in that a factor having an angiogenic effect or a gene thereof is administered to a patient.

31. The use of human granulocyte colony stimulating factor of claim 30, wherein the ischemic disease is trauma, rejection at transplantation, ischemic cerebrovascular disorder, ischemic renal disease, ischemic pulmonary disease, ischemic disease associated with infection, ischemic disease of limbs, or ischemic heart disease.

32. The use of human granulocyte colony stimulating factor of claim 30, wherein the ischemic disease is cerebral stroke, cerebral infarction, ischemic cardiomyopathy, myocardial infarction, ischemic cardiac insufficiency, or arteriosclerosis obliterans.

33. Use of a human granulocyte colony stimulating factor for treating ischemic diseases, characterized in that a factor having an angiogenesis activity or a gene thereof is administered in the vicinity of a diseased site.