WO2005097170A1

WO2005097170A1 - Angiogenesis promoter and angiogenic therapy

Info

Publication number: WO2005097170A1
Application number: PCT/JP2005/003333
Authority: WO
Inventors: Ken Toba; Kiminori Katou
Original assignee: Niigata Tlo Corporation
Priority date: 2004-03-30
Filing date: 2005-02-28
Publication date: 2005-10-20
Also published as: JP2007217283A

Abstract

It is intended to provide an angiogenesis promoter which can be easily applied, promotes an angiogenic effect and is highly efficacious in improving blood flow and an angiogenic therapy. By using an angiogenesis promoter, which contains an erythropoietin preparation comprising mammalian bone marrow cells and 5,000 to 50,000 IU/60 kg body weight per unit dose of erythropoietin and can be easily applied, erythroblasts induced by erythropoietin secret a vasoproliferative cytokine and thus the angiogenic effect can be strongly promoted. Since this angiogenic effect is potentiated in the presence of erythropoietin, additional angiogenic effect can be promoted. Thus, blood flow insufficiency which is fundamentally causative of the pathological conditions and symptoms of ischemic diseases and the thus induced ischemia can be ameliorated and clinical effects can be improved.

Description

Specification

Angiogenesis promoters and angiogenesis therapy

Technical field

[0001] The present invention relates to an angiogenesis-promoting agent and angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease.

Background art

[0002] In recent years, treatment of ischemic diseases such as extremities, heart, brain, and the like has become a major factor in clinical medicine, and this market size forms the largest disease group along with treatment of cancer diseases. Vasodilators, percutaneous transluminal coronary angioplasty, and vascular bypass are used to treat this ischemic disease, and vascular bypass surgery is conventionally used for patients with severe lower limb ischemic disease. There were problems, such as cutting of the lower limbs, if the force vessel was thin and bypassing was not possible. As a means to solve these problems, research on gene therapy and angiogenesis therapy is underway. However, gene therapy is not yet widely used due to safety and ethical issues that have not yet gone out of experimental therapy, so autologous cell transplantation, which is an angiogenic therapy, has become the mainstream. . This autologous cell transplantation treatment is a therapy in which bone marrow cells are transplanted into muscles near the affected area, and this is divided into blood vessels to form blood vessels, which will be treated in the future. Although it is necessary to evaluate the effect by increasing it, it is expected as a future treatment because it can treat severe cases.

[0003] In peripheral arterial diseases that are ischemic diseases (obstructive arteriosclerosis, Buerger's disease, ischemic heart disease, cerebrovascular disease, etc.) In order to form the basis of pathophysiology / symptoms, an attempt has been made to treat the disease by using a variety of techniques to generate blood vessels in the region's organ. Among these attempts, conventional technologies related to autologous cell transplantation include limb ischemia treatment using autologous bone marrow cell transplantation (Non-Patent Document 1) and ischemic heart disease treatment using autologous bone marrow cell transplantation. (Non-patent document 2), Treatment of limb ischemia using autologous peripheral blood cell transplantation (Non-patent document 3), Treatment of ischemic heart disease using autologous peripheral blood cell transplantation (Non-patent document 4), etc. It is done. Also blood vessels Endothelial progenitor cells are treated for cerebral vascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, and myocardial ischemia in a method for controlling neoplasia, ie, for enhancing or inhibiting angiogenesis. A method for administration to a patient having such a disease is disclosed in, for example, Patent Document 1 !. However, although these treatments have a certain clinical effect, they have not been able to obtain sufficient effects in terms of blood flow improvement effect and symptom reduction effect.

Patent Document 1: JP 2001-503427 Gazette

Special Reference 1: Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a ramdomised controlled trial "(Lancet, vol. 360, pp 427-435, 2002) Non-Patent Document 2: Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angio blasts, angiogenic ligands, and cytokines (and lrculation, vol. 104, pp 1046-1052, 2001)

Non-Patent Document 3: "Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs (circulation, vol. 106, pp

(2019-2025, 2002)

Non-Patent Document 4: "Improvement of collateral perfusion and regional function by implantation of peripheral olood mononuclear cells into ischemic hibernation myocardium" (Arteriosclerosis Thrombosis and Vascular Biology, vol. 22, pp 1804-1810, 2002)

Disclosure of the invention

Problems to be solved by the invention

[0004] There has been a strong demand for improvements in formulations and treatment methods that solve the above-mentioned drawbacks and can be applied more easily and have clinical effects.

[0005] Accordingly, an object of the present invention is to provide an angiogenesis promoting agent that can be easily applied, promotes angiogenic action, and has a high blood flow improving effect. Furthermore, an object of the present invention is to provide an angiogenesis therapy capable of enhancing a therapeutic effect by enhancing an angiogenesis action in an angiogenesis therapy for an ischemic disease. Means for solving the problem

[0006] As a result of diligent studies in view of the above problems, in mammalian bone marrow hematopoiesis, bone marrow cells other than erythroblasts have the ability to migrate and migrate to the blood vessels themselves as they mature and pass through the bone marrow blood vessels. While erythroblasts are recruited to peripheral blood, erythroblasts do not have the ability to migrate, so erythroblasts secrete vascular growth-directing site force-in (vascular endothelial growth factor: VEGF, placental growth factor: P1GF, etc.) We focused on the property of inducing new blood vessels to the hematopoietic foci and being mobilized to peripheral blood. It is widely adopted as a conventional technique for angiogenesis! For transplantation treatment of bone marrow cells or peripheral blood cells to an ischemic site! Because it contains many hematopoietic stem cells and stem cells that contain only vesicles, transient erythroblast hematopoiesis at the transplantation site can be achieved by mixing the transplanted cells with the erythropoietin erythropoietin. It was also found that efficient angiogenesis can be promoted by inducing erythroblast activation and secreting the above-mentioned vascular proliferative tropic force in the vicinity of vascular progenitor cells.

[0007] Further, nuclei exist in erythroblasts, and in the process of erythroblast maturation, the nuclei are removed (denucleated) by subendothelial macrophages around the microvessels of the bone marrow, At the same time as enucleation, intranuclear force moves through the gaps in the vascular endothelium, and enucleated erythroblasts (reticulocytes) move into the blood vessels. In other words, macrophages (in some of the CD14-positive cells) under the blood vessels of the blood vessels play a role in moving the reticulocytes into the blood vessels while taking the cell nuclei from the mature erythroblasts! This suggests that erythroblasts attract blood vessels by mechanisms such as cytodynamic in secretion, but do not come into direct contact with new blood vessels, and mediation by CD14-positive cells is necessary. It is induced in the erythroid hematopoiesis together with the new blood vessels by the cytodynamic force secreted by the blasts, and after induction, the CD14 positive cells adhere directly to the mature erythroblasts and directly to the vascular endothelium of the new blood vessels. To do. By stimulating these cell adhesions, it is expected that CD14 positive cells secrete various site force-ins and angiogenesis occurs as a whole of their temporal arrangement and spatial positional relationship. Therefore, efficient angiogenesis can be promoted by simultaneously administering bone marrow cells and erythropoietin with macrophage colony-stimulating factor (M-CSF), which is a site force-in for proliferating and activating CD14-positive cells. As a result, the present invention has been conceived. [0008] The angiogenesis-promoting agent according to claim 1 of the present invention comprises mammalian bone marrow cells and an erythropoietin preparation containing 5,000-50,000 international units Z body weight 60 kg of erythropoietin per dose. It is characterized by that.

[0009] The angiogenesis promoter according to claim 2 of the present invention is characterized in that in claim 1, it further comprises a macrophage colony stimulating factor.

[0010] The angiogenesis-promoting agent according to claim 3 of the present invention is characterized in that, in claim 1, the erythropoietin is a naturally-derived erythropoietin, a modified erythropoietin or a derivative thereof.

[0011] The angiogenesis promoting agent according to claim 4 of the present invention is the angiogenesis promoting agent according to claim 1, wherein the bone marrow cells are

It is characterized in that it is derived from a mammal whose autologous or histocompatibility antigen is identical or partially mismatched.

[0012] The angiogenesis-promoting agent according to claim 5 of the present invention is characterized in that, in claim 1, it is a dosage form of ampoules, syringes or vials.

[0013] The angiogenesis-promoting agent according to claim 6 of the present invention comprises erythroblasts derived from bone marrow, peripheral blood, umbilical cord blood or other cell resources, CD14-positive cells, and 5,000-50 per dose. , 000 international unit Z erythropoietin preparation containing 60 kg erythropoietin.

[0014] The angiogenesis promoter according to claim 7 of the present invention is characterized in that in claim 6, it further comprises a macrophage colony-stimulating factor.

[0015] The angiogenesis promoter according to claim 8 of the present invention is characterized in that, in claim 6, the erythropoietin is a naturally derived erythropoietin, a modified erythropoietin or a derivative thereof.

[0016] The angiogenesis promoting agent according to claim 9 of the present invention is the angiogenesis promoting agent according to claim 6, wherein the bone marrow, peripheral blood, umbilical cord blood or other cell resources are identical or partially mismatched with autologous or histocompatibility antigens. It is derived from a mammal.

[0017] The angiogenesis promoting agent according to claim 10 of the present invention is the angiogenesis promoting agent according to claim 6, wherein the erythroblast derived from the peripheral blood, umbilical cord blood or other cell resources is obtained by in vitro proliferation. It is characterized by that. [0018] The angiogenesis-promoting agent according to claim 11 of the present invention is characterized in that, in claim 6, it is a dosage form of ampoules, syringes or nominals.

[0019] The angiogenesis therapy according to claim 12 of the present invention is an angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease.

The angiogenesis promoting agent according to any one of 1 is administered intramuscularly to an ischemic site.

[0020] The angiogenesis therapy according to claim 13 of the present invention is characterized in that, in claim 12, the ischemic disease is a peripheral arterial disease.

[0021] The angiogenesis therapy according to claim 14 of the present invention is an angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease. Any one of the angiogenesis promoters described in any one of the above may be administered by introducing an intravascular force tail from a blood vessel at a remote site to reach the ischemic site transvascularly.

[0022] The angiogenesis therapy according to claim 15 of the present invention is characterized in that, in claim 14, the ischemic disease is a peripheral arterial disease.

The invention's effect

[0023] According to the angiogenesis promoting agent of claim 1 of the present invention, the angiogenesis action can be easily applied, and the erythroid hematopoietic cells induced by erythropoietin secrete vascular growth tropic site force in. Can be promoted.

[0024] According to the angiogenesis-promoting agent according to claim 2 of the present invention, the macrophage 'colony stimulating factor is a site force-in that activates the growth of macrophages, and therefore can further promote the angiogenesis action. it can.

[0025] According to the angiogenesis promoter of claim 3 of the present invention, various excellent effects such as enhancement of main effects and reduction of side effects can be expected by using modified erythropoietin or a derivative thereof. .

[0026] According to the angiogenesis-promoting agent of claim 4, the bone marrow cell resource can be easily obtained even when the same type (human vs. human, mouse vs. mouse, etc.) cell resource is used. be able to.

[0027] The angiogenesis promoter according to claim 5 of the present invention can be easily applied. [0028] According to the angiogenesis-promoting agent of claim 6 of the present invention, it can be easily applied, and the erythroid hematopoietic cell induced by erythropoietin secretes vascular growth-trophic site force-in and has a strong angiogenesis action. Can be promoted.

[0029] According to the angiogenesis promoting agent according to claim 7 of the present invention, the macrophage 'colony stimulating factor is a site force-in that activates the growth of macrophages, and therefore can further promote the angiogenesis action. it can.

[0030] According to the angiogenesis-promoting agent according to claim 8 of the present invention, various excellent effects such as enhancement of main effects and reduction of side effects can be expected by using modified erythropoietin or a derivative thereof. .

[0031] According to the angiogenesis promoting agent of claim 9 of the present invention, bone marrow, peripheral blood, umbilical cord blood or the like can be easily obtained even when the same type of cell resource (human vs. human, mouse vs. mouse, etc.) is used. Other cellular resources can be obtained.

[0032] According to the angiogenesis promoter of claim 10 of the present invention, erythroblasts can be easily obtained by in vitro growth.

[0033] The angiogenesis promoter according to claim 11 of the present invention can be easily applied.

[0034] According to the angiogenesis therapy of claim 12 of the present invention, it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.

[0035] According to the angiogenesis therapy of the thirteenth aspect of the present invention, it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.

[0036] According to the angiogenesis therapy of claim 14 of the present invention, it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.

[0037] According to the angiogenesis therapy of claim 15 of the present invention, it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.

Brief Description of Drawings

[0038] Fig. 1 is a graph showing a recovery curve (Fig. 1-1) of cyanosis force in an ischemic limb and a survival curve (Fig. 1-2) of an ischemic limb in Test Example 1 of the present invention.

FIG. 2 is a graph showing a recovery curve (FIG. 2-1) of cyanosis force of ischemic limbs and a survival curve (FIG. 2-2) of ischemic limbs in Test Example 2 of the present invention. FIG. 3 is a graph showing the results of blood flow ratio in Test Example 3 of the present invention.

[Fig. 4] Number of microvessels consisting only of vascular endothelial cells in Test Example 4 of the present invention (Fig. 4-1), number of arterioles with vascular smooth muscle (Fig. 4-2), only from vascular endothelial cells The total area of microvessels (Fig. 4-3), the total area of arterioles with vascular smooth muscle (Fig. 4-4), and the average area per microvessel consisting only of vascular endothelial cells (Fig. 4) -5) and the average area per arteriole with vascular smooth muscle (Figure 4-6).

FIG. 5 is a photomicrograph (FIGS. 5-1-6) showing a cultivated Hn vitro angiogenic sample in Test Example 5 of the present invention.

[Fig. 6] BFU-e, CFU-c, or EPO added (Fig. 6-1), VEG F, P1GF1, P1GF2, Angl, or EPO added (Fig. 6-2), sFltl, Fig. 6 is a graph showing the ratio of blood vessel area when sKDR or BFU-e is added (Figure 6-3).

FIG. 7 is a graph showing the results of secretion amounts of VEGF (FIG. 7-1) and P1GF (FIG. 7-2) in the culture supernatant measured by ELISA in Test Example 5 of the present invention.

FIG. 8 is a graph showing the measurement results of the amount of secreted protein (FIG. 8-1-2) by ELISA and the amount of mRNA (FIG. 8-3-4) by quantitative PCR in Test Example 6 of the present invention. .

FIG. 9 is a graph showing the results of spleen weight (FIG. 9-1), hemoglobin amount (FIG. 9-2), and hematocrit value (FIG. 9-3) in erythropoietin dose change in Test Example 7 of the present invention. It is. BEST MODE FOR CARRYING OUT THE INVENTION

[0039] Hereinafter, the present invention will be described in detail.

[0040] Ischemic diseases to which the angiogenesis-promoting agent and angiogenesis therapy of the present invention are applied are mainly peripheral arterial diseases (obstructive arteriosclerosis, Buerger's disease, ischemic heart disease, cerebrovascular disease, etc.). is there. However, in cases where the arterial stenosis is confined to a thick artery and the peripheral arterial blood flow is relatively maintained compared to this thick artery, surgical treatment such as autologous or human blood vessel transplantation It is preferable to apply the angiogenesis-promoting agent and the angiogenesis therapy of the present invention in cases in which main lesions of blood flow failure are observed in peripheral blood vessels.

In the present specification, the mammal means any mammal including humans, and specific examples include various mammals such as human, rabbit, mouse, guinea pig, chimpanzee, monkey and the like. The

[0042] As the bone marrow cells of mammals, bone marrow cells obtained by general anesthesia of mammals and collecting pelvic force (such as iliac and femoral bones) are also used. In addition, bone marrow cells are collected from autologous bone marrow cells of the patient (the patient's own), or bone marrow cells collected from mammalian relatives to whom an angiogenesis-promoting agent is administered, or matched or partially of histocompatibility antigen (HLA antigen) type. May be bone marrow cells collected from unmatched unrelated animals. The collected bone marrow cells are separated into nucleated cell components or low density cell components by a known method such as specific gravity centrifugation, and used as a cell floating solution for the angiogenesis promoter of the present invention. The cell suspension obtained from bone marrow cells contains cell populations containing erythroblasts (CD235a positive cells), monocytes / macrophages (CD14 positive cells), their progenitor cells, stem cells, and vascular endothelial progenitor cells. (CD34 positive cells).

[0043] As erythroblasts derived from the bone marrow of mammals, bone marrow and the like collected from a part of the pelvis (such as the iliac and femur) after general anesthesia of the mammal, such as by immunomagnetic bead method Purified erythroblast cell suspension may be used. Furthermore, as erythroblasts derived from mammalian peripheral blood, for example, peripheral blood mononuclear cell force collected by Leukapheresis, hematopoietic stem cells purified by immunomagnetic bead method can be used in vitro. You can also use cell suspension of erythroblasts obtained by liquid culture. In addition, as erythroblasts derived from mammalian umbilical cord blood, for example, umbilical cord blood force obtained from human umbilical cord and placenta, hematopoietic stem cells purified by immunomagnetic bead method are used outside the body, and together with various hematopoietic site power-in. Use cell suspension of erythroblasts obtained by liquid culture.

[0044] As a method for sorting CD14-positive cells, CD14-positive cells may be sorted from cells collected from mammalian bone marrow, peripheral blood or umbilical cord blood using, for example, an immunomagnetic bead method. . In addition, hematopoietic stem cells purified from mammalian bone marrow, peripheral blood, or umbilical cord blood by the immunomagnetic bead method are in vitro! Use floating liquid.

[0045] It should be noted that the bone marrow, peripheral blood or umbilical cord blood of a mammal is not limited to that of its own (patient itself), but, for example, bone marrow or blood of a mammal to which an angiogenesis-promoting agent is administered is collected. Peripheral blood, histocompatibility antigen (HLA antigen) type matched or partially mismatched unrelated It may be bone marrow or peripheral blood collected from an object, or neonatal force of related animals and Z or unrelated animals. The histocompatibility antigens are completely the same when using their own cell resources, but the histocompatibility antigens are completely the same when using cell resources of the same species (human vs. human, mouse vs. mouse, etc.). Obtaining cellular resources is not easy and is not necessary, so histocompatibility antigens (

The partial mismatch of the (HLA antigen) type includes cell resources of allogeneic individuals that differ in one or more histocompatibility antigens. In addition, in the angiogenesis promoter of the present invention, when using erythroblasts derived from mammalian bone marrow, peripheral blood, umbilical cord blood or other cell resources, strict antigen compatibility is not required. A cell suspension of erythroblasts or CD14-positive cells obtained by culturing hematopoietic stem cells isolated from non-self blood from a blood bank outside the body and liquid culture with various hematopoietic site force-in may be used. .

[0046] Erythropoietin (EPO) is an acidic glycoprotein hormone that promotes the differentiation and proliferation of erythroid progenitor cells, and mainly produces kidney strength. The most abundant red blood cells in the blood are destroyed in the spleen after functioning for a certain period of time, but are constantly supplied from the bone marrow, so that the total number of peripheral red blood cells is always kept constant under normal conditions. It is leaning. It is generally known that erythropoietin plays a central role in maintaining homeostasis of erythrocytes in the living body. In addition, the administration of erythropoietin to humans has already been performed on a large number of patients due to the “improvement of anemia caused by erythropoietin administration for renal anemia” listed in insurance, and there are sufficient problems such as the safety of the administration. As it has been confirmed, there seems to be no ethical problem regarding actual administration to patients with ischemia.

[0047] The erythropoietin contained in the erythropoietin preparation used for the angiogenesis-promoting agent of the present invention has substantially the same biological activity as that of mammals, particularly human erythropoietin, and is naturally derived erythropoietin. , Modified erythropoietin and z or its derivatives. The modified erythropoietin is a natural-type erythropoietin that has undergone sugar modification, such as sugar chain substitution and deletion 'addition, etc.' by means of modification with enzymes, etc. in vitro. Amino acid alterations that modify the amino acid sequence by deleting, substituting, or adding one or more of the amino acid sequences of erythropoietin. Modified erythropoietin such as lyslopoietin or erythropoietin derivatives obtained by modification of disulfide bond, etc., and those modified erythropoietin having substantially the same biological activity as human erythropoietin. By using these modified erythropoietins, there are advantages that, for example, main effects can be enhanced and side effects can be reduced. In addition, erythropoietin is a hydrophilic substance that is excreted even when administered, but remains in the body, but is modified erythropoietin (specifically, a modified erythropoietin with an addition of a henolin-binding domain). Etc.) can be used for a longer period of time in the body and more effective.

[0048] In addition, the erythropoietin can be produced in Escherichia coli, yeast, Chinese hamster ovary cells, primate cell lines, etc. by well-known genetic engineering techniques, which may be manufactured by any method. Extracted by various methods, separated and purified are used. Furthermore, the sugar chain structure etc. which were changed by enzymatic treatment or chemical treatment are used.

[0049] The erythropoietin preparation used in the present invention is a solution preparation containing erythropoietin obtained as described above. In addition to erythropoietin, components such as a stabilizer usually added to the solution preparation, For example, polyethylene glycol, saccharides, inorganic salts, organic salts, amino acids and the like may be included. As erythropoietin preparations that can also be obtained from Kayabacho, the product names are described, for example, Epogin Injection Syringe, Ampoule 750 (manufactured by Chugai Pharmaceutical Co., Ltd.), Epogin Injection Syringe, Ampoule Injection 1500, 3000 (Chugai Pharmaceutical Co., Ltd.) Epogin Injection Syringe, Ampoule 6000 (manufactured by Chugai Pharmaceutical Co., Ltd.), Epoegin Injection Syringe, Ampoule 9 000 '12000 (manufactured by Chugai Pharmaceutical Co., Ltd.), Espoo Subcutaneous 24000 Syringe (Epoetin α (gene) Recombination), 24000 international units 0.5 mL cylinder, Kirin Co., Ltd.), Espoo subcutaneous 12000 syringe (epoetin (genetical recombination), 12000 international units 0.5 mL cylinder, Kirin Co.).

[0050] The amount of erythropoietin contained in the erythropoietin preparation can be determined according to the type of disease to be treated, the severity of the disease, the age of the patient, etc. , 000 International Units Z Contains 60 kg erythropoietin It is preferable to do. As used herein, one dose means a dose once a day, and the number of administration is preferably 1 to 6 days, more preferably 5 to 6 days continuously. Specifically, it is preferable to administer 6,000 international units Z body weight 60 kg per dose for 5-6 consecutive days. Since erythropoietin is a hydrophilic substance that hardly stays at the site of administration, it is possible to maintain the effect by performing divided administration in this way. In addition, 5,000-50,000 international units Z per dose Z body weight 60 kg is a value converted to a patient with a body weight of 60 kg, and when converted to a weight per kg body weight 83.3-833.3 international unit Z Weight is 1kg. If the body weight is greater than or less than 60 kg, it is desirable to administer a dose converted to the patient's body weight.

[0051] Erythropoietin 5,000-50,000 international units per body dose Z body weight 60kg is a dosage that does not induce side effects such as polycythemia, and 50,000 international units per body weight Z body weight Above 60 kg, polycythemia may occur, and 5,000 international units per dose Z Body weight less than 60 kg cannot fully enhance the main effects.

[0052] The macrophage 'colony stimulating factor (M-CSF) used in the present invention acts on monocyte' macrophage cell (CD14 positive cell) and its progenitor cells, and promotes differentiation 'proliferation. It is. In addition, administration of M-CSF to humans has already been performed on a large number of patients by insurance (Trade name: Leukopro (registered trademark)), and problems such as safety of administration have been sufficiently confirmed. Therefore, there seems to be no ethical problem regarding actual administration to ischemic patients. Efficient angiogenesis can be promoted by administering M-CSF, bone marrow cells, and erythropoietin simultaneously. As M-CSF, an M-CSF preparation (Millimostim) can be used. Examples of M-CSF preparations that can be obtained from Kayabacho include, for example, “Leukoprol (registered trademark)” (manufactured by Kyowa Hakko Co., Ltd.). The amount of M-CSF preparation is a force that can be determined according to the severity of the disease, the age of the patient, etc., preferably 2 × 10 ⁶ — 20 × 10 ⁶ units / weight 60 kg per dose.

[0053] The angiogenesis promoter of the present invention is usually housed in a sealed or sterilized plastic or glass container. Examples of the container form include ampoules, syringes, and vials.

[0054] According to the angiogenesis promoting agent of the present invention, it can be easily applied and induced by erythropoietin. The induced erythroblasts secrete vascular growth-directing site force-in and can promote a strong angiogenic action. Furthermore, since this angiogenic action is enhanced in the presence of erythropoietin, it can promote further angiogenic action, improving the blood flow failure and the resulting ischemia that are the basis of the pathology of ischemic disease. Clinical effect can be improved. In addition, by increasing the number and area of microvessels consisting of vascular endothelium, the arteriole with vascular smooth muscles can be increased to induce larger blood vessels, that is, arteries with high blood supply. it can.

[0055] Next, the angiogenesis therapy of the present invention will be described. The angiogenesis therapy of the present invention is an angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease, and the angiogenesis promoting agent of the present invention is applied to the ischemic site at several tens of strengths. Intramuscular administration is included. Further, the present invention includes an angiogenesis-promoting agent of the present invention, which is administered by introducing an intravascular catheter from a blood vessel at a remote site to reach an ischemic site transvascularly. The route of administration of this preparation is as follows: (1) The preparation is intramuscularly administered directly to the ischemic site percutaneously; (2) The catheter is allowed to reach the ischemic site percutaneously transvascularly, and In addition, it is possible to inject this product. Specifically, for limb ischemia, it can be administered intramuscularly directly to the ischemic site, but for myocardial infarction, the chest is opened under general anesthesia and directly administered intramuscularly to the infarcted site. Alternatively, instead of performing thoracotomy under general anesthesia, the catheter can be inserted by puncturing a blood vessel such as the groin, and the tip of the catheter inside the heart ( The drug may also be injected into the ventricle (intraventricular or coronary). Especially for deep organs such as the heart and brain, the latter can be performed simply by local anesthesia alone.

[0056] Further, the angiogenesis therapy of the present invention comprises a step of collecting bone marrow cells from a mammal, and administering the bone marrow cells to the ischemic site intramuscularly at the ischemic site, or an intravascular catheter from a blood vessel at a remote site. And administering the erythrobotin preparation intramuscularly to the ischemic site, while administering it after transvascularly reaching the ischemic site. In addition, the angiogenesis therapy of the present invention includes a step of collecting bone marrow, peripheral blood or umbilical cord blood from a mammal, fractionating hematopoietic stem cells contained in the bone marrow, peripheral blood or umbilical cord blood, and hematopoietic into the hematopoietic stem cells. The process of liquid culture with the addition of site force-in and the culture obtained by the liquid culture The step of separating erythroblasts and CD14 positive cells from the nutrient solution, and the erythroblasts and CD14 positive cells are administered intramuscularly at several strengths and several strengths at the ischemic site, or from blood vessels at remote sites. Intravascular catheter was introduced and transvascularly reached to the ischemic site for administration, and erythropoietin preparation or erythropoietin preparation and M-CSF preparation were administered intramuscularly from several to several tens of places in the ischemic site. Alternatively, the method may include a step of introducing an intravascular catheter from a blood vessel at a remote site and transvascularly reaching the ischemic site for administration.

[0057] According to the angiogenesis therapy of the present invention, an erythrobotin preparation is simultaneously added to the administration site in the bone marrow or peripheral blood cells, which is a conventional technique as an angiogenesis therapy for an ischemic disease. The technical improvement measures of administration can significantly enhance the angiogenic effect, and improve the clinical effect by improving the blood flow failure and the resulting ischemia that form the basis of the patient's pathology and symptoms. Furthermore, since it is administered only to the ischemic site, there is little risk of side effects to others. In addition, administration of several strengths and several tens of strengths to the ischemic site can spread the formulation evenly in the ischemic site and promote the healing effect. Furthermore, since it is administered only to the ischemic site, there is little risk of side effects on other sites.

[0058] Furthermore, according to the angiogenesis therapy of the present invention, not only the number and area of microvessels composed solely of vascular endothelial cells are increased, but also the area of arterioles with vascular smooth muscles is increased, and more Large blood vessels, that is, arteries with high blood supply can be induced.

[0059] In addition, according to the angiogenesis therapy of the present invention using an angiogenesis promoter, the angiogenesis promoter contains erythroblasts, erythropoietin, CD14-positive cells, M-CSF and the like in one preparation. Therefore, compared to administering bone marrow cells and erythropoietin preparations to patients, it can be administered easily, and the burden on patients and medical staff can be reduced.

[0060] Hereinafter, the present invention will be specifically described with reference to test examples and examples, but the present invention is not limited thereto.

Test example 1

[0061] For the purpose of simulating actual clinical techniques for patients with human ischemic disease, experiments were conducted using a lower limb ischemia model of ICR mice.

[0062] (Method) An incision was made in the left lower limb thigh of an 8-week-old male ICR mouse and all the femoral artery groups were connected. A model of lower limb ischemia was prepared by resection of the heel. After collecting bone marrow from the femur of a syngeneic mouse and using it as a cell suspension, a cell suspension containing 1 x 10 ⁷ bone marrow cells per mouse ischemic limb is applied to the left lower limb thigh muscle at four locations. Intramuscular administration. In addition, 400 international units Z body weight (kg) erythropoietin preparation per dose was administered intramuscularly for 6 days.

[0063] (Comparative test) No administration to the ischemic limb, control group (C), bone marrow cell administration group (B), bone marrow cells after removal of erythroblasts by immunological technique A group (N) that received erythropoietin, a group that received erythropoietin alone (E), and a group that received erythropoietin at the same time as bone marrow cells (BE) were compared using 13 individuals each.

[0064] (Results) Macroscopic findings after administration: In the individuals in Groups C, E and N, necrosis of the lower limbs was mainly observed. In group B, some individuals were able to escape the necrosis of the lower limbs, but individuals who remained cyanosis (skin blue due to blood flow failure) were conspicuous. In the BE group, there were many individuals who escaped from necrosis of the lower limbs, and a good recovery without cyanosis was observed. In addition, a graph of the recovery curve from cyanosis in the ischemic limb is shown in Fig. 1-1, and a graph of the survival curve in the ischemic limb is shown in Fig. 1. The death of the lower limb was defined as the necrosis of all or part of the lower limb. From 1 in Fig. 1, it was confirmed that the recovery of cyanosis was better in the BE group than in the C, E and N groups. Based on 2 in Fig. 1, the BE group showed significant prolongation of lower limb survival compared to the C, E, and N groups.

[0065] From this result, it was shown that in the mouse lower limb ischemia model, simultaneous administration of bone marrow cells and erythropoietin preparation can significantly improve the affected lower limb and increase the survival of the lower limb. It was also shown that angiogenesis by bone marrow cell administration requires erythroblasts contained in the bone marrow, and that the erythropoietin preparation alone is less effective. In addition, elucidation of the effect mechanism in vitro and the actual therapeutic effect in vivo have demonstrated that co-administration of bone marrow cells and erythropoietin preparation significantly improves the inadequate effect of the prior art.

Test example 2

[0066] In the same manner as in Test Example 1 above, the group receiving only the erythroblast component purified from bone marrow (T), the group receiving the erythroblast component purified from bone marrow and CD14 positive cells simultaneously (TM The two groups were compared for 13 individuals. [0067] (Results) A graph of the recovery curve from cyanosis of the ischemic limb is shown in 1 of FIG. 2, and a graph of the survival curve of the ischemic limb is shown in 2 of FIG. The death of the lower limb was defined as the necrosis of all or part of the lower limb. From 1 and 2 in Fig. 2, TM group to which erythroblast component and CD14 positive cells were administered simultaneously showed recovery from cyanosis and prolonged survival of lower limbs. From this result, it was found that erythroblasts cannot show sufficient angiogenic ability in an environment where CD14 positive cells do not exist. In addition, the ability of angiogenesis can be further enhanced by co-administering a bone marrow cell and erythropoietin with a macrophage colony-stimulating factor (M-CSF), which is a site force-in that proliferates and activates CD14 positive cells.

Test example 3

[0068] For the purpose of simulating actual clinical techniques for patients with human ischemic disease, the blood flow of the ischemic limb was measured by the laser Doppler method using the ICR mouse lower limb ischemia model.

[0069] (Method) Prepared by basically the same method as in Test Example 1 (however, the femoral artery group was all ligated to remove it for the purpose of avoiding fallen-out necrosis of the lower limbs). Model mice were treated in a similar manner. The blood flow in the ischemic limb was measured by laser 'Doppler method' in an individual 6 days after ischemia creation.

[0070] (Comparative study) Control group (C), group administered only erythropoietin preparation (E), group administered bone marrow cells (B), and bone marrow cells A comparative study was conducted using 5 to 7 individuals in each of the 4 groups in the group (BE) to which erythropoietin was administered simultaneously.

(Results) Macroscopic findings after administration: Decreased blood flow or absence of blood flow was observed in the ischemic limb compared to the healthy lower limb, and the best recovery of blood flow was observed in the BE group. A graph of blood flow ratio in each of the 4 groups (C, E, B and BE) is shown in FIG. Only the BE group showed a significant improvement (p 0. 05) in blood flow. From this result, it was shown in the mouse lower limb ischemia model that the blood flow in the ischemic limb was improved by simultaneous administration of bone marrow cells and erythropoietin preparation.

Test example 4 [0072] For the purpose of simulating actual clinical techniques for patients with human ischemic disease, muscle tissue specimens of ICR mouse lower limb ischemia models were prepared, and the number of blood vessels and the area of blood vessels were calculated using image processing software.

(Method) The individual whose blood flow rate was measured in Test Example 3 was sacrificed as it was, and the biceps femoris of the ischemic limb was collected to prepare a muscle tissue sample. Vascular endothelium was stained with anti-CD31 antibody. In addition, vascular smooth muscle was stained with an anti-aSMA antibody. A digital image was created using a microscope with a video system, and the number of blood vessels and the blood vessel area (μm) were calculated using image processing software.

(Comparative Test) A comparative study was performed in the same manner as in Test Example 3 above.

[0075] (Results) FIG. 4 shows the results. In Fig. 4, 1, 3, 5 (left column) shows a comparison of vascular endothelial populations, and 2, 4, 6 (right column) of Fig. 4 shows a comparison of vascular smooth muscle populations. Number of microvessels consisting only of vascular endothelial cells (EC number) (1 in Fig. 4): Significant increase in the number of blood vessels in group B (p <0.05) and BE group (p <0.001) As seen, the blood vessel increasing effect in the BE group was significantly higher (p <0.05) than in the B group. Number of arterioles with vascular smooth muscle (SMC number) (2 in Fig. 4): Blood vessels with vascular smooth muscle were a small part of new blood vessels. Total area of microvessels consisting only of vascular endothelial cells (EC area) (Fig. 4-3): Significant (p <0.001) increase in total vascular area was observed only in BE group. Total arteriole area with vascular smooth muscle (SMC area) (Figure 4-4): Significant (p <0.001) increase in total vascular smooth muscle area was observed only in BE group. Compared with the group, it was significantly higher (p 0.01). Mean area per microvessel consisting only of vascular endothelial cells (Mean EC area) (5 in Fig. 4): There was no difference in the average area per blood vessel in each group. This is probably because most of the new blood vessels are microvessels without vascular smooth muscle. Mean SMC area per arteriole with vascular smooth muscle (Figure 4-6): On the other hand, for large blood vessels with vascular smooth muscle, significant in the BE group (P <0. 001) increased vascular smooth muscle mean area, and this effect was significantly higher (p <0. 05) than group B. This result shows that a stronger angiogenic effect can be obtained by co-administering an erythropoietin preparation compared to the single effect of bone marrow cell administration, but the cause is the simultaneous administration of an erythropoietin preparation. Increases the number of microvessels (similar to the effect of vascular endothelial growth factor (VEGF)) At the same time, it was shown that the diameter of large blood vessels with vascular smooth muscle was increased (similar to the effect of placental growth factor (P1GF)), and more effective blood flow was reconstructed.

Test Example 5

[0076] In order to demonstrate theoretical support for the effects of the present invention, in vitro experiments using human peripheral blood cells, human vascular endothelial cells and human fibroblasts were performed.

[0077] (Method) 2 weeks before the start of Hn in vitro angiogenesis culture, erythropoietic colony (BFU-e) in the presence of erythropoietin and leukocyte colony in the presence of G-CSF from peripheral blood of healthy subjects Culture of (CFU-c) was started. Immediately before the start of angiogenic culture, the prepared BFU-e and CFU-c were lifted and added to HI Hn vitro angiogenic culture, and the effect was observed. On day 9 after the start of Hn in vitro angiogenesis culture, the culture specimen was collected and fixed-stained. Blood vessels were stained by the same method as in Test Example 4 and evaluated by digital image processing. Some specimens were stained with two colors of blood vessels (VIIIRAg: colored with green fluorescence) and BFU-e (CD235a: colored with red fluorescence) and observed with a confocal laser microscope. In addition, VEGF and P1GF in the culture supernatant were measured by ELISA.

[0078] (Comparative test) No supplementation, control, supplemented with BFU-e, supplemented with CFU-c, direct addition of various vascular growth tropic site force-in Compared.

(Results) The results are shown in FIG. 5 and FIG. The vascular endothelial cells proliferated and took a tube-like structure, and the vascular-like structures that looked like filaments with weak expansion expanded to form a vascular network. In the control without any addition (1 in Fig. 5), angiogenesis was slightly observed, and when vascular endothelial growth factor (VEGF) was added (2 in Fig. 5), many angiogenesis was observed. Significant angiogenesis was induced by adding BFU-e (3 in Fig. 5). The angiogenesis caused by this BFU-e supplementation is seen to be slightly enlarged (Fig. 5-3), and many large colonies of the new blood vessels are observed. When this image is enlarged (Fig. 5-4), cells are located at the center of the village. A group was seen. When this is observed by double staining (5 and 6 in Fig. 5) of blood vessels (white network structure) and erythroblast populations (gray cell conglomerates), it can be seen that the blood vessel colonies are centered on BF U-e. all right.

[0080] As shown from 1 in Fig. 6, more angiogenesis occurs in the case of adding 10 BFU-e than in the case of adding 3 BFU-e (capacity dependence). This effect was enhanced by co-addition of erythropoietin (EPO) to e. Also, the effect of adding CFU-c Was weak. As shown in Figure 6-2, vascular endothelial growth factor (VEGF) and angiopoietin 1 (Angl) are strong in vascular growth-directing site force-in, and force that exhibits angiogenic action Placental growth factor (P1GF1, P1GF2) 16 international units (IU) Zml of erythropoietin (EPO), and 80 international units (IU) Zml of erythropoietin (EPO) itself had a weak angiogenic effect. As shown in Fig. 6-3, the angiogenic action of BFU-e supplemented and non-supplemented substances was significantly inhibited by the addition of sFlt 1 or sKDR VEGF receptors.

[0081] Fig. 7 shows the secretion amounts of VEGF and P1GF in the culture supernatant measured by ELISA. B FU-e supplementation secreted VEGF (see Figure 7-1) and P1GF (see Figure 7-2), and this effect was further enhanced by erythropoietin supplementation.

[0082] In an in vitro angiogenesis experiment using human vascular endothelial cells, the number of new blood vessels increased significantly due to the addition of erythroblast colonies prepared in the presence of erythropoietin derived from human peripheral blood. Was observed around this erythroblast colony, and it was further observed that this effect was enhanced by further supplementation of the erythropoietin preparation. This result shows that erythroblast colonies prepared from human peripheral blood strongly induce angiogenesis in Hn vitro angiogenesis culture, and that the effect can be further increased by simultaneous addition of erythropoietin preparation. It was.

[0083] In addition, the biological activity of erythropoietin on hematopoietic stem cells is mainly: 1. Inducing erythroblasts from bone marrow 'peripheral blood' umbilical cord blood and other hematopoietic progenitor cells and stem cells, and 2. 3. It is known to induce differentiation and activate proliferating erythroblasts. Therefore, as is clear from the results of this test example, the action point of erythropoietin on angiogenesis is as follows: 1. Bone marrow, peripheral blood, umbilical cord blood and other forces are induced by erythropoietin and erythroblasts have a strong angiogenic action. 2. Furthermore, it is considered that this erythroblast acts at least at these two points that it exerts a stronger angiogenic effect by stimulation of erythropoietin!

Test Example 6

[0084] In order to demonstrate theoretical support for the effects of the present invention, in vitro experiments using human bone marrow cells were performed. [0085] (Method) Erythroblasts and non-erythroblasts were isolated from healthy human bone marrow cells by immunomagnetic bead method and cultured in suspension for 4 days. Two days and four days later, the culture solution and the cultured cells were collected, and the protein amounts of VEGF and P1GF were measured from the culture solution by ELISA, and the mRNA of VEGF and P1GF were measured from the cells by quantitative PCR.

[0086] (Comparative test) Total bone marrow nucleated cells (MNC) before separation, isolated erythroblasts (GPA +), and non-erythroblast nucleated cells (GPA-) were compared. We also compared the effects of adding erythropoietin (EPO) to the culture system.

[0087] (Results) The main cell that produces and secretes vascular growth tropic cytoforce-in (vascular endothelial growth factor (VEGF) and placental growth factor (P1GF)) is erythroblast, and this effect is an erythropoietin preparation It was observed that it was enhanced by further supplementation. The results are shown in FIG. 1 and 2 in FIG. 8 are graphs showing the measurement results of the amount of secreted protein by ELISA. Further, 3 and 4 in FIG. 8 are graphs showing the measurement results of the mRNA amount by the quantitative PCR method. From these results, it is clear that the main cells that produce and secrete vascular growth tropic cytoforce-in (vascular endothelial growth factor (VEGF) and placental growth factor (P1GF)) are erythroblasts, and this effect is due to the effect of erythroid poetin. It was shown to be enhanced by further supplementation of the formulation.

Test Example 7

[0088] An erythropoietin preparation in which the content of erythropoietin was changed was administered to untreated normal mice.

[0089] (Method) An erythropoietin preparation of 40, 400, 4 000 international units Z body weight (kg) was administered intramuscularly to each untreated mouse without creating limb ischemia.

[0090] (Comparative study) No administration, control group (control), 40 international units per dose Group of erythropoietin with Z body weight (kg) administered intramuscularly for 6 days, 400 international units per dose Four groups were administered: intramuscularly administered Z body weight (kg) erythropoietin for 6 days daily, 4,000 international units per dose, and intramuscularly administered erythropoietin for Z body weight (kg) for 6 days daily. A comparative study was conducted using 8 individuals each.

[0091] (Results) The results are shown in FIG. Adverse effects of moderate polycythemia were observed in the group administered 400 erythropoietin preparations of 400 international units Z body weight (kg) per dose (24,000 international units Z body weight 60 kg when converted to 60 kg human adult) It was done. Also 40 international per dose In the group that received the erythropoietin preparation with a unit of Z body weight (kg), the hematocrit value, which is the ratio (volume) of red blood cells to a certain blood volume, was almost the same as the control. Most of the side effects such as polycythemia were not observed. In addition, significant symptomatic side effects were observed in the group receiving 4,000 international units Z body weight (kg) erythropoietin per dose.

Test Example 8

[0092] A human patient with ischemic disease was treated with a cell suspension containing 1 x 10 ⁸ bone marrow (kg) bone marrow cells on the first day of treatment, 6000 international units per dose Z weight 60 kg The erythrobotin preparation was administered for 5 consecutive days up to the 5th day on the first day of treatment. The dose of erythropoietin with a dose of 6,000 international units and Z body weight of 60 kg in this test example was within the range where the side effects of polycythemia were negligible in Test Example 7 above, and 24,000 international units per dose. This is a value calculated by multiplying a Z body weight of 60kg by taking a safety factor of 1Z4 in order to administer it in a smaller amount than that of mice in anticipation of further safety.

As a result, angiogenesis was observed, and side effects such as polycythemia were not observed.

From Test Examples 7 and 8 above, the minimum dose for erythropoietin is 6,000 international units Z weight 60 kg (5,000 international units Z weight 60 kg), which is effective in actual patients and has no side effects. I found it desirable to do it. In addition, since the clinical effect depends on the amount of erythropoietin to be administered, the upper limit of the dose can be 10 times the minimum dose (ie, 50,000 international units Z body weight 60 kg).

Example 1

[0094] (Formulation example)

Aseptic cell suspension containing 5 x 10 ⁹ human bone marrow cells obtained by aseptic manipulation [This is 6,000 international units / 60 kg body weight! Also, 24,000 international units / 60 kg erythropoietin preparation (Described in the pharmacopoeia) was added, and this was filled into a sterile syringe to obtain an angiogenesis promoting agent.

Example 2

[0095] (Formulation example)

Aseptic cell suspension containing 5 x 10 ⁹ human bone marrow cells obtained by aseptic manipulation Liquid [This is 6,000 international units / 60 kg body weight!] 24,000 international units / 60 kg erythropoietin preparation (pharmacopoeia description) and 8,000,000 units M-CSF preparation (millimostim: pharmacopoeia description) ) Was added and filled into a sterile syringe to obtain an angiogenesis-promoting agent.

Example 3

[0096] (Formulation example)

Hematopoietic stem cells purified by immunomagnetic bead method, such as human peripheral blood mononuclear cells obtained by Leu Caffelesis, are spread outside the body and liquid-cultured with various hematopoietic site-in to obtain erythroblast suspension. It was. After washing the cells, add erythrobotin preparation (described in Pharmacopeia) containing 6,000 international units Z body weight 60 kg or 2 4,000 international units Z body weight 60 kg genetically modified erythropoietin, and fill this into a sterile syringe Thus, an angiogenesis promoter was obtained.

Example 4

[0097] (Formulation example)

Hematopoietic stem cells purified by immunomagnetic bead method, such as human peripheral blood mononuclear cells obtained by Leucaulacesis, are spread outside the body and liquid-cultured with various hematopoietic site-in, erythroblasts and CD14 positive cells A mixed suspension was obtained. After washing the cells 6,000 international units Z body weight is 60 kg! /, Is 24,000 international units Z body weight 60 kg erythropoietin preparation containing genetically modified erythropoietin (described in pharmacopoeia) and 8,000, 000 units An angiogenesis-promoting agent was obtained by adding M-CSF preparation (Millimostim: described in the pharmacopoeia) and filling this into a sterile syringe.

Example 5

[0098] (Formulation example)

Umbilical cord blood force obtained from human umbilical cord and placenta was also cultured in vitro with hematopoietic stem cells purified by immunomagnetic bead method together with various hematopoietic site force ins to obtain erythroblast suspension. After washing the cells, add an erythropoietin preparation (described in the pharmacopoeia) of 6,000 international units Z body weight 60 kg or 24,000 international units Z body weight 60 kg, and fill this into a sterile syringe to obtain an angiogenesis promoter. It was. Example 6

(Formulation example)

Hematopoietic stem cells purified from human umbilical cord and placenta by immunomagnetic bead method were also cultured in liquid together with various hematopoietic site-in in vitro to obtain a mixed suspension of erythroblasts and CD14 positive cells. After washing the cells 6,000 international units Z body weight 60kg or 24,000 international units Z body weight 60kg erythropoietin preparation (pharmacopoeia description) and 8,000 0,000 units M-CSF preparation (Millimostim: Pharmacopoeia) And an angiogenesis-promoting agent was obtained by filling this into a sterile syringe.

Claims

The scope of the claims

[I] An angiogenesis-promoting agent comprising a mammalian bone marrow cell and an erythropoietin preparation containing erythropoietin having a weight of 5,000-50,000 international units Z body weight of 60 kg per dose.

[2] The angiogenesis promoter according to [1], further comprising a macrophage 'colony stimulating factor.

[3] The angiogenesis promoter according to [1], which is the erythropoietin power S, naturally-derived erythropoietin, modified erythropoietin or a derivative thereof.

[4] The angiogenesis promoter according to [1], wherein the bone marrow cells are derived from a mammal that is autologous or has a matched or partially mismatched histocompatibility antigen.

[5] The angiogenesis-promoting agent according to claim 1, which is in the form of ampoules, syringes or vials.

[6] erythroblasts derived from mammalian bone marrow, peripheral blood, umbilical cord blood or other cellular resources, CD 14 positive cells, 5,000-50,000 international units per dose Z erythropoietin weighing 60 kg And an erythropoietin preparation containing an angiogenesis promoter.

[7] The angiogenesis promoter according to [6], further comprising a macrophage 'colony stimulating factor.

[8] The angiogenesis promoter according to [6], which is the erythropoietin power S, naturally derived erythropoietin, modified erythropoietin or a derivative thereof.

[9] The angiogenesis promotion according to claim 6, wherein the bone marrow, peripheral blood, umbilical cord blood, or other cell resources are derived from a mammal that is autologous or has a matched or partially mismatched histocompatibility antigen. Agent.

10. The angiogenesis promoter according to claim 6, wherein the erythroblasts derived from the peripheral blood, umbilical cord blood or other cell resources are obtained by in vitro proliferation.

[II] The angiogenesis-promoting agent according to claim 6, which is a dosage form of ampoules, syringes or vials.

[12] An angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease, wherein the angiogenesis promoter according to any one of claims 1 to 11 is disabled. An angiogenesis therapy characterized by intramuscular administration to a blood site.

13. The angiogenesis therapy according to claim 12, wherein the ischemic disease is a peripheral arterial disease.

[14] An angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease, wherein the angiogenesis promoting agent according to any one of claims 1 to 11 is used at a remote site. An angiogenesis therapy characterized by introducing an intravascular catheter from a blood vessel and transvascularly reaching an ischemic site for administration.

15. The angiogenesis treatment according to claim 14, wherein the ischemic disease is a peripheral arterial disease.