WO2005097170A1 - Promoteur d’angiogenie et therapie angiogene - Google Patents

Promoteur d’angiogenie et therapie angiogene Download PDF

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Publication number
WO2005097170A1
WO2005097170A1 PCT/JP2005/003333 JP2005003333W WO2005097170A1 WO 2005097170 A1 WO2005097170 A1 WO 2005097170A1 JP 2005003333 W JP2005003333 W JP 2005003333W WO 2005097170 A1 WO2005097170 A1 WO 2005097170A1
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angiogenesis
erythropoietin
bone marrow
blood
cells
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PCT/JP2005/003333
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English (en)
Japanese (ja)
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Ken Toba
Kiminori Katou
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Niigata Tlo Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to an angiogenesis-promoting agent and angiogenesis therapy for promoting angiogenesis in a mammal having an ischemic disease.
  • ischemic diseases such as extremities, heart, brain, and the like
  • vascular bypass surgery is conventionally used for patients with severe lower limb ischemic disease.
  • problems such as cutting of the lower limbs, if the force vessel was thin and bypassing was not possible.
  • autologous cell transplantation which is an angiogenic therapy
  • 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.
  • ischemic diseases obstructive arteriosclerosis, Buerger's disease, ischemic heart disease, cerebrovascular disease, etc.
  • ischemic diseases obstructive arteriosclerosis, Buerger's disease, ischemic heart disease, cerebrovascular disease, etc.
  • 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
  • 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
  • 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)
  • 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
  • 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.
  • 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.
  • 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!
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 angiogenesis action can be easily applied, and the erythroid hematopoietic cells induced by erythropoietin secrete vascular growth tropic site force in. Can be promoted.
  • 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.
  • 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. .
  • 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.
  • the angiogenesis promoter according to claim 5 of the present invention can be easily applied.
  • 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.
  • 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.
  • angiogenesis-promoting agent 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. .
  • 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.
  • erythroblasts can be easily obtained by in vitro growth.
  • the angiogenesis promoter according to claim 11 of the present invention can be easily applied.
  • angiogenesis therapy of claim 12 of the present invention it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.
  • angiogenesis therapy of the thirteenth aspect of the present invention it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.
  • angiogenesis therapy of claim 14 of the present invention it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.
  • angiogenesis therapy of claim 15 of the present invention it is possible to enhance the therapeutic effect by enhancing the angiogenesis action.
  • 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
  • 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.
  • peripheral arterial diseases obstructive arteriosclerosis, Buerger's disease, ischemic heart disease, cerebrovascular disease, etc.
  • surgical treatment such as autologous or human blood vessel transplantation
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • an immunomagnetic bead method for example, 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.
  • 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.
  • 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. .
  • Erythropoietin 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the erythropoietin preparation used in the present invention is a solution preparation containing erythropoietin obtained as described above.
  • 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.
  • Epogin Injection Syringe 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 ⁇ (gene) Recombination), 24000 international units 0.5 mL cylinder, Kirin Co., Ltd.), Espoo subcutaneous 12000 syringe (epoetin (genetical recombination), 12000 international units
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • M-CSF monocyte' macrophage cell
  • progenitor cells CD14 positive cell
  • M-CSF monocyte' macrophage cell
  • 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.
  • an M-CSF preparation can be used as M-CSF.
  • 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.
  • the angiogenesis promoter of the present invention is usually housed in a sealed or sterilized plastic or glass container.
  • the container form include ampoules, syringes, and vials.
  • the angiogenesis promoting agent of the present invention 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.
  • 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.
  • the arteriole with vascular smooth muscles can be increased to induce larger blood vessels, that is, arteries with high blood supply. it can.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • M-CSF macrophage colony-stimulating factor
  • the blood flow of the ischemic limb was measured by the laser Doppler method using the ICR mouse lower limb ischemia model.
  • Test example 4 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.
  • 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 ( ⁇ m) were calculated using image processing software.
  • FIG. 4 shows the results.
  • 1, 3, 5 shows a comparison of vascular endothelial populations
  • 2, 4, 6 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.
  • EC area Total area of microvessels consisting only of vascular endothelial cells (Fig. 4-3): Significant (p ⁇ 0.001) increase in total vascular area was observed only in BE group.
  • SMC area 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.
  • VEGF vascular endothelial growth factor
  • VIIRAg colored with green fluorescence
  • BFU-e CD235a: colored with red fluorescence
  • VEGF and P1GF in the culture supernatant were measured by ELISA.
  • 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.
  • P1GF1, P1GF2 Placental growth factor
  • EPO erythropoietin
  • IU erythropoietin
  • 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.
  • 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!
  • vascular growth tropic cytoforce-in vascular endothelial growth factor (VEGF) and placental growth factor (P1GF)
  • VEGF vascular endothelial growth factor
  • P1GF placental growth factor
  • vascular growth tropic cytoforce-in vascular endothelial growth factor (VEGF) and placental growth factor (P1GF)
  • VEGF vascular endothelial growth factor
  • P1GF placental growth factor
  • 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.
  • angiogenesis was observed, and side effects such as polycythemia were not observed.
  • 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.
  • the upper limit of the dose can be 10 times the minimum dose (ie, 50,000 international units Z body weight 60 kg).
  • 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.
  • 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.
  • 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.
  • erythrobotin preparation described in Pharmacopeia
  • 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!
  • angiogenesis-promoting agent was obtained by adding M-CSF preparation (Millimostim: described in the pharmacopoeia) and filling this into a sterile syringe.
  • 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.
  • 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.

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Abstract

On entend proposer un promoteur d’angiogénie facile à appliquer, promouvant un effet angiogène et extrêmement efficace pour améliorer la circulation sanguine et une thérapie angiogène. A l’aide d’un promoteur d’angiogénie, contenant une préparation d’érythropoïétine contenant des cellules de moelle osseuse de mammifère et de 5.000 à 50.000 IU/60 kg de poids de corps par dose unitaire d’érythropoïétine et facile à appliquer, des érythroblastes induits par érythropoïétine sécrètent une cytokine vasoproliférative et ainsi l’effet angiogène peut être considérablement favorisé. Comme l’effet angiogène est augmenté en présence d’érythropoïétine, l'effet angiogène s'en trouve encore renforcé. Ainsi, on peut améliorer une faiblesse de la circulation sanguine essentiellement responsable des conditions pathologiques et des symptômes de maladies ischémiques et donc l’ischémie induite et les effets cliniques.
PCT/JP2005/003333 2004-03-30 2005-02-28 Promoteur d’angiogenie et therapie angiogene WO2005097170A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2047859A1 (fr) * 2006-06-07 2009-04-15 The University of Tokushima Traitement d'une maladie ischémique à l'aide d'érythropoïétine
EP2047859A4 (fr) * 2006-06-07 2010-05-05 Univ Tokushima Traitement d'une maladie ischémique à l'aide d'érythropoïétine
AU2007255717B2 (en) * 2006-06-07 2013-07-11 Chugai Seiyaku Kabushiki Kaisha Treatment of ischemic disease using erythropoietin

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