WO2011030847A1 - Inducer composition for hematopoietic stem cells - Google Patents

Inducer composition for hematopoietic stem cells Download PDF

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WO2011030847A1
WO2011030847A1 PCT/JP2010/065594 JP2010065594W WO2011030847A1 WO 2011030847 A1 WO2011030847 A1 WO 2011030847A1 JP 2010065594 W JP2010065594 W JP 2010065594W WO 2011030847 A1 WO2011030847 A1 WO 2011030847A1
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hematopoietic stem
stem cell
stem cells
composition according
composition
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PCT/JP2010/065594
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French (fr)
Japanese (ja)
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丘司 長澤
立樹 杉山
芳樹 尾松
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to a hematopoietic stem cell inducer composition.
  • Hematopoietic stem cells used for hematopoietic stem cell transplantation are usually those collected from donor bone marrow or peripheral blood. Although hematopoietic stem cells can be obtained from preserved umbilical cord blood, transplantation to adults has problems of insufficient number of cells and delayed recovery of hematopoiesis.
  • the donor When hematopoietic stem cells are collected from the bone marrow of the donor, the donor needs to be hospitalized for 1 week to 10 days, is highly invasive, such as being collected under general anesthesia, and the amount of bone marrow fluid collected is large, This puts a heavy burden on the donor.
  • Peripheral blood contains very few hematopoietic stem cells, so when transplanting peripheral blood hematopoietic stem cells, a stem cell mobilizing agent is administered to the donor in advance, and hematopoietic stem cells are induced from the bone marrow into the peripheral blood, and peripheral blood is collected. Done.
  • a stem cell mobilizing agent granulocyte stimulating factor (G-CSF) has already been put into practical use (Non-patent Document 1), and plurixafor (development name: AMD3100, an inhibitor of chemokine receptor CXCR4).
  • Non-patent Document 2 Trade name MOZOBIL, Genzyme
  • Patent Document 1 N-acetylneuraminic acid sulfate, N-acetylneuraminic acid homopolymer sulfate or a pharmaceutically acceptable salt thereof (Patent Document 1) and placenta A constituent cell pulverized product (Patent Document 2) and the like have been proposed.
  • the stem cell mobilization agent is pre-administered at the time of peripheral blood hematopoietic stem cell transplantation, the number of hematopoietic stem cells may not be ensured sufficiently, or apheresis may need to be repeated many times to ensure a sufficient number of hematopoietic stem cells. It is desired to increase the number of hematopoietic stem cells that can be collected during hematopoietic stem cell transplantation.
  • Nuclear receptor PPAR ⁇ is expressed before fetal mesoderm induction, in addition to adipocytes and vascular endothelial cells, fetal liver and adult bone marrow blood progenitor cells, adult peripheral blood leukocytes, It has been reported that it is expressed in the cell stroma of bone marrow (Non-patent Document 4).
  • Thiazolidine compounds such as troglitazone, pioglitazone, rosiglitazone, and the like are known as agonists of PPAR ⁇ receptors. There are various reports on the action of such thiazolidine compounds on hematopoietic stem cells.
  • Non-Patent Document 5 reports that troglitazone stops proliferation of myeloid leukemia-derived cells and hematopoietic stem cells in vitro at the G1 phase and inhibits hematopoietic stem cell proliferation.
  • Non-patent document 6 shows that administration of rosiglitazone increases granulocyte monocyte colony-forming cells (CFU-GM) in mice after administration of anticancer agents, but the effect on the number of CFU-GM even when administered to normal mice Report no.
  • Non-patent document 7 describes the presence of pioglitazone in hematopoietic progenitor cells such as granulocyte monocyte colony forming cells (CFU-GM), eosinophil colony forming cells (CFU-E), and erythroblast precursor cells (BFU-E).
  • CFU-GM granulocyte monocyte colony forming cells
  • CFU-E eosinophil colony forming cells
  • BFU-E erythroblast precursor cells
  • Non-Patent Document 8 reports that administration of pioglitazone to a diabetic patient with coronary artery disease increases the number of vascular endothelial progenitor cells (CD34 + / Kinase insert domain receptor + ).
  • Non-Patent Document 9 reports that administration of pioglitazone to a type 2 diabetic patient increases CD34 positive cells (bone marrow-derived vascular endothelial progenitor cells) in peripheral blood. There is no unified view of the relationship between thiazolidine compounds and hematopoietic systems.
  • pioglitazone hydrochloride has already been used in the clinical setting for the treatment of diabetes as an insulin sensitizer (Patent Document 3 and Non-Patent Document 10). There is no report showing the effect of pioglitazone or a salt thereof on hematopoietic stem cells.
  • An object of the present invention is to provide a hematopoietic stem cell inducer.
  • the present invention provides a hematopoietic stem cell inducer for increasing the number of hematopoietic stem cells in a subject, comprising a thiazolidine compound having a nuclear receptor PPAR ⁇ agonist activity or a salt thereof as an active ingredient.
  • a thiazolidine compound having a nuclear receptor PPAR ⁇ agonist activity or salts thereof include pioglitazone, rosiglitazone, troglitazone and salts thereof, and particularly preferably include pioglitazone and salts thereof.
  • the hematopoietic stem cell inducer of the present invention is preferably used for induction of hematopoietic stem cells in donors of allogeneic hematopoietic stem cell transplantation or autologous hematopoietic stem cell transplantation.
  • Hematopoietic stem cell transplantation may be bone marrow transplantation or peripheral blood hematopoietic stem cell transplantation.
  • the hematopoietic stem cell inducing agent of the present invention is particularly suitable for peripheral blood hematopoietic stem cell transplantation in which it is difficult to secure the necessary number of cells. It is preferably used to increase the number of stem cells.
  • the hematopoietic stem cell inducer of the present invention When used for peripheral blood hematopoietic stem cell transplantation, it is preferable to use the hematopoietic stem cell inducer of the present invention in combination with a hematopoietic stem cell mobilizing agent.
  • the hematopoietic stem cell inducer of the present invention can also be used for the treatment of diseases accompanied by a decrease in hematopoietic stem cells.
  • the hematopoietic stem cell inducer of the present invention is further used for treatment of hematopoietic stem cell reduction in patients undergoing treatment in which hematopoietic stem cell reduction is induced, for example, patients undergoing chemotherapy or radiation therapy for cancer or autoimmune diseases.
  • treatment includes all management such as prevention, treatment, symptom reduction, symptom reduction, progression stoppage and the like.
  • the present invention also increases the number of hematopoietic stem cells in a subject, comprising administering an effective amount of a thiazolidine compound having a nuclear receptor PPAR ⁇ agonist activity or a salt thereof to a subject in need of increasing the number of hematopoietic stem cells. Provide a way to make it happen.
  • subjects that need to increase the number of hematopoietic stem cells include donors for allogeneic hematopoietic stem cell transplantation or autologous hematopoietic stem cell transplantation, and patients suffering from diseases associated with decreased hematopoietic stem cells and such Examples are patients who are highly likely to have a disease, patients undergoing chemotherapy for cancer or autoimmune disease, or cancer radiotherapy.
  • the present invention further relates to the use of a thiazolidine compound having a nuclear receptor PPAR ⁇ agonist activity or a salt thereof for producing a medicament for increasing the number of hematopoietic stem cells.
  • the number of hematopoietic stem cells in the bone marrow increases by administering the hematopoietic stem cell inducer composition of the present invention.
  • the number of hematopoietic stem cell inducer composition of the present invention can be increased at the time of peripheral blood hematopoietic stem cell transplantation. It becomes easy to secure a number of hematopoietic stem cells.
  • the hematopoietic stem cell inducer composition of the present invention is administered to a subject having a reduced number of hematopoietic stem cells or a subject in which a decrease in hematopoietic stem cells is predicted to increase hematopoietic stem cells generated in the bone marrow of the subject. be able to. By such an action, it is possible to effectively prevent or treat diseases accompanied by a decrease in hematopoietic stem cells.
  • HSC Hematopoietic stem cell
  • Example 2 The result of having analyzed HSC fraction of the peripheral blood injection of the mouse
  • Example 3 C shows a control, P shows the result of a pioglitazone administration group.
  • the hematopoietic stem cell inducer composition of the present invention contains a thiazolidine compound having a nuclear receptor PPAR ⁇ agonist activity or a salt thereof as an active ingredient.
  • thiazolidine compounds having agonist activity of the nuclear receptor PPAR ⁇ include compounds disclosed as “thiazolidinedione derivatives” or “insulin resistance improvers” in JP-A-5-70633 and JP-A-2000-355550, for example. Is done.
  • 5- [4- [2- (5-ethyl-2-pyridyl) ethoxy] benzyl] -2,4-thiazolidinedione (generic name: pioglitazone); 5-[[4-[(3, 4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) methoxy] phenyl] methyl] -2,4-thiazolidinedione (generic name: troglitazone); 5 -[[4- [2- (methyl-2-pyridinylamino) ethoxy] phenyl] methyl] -2,4-thiazolidinedione (generic name: rosiglitazone); 5- [3- [4- (5-methyl-2 -Phenyl-4-thiazolylmethoxy) phenyl] propyl] -2,4-oxazolidinedione; 5- [4- (6-meth
  • Salts of thiazolidine compounds include pharmacologically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids.
  • pharmacologically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids.
  • Preferable examples of salts with inorganic bases include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and salts with aluminum and ammonium.
  • Preferable examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like.
  • Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like.
  • Preferable examples of the salt with organic acid include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p -Salts with toluenesulfonic acid and the like.
  • salts with basic amino acids include salts with arginine, lysine, ornithine and the like
  • salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
  • the thiazolidine compound or a salt thereof is preferably pioglitazone, troglitazone, rosiglitazone or a salt thereof, more preferably pioglitazone or a hydrochloride thereof, troglitazone, rosiglitazone or a maleate thereof, particularly preferably pioglitazone.
  • the hydrochloride salt is preferably pioglitazone, troglitazone, rosiglitazone or a salt thereof, more preferably pioglitazone or a hydrochloride thereof, troglitazone, rosiglitazone or a maleate thereof, particularly preferably pioglitazone.
  • the hydrochloride salt is preferably pioglitazone, troglitazone, rosiglitazone or a salt thereof, more preferably pioglitazone or a hydrochloride thereof, troglitazone, rosiglitazone or a maleate thereof, particularly preferably pio
  • Examples of the dosage form of the hematopoietic stem cell inducer composition of the present invention include oral agents such as tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, and films. Moreover, it can be administered as parenteral agents such as injections, external preparations (eg, transdermal preparations), suppositories, pellets, nasal preparations and the like. Oral preparations are more preferably used.
  • Such a pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, for example, a method described in the Japanese Pharmacopoeia.
  • the hematopoietic stem cell inducer composition of the present invention contains a pharmacologically acceptable carrier in addition to a thiazolidinedione derivative having a nuclear receptor PPAR ⁇ agonist activity or a pharmaceutically acceptable salt thereof.
  • a pharmacologically acceptable carrier various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, dissolution aids It is formulated as an agent, suspending agent, isotonic agent, buffering agent, soothing agent and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used. As such components, those described in general textbooks of pharmaceutics can be appropriately used depending on the purpose.
  • Pioglitazone hydrochloride which is a preferred active ingredient of the hematopoietic stem cell inducer composition of the present invention, is currently marketed as an insulin resistance improving drug (Actos (registered trademark) Takeda Pharmaceutical Co., Ltd.), but the hematopoietic stem cell of the present invention.
  • an insulin resistance improving drug Actos (registered trademark) Takeda Pharmaceutical Co., Ltd.
  • the inducer composition such a commercially available drug may be used.
  • hematopoietic stem cells are pluripotent cells that can differentiate into all blood cells.
  • the hematopoietic stem cell inducer composition of the present invention is suitably used for increasing the number of hematopoietic stem cells in a transplant donor in transplantation of hematopoietic stem cells.
  • the composition of the present application is suitably used for both allogeneic hematopoietic stem cell transplant donors and patients who are autologous hematopoietic stem cell transplant donors.
  • the term “hematopoietic stem cell transplant donor” or “donor” is used to include both allogeneic and autologous donors.
  • the dose and administration schedule of the hematopoietic stem cell inducer composition of the present invention are not limited, and may be appropriately set depending on the purpose of inducing hematopoietic stem cells and the age, weight, sex, etc. of the administration target.
  • administration to a hematopoietic stem cell transplant donor it may be appropriately set depending on the donor's age, weight, sex, general health status, bone marrow transplant or peripheral blood hematopoietic stem cell transplant, desired hematopoietic stem cell amount, and the like.
  • decisions should be made to meet the overall treatment plan.
  • the daily dose of pioglitazone or a salt thereof is 0.1-100 mg, preferably 1-50 mg, The dose is preferably administered once a day or in multiple doses so that the dose is 45 mg or less.
  • the administration period is not limited and may be determined as appropriate. For example, administration may be performed for 2 to 80 days.
  • the number of hematopoietic stem cells in the bone marrow increases by administering the hematopoietic stem cell inducer composition of the present invention to a bone marrow transplant or peripheral blood hematopoietic stem cell transplant donor.
  • the number of hematopoietic stem cells in the bone marrow increases, in the case of bone marrow transplantation, it is expected that the amount of bone marrow fluid collected from the donor can be reduced and the burden on the donor can be reduced.
  • the number of hematopoietic stem cells in peripheral blood can be increased by administering the hematopoietic stem cell inducer composition of the present invention and then administering the hematopoietic stem cell mobilizing agent.
  • hematopoietic stem cell mobilizing agent refers to a substance having an effect of mobilizing hematopoietic stem cells in bone marrow into peripheral blood.
  • Hematopoietic stem cell mobilization agents are not limited to known ones, and those that will be developed in the future as hematopoietic stem cell mobilization agents may be used in combination with the pharmaceutical composition of the present invention.
  • hematopoietic stem cell mobilizing agent granulocyte colony-stimulating factor (G-CSF), plerixafor hydrochloride, N-acetylneuraminic acid sulfate, N-acetylneuraminic acid homopolymer sulfate or pharmaceuticals thereof
  • G-CSF granulocyte colony-stimulating factor
  • plerixafor hydrochloride N-acetylneuraminic acid sulfate
  • N-acetylneuraminic acid homopolymer sulfate or pharmaceuticals thereof examples of such a salt are acceptable, (Patent Document 1), pulverized placenta-constituting cells (Patent Document 2) and the like, but are not limited thereto.
  • one or two or more hematopoietic stem cell mobilizing agents may be used in combination.
  • the dose and administration schedule of the hematopoietic stem cell mobilizing agent may be appropriately determined according to the age, weight, sex, general health status, desired hematopoietic stem cell amount, etc. of the donor.
  • G-CSF When G-CSF is used, it may be administered according to the current clinical protocol. As a typical example, about 10 ⁇ g / kg / day of G-SCF per donor body weight is divided into once or twice a day. Subcutaneous administration may be continued every day for 4 to 6 days, and then peripheral blood may be collected.
  • the dose and administration period of G-CSF may be lower than those currently used in clinical practice as hematopoietic stem cells increase with the hematopoietic stem cell inducer of the present invention.
  • prelixaphor or other hematopoietic stem cell mobilizing agent it may be adjusted based on the known usage and dose of each hematopoietic stem cell mobilizing agent.
  • any known method may be employed for collecting hematopoietic stem cells from a donor.
  • bone marrow transplantation bone marrow fluid is collected from a donor and, if necessary, the one from which impurities are removed with a mesh or filter is used for transplantation. If necessary, red blood cells or plasma components may be removed from the bone marrow fluid.
  • contamination with cancer cells or the like is predicted, such as in autologous transplantation, it is preferable to remove the cancer cells or transplant after further purifying the hematopoietic stem cell fraction with a surface marker or the like.
  • CD34 As a surface marker that can be used for purification of hematopoietic stem cells, CD34 is exemplified, and it is known that hematopoietic stem cells are CD34 positive. Other markers may be used as long as hematopoietic stem cells can be purified.
  • peripheral blood from a donor is subjected to extracorporeal circulation (apheresis) using a continuous blood component separation device to isolate a mononuclear cell fraction in the peripheral blood, and the remaining blood is returned to the donor.
  • the isolated mononuclear cell fraction may be further purified as necessary.
  • any of the conventionally known methods may be adopted, but for example, purification may be performed based on a surface marker such as CD34.
  • purification may be performed based on a surface marker such as CD34.
  • known means such as a flow cytometer, an immunomagnetic bead method using an anti-CD34 antibody, and an immune avidin bead method may be used.
  • the hematopoietic stem cells obtained from a donor using the composition of the present invention are suitably used for treatment of various diseases that can be treated by hematopoietic stem cell transplantation.
  • diseases include, but are not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, acute myeloid leukemia, neuroblastoma, rhabdomyosarcoma, brain tumor ( Autologous diseases such as collagenous disease, and so on. Examples thereof include amyloidosis.
  • Diseases for which allogeneic hematopoietic stem cell transplantation is effective include acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, kidney Malignant tumors such as cell cancer, pancreatic cancer, colon cancer, breast cancer and aplastic anemia, paroxysmal nocturnal hemoglobinuria, severe combined immunodeficiency, Fanconi anemia, Diamond-Blackfan anemia, sickle cell disease, Wiskott-Aldrich syndrome Examples include marble bone disease, inborn metabolic diseases and autoimmune diseases.
  • the composition of the present invention is also useful for the treatment of diseases accompanied by a decrease in hematopoietic stem cells.
  • Diseases with decreased hematopoietic stem cells include chronic inflammation, malignant tumor, severe infection, autoimmune disease, diabetes, sickle cell disease, uremia, myelodysplastic syndrome, Fanconi anemia, aplastic anemia, chronic liver injury And renal failure, myelopathic thrombocytopenia, idiopathic thrombocytopenic purpura and the like.
  • the compositions of the present invention are further usefully used to increase the number of hematopoietic stem cells in the bone marrow of patients undergoing cancer chemotherapy, cancer radiotherapy, autoimmune disease chemotherapy, or autoimmune disease radiation therapy. It is done.
  • the dosage of the hematopoietic stem cell inducer composition of the present invention is not limited, and may be appropriately set depending on the age, weight, sex, etc. of the administration subject.
  • the daily dose of pioglitazone or a salt thereof is 0.1 to 100 mg, preferably 1 to 50 mg, more preferably 45 mg or less. Dosage once to several times a day.
  • mice Hematopoietic stem cell inducing ability of pioglitazone hydrochloride 15-25 week old mice were used. Feed was prepared by mixing pioglitazone hydrochloride (Takeda Pharmaceutical Co., Ltd.) into a standard chow for mice for 0.7 mg / g solid feed for 8 weeks. The control group was fed the same chow diet except that it did not contain pioglitazone hydrochloride. Four mice were used for each group.
  • the mice were killed and bone marrow cells were washed out from the femur and tibia to obtain a bone marrow single cell suspension.
  • the resulting bone marrow cell suspension is known as CD45, CD3 ⁇ , B220, c-Kit, CD48, Gr-1, CD11b, CD34, CD150, Sca-1, Ter119, Lin, C03, C011b, Br-1 antibodies. Staining was performed by the method (M. Osawa et al., Science 273, 242 (1996)).
  • the obtained cells were analyzed by flow cytometry (BD FACSAria (BD Biosystems)), and CD34 ( ⁇ ) CD48 ( ⁇ ) c-Kit (+) Sca-1 (+) Lin ( ⁇ ) cells in bone marrow cells.
  • the fraction fraction was measured. It is known that about 40% of this cell fraction is hematopoietic stem cells (HSC) in mice.
  • HSC hematopoietic stem cells
  • the antibodies used are as follows: CD45 (30-F11), CD3 ⁇ (145-2C11), B220 (RA3-6B2), c-Kit (2B8), CD48 (HM48-1), Gr-1 (RB6-8C5), CD11b (M1 / 70) CD34 (RAM34), CD150 (TC15-12F12.2), Sca-1 (E13-1617.7, D7), Ter119 (Ter119)
  • the number of hematopoietic stem cells in the bone marrow was significantly increased in the mice administered with the composition of the present invention compared to the control mice.
  • Hematopoietic stem cells were induced into peripheral blood using pioglitazone hydrochloride and G-CSF. 15-25 week old mice were used.
  • a feed containing pioglitazone hydrochloride was administered for 8 weeks. Thereafter, the mice were fed with food containing no pioglitazone hydrochloride, and G-CSF was subcutaneously administered once a day for 5 days at 100 ng / g / day.
  • Control mice were fed a diet containing no pioglitazone hydrochloride for 8 weeks, and then G-CSF was administered according to the same protocol as in the test group.
  • mice peripheral blood was collected, and a mononuclear cell fraction was obtained by hemolysis. This was stained in the same manner as in Example 1, and hematopoietic stem cell fraction CD34 was obtained by flow cytometry. The proportion of ( ⁇ ) CD48 ( ⁇ ) c-Kit (+) Sca-1 (+) Lin ( ⁇ ) cells was examined. Three mice were used for each group. The results are shown in FIG.
  • the hematopoietic stem cell fraction in the peripheral blood of mice administered with pioglitazone hydrochloride increased compared to the control group.
  • Hematopoietic stem cell induction into peripheral blood was performed using pioglitazone hydrochloride and AMD3100 (prerixafor). 15-25 week old mice were used.
  • a feed containing pioglitazone hydrochloride was administered for 8 weeks. Thereafter, the feeding to the mice was changed to one containing no pioglitazone hydrochloride, and 5 mg / kg of AMD3100 was subcutaneously administered.
  • Control mice were fed a diet containing no pioglitazone hydrochloride for 8 weeks and then administered AMD3100 in the same protocol as the test group.
  • One hour after administration of AMD3100 mouse peripheral blood was collected, and a mononuclear cell fraction was obtained by hemolysis.

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Abstract

Provided is an inducer composition for hematopoietic stem cells, which contains a thiazolidine compound or salts thereof having nuclear receptor PPARγ agonist activity, and which serves to increase the number of hematopoietic stem cells in a subject. The inducer composition for hematopoietic stem cells can increase the number of hematopoietic stem cells for bone-marrow transplantation, and also increase the number of peripheral blood hematopoietic stem cells in combination with a hematopoietic stem cell mobilizer. The inducer composition for hematopoietic stem cells is useful for increasing the number of hematopoietic stem cells of a hematopoietic stem cell donor, and may be used for any donor of autologous hematopoietic stem-cell transplantation and allogeneic hematopoietic stem cell transplantation. In addition, the inducer composition for hematopoietic stem cells is also useful for treatment of conditions associated with a decrease in hematopoietic stem cells and treatment of patients undergoing chemotherapy or radiation therapy.

Description

造血幹細胞誘導剤組成物Hematopoietic stem cell inducer composition
　本発明は、造血幹細胞誘導剤組成物に関する。 The present invention relates to a hematopoietic stem cell inducer composition.
　強力な抗がん剤投与で悪性腫瘍を治療した後、抗がん剤の副作用で正常な造血幹細胞が枯渇する患者等に対して、患者自身または組織適合抗原より選択された他人の提供者より採取した造血幹細胞を移植して補う自家造血幹細胞移植および同種造血幹細胞移植は血液系を中心とした悪性腫瘍の治療などで実践され、大きな成果をあげている。 For patients with normal hematopoietic stem cells depleted due to side effects of anticancer drugs after treating malignant tumors with powerful anticancer drugs, from patients themselves or other donors selected from histocompatibility antigens Autologous hematopoietic stem cell transplantation and allogeneic hematopoietic stem cell transplantation, in which collected hematopoietic stem cells are transplanted and supplemented, have been practiced in the treatment of malignant tumors centered on the blood system, and have achieved great results.
　造血幹細胞移植に使用される造血幹細胞は通常、ドナーの骨髄もしくは末梢血から採取されたものが用いられる。保存臍帯血から造血幹細胞を得ることも可能であるが、成人への移植には、細胞数の不足と造血の回復遅延の問題がある。 Hematopoietic stem cells used for hematopoietic stem cell transplantation are usually those collected from donor bone marrow or peripheral blood. Although hematopoietic stem cells can be obtained from preserved umbilical cord blood, transplantation to adults has problems of insufficient number of cells and delayed recovery of hematopoiesis.
　造血幹細胞をドナーの骨髄から採取する場合は、ドナーは１週間から１０日の入院が必要となり、また全身麻酔下での採取となるなど侵襲性が高く、また骨髄液採取量も多量であり、ドナーに大きな負担をかけることになる。 When hematopoietic stem cells are collected from the bone marrow of the donor, the donor needs to be hospitalized for 1 week to 10 days, is highly invasive, such as being collected under general anesthesia, and the amount of bone marrow fluid collected is large, This puts a heavy burden on the donor.
　ドナーから造血幹細胞を採取する方法として、ドナーの末梢血造血幹細胞を採取する方法も実用化されている。かかる方法においては全身麻酔の必要はなく、ドナーへの侵襲性は骨髄移植に比してかなり低い。 As a method of collecting hematopoietic stem cells from a donor, a method of collecting peripheral blood hematopoietic stem cells of a donor has been put into practical use. Such methods do not require general anesthesia and are much less invasive to donors than bone marrow transplants.
　末梢血中に含まれる造血幹細胞は非常に少ないことから、末梢血造血幹細胞移植に際してはドナーへ予め幹細胞動員剤を投与し、骨髄から末梢血中へ造血幹細胞を誘導した上で末梢血の採取が行われる。かかる造血幹細胞動員剤としては、顆粒球刺激因子（Ｇ－ＣＳＦ）が既に実用化されており（非特許文献１）、また、ケモカイン受容体CXCR4の阻害剤であるプレリキサフォル（plerixafor 開発名ＡＭＤ３１００、商品名ＭＯＺＯＢＩＬ、ジェンザイム社）が米国において末梢血造血幹細胞自家移植における造血幹細胞動員剤としてＧ－ＣＳＦと組み合わせての使用が承認されている（非特許文献２）。また、プレリキサフォル単独投与にても造血幹細胞の末梢血への動員が可能であるとの報告もある（非特許文献３）。 Peripheral blood contains very few hematopoietic stem cells, so when transplanting peripheral blood hematopoietic stem cells, a stem cell mobilizing agent is administered to the donor in advance, and hematopoietic stem cells are induced from the bone marrow into the peripheral blood, and peripheral blood is collected. Done. As such a hematopoietic stem cell mobilizing agent, granulocyte stimulating factor (G-CSF) has already been put into practical use (Non-patent Document 1), and plurixafor (development name: AMD3100, an inhibitor of chemokine receptor CXCR4). (Trade name MOZOBIL, Genzyme) has been approved for use in combination with G-CSF as a hematopoietic stem cell mobilizing agent in autologous transplantation of peripheral blood hematopoietic stem cells (Non-patent Document 2). There is also a report that hematopoietic stem cells can be mobilized to peripheral blood even by administering pririxafor alone (Non-patent Document 3).
　また、造血幹細胞の末梢血中への誘導剤としては、Ｎ－アセチルノイラミン酸硫酸エステル、Ｎ－アセチルノイラミン酸ホモポリマー硫酸エステルまたはその薬学的に許容される塩（特許文献１）および胎盤構成細胞粉砕物（特許文献２）などが提案されている。 In addition, as an inducer of hematopoietic stem cells into peripheral blood, N-acetylneuraminic acid sulfate, N-acetylneuraminic acid homopolymer sulfate or a pharmaceutically acceptable salt thereof (Patent Document 1) and placenta A constituent cell pulverized product (Patent Document 2) and the like have been proposed.
　しかしながら、末梢血造血幹細胞移植に際して幹細胞動員剤を事前投与したとしても造血幹細胞数が十分確保できない場合や、十分な造血幹細胞数確保のためにアフェレーシスを何度も繰り返す必要がある場合があり、末梢血造血幹細胞移植に際して採取されうる造血幹細胞を増やすことが望まれている。 However, even if the stem cell mobilization agent is pre-administered at the time of peripheral blood hematopoietic stem cell transplantation, the number of hematopoietic stem cells may not be ensured sufficiently, or apheresis may need to be repeated many times to ensure a sufficient number of hematopoietic stem cells. It is desired to increase the number of hematopoietic stem cells that can be collected during hematopoietic stem cell transplantation.
　また造血幹細胞の減少を伴う疾患の予防および治療、並びに癌や自己免疫疾患の患者における化学療法や放射線療法による造血幹細胞の減少の予防および治療には、体内における造血幹細胞産生を効果的に増やすことのできる処置が求められている。 In addition, for the prevention and treatment of diseases associated with the reduction of hematopoietic stem cells, as well as the prevention and treatment of hematopoietic stem cell depletion caused by chemotherapy and radiation therapy in patients with cancer and autoimmune diseases, it is necessary to effectively increase hematopoietic stem cell production in the body. There is a need for an action that can be performed.
　核内受容体ＰＰＡＲγは胎児期の中胚葉誘導前から発現が認められ、脂肪細胞や血管内皮細胞の他、胎児肝や成体骨髄の血液前駆細胞の一部や成体末梢血の白血球の一部、骨髄の細胞間質などで発現していることが報告されている（非特許文献４）。 Nuclear receptor PPARγ is expressed before fetal mesoderm induction, in addition to adipocytes and vascular endothelial cells, fetal liver and adult bone marrow blood progenitor cells, adult peripheral blood leukocytes, It has been reported that it is expressed in the cell stroma of bone marrow (Non-patent Document 4).
　ＰＰＡＲγ受容体のアゴニストとしてトログリタゾン（troglitazone）、ピオグリタゾン(pioglitazone)、ロシグリタゾン(rosiglitazone)などのチアゾリジン系化合物が知られている。かかるチアゾリジン系化合物の造血幹細胞への作用としては、種々の報告がある。非特許文献５は、トログリタゾンがインビトロで骨髄性白血病由来細胞や造血幹細胞の増殖をG1期で停止させ、造血幹細胞の増殖を阻害することを報告する。非特許文献６はロシグリタゾンの投与によって抗がん剤投与後のマウスの顆粒球単球コロニー形成細胞（CFU-GM)が増加するが、正常マウスに投与してもCFU-GM数への影響がないことを報告する。非特許文献７は顆粒球単球コロニー形成細胞（CFU-GM)、好酸球コロニー形成細胞（CFU-E)、前記赤芽球系前駆細胞（BFU-E)等の造血前駆細胞をピオグリタゾン存在下で培養すると、コロニー数が減少する、即ちピオグリタゾンが造血前駆細胞の増殖を阻害することを報告する。非特許文献８は冠動脈疾患を伴う糖尿病患者へピオグリタゾンを投与すると、血管内皮前駆細胞（ＣＤ３４＋／Kinase insert domain receptor+)数が増加することを報告する。非特許文献９は２型糖尿病患者へピオグリタゾンを投与すると、末梢血中のＣＤ３４陽性細胞（骨髄由来血管内皮前駆細胞）が増加することを報告する。チアゾリジン系化合物と造血系の関係については、統一した見解は得られていない。 Thiazolidine compounds such as troglitazone, pioglitazone, rosiglitazone, and the like are known as agonists of PPARγ receptors. There are various reports on the action of such thiazolidine compounds on hematopoietic stem cells. Non-Patent Document 5 reports that troglitazone stops proliferation of myeloid leukemia-derived cells and hematopoietic stem cells in vitro at the G1 phase and inhibits hematopoietic stem cell proliferation. Non-patent document 6 shows that administration of rosiglitazone increases granulocyte monocyte colony-forming cells (CFU-GM) in mice after administration of anticancer agents, but the effect on the number of CFU-GM even when administered to normal mice Report no. Non-patent document 7 describes the presence of pioglitazone in hematopoietic progenitor cells such as granulocyte monocyte colony forming cells (CFU-GM), eosinophil colony forming cells (CFU-E), and erythroblast precursor cells (BFU-E). We report that the number of colonies decreases when cultured under, ie, pioglitazone inhibits the growth of hematopoietic progenitor cells. Non-Patent Document 8 reports that administration of pioglitazone to a diabetic patient with coronary artery disease increases the number of vascular endothelial progenitor cells (CD34 + / Kinase insert domain receptor + ). Non-Patent Document 9 reports that administration of pioglitazone to a type 2 diabetic patient increases CD34 positive cells (bone marrow-derived vascular endothelial progenitor cells) in peripheral blood. There is no unified view of the relationship between thiazolidine compounds and hematopoietic systems.
　なお、チアゾリジン系化合物のうち、ピオグリタゾン塩酸塩はインスリン抵抗性改善薬として糖尿病の治療に臨床現場で既に用いられている（特許文献３および非特許文献１０）。ピオグリタゾンまたはその塩について、造血幹細胞への作用を示す報告は無い。 Of the thiazolidine compounds, pioglitazone hydrochloride has already been used in the clinical setting for the treatment of diabetes as an insulin sensitizer (Patent Document 3 and Non-Patent Document 10). There is no report showing the effect of pioglitazone or a salt thereof on hematopoietic stem cells.
特開２００６－３４２１３１号公報JP 2006-342131 A 特開２００７－１０６７６０号公報JP 2007-106760 A 特開昭６０－２０８９８０号公報JP 60-208980 A
　本発明は、造血幹細胞誘導剤を提供することを目的とする。 An object of the present invention is to provide a hematopoietic stem cell inducer.
　本発明は、核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩を有効成分とする、対象における造血幹細胞数を増加させるための造血幹細胞誘導剤を提供する。核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩としては、好ましくはピオグリタゾン、ロシグリタゾン、トログリタゾンおよびその塩が例示され、特に好ましくはピオグリタゾンおよびその塩が例示される。 The present invention provides a hematopoietic stem cell inducer for increasing the number of hematopoietic stem cells in a subject, comprising a thiazolidine compound having a nuclear receptor PPARγ agonist activity or a salt thereof as an active ingredient. Preferable examples of thiazolidine compounds having a nuclear receptor PPARγ agonist activity or salts thereof include pioglitazone, rosiglitazone, troglitazone and salts thereof, and particularly preferably include pioglitazone and salts thereof.
　本発明の造血幹細胞誘導剤は好適には同種造血幹細胞移植または自家造血幹細胞移植のドナーにおける造血幹細胞の誘導に用いられる。造血幹細胞移植は、骨髄移植でも、末梢血造血幹細胞移植でもよいが、本発明の造血幹細胞誘導剤は、特に必要細胞数の確保が困難である末梢血造血幹細胞移植において、ドナー末梢血中の造血幹細胞数を増加させるのに好適に用いられる。 The hematopoietic stem cell inducer of the present invention is preferably used for induction of hematopoietic stem cells in donors of allogeneic hematopoietic stem cell transplantation or autologous hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation may be bone marrow transplantation or peripheral blood hematopoietic stem cell transplantation. However, the hematopoietic stem cell inducing agent of the present invention is particularly suitable for peripheral blood hematopoietic stem cell transplantation in which it is difficult to secure the necessary number of cells. It is preferably used to increase the number of stem cells.
　本発明の造血幹細胞誘導剤を末梢血造血幹細胞移植に用いる場合には、本発明の造血幹細胞誘導剤を造血幹細胞動員剤と組み合わせて用いるのが好適である。 When the hematopoietic stem cell inducer of the present invention is used for peripheral blood hematopoietic stem cell transplantation, it is preferable to use the hematopoietic stem cell inducer of the present invention in combination with a hematopoietic stem cell mobilizing agent.
　本発明の造血幹細胞誘導剤はまた、造血幹細胞の減少を伴う疾患の処置のために用いることができる。本発明の造血幹細胞誘導剤はさらに、造血幹細胞の減少が誘導される治療を受けている患者、例えば癌や自己免疫疾患の化学療法や放射線療法を受けている患者において、造血幹細胞減少の処置のために用いることができる。本発明において「処置」には予防、治療、症状の軽減、症状の減退、進行停止等、あらゆる管理が含まれる。 The hematopoietic stem cell inducer of the present invention can also be used for the treatment of diseases accompanied by a decrease in hematopoietic stem cells. The hematopoietic stem cell inducer of the present invention is further used for treatment of hematopoietic stem cell reduction in patients undergoing treatment in which hematopoietic stem cell reduction is induced, for example, patients undergoing chemotherapy or radiation therapy for cancer or autoimmune diseases. Can be used for In the present invention, “treatment” includes all management such as prevention, treatment, symptom reduction, symptom reduction, progression stoppage and the like.
　本発明はまた、造血幹細胞数を増加させることが必要な対象に、有効量の核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩を投与することを含む、対象における造血幹細胞数を増加させる方法を提供する。本発明の方法において、「造血幹細胞数を増加させることが必要な対象」としては、同種造血幹細胞移植または自家造血幹細胞移植のドナー、並びに造血幹細胞の減少を伴う疾患に罹患している患者およびかかる疾患に罹患する可能性が高い患者、癌や自己免疫疾患の化学療法または癌放射線療法を受けている患者が例示される。 The present invention also increases the number of hematopoietic stem cells in a subject, comprising administering an effective amount of a thiazolidine compound having a nuclear receptor PPARγ agonist activity or a salt thereof to a subject in need of increasing the number of hematopoietic stem cells. Provide a way to make it happen. In the method of the present invention, "subjects that need to increase the number of hematopoietic stem cells" include donors for allogeneic hematopoietic stem cell transplantation or autologous hematopoietic stem cell transplantation, and patients suffering from diseases associated with decreased hematopoietic stem cells and such Examples are patients who are highly likely to have a disease, patients undergoing chemotherapy for cancer or autoimmune disease, or cancer radiotherapy.
　本発明はさらに、核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩の、造血幹細胞数を増加させるための医薬の製造のための使用に関する。 The present invention further relates to the use of a thiazolidine compound having a nuclear receptor PPARγ agonist activity or a salt thereof for producing a medicament for increasing the number of hematopoietic stem cells.
　本発明の造血幹細胞誘導剤組成物を投与することによって、骨髄中の造血幹細胞数が増加する。本発明の造血幹細胞誘導剤組成物を造血幹細胞動員剤と組み合わせることによって、末梢血造血幹細胞移植に際してドナーの末梢血中に含まれる造血幹細胞数を増加させることができ、末梢血造血幹細胞移植に際して必要数の造血幹細胞を確保することが容易となる。 The number of hematopoietic stem cells in the bone marrow increases by administering the hematopoietic stem cell inducer composition of the present invention. By combining the hematopoietic stem cell inducer composition of the present invention with a hematopoietic stem cell mobilizing agent, the number of hematopoietic stem cells contained in the peripheral blood of the donor can be increased at the time of peripheral blood hematopoietic stem cell transplantation. It becomes easy to secure a number of hematopoietic stem cells.
　また、本発明の造血幹細胞誘導剤組成物を、造血幹細胞数が減少した対象、あるいは造血幹細胞の減少が予測される対象に投与することによって、対象の骨髄にて生成される造血幹細胞を増加させることができる。かかる作用によって、造血幹細胞の減少を伴う疾患の予防や治療を効果的に行うことができる。 In addition, the hematopoietic stem cell inducer composition of the present invention is administered to a subject having a reduced number of hematopoietic stem cells or a subject in which a decrease in hematopoietic stem cells is predicted to increase hematopoietic stem cells generated in the bone marrow of the subject. be able to. By such an action, it is possible to effectively prevent or treat diseases accompanied by a decrease in hematopoietic stem cells.
ピオグリタゾン塩酸塩を８週間投与したマウス（piglitazone）と非投与マウス（control）の骨髄細胞中の造血幹細胞（ＨＳＣ）分画（ＣＤ３４（－）ＣＤ４８（－）ｃ－Ｋｉｔ（＋）Ｓｃａ－１（＋）Ｌｉｎ（－））をフローサイトメトリーにて解析した結果を示す。（実施例１）Hematopoietic stem cell (HSC) fractions (CD34 (−) CD48 (−) c-Kit (+) Sca-1 () in bone marrow cells of mice administered with pioglitazone hydrochloride for 8 weeks (piglitazone) and mice not administered (control) (+) Lin (-)) is analyzed by flow cytometry. Example 1 ピオグリタゾン塩酸塩を８週間投与した後、Ｇ－ＣＳＦを５日間投与したマウスの末梢血中のＨＳＣ分画をフローサイトメトリーにて解析した結果を示す。（実施例２）The results of analysis of HSC fraction in peripheral blood of mice administered with pioglitazone hydrochloride for 8 weeks and then administered with G-CSF for 5 days by flow cytometry are shown. (Example 2) ピオグリタゾン塩酸塩を８週間投与した後、ＡＭＤ３１００を１回投与したマウスの抹消血注のＨＳＣ分画をフローサイトメトリーにて解析した結果を示す。（実施例３）Ｃはコントロールを、Ｐはピオグリタゾン投与群の結果を示す。The result of having analyzed HSC fraction of the peripheral blood injection of the mouse | mouth which administered AMD3100 once after administering pioglitazone hydrochloride for 8 weeks is shown by the flow cytometry. (Example 3) C shows a control, P shows the result of a pioglitazone administration group.
　本発明の造血幹細胞誘導剤組成物は、核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩を有効成分として含有する。核内受容体ＰＰＡＲγのアゴニスト活性を有するチアゾリジン系化合物としては、例えば特開平５－７０６３３および特開２０００－３５５５５０に「チアゾリジンジオン誘導体」あるいは「インスリン抵抗性改善薬」として開示されている化合物が例示される。 The hematopoietic stem cell inducer composition of the present invention contains a thiazolidine compound having a nuclear receptor PPARγ agonist activity or a salt thereof as an active ingredient. Examples of thiazolidine compounds having agonist activity of the nuclear receptor PPARγ include compounds disclosed as “thiazolidinedione derivatives” or “insulin resistance improvers” in JP-A-5-70633 and JP-A-2000-355550, for example. Is done.
　具体的には例えば５－〔４－〔２－（５－エチル－２－ピリジル）エトキシ〕ベンジル〕－２,４－チアゾリジンジオン （一般名：ピオグリタゾン）；５－〔〔４－〔（３,４－ジヒドロ－６－ヒドロキシ－２,５,７,８－テトラメチル－２Ｈ－１－ベンゾピラン－２－イル）メトキシ〕フェニル〕メチル〕－２,４－チアゾリジンジオン （一般名：トログリタゾン）；５－〔〔４－〔２－（メチル－２－ピリジニルアミノ）エトキシ〕フェニル〕メチル〕－２,４－チアゾリジンジオン （一般名：ロシグリタゾン）；５－〔３－〔４－（５－メチル－２－フェニル－４－チアゾリルメトキシ）フェニル〕プロピル〕－２,４－オキサゾリジンジオン；５－〔４－（６－メトキシ－１－Ｈ－ベンズイミダゾール－２－イルメトキシ）ベンジル〕－２,４－チアゾリジンジオン；５－〔４－（６－メトキシ－１－メチルベンズイミダゾール－２－イルメトキシ）ベンジル〕－２,４－チアゾリジンジオン；などが挙げられる。 Specifically, for example, 5- [4- [2- (5-ethyl-2-pyridyl) ethoxy] benzyl] -2,4-thiazolidinedione (generic name: pioglitazone); 5-[[4-[(3, 4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) methoxy] phenyl] methyl] -2,4-thiazolidinedione (generic name: troglitazone); 5 -[[4- [2- (methyl-2-pyridinylamino) ethoxy] phenyl] methyl] -2,4-thiazolidinedione (generic name: rosiglitazone); 5- [3- [4- (5-methyl-2 -Phenyl-4-thiazolylmethoxy) phenyl] propyl] -2,4-oxazolidinedione; 5- [4- (6-methoxy-1-H-benzimidazol-2-ylmethoxy) benzyl] 2,4-thiazolidinedione; 5- [4- (6-methoxy-1-methyl-benzimidazol-2-ylmethoxy) benzyl] -2,4-thiazolidinedione; and the like.
　チアゾリジン系化合物の塩としては、薬理学的に許容し得る塩、例えば無機塩基との塩、有機塩基との塩、無機酸との塩、有機酸との塩、塩基性または酸性アミノ酸との塩などが挙げられる。無機塩基との塩の好適な例としては、例えばナトリウム，カリウムなどのアルカリ金属、カルシウム，マグネシウムなどのアルカリ土類金属、ならびにアルミニウム、アンモニウムなどとの塩が挙げられる。有機塩基との塩の好適な例としては、例えばトリメチルアミン、トリエチルアミン、ピリジン、ピコリン、エタノールアミン、ジエタノールアミン、トリエタノールアミン、ジシクロヘキシルアミン、Ｎ，Ｎ－ジベンジルエチレンジアミンなどとの塩が挙げられる。無機酸との塩の好適な例としては、例えば塩酸、臭化水素酸、硝酸、硫酸、リン酸などとの塩が挙げられる。有機酸との塩の好適な例としては、例えばギ酸、酢酸、トリフルオロ酢酸、フマール酸、シュウ酸、酒石酸、マレイン酸、クエン酸、コハク酸、リンゴ酸、メタンスルホン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩の好適な例としては、例えばアルギニン、リジン、オルニチンなどとの塩が挙げられ、酸性アミノ酸との塩の好適な例としては、例えばアスパラギン酸、グルタミン酸などとの塩が挙げられる。 Salts of thiazolidine compounds include pharmacologically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids. Etc. Preferable examples of salts with inorganic bases include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, and salts with aluminum and ammonium. Preferable examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p -Salts with toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.
　チアゾリジン系化合物またはその塩としては、好ましくはピオグリタゾン、トログリタゾン、ロシグリタゾンまたはそれらの塩であり、さらに好ましくは、ピオグリタゾンまたはその塩酸塩、トログリタゾン、ロシグリタゾンまたはそのマレイン酸塩であり、特に好ましくはピオグリタゾン塩酸塩である。 The thiazolidine compound or a salt thereof is preferably pioglitazone, troglitazone, rosiglitazone or a salt thereof, more preferably pioglitazone or a hydrochloride thereof, troglitazone, rosiglitazone or a maleate thereof, particularly preferably pioglitazone. The hydrochloride salt.
　本発明の造血幹細胞誘導剤組成物の剤形としては、例えば、錠剤、カプセル剤、顆粒剤、散剤、トローチ剤、シロップ剤、乳剤、懸濁剤、フィルム剤等の経口剤が例示される。また、注射剤、外用剤（例、経皮製剤）、坐剤、ペレット、経鼻剤等の非経口剤として投与することもあり得る。経口剤がより好適に用いられる。かかる医薬組成物は製剤技術分野において慣用の方法、例えば、日本薬局方に記載の方法等により製造することができる。 Examples of the dosage form of the hematopoietic stem cell inducer composition of the present invention include oral agents such as tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, and films. Moreover, it can be administered as parenteral agents such as injections, external preparations (eg, transdermal preparations), suppositories, pellets, nasal preparations and the like. Oral preparations are more preferably used. Such a pharmaceutical composition can be produced by a method commonly used in the field of pharmaceutical technology, for example, a method described in the Japanese Pharmacopoeia.
　本発明の造血幹細胞誘導剤組成物は、核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジンジオン誘導体またはその医薬上許容される塩に加えて、薬理学的に許容し得る担体を含有する。薬理学的に許容し得る担体としては、製剤素材として慣用の各種有機あるいは無機担体物質が用いられ、固形製剤における賦形剤、滑沢剤、結合剤、崩壊剤；液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤等として配合される。また必要に応じて、防腐剤、抗酸化剤、着色剤、甘味剤等の製剤添加物を用いることもできる。かかる成分は、製剤学の一般教科書に記載されているものを目的に応じて適宜用いることができる。 The hematopoietic stem cell inducer composition of the present invention contains a pharmacologically acceptable carrier in addition to a thiazolidinedione derivative having a nuclear receptor PPARγ agonist activity or a pharmaceutically acceptable salt thereof. As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, lubricants, binders, disintegrants in solid preparations; solvents in liquid preparations, dissolution aids It is formulated as an agent, suspending agent, isotonic agent, buffering agent, soothing agent and the like. If necessary, preparation additives such as preservatives, antioxidants, colorants, sweeteners and the like can also be used. As such components, those described in general textbooks of pharmaceutics can be appropriately used depending on the purpose.
　本発明の造血幹細胞誘導剤組成物の好ましい有効成分であるピオグリタゾン塩酸塩としては現在インスリン抵抗性改善薬（アクトス（登録商標）武田医薬品工業株式会社）として市販されているが、本発明の造血幹細胞誘導剤組成物としては、かかる市販の医薬品を用いてもよい。 Pioglitazone hydrochloride, which is a preferred active ingredient of the hematopoietic stem cell inducer composition of the present invention, is currently marketed as an insulin resistance improving drug (Actos (registered trademark) Takeda Pharmaceutical Co., Ltd.), but the hematopoietic stem cell of the present invention. As the inducer composition, such a commercially available drug may be used.
　本発明の造血幹細胞誘導剤組成物を対象へ投与することにより、対象の骨髄中の造血幹細胞数が増加する。ここで造血幹細胞とは、全ての血球系細胞へ分化し得る多能性を有する細胞である。 The number of hematopoietic stem cells in the bone marrow of the subject increases by administering the hematopoietic stem cell inducer composition of the present invention to the subject. Here, hematopoietic stem cells are pluripotent cells that can differentiate into all blood cells.
　本発明の造血幹細胞誘導剤組成物は、造血幹細胞移植における、移植ドナーにおいて造血幹細胞数を増加させるのに好適に用いられる。本願の組成物は同種造血幹細胞移植のドナーおよび自家造血幹細胞移植のドナーとなる患者のいずれにも好適に用いられる。本明細書および特許請求の範囲において「造血幹細胞移植ドナー」あるいは「ドナー」と特に区別無く記載する場合は、同種移植並びに自家移植の両方のドナーの両方を包含する。 The hematopoietic stem cell inducer composition of the present invention is suitably used for increasing the number of hematopoietic stem cells in a transplant donor in transplantation of hematopoietic stem cells. The composition of the present application is suitably used for both allogeneic hematopoietic stem cell transplant donors and patients who are autologous hematopoietic stem cell transplant donors. In the present specification and claims, the term “hematopoietic stem cell transplant donor” or “donor” is used to include both allogeneic and autologous donors.
　本発明の造血幹細胞誘導剤組成物の投与量、投与スケジュールは限定的でなく、造血幹細胞誘導の目的並びに投与対象の年齢、体重、性別等によって適宜設定すればよい。造血幹細胞移植ドナーへ投与する場合は、ドナーの年齢、体重、性別、一般的健康状況、骨髄移植か末梢血造血幹細胞移植か、所望の造血幹細胞量等によって適宜設定すればよい。また自家移植の場合には、全般的な治療計画に合致するよう決定すべきである。例えば有効成分としてピオグリタゾンを用い、ドナーが健常成人男性であって、経口投与する場合には、ピオグリタゾンまたはその塩を１日投与量が０．１～１００ｍｇとなるよう、好ましくは１～５０ｍｇ、より好適には４５ｍｇ以下となるよう１日１回～複数回に分けて投与する。投与期間は限定的ではなく、適宜定めればよいが例えば２日～８０日間程度投与すればよい。 The dose and administration schedule of the hematopoietic stem cell inducer composition of the present invention are not limited, and may be appropriately set depending on the purpose of inducing hematopoietic stem cells and the age, weight, sex, etc. of the administration target. In the case of administration to a hematopoietic stem cell transplant donor, it may be appropriately set depending on the donor's age, weight, sex, general health status, bone marrow transplant or peripheral blood hematopoietic stem cell transplant, desired hematopoietic stem cell amount, and the like. In the case of autologous transplants, decisions should be made to meet the overall treatment plan. For example, when pioglitazone is used as an active ingredient and the donor is a healthy adult male and is orally administered, the daily dose of pioglitazone or a salt thereof is 0.1-100 mg, preferably 1-50 mg, The dose is preferably administered once a day or in multiple doses so that the dose is 45 mg or less. The administration period is not limited and may be determined as appropriate. For example, administration may be performed for 2 to 80 days.
　本発明の造血幹細胞誘導剤組成物を、骨髄移植または末梢血造血幹細胞移植ドナーへ投与することによって骨髄中の造血幹細胞数が増加する。骨髄中の造血幹細胞数が増えることより、骨髄移植の場合には、ドナーから採取する骨髄液の量を減らすことができ、ドナーの負担を軽減することが期待される。また、本発明の造血幹細胞誘導剤組成物を投与した後、造血幹細胞動員剤を投与することによって、末梢血中の造血幹細胞数を増加させることができる。 The number of hematopoietic stem cells in the bone marrow increases by administering the hematopoietic stem cell inducer composition of the present invention to a bone marrow transplant or peripheral blood hematopoietic stem cell transplant donor. As the number of hematopoietic stem cells in the bone marrow increases, in the case of bone marrow transplantation, it is expected that the amount of bone marrow fluid collected from the donor can be reduced and the burden on the donor can be reduced. Moreover, the number of hematopoietic stem cells in peripheral blood can be increased by administering the hematopoietic stem cell inducer composition of the present invention and then administering the hematopoietic stem cell mobilizing agent.
　ここで、「造血幹細胞動員剤」とは、骨髄中の造血幹細胞を末梢血中へ動員する効果を有する物質をいう。造血幹細胞動員剤としては公知のものに限らず、将来造血幹細胞動員剤として開発されるものを本願発明の医薬組成物と組み合わせて用いても良い。造血幹細胞動員剤としては、顆粒球コロニー刺激因子（Ｇ－ＣＳＦ）、プレリキサフォール塩酸塩（Plerixafor hydrochloride）、Ｎ－アセチルノイラミン酸硫酸エステル、Ｎ－アセチルノイラミン酸ホモポリマー硫酸エステルまたはその薬学的に許容される塩、（特許文献１）、胎盤構成細胞粉砕物（特許文献２）などが例示されるがこれらに限定されるものではない。必要に応じて、１または２以上の造血幹細胞動員剤を組み合わせて用いてもよい。 Here, “hematopoietic stem cell mobilizing agent” refers to a substance having an effect of mobilizing hematopoietic stem cells in bone marrow into peripheral blood. Hematopoietic stem cell mobilization agents are not limited to known ones, and those that will be developed in the future as hematopoietic stem cell mobilization agents may be used in combination with the pharmaceutical composition of the present invention. As a hematopoietic stem cell mobilizing agent, granulocyte colony-stimulating factor (G-CSF), plerixafor hydrochloride, N-acetylneuraminic acid sulfate, N-acetylneuraminic acid homopolymer sulfate or pharmaceuticals thereof Examples of such a salt are acceptable, (Patent Document 1), pulverized placenta-constituting cells (Patent Document 2) and the like, but are not limited thereto. If necessary, one or two or more hematopoietic stem cell mobilizing agents may be used in combination.
　造血幹細胞動員剤の投与量、投与スケジュールは、ドナーの年齢、体重、性別、一般的健康状況並びに所望の造血幹細胞量等に応じて適宜定めればよい。Ｇ－ＣＳＦを用いる場合は現在の臨床プロトコルに沿って投与すればよく、典型的な例としては、ドナー体重当たり約１０μｇ／ｋｇ／日のＧ－ＳＣＦを１日１回または２回に分けて、毎日４～６日間続けて皮下投与し、その後末梢血を採取すればよい。Ｇ－ＣＳＦの投与量や投与期間は本発明の造血幹細胞誘導剤により造血幹細胞が増加することに伴い、現在臨床で使われているものより下げることができる可能性がある。プレリキサフォールその他の造血幹細胞動員剤を用いる場合も、各造血幹細胞動員剤の公知の用法、用量を元に調節すればよい。 The dose and administration schedule of the hematopoietic stem cell mobilizing agent may be appropriately determined according to the age, weight, sex, general health status, desired hematopoietic stem cell amount, etc. of the donor. When G-CSF is used, it may be administered according to the current clinical protocol. As a typical example, about 10 μg / kg / day of G-SCF per donor body weight is divided into once or twice a day. Subcutaneous administration may be continued every day for 4 to 6 days, and then peripheral blood may be collected. The dose and administration period of G-CSF may be lower than those currently used in clinical practice as hematopoietic stem cells increase with the hematopoietic stem cell inducer of the present invention. In the case of using prelixaphor or other hematopoietic stem cell mobilizing agent, it may be adjusted based on the known usage and dose of each hematopoietic stem cell mobilizing agent.
　ドナーからの造血幹細胞の採取は公知の方法いずれを採用してもよい。骨髄移植の場合は、ドナーより骨髄液を採取し、必要に応じてメッシュ、フィルターにて不純物を除いたものを移植に用いる。必要に応じて骨髄液から赤血球あるいは血漿成分を除去してもよい。また、自家移植の場合等、癌細胞等の混入が予測される場合には、癌細胞を除去する、あるいは表面マーカー等により造血幹細胞分画をさらに精製した上で移植するのが好ましい。造血幹細胞の精製に用いうる表面マーカーとしては、ＣＤ３４が例示され、造血幹細胞はＣＤ３４陽性であることが知られている。造血幹細胞の精製が可能であれば他のマーカーを用いてもよい。 Any known method may be employed for collecting hematopoietic stem cells from a donor. In the case of bone marrow transplantation, bone marrow fluid is collected from a donor and, if necessary, the one from which impurities are removed with a mesh or filter is used for transplantation. If necessary, red blood cells or plasma components may be removed from the bone marrow fluid. In addition, when contamination with cancer cells or the like is predicted, such as in autologous transplantation, it is preferable to remove the cancer cells or transplant after further purifying the hematopoietic stem cell fraction with a surface marker or the like. As a surface marker that can be used for purification of hematopoietic stem cells, CD34 is exemplified, and it is known that hematopoietic stem cells are CD34 positive. Other markers may be used as long as hematopoietic stem cells can be purified.
　末梢血造血幹細胞移植の場合は、ドナーから末梢血を連続血液成分分離装置を用いて体外循環（アフェレーシス）を行い末梢血中の単核球分画を単離し、残りをドナーへ返血する。単離した単核球分画は、必要に応じてさらに精製してもよい。造血幹細胞の精製は、従来公知の方法のいずれを採用してもよいが、例えばＣＤ３４などの表面マーカーに基づいて精製すればよい。ＣＤ３４陽性細胞を精製するには、フローサイトメーター、抗ＣＤ３４抗体を利用した免疫磁気ビーズ法、免役アビジンビーズ法等の公知の手段を用いればよい。 In the case of peripheral blood hematopoietic stem cell transplantation, peripheral blood from a donor is subjected to extracorporeal circulation (apheresis) using a continuous blood component separation device to isolate a mononuclear cell fraction in the peripheral blood, and the remaining blood is returned to the donor. The isolated mononuclear cell fraction may be further purified as necessary. For purification of hematopoietic stem cells, any of the conventionally known methods may be adopted, but for example, purification may be performed based on a surface marker such as CD34. In order to purify CD34-positive cells, known means such as a flow cytometer, an immunomagnetic bead method using an anti-CD34 antibody, and an immune avidin bead method may be used.
　本発明の組成物を用いてドナーより得られる造血幹細胞は、造血幹細胞移植により治療し得る種々の疾患の治療に好適に用いられる。かかる疾患としては、これらに限定されないが、自家造血幹細胞移植が有効な疾患としては非ホジキンリンパ腫、ホジキンリンパ腫、多発性骨髄腫、急性骨髄性白血病、神経芽細胞腫、横紋筋肉腫、脳腫瘍（随芽腫、上衣細胞腫、ＰＮＥＴ等）、肝芽腫、ウイルスムス腫、ユーイング肉腫、骨肉腫、肺細胞腫瘍、乳癌、卵巣癌、肺ガンなどの悪性腫瘍、および膠原病等の自己免疫疾患並びにアミロイドーシスなどが例示される。また同種造血幹細胞移植が有効な疾患としては、急性骨髄性白血病、急性リンパ性白血病、非ホジキンリンパ腫、ホジキンリンパ腫、骨髄異形成症候群、多発性骨髄腫、慢性骨髄性白血病、慢性リンパ性白血病、腎細胞癌、膵癌、大腸癌、乳癌等の悪性腫瘍並びに再生不良性貧血、発作性夜間血色素尿症、重症複合型免疫不全症、Fanconi貧血、Diamond-Blackfan貧血、鎌状赤血球症、Wiskott-Aldrich症候群、大理石骨病、先天性代謝性疾患並びに自己免疫疾患が例示される。 The hematopoietic stem cells obtained from a donor using the composition of the present invention are suitably used for treatment of various diseases that can be treated by hematopoietic stem cell transplantation. Such diseases include, but are not limited to, non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, acute myeloid leukemia, neuroblastoma, rhabdomyosarcoma, brain tumor ( Autologous diseases such as collagenous disease, and so on. Examples thereof include amyloidosis. Diseases for which allogeneic hematopoietic stem cell transplantation is effective include acute myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, kidney Malignant tumors such as cell cancer, pancreatic cancer, colon cancer, breast cancer and aplastic anemia, paroxysmal nocturnal hemoglobinuria, severe combined immunodeficiency, Fanconi anemia, Diamond-Blackfan anemia, sickle cell disease, Wiskott-Aldrich syndrome Examples include marble bone disease, inborn metabolic diseases and autoimmune diseases.
　本発明の組成物はまた、造血幹細胞の減少を伴う疾患の処置に有用に用いられる。造血幹細胞の減少を伴う疾患としては、慢性炎症、悪性腫瘍、重症感染症、自己免疫疾患、糖尿病、鎌状赤血球症、尿毒症、骨髄異形成症候群、Fanconi貧血、再生不良性貧血、慢性肝障害、腎不全、骨髄障害性血小板減少症、特発性血小板減少性紫斑症等が例示される。本発明の組成物はさらに、癌化学療法、癌放射線療法、自己免疫疾患の化学療法、または自己免疫疾患の放射線療法を受けている患者の骨髄中の造血幹細胞数を増加させるために有用に用いられる。本発明の組成物を造血幹細胞の減少を伴う疾患の処置に用いる場合、並びに化学療法もしくは癌放射線療法を受けている患者の処置に用いる場合、本発明の造血幹細胞誘導剤組成物の投与量、投与スケジュールは限定的でなく、投与対象の年齢、体重、性別等によって適宜設定すればよい。例えば有効成分としてピオグリタゾンを用い、経口投与する場合には、ピオグリタゾンまたはその塩を１日投与量が０．１～１００ｍｇとなるよう、好ましくは１～５０ｍｇ、より好適には４５ｍｇ以下となるよう１日１回～複数回に分けて投与する。 The composition of the present invention is also useful for the treatment of diseases accompanied by a decrease in hematopoietic stem cells. Diseases with decreased hematopoietic stem cells include chronic inflammation, malignant tumor, severe infection, autoimmune disease, diabetes, sickle cell disease, uremia, myelodysplastic syndrome, Fanconi anemia, aplastic anemia, chronic liver injury And renal failure, myelopathic thrombocytopenia, idiopathic thrombocytopenic purpura and the like. The compositions of the present invention are further usefully used to increase the number of hematopoietic stem cells in the bone marrow of patients undergoing cancer chemotherapy, cancer radiotherapy, autoimmune disease chemotherapy, or autoimmune disease radiation therapy. It is done. When the composition of the present invention is used for the treatment of a disease associated with a decrease in hematopoietic stem cells, and when used for the treatment of a patient undergoing chemotherapy or cancer radiotherapy, the dosage of the hematopoietic stem cell inducer composition of the present invention, The administration schedule is not limited, and may be appropriately set depending on the age, weight, sex, etc. of the administration subject. For example, when pioglitazone is used as an active ingredient and is orally administered, the daily dose of pioglitazone or a salt thereof is 0.1 to 100 mg, preferably 1 to 50 mg, more preferably 45 mg or less. Dosage once to several times a day.
　以下、本発明を実施例を用いてさらに本発明を説明する。 Hereinafter, the present invention will be further described with reference to examples.
　ピオグリタゾン塩酸塩の造血幹細胞誘導能
　１５－２５週齢のマウスを用いた。マウス用標準固形飼料へ０．７ｍｇ／ｇ固形飼料となるようピオグリタゾン塩酸塩（武田医薬品工業株式会社）を混合した餌を８週間給餌した。コントロール群にはピオグリタゾン塩酸塩を含まない以外は同じ固形飼料を給餌した。各群それぞれ４匹のマウスを用いた。 Hematopoietic stem cell inducing ability of pioglitazone hydrochloride 15-25 week old mice were used. Feed was prepared by mixing pioglitazone hydrochloride (Takeda Pharmaceutical Co., Ltd.) into a standard chow for mice for 0.7 mg / g solid feed for 8 weeks. The control group was fed the same chow diet except that it did not contain pioglitazone hydrochloride. Four mice were used for each group.
　８週間後にマウスを殺し、大腿骨並びに脛骨より骨髄細胞を流し出し、骨髄単細胞浮遊液を得た。得られた骨髄細胞浮遊液をＣＤ４５、ＣＤ３ε、Ｂ２２０、ｃ－Ｋｉｔ、ＣＤ４８、Ｇｒ－１、ＣＤ１１ｂ、ＣＤ３４、ＣＤ１５０、Ｓｃａ－１、Ｔｅｒ１１９、Ｌｉｎ、Ｃ０３，Ｃ０１１ｂ、Ｂｒ－１抗体にて公知の方法(M. Osawa et al., Science 273, 242 (1996))にて染色した。 After 8 weeks, the mice were killed and bone marrow cells were washed out from the femur and tibia to obtain a bone marrow single cell suspension. The resulting bone marrow cell suspension is known as CD45, CD3ε, B220, c-Kit, CD48, Gr-1, CD11b, CD34, CD150, Sca-1, Ter119, Lin, C03, C011b, Br-1 antibodies. Staining was performed by the method (M. Osawa et al., Science 273, 242 (1996)).
　得られた細胞をフローサイトメトリー（ＢＤ　ＦＡＣＳＡｒｉａ（BD Biosystems)）で解析を行い、骨髄細胞中のＣＤ３４（－）ＣＤ４８（－）ｃ－Ｋｉｔ（＋）Ｓｃａ－１（＋）Ｌｉｎ（－）細胞分画の割合を測定した。マウスにおいてはこの細胞分画の約４０％が造血幹細胞（ＨＳＣ）であることが知られている。結果を図１に示す。 The obtained cells were analyzed by flow cytometry (BD FACSAria (BD Biosystems)), and CD34 (−) CD48 (−) c-Kit (+) Sca-1 (+) Lin (−) cells in bone marrow cells. The fraction fraction was measured. It is known that about 40% of this cell fraction is hematopoietic stem cells (HSC) in mice. The results are shown in FIG.
　なお、使用した抗体は下記のとおりである：
　ＣＤ４５（３０－Ｆ１１）、ＣＤ３ε（１４５－２Ｃ１１）、Ｂ２２０（ＲＡ３－６Ｂ２）、ｃ－Ｋｉｔ（２Ｂ８）、ＣＤ４８（ＨＭ４８－１）、Ｇｒ－１（ＲＢ６－８Ｃ５）、ＣＤ１１ｂ（Ｍ１／７０）、ＣＤ３４（ＲＡＭ３４）、ＣＤ１５０（ＴＣ１５－１２Ｆ１２．２）、Ｓｃａ－１（Ｅ１３－１６１７．７、Ｄ７）、Ｔｅｒ１１９（Ｔｅｒ１１９） The antibodies used are as follows:
CD45 (30-F11), CD3ε (145-2C11), B220 (RA3-6B2), c-Kit (2B8), CD48 (HM48-1), Gr-1 (RB6-8C5), CD11b (M1 / 70) CD34 (RAM34), CD150 (TC15-12F12.2), Sca-1 (E13-1617.7, D7), Ter119 (Ter119)
　図１より明らかなように、本発明の組成物を投与したマウスでは骨髄中の造血幹細胞数がコントロールマウスに比して有意に増加していた。 As is clear from FIG. 1, the number of hematopoietic stem cells in the bone marrow was significantly increased in the mice administered with the composition of the present invention compared to the control mice.
　ピオグリタゾン塩酸塩およびＧ－ＣＳＦを用い、末梢血中への造血幹細胞誘導を行った。
　１５－２５週齢のマウスを用いた。実施例１と同様にしてピオグリタゾン塩酸塩を含有する餌を８週間投与した。その後、マウスへの給餌をピオグリタゾン塩酸塩を含まないものに換え、Ｇ－ＣＳＦを１００ｎｇ／ｇ／日、１日１回、５日間皮下投与した。コントロールマウスに対しては、ピオグリタゾン塩酸塩を含有しない餌を８週間給餌した後、Ｇ－ＣＳＦを試験群と同様のプロトコルにて投与した。最後のＧ－ＣＳＦ投与から4時間後、マウス末梢血を採取し、溶血により単核球分画を得、これを実施例１と同様にして染色し、フローサイトメトリーにて造血幹細胞分画ＣＤ３４（－）ＣＤ４８（－）ｃ－Ｋｉｔ（＋）Ｓｃａ－１（＋）Ｌｉｎ（－）細胞の割合を調べた。各群それぞれ３匹のマウスを用いた。結果を図２に示す。 Hematopoietic stem cells were induced into peripheral blood using pioglitazone hydrochloride and G-CSF.
15-25 week old mice were used. In the same manner as in Example 1, a feed containing pioglitazone hydrochloride was administered for 8 weeks. Thereafter, the mice were fed with food containing no pioglitazone hydrochloride, and G-CSF was subcutaneously administered once a day for 5 days at 100 ng / g / day. Control mice were fed a diet containing no pioglitazone hydrochloride for 8 weeks, and then G-CSF was administered according to the same protocol as in the test group. Four hours after the last G-CSF administration, mouse peripheral blood was collected, and a mononuclear cell fraction was obtained by hemolysis. This was stained in the same manner as in Example 1, and hematopoietic stem cell fraction CD34 was obtained by flow cytometry. The proportion of (−) CD48 (−) c-Kit (+) Sca-1 (+) Lin (−) cells was examined. Three mice were used for each group. The results are shown in FIG.
　図２に示されるように、コントロール群に比してピオグリタゾン塩酸塩を投与したマウス末梢血中の造血幹細胞分画が増加した。 As shown in FIG. 2, the hematopoietic stem cell fraction in the peripheral blood of mice administered with pioglitazone hydrochloride increased compared to the control group.
　ピオグリタゾン塩酸塩およびＡＭＤ３１００（プレリキサフォル）を用い、末梢血中への造血幹細胞誘導を行った。
　１５－２５週齢のマウスを用いた。実施例１と同様にしてピオグリタゾン塩酸塩を含有する餌を８週間投与した。その後、マウスへの給餌をピオグリタゾン塩酸塩を含まないものに換え、ＡＭＤ３１００の５ｍｇ／ｋｇを皮下投与した。コントロールマウスに対しては、ピオグリタゾン塩酸塩を含有しない餌を８週間給餌した後、ＡＭＤ３１００を試験群と同様のプロトコルにて投与した。ＡＭＤ３１００投与から１時間後、マウス末梢血を採取し、溶血により単核球分画を得、これを実施例１と同様にして染色し、フローサイトメトリーにて造血幹細胞分画ＣＤ３４（－）ＣＤ４８（－）ｃ－Ｋｉｔ（＋）Ｓｃａ－１（＋）Ｌｉｎ（－）細胞の割合を調べた。各群それぞれ３匹のマウスを用いた。結果を図３に示す。 Hematopoietic stem cell induction into peripheral blood was performed using pioglitazone hydrochloride and AMD3100 (prerixafor).
15-25 week old mice were used. In the same manner as in Example 1, a feed containing pioglitazone hydrochloride was administered for 8 weeks. Thereafter, the feeding to the mice was changed to one containing no pioglitazone hydrochloride, and 5 mg / kg of AMD3100 was subcutaneously administered. Control mice were fed a diet containing no pioglitazone hydrochloride for 8 weeks and then administered AMD3100 in the same protocol as the test group. One hour after administration of AMD3100, mouse peripheral blood was collected, and a mononuclear cell fraction was obtained by hemolysis. This was stained in the same manner as in Example 1, and hematopoietic stem cell fraction CD34 (−) CD48 was obtained by flow cytometry. The proportion of (−) c-Kit (+) Sca-1 (+) Lin (−) cells was examined. Three mice were used for each group. The results are shown in FIG.
　図３に示されるように、ＡＭＤ３１００を用いた場合にも、コントロール群に比してピオグリタゾン塩酸塩を投与したマウス末梢血中の造血幹細胞分画が増加した。 As shown in FIG. 3, even when AMD3100 was used, the hematopoietic stem cell fraction in the peripheral blood of mice administered with pioglitazone hydrochloride increased compared to the control group.

Claims (16)

  1. 核内受容体ＰＰＡＲγアゴニスト活性を有するチアゾリジン系化合物またはその塩を含有する、対象における造血幹細胞数を増加させるための造血幹細胞誘導剤組成物。 A hematopoietic stem cell inducer composition for increasing the number of hematopoietic stem cells in a subject, comprising a thiazolidine compound having a nuclear receptor PPARγ agonist activity or a salt thereof.
  2. チアゾリジン系化合物が、ピオグリタゾン、トログリタゾン、ロシグリタゾンまたはそれらの塩からなる群から選択される、請求項１記載の組成物。 The composition according to claim 1, wherein the thiazolidine-based compound is selected from the group consisting of pioglitazone, troglitazone, rosiglitazone, or a salt thereof.
  3. チアゾリジン系化合物が、ピオグリタゾンまたはその塩である、請求項１記載の組成物。 The composition according to claim 1, wherein the thiazolidine-based compound is pioglitazone or a salt thereof.
  4. ピオグリタゾン塩酸塩を含有する、請求項３記載の組成物。 4. A composition according to claim 3, comprising pioglitazone hydrochloride.
  5. 対象が、造血肝細胞移植ドナーである、請求項１～４何れかに記載の組成物。 The composition according to any one of claims 1 to 4, wherein the subject is a hematopoietic hepatocyte transplant donor.
  6. 対象が同種造血幹細胞移植ドナーである、請求項５記載の組成物。 6. The composition of claim 5, wherein the subject is an allogeneic hematopoietic stem cell transplant donor.
  7. 対象が自家造血幹細胞移植ドナーである、請求項５記載の組成物。 6. The composition of claim 5, wherein the subject is an autologous hematopoietic stem cell transplant donor.
  8. 造血幹細胞移植が、骨髄移植である、請求項５～７いずれかに記載の組成物。 The composition according to any one of claims 5 to 7, wherein the hematopoietic stem cell transplantation is bone marrow transplantation.
  9. 造血幹細胞移植が、末梢血造血幹細胞移植である、請求項５～７いずれかに記載の組成物。 The composition according to any one of claims 5 to 7, wherein the hematopoietic stem cell transplantation is peripheral blood hematopoietic stem cell transplantation.
  10. さらに造血幹細胞動員剤を別個の製剤として含有する、請求項９記載の組成物。 The composition according to claim 9, further comprising a hematopoietic stem cell mobilizing agent as a separate preparation.
  11. 造血幹細胞動員剤がG-CSFおよび／またはプレリキサフォルである、請求項１０記載の組成物。 The composition according to claim 10, wherein the hematopoietic stem cell mobilizing agent is G-CSF and / or plerixafor.
  12. 造血幹細胞動員剤がG-CSFである、請求項１１記載の組成物。 The composition according to claim 11, wherein the hematopoietic stem cell mobilizing agent is G-CSF.
  13. 造血幹細胞動員剤がプレリキサフォルである、請求項１０記載の組成物。 The composition according to claim 10, wherein the hematopoietic stem cell mobilizing agent is prilixafor.
  14. 対象が、造血幹細胞の減少に関連する疾患に罹患しているか、または該疾患に罹患する可能性が高い患者である、１～４何れかに記載の組成物。 5. The composition according to any one of 1 to 4, wherein the subject is a patient suffering from a disease associated with a decrease in hematopoietic stem cells or a patient likely to suffer from the disease.
  15. 慢性炎症、悪性腫瘍、重症感染症、自己免疫疾患、糖尿病、鎌状赤血球症、尿毒症、骨髄異形成症候群、Fanconi貧血、再生不良性貧血、慢性肝障害、腎不全、骨髄障害性血小板減少症および特発性血小板減少性紫斑症からなる群から選択される造血幹細胞の減少を伴う疾患の処置のためのものである、請求項１４記載の組成物。 Chronic inflammation, malignant tumor, severe infection, autoimmune disease, diabetes, sickle cell disease, uremia, myelodysplastic syndrome, Fanconi anemia, aplastic anemia, chronic liver injury, renal failure, myelopathic thrombocytopenia 15. The composition of claim 14, wherein the composition is for the treatment of a disease associated with a reduction in hematopoietic stem cells selected from the group consisting of idiopathic thrombocytopenic purpura.
  16. 対象が、化学療法または放射線療法を受ける患者である、請求項１～４何れかに記載の組成物。 The composition according to any of claims 1 to 4, wherein the subject is a patient receiving chemotherapy or radiation therapy.
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