Hematopoietic stimulants

Abstract

The present invention aims to provide a novel hematopoiesis-stimulating agent and a pharmaceutical for preventing or treating anemia, particularly refractory anemia, which contains the hematopoiesis-stimulating agent as an active ingredient. The present invention provides a hematopoiesis-stimulating agent containing an S-adenosylmethionine synthase inhibitor.

Description

[Technical Field]

[0001] The present invention relates to a hematopoiesis-stimulating agent, a pharmaceutical agent for preventing or treating anemia containing the hematopoiesis-stimulating agent, a method for producing hematopoietic cells using the hematopoietic-stimulating agent, etc. The present invention also relates to a screening method for hematopoiesis-stimulating agents using the measured value of S-adenosylmethionine synthetase activity as an index, a method for evaluating hematopoiesis-stimulating agents, etc. [Background technology]

[0002] Until now, there have been no effective hematopoietic products that directly stimulate erythropoiesis in refractory anemia, such as aplastic anemia and myelodysplastic syndrome. The only treatments available, which carry a high risk of complications, include immunosuppressive therapy, chemotherapy, and hematopoietic stem cell transplantation. Furthermore, there are many cases in which these treatments are resistant to these therapies or are inappropriate for these treatments due to age or other factors (Non-Patent Documents 1 and 2). While erythropoietin preparations, which have been known to promote hematopoiesis, have shown some efficacy in treating renal anemia, they are ineffective in treating anemia associated with blood disorders in which endogenous erythropoietin production is already stimulated. Therefore, many cases require red blood cell transfusions, one of the most effective symptomatic treatments. However, due to the cost associated with long-term treatment and the potential for organ damage due to iron overload, eliminating transfusion dependency is one of the goals of treating refractory anemia.

[0003] On the other hand, MAT is an S-adenosylmethionine synthase, and its inhibitors are considered as candidates for cancer treatment (Non-Patent Documents 3 and 4). However, the hematopoietic effect of S-adenosylmethionine synthase inhibitors was not known at all. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Bacigalupo A., Blood 2017 Mar 16;129(11):1428-1436 [Non-patent document 2] Almeida A et al., Leuk Res 2017 Jan;52:50-57. [Non-patent document 3] Jackson B. Hester, Jr., J. Med. Chem. 1980, 23, 392-402 [Non-patent document 4] Casey L Quinlan et al., Nature Chemical Biology 13, 785-792 (2017) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made in view of the above-mentioned situation in the prior art, and aims to provide a novel hematopoiesis-stimulating agent and a medicament for preventing or treating anemia, particularly refractory anemia, which contains the hematopoiesis-stimulating agent as an active ingredient. [Means for solving the problem]

[0006] S-adenosylmethionine synthase (MAT) (there are two isozymes, MAT1 and MAT2, with MAT2 consisting of two subunits, MAT2A and MAT2B) synthesizes the methyl group donor S-adenosylmethionine from ATP and methionine and is an important enzyme for epigenetic modification. The present inventors have found that the MAT inhibitor cycloleucine (CLEU) significantly accelerates the maturation of bone marrow erythroblasts and significantly increases peripheral blood hemoglobin levels in mice. The present invention is based on this finding. Specifically, the present invention provides a novel therapeutic agent for anemia associated with hematopoietic diseases that has been difficult to treat with conventional hematopoietic stimulating agents using MAT inhibitors. Furthermore, the present invention provides a high-throughput screening system for the discovery of novel hematopoietic stimulating agents by identifying various compounds that target MAT and are more suitable for clinical application using a high-throughput screening system, and synthesizing lead compounds from the hit compounds.

[0007] That is, the present invention relates to the following. [1] A hematopoietic promoter containing an S-adenosylmethionine synthase inhibitor. [2] The hematopoiesis-stimulating agent according to [1], wherein the S-adenosylmethionine synthase is MAT2A. [3] The hematopoiesis-promoting agent according to [1] or [2], wherein the S-adenosylmethionine synthase inhibitor is selected from cycloleucine, its derivatives, 4H-s-triazolo[4,3-a][1,4]benzodiazepine derivatives, and pharmaceutically acceptable salts thereof. [4] The hematopoiesis-stimulating agent according to [1] or [2], wherein the S-adenosylmethionine synthase inhibitor is selected from 3-acetyl-11-keto-β-boswellic acid, its derivatives, and pharmaceutically acceptable salts thereof. [5] A pharmaceutical for preventing or treating anemia, comprising the hematopoiesis-stimulating agent according to any one of [1] to [4]. [6] A method for producing hematopoietic cells, comprising the step of culturing hematopoietic progenitor cells in the presence of the hematopoietic promoter according to any one of [1] to [4]. [7] measuring S-adenosylmethionine synthetase activity in the presence or absence of a candidate substance for a hematopoietic stimulator; and selecting a candidate substance for hematopoiesis-stimulating agent in which the measured value of S-adenosylmethionine synthetase activity in the presence of the candidate substance is lower than the measured value in the absence of the substance; A method for screening for a hematopoietic stimulator, comprising: [8] measuring S-adenosylmethionine synthetase activity in the presence of an evaluation substance for hematopoiesis-stimulating agents; and a step of evaluating the hematopoiesis-promoting effect using the measured value of S-adenosylmethionine synthetase activity in the presence of an evaluation substance for the hematopoiesis-promoting agent as an index; A method for evaluating a hematopoietic stimulator, comprising: [9] A method for promoting hematopoiesis, comprising administering an S-adenosylmethionine synthase inhibitor to a subject in need of hematopoiesis promotion.

[10] S-adenosylmethionine synthetase inhibitor for use in promoting hematopoiesis. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a hematopoiesis-stimulating agent having an excellent hematopoietic promoting effect, which has an action mechanism completely different from that of erythropoietin, a conventional hematopoietic stimulating agent, and also to provide a pharmaceutical agent for preventing or treating anemia, particularly refractory anemia. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows experimental results demonstrating that SAM (S-adenosylmethionine) synthesis is downregulated during erythroblast maturation. A: The abundance of methionine, SAH (S-adenosylhomocysteine), and SAM in each subset of cells sorted from bone marrow. These abundances were quantitatively analyzed using mass spectrometry. B: The ratio of SAM to methionine in each subset. The abundance of SAM and methionine in each sample was adjusted to their abundance in the non-erythroblast subset. C: The RNA expression levels of MAT2A and MAT2B analyzed by RT-PCR. Expression levels were normalized by Actb. Each point in the box plot represents an individual mouse sample. Asterisks indicate *P<0.05, **P<0.01, and ***P<0.001. [Figure 2]Figure 2 shows experimental results demonstrating that CLEU-induced reduction of SAMs induced significant erythropoietin-independent erythropoiesis. A: SAM abundance in cells sorted from myeloid subset II with PBS or CLEU treatment (day 2). B: Peripheral blood hemoglobin concentration on the indicated days after PBS or CLEU treatment. C: Total bone marrow cells in the left and right femurs and tibias of PBS- or CLEU-treated mice (day 2). D: Representative images (photographs) of tibias from PBS- or CLEU-treated mice (day 2). E and F: Myeloid mature cells in each group analyzed by flow cytometry (day 2). Representative results of flow cytometry analysis are shown in E. Cumulative cell counts for the indicated groups are shown in F. Erythroid (Ter119+Gr1-B220-CD4-CD8-) and myeloid (Gr1+Ter119-B220-CD4-CD8-). B cells (B220+Ter119-Gr1-CD4-CD8-). G and H: Bone marrow erythroblasts in each subset analyzed by flow cytometry (day 2). Representative results of flow cytometry analysis are shown in G. Cumulative cell counts of the indicated subsets are shown in H. I: Erythroid maturation index (EMI = III + IV / I + II) of bone marrow in PBS- or CLEU-treated mice (day 2). J: Hematoxylin and eosin-stained images (photographs) of bone marrow in PBS- or CLEU-treated mice (day 2). K: May-Giemsa-stained images (photographs) of peripheral blood smears from PBS- or CLEU-treated mice (day 2). L: Serum erythropoietin levels analyzed by ELISA. Each point in the boxplot represents an individual mouse sample. Asterisks indicate *P<0.05, **P<0.01, and ***P<0.001. [Figure 3]Figure 3 shows experimental results demonstrating that CLEU treatment strongly induces hemoglobin-related gene expression in bone marrow erythroblast subset II. A: RNA-seq analysis of bone marrow erythroblast subset II in mice treated with PBS or CLEU (day 2) (CLEU; n = 4, PBS; n = 4). Only genes whose expression levels were altered (in at least one of the two conditions) with FPKM > 10 and CV < 0.5 (log2FC < 0.5 or log2FC > 0.5, P < 0.05) are shown as dots. B: Gene Ontology (GO) analysis of genes significantly induced by CLEU treatment using DAVID 6.8. C: RNA expression levels of the indicated genes analyzed by RT-PCR to confirm RNA-seq expression levels were normalized by Actb. D: RNA expression levels of the indicated genes in the indicated bone marrow erythroblast subsets treated with PBS or CLEU (day 2). Expression levels were normalized by Actb. Each point in the box plot represents an individual mouse sample. Asterisks indicate *P<0.05, **P<0.01, and ***P<0.001. [Figure 4]Figure 4 shows experimental results demonstrating that epigenetic demethylation induces terminal maturation of erythroblasts. A: Whole-genome bisulfite sequencing (WGBS) results (day 2) of bone marrow erythroblast subset II in mice treated with CLEU or PBS (CLEU; n=2, PBS; n=2). B: Differences in expression versus promoter CpG methylation for all expressed genes (one of at least one conditions: FPKM>10 and CV<0.5). Correlation (Cor) was -0.08. C: Differences in expression versus promoter CpG methylation for significantly induced genes. Correlation (Cor) was -0.11. D: Images (photographs) showing Western blot (WB) results of bone marrow erythroblast subset II in mice treated with CLEU or PBS (day 2). EG: In vitro erythroid differentiation of human CD34+ umbilical cord blood cells. E: Representative flow cytometry analysis results. F: Cumulative results for the indicated cell populations. G: Cumulative results of CD34 EMI (CD34 erythroid maturation index CD71+GlyA+ / CD71+GlyA-). Each point shown in the box plot represents a technical replicate. Asterisks indicate P<0.001. [Figure 5] Figure 5 shows experimental results demonstrating that the abundance of SAM-related metabolites does not change during bone marrow erythroblast differentiation and maturation. A and B: The abundance of the indicated metabolites in each subset of cells sorted from bone marrow was analyzed using mass spectrometry. A: cystathionine, B: homocysteine. Each point in the box plot represents an individual mouse sample. [Figure 6] Figure 6 shows experimental results showing complete blood counts in mice treated with CLEU or PBS. A-D: Complete peripheral blood counts (CBCs) in mice treated with CLEU or PBS (days 1-6). A: White blood cell count (103 / μL). B: Platelet count (105 / μL). C: Red blood cell count (106 / μL). D: Hematocrit (%). E: May-Giemsa stained images (photographs) of nucleated cells sorted from bone marrow erythroblast subset II in PBS- or CLEU-treated mice (day 2). F: Principal component (PC) analysis results of the RNA sequencing data shown in Figure 3A. [Figure 7]Figure 7 shows experimental results demonstrating that CLEU treatment induces a gene bundle containing ribonucleotide- and erythroid transcription factor-related genes. A-C: Gene Ontology (GO) analysis using DAVID 6.8 for genes significantly induced by CLEU treatment. A: DNA / nucleus-related GO terms. B: chromatin-related GO terms. C: RNA-related GO terms. D: RNA-sequencing results for individual genes in the indicated samples. Expression of each gene in each sample was normalized by the average FPKM of each gene in eight samples. E: RNA expression levels of individual genes in the indicated erythroblast subsets of PBS- or CLEU-treated bone marrow (day 2). Expression levels were normalized by Actb. Each point in the boxplot represents an individual mouse sample. Asterisks indicate *P<0.05, **P<0.01, and ***P<0.001. [Figure 8] Figure 8 shows experimental results demonstrating that CLEU treatment induces a gene bundle containing hematopoietic stem / progenitor cell-related genes. A: RNA sequencing results for the indicated genes in the indicated samples. Expression of each gene in each sample was normalized by the average FPKM of each gene in the eight samples. B: RNA sequencing. RNA expression levels of the indicated genes analyzed by RT-PCR to confirm expression levels were normalized by Actb. Each point in the boxplot represents an individual mouse sample. Asterisks indicate P<0.001. C: RNA sequencing results for Alas2 in the indicated samples. Expression of each gene in each sample was normalized by the average FPKM of each gene in the eight samples. [Figure 9] Figure 9 shows experimental results demonstrating that histone methyltransferases were downregulated by CLEU treatment. RNA sequencing results for the indicated genes in the indicated samples. Expression of each gene in each sample was normalized by the average FPKM of each gene across eight samples. [Figure 10]Figure 10 shows experimental results demonstrating the promotion of erythroblast maturation by PF9366. A and B: Bone marrow erythroblasts in each subset analyzed by flow cytometry (day 2). Representative results of flow cytometry analysis are shown in A. Cumulative cell counts for the indicated subsets are shown in B. C: Erythroblast maturation index is shown. Asterisks indicate *P<0.05. [Figure 11] FIG. 11 is a schematic diagram showing the methionine cycle. [Figure 12] Figure 12 shows experimental results demonstrating the promotion of erythroblast maturation by AKBA. Bone marrow erythroblasts in each subset analyzed by flow cytometry (day 2). Representative results of flow cytometry analysis are shown in the left panel. Cumulative cell counts for the indicated subsets are shown in the right panel. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention will be described below.

[0011] The main abbreviations used in this specification are as follows: MAT: S-adenosylmethionine synthase EPO: erythropoietin SAM: S-adenosylmethionine SAH: S-adenosylhomocysteine CLEU: Cycloleucine AKBA: 3-acetyl-11-keto-β-boswellic acid (CAS Number: 67416-61-9)

[0012] (Hematopoietic promoter) One aspect of the present invention relates to a hematopoiesis-stimulating agent containing an S-adenosylmethionine synthase inhibitor. As described above, the hematopoiesis-stimulating agent of the present invention exhibits the hematopoiesis-stimulating effect due to the S-adenosylmethionine synthase inhibitor.

[0013] The term "S-adenosylmethionine synthase" as used herein is not particularly limited as long as it is an enzyme that catalyzes the biosynthesis of S-adenosylmethionine using methionine and ATP as substrates. In addition, "S-adenosylmethionine synthase" is also commonly referred to as "adenosylmethionine synthase" or "methionine adenosyltransferase," but these terms have the same meaning in this specification.

[0014] Specific examples of S-adenosylmethionine synthetase include MATI / III (composed of an active subunit, MAT1A; generally, it is sometimes referred to as "MAT1A," but both have the same meaning in this specification), MATII (composed of an active subunit, MAT2A, and a regulatory subunit, MAT2B; generally, it is sometimes referred to as "MAT2A" or "MAT2," but both have the same meaning in this specification), etc. The preferred S-adenosylmethionine synthetase is MAT2A.

[0015] Furthermore, the term "methionine" used herein refers to L-methionine unless otherwise specified.

[0016] Furthermore, the term "S-adenosylmethionine synthetase inhibitor" as used herein is not particularly limited as long as it has the effect of inhibiting S-adenosylmethionine synthetase in mammals. Preferably, the S-adenosylmethionine synthetase inhibitor is a substance that has S-adenosylmethionine synthetase inhibitory activity in any mammalian cell (preferably hematopoietic stem cells, myeloid progenitor cells, erythroid progenitor cells, proerythroblasts, erythroblasts, etc.). The mechanism of the S-adenosylmethionine synthetase inhibitory activity is not particularly limited, and may be, for example, suppression of S-adenosylmethionine synthetase expression at any stage of S-adenosylmethionine synthetase gene transcription, post-transcriptional regulation, translation, post-translational modification, etc.; expression inhibition of S-adenosylmethionine synthetase protein other than those mentioned above; competitive inhibition; non-competitive inhibition such as inhibition of subunit association; uncompetitive inhibition; or other enzyme inhibition.

[0017] As described above, a strong correlation between S-adenosylmethionine synthase inhibitory effects and hematopoiesis promotion has been confirmed. Therefore, various S-adenosylmethionine synthase inhibitors can be screened using S-adenosylmethionine synthase activity as an indicator, and the inhibitors can be used as hematopoiesis-promoting agents. That is, S-adenosylmethionine synthase inhibitors are not limited to specific compounds, as long as they have MAT inhibitory effects. They may include low-molecular-weight compounds, peptides, antibodies, nucleic acids (including antisense oligonucleotides and siRNAs), extracts, and the like. S-adenosylmethionine synthase inhibitors may be known compounds known as S-adenosylmethionine synthase inhibitors or compounds obtained by screening. Specific examples include, but are not limited to, cycloleucine and its derivatives, 4H-s-triazolo[4,3-a][1,4]benzodiazepine derivatives, 3-acetyl-11-keto-β-boswellic acid and its derivatives, or salts thereof. The S-adenosylmethionine synthase inhibitor may be a commercially available product or may be synthesized by a conventional organic synthesis method. The S-adenosylmethionine synthase inhibitor may be used alone or in combination of two or more.

[0018] Cycloleucine derivatives refer to reactive derivatives chemically derived from cycloleucine that exhibit the same effects as cycloleucine in the human body. Examples include, but are not limited to, N-Boc-L-valine, 3-aminopropionic acid, 3-(carboxymethyl)-2-(2-pentenyl)cyclopentanone, D-proline, and other unnatural amino acids. Pharmaceutically non-toxic compounds with little or no side effects in the human body are preferred. Derivatives may also include prodrugs of cycloleucine. Examples of 4H-s-triazolo[4,3-a][1,4]benzodiazepine derivatives include, but are not limited to, PF9366 (2-(7-Chloro-5-phenyl-[1,2,4]triazolo[4,3-a]quinolin-1-yl)-N,N-dimethylethan-1-amine) (Non-Patent Documents 3 and 4). Derivatives of 3-acetyl-11-keto-β-boswellic acid include, but are not limited to, 11-keto-β-boswellic acid and compounds disclosed in literature such as Mediterranean Journal of Chemistry 2017, 6(5), 180-190, Nat Prod Res. 2019 Jan 29:1-8, and Anticancer Agents Med Chem. 2017;17(8):1153-1167.

[0019] Further examples of S-adenosylmethionine synthase inhibitors include fluorinated N,N-dialkylaminostilbene compounds described in Zhang et al., ACS Chem Biol, 2013, 8(4):796-803, 2',6'-dihalostyrylaniline, pyridine, or pyrimidine compounds described in Sviripa et al., J Med Chem, 2014, 57:6083-6091, and compounds described in WO2012103457.

[0020] Specific examples of pharmaceutically acceptable salts include, but are not limited to, inorganic salts (sodium salts, potassium salts, lithium salts; calcium salts, magnesium salts; aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; ammonium salts, etc.), various organic salts, hydrohalide salts, inorganic acid salts, organic acid salts, and amino acid salts.

[0021] The inhibitory effect of S-adenosylmethionine synthetase can be measured by known methods, for example, in an in vitro enzyme activity measurement system. Specifically, MAT2A inhibitory activity can be measured using the reaction (Figure 11) in which S-adenosylmethionine synthase (MAT2A) synthesizes S-adenosylmethionine from ATP and methionine. For example, but not limited to, the enzymatic activity of MAT2A is evaluated using a measurement system such as a luciferase assay system to measure the ATP concentration remaining after the reaction, and the inhibitory activity of the substance to be measured is measured. Cycloleucine, for example, can be used as a positive control inhibitor.

[0022] The S-adenosylmethionine synthetase inhibitor used in the present invention is not limited to, but may be, for example, an inhibitor of S-adenosylmethionine synthetase activity, such as IC 50 Substances having a concentration of 10 mM or less, 1 mM or less, 100 μM or less, or 10 μM or less can be preferably used.

[0023] Furthermore, the term "promoting hematopoiesis" as used herein means that an S-adenosylmethionine synthase inhibitor induces differentiation of hematopoietic progenitor cells (myeloid progenitor cells, erythroid progenitor cells, proerythroblasts, erythroblasts, etc.) into hematopoietic cells (proerythroblasts, erythroblasts, erythrocytes, etc.), resulting in an increase in the number of hematopoietic cells after administration of the S-adenosylmethionine synthase inhibitor compared to before administration, or an improvement in the numerical values ​​corresponding to the increase in the number of hematopoietic cells. The hematopoietic-promoting effect can be measured using conventional biological techniques such as blood count and flow cytometry, molecular biological techniques, etc. The term "having a hematopoietic-promoting effect" is not limited to, but can refer to, for example, a 1.2- to 50-fold, preferably 1.2- to 5-fold, and more preferably 1.2- to 1.5-fold increase in the number of hematopoietic cells compared to a control (absence of a hematopoietic promoter).

[0024] (Pharmaceuticals, food and beverages) A further aspect of the present invention relates to a pharmaceutical for preventing or treating anemia, comprising the hematopoietic stimulator of the present invention. As described above, the hematopoietic stimulator of the present invention has a hematopoietic promoting effect by inducing differentiation of hematopoietic cells from hematopoietic progenitor cells, and therefore can be suitably used as a pharmaceutical for preventing or treating diseases caused by blood loss, decreased hematopoietic function, etc. Furthermore, the hematopoietic stimulator can be suitably used as a food or drink to be taken daily, a reagent for research purposes, etc., for improving decreased hematopoietic function (including the prevention of the above-mentioned diseases).

[0025] As will be shown in the Examples below, S-adenosylmethionine synthetase inhibitors are thought to be novel hematopoietic stimulators with a mechanism of action completely different from that of erythropoietin preparations, which are conventional hematopoietic stimulators. Many cases of refractory anemia due to blood diseases are refractory to erythropoietin preparations, and the discovery that erythropoiesis is promoted by S-adenosylmethionine synthetase inhibition is expected to lead to a significant reduction in complications and social costs associated with transfusion dependence.

[0026] Diseases caused by blood loss or decreased hematopoietic function include, but are not limited to, renal anemia (preferably erythropoietin-unresponsive renal anemia), iron deficiency anemia, anemia caused by anticancer drug treatment, refractory anemia (aplastic anemia, myelodysplastic syndrome, myelofibrosis, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, congenital bone marrow failure syndrome, etc.), hemolytic anemia, blood loss anemia, etc.

[0027] The medicament of the present invention can be formulated into various pharmaceutical preparations by known pharmaceutical methods. For example, it can be formulated into a preparation suitable for administration by oral administration, intraperitoneal administration, transdermal administration, subcutaneous administration, intravenous administration, inhalation administration, etc. In the formulation, it can be appropriately combined with various pharmaceutically acceptable carriers, such as excipients, disintegrants, lubricants, binders, surfactants, flow enhancers, colorants, flavors, etc.

[0028] Furthermore, the food and drink of the present invention can be produced in various forms by known food and drink production methods. The medicines and foods and drinks of the present invention may contain known medicinal ingredients used for the prevention and treatment of anemia in combination, and may also contain other known pharmacologically active ingredients.

[0029] The hematopoiesis-promoting agent, medicine, and food and beverage product of the present invention can be used for mammals including humans, but there is no particular limitation on animals other than humans, and various livestock, poultry, pets, laboratory animals, etc. can be used for the hematopoiesis-promoting agent, medicine, and food and beverage product of the present invention.

[0030] When the hematopoiesis-stimulating agent, medicine, and food and beverage of the present invention are administered or ingested, the dosage or intake amount is appropriately selected depending on the age, body weight, symptoms, health condition of the subject, type of composition (medicine, food and beverage, etc.), etc. For example, the dosage or intake amount of the hematopoiesis-stimulating agent, medicine, and food and beverage of the present invention per administration is not limited, but is, for example, 0.01 mg / kg body weight to 1000 mg / kg body weight in terms of the amount of hematopoiesis-stimulating agent.

[0031] A further aspect of the present invention relates to a method for promoting hematopoiesis in a subject, comprising administering to or ingesting the hematopoiesis-stimulating agent, medicament, or food or drink of the present invention to the subject. Also, a further aspect of the present invention relates to a method for preventing or treating anemia in a subject, comprising administering to or ingesting the hematopoiesis-stimulating agent, medicament, or food or drink of the present invention to the subject.

[0032] (Manufacturing method) A further aspect of the present invention relates to a method for producing hematopoietic cells, which comprises the step of culturing hematopoietic progenitor cells in the presence of the hematopoietic-stimulating agent of the present invention. In other words, this method is a method for inducing differentiation of hematopoietic cells, which comprises the step of culturing hematopoietic progenitor cells in the presence of the hematopoietic-stimulating agent of the present invention.

[0033] Examples of hematopoietic progenitor cells include myeloid progenitor cells, erythroid progenitor cells, proerythroblasts, erythroblasts, etc. Hematopoietic progenitor cells are not limited to those derived from animals, and those obtained by inducing differentiation from iPS cells, etc. can also be used. The culture method in the hematopoietic cell production method of the present invention is not particularly limited as long as it allows the maintenance and survival of hematopoietic progenitor cells (preferably myeloid progenitor cells, erythroid progenitor cells, proerythroblasts, erythroblasts) and erythrocytes, and the maintenance, survival, and differentiation of hematopoietic progenitor cells, except for the addition of the hematopoiesis-stimulating agent of the present invention, and can be performed using conventional cell culture methods.

[0034] Specific examples of culture media for hematopoietic progenitor cells include, but are not limited to, MEMα, DMEM, RPMI-1640, Ham's F-12, and IMDM. Additives typically used in cell culture can be used in the culture media without any particular limitation, as long as they do not interfere with the effects of the present invention. It is particularly preferable to use serum or serum substitutes. Further examples of additives include 2-mercaptoethanol, sodium pyruvate, amino acids, antibiotics, and N-acetylcysteine.

[0035] To the culture medium for hematopoietic progenitor cells, an S-adenosylmethionine synthase inhibitor, which serves as a differentiation inducer for hematopoietic cells, i.e., the hematopoietic promoter of the present invention, is added. The concentration of the hematopoietic promoter in the culture medium can be appropriately adjusted depending on the cell source, cell number, culture medium volume, etc., and is not limited, but is usually about 1 to 10,000 μg / mL, preferably about 1 to 5,000 ng / mL.

[0036] The incubator used for the culture may be any one generally used for animal cell culture. Culture conditions include, for example, 37 to 39°C, a gas phase of 5% carbon dioxide and 95% air, and high humidity, and an incubator capable of setting such conditions is used.

[0037] During the culture period, it is preferable to replace a portion of the culture medium with fresh medium every 2 to 4 days. The culture period can be changed depending on the animal species used and the condition of the collected cells, but is generally about several days to a week.

[0038] During or after the culture, it is also preferable to confirm that the differentiation of hematopoietic cells has been induced by blood count measurement, flow cytometry, etc. The culture period can also be changed depending on the degree of differentiation induction of hematopoietic cells.

[0039] (Screening method) A further aspect of the present invention relates to a method for screening for an S-adenosylmethionine synthetase inhibitor, comprising the steps of measuring S-adenosylmethionine synthetase activity in the presence or absence of a candidate substance for the S-adenosylmethionine synthetase inhibitor, and selecting a candidate substance for which the measured value of S-adenosylmethionine synthetase activity in the presence of the candidate substance for the enzyme inhibitor is lower than the measured value in the absence of the candidate substance.

[0040] Furthermore, another aspect of the present invention relates to a method for screening for hematopoiesis-stimulating agents, comprising the steps of measuring S-adenosylmethionine synthetase activity in the presence or absence of a candidate substance for the hematopoiesis-stimulating agent, and selecting a candidate substance for which the measured S-adenosylmethionine synthetase activity in the presence of the candidate substance is lower than that in the absence of the candidate substance. That is, since a strong correlation was confirmed between the S-adenosylmethionine synthetase inhibitory effect and the promotion of hematopoiesis as described above, hematopoiesis-stimulating agents can be screened using S-adenosylmethionine synthetase activity as an indicator. The "step of measuring S-adenosylmethionine synthetase activity in the presence or absence of a candidate substance for an S-adenosylmethionine synthetase inhibitor (or hematopoiesis-stimulating agent)" may be carried out in an embodiment in which S-adenosylmethionine synthetase is expressed in the presence or absence of a candidate substance for an S-adenosylmethionine synthetase inhibitor (or hematopoiesis-stimulating agent), and then the expression level of S-adenosylmethionine synthetase or S-adenosylmethionine synthetase activity is measured.

[0041] Here, the phrase "the measured value of S-adenosylmethionine synthetase activity in the presence of a candidate substance for an S-adenosylmethionine synthetase inhibitor (or hematopoiesis-stimulating agent) is lower than the measured value in its absence" is not limited to, but may mean, for example, that the measured value of S-adenosylmethionine synthetase activity in the presence of a candidate substance for an S-adenosylmethionine synthetase inhibitor (or hematopoiesis-stimulating agent) is 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the measured value in its absence.

[0042] The activity of S-adenosylmethionine synthetase can be measured, for example, in an in vitro enzyme activity measurement system. Specifically, MAT2A inhibitors can be identified using the reaction (Figure 11) in which S-adenosylmethionine synthase (MAT2A) synthesizes S-adenosylmethionine from ATP and methionine. For example, the enzyme activity of recombinant mouse MAT2A after the addition of a candidate hematopoietic stimulator is assessed using a luciferase assay or other measurement system to measure the remaining ATP concentration after the reaction, and the inhibitory activity of the candidate substance is evaluated. This method allows compounds with MAT2A inhibitory activity to be identified using a high-throughput screening system.

[0043] Because MAT2A synthesizes S-adenosylmethionine from methionine and ATP, its inhibitory effect can be evaluated by measuring the amount of remaining ATP in a test tube using luciferase activity, an ATP-dependent enzyme. While either native or recombinant MAT2A can be used, the present inventors have already produced recombinant MAT2A. High-throughput screening using luciferase assays in 384-well plates is widely performed, so a screening system should be rapidly established. Positive controls for inhibitors include, for example, cycloleucine, PF9366, and 3-acetyl-11-keto-β-boswellic acid. It is also preferable to confirm the hematopoietic activity and safety of the hematopoietic promoters obtained by screening. Hematopoietic activity can be measured using, for example, the methods for measuring hematopoietic effects described above.

[0044] (Evaluation method) A further aspect of the present invention relates to a method for evaluating a hematopoietic stimulator, comprising the steps of measuring S-adenosylmethionine synthetase activity in the presence of a substance to be evaluated as a hematopoietic stimulator, and evaluating the hematopoietic stimulating effect using the measured value of S-adenosylmethionine synthetase activity in the presence of the substance to be evaluated as an index. That is, as described above, it has been confirmed that there is a strong correlation between the S-adenosylmethionine synthetase inhibitory effect and hematopoietic promotion, and therefore it is possible to evaluate the presence or degree of the hematopoietic stimulating effect of a substance to be evaluated as a hematopoietic stimulator using S-adenosylmethionine synthetase inhibitory activity as an index.

[0045] The method for measuring S-adenosylmethionine synthase activity can be the same as that described above in the screening method of the present invention. Positive controls that act as inhibitors include, for example, cycloleucine, PF9366, and 3-acetyl-11-keto-β-boswellic acid. [Example]

[0046] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples as long as it does not depart from the gist of the invention.

[0047] Materials and Methods (mouse) Wild-type mice on a C57BL / 6J genetic background were purchased from Charles River Laboratories, Inc. Mice were analyzed at 8 to 14 weeks of age. For CLEU treatment (50 mg / body), cycloleucine (CLEU) (Sigma) was diluted with PBS (50 mg / ml) to prepare a CLEU solution, and 1 ml of the CLEU solution was administered intraperitoneally. For AKBA treatment (2 mg / body), 2 mg of AKBA (Enzo Life Sciences) in 20% DMSO in 1 ml of PBS was administered intraperitoneally. Vehicle without AKBA served as a control. For peripheral blood complete blood counts, peripheral blood was collected from the facial vein and counted using an automated animal hemocytometer (microsemi LC-662). All experiments performed in this study were approved by the Animal Care and Use Committee of the Tohoku University Environmental Safety Committee.

[0048] (Flow cytometry and cell sorting) BM cells and spleen cells were routinely harvested and stained with specific antibody combinations to identify erythroblasts and mature cells. Visualization was performed with streptavidin-peridinin chlorophyll protein-cyanin 5.5 antibodies. The antibodies used in this study were as follows: anti-CD3e (145-2C11), anti-CD4 (GK1.5 or RM4.5), anti-CD8a (53-6.7), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-Ter-119 (TER-119), and anti-CD71 (R17217 or C2). DAPI (Sigma) staining was performed to exclude dead cells for analysis. Flow cytometry analysis and cell sorting were performed using a FACS Aria II (BD) according to the manufacturer's protocol, and the resulting data were analyzed using FlowJo software (TreeStar).

[0049] (RNA sequencing data sampling and analysis) Total RNA was isolated from sorted samples of myeloid subset II erythroblasts using the RNeasy kit (Qiagen) (PBS; n = 4, CLEU; n = 4). The quality of the isolated RNA (RIN > 0.8) was confirmed using a 2100 bioanalyzer (Agilent), and 250 ng of RNA was used for library preparation using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions. High-throughput sequencing (51 bp, single-end) was performed using Hiseq2500 Rapid mode v2 (Illumina). Sequencing reads were mapped to the reference mouse genome (mm9) using bowtie2 version 2.2.5 and tophat version 2.1.0, and transcript abundance was then estimated using cufflinks version 2.2.1. Gene expression levels were normalized by the average fragments per kilobase of transcript per million mapped reads (FPKM). Only differentially expressed genes with FPKM > 10 and CV < 0.5 (in at least one of the two conditions) (log2FC < 0.5 or log2FC > 0.5, P < 0.05 between conditions) were used for further analysis. The DAVID 6.8 web tool was used to find gene ontology terms enriched in significantly up- or down-regulated genes.

[0050] (Sampling and analysis of whole genome bisulfite sequencing (WGBS) data) DNA was isolated from sorted samples of myeloid subset II erythroblasts using phenol / chloroform extraction (PBS; n=2, CLEU; n=2). DNA quality control, bisulfite conversion, library preparation, high-throughput sequencing, and data mapping were performed at Macrogen, Inc. Briefly, the TruSeq DNA Methylation Kit and EZ DNA Methylation Gold (Illumina and Zymo Research) were used for library preparation according to the manufacturer's protocol (TruSeq DNA Methylation Library Preparation Guide), followed by high-throughput sequencing using HiseqX (Illumina). After sequencing, raw sequence reads were filtered based on quality and adapter sequences were trimmed. The remaining reads were mapped to the reference genome (mm9) using BSMAP based on SOAP (Short Oligo Alignment Program). SAMBAMBA version 0.5.9 was used to remove indexes and PCR duplicates to select only modified reads. The methylation rate of every single cytosine is calculated from the mapping results using BSMAP version 2.87. Only cytosine positions with >=5 reads were used for further analysis.

[0051] (human CD34 + In vitro erythroid differentiation of umbilical cord blood cells Human CD34 + Umbilical cord blood cells were obtained from the Kanto-Koshinetsu Block Blood Center (Tokyo, Japan). The use of umbilical cord blood samples for research was approved by the Ethics Committee of Tohoku University. The technical details of this method have been described previously. Briefly, CD34 + Cord blood cells were cultured in growth medium for 5 days, and then the cells (1 × 10 5The cells were cultured in differentiation medium for 7 days. For CLEU treatment, the concentration was 2.5 mg / ml. The expansion medium was StemMACS HSC Expansion Media XF Human (MACS) containing StemMACS HSC Expansion Cocktail Human (MACS). The differentiation medium was StemSpan SFEM (STEMCELL) containing hSCF (100 ng / ml; Peprotech), hFLT3-L (33.3 ng / ml; Peprotech), hIL3 (13.3 ng / ml; Peprotech), hBMP4 (13.3 ng / ml, Peprotech), hEPO (2.67 IU / ml; Kyowa Hakko Kirin Co., Ltd.), and hydrocortisone (1 μM; Sigma).

[0052] The present inventors hypothesized that epigenetic modifications play a central role in blood cell development. Erythroblast maturation is a dynamic process in which erythroblasts undergo significant morphological changes, including nuclear aggregation and enucleation. Previous studies have demonstrated that global DNA demethylation and posttranslational histone modifications occur during erythroblast maturation, indicating that appropriate epigenetic modifications are essential for erythroblast maturation. DNA and histone methylation require S-adenosylmethionine (SAM) as a potent methyl donor. Therefore, SAM and methionine adenosyltransferase (MAT), which catalyzes SAM from methionine and ATP, play an important role in erythroblast maturation. However, little is known about the functions of these factors in this process. Therefore, to clarify the functions of these factors, the present inventors conducted the following experiments.

[0053] Example 1 We investigated changes in the abundance of SAM-related metabolites during erythroblast maturation in bone marrow (BM). Mass spectrometry analysis revealed that the abundance of SAM gradually decreased during erythroblast maturation, whereas methionine and other related amino acids did not significantly differ (Figure 1A). Therefore, SAM synthesis appears to decrease during erythroblast maturation (Figure 1B). Consistently, MAT2A, the major MAT isozyme in tissues other than the liver, and its cofactor MAT2B, decreased during erythroblast maturation (Figure 1C). Thus, SAM synthesis is negatively regulated during erythroblast maturation.

[0054] <Example 2> Next, to clarify the importance of SAM in erythropoiesis, mice were treated with the MAT inhibitor cycloleucine (CLEU; 50 mg / body). CLEU was able to actually reduce the abundance of SAM in erythroblasts (Figure 2A). Surprisingly, CLEU treatment significantly increased hemoglobin, erythrocytes, and hematocrit, although there were no significant differences in white blood cell and platelet counts (Figure 2B, Figure 6A-D). BM cell counts significantly increased, and the BM color turned red with CLEU treatment (Figure 2C-D). Flow cytometry analysis revealed that this phenotype was due to a particular increase in erythroid cells in the BM (Figure 2E-F). Furthermore, Ter119 / CD71 double staining revealed that erythroblasts began to increase from subset II, and erythroblast maturation was significantly upregulated by CLEU treatment (Figure 2G-I). Pathological findings of BM and peripheral blood also supported these findings (Fig. 2J-K). Interestingly, serum erythropoietin levels were significantly downregulated by CLEU treatment (Fig. 2L), which may be due to a negative feedback loop. Taken together, CLEU treatment significantly increased erythroblast maturation in the BM in an erythropoietin-independent manner and decreased SAM synthesis and erythropoietin.

[0055] We also examined the effects of PF9366 (PF), a MAT inhibitor, in mice. Vehicle (DMSO) (control) or 10 mM PF9366 was administered in a 40 μl / 40 ml water bottle at 0 h. At 0 h and 24 h, 80 μl of vehicle (DMSO) or 10 mM PF9366 dissolved in 1 ml PBS was administered intraperitoneally. Analysis was performed at day 2 (48 h). Erythroblasts began to increase from subset III, demonstrating that erythroblast maturation was significantly upregulated by PF treatment (Figure 10, A-B). Significant differences were also observed in the erythroblast maturation index (Figure 10, C).

[0056] Example 3 To clarify the mechanism of erythroblast maturation induced by CLEU treatment, we performed RNA sequencing analysis of BM erythroblast subset II. Although there were no significant morphological differences between nucleated cells from both conditions (Figure 6E), significant gene expression changes occurred (Figure 6F). Interestingly, most differentially regulated genes were downregulated (Figure 3A). Gene ontology analysis of these downregulated genes revealed a significant enrichment of DNA / nucleus- and / or chromatin-related genes (Figure 7A-B), suggesting that these gene changes are involved in the dynamic chromatin remodeling that erythroblasts undergo during terminal maturation for final enucleation. Furthermore, RNA-related genes were also enriched among these downregulated genes (Figure 4C), suggesting that RNA processing plays an important role in erythroblast maturation. Furthermore, transcription factors important for erythropoiesis, such as Gata1, Tal1, and Klf1, were significantly downregulated (Figure 7D), suggesting the existence of other important factors governing final erythroblast maturation. Meanwhile, 72 genes were identified as significantly upregulated (Figure 8A). Globin genes were most abundantly expressed in CLEU-treated erythroblasts (Figure 3A). Gene ontology analysis of these upregulated genes revealed that not only hemoglobin-related genes and erythroid development-related genes, but also extracellular matrix- and immune system-related genes were significantly enriched (Figure 3B), suggesting that the contribution of these factors was necessary for effective erythroblast maturation. For example, validation analysis confirmed that not only hemoglobin-related genes but also CD163 (a high-affinity scavenger receptor for the hemoglobin-haptoglobin complex) and Apoe were significantly upregulated (Figure 3C). According to previous reports indicating their contributory role in erythropoiesis, upregulation of CD163 and / or Apoe is essential for proper heme utilization and / or lipid membrane synthesis during erythroblast maturation.Furthermore, the expression of Myb and Cited2, known to be important transcription factors in hematopoietic stem / progenitor cell differentiation, as well as the linker histone component H1f0, was significantly upregulated by CLEU treatment (Figure 8B), suggesting their unexplored functions in definitive erythroblast maturation. Interestingly, the expression of hemoglobin-related genes in subset II was significantly upregulated by CLEU treatment compared with that in subset III. These genes included Alas2, which was not included in the upregulated genes identified above due to its relatively small difference in expression levels between PBS and CLEU treatment (Figure 8C), indicating that the upregulation of these genes was stronger than the natural course of erythroblast maturation. Considering that abnormalities in globin genes cause erythroblast maturation defects in conditions such as thalassemia, the upregulation of these genes may, on the other hand, be the driving force behind definitive erythroblast maturation.

[0057] Example 4 Because the global transcriptional changes observed above may be due to epigenetic modifications caused by the reduction in intracellular SAM abundance (Figure 2A), we performed whole-genome bisulfite sequencing (WGBS) to reveal DNA methylation changes during induced erythroblast maturation. As expected, CLEU treatment reduced DNA CpG methylation by approximately 2% overall (Figure 4A), and the reduction in CpG methylation was more pronounced in highly methylated genomic regions (Figure 4A, red box). As previously observed, highly methylated genomic regions may be more susceptible to demethylation during terminal erythroblast maturation. Consistent with the idea that DNA methylation represses gene expression, differences in RNA expression were weakly but negatively correlated with gene promoter CpG methylation (Figure 4B). These correlations were also observed for significantly upregulated genes, such as the β-globin gene and Hmox1 (Figure 4C). Furthermore, trimethylation of H3K9, a typical histone methylation marker, was reduced by CLEU treatment (Fig. 4D). Considering that the expression levels of several histone methyltransferases were also significantly downregulated by CLEU treatment (Fig. 9), the reduction of SAM and the suppression of histone methyltransferases may also have contributed to the global transcriptional changes.

[0058] <Example 5> Finally, human umbilical cord blood CD34 + The effects of in vitro erythroid differentiation conditions on CLEU cells were investigated. In this model, CLEU not only stimulated erythroid differentiation but also stimulated human umbilical cord blood CD34 cells. + It was also able to induce maturation from erythroblasts (FIGS. 4E-G). This observation indicates a direct effect of CLEU on erythroblast maturation and that this mechanism may be applicable to human erythropoiesis.

[0059] Example 6 Mice were intraperitoneally administered the MAT inhibitor 3-acetyl-11-keto-β-boswellic acid (AKBA; 2 mg / body) at 0 and 24 hours, and analyzed 48 hours later. Ter119 / CD71 double staining revealed that erythroblast maturation was significantly upregulated by AKBA treatment (Figure 12). AKBA, in addition to CLEU and PF-9366, also induced erythrocyte maturation, and similar results were obtained with these three structurally distinct agents, suggesting that MAT2A inhibitors promote erythropoiesis.

[0060] In summary, this study revealed that the final maturation of erythroblasts is controlled by intracellular SAM levels accompanied by epigenetic modifications. By utilizing this system, it is possible to induce erythropoiesis in an erythropoietin-independent manner by treatment with an S-adenosylmethionine synthase inhibitor such as CLEU. These findings open new avenues for future research into erythropoiesis to overcome anemic diseases.

[0061] These results suggest that MAT2A inhibitors may be a potential new treatment for anemia associated with blood disorders, which has been difficult to treat with conventional hematopoietic stimulators. In addition to cycloleucine, we will identify compounds that target MAT2A and are more suitable for clinical application using a high-throughput screening system, synthesize lead compounds from the hit compounds, and administer them to mice to verify their erythropoiesis-stimulating activity and safety, thereby developing additional hematopoietic stimulators. [Industrial Applicability]

[0062] The present invention relates to a hematopoiesis-stimulating agent useful in medicines, foods and beverages, etc. This research has led to the development of a new treatment for refractory anemia, which is expected to reduce the number of patients dependent on red blood cell transfusions due to refractory anemia and the number of patients with associated iron overload, as well as reduce the social costs associated with transfusion medicine.

Claims

1. A composition for promoting erythropoiesis, comprising an S-adenosylmethionine synthase inhibitor selected from cycloleucine, PF9366, and 3-acetyl-11-keto-β-boswellic acid.

2. The composition of claim 1, wherein the S-adenosylmethionine synthase is MAT2A.

3. A medicament for preventing or treating anemia, comprising the composition according to claim 1 or 2.

4. A method for producing red blood cells, comprising a step of culturing red blood cell precursor cells in the presence of a composition described in claim 1 or 2.

5. A step of measuring S-adenosylmethionine synthase activity in the presence or absence of a candidate substance for a substance having an erythropoiesis-stimulating effect; and selecting a candidate substance having an erythropoiesis-stimulating effect, the measured value of which is lower in the presence of the candidate substance than in the absence of the candidate substance; A method for screening for a substance having an erythropoiesis-stimulating effect, comprising:

Images