JP2021059495A - Albendazole-containing monocyte differentiation inducing agent - Google Patents

Abstract

To provide a hematopoietic cell differentiation inducing agent.SOLUTION: The present invention relates to a monocyte differentiation inducing agent containing albendazole as an active ingredient.SELECTED DRAWING: None

Description

The present application relates to a monocyte differentiation inducer containing albendazole as an active ingredient.

Among blood diseases, there are diseases caused by inhibition of the differentiation process of hematopoietic stem cells, such as leukemia, aplastic anemia, and Costman's syndrome.

Leukemia is a disease that is thought to be caused by the tumorigenesis of hematopoietic stem cells (or slightly differentiated cells). Chemotherapy is the basis of curative treatment for leukemia, but in order to perform chemotherapy, it is necessary to maintain sufficient general condition and organ function to withstand organ toxicity and complications, so chemotherapy is used in elderly patients. In many cases, therapy cannot be applied, and there are elderly leukemia patients who have no cure. For such patients, new therapeutic agents with few side effects are needed.

Differentiation induction therapy is known as a treatment method for leukemia with few side effects. In differentiation induction therapy, leukemia cells that continue to proliferate while remaining in the state of progenitor cells are differentiated and matured to kill them at the original lifespan of normal leukocytes, so that leukemia cells such as many anticancer drugs and chemotherapy There are fewer side effects compared to treatments that directly destroy the disease. However, in acute promyelocytic leukemia, the effectiveness of differentiation-inducing therapy using all-trans retinoic acid (ATRA) and / or arsenic as a differentiation-inducing agent for neutrophils has been established, but for other leukemias. At present, no effective differentiation-inducing therapy has been established. In addition, acute promyelocytic leukemia is resistant to ATRA. There is a strong demand in the medical field for new differentiation-inducing therapies that are effective against a wide range of leukemias.

Aplastic anemia is a disease in which the differentiation process is inhibited by some abnormality in hematopoietic stem cells, and Costman syndrome (also known as severe congenital neutropenia) is favorable because it is found to inhibit the maturation of bone marrow granulocytic cells. It is a group of diseases in which neutrophils decrease.
In diseases such as leukemia, aplastic anemia, and costman syndrome, which are caused by inhibition of the differentiation process of hematopoietic stem cells, hematopoietic function is reduced, resulting in a decrease in neutrophils and monocytes, resulting in cell infection. It becomes a problem. At present, neutrophil differentiation-inducing agents such as granulocyte colony-stimulating factor (G-CSF) are administered as a treatment for bacterial infections in these diseases, but neutrophils are still administered after G-CSF administration. There are cases where it does not increase. In order to improve the symptoms in these diseases, an effective blood cell differentiation inducer that promotes the differentiation process from hematopoietic stem cells is desired.

Mol Cell Biol. 2015 Dec 7; 36 (4): 559-73. Doi: 10.1128 / MCB.00712-15. Deregulated KLF4 Expression in Myeloid Leukemias Alters Cell Proliferation and Differentiation through MicroRNA and Gene Targets. Morris VA, Cummings CL, Korb B, Boaglio S, Oehler VG. 2016 American Society of Hematology Aabstruct: http://www.bloodjournal.org/content/128/22/1546?sso-checked=true

All disclosures of the prior art documents cited herein are incorporated herein by reference.

An object of the present application is to provide a novel blood cell lineage cell differentiation inducer.

As a result of diligent studies to solve the above problems, the present inventors have found that albendazole, which is a parasite repellent, unexpectedly strongly induces leukemia cells to differentiate into monocyte-like cells. It came to this application. Furthermore, surprisingly, albendazole has been found to induce the differentiation of normal blood cell progenitor cells into monocytes, and has reached the present application. That is, the present application provides a monocyte differentiation inducer containing albendazole.
The invention of the present application will be described in detail below.

[1] A monocyte differentiation inducer containing albendazole as an active ingredient.
[2] The monocyte differentiation inducer according to [1] for treating a disease associated with inhibition of the differentiation process of hematopoietic stem cells.
[3] The monocyte differentiation inducing agent according to [2], wherein the disease associated with inhibition of the differentiation process of the hematopoietic stem cell is selected from aplastic anemia, Costman syndrome, leukemia or a related disease thereof.
[4] The monocyte differentiation-inducing agent according to [2] or [3], wherein the disease associated with inhibition of the differentiation process of the hematopoietic stem cell is leukemia or a related disease thereof.
[5] The monocyte differentiation inducing agent according to any one of [2] to [4], wherein the leukemia or a related disease thereof is chronic myelogenous leukemia, acute myelogenous leukemia or myelodysplastic syndrome.
[6] The monocyte differentiation inducer according to [4] or [5], which is used as a differentiation induction therapy.

[7] A method for inducing differentiation into monocytes, which comprises administering an effective amount of albendazole to a subject in need thereof.
[8] The method according to [7], wherein the method is used in the treatment of a disease associated with inhibition of the differentiation process of hematopoietic stem cells.

[9] Use of albendazole for producing monocyte differentiation inducers.
[10] The use according to [9], wherein the monocyte differentiation inducer is used to treat a disease associated with inhibition of the differentiation process of hematopoietic stem cells.

[11] Albendazole for use in inducing monocyte differentiation.
[12] The albendazole according to [11] for use in inducing monocyte differentiation in the treatment of diseases associated with inhibition of the differentiation process of hematopoietic stem cells.

Albendazole has high monocyte differentiation-inducing activity and can be used as a monocyte differentiation inducer. Furthermore, albendazole is already commercially available as a therapeutic agent for echinococcosis, and since data on its safety to humans have been accumulated, it is possible to provide a safe monocyte differentiation inducing agent.

Representative microscopic images (Diff-Quik staining) of THP-1 cells transduced with KLF4 or the lentivirus encoding the control (magnification 20x). Results of transducing THP-1 cells with KLF4 or a lentivirus encoding the control and analyzing the expression level of the candidate gene by real-time RT-PCR. The data are mean ± SEM values. ** P <0.01, by two-sided student's t-test. Results of transducing THP-1 cells with KLF4 or a lentivirus encoding the control and analyzing the expression level of the candidate gene by real-time RT-PCR. The data are mean ± SEM values. ** P <0.01, by two-sided student's t-test. Immunoblots of KLF4, DPYSL2A (DPYSL2A) and GAPDH in THP-1 and MOLM-13 cells transformed with KLF4 or the lentivirus encoding the control. Immunoblots of KLF4, DPYSL2A and GAPDH in THP-1 cells transduced with a leathyvirus transducing shRNA encoding shRNA targeting KLF4 and DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.). The THP-1 cells of Test Example 3-1 were treated with 3 μM doxycycline for 48 hours and collected, and the cell surface expression of CD11b and CD14 was measured by flow cytometry. Cell proliferation curve of THP-1 cells of Test Example 3-1. The data are mean ± SEM values. * P <0.05, *** P <0.001, by two-sided student's t-test. Representative microscopic images of THP-1 cells of Test Example 3-1. (Enlargement 20 x, scale bar: 50 μm) Drug screening to identify KLF4-inducible compounds. Relative expression of KLF4 in THP-1 cells treated with the 38 candidate compounds identified in Test Example 4-1. Relative expression of KLF4 and DPYSL2A in albendazole-treated THP-1 cells. Left: Before processing. Right: After processing. The data are mean ± SEM values. ** P <0.01, *** P <0.001, by two-sided student's t-test. The cell surface expression of CD11b and CD14 in albendazole-treated THP-1 cells was measured by flow cytometry. Representative microscopic images of THP-1 cells of Test Example 4-4. Growth curve of THP-1 cells transduced with shRNA targeting KLF4 (sh_KLF4 # 1 and # 2), DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.) In the presence of 1nMPMA. Dose-response curve of albendazole in THP-1 and MOLM-13 cells. Dose-response curve of albendazole in ATRA-resistant UF-1 cells. Dose-response curve of albendazole in ATRA-resistant NB4 cells. Overall survival of NOG mice transplanted with THP-1 cells and treated with albendazole or controls. P <0.001, log-rank (Mantel-Cox) test. Representative microscopic images of bone marrow of AML (THP-1 cell) xenograft mice (30 days after transplantation). (Enlarged 4x and 20x, scale bar 100 μm). (D) Representative images of the spleen and liver of AML (THP-1 cell) xenograft mice (30 days after transplantation) of (C). Albendazole was administered to c-kit surface antigen-positive cells collected from the bone marrow of wild-type C57BL / 6 mice at 0 nM, 100 nM, and 500 nM, and colonization was confirmed one week later. The vertical axis shows pieces / dishes.

The present application provides a monocyte differentiation inducer containing albendazole as an active ingredient. The monocyte differentiation inducing agents of the present application can be used, for example, to treat diseases associated with inhibition of the differentiation process of hematopoietic stem cells. In addition, the monocyte differentiation inducing agent of the present application can also be used for patients with neutrophil or monocyte depletion regardless of the presence or absence of a disease associated with inhibition of the differentiation process of hematopoietic stem cells, and is neutrophilic. It can be used to prevent or ameliorate symptoms (eg, bacterial infections) that can be caused by a decrease in spheres or monocytes.

Albendazole [Chemical name: (methyl 5- (propylthio) -2-benzimidazolecarbamate)]
Is known as an anthelmintic benzimidazole, therapeutics Tsutsumimushisho (echinococcosis) (e.g. in Japan Esukazoru ^(R) tablets) have already been marketed as. Its action is thought to be due to the inhibition of tubulin's binding to the colchicine binding site and the formation of microtubules.

In Japanese Patent Publication No. 2004-525140, Japanese Patent Publication No. 2008-522984, and Japanese Patent Publication No. 2016-535014, solid cancers such as liver cancer, colon cancer, pancreatic cancer, and ovarian cancer using albendazole known as an anthelmintic agent Although it describes the treatment method for leukemia, it does not describe the specific effect on leukemia. Moreover, these documents do not describe the induction of differentiation of blood cell lineage cells by albendazole.

In the present application, albendazole may be synthesized by a known method, or may be obtained as a commercially available product (for example, Tokyo Chemical Industry Co., Ltd.). Albendazole may also be in the form of a pharmaceutically acceptable salt or hydrate.

In the present application, "monocyte differentiation induction" refers to hematopoietic stem cells (strictly speaking, cells that are not hematopoietic stem cells but are immature and have the ability to differentiate into both myeloid and lymphoid systems (for example, c-kit surface antigen-positive cells)). Includes) and / or induces the differentiation of myeloid hematopoietic progenitor cells into monocytes. The monocytes differentiated by the monocyte differentiation inducer of the present application have at least one of the morphological features commonly used to identify monocytes (eg, notched or partially recessed). Has nuclei; has basic cytoplasmic granules (azur granules); has vacuoles in the cytoplasm) and / or has features based on surface antigen classification (eg, CD11b positive and CD14 positive) and is self-propagating. And means cells that do not have the ability to immortalize. The genetic information of the differentiation-induced monocytes is not particularly limited, and may contain, for example, genetic information derived from a disease.

In the present application, "myeloid hematopoietic progenitor cells" means progenitor cells found in the process of differentiation of hematopoietic stem cells into myeloid blood cells, for example, myeloid hematopoietic stem cells and myeloid hematopoietic stem cells to mature differentiated cells. It means progenitor cells in the process of differentiating into (eg, obese cells, eosinophils, eosinophils, neutrophils, monospheres, macronuclear cells, or erythrocytes). Examples of myeloid blood cell precursors are myeloid hematopoietic stem cells, granule / monocyte stem cells, erythroblast / megakaryocyte stem cells, myeloid blasts, anterior myeloid cells, myeloid cells, posterior myeloid cells, rods. Examples thereof include nuclei, lobulated nuclei, monocytes, anterior monocytes, anterior erythroblasts, erythroblasts, and anterior megakaryocytes. Hematopoietic stem cells (including cells that are not strictly hematopoietic stem cells but are immature cells that have the ability to differentiate into both myeloid and lymphatic systems) and myeloid hematopoietic progenitor cells are abnormal even if they are normal cells. (For example, canceration) may be possessed.

The monocyte differentiation-inducing agent of the present application includes hematopoietic stem cells (including cells that are not strictly hematopoietic stem cells but are immature cells capable of differentiating into both myeloid and lymphoid systems) and / or myeloid hematopoietic precursors. By inducing the differentiation of cells into monocytes, diseases associated with inhibition of the differentiation process of hematopoietic stem cells (eg, regenerative anemia, Costman syndrome, or leukemia or related diseases (eg, bone marrow dysplasia syndrome)) can be treated. ..

In aplastic anemia, the differentiation process is inhibited by some abnormality in hematopoietic stem cells, and blood cells in the blood, especially neutrophils, decrease, so that severe bacterial infections such as pneumonia and sepsis are likely to occur.

Costman's syndrome (also known as severe congenital neutropenia) is a group of diseases in which severe chronic neutropenia is observed due to gene mutation, and bacterial infections occur repeatedly. Diagnosis based on the bone marrow image shows inhibition of maturation of bone marrow granulocytic cells.

In the treatment of aplastic anemia or Costman's syndrome, the monocyte differentiation-inducing agent of the present application, for example, in addition to the radical treatment thereof, causes symptoms (for example, bacterial infection) caused by a decrease in blood cells (particularly neutropenia). Can be used to treat. The function of macrophages that are more mature from monocytes against bacterial infection is similar to that of neutrophils, and by inducing monocyte differentiation with the monocyte differentiation inducer of the present application, symptoms such as bacterial infections can be treated. Can be done.

Leukemia is a disease that is thought to occur when hematopoietic stem cells (or slightly differentiated cells) become tumors. Tumorized cells (leukemia cells) overgrow and occupy the bone marrow, significantly impairing normal hematopoietic function. As a result, red blood cells, normal white blood cells and platelets are reduced, and as the condition progresses, spleen, liver and lymph nodes become swollen due to infiltration of leukemia cells.

Leukemia is a case where leukemia cells of blood cell progenitor cells that have progressed to a certain stage and stopped proliferating (acute leukemia) and a case where leukemia cells retain the ability to differentiate and mature (chronic leukemia). It is roughly divided into. In addition, it is classified into myelogenous leukemia and lymphocytic leukemia according to the cancerous cell lineage.

FAB classification based on the morphological classification method is known as one of the classifications of acute leukemia. In the FAB classification, peroxidase staining is roughly classified into acute myeloid leukemia and acute lymphocytic leukemia, and acute myeloid leukemia is further classified into the following M0 to M7.

The "leukemia" that can be treated with the monocyte differentiation inducer of the present application is not particularly limited as long as it is judged to be leukemia based on general criteria used by doctors or researchers. Since the monocyte differentiation-inducing agent of the present application has the ability to induce differentiation from myeloid blood cell progenitor cells into monocytes, it is preferably used for the treatment of acute and / or chronic myelogenous leukemia, and particularly preferably acute myelogenous. Used to treat leukemia.

The monocyte differentiation-inducing agent of the present application can induce the differentiation of abnormally proliferating leukemia cells into monocytes in the treatment of leukemia. Leukemic cells can be induced to differentiate into monocytes lacking autoproliferative and immortalizing abilities by the monocyte differentiation inducer of the present application, and can be gently killed in the original short life span of normal monocytes. That is, the monocyte differentiation inducer of the present application can be used as a differentiation induction therapy.

Since the monocytic differentiation inducer of the present application can gently kill leukemia cells, side effects that can occur with conventionally used chemotherapeutic agents with cell-killing action (for example, digestive organs such as loss of appetite, vomiting, vomiting, diarrhea, etc.) Symptoms, fever, general malaise, sepsis, hair loss, acute respiratory distress syndrome, interstitial pneumonia, liver dysfunction, jaundice, arrhythmia, heart failure, gastrointestinal disorders, central nervous system disorders, liver abscess, acute pancreatitis, pulmonary edema, Painful jaundice, elevated CRP, elevated ALT (GPT), elevated AST (GOT), etc.) can be suppressed.

An example of a related disease of leukemia (specifically, acute myeloid leukemia) is myelodysplastic syndrome (MDS). This is caused by abnormalities in hematopoietic stem cells, and blood cells proliferate abnormally as immature cells in the process of differentiation. As it progresses, it may progress to acute myeloid leukemia.

The monocyte differentiation-inducing agent of the present application can induce the differentiation of immature abnormal cells and gently kill them in the treatment of myelodysplastic syndrome as well as leukemia.

The monocyte differentiation inducers of the present application can improve monocyte levels in the treatment of leukemia or related diseases and prevent various symptoms (eg, bacterial infections, etc.) caused by neutrophil / monocyte depletion. Can also be used for improvement.

As used herein, the term "treatment" means, in addition to treating a disease, preventing the disease, alleviating the symptoms of the disease, slowing the progression of the disease, suppressing the symptoms of the disease, or relieving the symptoms of the disease. And may include inducing improvement in the symptoms of the disease.

In the present application, the "effective amount" is the amount of the active agent required to give the patient the benefit of inducing monocyte differentiation.

In the present application, albendazole can be administered simultaneously with other agents, either separately or in combination.
When administered simultaneously, albendazole and other agents may be contained in the same formulation or in separate formulations. When contained in separate formulations, they may be administered simultaneously via the same or different routes of administration.
When administered separately, albendazole and other agents may be administered separately according to different dosing regimens or by different routes of administration.
When administered continuously, any drug may be administered first. The time between administration of one therapeutic agent and administration of the other is preferably less than 8 hours. More preferably less than 4 hours, even more preferably less than 1 hour.

In the treatment of leukemia or related diseases (eg, myeloid dysplasia syndrome), examples of such other drugs are conventionally used drugs (eg, carmustin, chlorambusyl, romustine, methotrexate, azacitidine, acyclovir, L-asparaginase, etc. Hypoic acid, alemtuzumab, idarubicin, cyclophosphamide, ibritsumomabutiuxetane, imatinib, irinotecan, interferon α, etopocid, enocitabine, epirubicin, erythropoetin, ofatumumab, all-trans-type retinoic acid, carboplatin Mycin, salidomide, cyclosporin, cyclophosphamide, cisplatin, citarabin, didobusin, sobzoxane, daunorubicin, dacarbazine, dasatinib, tamibarotene, thioguanine, dexamethasone, doxorubicin, nirotinib, nerarabin, hydroxyurea, pyralubicin, vincristine Fludalabine, bleomycin, prednisolone, procarbazine, bendamustine, pentostatin, bortezomib, pomalidemid, mitoxanthrone, methylprednisolone, methotrexate, mercartoprin, melphalan, mogamurizumab, lanimustine, rituximab, lenalidomide, etc.

The albendazole-containing monocytic differentiation inducer in the present application is prepared by mixing one or more pharmaceutically acceptable carriers, for example, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions and the like. Oral preparations, or parenteral preparations such as external preparations, suppositories, injections, eye drops, nasal preparations, and pulmonary preparations can be prepared by conventional methods.

Preferred formulations of the monocyte differentiation inducer according to the present application include, for example, tablets and injections.

The carrier / additive contained in the oral preparation of the present application is not particularly limited as long as it is commonly used and pharmaceutically acceptable, but for example,
Excipients (eg, lactose, sucrose, D-mannitol, D-sorbitol, corn starch, dextrin, microcrystalline cellulose, crystalline cellulose, carmellose, carmellose calcium, sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, and Arabia. Rubber);
Disintegrants (eg, carmellose, carmellose calcium, carmellose sodium, carboxymethyl starch sodium, croscarmellose sodium, crospovidone, low degree of substitution hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and crystalline cellulose);
Binders (eg, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone, crystalline cellulose, sucrose, dextrin, starch, gelatin, sodium carmellose, and gum arabic);
Fluidizers (eg, light silicon dioxide and magnesium stearate);
Lubricants such as magnesium stearate, calcium stearate, and talc can be used.

The carrier / additive contained in the liquid preparation (including injection) of the present application is not particularly limited as long as it is commonly used and pharmaceutically acceptable, but for example,
Solvents (eg water, ethanol, propylene glycol, macrogol, glycerin, etc.);
Dissolution aids (eg, propylene glycol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, tromethamole (tris [hydroxymethyl] aminomethane), meglumine, etc.);
Isotonic agents (eg glucose, glycerin, D-mannitol, D-sorbitol, sodium chloride, sucrose, potassium chloride, etc.);
Buffering agents (eg trisodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, boric acid, citric acid, sodium citrate, tartaric acid, acetic acid, sodium acetate, epsilon-aminocaproic acid, sodium glutamate) Such);
Acidity regulators (eg, sodium hydrogen phosphate, sodium acetate, sodium carbonate, sodium citrate, sodium hydroxide, hydrochloric acid, etc.);
Dissolving agents (eg, meglumine) and the like can be used.

The amount of albendazole contained in the monocyte differentiation inducer according to the present application is not particularly limited and is widely selected. For example, in the case of tablets, it is about 0.05% by weight to about 90% by weight with respect to the composition. In the case of an injection, it is preferably about 0.01% by weight to about 50% by weight, more preferably about 0.05% by weight to about 20% by weight, still more preferably about 0.2% by weight to about 10% by weight. Yes, it can be administered by appropriately diluting it with a diluting solution (for example, physiological saline) at the time of use (for example, intravenous administration).

The dose of albendazole of the present application is appropriately selected depending on the administration method, subject, age of subject, degree of disease, symptom, dosage form, administration route, etc., and for example, 0.1 mg orally per day. It can be administered in a dose of ~ 100 g, preferably 1 mg to 50 g, more preferably 10 mg to 20 g, more preferably 100 mg to 10 g. Examples of the lower limit of the daily dose of albendazole include 0.1 mg, 1 mg, 10 mg, 100 mg, and 400 mg, and examples of the upper limit include 700 mg, 1 g, 3 g, 5 g, 10 g, 20 g, 50 g and 100 g are mentioned, and the preferable range of the daily dose of albendazole can be indicated by the combination of the upper limit value and the lower limit value. The amount of albendazole per day may be administered in 1 to several divided doses.

The administration target of the monocyte differentiation inducing agent of the present application is not particularly limited, but is preferably mammals (humans and mammals other than humans (for example, cows, horses, pigs, dogs, cats, mice, rats, rabbits, monkeys)). , More preferably human.

Kruppel-like transcription factor 4 (KLF4) is one of the KLF family transcription factors, and its expression is known to be suppressed in hematopoietic tumors (Non-Patent Document 1). As shown in Test Examples 1 to 3 below, the inventors of the present application have clarified that the signal pathway by KLF4 and its downstream factor DPYSL2A contributes to the differentiation of hematological progenitor cells into monocytes ( Presented at the 2016 American Society of Hematology: Non-Patent Document 2).
Based on the above findings, as shown in Test Examples 4 to 7 below, the inventors of the present application prepared a cell line in which a reporter construct in which luciferase was ligated to the promoter of KLF4 was constitutively introduced into HEK293T cells, and these cells were prepared. A screening system was constructed in which various compounds were added to the strain to measure luciferase activity, and this screening system increased the expression of KLF4 and showed high activity in inducing differentiation into monocytes. Was clarified for the first time, leading to the invention of the present application.
Hereinafter, the present invention will be described with reference to test examples, but the present invention is not limited to these test examples.

Method <cell line>
Acute myeloid leukemia (AML) -derived THP-1 and KG-1a cells were purchased from the RIKEN biological resource center (BRC) (Japan).
SKNO-1 cells and fetal kidney-derived HEK293T cells were purchased from the Japanese Collection of Research Bioresources (JCRB) (Japan).
AML-derived OCI-AML3 and MOLM-13 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Germany).
AML-derived MV4-11 and KG-1a cells were purchased from the American Type Culture Collection (ATCC) (USA).
NB4 and UF-1 cells derived from acute promyelocytic leukemia (APL) were donated by Dr. Y. Ikeda (Keio University School of Medicine, Japan).
AML-derived ME-1 cells were donated by Dr. PP Liu (National Human Genome Research Institute, National Institutes of Health, USA).
HEK293T cells are in a humid environment of _{5% CO 2} and 95% air in Dulbecco's Modified Eagle's Medium (DMEM) (10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) supplementation), 37. Maintained in the incubator at ° C.
Other cells were cultured in Rosswell Park Memorial Institute (RPMI) 1640 medium (containing 10% FBS and 1% PS) at _{37 ° C. under 5% CO 2} and 95% air.

＊：ＭＯＬＭ−１３は骨髄異形成症候群（ＭＤＳ）から発症した白血病である。
＊＊：ＮＢ４およびＵＦ−１はオールトランス型レチノイン酸（ＡＴＲＡ）耐性である。 The above leukemia cell lines are classified as shown in the table below in the FAB classification.

*: MOLM-13 is a leukemia that develops from myelodysplastic syndrome (MDS).
**: NB4 and UF-1 are all-trans retinoic acid (ATRA) resistant.

<IC ₅₀ evaluation>
For cell survival analysis, cells were seeded at a concentration of 1 × 10 ⁵ cells / mL. A predetermined concentration of drug was added to the culture medium, and the cells were cultured for 48 hours. Followed by WST assay Cell viability, Cell Count Reagent (nacalai tesque, Inc.) and Infinite was evaluated using as described in ^(TM) 200 PRO multimode reader the (TECAN) manufacturer. Depict percent inhibition curve, an IC ₅₀ value of the drug median-effect method (Chou TC, Talalay P. Quantitative analysis of dose-effect relationships:.. The combined effects of multiple drugs or enzyme inhibitors Adv Enzyme Regul 1984; 22 : 27-55) Calculated based on.

<Real-time quantitative PCR (qRT-PCR)>
Total RNA was isolated using the RNeasy mini kit (Qiagen) and reverse transcribed using the Reverse script kit (TOYOBO) to prepare cDNA. Real-time quantitative polymerase chain reaction (PCR) was performed by the 7500 Real-Time PCR System (Applied Biosystems) according to the manufacturer's recommendations. Results were standardized for GAPDH levels. Relative expression levels were calculated using the 2-ΔΔCt method. The primers used for qRT-PCR are shown in the table below.

<ChIP-qPCR>
Chromatin immunoprecipitation assay (ChIP), was performed according SimpleChIP ^{(TM) Plus Enzymatic Chromatin IP Kit (Cell} Signaling Technology, USA) manufacturer's protocol using the. Briefly, cells were cross-linked in 1% formaldehyde / PBS for 10 minutes at room temperature. Quenched with glycine, cell pellets were collected, lysed, and then sonicated with the Q55 sonicator system (QSONICA, USA). The supernatant was diluted with the same sonicated buffer and immunoprecipitated with anti-KLF4 antibody (ab106629, abcam) at 4 ° C. overnight. Agarose beads were then added and washed with wash buffer as recommended. Chromatin DNA was reverse cross-linked and purified by ethanol precipitation. After ChIP, the precipitated DNA was quantified by qPCR using a 7500 Real-Time PCR System (Applied Biosystems) using standard procedures. The primers for ChIP-qPCR are shown in the table below.

<SiRNA interference>
Specific shRNAs targeting human KLF4 and DPYSL2A were designed and subcloned into pENTR4-H1tetOx1 and CS-RfA-ETV vectors. These vectors were provided by Dr. H. Miyoshi (RIKEN, BRC, Japan). A non-targeted control shRNA was designed for luciferase (sh_Luc). The target sequences are shown in the table below.

<Expression plasmid>
Human KLF4, DPYSL2A and DPYSL2B cDNAs were amplified by PCR and then inserted into the pENTR1A dual selection vector (Thermo Fisher Scientific), CSIV-TRE-Ubc-KT, and pLenti CMV Puro DEST (addgene) expression vectors. CSIV-TRE-Ubc-KT was provided by Dr. H. Miyoshi (RIKEN, BRC, Japan). All PCR products were confirmed by DNA sequencing.

<Production and transduction of wrench virus>
Produced wrench virus as described in Morita K, Suzuki K, Maeda S, et al. Genetic regulation of the RUNX transcription factor family has antitumor effects. J Clin Invest. 2017; 127 (7): 2815-2828. It was. Briefly, HEK293T cells were transiently co-introduced with a lentiviral vector (eg psPAX2 and pMD2.G) by polyethyleneimine (PEI, Sigma-Aldrich). Forty-eight hours after transfection, the virus supernatant was collected and used immediately for infection, followed by immunofluorescence (Kusabira-Orange or Venus) with a flow cytometer Aria III (BD Biosciences) for successfully transduced cells. Sorted based on.

<Immune blot>
Immunoblots were performed as described in Morita K, Masamoto Y, Kataoka K, et al. BAALC potentiates oncogenic ERK pathway through interactions with MEKK1 and KLF4. Leukemia. 2015; 29 (11): 2248-2256. Membrane with the following primary antibodies: anti-KLF4 (# 4038, Cell Signaling Technology), anti-GAPDH (FL-335, Santa Cruz Biotechnology, Inc.), anti-DPYSL2 (HPA002381, Sigma-Aldrich), and anti-TCP-1γ (CCT3) ) Antibody (F-3) (sc-271336, Santa Cruz Biotechnology) Antibodies were reacted and HRP-conjugated anti-rabbit IgG and anti-mouse IgG (Cell Signaling Technology) were used as secondary antibodies. Blots were visualized using Chemi-Lumi One Super (nacalai tesque, Inc.) and ChemiDoc ^™ XRS + Imager (Bio-Rad Laboratories, Inc.) as recommended by the manufacturer. Protein levels were quantified using Image Lab Software (Bio-Rad Laboratories, Inc.).

<Statistics>
The statistical significance of the difference between the control group and the test group was evaluated by a two-sided unpaired Student's t-test, and was considered significant when the p value was less than 0.05. Homoscedasticity in the two populations was calculated by F-test. Results were expressed as mean ± SEM values obtained from three independent trials. In the transplantation experiment, animals were randomly divided into test groups and treated blindly. The survival rates of each group were compared using the log-rank test.

<Mouse>
NOD / Shi-sid, IL-2RγKO (NOG) mice were purchased from the Central Institute for Experimental Animals (Japan). Littermates were used as controls in all studies.

<Xenograft mouse model>
A xenograft mouse model of a human cancer cell line was created using NOG mice. For AML mouse model, the THP-1 cells (2 × 10 ⁶ cells / mouse) was administered intravenously. Peripheral blood (PB) was collected weekly and chimerization was checked with a flow cytometer using anti-human CD45 antibody (BD Biosciences). One week after injection, mice received albendazole (50 mg / kg body weight, twice / week, oral (po)) or an equal dose of dimethyl sulfoxide (DMSO).

<Test Example 1>: Overexpression of KLF4 induces differentiation of AML cells.
THP-1 cells transduced with KLF4 or the lentivirus encoding the control were treated with 3 μM doxycycline for 48 hours, then collected and cytospinned onto glass slides. Each slide was Diff-Quik stained (modified Giemsa stain). A representative microscopic image of THP-1 cells is shown in FIG.
As shown in FIG. 1, when KLF4 was overexpressed in an AML cell line, abnormal cells were induced to differentiate into monocyte-like cells.

<Test Example 2>: DPYSL2A is a direct downstream factor of KLF4.

Test Example 2-1: Expression level of candidate gene in KLF4 high-expressing THP-1 cells THP-1 cells are transduced with KLF4 or a lentivirus encoding a control, treated with 3 μM doxycycline for 48 hours, and then total RNA is prepared. And analyzed by real-time RT-PCR. Values were standardized for control vector transduced cells (n = 3). The results are shown in FIG. 2-1. As shown in FIG. 2-1 the expression of DPYSL2A increased by more than 2000 times under the overexpression of KLF4.

Test Example 2-2: KLF4 upregulates the gene expression of DPYSL2A.
(1) THP-1 cells were treated in the same manner as in Test Example 2-1 and the expression level of the DPYSL family was analyzed by real-time RT-PCR. Values were standardized for control vector transduced cells (n = 3). The results are shown in Figure 2-2.
(2) THP-1 and MOLM-13 cells transformed with KLF4 or control-encoding lentivirus were treated with 3 μM doxycycline for 48 hours, lysed for protein extraction, and immunoblotted with KLF4, DPYSL2A, and GAPDH. Was done. The results are shown in Figure 2-3.

As shown in FIGS. 2-2 and 2-3, KLF4 upregulated the gene expression of DPYSL2A.

<Test Example 3>: DPYSL2A is an important mediator of KLF4-induced differentiation.

Test Example 3-1: THP-1 cells transduced with a GaAs virus encoding shRNA targeting KLF4 and DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.) Are treated with 3 μM doxycycline for 48 hours. It was then lysed for protein extraction and immunoblotted for KLF4, DPYSL2A and GAPDH. The results are shown in FIG. 3-1.

Test Example 3-2: THP-1 cells of Test Example 3-1 were treated with 3 μM doxycycline for 48 hours and collected, and the cell surface expression of CD11b and CD14 was measured by flow cytometry. The results are shown in Figure 3-2.

Test Example 3-3: The cell proliferation curve of the THP-1 cells of Test Example 3-1 is shown in FIG. 3-3.
Cells were cultured in the presence of 3 μM doxycycline (n = 3).

Test Example 3-4: A representative microscopic image of the THP-1 cells of Test Example 3-1 is shown in FIG. 3-4.
Cells were treated with 3 μM doxycycline for 48 hours, collected and cytospinned onto glass slides. Each slide was Diff-Quik stained (modified Giemsa stain).

As shown in FIGS. 3-1 to 4, THP-1 cells expressing KLF4 and DPYSL2A differentiated into monocyte-like cells, and after 5 days of culture, the cells were significantly compared to the cells in which DPYSL2A was knocked down. The number has decreased.

<Test Example 4>: Albendazole strongly differentiates AML cells by stimulating the KLF4-DPYSL2A system.

Test Example 4-1: Drug screening to identify KLF4-inducible compounds.
HEK293T cells were transfected with a KLF4 promoter reporter vector (a plasmid in which the KLF4 promoter sequence was inserted on the N-terminal side of the luciferase gene), and then exposed to 10 μM of each compound for 24 hours. The cells were then collected and lysed and the luciferase activity was measured with a luminometer. In the first round, 2590 compounds (purchased from the Medical Research Support Center, Graduate School of Medicine, Kyoto University) were screened. The top 342 compounds were selected for the second round, and finally 38 compounds that could potentially activate KLF4 expression were selected for further assay. A schematic diagram of the results is shown in FIG. 4-1.

Test Example 4-2: Relative expression of KLF4 in THP-1 cells treated with the 38 candidate compounds identified in Test Example 4-1.
Cells were treated with 10 μM of these compounds for 24 hours, then total RNA was prepared and analyzed by real-time RT-PCR. Values were standardized for control vector transduced cells (n = 3). The results are shown in FIG. 4-2.

Test Example 4-3: Relative expression of KLF4 and DPYSL2A in albendazole-treated THP-1 cells.
Cells were treated with albendazole (1 μM) for 24 hours, then total RNA was prepared and analyzed by real-time RT-PCR. Values were standardized for control vector transduced cells (n = 3). The results are shown in FIG. 4-3.

Test Example 4-4: Cell surface expression of CD11b and CD14 in albendazole-treated THP-1 cells was measured by flow cytometry.
Cells were treated with albendazole (1 μM) for 24 hours and collected for flow cytometric analysis. The results are shown in FIG. 4-4.

Test Example 4-5: Representative microscopic images of THP-1 cells of Test Example 4-4.
Diff-Quik staining (modified Giemsa staining) was performed on each slide. (Enlargement 20 x, scale bar: 50 μm). The results are shown in FIG. 4-5.

Test Example 4-6: Presence of 1nMPMA transduced with shRNA targeting KLF4 (sh_KLF4 # 1 and # 2), DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.) Growth curve below.
shRNA expression was induced with 3 μM doxycycline. The results are shown in FIG. 4-6.

As shown in FIGS. 4-1 to 5, albendazole increased the expression of KLF4 and DPYSL2A and differentiated THP-1 cells into monocyte-like cells. As shown in FIG. 4-6, the antitumor effect of albendazole was suppressed in cells in which KLF4 or DPYSL2A was knocked down, indicating that the expression of KLF4 by albendazole produces such an antitumor effect. Was done.

<Test Example 5>: _{IC 50} evaluation -I

Test Example 5-1: Dose-response curve of albendazole in THP-1 cells and MOLM-13 cells.
Cells were treated with a given concentration of albendazole for 48 hours (n = 3). The results are shown in FIG. 5-1.

Test Example 5-2: Dose-response curve of albendazole and ATRA in ATRA-resistant NB4 and UF-1 cells.
Cells were treated with a given concentration of albendazole or ATRA for 48 hours (n = 3). The results are shown in FIGS. 5-2 and 5-3. The data are mean ± SEM values.

For each cell in the table below shows ATRA and / or _{IC 50} of albendazole. Albendazole showed a very low IC ₅₀ for each leukemic cells.

<Test Example 6>: _{IC 50} evaluation -II
AML cell lines were treated with various concentrations of albendazole for 48 hours to calculate _{IC 50.} The results are shown in the table below.
Albendazole showed a very low _{IC 50} with respect to a wide variety of AML cells.

<Test Example 7> In vivo test: xenograft mouse model

Test Example 7-1
THP-1 cells were transplanted into NOG mice, and 7 days after transplantation, the mice were randomly divided into two groups (albendazole group and DMSO group) (n = 7 each). The albendazole and DMSO groups were treated with albendazole (50 mg / kg bw, twice / week, po) or an equal dose of DMSO (control). As shown in FIG. 6-1, overall survival in the albendazole group was significantly prolonged compared to the DMSO group.

Test Example 7-2
Mice treated according to Test Example 7-1 were appropriately anesthetized and sacrificed. Bone marrow tissue was harvested and hematoxylin-eosin (H & E) staining and immunohistochemical staining with anti-human CD45 antibody were performed on each slide. Representative images of the spleen and liver of AML (THP-1 cell) xenograft mice (30 days after transplantation) are shown in FIG. 6-3. As shown in FIG. 6-2, the proliferation of THP-1 cells was suppressed. As shown in FIG. 6-3, albendazole suppressed swelling of the liver and spleen.

Test Example 8
From the bone marrow cells of wild-type C57BL / 6 mice, c-Kit surface antigen was stained and positive cells (these cells are considered to be immature cells that are hardly differentiated and enter both the myeloid system and lymphatic system. (Has the ability to differentiate) was recovered using a flow cytometer. 5000 c-kit surface antigen-positive cells were seeded on a methylcellulose semi-solid medium, and albendazole was administered at 0 nM, 100 nM, and 500 nM, and cultured at 37 ° C. for 1 week. After 1 week, colonization was observed under a microscope and counted. The results are shown in FIG. As is clear from FIG. 7, the number of monocyte colonies increased more than that of granulocytic colonies.

Claims (6)

A monocyte differentiation inducer containing albendazole as an active ingredient. The monocyte differentiation inducer according to claim 1, for treating a disease associated with inhibition of the differentiation process of hematopoietic stem cells. The monocyte differentiation-inducing agent according to claim 2, wherein the disease associated with inhibition of the differentiation process of the hematopoietic stem cell is selected from aplastic anemia, Costman syndrome, leukemia or a related disease thereof. The monocyte differentiation-inducing agent according to claim 2 or 3, wherein the disease associated with inhibition of the differentiation process of hematopoietic stem cells is leukemia or a related disease thereof. The monocyte differentiation-inducing agent according to any one of claims 2 to 4, wherein the leukemia or a related disease thereof is chronic myelogenous leukemia, acute myelogenous leukemia or myelodysplastic syndrome. The monocyte differentiation inducer according to claim 4 or 5, which is used as a differentiation induction therapy.

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