WO2019151514A1

WO2019151514A1 - Monocyte differentiation inducer containing albendazole

Info

Publication number: WO2019151514A1
Application number: PCT/JP2019/003802
Authority: WO
Inventors: 剣森田; 壯一足立; 正敏萩原
Original assignee: 国立大学法人京都大学
Priority date: 2018-02-05
Filing date: 2019-02-04
Publication date: 2019-08-08
Also published as: JP2021059495A

Abstract

Provided is a monocyte differentiation inducer which contains albendazole as an active ingredient.

Description

Albendazole-containing monocyte differentiation inducer

This 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, and examples include leukemia, aplastic anemia, and Costman syndrome.

Leukemia is a disease thought to occur when hematopoietic stem cells (or slightly differentiated cells) become tumors. Chemotherapy is the basis of definitive treatment in leukemia. However, chemotherapy requires a sufficient general state and organ function to withstand organ toxicity and complications. There are many cases where elderly patients with leukemia who are unable to apply therapy and have no cure. There is a need for new therapeutic agents with few side effects for such patients.

”Differentiation induction therapy is known as a treatment for leukemia with few side effects. In differentiation-inducing therapy, leukemia cells that continue to proliferate while remaining in the state of progenitor cells are killed by the normal life of normal leukocytes by differentiation and maturation, so leukemia cells such as many anticancer drugs and chemotherapy There are fewer side effects compared to treatments that directly destroy 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. Currently, no effective differentiation induction therapy has been established. Acute promyelocytic leukemia is ATRA-resistant. A new differentiation induction therapy effective for a wide range of leukemias is strongly desired in the medical field.

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 found to inhibit the maturation of myeloid granulocyte cells. A group of diseases in which neutrophils decrease.
In diseases caused by inhibition of the differentiation process of hematopoietic stem cells, such as leukemia, aplastic anemia, and Costman syndrome, hematopoietic function is reduced, resulting in decreased neutrophils and monocytes and cell infection. It becomes a problem. As a countermeasure against bacterial infections in these diseases, neutrophil differentiation inducers such as granulocyte colony stimulating factor (G-CSF) are currently administered. There are cases where it does not increase. In order to improve the symptoms in these diseases, an effective hematopoietic differentiation inducer that promotes the differentiation process from hematopoietic stem cells is desired.

All disclosures of prior art documents cited in this specification are incorporated herein by reference.

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

As a result of diligent studies to solve the above-mentioned problems, the present inventors have found that albendazole, a parasite-controlling agent, unexpectedly strongly induces differentiation of leukemia cells into monocyte-like cells, It came to this application. Surprisingly, it has been found that albendazole induces differentiation of normal progenitor cells into monocytes. 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 agent for inducing differentiation of monocytes according to [1] for treating a disease accompanied by inhibition of a differentiation process of hematopoietic stem cells.
[3] The agent for inducing differentiation of monocytes according to [2], wherein the disease accompanied by inhibition of the differentiation process of hematopoietic stem cells is selected from aplastic anemia, Costman syndrome, or leukemia or related diseases.
[4] The monocyte differentiation inducer according to [2] or [3], wherein the disease accompanied by inhibition of the differentiation process of the hematopoietic stem cells is leukemia or a related disease thereof.
[5] The agent for inducing differentiation of monocytes according to any one of [2] to [4], wherein the leukemia or a related disease thereof is chronic myelogenous leukemia, acute myeloid 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, comprising 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 involving inhibition of the differentiation process of hematopoietic stem cells.

[9] Use of albendazole for producing a monocyte differentiation inducer.
[10] The use according to [9], wherein the agent for inducing differentiation of monocytes is used for treating a disease accompanied by inhibition of a 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 a disease accompanied by inhibition of the differentiation process of hematopoietic stem cells.

Albendazole has a high differentiation-inducing activity toward monocytes and can be used as a monocyte differentiation-inducing agent. Furthermore, albendazole is already on the market as a therapeutic agent for hydatid disease (echinococcosis), and since data on safety to humans is accumulated, a safe monocyte differentiation inducer can be provided.

Representative microscopic image of THP-1 cells transduced with KLF4 or control encoding lentivirus (Diff-Quik staining) (magnification 20 ×). Immunoblots of KLF4, DPYSL2A (DPYSL2A) and GAPDH in THP-1 and MOLM-13 cells transformed with KLF4 or lentivirus encoding control. Immunoblots of KLF4, DPYSL2A and GAPDH in THP-1 cells transduced with lentivirus encoding shRNA targeting KLF4 and DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.). THP-1 cells of Test Example 3-1 were collected by treatment with 3 μM doxycycline for 48 hours, and cell surface expression of CD11b and CD14 was measured by flow cytometry. The cell growth curve of the THP-1 cell of Test Example 3-1. Data are mean ± SEM values. * P <0.05, *** P <0.001, two-sided student t test. A representative microscopic image of THP-1 cells of Test Example 3-1. (Enlarged 20 ×, scale bar: 50 μm) Relative expression of KLF4 and DPYSL2A in THP-1 cells treated with albendazole. Left: Before processing. Right: After processing. Data are mean ± SEM values. ** P <0.01, *** P <0.001, two-sided Student t test. Cell surface expression of CD11b and CD14 in THP-1 cells treated with albendazole was measured by flow cytometry. Representative microscopic images of THP-1 cells of Test Example 4-3. Growth curves in the presence of 1 nMPMA 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.). Albendazole dose response curves in THP-1 and MOLM-13 cells. Albendazole dose response curve in ATRA resistant UF-1 cells. Albendazole dose response curve in ATRA resistant NB4 cells. Overall survival of NOG mice transplanted with THP-1 cells and treated with albendazole or control. P <0.001, log-rank (Mantel-Cox) test. Representative microscopic image of bone marrow of AML (THP-1 cell) xenograft mice (30 days after transplantation). (Magnification 4 × and 20 ×, scale bar 100 μm). (D) Representative images of spleen and liver of AML (THP-1 cells) xenograft mice (30 days after transplant) in (C). Albendazole was administered at 0 nM, 100 nM, and 500 nM to c-kit surface antigen positive cells collected from bone marrow of wild type C57BL / 6 mice, and colony formation was confirmed after 1 week. The vertical axis represents pieces / dish.

This application provides a monocyte differentiation inducer containing albendazole as an active ingredient. The monocyte differentiation inducer of the present application can be used, for example, to treat a disease accompanied by inhibition of the differentiation process of hematopoietic stem cells. In addition, the monocyte differentiation inducer of the present application can be used for patients with reduced neutrophils or monocytes regardless of the presence or absence of a disease accompanied by inhibition of the differentiation process of hematopoietic stem cells. It can be used to prevent or ameliorate symptoms that can be caused by a decrease in spheres or monocytes (eg, bacterial infections).

Albendazole [Chemical name: (methyl 5- (propylthio) -2-benzimidazolecarbamate)]
Is known as a benzimidazole-type anthelmintic agent and has already been marketed as a therapeutic agent for encaustic diseases (eg, echinococcosis) (for example, Escazole ^{(registered trademark)} tablets in Japan). The action is thought to be due to the inhibition of tubulin binding to the colchicine binding site and the formation of microtubules.

JP-T-2004-525140, JP-T-2008-522984, and JP-T-2016-535014 disclose solid cancers such as liver cancer, colon cancer, pancreatic cancer, ovarian cancer and the like using albendazole known as an anthelmintic agent. However, it does not describe a specific effect on leukemia. Moreover, these literatures do not describe induction of blood cell differentiation by albendazole.

In the present application, albendazole may be synthesized by a known method, or may be obtained as a commercial 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” means a hematopoietic stem cell (strictly not a hematopoietic stem cell but an immature cell group that has the ability to differentiate into both myeloid and lymphoid cells (eg, c-kit surface antigen positive cells) And / or induce differentiation of myeloid hematopoietic progenitor cells into monocytes. Monocytes differentiated by the monocyte differentiation inducer of the present application are at least one of the morphological features commonly used to identify monocytes (eg, notched or partially depressed) With nuclei; with basophil cytoplasmic granules (azurole granules); with vacuoles in the cytoplasm) and / or with features based on surface antigen classification (eg CD11b positive and CD14 positive) and self-proliferating ability And cells that do not have the ability to immortalize. The gene information of the monocytes induced to differentiate is not particularly limited, and for example, gene information derived from a disease can be retained.

In the present application, “myeloid hematopoietic progenitor cell” means a progenitor cell found in the process of differentiation from a hematopoietic stem cell into a myeloid blood cell, such as a myeloid hematopoietic stem cell and a mature differentiated cell from a myeloid hematopoietic stem cell. It means a progenitor cell in the process of differentiation into (for example, mast cell, basophil, eosinophil, neutrophil, monocyte, megakaryocyte, or erythrocyte). Examples of myeloid hematopoietic progenitor cells include: myeloid hematopoietic stem cells, granule / monocytic stem cells, erythroblast / megakaryocytic stem cells, myeloblasts, promyelocytes, myelospheres, postmyelocytes, rods Examples thereof include nuclear spheres, segmented nucleus nuclei, monoblasts, pre-monocytes, pre-erythroblasts, erythroblasts, and pre-megakaryocytes. Hematopoietic stem cells (which are not strictly hematopoietic stem cells but include immature cells that have the ability to differentiate into both myeloid and lymphoid) and myeloid progenitor cells are normal even if they are normal cells. (For example, canceration).

The monocyte differentiation inducer of the present application includes hematopoietic stem cells (including not strictly hematopoietic stem cells but immature cells and cells capable of differentiating into both myeloid and lymphoid systems) and / or myeloid progenitors. Inducing differentiation of cells into monocytes can treat diseases involving inhibition of the differentiation process of hematopoietic stem cells (eg, aplastic anemia, Costman syndrome, or leukemia or related diseases (eg myelodysplastic syndrome)) .

Aplastic anemia is susceptible to severe bacterial infections such as pneumonia and sepsis because the differentiation process is inhibited by some abnormality in hematopoietic stem cells, and blood cells in blood, particularly neutrophils, decrease.

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

In the treatment of aplastic anemia or costman syndrome, the monocyte differentiation inducer of the present application is a symptom (for example, bacterial infection) that is accompanied by a decrease in blood cells (particularly neutrophils) in addition to their fundamental treatment, for example. Can be used to treat. The functions of macrophages matured from monocytes to bacterial infection are similar to those of neutrophils. By inducing differentiation into monocytes using the monocyte differentiation inducer of the present application, symptoms such as bacterial infections can be treated. Can be done.

Leukemia is a disease thought to occur when hematopoietic stem cells (or slightly differentiated cells) become tumors. Tumorized cells (leukemia cells) grow abnormally and occupy the bone marrow, and normal hematopoietic function is significantly inhibited. As a result, red blood cells, normal white blood cells, and platelets decrease, and as the disease progresses, enlargement of the spleen, liver, and lymph nodes due to infiltration of leukemia cells begins to occur.

In leukemia, leukemia cells, which are progenitor cells that have been differentiated to a certain stage, have proliferated (acute leukemia) and have the ability to differentiate and mature (chronic leukemia). It is divided roughly into. They are also classified into myeloid leukemia and lymphocytic leukemia depending on the cancerous cell lineage.

As one of the classifications of acute leukemia, FAB classification based on morphological classification is known. In the FAB classification, acute myeloid leukemia and acute lymphoblastic leukemia are roughly classified by peroxidase staining, 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 determined as leukemia based on general criteria used by doctors or researchers. Since the monocyte differentiation inducer of the present application has the ability to induce differentiation from myeloid hematopoietic progenitor cells to monocytes, it is preferably used for the treatment of acute and / or chronic myeloid leukemia, particularly preferably acute myeloid Used to treat leukemia.

The monocyte differentiation inducer of the present application can induce differentiation of abnormally proliferating leukemia cells into monocytes in the treatment of leukemia. With the monocyte differentiation inducer of the present application, leukemia cells can be induced to differentiate into monocytes lacking the ability of self-proliferation and immortalization, and can be killed gently with the original short life span of normal monocytes. That is, the monocyte differentiation inducer of the present application can be used as differentiation induction therapy.

Since the monocyte differentiation inducer of the present application can gently kill leukemia cells, side effects (eg, anorexia, nausea, vomiting, diarrhea, etc.) that may occur with conventional chemotherapeutic agents with cell killing action Symptoms, fever, general malaise, sepsis, hair loss, acute respiratory distress syndrome, interstitial pneumonia, liver dysfunction, jaundice, arrhythmia, heart failure, gastrointestinal dysfunction, central nervous system disorder, liver abscess, acute pancreatitis, lung edema, Painful erythema, CRP elevation, ALT (GPT) elevation, AST (GOT) elevation, etc.) can be suppressed.

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

The monocyte differentiation inducer of the present application can induce the differentiation of immature abnormal cells and gently kill them in the treatment of myelodysplastic syndromes as well as leukemia.

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

As used herein, "treatment" refers to treating disease, preventing disease, alleviating disease symptoms, delaying disease progression, suppressing disease symptoms, ameliorating disease symptoms, And inducing amelioration of disease symptoms.

In the present application, the “effective amount” is the amount of the active agent necessary to give the patient the benefit of inducing differentiation of monocytes.

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

In the treatment of leukemia or related diseases (for example, myelodysplastic syndrome), as other examples of such drugs, conventionally used drugs (for example, carmustine, chlorambucil, lomustine, mechloretamine, azacitidine, acyclovir, L-asparaginase, Arsenite, alemtuzumab, idarubicin, ifosfamide, ibritumomab tiuxetan, imatinib, irinotecan, interferon alpha, etoposide, enocitabine, epirubicin, erythropoietin, ofatatumumab, all-trans retinoic acid, carbogetum zobin, Mycin, thalidomide, cyclosporine, cyclophosphamide, cisplatin, cytarabine, zidobucin, sobuzoxane, daunorubicin, dacarbazine, Dasatinib, tamibarotene, thioguanine, dexamethasone, doxorubicin, nilotinib, nelarabine, hydroxyurea, pirarubicin, vincristine, vindesine, vinblastine, busulfan, fludarabine, bleomycin, prednisolone, procarbazine, bendamustine, pentomistatone, bortezidotron , Methotrexate, mercatopurine, melphalan, mogamulizumab, ranimustine, rituximab, ruxolitinib, lenalidomide, etc.).

The albendazole-containing monocyte differentiation inducer in the present application is mixed with one or more pharmaceutically acceptable carriers, for example, tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, etc. Or a parenteral preparation such as an external preparation, a suppository, an injection, an eye drop, a nasal preparation, a pulmonary preparation and the like.

Examples of preferable dosage forms of the monocyte differentiation inducer according to the present application include tablets and injections.

The carrier / additive contained in the oral preparation of the present application is not particularly limited as long as it is a pharmaceutically acceptable one that is usually used.
Excipients such as lactose, sucrose, D-mannitol, D-sorbitol, corn starch, dextrin, microcrystalline cellulose, crystalline cellulose, carmellose, carmellose calcium, sodium carboxymethyl starch, low substituted hydroxypropylcellulose, and arabic Rubber);
Disintegrants (eg, carmellose, carmellose calcium, carmellose sodium, carboxymethyl starch sodium, croscarmellose sodium, crospovidone, low substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, and crystalline cellulose);
Binders (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone, crystalline cellulose, sucrose, dextrin, starch, gelatin, carmellose sodium, and gum arabic);
Fluidizing agents (eg, light anhydrous silicic acid, 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 a pharmaceutically acceptable one that is usually used.
Solvent (eg, water, ethanol, propylene glycol, macrogol, glycerin, etc.);
Solubilizers (eg, propylene glycol, benzyl benzoate, ethanol, triethanolamine, sodium carbonate, sodium citrate, trometamol (tris [hydroxymethyl] aminomethane), meglumine, etc.);
Isotonic agents (eg, glucose, glycerin, D-mannitol, D-sorbitol, sodium chloride, sucrose, potassium chloride, etc.);
Buffers (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);
pH adjusting agents (for example, sodium hydrogen phosphate, sodium acetate, sodium carbonate, sodium citrate, sodium hydroxide, hydrochloric acid, etc.);
Solubilizers (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 selected in a wide range. For example, in the case of a tablet, it is about 0.05% to about 90% by weight based on the composition. In the case of an injection, it is preferably about 0.01 wt% to about 50 wt%, more preferably about 0.05 wt% to about 20 wt%, more preferably about 0.2 wt% to about 10 wt%. Yes, at the time of use, it can be appropriately diluted with a diluting solution (eg, physiological saline) and administered (eg, intravenous administration).

The dose of albendazole of the present application is appropriately selected depending on the administration method, the subject, the age of the subject, the degree of the disease, symptoms, dosage form, administration route, etc., for example, orally 0.1 mg per day It can be administered at 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 can be mentioned, and a preferable range of the daily dose of albendazole can be indicated by a combination of the upper limit value and the lower limit value. The amount of albendazole per day may be administered in one to several divided doses.

The subject of administration of the monocyte differentiation inducer of the present application is not particularly limited, but is preferably a mammal (human and non-human mammals (eg, cows, horses, pigs, dogs, cats, mice, rats, rabbits, monkeys)). More preferably, it is a human.

Kruppel-like transcription factor 4 (KLF4) is one of the KLF family transcription factors and is known to be suppressed in hematopoietic tumors (Non-patent Document 1). As shown in Test Examples 1 to 3 below, the present inventors have clarified that the signal pathway by KLF4 and its downstream factor DPYSL2A contributes to the differentiation from hematopoietic progenitor cells to monocytes ( Presented at American Society of Hematology in 2016: Non-Patent Document 2).
Based on the above findings, as shown in Test Examples 1 to 7 below, the inventors of the present application show that albendazole (ABZ) increases the expression of KLF4 and is highly active in inducing differentiation into monocytes through stimulation of DPYSL2A. It was revealed for the first time that the present invention was achieved.
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 RIKEN biological resource center (BRC) (Japan).
SKNO-1 cells and fetal kidney-derived HEK293T cells were purchased from 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 American Type Culture Collection (ATCC) (USA).
NB4 and UF-1 cells derived from acute promyelocytic leukemia (APL) were provided by Dr. Y. Ikeda (Keio University School of Medicine, Japan).
AML-derived ME-1 cells were provided by Dr. PP Liu (National Human Genome Research Institute, National Institutes of Health, USA).
HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (supplemented with 10% heat-inactivated fetal calf serum (FBS) and 1% penicillin-streptomycin (PS)) in a humidified environment of 5% CO ₂ and 95% air, 37 Maintained in an incubator at 0 ° C.
Other cells were cultured at 37 ° C. in 5% CO ₂ and 95% air in Roswell Park Memorial Institute (RPMI) 1640 medium (containing 10% FBS and 1% PS).

The above leukemia cell lines are classified as shown in the following table in the FAB classification.

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

<IC ₅₀ evaluation>
For cell viability 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).

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

<SiRNA interference>
Specific shRNAs targeting human KLF4 and DPYSL2A were designed and subcloned into the 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 against 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 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.

<Lentivirus production and transduction>
Lentivirus production is performed 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-transfected with polyethyleneimine (PEI, Sigma-Aldrich) with lentiviral vectors (eg psPAX2 and pMD2.G). Forty-eight hours after transfection, viral supernatants are collected and used immediately for infection, and then successfully transduced cells are immunofluorescent (Kusabira-Orange or Venus) with a flow cytometer Aria III (BD Biosciences). Sorted based on.

<Immunoblotting>
Immunoblots were performed as described by 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. The membrane was treated with the following primary antibodies: anti-KLF4 (# 4038, Cell Signaling Technology), anti-GAPDH (FL-335, Santa Cruz Biotechnology, Inc.), anti-DPYSL2 (HPA002381, Sigma-Aldrich), anti-DPYSL antibody (HPA002381, Sigma) -Aldrich) 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.) according to the manufacturer's recommendations. 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 t test, and was considered significant when the p value was less than 0.05. Equal variance in the two populations was calculated by F test. Results were expressed as mean ± SEM values obtained from three independent tests. In the transplantation experiment, the animals were randomly divided into test groups, and the treatment was performed in a blinded manner. The survival rate of each group was compared using a log-rank test.

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

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

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

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

THP-1 and MOLM-13 cells transformed with lentivirus encoding KLF4 or control (Ctrl) were treated with 3 μM doxycycline for 48 hours, lysed for protein extraction, and immunoblotting of KLF4, DPYSL2A, and GAPDH Went. The results are shown in FIG.

As shown in FIG. 2, KLF4 up-regulated DPYSL2A gene expression.

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

Test Example 3-1 THP-1 cells transduced with a lentivirus encoding shRNA targeting KLF4 and DPYSL2A (sh_DPYSL2A # 1 and # 2) or control luciferase (sh_Luc.) Were treated with 3 μM doxycycline for 48 hours And 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 collected by treatment with 3 μM doxycycline for 48 hours, and cell surface expression of CD11b and CD14 was measured by flow cytometry. The results are shown in Fig. 3-2.

Test Example 3-3: The cell growth curve of the THP-1 cell 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 cell of Test Example 3-1 is shown in FIG. 3-4.
Cells were collected by treatment with 3 μM doxycycline for 48 hours and cytospun onto glass slides. Each slide was subjected to Diff-Quik staining (modified Giemsa staining).

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 different from those 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: Relative expression of KLF4 and DPYSL2A in THP-1 cells treated with albendazole.
Cells were treated with albendazole (1 μM) for 24 hours, then total RNA was prepared and analyzed by real-time RT-PCR. Values were normalized for each sample with GAPDH and then normalized to control vector transduced cells (n = 3). The results are shown in Fig. 4-1.

Test Example 4-2: Cell surface expression of CD11b and CD14 in THP-1 cells treated with albendazole was measured by flow cytometry.
Cells were treated with albendazole (1 μM) for 24 hours and harvested for flow cytometric analysis. The results are shown in Fig. 4-2.

Test Example 4-3: Representative microscopic image of THP-1 cells of Test Example 4-2.
Diff-Quik staining (modified Giemsa staining) was performed on each slide. (Enlargement 20 ×, scale bar: 50 μm). The results are shown in Fig. 4-3.

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

As shown in FIGS. 4-1 to 3, albendazole increased the expression of KLF4 and DPYSL2A and differentiated THP-1 cells into monocyte-like cells. As shown in FIG. 4-4, since the antitumor effect of albendazole was suppressed in cells knocked down with KLF4 or DPYSL2A, the expression of KLF4 by albendazole shows that such an antitumor effect appears. It 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 predetermined concentration of albendazole for 48 hours (n = 3). The results are shown in FIG.

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

The table below shows the IC ₅₀ of ATRA and / or albendazole for each cell. Albendazole showed a very low IC ₅₀ for each leukemia cell.

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

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

Test Example 7-1
NOG mice were transplanted with THP-1 cells, and 7 days after transplantation, the mice were randomly divided into 2 groups (albendazole group and DMSO group) (n = 7 each). The albendazole and DMSO groups were treated with albendazole (50 mg / kg body weight, 2 times / week, po) or an equivalent amount of DMSO (control). As shown in FIG. 6-1, overall survival of the albendazole group was significantly prolonged compared to the DMSO group.

Test Example 7-2
Mice treated as in Test Example 7-1 were appropriately anesthetized and sacrificed. Bone marrow tissue was collected and hematoxylin and eosin (H & E) staining and immunohistochemical staining with anti-human CD45 antibody were performed on each slide. A representative image of the spleen and liver of an AML (THP-1 cell) xenograft mouse (30 days after transplantation) is 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 the enlargement 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 (this cell is considered to be an immature cell that has hardly differentiated, and to both the myeloid and lymphatic systems. Were collected using a flow cytometer. 5000 collected c-kit surface antigen positive cells were seeded on a methylcellulose semi-solid medium, and albendazole was administered thereto at 0 nM, 100 nM, and 500 nM, and cultured at 37 ° C. for 1 week. One week later, colony formation was observed with a microscope and counted. The results are shown in FIG. As apparent from FIG. 7, the number of monocytic colonies increased from that of granulocyte colonies.

Claims

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