Pharmaceutical composition for treating hematopoietic tumor-related diseases

Abstract

To provide a pharmaceutical composition for treating hematopoietic tumor-related diseases.SOLUTION: The present invention provides a pharmaceutical composition for treating hematopoietic tumor-related diseases, the pharmaceutical composition comprising an active ingredient selected from the group consisting of a kinase inhibitor, derivatives thereof, and pharmaceutically acceptable salts thereof.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating hematopoietic tumor-related diseases.

Background Art

[0002] Hematopoietic tumors are tumors derived from hematopoietic stem cells or hematopoietic progenitor cells that are present in the bone marrow that produces blood cells (such as white blood cells, red blood cells, and platelets).

[0003] As an example of a hematopoietic tumor-related disease, myelodysplastic syndrome (MDS) can be mentioned. Myelodysplastic syndrome is a hematopoietic tumor that presents various symptoms such as transfusion dependence associated with progressive anemia and thrombocytopenia, and a state of susceptibility to infection associated with leukopenia and immune abnormalities. When the platelet count is low, it poses a serious bleeding risk directly related to life survival. Small megakaryocytes are observed in the bone marrow of patients with hematopoietic tumor-related diseases, suggesting insufficient maturation (for example, Non-Patent Documents 1 to 3).

[0004] So far, as a radical treatment method for thrombocytopenia associated with hematopoietic tumor-related diseases, for example, stem cell transplantation has been proposed. Also, as symptomatic treatments, for example, platelet transfusion, administration of thrombopoietin receptor agonists, splenectomy, etc. have been proposed.

[0005] Here, in addition to the fact that stem cell transplantation cannot be carried out unless multiple conditions such as the availability and age of a donor are satisfied, the onset of myelodysplastic syndrome is at an advanced age and is often out of the scope of indication. Also, the physical burden on the donor and the patient, as well as the burden on medical staff, is not small.

[0006] Platelet transfusion is a concern because anti - platelet antibodies may be induced, leading to refractoriness and loss of effectiveness, and because of the heavy burden on patients. Furthermore, thrombopoietin receptor agonists may stimulate stem cell proliferation and carry risks because there is a possibility of progression to acute leukemia. Specifically, a recombinant protein or compound having a thrombopoietin receptor agonist effect promotes the proliferation of hematopoietic stem cells and differentiation into megakaryocytes at the animal level, and promotes an increase in platelets (Patent Document 1). However, there is concern about the risk of progression to acute leukemia because it proliferates myelodysplastic syndrome stem cells.

[0007] Therefore, it has been desired to expand the options for treatment methods with reduced burden and risk for patients with hematopoietic tumor - related diseases.

Prior Art Documents

Non - Patent Documents

[0008]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Patent Documents

[0009]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a pharmaceutical composition for treating hematopoietic tumor - related diseases. [Means for Solving the Problem]

[0011] In the research on identifying treatment targets for myelodysplastic syndromes, the present inventors surprisingly found that crizotinib, which is approved for the treatment of ALK fusion gene-positive non-small cell lung cancer, promotes megakaryocyte maturation derived from patients with myelodysplastic syndromes. On the other hand, a useful animal model of myelodysplastic syndrome (MDS) was required to examine whether such a megakaryocyte maturation-promoting substance could increase the platelet count at the animal level. In 2022, the present inventors reported the establishment of an animal model of myelodysplastic syndrome in the world's leading academic journal Cancer Discovery (Aoyagi, Y., et al., Cancer Discovery, 12, 250-269, 2022 (Reference 1)). Using this animal model and testing whether crizotinib could increase the platelet count at the animal level, the result was obtained that crizotinib improved thrombocytopenia in myelodysplastic syndrome (MDS) model animals (Figure 2). That is, the present inventors first found, as a new activity of pharmaceutically approved crizotinib, the effect of restoring the platelet count at the animal level through megakaryocyte maturation. Furthermore, the present inventors found that crizotinib also promotes megakaryocyte maturation in human-derived bone marrow cells (Figure 3). Furthermore, the present inventors discovered that kinase inhibitors other than crizotinib also promote megakaryocyte maturation, and found that the platelet count can be restored by using these alone or in combination (Figure 5). The present invention was made based on these findings.

[0012] That is, the present invention provides: [1] A pharmaceutical composition for the treatment of hematopoietic tumor-related diseases, comprising an active ingredient selected from the group consisting of a kinase inhibitor, its derivatives, and pharmaceutically acceptable salts thereof; [2] The pharmaceutical composition according to [1], wherein the kinase inhibitor is one or more selected from the group consisting of an ALK inhibitor, an aurora kinase inhibitor, and a PLK1 inhibitor; [3] The ALK inhibitor is selected from the group consisting of crizotinib and PF-06439015, and the aurora kinase inhibitor is selected from the group consisting of barasertib, alisertib, tozasertib, ZM-447439, MLN8054, danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108, and AMG-900. The pharmaceutical composition according to [2], wherein the PLK1 inhibitor is selected from the group consisting of volasertib and BI2536; [4] The pharmaceutical composition according to any one of [1] to [3], wherein the hematopoietic tumor-related disease is selected from the group consisting of myelodysplastic syndrome, aplastic anemia, and platelet diseases; [5] The pharmaceutical composition according to [4], wherein the platelet disease is selected from the group consisting of familial platelet disorder and thrombocytopenia; [6] The pharmaceutical composition according to [5], wherein the thrombocytopenia is selected from the group consisting of idiopathic thrombocytopenic purpura and thrombocytopenia in platelet transfusion refractoriness associated with remission induction therapy for acute leukemia; [7] The pharmaceutical composition according to any one of [1] to [6], which is for oral administration; [8] A platelet formation and / or megakaryocyte maturation promoter comprising an active ingredient selected from the group consisting of a kinase inhibitor, a derivative thereof, and a pharmaceutically acceptable salt thereof; [9] A composition for promoting megakaryocyte maturation and / or platelet formation in a subject in which megakaryocyte maturation and / or platelet formation is suppressed, A composition comprising an active ingredient selected from the group consisting of a kinase inhibitor, a derivative thereof, and a pharmaceutically acceptable salt thereof;

[10] The composition according to [9], wherein the subject is a mammal;

[11] The composition according to [9] or

[10] , wherein the mammal is selected from the group consisting of humans, monkeys, mice, rats, cows, horses, sheep, dogs, and cats related thereto.

Advantages of the Invention

[0013] According to the present invention, a pharmaceutical composition for treating hematopoietic tumor-related diseases can be provided.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Embodiments for Carrying Out the Invention

[0015] Hereinafter, embodiments for carrying out the present invention will be specifically described. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof.

[0016] A first aspect of the present invention relates to a pharmaceutical composition for the treatment of hematopoietic tumor-related diseases. This pharmaceutical composition contains an active ingredient selected from the group consisting of a kinase inhibitor, its derivatives, and pharmaceutically acceptable salts thereof.

[0017] Examples of the kinase inhibitor include, but are not limited to, anaplastic lymphoma kinase (ALK) inhibitor, Aurora kinase inhibitor, Polo-like kinase 1 (PLK1) inhibitor, Polo-like kinase 4 (PLK4) inhibitor, and LIM kinase (LIMK) inhibitor, etc. The kinase inhibitor is preferably at least one selected from the group consisting of an ALK inhibitor, an Aurora kinase inhibitor, and a PLK1 inhibitor.

[0018] As used herein, the "ALK inhibitor" refers to a drug that inhibits anaplastic lymphoma kinase (ALK). The ALK inhibitor may have an effect of inhibiting kinases other than ALK in the present invention. Examples of the ALK inhibitor include, but are not limited to, crizotinib and derivatives of crizotinib detailed below.

[0019] Crizotinib is a compound with the chemical name 3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine. In this specification, crizotinib is considered to include (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine and (S)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine. Crizotinib is preferably (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine. Crizotinib may be synthetic or commercially available, and examples of commercially available products include, but are not limited to, PF-02341066.

Chem.

[0020] In this specification, derivatives of crizotinib are considered to include a group of compounds containing a pyridine ring substituted with an amine. Specifically, derivatives of crizotinib are a group of compounds containing 3-isopropoxypyridin-2-amine, and more specifically, a group of compounds containing 3-(1-phenylethoxy)pyridin-2-amine.

Chem.

[0021] Derivatives of crizotinib are represented, for example, by the above formula (I). In this specification, derivatives of crizotinib also include both the (R) form and the (S) form, similar to crizotinib. In formula (I), the pyridine ring is substituted with an -NR1R2 group. R1 and R2 may be the same or different and are each selected from the group consisting of hydrogen, cyano, -CHO, -OH, C1-C4-alkyl, C1-C4-halogenoalkyl having 1 to 9 halogen atoms, C1-C4-alkoxy-C1-C4-alkyl, C1-C4-alkoxy, C1-C4-halogenoalkoxy having 1 to 9 halogen atoms, C2-C4-alkenyl, and C2-C4-alkynyl (wherein the hydrogen atoms of the alkyl, alkoxy, alkenyl, and alkynyl may be replaced by -OH).

[0022] The pyridine ring may further be substituted with a substituent A selected from 5- to 7-membered aromatic groups and cycloaliphatic groups. In this case, for example, the 1-position, 2-position, 3-position, 4-position, 5-position, or 6-position of the pyridine ring is substituted by the substituent A. The substituent A is preferably selected from 5- to 6-membered aromatic groups and cycloaliphatic groups, and more preferably selected from 5-membered aromatic groups. The aromatic groups and cycloaliphatic groups include heterocyclic groups, and one or more heteroatoms selected from S, N, and O form the heterocyclic ring together with carbon atoms.

[0023] Examples of the substituent A include, but are not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, thiophenyl, phenyl, pyridinyl, pyrimidinyl, pyrazinyl.

[0024] The substituent A is unsubstituted or substituted by one or more substituents B. When a plurality of substituents B are present, they may be the same or different. The substituent B is selected from the following (b1) and (b2): (b1) A chain hydrocarbon group selected from the group consisting of halogen, cyano, -CHO, -OH, -COOH, C1-C4 alkyl, C1-C4 halogenoalkyl having 1 to 9 halogen atoms, C1-C4 alkoxy-C1-C4 alkyl, C1-C4 alkoxy, C1-C4 halogenoalkoxy having 1 to 9 halogen atoms, C2-C4 alkenyl, and C2-C4 alkynyl (wherein the hydrogen atoms of the alkyl, alkoxy, alkenyl, and alkynyl may be replaced by -OH). (b2) A cyclic hydrocarbon group selected from the group consisting of aromatic groups and cycloaliphatic groups having 4 to 7 members. Here, the aromatic group and the cycloaliphatic group include heterocyclic groups, and one or more heteroatoms selected from S, N, and O form the heterocyclic ring together with carbon atoms.

[0025] When the substituent B is a cyclic hydrocarbon group (in the case of (b2) above), it may be unsubstituted or substituted by one or more selected from the group consisting of cyano, -CHO, -OH, C1-C4 alkyl, C1-C4 halogenoalkyl having 1 to 9 halogen atoms, C1-C4 alkoxy, C1-C4 halogenoalkoxy having 1 to 9 halogen atoms, C2-C4 alkenyl, C2-C4 alkynyl, and C1-C4 alkoxy-C1-C4 alkyl.

[0026] Examples of the substituent B include, but are not limited to, piperidinyl group, pyridinyl group, pyrrolyl group, pyrazolyl group, methyl group, ethyl group, propyl group, isopropyl group, and butyl group. Here, preferably, 1 to 4 hydrogen atoms of the alkyl group are replaced by -OH.

[0027] In the derivative of crizotinib, the benzene ring may be substituted by one or more substituents X. When a plurality of substituents X are present, they may be the same or different. The substituent X is selected from the following (x1) and (x2): (x1) A chain hydrocarbon group selected from the group consisting of halogen, cyano, -CHO, -OH, -COOH, C1-C4 alkyl, C1-C4 halogenoalkyl having 1 to 9 halogen atoms, C1-C4 alkoxy-C1-C4 alkyl, C1-C4 alkoxy, C1-C4 halogenoalkoxy having 1 to 9 halogen atoms, C2-C4 alkenyl, and C2-C4 alkynyl (wherein the hydrogen atoms of the alkyl, alkoxy, alkenyl, and alkynyl may be replaced by -OH); (x2) A cyclic hydrocarbon group selected from the group consisting of an aromatic group and a cycloaliphatic group having 4 to 7 members. Here, the aromatic group and the cycloaliphatic group include a heterocyclic group, and one or more heteroatoms selected from S, N, and O form the heterocyclic ring together with carbon atoms.

[0028] Examples of the substituent X include, but are not limited to, an imidazolyl group, a pyrazolyl group, a thiadiazolyl group, a thiazolyl group, a triazolyl group, F, Cl, Br, I, a methyl group, an ethyl group, a propyl group, an isopropyl group, and a butyl group.

[0029] Examples of the derivative of crizotinib include, but are not limited to, PF-06439015.

[0030] As used herein, the "Aurora kinase inhibitor" refers to an agent that inhibits Aurora kinase. The Aurora kinase inhibitor may have an effect of inhibiting kinases other than Aurora kinase in the present invention. Examples of the Aurora kinase inhibitor include, but are not limited to, Barasertib, Alisertib, Tozasertib, ZM-447439, MLN8054, Danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108, and AMG-900.

[0031] As used herein, the term "PLK1 inhibitor" refers to an agent that inhibits Polo-like kinase 1 (PLK1). The PLK1 inhibitor may have an effect of inhibiting kinases other than PLK1 in the present invention. Examples of the PLK1 inhibitor include, but are not limited to, Volasertib and BI2536.

[0032] In one aspect, the kinase inhibitor is at least one selected from the group consisting of an ALK inhibitor, an aurora kinase inhibitor, and a PLK1 inhibitor. For example, the ALK inhibitor is selected from the group consisting of crizotinib and PF-06439015, the aurora kinase inhibitor is selected from the group consisting of barasertib, alisertib, tozasertib, ZM-447439, MLN8054, danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108, and AMG-900, and the PLK1 inhibitor is selected from the group consisting of volasertib and BI2536.

[0033] In another aspect, the kinase inhibitor is at least one selected from the group consisting of crizotinib, PF-06439015, barasertib, alisertib, volasertib, and BI2536.

[0034] Blood cells such as white blood cells, red blood cells, and platelets are produced from hematopoietic stem cells in the bone marrow. The hematopoietic stem cells or tissues at the stage where the hematopoietic stem cells have differentiated and / or matured and become tumorous are collectively referred to as hematopoietic tumors. Examples of hematopoietic tumors include leukemia, malignant lymphoma, and multiple myeloma. Symptoms of hematopoietic tumors include anemia, increased risk of infection due to decreased resistance caused by normal leukopenia, and bleeding due to thrombocytopenia.

[0035] Examples of hematopoietic tumor-related diseases include myelodysplastic syndrome, aplastic anemia, and platelet diseases. In the present invention, among these diseases and symptoms, those in which the maturation of megakaryocytes is suppressed and / or the formation of platelets is suppressed are targeted for treatment.

[0036] Examples of hematopoietic tumor-related diseases include, for example, myelodysplastic syndrome, aplastic anemia, and platelet diseases. Therefore, in one aspect, the pharmaceutical composition according to the present invention is a pharmaceutical composition for the treatment of a hematopoietic tumor-related disease selected from the group consisting of myelodysplastic syndrome, aplastic anemia, and platelet diseases.

[0037] Examples of platelet diseases include, for example, familial platelet disorder and thrombocytopenia. Therefore, in one aspect, the pharmaceutical composition according to the present invention is a pharmaceutical composition for the treatment of a platelet disease selected from the group consisting of familial platelet disorder and thrombocytopenia.

[0038] Furthermore, examples of thrombocytopenia include, for example, idiopathic thrombocytopenic purpura and thrombocytopenia in platelet transfusion refractoriness associated with remission induction therapy for acute leukemia. Therefore, in one aspect, the pharmaceutical composition according to the present invention is a pharmaceutical composition for the treatment of a thrombocytopenia selected from the group consisting of idiopathic thrombocytopenic purpura and thrombocytopenia in platelet transfusion refractoriness associated with remission induction therapy for acute leukemia.

[0039] As used herein, the term "treatment" encompasses both therapeutic treatment and prevention, the purpose of which is to prevent or delay (mitigate) undesirable physiological changes or disorders. In the context of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, stabilization of the disease state (i.e., not worsening), delay or deceleration of disease progression, improvement or temporary remission of the disease state, and remission (including partial remission and complete remission), whether or not detectable. In a preferred aspect, "treatment" is, for example, promotion of megakaryocyte maturation or platelet formation as compared to a control.

[0040] Preferably, the pharmaceutical composition according to the present invention is for oral administration. The pharmaceutical composition according to the present invention may be formulated, for example, into tablets, powders, granules, capsules, solutions, etc., but is not limited thereto.

[0041] The pharmaceutical composition according to the present invention may typically contain pharmaceutically acceptable additives, such as excipients, binders, lubricants, solvents, diluents, stabilizers, isotonic agents, etc. known in the art.

[0042] The composition according to the present invention may also be used as a combination agent combined with a further pharmaceutical composition. Regarding the combination agent, the components (i.e., the first active ingredient and the second active ingredient) in each pharmaceutical composition may be administered together, sequentially or individually, in one combined unit dosage form or two separate unit dosage forms. A therapeutically effective amount of each active ingredient of the combination agent according to the present invention may be administered simultaneously, individually, or in any order, sequentially or continuously. In the combination agent, the first active ingredient and the second active ingredient may be present in multiple units. The combination agent includes, for example, a kit containing each active ingredient and instructions for use.

[0043] A second aspect of the present invention relates to a thrombopoiesis and / or megakaryocyte maturation promoter comprising an active ingredient selected from the group consisting of a kinase inhibitor, its derivatives and / or pharmaceutically acceptable salts thereof.

[0044] In one embodiment, the kinase inhibitor is at least one selected from the group consisting of an ALK inhibitor, an aurora kinase inhibitor and a PLK1 inhibitor. For example, the ALK inhibitor is selected from the group consisting of crizotinib and PF-06439015, the aurora kinase inhibitor is selected from the group consisting of barasertib, alisertib, tozasertib, ZM-447439, MLN8054, danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108 and AMG-900, and the PLK1 inhibitor is selected from the group consisting of volasertib and BI2536.

[0045] In another aspect, the kinase inhibitor is one or more selected from the group consisting of crizotinib, PF-06439015, barasertib, alisertib, volasertib, and BI2536.

[0046] As used herein, the term "thrombopoiesis" shall include, in addition to forming or producing platelets, increasing the number of platelets. Thus, the term "inhibition of thrombopoiesis" shall include, in addition to the inhibition of platelet formation and production, a decrease and no change in the number of platelets.

[0047] As used herein, the term "megakaryocyte maturation" shall include megakaryocyte differentiation in addition to megakaryocyte maturation. Megakaryocyte maturation can be confirmed, for example, by evaluating the nuclear ploidy of megakaryocytes in bone marrow cells obtained by bone marrow aspiration. Specifically, the cells of interest are fixed, stained with a nuclear staining reagent, and it can be confirmed by using a flow cytometer that the nuclear ploidy of the cells is increased. Conversely, the inhibition of megakaryocyte maturation can be confirmed by the same method, using a flow cytometer, that the nuclear ploidy of the cells is decreased. In addition, the inhibition of megakaryocyte maturation compared to healthy subjects can also be confirmed by morphological and immunological observations of bone marrow cells obtained by bone marrow aspiration.

[0048] A third aspect of the present invention relates to a composition for promoting megakaryocyte maturation and / or thrombopoiesis in a subject in which megakaryocyte maturation and / or thrombopoiesis is inhibited, the composition comprising an active ingredient selected from the group consisting of a kinase inhibitor, its derivatives, and / or pharmacologically acceptable salts thereof.

[0049] In one aspect, the kinase inhibitor is at least one selected from the group consisting of an ALK inhibitor, an aurora kinase inhibitor, and a PLK1 inhibitor. For example, the ALK inhibitor is selected from the group consisting of crizotinib and PF-06439015, the aurora kinase inhibitor is selected from the group consisting of barasertib, alisertib, tozasertib, ZM-447439, MLN8054, danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108, and AMG-900, and the PLK1 inhibitor is selected from the group consisting of volasertib and BI2536.

[0050] In another aspect, the kinase inhibitor is one or more selected from the group consisting of crizotinib, PF-06439015, barasertib, alisertib, volasertib, and BI2536.

[0051] The subject is a vertebrate, preferably a mammal. As used herein, the term "mammal" is used to refer to any animal classified as a mammal, for example, but not limited to, selected from the group consisting of humans, monkeys, mice, rats, cows, horses, sheep, dogs, and cats. In a preferred aspect, the subject is a human.

[0052] In one aspect, "megakaryocyte maturation is suppressed" in a subject means that megakaryocyte maturation and / or differentiation is suppressed in the subject. That megakaryocyte maturation and / or differentiation is suppressed can be confirmed by evaluation of megakaryocyte nuclear ploidy or morphological and immunological observations, as described above.

[0053] In one aspect, "megakaryocyte maturation is suppressed" in a subject means that the subject has a hematopoietic tumor-related disease.

[0054] The kinase inhibitor contained in the pharmaceutical composition according to the present invention can promote the maturation of megakaryocytes and restore the platelet count. Therefore, by administering the pharmaceutical composition according to the present invention to a patient with a hematopoietic tumor-related disease, particularly a patient with thrombocytopenia associated with a hematopoietic tumor-related disease, an increase in the platelet count is expected. That is, it becomes possible to treat thrombocytopenia by using an existing kinase inhibitor alone or in combination of a plurality thereof.

[0055] In addition, crizotinib contained in the pharmaceutical composition according to the present invention can promote the maturation of megakaryocytes and restore the platelet count. Therefore, by administering the pharmaceutical composition according to the present invention to a patient with a hematopoietic tumor-related disease, particularly a patient with thrombocytopenia associated with a hematopoietic tumor-related disease, an increase in the platelet count is expected. That is, it becomes possible to treat thrombocytopenia by drug repositioning.

[0056] The kinase inhibitor contained in the pharmaceutical composition according to the present invention can also be orally administered, so that the burden on patients and medical staff can be reduced. In addition, since it has an action of differentiating megakaryocytes from hematopoietic progenitor cells, the risk of stimulating stem cell proliferation and progression to acute leukemia is low.

[0057] So far, a megakaryocyte maturation promoting substance has only been confirmed to be effective at the cultured cell level (for example, WO 2014 / 168255). The present inventors have first found that by using a substance with guaranteed solubility, in vivo stability and safety, acting at the animal level, and promoting an increase in platelets. Therefore, the present invention is considered to be superior to the prior art in terms of practicality and immediate effectiveness.

[0058] In addition, although a recombinant protein or compound having thrombopoietin receptor agonist activity promotes the proliferation of hematopoietic stem cells and differentiation into megakaryocytes at the animal level, thereby promoting platelet increase (for example, see International Publication No. 2014 / 003155), the proliferation of myelodysplastic syndrome stem cells poses a risk of progression to acute leukemia. In contrast, the present invention has the effect of differentiating immature megakaryocytes and has not been recognized to have a promoting effect on the proliferation of stem cells, and thus has an advantage over the prior art in that the risk of progression to acute leukemia is low.

Examples

[0059] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples at all.

[0060] Samples and test animals used in the examples were prepared and produced, and each measurement was performed according to the following procedures.

[0061] [Preparation of MDS model mice] MDS model mice were prepared by modifying the method described in Reference 1. Hematopoietic stem cells and hematopoietic progenitor cells (c-Kit positive cells) were enriched from the bone marrow cells of the femurs and tibias of 7- to 8-week-old experimental mice C57BL / 6 using the EasySep mouse c-Kit Positive Selection Kit (STEMCELL Technologies). The enriched c-Kit positive cells were cultured overnight in IMDM medium (Wako) containing 20% FBS, 50 ng / mL each of mouse SCF (Biolegend), mouse FLT3-L (Biolegend), mouse IL-6 (Biolegend), and mouse TPO (Biolegend), and a gene expression system of a mutant RUNX1 gene (RUNX1 S291fs mutation: a frameshift caused by a single nucleotide deletion starting from the 291st amino acid) and a mutant CBL gene (CBL ΔE8 / 9: a deletion mutant of exons 8 and 9) was introduced by retrovirus. The gene-introduced cells were transplanted via the tail vein into C57BL / 6 mice irradiated with a sublethal dose of radiation to prepare MDS model mice.

[0062] [Ex vivo culture of hematopoietic stem cells and hematopoietic progenitor cells] Hematopoietic stem cells and hematopoietic progenitor cells obtained from the bone marrow of the prepared model mice were cultured in Ham's F12 culture medium (Wako) containing 1% ITSX (Gibco), 10 mM HEPES, 1% P / S, 100 ng / mL mouse TPO (Biolegend), 10 ng / mL mouse SCF (Biolegend), and 0.1% polyvinyl alcohol.

[0063] [Preparation of MDS model cells] According to the method described in the section of [Preparation of MDS model mice], hematopoietic stem cells and hematopoietic progenitor cells were cultured, and gene expression systems of mutant RUNX1 gene (RUNX1 S291fs mutation: a frameshift mutation caused by a single-base deletion starting from the 291st amino acid) and mutant CBL gene (CBL ΔE8 / 9: a deletion mutant of exons 8 and 9) were introduced by retrovirus. After that, the gene-introduced cells were isolated by FACS (Fluorescence Activated Cell Sorting) 48 hours after retrovirus infection, and the cells cultured in Ham's F12 culture medium (Wako) containing 1% ITSX (Gibco), 10 mM HEPES, 1% P / S, 100 ng / mL mouse TPO (Biolegend), 10 ng / mL mouse SCF (Biolegend), and 0.1% polyvinyl alcohol were used as MDS model cells.

[0064] [Measurement of platelet count] Blood was collected from the orbital venous plexus of mice under anesthesia, and the platelet count was measured using an automatic multi-item hematology analyzer pocH(trademark)-100iV Diff (Sysmex) for animals.

[0065] [Evaluation of megakaryocyte nuclear ploidy] Bone marrow cells or cultured bone marrow cells were fixed with ice-cold 70% ethanol. After washing the fixed cells with a buffer (PBS containing 2% FBS and 2 mM EDTA), they were stained with anti-CD41 antibody (Biolegend or Miltenyi Biotec). Subsequently, they were stained with PI or DAPI, which are nuclear staining reagents, and the nuclear ploidy of CD41-positive cells in the bone marrow cells or cultured bone marrow cells was evaluated using a flow cytometer, FACSCelesta (BD Biosciences) or CytoFLEX (Beckman Coulter). Also, MDS model cells were fixed with ice-cold 70% ethanol, washed with a buffer (2% FBS, 2 mM PBS), stained with anti-CD41 antibody (Biolegend) and the nuclear staining reagent PI, and the nuclear ploidy of CD41-positive megakaryocytes in the MDS model cells was analyzed and evaluated using a flow cytometer, CytoFLEX (Beckman Coulter).

[0066] <1>Myeloid megakaryocyte maturation and platelet count in MDS model mice The maturity of bone marrow megakaryocytes in control mice and MDS model mice prepared according to the above method was evaluated by flow cytometry using nuclear ploidy as an index. Also, the platelet count was evaluated using the above hemocytometer. The results are shown in FIGS. 1A and 1B.

[0067] From FIG. 1A, it was shown that the MDS model mice had a lower nuclear ploidy of bone marrow megakaryocytes and suppressed megakaryocyte maturation compared to the control mice. From FIG. 1B, it was shown that the MDS model mice had a decreased platelet count compared to the control mice. From FIGS. 1A and 1B, it was shown that in MDS model animals, myeloid megakaryocyte maturation was suppressed and the platelet count was decreased.

[0068] <2>Crizotinib administration to MDS model mice Hematopoietic stem cells and hematopoietic progenitor cells cultured ex vivo according to the above method were treated with DMSO, a solvent, or crizotinib (MedChemExpress (MCE)) to evaluate the nuclear ploidy of CD41-positive cells. The evaluation results are shown in Fig. 2A. It was shown from Fig. 2A that crizotinib promoted megakaryocyte maturation.

[0069] Furthermore, solvent (water containing 0.5% methylcellulose and 0.5% Tween-80) or crizotinib was administered to MDS model mice. As the administration schedule, 100 mg / kg was orally administered once a day, 5 days a week, and the cycle of 2 days of drug withdrawal was repeated for 4 weeks. The administration started on the 17th day after the production of model mice by transplantation. In MDS model mice administered with the solvent and MDS model mice administered with crizotinib, the maturation of bone marrow megakaryocytes and the platelet count were evaluated. The results are shown in Figs. 2B and 2C. It was shown from Figs. 2B and 2C that crizotinib administration promoted megakaryocyte maturation and increased the platelet count.

[0070] From Figs. 2A to 2C, it was shown that crizotinib promoted megakaryocyte maturation ex vivo and in vivo in MDS model animals and restored the platelet count in MDS model animals.

[0071] <3>Crizotinib treatment of bone marrow cells derived from MDS patients Bone marrow mononuclear cells were prepared from human bone marrow fluid using Lymphoprep (Serumwerk Bernburg AG). The prepared cells were cultured in StemSpan SFEM II (STEMCELL Technologies) containing Stem Span Megakaryocyte Expansion Supplement (STEMCELL Technologies) for 9 - 10 days while changing the culture medium every two days. Thereafter, they were treated with 0.1% DMSO as the solvent or 1 μM crizotinib (MedChemExpress (MCE)) for 3 days. Furthermore, after culturing the cells in a drug-free culture medium for 24 hours, the cells were collected and fixed with ice-cold 70% ethanol. The fixed cells were washed with a buffer (PBS containing 2% FBS and 2 mM EDTA) and then stained with anti-CD41 / CD61 antibody (Biolegend). Staining was performed with PI, a nuclear staining reagent, and the nuclear ploidy of CD41 / CD61 positive cells was evaluated using a flow cytometer, F CytoFLEX (Beckman Coulter). The results are shown in Figure 3.

[0072] As shown in Figure 3, it was shown that crizotinib promoted megakaryocyte maturation of bone marrow cells derived from MDS patients.

[0073] <4>Treatment of hematopoietic stem cells and hematopoietic progenitor cells derived from MDS model mice with inhibitors Hematopoietic stem cells and hematopoietic progenitor cells cultured ex vivo according to the above method were treated with the following: - DMSO as the solvent, - crizotinib, - ALK / ROS1 inhibitor lorlatinib, - ALK inhibitor alectinib, - c-Met inhibitor capmatinib, - capmatinib and lorlatinib, - capmatinib and alectinib were added to evaluate the nuclear ploidy of CD41 positive cells. The evaluation results are shown in Figure 4.

[0074] Figure 4 showed that crizotinib promoted megakaryocyte maturation, while inhibitors other than crizotinib showed no megakaryocyte maturation activity. These results indicated that crizotinib induced megakaryocyte maturation independently of ALK inhibition, ROS1 inhibition, and c-Met inhibition.

[0075] <5>Treatment of MDS model cells with inhibitors The MDS model cells prepared according to the above method were treated with the following: - DMSO as a solvent, - 1 μM crizotinib, - 100 nM centrinone, - 10 μM barasertib, - 0.1 μM alisertib, - 1.5 μM BMS-5, - 30 nM volasertib, - 25 nM BI2536 and the nuclear ploidy of CD41-positive cells in the MDS model cells was evaluated by flow cytometry according to the method described above. The results are shown in Figure 5.

[0076] From Figure 5, it was shown that centrinone, a PLK4 inhibitor, and BMS-5, a LIMK inhibitor, had no effect on megakaryocyte ploidy, while barasertib and alisertib, Aurora kinase inhibitors, and volasertib and BI2536, PLK1 inhibitors, increased megakaryocyte ploidy. These results indicated that Aurora kinase inhibitors and PLK1 inhibitors promoted megakaryocyte maturation in MDS model cells.

[0077] Aurora kinase activates PLK1 to promote cytokinesis, and crizotinib inhibits Aurora kinase to increase megakaryocyte nuclear polyploidy (Figure 6). In other words, Aurora kinase advances the cell cycle through the activation of PLK1 (References 2 and 3), and Aurora kinase inhibitors and PLK1 inhibitors increase nuclear polyploidy by suppressing this. In addition, previous literature has shown that crizotinib binds to and inhibits Aurora kinase (References 4 and 5). Taking the results of Figures 4 and 5 together, it is thought that crizotinib promotes megakaryocyte maturation by inhibiting Aurora kinase.

[0078] (References) Reference 1: Aoyagi, Y., et al., Cancer Discovery, 12, 250-269, 2022 Reference 2: Seki, A., et al., Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science. 320, 1655-1658 (2008). Reference 3: Macurek, L., et al., Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature. 455, 119-123 (2008). Reference 4: Davis, M., et al., Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1046-1051 (2011). Literature 5: Kong, Y., et al., Identification of Novel Aurora Kinase A (AURKA) Inhibitors via Hierarchical Ligand-Based Virtual Screening. J. Chem. Inf. Model. 58, 36-47 (2018).

Industrial Applicability

[0079] By administering the composition according to the present invention, it is possible to provide a treatment method with reduced burden and risk in patients with hematopoietic tumor-related diseases.

Claims

1. A pharmaceutical composition for the treatment of a hematopoietic tumor-related disease, comprising an active ingredient selected from the group consisting of a kinase inhibitor, a derivative thereof, and a pharmacologically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the kinase inhibitor is one or more selected from the group consisting of an ALK inhibitor, an aurora kinase inhibitor, and a PLK1 inhibitor.

3. The ALK inhibitor is selected from the group consisting of crizotinib and PF-06439015, The aurora kinase inhibitor is selected from the group consisting of barasertib, alisertib, tozasertib, ZM-447439, MLN8054, danusertib, AT9283, JNJ-7706621, TCS7010, MK-5108, and AMG-900, The PLK1 inhibitor is selected from the group consisting of volasertib and BI2536, the pharmaceutical composition according to claim 2.

4. The pharmaceutical composition according to claim 1 or 2, wherein the hematopoietic tumor-related disease is selected from the group consisting of myelodysplastic syndrome, aplastic anemia, and platelet diseases.

5. The pharmaceutical composition according to claim 4, wherein the platelet disease is selected from the group consisting of familial platelet disorder and thrombocytopenia.

6. The pharmaceutical composition according to claim 5, wherein the thrombocytopenia is selected from the group consisting of idiopathic thrombocytopenic purpura and thrombocytopenia in platelet transfusion refractoriness associated with remission induction therapy for acute leukemia.

7. The pharmaceutical composition according to claim 1 or 2, which is for oral administration.

8. A platelet formation and / or megakaryocyte maturation promoter, comprising an active ingredient selected from the group consisting of a kinase inhibitor, a derivative thereof, and a pharmacologically acceptable salt thereof.

9. A composition for promoting megakaryocyte maturation and / or platelet formation in a subject in which megakaryocyte maturation and / or platelet formation is suppressed, The composition comprising an active ingredient selected from the group consisting of a kinase inhibitor, a derivative thereof, and a pharmacologically acceptable salt thereof.

10. The composition according to claim 9, wherein the subject is a mammal.

11. The composition according to claim 9 or 10, wherein the mammal is selected from the group consisting of humans, monkeys, mice, rats, cows, horses, sheep, dogs, and cats.

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