Application of AZ505 and Imatinib in preparation of pharmaceutical composition for treating leukemia

Abstract

The invention discloses an application of AZ505 and Imatinib in preparation of a pharmaceutical composition for treating leukemia, and belongs to the technical field of biology, experiments prove that the AZ505 and Imatinib combined medication can induce apoptosis of leukemia cells and can inhibit the vitality of the leukemia cells at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of AZ505 combined with Imatinib in preparing a pharmaceutical composition for treating leukemia. Background Art

[0002] Leukemia, also known as blood cancer, is a group of progressive malignant tumor diseases of the hematopoietic organs. The cause is that hematopoietic stem cells or progenitor cells undergo differentiation blockage, apoptosis disorder and malignant proliferation at different stages of development and maturation, which in turn causes heterogeneous hematopoietic system malignancies. The main characteristics of leukemia are the qualitative and quantitative abnormalities of white blood cells and their immature cells (precursor cells or leukemia cells) in the blood and bone marrow, which inhibit the normal hematopoietic function of the bone marrow and infiltrate tissues and organs such as the liver, spleen, and lymph nodes. Symptoms occur due to the lack of normal blood cells and may include: bleeding and bruising, fatigue, and increased risk of infection.

[0003] The compound AZ505 is a histone lysine methyltransferase (SMYD2) inhibitor that can specifically inhibit SMYD2 activity. The molecular formula of AZ505 is C33H40Cl2F6N4O8, and the CAS number is 1035227-44-1. SMYD2 is a protein lysine methyltransferase that can regulate histone and non-histone lysine methylation modifications and participate in regulating tumor epigenetic signals and cell physiology. The target of AZ505's anti-tumor effect is the lysine methyltransferase SMYD2. It specifically binds to SMYD2 and competitively binds to SMYD2 with peptide substrates, thereby inhibiting the function of SMDY2's methylated substrates, resulting in a series of effects such as inhibiting proliferation and inducing cell apoptosis.

[0004] Imatinib is a small molecule tyrosine kinase inhibitor that mainly targets and inhibits the activity of three tyrosine kinases: abl, c-Kit, and platelet-derived growth factor receptor (PDGFR). It can competitively inhibit the binding of tyrosine kinase to ATP to block the signal transduction caused by the phosphorylation of tyrosine kinase, thereby achieving the purpose of anti-tumor. So far, imatinib has been approved by the US FDA for the treatment of various cancers. Imatinib was initially used mainly to treat chronic myeloid leukemia and gastrointestinal stromal tumors. Clinical studies have found that imatinib can also be used to control the development of chordoma. Summary of the invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0006] The present invention provides the use of AZ505 combined with Imatinib in preparing a pharmaceutical composition for preventing, alleviating and treating leukemia.

[0007] Furthermore, the pharmaceutical composition is administered orally, by suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

[0008] Furthermore, AZ505 and Imatinib in the pharmaceutical composition are administered sequentially or simultaneously.

[0009] Furthermore, the concentration of AZ505 is 5-10 μM.

[0010] In some embodiments, the concentration of AZ505 is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0011] Furthermore, the concentration of AZ505 is 5 μM and 10 μM.

[0012] Furthermore, the concentration of Imatinib is 0.05-0.1 μM.

[0013] In some embodiments, the concentration of Imatinib is 0.05 μM, 0.06 μM, 0.07 μM, 0.08 μM, 0.09 μM, or 0.1 μM.

[0014] Furthermore, the concentration of Imatinib is 0.05 μM and 0.1 μM.

[0015] Furthermore, the concentration ratio of AZ505 to Imatinib is 50:1-200:1.

[0016] In some embodiments, the concentration ratio of AZ505:1 is 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 105:1, 110:1, 115:1, 120:1, 125:1, 130:1, 135:1, 140:1, 145:1, 150:1, 155:1, 160:1, 165:1, 170:1, 175:1, 180:1, 185:1, 190:1, 195:1, and 200:1.

[0017] Furthermore, the AZ505 and Imatinib also include pharmaceutically acceptable salts thereof.

[0018] Furthermore, the pharmaceutically acceptable salts include acid addition salts and base addition salts.

[0019] Further, the acid addition salt includes, but is not limited to, any one of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, caproate, argininate, fumarate, nicotinate, phthalate or oxalate, or a combination of at least two thereof.

[0020] Furthermore, the base addition salt includes but is not limited to lithium salt, sodium salt, potassium salt, barium salt, calcium salt, magnesium salt, aluminum salt, iron salt, ferrous salt, copper salt, zinc salt, or salts with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histidine.

[0021] Furthermore, the leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia.

[0022] Furthermore, the acute leukemia includes L1 acute lymphocytic leukemia, L2 acute lymphocytic leukemia, L3 acute lymphocytic leukemia, and acute myeloid leukemia.

[0023] Furthermore, the acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

[0024] Furthermore, the chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia.

[0025] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0026] In some embodiments, the "administration" refers to the actual physical introduction of the agent into or onto a target biological target (e.g., a host and / or a subject) (where appropriate). The composition can be administered to a cell (e.g., "contacting") in vitro or in vivo. The composition can be administered to a subject in vivo via an appropriate route of administration. Any and all methods of introducing the composition into a host are contemplated according to the present invention. The methods do not rely on any particular mode of introduction and should not be so interpreted. Modes of introduction are well known to those skilled in the art and are also exemplified herein. The term includes routes of administration that allow the agent to perform its intended function.

[0027] The term "prevention" used in the present invention refers to the prophylactic administration of the combination to healthy patients to prevent the occurrence of the diseases and disorders described herein. In addition, the term "prevention" can also refer to the prophylactic administration of the combination to patients in the early stages of the disease to be treated.

[0028] The present invention provides a pharmaceutical composition, which comprises an effective amount of AZ505 and Imatinib in the above application.

[0029] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable adjuvant.

[0030] Furthermore, the auxiliary agent includes excipients, diluents, dispersing aids, suspending aids, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, and lubricants.

[0031] In some embodiments, the pharmaceutical composition depends on the route of administration, and the pharmaceutical composition can be coated with or arranged in a selected material to protect it from natural conditions that can adversely affect its ability to perform its intended function. The pharmaceutical composition can be administered alone or in combination with a pharmaceutically acceptable adjuvant. The pharmaceutical composition can also be administered as a prodrug, which is converted into its active form in vivo.

[0032] In some embodiments, the excipients added to the pharmaceutical composition include but are not limited to: diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, etc.

[0033] Furthermore, the pharmaceutical composition can be prepared into powders, tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc. for oral administration; powders or liquid aerosols suitable for inhalation; creams, ointments, gels, aqueous or oily solutions, aqueous or oily suspensions, etc. suitable for topical use; sterile aqueous or oily injections or lyophilized powder injections, suppositories, etc. suitable for intravenous, subcutaneous or intramuscular injections for parenteral administration.

[0034] Furthermore, the pharmaceutical composition also includes anti-apoptotic agents, mitotic inhibitors, immunomodulators, nucleic acids for gene therapy, alkylating agents, anti-angiogenic agents, anti-metabolites, boron-containing agents, chemotherapeutic protective agents, hormone agents, anti-hormonal agents, antiviral drugs, photosensitizing therapeutic agents, oligonucleotides, radionuclide agents, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and antibiotic drugs.

[0035] The term "effective amount" used in the present invention refers to an amount that can produce a function or activity in humans and / or animals and can be accepted by humans and / or animals. The effective amount can be changed due to the following factors: the intended application (in vitro or in vivo), or the subject and disease condition to be treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., which can be easily determined by a person of ordinary skill in the art. The specific dosage will vary depending on the following factors: the specific compound selected, the dosage regimen based on, whether it is co-administered with other compounds, the time arrangement of administration, the tissue to be administered, and the physical delivery system carried.

[0036] The term "pharmaceutically acceptable" as used in the present invention refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0037] The present invention provides a medicine kit, comprising: a first preparation containing AZ505, and a second preparation containing Imatinib.

[0038] Furthermore, the concentration of AZ505 is 5-10 μM.

[0039] Furthermore, the concentration of AZ505 is 5 μM and 10 μM.

[0040] Furthermore, the concentration of Imatinib is 0.05-0.1 μM.

[0041] Furthermore, the concentration of Imatinib is 0.05 μM and 0.1 μM.

[0042] In some embodiments, the kit can have a time interval when using the first preparation and the second preparation, and the interval can be 1min, 5min, 10min, 30min, 1h, 2h, 5h, 10h, 20h, 1d, 2d, 3d, 5d, 7d. In some embodiments, the kit can be used in combination with the first preparation and the second preparation.

[0043] In some embodiments, the drug kit may be a pretreatment drug kit for other leukemia therapeutic drugs, which is used to accelerate the induction of apoptosis of leukemia cells and inhibit the activity of leukemia cells.

[0044] The present invention provides the use of AZ505 combined with Imatinib in preparing a product for inducing apoptosis of leukemia cells.

[0045] Furthermore, the leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia.

[0046] Furthermore, the acute leukemia includes L1 acute lymphocytic leukemia, L2 acute lymphocytic leukemia, L3 acute lymphocytic leukemia, and acute myeloid leukemia.

[0047] Furthermore, the acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

[0048] Furthermore, the chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia.

[0049] The present invention provides a method for inducing apoptosis of leukemia cells in vitro for non-therapeutic purposes, and the method comprises the step of using the above-mentioned pharmaceutical composition.

[0050] The present invention provides an in vitro non-therapeutic method for inhibiting leukemia pathological lymph and bone marrow cell tissues, the method comprising the steps of using the aforementioned pharmaceutical composition and the aforementioned medicine kit.

[0051] In some embodiments, the "treatment" refers to the improvement, prevention, or reversal of a disease or condition or at least one distinguishable symptom thereof. In certain specific embodiments, the "treatment" refers to the improvement, prevention, or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. In some embodiments, the "treatment" refers to the inhibition or slowing of a disease or disease process, and this inhibition or slowing can be physical, such as certain distinguishable adverse symptoms. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of diseases or conditions in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease.

[0052] The term "alleviation" as used in the present invention refers to a process in which the severity of the signs or symptoms of a disease is reduced. The signs or symptoms may be alleviated but not eliminated.

[0053] The term "non-therapeutic purpose" used in the present invention is synonymous with "non-treatment" and "non-therapeutic", and means that the method and / or application of the present invention excludes the diagnosis and treatment methods for diseases stipulated in Article 25 of the Chinese Patent Law.

[0054] The term "apoptosis" or "programmed cell death" used in the present invention refers to the physiological process of eliminating unwanted or useless cells during development and other normal biological processes. Apoptosis is a mode of cell death that occurs under normal physiological conditions, and cells are active participants in their own death ("cell suicide"). It is most common in the renewal of normal cells and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system, and endocrine-dependent tissue atrophy. Apoptosis can also be triggered by external events or stimuli, such as the induction of apoptosis of leukemic cells by drug combination therapy in the context of certain preferred embodiments of the present invention.

[0055] The term "leukemia" used in the present invention refers to a class of malignant clonal diseases of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in large quantities in the bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, differentiation disorders, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function. According to the degree of differentiation of leukemia and the length of the natural course, it can be divided into acute and chronic leukemia. Acute leukemia cells are efficiently differentiated and stagnated in the early stages, mainly primitive and early immature cells, and the disease develops rapidly with a course of several months. Chronic leukemia cells are efficiently differentiated better, mainly immature or mature cells, develop slowly, and the course of several years. According to the classification of the diseased cell series, it includes the myeloid granulocyte, monocytic, erythrocytic, megakaryocytic system and the lymphoid system T and B cell system. Clinically, leukemia is often divided into lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, etc. For example, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemic leukemia, blast cell leukemia, bovine leukemia, chronic granulocytic leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, Lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloid leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myelomonocytic leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, stem cell leukemia, subleukemic leukemia or undifferentiated cell leukemia, etc. In certain embodiments of the present disclosure, the leukemia is preferably chronic myeloid leukemia.

[0056] The term "μM" used in the present invention is a concentration unit, which refers to the concentration expressed as the mass of the solute as parts per million of the mass of the total solution, also known as parts per million concentration, 1μM=1μmol / L=1pmol / μL.

[0057] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly indicated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" etc. will be understood to include the stated elements or components without excluding other elements or other components. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the result of AZ505 combined with Imatinib inducing apoptosis of chronic myeloid leukemia cells.

[0059] Figure 2 This is the result of AZ505 and Imatinib combining to inhibit the viability of chronic myeloid leukemia cells. DETAILED DESCRIPTION

[0060] The disclosure above generally describes the present invention. A more complete understanding can be obtained with reference to the following specific embodiments. The purpose of describing these embodiments is only for illustration and is not intended to limit the scope of the present invention. Form changes and equivalent substitutions are considered to be circumstances that may suggest or confer convenience. Although specific terms are used in this article, the purpose of these terms is descriptive rather than limiting.

[0061] Example 1

[0062] 1. Experimental materials and experimental groups Experimental materials: KU812 cells and BV173 cells.

[0063] Experimental group: control group.

[0064] AZ505 monotherapy group 1: 2.5 μM AZ505 (abbreviated as: AZ 2.5 μM).

[0065] AZ505 monotherapy group 2: 5 μM AZ505 (abbreviated as: AZ 5 μM).

[0066] AZ505 monotherapy group 3: 10 μM AZ505 (abbreviated as: AZ 10 μM).

[0067] Imatinib monotherapy group 1: 0.025 μM Imatinib (abbreviation: IM 0.025 μM).

[0068] Imatinib monotherapy group 2: 0.05 μM of Imatinib (abbreviation: IM 0.05 μM).

[0069] Imatinib monotherapy group 3: 0.1 μM Imatinib (abbreviation: IM 0.1 μM).

[0070] AZ505 combined with Imatinib group 1: 5 μM AZ505 and 0.05 μM Imatinib (abbreviated as: AZ+IM 5+0.05 μM).

[0071] AZ505 combined with Imatinib group 2: 5 μM AZ505 and 0.1 μM Imatinib (abbreviated as: AZ+IM 5+0.1 μM).

[0072] AZ505 combined with Imatinib group 3: 10 μM AZ505 and 0.05 μM Imatinib (abbreviated as: AZ+IM 10+0.05 μM).

[0073] AZ505 combined with Imatinib group 4: 10 μM AZ505 and 0.1 μM Imatinib (abbreviated as: AZ+IM 10+0.1 μM).

[0074] 2. Experimental methods The CML cell line KU812 (3 × 10) was treated with ctrl, AZ505 (2.5, 5, 10 µM), Imatinib (25, 50, 100 nM), AZ+IM (5 µM+50nM, 5 µM+100 nM, 10 µM+50 nM, 10 µM+100 nM), respectively. 5 / mL) cells for 24h, collect the cells, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, wash the cell pellet with 1 mL pre-cooled PBS, transfer to a 1.5mL EP tube, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant as much as possible. Add 50 μL of binding buffer to the 1.5 mL EP tube, then add 5 μL Annexin-FITC and 5 μL propidium iodide (PI) and mix well, and stain at room temperature in the dark for 15 minutes. Add 450 μL of binding buffer to terminate the reaction, filter the sample with a double-layer 200-mesh nylon mesh, detect it with a flow cytometer within 1 hour, save the data file, and process the data with GraphPad Prism software.

[0075] CML cell line BV173 (3 × 10 cells / mL) was treated with ctrl, AZ505 (2.5, 5, 10 µM), Imatinib (25, 50, 100 nM), AZ+IM (5 µM+50nM, 5 µM+100 nM, 10 µM+50 nM, 10 µM+100 nM), respectively. 5 / mL) cells for 24 h, and the cell apoptosis was detected using the same method as described above.

[0076] 3. Experimental results The experimental results are as follows Figure 1 As shown, the results showed that after treating leukemia with different concentrations of AZ505 and Imatinib, the combined treatment induced apoptosis of leukemia cells.

[0077] in addition, Figure 2 The results showed that AZ505 combined with Imatinib inhibited the viability of CML cells.

Claims

1. Application of AZ505 combined with Imatinib in the preparation of a pharmaceutical composition for preventing, alleviating and treating leukemia.

2. The use according to claim 1, wherein the pharmaceutical composition is administered orally, by suppository, around the lesion, on the lesion surface, topically, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously; Preferably, AZ505 and Imatinib in the pharmaceutical composition are administered sequentially or simultaneously; Preferably, the concentration of AZ505 is 5-10 μM; Preferably, the concentration of AZ505 is 5 μM, 10 μM; Preferably, the concentration of Imatinib is 0.05-0.1 μM; Preferably, the concentration of Imatinib is 0.05 μM or 0.1 μM.

3. The use according to claim 1, wherein the leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia; Preferably, the acute leukemia includes L1 acute lymphoblastic leukemia, L2 acute lymphoblastic leukemia, L3 acute lymphoblastic leukemia, and acute myeloid leukemia; Preferably, the acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia; Preferably, the chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

4. A pharmaceutical composition comprising an effective amount of AZ505 and Imatinib for use according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable adjuvant; Preferably, the auxiliary agent includes an excipient, a diluent, a dispersing aid, a suspending aid, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, and a lubricant.

6. A medicine box, comprising: A first formulation containing AZ505 and a second formulation containing Imatinib; Preferably, the concentration of AZ505 is 5-10 μM; Preferably, the concentration of AZ505 is 5 μM, 10 μM; Preferably, the concentration of Imatinib is 0.05-0.1 μM; Preferably, the concentration of Imatinib is 0.05 μM or 0.1 μM.

7. Application of AZ505 combined with Imatinib in the preparation of products for inducing leukemia cell apoptosis and inhibiting leukemia cell viability.

8. The use according to claim 7, wherein the leukemia includes acute leukemia, chronic leukemia, hairy cell leukemia, and prolymphocytic leukemia; Preferably, the acute leukemia includes L1 acute lymphoblastic leukemia, L2 acute lymphoblastic leukemia, L3 acute lymphoblastic leukemia, and acute myeloid leukemia; Preferably, the acute myeloid leukemia includes granulocytic leukemia, erythroleukemia, and megakaryocytic leukemia; Preferably, the chronic leukemia includes chronic lymphocytic leukemia and chronic myeloid leukemia.

9. An in vitro method for inducing apoptosis of leukemia cells for non-therapeutic purposes, comprising the step of using the pharmaceutical composition according to claim 4 or 5.

10. An in vitro method for inhibiting the activity of leukemic lymphocyte and bone marrow cell tissues for non-therapeutic purposes, the method comprising the steps of using the pharmaceutical composition of claim 4 or 5 and the drug kit of claim 6.

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