Use of az505 in combination with imatinib in the preparation of a pharmaceutical composition for treating leukemia

Abstract

The application discloses application of AZ505 combined with Imatinib in preparation of a drug composition for treating leukemia, and belongs to the technical field of biotechnology. Experiments prove that the combined use of AZ505 and Imatinib can induce apoptosis of leukemia cells, and meanwhile, can inhibit the activity of leukemia cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to application of AZ505 combined with Imatinib in preparation of a pharmaceutical composition for treating leukemia. BACKGROUND

[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 at different stages of development and maturation have differentiation arrest, apoptosis disorder and malignant proliferation, thereby causing heterogeneous hematopoietic system malignancies. The main feature of leukemia is the abnormality in quality and quantity of white blood cells and their immature cells (precursor cells or leukemia cells) in blood and bone marrow, which can inhibit normal hematopoiesis of bone marrow and infiltrate tissues and organs such as liver, spleen and lymph nodes. Symptoms can occur due to lack of normal blood cells, which can include: bleeding and ecchymosis, fatigue and increased risk of infection.

[0003] Compound AZ505 is a histone lysine methyltransferase (SMYD2) inhibitor, which can specifically inhibit the activity of SMYD2. The molecular formula of AZ505 is C33H40Cl2F6N4O8, and the CAS number is 1035227-44-1. SMYD2 is a protein lysine methyltransferase, which can regulate histone and non-histone lysine methylation modification, and is involved in regulating tumor epigenetic signals and cell physiology. The target of AZ505 to play an anti-tumor effect is lysine methyltransferase SMYD2, which binds to SMYD2 specifically, and competes with the peptide substrate to bind to SMYD2, thereby inhibiting the function of the methylation substrate of SMDY2, producing a series of effects such as inhibiting proliferation and inducing apoptosis.

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

[0005] In order to solve the technical problems existing in the prior art, the technical solutions of the present application are provided.

[0006] The application provides application of AZ505 combined with Imatinib in preparation of a pharmaceutical composition for preventing, alleviating and treating leukemia.

[0007] Further, the pharmaceutical composition is administered orally, by suppository, around a lesion, on a lesion surface, locally, intravenously, parenterally, intraperitoneally, intramuscularly, into a lesion, intrathecally, intranasally or subcutaneously.

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

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

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

[0011] Further, the concentration of the AZ505 is 5 μM or 10 μM.

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

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

[0014] Further, the concentration of the Imatinib is 0.05 μM or 0.1 μM.

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

[0016] In some embodiments, the concentration ratio of the AZ505 and Imatinib 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 or 200:1.

[0017] Further, the AZ505 and Imatinib further include pharmaceutically acceptable salts thereof.

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

[0019] Further, the acid addition salts include, but are not limited to, any one of or a combination of hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, nitrate, ethanesulfonate, toluenesulfonate, benzenesulfonate, acetate, maleate, tartrate, succinate, citrate, benzoate, ascorbate and salicylate, malonate, adipate, hexanoate, arginate, fumarate, nicotinate, phthalate or oxalate.

[0020] Further, the base addition salts include, but are not limited to, lithium, sodium, potassium, barium, calcium, magnesium, aluminum, iron, ferrous, copper, zinc, or salts with morpholine, diethylamine, triethylamine, isopropylamine, trimethylamine, lysine or histamine acid.

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

[0022] Further, the acute leukemia includes L1 type acute lymphoblastic leukemia, L2 type acute lymphoblastic leukemia, L3 type acute lymphoblastic leukemia, acute myeloid leukemia.

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

[0024] Further, the chronic leukemia includes chronic lymphocytic leukemia, chronic myelogenous leukemia.

[0025] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

[0026] In some embodiments, the "administering" involves the actual physical introduction of an agent into or onto (as appropriate) a target biological subject (e.g., a host and / or a subject). A composition can be administered (e.g., "contacted") to a cell in vitro or in vivo. A composition can be administered to a subject in vivo via an appropriate administration route. Any and all methods of introducing a composition into a host are encompassed by the present application. The methods are not dependent on any particular mode of introduction and should not be so construed. Modes of introduction are well known to those of skill in the art and are also exemplified herein. The term includes administration routes that allow the agent to perform its intended function.

[0027] The term "prevention" as used in the present application means prophylactic administration of the combination to a healthy patient to prevent the occurrence of a disease or condition as described herein. In addition, the term "prevention" can also mean prophylactic administration of the combination to a patient in a pre-stage of the disease to be treated.

[0028] The present application provides a pharmaceutical composition comprising an effective amount of AZ505 and Imatinib as described above in the uses.

[0029] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

[0030] Further, the adjuvant comprises excipient, diluent, dispersion aid, suspension aid, surfactant, isotonic agent, thickening agent, emulsifying agent, preservative, lubricant.

[0031] In some embodiments, the pharmaceutical composition, depending on the route of administration, can be coated with or otherwise formulated with selected materials to protect it from the action of natural conditions that can detrimentally affect its ability to perform its intended function. The pharmaceutical composition can be administered alone or in combination with pharmaceutically acceptable adjuvants. The pharmaceutical composition can also be administered as a prodrug, which is converted in vivo to its active form.

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

[0033] Further, the pharmaceutical composition can be made into a powder for oral administration, tablets, (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, pellets, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc.; 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 parenteral administration.

[0034] Further, the pharmaceutical composition further comprises an anti-apoptotic agent, a mitotic inhibitor, an immunomodulator, a nucleic acid for gene therapy, an alkylating agent, an anti-angiogenic agent, an anti-metabolite, a boron-containing agent, a chemoprotective agent, a hormonal agent, an anti-hormonal agent, an antiviral drug, a photosensitizing therapeutic agent, an oligonucleotide, a radionuclide agent, a radiosensitizer, a topoisomerase inhibitor, a tyrosine kinase inhibitor, an antibiotic drug.

[0035] The term "effective amount" used in the present application means an amount that can produce a function or activity and can be accepted by a human and / or an animal. The effective amount can vary depending on 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 dose will vary depending on the following factors: the selected specific compound, the administration schedule, whether it is administered in combination with other compounds, the time schedule of administration, the tissue to be administered, and the physical delivery system to be carried.

[0036] The term "pharmaceutically acceptable" used in the present application means a substance that is suitable for use in a human and / or a mammal without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), i.e., having a reasonable benefit / risk ratio.

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

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

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

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

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

[0042] In some embodiments, the kit can have a time interval when using the first preparation and the second preparation, the interval can be 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 5 h, 10 h, 20 h, 1 d, 2 d, 3 d, 5 d, 7 d. In some embodiments, the kit can be combined for co-use when using the first preparation and the second preparation.

[0043] In some embodiments, the kit can be a pre-treatment kit of other leukemia treatment drugs, for accelerating the induction of apoptosis of leukemia cells and inhibiting the viability of leukemia cells.

[0044] The present application provides the use of AZ505 in combination with Imatinib in the preparation of a product for inducing apoptosis of leukemia cells.

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

[0046] Further, the acute leukemia includes Ll type acute lymphocytic leukemia, L2 type acute lymphocytic leukemia, L3 type acute lymphocytic leukemia, acute myeloid leukemia.

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

[0048] Further, the chronic leukemia includes chronic lymphocytic leukemia, chronic myelogenous leukemia.

[0049] The present application provides a method for inducing apoptosis of leukemia cells for non-therapeutic purposes in vitro, the method comprising the step of using the pharmaceutical composition as described above.

[0050] The present application provides a method for inhibiting leukemia lesion lymphocyte, bone marrow cell tissue for non-therapeutic purposes in vitro, the method comprising the step of using the pharmaceutical composition as described above, the pharmaceutical kit as described above.

[0051] In some embodiments, the "treatment" refers to the improvement, prevention, or reversal of a disease or disorder or at least one discernible 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 disorder to be treated, which parameter need not necessarily be discernible or recognized by the mammal. In some embodiments, the "treatment" refers to the inhibition or slowing of a disease or disease process, which inhibition or slowing can be physical, such as a reduction in some discernible adverse symptom. As used herein, "treatment" of a disease in a mammal, particularly a human, encompasses: (a) preventing the disease or disorder from occurring in an individual that is predisposed or does not yet experience symptoms of the disease; (b) inhibiting the disease, for example, arresting its development; or (c) relieving the disease, for example, causing regression of symptoms associated with the disease.

[0052] As used herein, the term "relief" refers to the process of reducing the severity of a sign or symptom of a disease. The sign or symptom can be reduced, but not necessarily eliminated.

[0053] As used herein, the term "non-therapeutic purposes" is synonymous with "non-therapeutic", "non-therapeutically" and means that the methods and / or uses of the present application exclude the methods of diagnosis and treatment of diseases as provided in Article 25 of the Chinese Patent Law.

[0054] The term "apoptosis" or "programmed cell death" as used herein 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, with the cell being an active participant in its own demise ("cellular suicide"). It is most commonly seen in the turnover of normal cells, tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system, and endocrine-dependent tissue atrophy. Apoptosis can also be induced by external events or stimuli, such as the drug combination therapy of certain preferred embodiments of the present application to induce apoptosis in leukemia cells.

[0055] The term "leukemia" as used herein refers to a class of malignant clonal diseases of hematopoietic stem cells. The clonal leukemia cells proliferate and accumulate in large numbers in the bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, differentiation disorder, and apoptosis blockage, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoiesis. According to the differentiation degree of leukemia and the length of natural course, leukemia can be divided into acute and chronic leukemia. The cells of acute leukemia are highly differentiated and arrested at the early stage, mainly consisting of primitive and early myeloblast cells, and the disease develops rapidly with a course of several months. The cells of chronic leukemia are well differentiated, mainly consisting of immature or mature cells, and the disease develops slowly with a course of several years. According to the series of diseased cells, leukemia includes granulocyte, monocyte, erythrocyte, megakaryocyte of myeloid series and T and B cell series of lymphoid series. Clinically, leukemia is often divided into lymphocyte leukemia, myelocyte leukemia, mixed cell leukemia, etc. For example, acute non-lymphocyte leukemia, chronic lymphocyte leukemia, acute granulocyte leukemia, chronic granulocyte leukemia, acute promyelocytic leukemia, adult T-cell leukemia, non-leukemia leukemia, blast cell leukemia, bovine leukemia, chronic granulocytic leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocyte leukemia, leukopenic leukemia, lymphoblastic leukemia, lymphoblastoid leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyelocytic leukemia, monocyte leukemia, myeloblastic leukemia, myelocytic leukemia, myelogenous leukemia, myelomonocytic leukemia, plasma cell leukemia, multiple myeloma, plasmacytic 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 myelocytic leukemia.

[0056] The term "μM" used in the present application is a concentration unit, which means the concentration expressed by the mass of solute in one million of the mass of the solution, also called the molar concentration, 1 μM = 1 μmol / L = 1 pmol / μL.

[0057] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. In the entire specification and claims, the term "comprise" or its variants such as "comprises" or "comprising" will be understood to encompass the stated elements or components, but not to exclude the presence of other elements or components. The terms "such as", "for example", and the like are intended to indicate exemplary embodiments, and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

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

[0059] Figure 2 Figure 2 is a result graph of AZ505 combined with Imatinib inhibiting the viability of chronic myeloid leukemia cells. DETAILED DESCRIPTION

[0060] The above disclosure describes the present application in general terms. A more complete understanding can be obtained by reference to the following specific examples. The purpose of the description of these examples is merely to illustrate and not to limit the scope of the present application. Form variations and equivalent replacements are considered as circumstances may suggest or impart convenience. Although specific terms are used herein, the purpose of these terms is descriptive rather than limiting.

[0061] Example 1

[0062] 1. Experimental materials and experimental grouping

[0063] Experimental materials, KU812 cells and BV173 cells.

[0064] Experimental grouping: control group.

[0065] AZ505 single drug group 1: 2.5 μM of AZ505 (abbreviation: AZ 2.5 μM).

[0066] AZ505 single drug group 2: 5 μM of AZ505 (abbreviation: AZ 5 μM).

[0067] AZ505 single drug group 3: 10 μM of AZ505 (abbreviation: AZ 10 μM).

[0068] Imatinib single drug group 1: 0.025 μM of Imatinib (abbreviation: IM 0.025 μM).

[0069] Imatinib single drug group 2: Imatinib at 0.05 μM (abbreviation: IM 0.05 μM).

[0070] Imatinib single drug group 3: Imatinib at 0.1 μM (abbreviation: IM 0.1 μM).

[0071] AZ505 combined with Imatinib group 1: AZ505 at 5 μM and Imatinib at 0.05 μM (abbreviation: AZ+IM 5+0.05 μM).

[0072] AZ505 combined with Imatinib group 2: AZ505 at 5 μM and Imatinib at 0.1 μM (abbreviation: AZ+IM 5+0.1 μM).

[0073] AZ505 combined with Imatinib group 3: AZ505 at 10 μM and Imatinib at 0.05 μM (abbreviation: AZ+IM 10+0.05 μM).

[0074] AZ505 combined with Imatinib group 4: AZ505 at 10 μM and Imatinib at 0.1 μM (abbreviation: AZ+IM 10+0.1 μM).

[0075] 2. Experimental method

[0076] KU812 (3 × 10 5 / mL) cells were treated with ctrl, AZ505 (2.5, 5, 10 μM), Imatinib (25, 50, 100 nM), AZ+IM (5 μM+50 nM, 5 μM+100 nM, 10 μM+50 nM, 10 μM+100 nM) respectively for 24 h, then the cells were collected and centrifuged at 2000 rpm for 5 min, the supernatant was discarded, the cell pellet was washed with 1 mL of pre-cooled PBS, transferred to a 1.5 mL EP tube, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded as much as possible. 50 μL of binding buffer was added to the 1.5 mL EP tube, followed by 5 μL of Annexin-FITC and 5 μL of propidium iodide (PI) and mixed well, and the staining was performed at room temperature for 15 min in the dark. The reaction was terminated by adding 450 μL of binding buffer, and the sample was filtered with double-layer 200 mesh nylon net, and the data file was detected by flow cytometry within 1 h and saved. The data were processed by GraphPad Prism software.

[0077] CML cell line BV173 (3 x 10 5 / mL) cells were treated with Ctrl, AZ505 (2.5, 5, 10 μΜ), Imatinib (25, 50, 100 nM), AZ + IM (5 μΜ + 50 nM, 5 μΜ + 100 nM, 10 μΜ + 50 nM, 10 μΜ + 100 nM) for 24 h, and the apoptosis of the cells was detected using the same method as described above.

[0078] 3. Experimental results

[0079] The experimental results are shown in Figure 1 Table 3. The results show that the combination treatment of AZ505 and Imatinib induces apoptosis of leukemia cells after treatment of leukemia with different concentrations of AZ505 and Imatinib.

[0080] In addition, Figure 2 The results show that AZ505 and Imatinib inhibit the viability of CML cells in combination.

Claims

1. Use of AZ505 in combination with Imatinib in the preparation of a pharmaceutical composition for preventing, alleviating or treating chronic myelogenous leukemia.

2. The use according to claim 1, wherein the pharmaceutical composition is administered orally, by suppository, perilesionally, intravenously, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally or subcutaneously.

3. The use according to claim 1, wherein the AZ505 and Imatinib in the pharmaceutical composition are administered sequentially or simultaneously.

4. The use according to claim 1, wherein the concentration of AZ505 is 5-10 μM.

5. The use according to claim 4, wherein the concentration of AZ505 is 5 μM or 10 μM.

6. The use according to claim 1, wherein the concentration of Imatinib is 0.05-0.1 μM.

7. The use according to claim 6, wherein the concentration of Imatinib is 0.05 μM or 0.1 μM.

8. The use according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

9. A pharmaceutical composition comprising an effective amount of AZ505 and Imatinib for use according to any one of claims 1-8.

10. The pharmaceutical composition according to claim 9, further comprising a pharmaceutically acceptable adjuvant.

11. The pharmaceutical composition according to claim 10, wherein the adjuvant comprises a diluent, a dispersion aid, a surfactant, an isotonic agent, a thickening agent, a preservative, a lubricant.

12. A kit comprising: a first preparation comprising AZ505, and a second preparation comprising Imatinib.

13. The kit according to claim 12, wherein the concentration of AZ505 is 5-10 μM.

14. The kit according to claim 13, wherein the concentration of AZ505 is 5 μM or 10 μM.

15. The kit according to claim 12, wherein the concentration of Imatinib is 0.05-0.1 μM.

16. The kit according to claim 15, wherein the concentration of Imatinib is 0.05 μM or 0.1 μM.

17. Use of AZ505 in combination with Imatinib in the preparation of a product for inducing apoptosis and inhibiting the viability of chronic myelogenous leukemia cells.

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