A pharmaceutical composition and its use in the treatment of cancer

Abstract

The application discloses a kind of pharmaceutical composition and its application in treating cancer, the pharmaceutical composition includes MK-2206 or its pharmaceutically acceptable salt and SCH772984 or its pharmaceutically acceptable salt.The application first discovers that MK-2206 and SCH772984 both have synergistic effect on the treatment of colon cancer or breast cancer, and the application provides effective drug combination strategy for the treatment of cancer, and has wide application prospect in the technical field of development of cancer treatment drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition and application thereof in treating cancer. BACKGROUND

[0002] MK-2206 is a selective, orally bioavailable AKT inhibitor, which mainly functions to inhibit AKT phosphorylation, and has a better blocking effect than traditional competitive inhibitors, higher specificity, relatively small side effects and toxicity, and the advantage of being able to identify and inhibit all three AKT isozymes. MK-2206 is mainly used for research and potential treatment of various cancers, has passed the phase II clinical trial of pancreatic solid tumors and renal cell carcinoma, and the safety has been further verified. MK-2206 single agent can exert antitumor activity in vivo and in vitro.

[0003] SCH772984 is an ATP competitive inhibitor, which can bind to non-phosphorylated inactive ERK1 / 2 and strongly and selectively inhibit ERK1 and ERK2 activity. SCH772984 can inhibit BRAF / RAS mutant cancer cell proliferation and increase cell apoptosis in vitro and in vivo.

[0004] At present, there is no research report on the combination of MK-2206 and SCH772984 for the treatment of cancer, especially colon cancer and breast cancer. SUMMARY

[0005] The present application aims to provide a pharmaceutical composition and application thereof in treating cancer, and to provide an effective drug combination strategy for the treatment of cancer. The drug combination composition comprises MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof. The present application first discovers that the combination of MK-2206 and SCH772984 has a synergistic effect on the treatment of cancer.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The first aspect of the present application provides a pharmaceutical composition and application thereof in treating cancer.

[0008] Further, the pharmaceutical composition comprises MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof.

[0009] Further, the concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.0137-30):(0.0137-30).

[0010] Further, the concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.1235 - 30):(0.1235 - 30) or (0.0137 - 3.3333):(0.0137 - 3.3333).

[0011] Further, the concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704 - 10):(0.1235 - 10) or (0.3704 - 3.3333):(0.0137 - 0.1235).

[0012] Further, the concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704 - 3.3333):(1.1111 - 10) or (1.1111 - 10):(1.1111 - 10) or (0.3704 - 3.3333):(0.1235 - 1.1111) or (0.3704 - 3.3333):(0.0137 - 0.1235).

[0013] Further, the concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1:1 or 4:1.

[0014] Further, the specific concentration of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, which is confirmed by experiments, is (0.1235 μM - 30 μM):(0.1235 μM - 30 μM) or (0.0137 μM - 3.3333 μM):(0.0137 μM - 3.3333 μM).

[0015] Further, the specific concentration of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, which is confirmed by experiments, is (0.3704 μM - 10 μM):(0.1235 μM - 10 μM) or (0.3704 μM - 3.3333 μM):(0.0137 μM - 0.1235 μM).

[0016] Further, the specific concentrations of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, which have been experimentally verified, are (0.3704 μM - 3.3333 μM):(1.1111 μM - 10 μM) or (1.1111 μM - 10 μM):(1.1111 μM - 10 μM) or (0.3704 μM - 3.3333 μM):(0.1235 μM - 1.1111 μM) or (0.3704 μM - 3.3333 μM):(0.0137 μM - 0.1235 μM).

[0017] Further, the specific concentrations of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition, which have been experimentally verified, are 0.1 μM:0.1 μM or 0.3 μM:0.3 μM or 100 mg / kg:25 mg / kg.

[0018] In the present application, the MK-2206 is a selective, orally bioavailable AKT inhibitor with a molecular formula of C 25 H 21 N5O, a molecular weight of 443.93, and is mainly used for research and potential treatment of various cancers.

[0019] In the present application, the SCH772984 is a highly selective and ATP-competitive ERK1 / 2 inhibitor with a molecular formula of C 33 H 33 N9O2, a molecular weight of 587.67, and has good antitumor activity in cells with BRAF or RAS mutations against native MAPK inhibition and drug-resistant MAPK inhibition.

[0020] In some embodiments, the pharmaceutically acceptable salt is safe and effective for topical application in mammals and has the desired biological activity. The pharmaceutically acceptable salt includes a salt of an acidic or basic group present in the specified compound. The pharmaceutically acceptable acid addition salt includes, but is not limited to, a hydrochloride, a hydrobromide, a hydroiodide, a nitrate, a sulfate, a bisulfate, a phosphate, a hydrogen phosphate, an isonicotinate, an acetate, a lactate, a salicylate, a citrate, a tartrate, a pantothenate, a bitartrate, an ascorbate, a succinate, a maleate, a gentisinate, a fumarate, a gluconate, a glucaronate, a saccharate, a formate, a benzoate, a glutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate, a p-toluenesulfonate, and a pamoate (i.e., 1,1' Methylene Di (2 hydroxyl 3 naphthoate). Certain compounds used in the present application can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.

[0021] In some embodiments, the MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition can be administered simultaneously, separately or sequentially. By simultaneously, it is meant that the two drugs are administered at the same time. If not administered simultaneously, the administration is sequential within a time frame such that both are therapeutically active within the same time frame. Thus, sequential administration can allow for administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours or several hours after administration of the other, provided that the circulating half-life of the first administered drug is such that both are present in therapeutically effective amounts at the same time. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction between them, and their respective half-lives. Separate, unlike simultaneous or sequential, means that there is a significant interval between administration of one drug and the other, i.e., when the second drug is administered, the first administered drug can no longer be present in the bloodstream in a therapeutically effective amount.

[0022] In some embodiments, the pharmaceutical composition described in the present application can also be used in combination with other therapeutic compounds that can be used in the treatment and / or prevention and / or adjuvant treatment and / or adjuvant prevention of cancer, which can be administered simultaneously with the main active ingredients in the pharmaceutical composition described in the present application, even in the same composition. The other therapeutic compounds can also be administered separately in a separate composition or in a different dosage form from the main active ingredients. Part of the dose of the main ingredients can be administered simultaneously with the other therapeutic compounds, while the other doses can be administered separately. During the course of treatment, the dose of the pharmaceutical composition described in the present application can be adjusted according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

[0023] In the present application, the treatment refers to the prevention and reduction of the occurrence or development of the disease, so that the development or aggravation of the disease course is inhibited, suppressed, reduced, improved, slowed down, stopped, delayed or reversed, various indicators of the described disease, disorder or pathological state during and / or after medication, including alleviation or reduction of symptoms or complications, or cure or elimination of the disease, disorder or condition, in specific embodiments of the present application, the disease is preferably colon cancer, breast cancer.

[0024] In the present application, the prevention refers to various means or measures for preventing the occurrence or development of diseases before the diseases are recognized by clinical standards, including medical, physical or chemical methods, to prevent and reduce the occurrence or development of various symptoms of diseases. In specific embodiments of the present application, the diseases are preferably colon cancer and breast cancer.

[0025] The second aspect of the present application provides a pharmaceutical preparation.

[0026] Further, the pharmaceutical preparation comprises the pharmaceutical composition according to the first aspect of the present application.

[0027] Further, the dosage form of the pharmaceutical preparation includes injection administration dosage form, respiratory tract administration dosage form, cavity administration dosage form, mucosa administration dosage form, skin administration dosage form.

[0028] Further, the pharmaceutical preparation is a single complex preparation or a combination of two separate single preparations.

[0029] Further, the complex preparation is a complex preparation comprising MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof.

[0030] Further, the combination of the single preparations is a combination of a single preparation comprising MK-2206 or a pharmaceutically acceptable salt thereof and a single preparation comprising SCH772984 or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the dosage form of the pharmaceutical preparation according to the present application is a dosage form that is prepared by a conventional method and is advantageous for administration, including but not limited to: parenteral administration dosage form, enteral administration dosage form, and specific examples include but are not limited to: aqueous solution injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder spray, sustained-release preparation and controlled-release preparation, etc.

[0032] In some embodiments, the pharmaceutically acceptable carrier and / or excipient according to the present application includes but is not limited to: diluent, binder, surfactant, wetting agent, adsorption carrier, lubricant, filler, disintegrant.

[0033] In specific embodiments, the diluent includes but is not limited to: lactose, sodium chloride, glucose, urea, starch, water, etc.

[0034] In specific embodiments, the binder includes but is not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginic acid salt, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc.

[0035] In specific embodiments, the surface active agent includes, but is not limited to, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, stearic acid monoglyceride, cetyl alcohol, etc.

[0036] In specific embodiments, the wetting agent includes, but is not limited to, glycerol, starch, etc.

[0037] In specific embodiments, the adsorbing carrier includes, but is not limited to, starch, lactose, bentonite, silica gel, kaolin, soap clay, etc.

[0038] In specific embodiments, the lubricant includes, but is not limited to, zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauryl sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.

[0039] In specific embodiments, the filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupled sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.

[0040] In specific embodiments, the disintegrant includes, but is not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0041] In some embodiments, the pharmaceutical composition of the present application can further comprise stabilizers, buffers, bactericides, isotonic agents, pH control agents, surfactants, and chelating agents, etc.

[0042] In some embodiments, the complex preparation refers to a preparation with two or more active pharmaceutical ingredients, for example, the combination drug composition of the present application is a complex preparation, which can represent that it simultaneously comprises MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, the single preparation refers to a preparation with a single active pharmaceutical ingredient, for example, the combination drug composition of the present application is a single preparation combination, which can represent a combination of a single preparation comprising MK-2206 or a pharmaceutically acceptable salt thereof, and a single preparation comprising SCH772984 or a pharmaceutically acceptable salt thereof.

[0044] In some embodiments, the administration mode of the two single formulations in the combination of the single formulations comprises: simultaneous administration, sequential administration. When the administration mode of the two single formulations in the combination of the single formulations is sequential administration, the administration mode comprises: administering the single formulation comprising MK-2206 or a pharmaceutically acceptable salt thereof first, and then administering the single formulation comprising SCH772984 or a pharmaceutically acceptable salt thereof; administering the single formulation comprising SCH772984 or a pharmaceutically acceptable salt thereof first, and then administering the single formulation comprising MK-2206 or a pharmaceutically acceptable salt thereof.

[0045] The third aspect of the present application provides any one of the following applications, which comprises:

[0046] (1) The use of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating and / or preventing cancer;

[0047] (2) The use of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical preparation for treating and / or preventing cancer;

[0048] (3) The use of MK-2206 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the therapeutic effect of SCH772984 or a pharmaceutically acceptable salt thereof in treating cancer;

[0049] (4) The use of SCH772984 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for improving the therapeutic effect of MK-2206 or a pharmaceutically acceptable salt thereof in treating cancer;

[0050] (5) The use of the pharmaceutical composition of the first aspect of the present application or the pharmaceutical preparation of the second aspect of the present application in the preparation of an agent for inhibiting the growth of cancer cells and promoting the apoptosis of cancer cells.

[0051] Further, the cancer comprises colon cancer, breast cancer.

[0052] Further, the cancer cells comprise colon cancer cells, breast cancer cells.

[0053] Further, the cancer cells comprise human colon cancer cell line HCT116, triple-negative breast cancer cell line HCC1806.

[0054] The fourth aspect of the present application provides a method for inhibiting the growth of cancer cells and promoting the apoptosis of cancer cells for non-therapeutic purposes.

[0055] Further, the cancer comprises colon cancer, breast cancer.

[0056] Further, the method comprises the following steps: treating colon cancer cells, breast cancer cells with the pharmaceutical composition of the first aspect of the present application or the pharmaceutical preparation of the second aspect of the present application.

[0057] In a specific embodiment of the present application, the present application first discovered by experiment that the combination of MK-2206 and SCH772984 significantly inhibits the growth of colon cancer cells and breast cancer cells. Therefore, the combination drug composition can be used as an inhibitor for non-therapeutic purposes to inhibit the growth and proliferation of colon cancer cells and breast cancer cells in the field of scientific research, such as further studying the growth and metabolism mechanism or behavior of colon cancer cells and breast cancer cells, screening potential drugs for treating colon cancer and breast cancer, etc.

[0058] In a specific embodiment of the present application, the present application further verifies the synergistic effect of the combination of MK-2206 and SCH772984 by using the combination drug HSA model, Loewe model, Bliss model and ZIP model. The combination drug HSA model is a mathematical model for evaluating the effect of drug combination. In drug combination experiments, the HSA model evaluates synergism by comparing the actual effect of drug combination with the expected effect of the highest single drug. If the calculated HSA value is greater than 0, it is considered that the drug combination has synergistic effect; if the HSA value is less than 0, it may indicate that there is antagonism between drugs. Loewe additivity model is a mathematical model for analyzing drug interactions, which can be used to predict the expected effect of the combination of two or more drugs without interaction, and can help researchers evaluate the potential synergistic effect of drug combination in drug development and clinical application. Bliss synergy model is a mathematical model for evaluating drug interactions, which is based on the assumption that two drugs act independently on the biological system without interaction. A key feature of the Bliss model is that it considers the probability of independent drug action, and calculates the expected joint effect by multiplying the probabilities. This method is very useful in drug combination research, especially in high-throughput screening (HTS) experiments, which can quickly evaluate the potential synergistic effect of a large number of drug combinations. ZIP (Zero Interaction Potency) model is a reference model for evaluating drug combination interactions. It is based on the assumption that if there is no interaction between two drugs, their potency on the dose-response curve will not change. ZIP model quantifies the strength of drug interaction by comparing the dose-response curves of single drugs and drug combinations. In practical applications, ZIP model is often used to analyze the synergistic effect of drug combination, especially in high-throughput drug screening. It combines the advantages of Loewe additivity and Bliss model, aiming to systematically evaluate various possible drug interaction patterns.

[0059] In addition, the present application also provides a method for treating colon cancer or breast cancer, comprising the following steps: administering a therapeutically effective amount of the pharmaceutical composition of the first aspect of the present application or the pharmaceutical preparation of the second aspect of the present application to a subject in need.

[0060] In the present application, the effective amount refers to the amount of the compound that is effective to produce the desired prevention, alleviation or treatment effect. Depending on the compound, the severity of the symptoms, the age of the mammal being treated and other factors, the amount of the pharmaceutical composition or the pharmaceutical preparation of the present application to achieve the effective amount will vary, but can be routinely determined by one of ordinary skill in the art with due consideration of the knowledge in the art and the disclosure of the present application.

[0061] In some embodiments, the pharmaceutical composition or the pharmaceutical preparation can be administered to the subject by injection, topical administration or oral administration. For example, the method can comprise administering the pharmaceutical composition or the pharmaceutical preparation to the subject three times a day, once a day, once every two days, etc. In some embodiments, the injection can comprise subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, the injection can comprise injecting the pharmaceutical composition directly into the lesion or the area close to the lesion, and in some embodiments, the topical administration can comprise rectal administration, nasal administration, ear administration, intramedullary administration, intra-articular administration, intrapleural administration, etc., or any combination thereof. In some embodiments, the pharmaceutical composition or the pharmaceutical preparation can be administered to the subject via a combination of different administration methods.

[0062] In some embodiments, the subject is an animal, preferably a mammal (human and non-human animals), including but not limited to: human, non-human primates (especially higher primates, such as macaques, cynomolgus monkeys, marmosets, lorises, guenons, golden snub-nosed monkeys and tree shrews), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, any livestock or pet, etc., and in preferred embodiments, the subject is a human.

[0063] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0064] For the first time, the present application discovers that the combination of MK-2206 and SCH772984 has a synergistic inhibitory effect on the proliferation of colon cancer cell lines and triple-negative breast cancer cell lines, i.e., the combination has a significant synergistic effect, and the inhibitory effect of the combined pharmaceutical composition on tumor cells is significantly better than that of MK-2206 or SCH772984 alone. The present application provides an effective drug combination strategy for the treatment of cancer and has a broad application prospect in the technical field of the development of cancer treatment drugs.

[0065] As used above, the terms "have", "comprise" or "include" or any grammatical variants thereof are used in a non-exclusive way. Thus, these terms can both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As used above, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, i.e. features which are not indispensable in order to practice the application. Thus, these terms do not exclude that any of these features, or any combination thereof, is / are present in any embodiment of the application.

[0066] As used above, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, i.e. features which are not indispensable in order to practice the application. Thus, these terms do not exclude that any of these features, or any combination thereof, is / are present in any embodiment of the application. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 Figure 1 is a graph showing the results of MK-2206 and SCH772984 synergistically inhibiting the growth of colon cancer cell line HCT116, wherein A: IC50 curve of MK-2206 in HCT116 cells; B: IC50 curve of SCH772984 in HCT116 cells; C: experimental scheme of CCK8 activity detection of MK-2206 and SCH772984 combined administration; D: IC50 curve of MK-2206 and SCH772984 combined administration compared with MK-2206 alone in HCT116 cells; E: IC50 curve of MK-2206 and SCH772984 combined administration compared with SCH772984 alone in HCT116 cells; F: Bliss model synergy index score and three-dimensional surface graph of MK-2206 and SCH772984 combined administration in HCT116 cells; G: HSA model synergy index score and three-dimensional surface graph of MK-2206 and SCH772984 combined administration in HCT116 cells; H: Loewe model synergy index score and three-dimensional surface graph of MK-2206 and SCH772984 combined administration in HCT116 cells; I: ZIP model synergy index score and three-dimensional surface graph of MK-2206 and SCH772984 combined administration in HCT116 cells; J: synergy index analysis result table of F-I.

[0068] Figure 2 Figure 2 is a graph showing the results of MK-2206 and SCH772984 synergistically inhibiting the colony formation of colon cancer cell line HCT116.

[0069] Figure 3Figure 6 is a graph showing MK-2206 and SCH772984 synergistically inhibit the growth of triple negative breast cancer cell line HCC1806, wherein A: IC50 curve of MK-2206 in HCC1806 cells; B: IC50 curve of SCH772984 in HCC1806 cells; C: experimental schematic of CCK8 activity detection of MK-2206 and SCH772984 combined administration; D: IC50 curve of MK-2206 and SCH772984 combined administration compared with MK-2206 alone in HCC1806 cells; E: IC50 curve of MK-2206 and SCH772984 combined administration compared with SCH772984 alone in HCC1806 cells; F: ZIP model synergy index score and three-dimensional surface plot of MK-2206 and SCH772984 combined administration in HCC1806 cells; G: Bliss model synergy index score and three-dimensional surface plot of MK-2206 and SCH772984 combined administration in HCC1806 cells; H: HSA model synergy index score and three-dimensional surface plot of MK-2206 and SCH772984 combined administration in HCC1806 cells; I: Loewe model synergy index score and three-dimensional surface plot of MK-2206 and SCH772984 combined administration in HCC1806 cells; J: synergy index analysis results table of F-I.

[0070] Figure 4 Figure 7 is a graph showing MK-2206 and SCH772984 synergistically inhibit the colony formation and tumor growth of triple negative breast cancer cell line HCC1806, wherein A: results graph of MK-2206 and SCH772984 combined administration inhibiting the colony formation of HCC1806 cells; B: mouse tumor volume quantification graph of MK-2206 and SCH772984 combined administration; C: mouse tumor photo of MK-2206 and SCH772984 combined administration; D: mouse tumor volume Log2FC conversion quantification graph of MK-2206 and SCH772984 combined administration; E: mouse tumor weight quantification graph of MK-2206 and SCH772984 combined administration; F: mouse body weight quantification graph of MK-2206 and SCH772984 combined administration.

[0071] Figure 5The results of blood index toxicity analysis for the synergistic administration of MK-2206 and SCH772984 are shown in the following figures, in which A: aspartate aminotransferase (AST); B: alanine aminotransferase (ALT); C: total bilirubin (BILT); D: direct bilirubin (BILD); E: albumin (Alb); F: creatinine (CREA); G: blood urea nitrogen (UREA); H: uric acid (UA); I: total serum protein (TP); J: creatine kinase (CK); K: lactate dehydrogenase isozyme 1 (LDH1); L: lactate dehydrogenase isozyme 2 (LDH2); M: red blood cells (RBC); N: hemoglobin (Hemoglobin); O: white blood cells (WBC); P: platelets (PLT). DETAILED DESCRIPTION

[0072] The present application will be further described below in conjunction with specific examples, which are only used to explain the present application and cannot be understood as limiting the present application. Those skilled in the art can understand that various changes, modifications, replacements and variations can be made to these examples without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

[0073] The drugs, reagents and raw materials used in the present application are readily available to those skilled in the art, and can be obtained from commercial channels if not otherwise specified, and the experimental methods not specified in the present application are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturers. In particular, the following examples are only used to illustrate the present application and should not be used to limit the scope of the present application in any way.

[0074] Example 1 Synergistic inhibition of MK-2206 and SCH772984 on the growth of human colon cancer cell line HCT116

[0075] 1. Experimental materials

[0076] MK-2206 (TargetMol, T1952), SCH772984 (TargetMol, T6066), CCK8 kit, Synergy Finder online platform, crystal violet solution, paraformaldehyde.

[0077] 2. Experimental methods

[0078] (1) CCK8 cell activity detection

[0079] HCT116 cells were seeded in a 96-well plate at a density of 4000 cells / well and cultured in a 37℃, 5% CO2 incubator overnight until the cells adhered, and then the cells were divided into 4 groups and administered with drugs:

[0080] 1) Control group, no MK-2206 and SCH772984, i.e. no drug added solvent group;

[0081] 2) MK-2206 alone group, 10 concentrations of 0.0015, 0.0046, 0.0137, 0.0411, 0.1235, 0.3704, 1.1111, 3.3333, 10, 30 μM were set to verify the killing ability of the drug on cells more fully;

[0082] 3) SCH772984 alone group, 10 concentrations of 0.0015, 0.0046, 0.0137, 0.0411, 0.1235, 0.3704, 1.1111, 3.3333, 10, 30 μM were set to verify the killing ability of the drug on cells more fully;

[0083] 4) MK-2206 and SCH772984 combination group, 6 x 6 combination experiments were performed (drug concentrations of 0.1235, 0.3704, 1.1111, 3.3333, 10, 30 μM).

[0084] Continue to culture for 72 hours, aspirate the old culture solution, add 100 μl of new culture solution containing 10% CCK8 to each well, incubate for two hours, and then read at 450 nm wavelength. Cell viability = (experimental group OD value - blank control OD value) / (control group OD value - blank control OD value) x 100%.

[0085] (2) Process CCK8 data, calculate cell viability under different concentrations of drugs, log in to Synergy Finder online platform (https: / / synergyfinder.aittokallio.group), upload data, and calculate synergy index.

[0086] (3) Cloning experiment

[0087] HCT116 cells were seeded in a 12-well plate at a density of 1000 cells / well and cultured in a 37°C, 5% CO2 incubator overnight until the cells adhered, then the cells were divided into 4 groups and added with drugs:

[0088] 1) Control group, no MK-2206 and SCH772984, i.e. no drug added solvent group;

[0089] 2) MK-2206 alone group, the dose of MK-2206 was 0.3 μM;

[0090] 3) SCH772984 group alone, the dose of SCH772984 is 0.3 μΜ;

[0091] 4) MK-2206 and SCH772984 combination group, the dose of MK-2206 is 0.3 μΜ, the dose of SCH772984 is 0.3 μΜ.

[0092] After 14 days of culture, the cells were fixed with 4% paraformaldehyde and stained with crystal violet, and then photographed for counting.

[0093] (4) Statistical analysis

[0094] The data were analyzed by One way ANOVA Tukey's multiple comparisons test.

[0095] 3, Experimental results

[0096] (1) CCK8 assay to detect the effect of MK-2206 and SCH772984 combination on HCT116 cell growth

[0097] The results of cell viability detection are shown in Figure 1 A-B, both MK-2206 and SCH772984 alone can inhibit the proliferation of HCT116 cells to a certain extent, and we designed a combination drug experiment scheme as shown in Figure 1 C, the combination of MK-2206 and SCH772984 significantly enhances the inhibition of HCT116 cells (1D-1E), achieving a synergistic effect.

[0098] (2) Synergy Finder online platform is used to calculate and analyze whether MK-2206 and SCH772984 have synergistic effect on the inhibition of HCT116 cell growth

[0099] The results are shown in Figure 1 F-J, the four synergy indexes of MK-2206 and SCH772984, including Bliss synergy model, HSA (Highest Single Agent) synergy index, Loewe additivity model, ZIP (Zero Interaction Potency) model, are all greater than 10, indicating that the two drugs have strong synergistic effect from different aspects.

[0100] (3) Clonogenic assay to detect the effect of MK-2206 and SCH772984 combination on HCT116 cell growth.

[0101] The results of clonogenic assay are shown in Figure 2As shown, both MK-2206 and SCH772984 alone can inhibit the colony formation ability of HCT116 cells to a certain extent, and the combination of 0.3 μM MK-2206 and 0.3 μM SCH772984 significantly enhances the colony formation ability of inhibiting the growth of HCT116 cells, further proving that the combination of MK-2206 and SCH772984 has a synergistic effect.

[0102] Example 2 MK-2206 and SCH772984 synergistically inhibit the growth of triple-negative breast cancer cell line HCC1806

[0103] 1. Experimental materials

[0104] (1) Experimental animals

[0105] Female nude mice, 6 weeks old, about 20g, purchased from Beijing Speroff Biotechnology Co., Ltd.

[0106] (2) Experimental reagents

[0107] MK-2206 (TargetMol, T1952), SCH772984 (TargetMol, T6066), CCK8 kit, Synergy Finder online platform, crystal violet solution, paraformaldehyde.

[0108] 2. Experimental methods

[0109] (1) CCK8 cell activity detection

[0110] HCC1806 cells were seeded in a 96-well plate at a density of 3000 cells / well and cultured in a 37°C, 5% CO2 incubator overnight until the cells adhered. The cells were divided into 4 groups and dosed:

[0111] 1) Control group, no MK-2206 and SCH772984 were used, i.e. no drug was added to the solvent group;

[0112] 2) MK-2206 alone group, in order to more fully verify the killing ability of the drug on the cells, 10 dosing concentrations were set, which were 0.0015, 0.0046, 0.0137, 0.0411, 0.1235, 0.3704, 1.1111, 3.3333, 10, 30 μM;

[0113] 3) SCH772984 alone group, in order to more fully verify the killing ability of the drug on the cells, 10 dosing concentrations were set, which were 0.0015, 0.0046, 0.0137, 0.0411, 0.1235, 0.3704, 1.1111, 3.3333, 10, 30 μM;

[0114] 4) MK-2206 combined with SCH772984 group, 6 x 6 way to combine the experiment (drug concentration of 0.0134, 0.0412, 0.1235, 0.3704, 1.1111, 3.3333 μM).

[0115] Continue to culture for 72 hours, the old culture fluid suction, each hole 100 μl containing 10% CCK8 new culture fluid, incubated for two hours, read at 450 nm wavelength. Cell viability = (experimental group OD value - blank control OD value) / (control group OD value - blank control OD value) x 100%.

[0116] (2) processing CCK8 data, calculate the cell viability under different concentrations of drugs, login Synergy Finder online platform (https: / / synergyfinder.aittokallio.group), upload data, calculate synergy index.

[0117] (3) colony formation experiment

[0118] HCC1806 cells were seeded in 12-well plates at a density of 1000 cells / well, incubated in a 37°C, 5% CO2 incubator overnight until the cells were adherent, the cells were divided into 4 groups and added drugs:

[0119] 1) control group, no MK-2206 and SCH772984, namely no drug solvent group;

[0120] 2) MK-2206 alone group, MK-2206 dose of 0.1 μM;

[0121] 3) SCH772984 alone group, SCH772984 dose of 0.1 μM;

[0122] 4) MK-2206 combined with SCH772984 group, MK-2206 dose of 0.1 μM, SCH772984 dose of 0.1 μM.

[0123] Each group was cultured for 14 days, fixed with 4% paraformaldehyde, crystal violet staining and taking pictures for statistics.

[0124] (4) tumor formation experiment in nude mice

[0125] Five million HCC1806 cells were inoculated into nude mice, and when the tumor grew to 100 mm 3 The mice were divided into 4 groups and started to give drugs:

[0126] 1) Control group, no MK-2206 and SCH772984, namely no drug solvent group;

[0127] 2) MK-2206 alone group, MK-2206 administration method is gavage, the dose is 100 mg / kg;

[0128] 3) SCH772984 alone group, SCH772984 administration method is intraperitoneal injection, the dose is 25 mg / kg;

[0129] 4) MK-2206 combined with SCH772984 group, MK-2206 administration method is gavage, the dose is 100 mg / kg, SCH772984 administration method is intraperitoneal injection, the dose is 25 mg / kg.

[0130] Once every 3 days, weigh and measure the size of the tumor. When reaching the experimental endpoint, before killing the mice, randomly select three mice from the group, collect mouse blood with an anticoagulant tube, perform blood cell analysis, and then immediately separate the plasma sample for liver and kidney dynamic energy detection analysis. Roche cobas 5800 is used for sample detection.

[0131] (5) Statistical analysis

[0132] Cell experiment data uses One way ANOVA Tukey's multiple comparisons test, and animal experiment data two-group analysis uses Unpaired t test for statistical analysis.

[0133] 3. Experimental results

[0134] (1) CCK8 experiment method detects the effect of MK-2206 and SCH772984 combined use on HCC1806 cell growth

[0135] The results of cell viability detection are shown in A-B, and both MK-2206 and SCH772984 alone can inhibit the proliferation of HCC1806 cells to a certain extent, and we designed a combined drug experiment scheme as shown in C. Figure 3 MK-2206 and SCH772984 combined use significantly enhances the inhibition of HCT116 cells (3D-3E), achieving a synergistic effect. Figure 3

[0136] (2) Synergy Finder online platform is used to calculate and analyze whether MK-2206 and SCH772984 have a synergistic effect on inhibiting the growth of HCC1806 cells

[0137] The results are shown in​Figure 3 F-3J, four synergistic indexes of MK-2206 and SCH772984, including Bliss synergy model, HSA (Highest Single Agent) synergy index, Loewe additivity model, ZIP (Zero Interaction Potency) model, three of which have synergistic coefficients greater than 10, indicating that the two have a strong synergistic effect from different aspects.

[0138] (3) Cloning formation experiment method was used to detect the effect of MK-2206 and SCH772984 combined use on the growth of HCC1806 cells.

[0139] The cloning formation results are shown in Figure 4 A, MK-2206 and SCH772984 alone can inhibit the cloning formation ability of HCC1806 cells to a certain extent, and the combination of 0.1 μM MK-2206 and 0.1 μM SCH772984 significantly enhances the cloning formation ability of HCC1806 cells to inhibit cell growth, further proving that MK-2206 and SCH772984 have a synergistic effect.

[0140] (4) Tumor formation experiment in nude mice was used to verify that MK-2206 and SCH772984 can inhibit the growth of HCC1806 tumors in vivo

[0141] The results are shown in Figure 4 B-E, MK-2206 and SCH772984 alone can inhibit tumor growth in vivo to a certain extent, and the tumor size and weight are significantly lower than the solvent group. The combination of MK-2206 and SCH772984 significantly enhances the inhibition of tumor growth in mice in vivo, and the tumor size and weight are more significantly reduced than the single drug administration group, further proving that MK-2206 and SCH772984 have a synergistic effect.

[0142] (5) The effect of MK-2206 and SCH772984 combined administration on mouse organs

[0143] The results are shown in Figure 5 , combined administration did not cause significant liver and kidney toxicity, and blood cell and plasma analysis data did not change significantly in the single drug administration and combined administration groups, proving that MK-2206 and SCH772984 combined administration did not increase toxicity.

[0144] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A pharmaceutical composition for treating cancer, characterized by, The active ingredient of the pharmaceutical composition consists of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof; The cancer is colon cancer, breast cancer.

2. The pharmaceutical composition of claim 1, wherein, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

3. The pharmaceutical composition of claim 2, wherein, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

4. The pharmaceutical composition of claim 3, wherein, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

5. The pharmaceutical composition of claim 4, wherein, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

6. The pharmaceutical composition of claim 5, wherein, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1:1 or 4:

1.

7. A pharmaceutical preparation, characterized by, The pharmaceutical preparation comprises the pharmaceutical composition of any one of claims 1-6.

8. The pharmaceutical preparation according to claim 7, characterized in that, The dosage form of the pharmaceutical preparation includes an injection administration dosage form, an oral administration dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, a skin administration dosage form.

9. The use of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical composition for treating cancer; The cancer is colon cancer, breast cancer.

10. Use according to claim 9, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

11. Use according to claim 10, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

12. Use according to claim 11, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

13. Use according to claim 12, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is (0.3704-3.3333):(1.1111-10) or (1.1111-10):(1.1111-10) or (0.3704-3.3333):(0.1235-1.1111) or (0.3704-3.3333):(0.0137-0.1235).

14. Use according to claim 13, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 1:1 or 4:

1.

15. Use of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in combination in the preparation of a pharmaceutical preparation for treating cancer. The cancer is colon cancer, breast cancer.

16. The use according to claim 15, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical preparation is (0.0137-30):(0.0137-30).

17. Use according to claim 16, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical preparation is (0.1235-30):(0.1235-30) or (0.0137-3.3333):(0.0137-3.3333).

18. The use according to claim 17, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical preparation is (0.3704-10):(0.1235-10) or (0.3704-3.3333):(0.0137-0.1235).

19. Use according to claim 18, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical preparation is (0.3704-3.3333):(1.1111-10) or (1.1111-10):(1.1111-10) or (0.3704-3.3333):(0.1235-1.1111) or (0.3704-3.3333):(0.0137-0.1235).

20. The use according to claim 19, characterized in that, The concentration ratio of MK-2206 or a pharmaceutically acceptable salt thereof and SCH772984 or a pharmaceutically acceptable salt thereof in the pharmaceutical preparation is 1:1 or 4:

1.

21. Use of the pharmaceutical composition of any one of claims 1-6 or the pharmaceutical preparation of claim 7 or 8 in the preparation of a reagent for inhibiting the growth of cancer cells and / or promoting the apoptosis of cancer cells in vitro. The cancer cells are colon cancer cells, breast cancer cells.

22. The use according to claim 21, characterized in that, The cancer cells are human colon cancer cell line HCT116, triple-negative breast cancer cell line HCC1806.

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