Application of medicine combination of CD47 antibody and sodium-potassium ATPase inhibitor in tumor treatment

Abstract

The invention belongs to the technical field of anti-cancer drugs, discloses an application of a drug combination of a CD47 antibody and a sodium-potassium ATPase inhibitor in tumor treatment, and particularly discloses an application of the sodium-potassium ATPase inhibitor in preparation of a product for promoting phagocytosis of the CD47 antibody. On the basis of early-stage drug screening results, it is found for the first time that sodium-potassium ATP enzyme inhibitor drugs (ouabain and digoxin) significantly improve the phagocytic effect of the CD47 antibody, and other anti-tumor compounds do not have the phagocytic effect in the screening results. According to the invention, the improvement of the phagocytic function of macrophages is dominated by a sodium-potassium ATP enzyme inhibitor such as ouabain and digoxin and a CD47 antibody combined drug, and the subsequent anti-tumor immune response is further activated.

Description

Technical Field

[0001] The present invention belongs to the technical field of anticancer drugs, and specifically relates to an application of a drug combination of a CD47 antibody and a sodium-potassium ATPase inhibitor in tumor treatment. Background Art

[0002] Malignant tumors remain a major public health problem in China. In recent years, tumor immunotherapy has gradually gained an important position in tumor treatment due to its remarkable efficacy in treating some patients. At the same time, more and more immunotherapies have emerged. Among them, immunotherapy targeting macrophages is showing unique advantages. In some solid tumors, tumor-associated macrophages account for the highest proportion of immune cells in the tumor microenvironment. It would be of great significance if these macrophages can be targeted and their phagocytic functions can be used to kill tumors. CD47 is an important immune escape signal on tumor cells, which inhibits macrophage phagocytosis by interacting with the macrophage surface protein SIRPα. Therefore, blocking the CD47-SIRPα interaction with antibodies targeting CD47 may partially restart the macrophage phagocytosis effect. However, tumor cells proliferate rapidly, and inhibiting their growth often requires a stronger macrophage phagocytosis effect. This effect can bring more antigen presentation after engulfing the tumor, activate subsequent T cell immune responses, and cause greater killing effects on the tumor. Therefore, a screening model based on macrophage engulfing tumor cells can be used to find drugs that can increase the phagocytic effect of CD47 antibodies, thereby enhancing their anti-tumor effects. Summary of the invention

[0003] The first aspect of the present invention aims to provide a use of a sodium-potassium ATPase inhibitor in the preparation of a product for promoting the phagocytosis of CD47 antibodies.

[0004] The second aspect of the present invention aims to provide a drug combination.

[0005] The third aspect of the present invention is to provide a preparation.

[0006] The purpose of the fourth aspect of the present invention is to provide the use of the drug combination of the second aspect of the present invention and the preparation of the third aspect of the present invention in the preparation of products for preventing and / or treating tumors and / or inhibiting tumor growth.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides the use of a sodium-potassium ATPase inhibitor in the preparation of a product for promoting the phagocytosis of a CD47 antibody.

[0009] In some embodiments of the present invention, the sodium potassium ATPase inhibitor includes at least one of ouabain, digoxin, bufalin and cinobucini.

[0010] In some preferred embodiments of the present invention, the sodium potassium ATPase inhibitor is ouabain or digoxin.

[0011] Based on the previous drug screening results, the present invention discovered for the first time that sodium-potassium ATPase inhibitor drugs (ouabain, digoxin) significantly enhance the phagocytic effect of CD47 antibodies, while other anti-tumor compounds in the screening results do not have this effect. The present invention is based on the combination of sodium-potassium ATPase inhibitors such as ouabain and digoxin and CD47 antibodies to enhance the phagocytic function of macrophages, which will further activate subsequent anti-tumor immune responses.

[0012] The second aspect of the present invention provides a drug combination comprising a CD47 antibody and a sodium-potassium ATPase inhibitor.

[0013] In some embodiments of the present invention, the sodium potassium ATPase inhibitor includes at least one of ouabain, digoxin, bufalin and cinobucini.

[0014] In some embodiments of the present invention, the pharmaceutical combination comprises a CD47 antibody and ouabain.

[0015] In some embodiments of the present invention, the drug combination comprises CD47 antibody and digoxin.

[0016] In some embodiments of the present invention, the drug combination contains 25-100 nM ouabain.

[0017] In some preferred embodiments of the present invention, the drug combination contains 40-70 nM ouabain.

[0018] In some more preferred embodiments of the present invention, the pharmaceutical combination contains 50 nM ouabain.

[0019] In some embodiments of the present invention, the drug combination contains 100-300 nM digoxin.

[0020] In some preferred embodiments of the present invention, the drug combination contains 100-250 nM digoxin.

[0021] In some more preferred embodiments of the present invention, the drug combination contains 200 nM digoxin.

[0022] The third aspect of the present invention provides a preparation comprising the drug combination of the second aspect of the present invention.

[0023] In some embodiments of the present invention, the preparation further comprises a pharmaceutically acceptable excipient.

[0024] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0025] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose; the binder is selected from cellulose derivatives, alginate, starch, water, dextrin, gelatin or polyvinyl pyrrolidone; the disintegrant is selected from microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose or cross-linked sodium carboxymethyl cellulose; the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, micro-powder silica gel, talc or magnesium stearate; the suspending agent is selected from micro-powder silica gel, beeswax, cellulose, solid polyethylene glycol : The wetting agent is selected from glycerol, Tween-80, hydrogenated castor oil or lecithin; the solvent is selected from ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, propylene glycol or vegetable oil, and the vegetable oil is selected from soybean oil, castor oil, peanut oil, blending oil, etc.; the surfactant is selected from sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate (Tween), etc.; the flavoring agent is selected from aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium; the preservative is selected from at least one of methyl paraben or propyl paraben.

[0026] In some embodiments of the present invention, the dosage form of the preparation is one of drops, oral liquid, tablets, capsules, granules, granules, films, gels, powders, emulsions, pills, suppositories, aerosols, sprays, powder mists, patches, plasters, solutions, ointments or creams.

[0027] The fourth aspect of the present invention provides use of the drug combination of the second aspect of the present invention and the preparation of the third aspect of the present invention in the preparation of a product for preventing and / or treating tumors and / or inhibiting tumor growth.

[0028] In some embodiments of the present invention, the product contains 25-100 nM ouabain.

[0029] In some preferred embodiments of the present invention, the product contains 40-70 nM ouabain.

[0030] In some more preferred embodiments of the present invention, the product contains 50 nM ouabain.

[0031] In some embodiments of the present invention, the product contains 100-300 nM digoxin.

[0032] In some preferred embodiments of the present invention, the product contains 100-250 nM digoxin.

[0033] In some more preferred embodiments of the present invention, the product contains 200 nM digoxin.

[0034] In some embodiments of the present invention, the tumor comprises lung cancer, colorectal cancer, breast cancer, liver cancer, kidney cancer, or melanoma.

[0035] The beneficial effects of the present invention are:

[0036] The present invention discovers for the first time that sodium-potassium ATPase inhibitors can significantly increase the phagocytic rate of CD47 antibodies at non-toxic doses and enhance the anti-tumor efficacy of CD47 antibodies in vivo.

[0037] The present invention obtains a new drug combination through screening, including CD47 antibody combined with sodium-potassium ATPase inhibitor ouabain, or CD47 antibody combined with sodium-potassium ATPase inhibitor digoxin. The drug combination will overcome the shortcomings of the existing CD47 antibody alone, such as low phagocytosis rate and low anti-solid tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Drug screening results obtained for the phagocytosis drug screening model.

[0039] Figure 2 The combined administration of ouabain and CD47 antibody enhances the inhibitory effect of CD47 antibody on tumor; wherein, A is the volume of mouse tumor (H1975) in different treatment groups, B is the weight of mice in different treatment groups (H1975), C is the actual picture of mouse tumor (H1975) in different treatment groups, D is the volume of mouse tumor (A549) in different treatment groups, E is the weight of mice in different treatment groups (A549), F is the actual picture of mouse tumor (A549) in different treatment groups, in the figure, Ctrl: control group, Ouabain: ouabain group, anti-CD47 antibody: CD47 antibody group, Combo: ouabain combined with CD47 antibody group, * represents P<0.05, ** represents P<0.01, *** represents P<0.001. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below through specific examples.

[0041] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1 Extraction and culture of mouse macrophages

[0045] Select 6-8 week old Balb / c mice, use scissors and forceps to remove the leg bones and humerus of the mice, and place them on ice. Then, cut off both ends of the bones, and use serum-free DMEM medium to flush the bone marrow out of the middle section. Then add red blood cell lysis buffer to remove red blood cells. After centrifugation, remove the supernatant, add DMEM complete medium containing 25% L929 cell supernatant to the remaining cells, and transfer them to non-adherent culture dishes for 7 days (replace the medium on the 3rd day) to differentiate the cells into macrophages. After 7 days, plant them in a 24-well plate overnight to adhere to the wall. Before phagocytosis, replace the original complete medium with serum-free DMEM medium and starve for 2 hours.

[0046] Example 2 Drug screening based on macrophage phagocytosis

[0047] The inventors established a drug screening model based on macrophage phagocytosis of tumor cells, and screened a compound library (including drugs that have been reported to have anti-tumor effects, such as ouabain, digoxin, irinotecan, trametinib, etc.). The specific process is as follows: A549 cells stained with CFSE cell tracer, mouse macrophages stained with Cell trace orange, and drugs diluted to a uniform concentration (diluted at 1:1000 according to the given drug concentration mother solution), and 1 μg / mL CD47 antibody (B6H12, #BE0019-1, InVivoMAb) were added to a 96-well plate at the same time. After co-culture for 3 hours, they were placed in a high-content screening instrument for photography and analysis. Cells with overlapping Cell trace Orange and CFSE staining were regarded as having a phagocytic effect between cells. The phagocytic rate was calculated according to the analysis system provided by the instrument. The relative phagocytic rate obtained was the ratio of the phagocytic rate of the drug combined with CD47 antibody group and the CD47 antibody alone group.

[0048] The results showed that only two drugs belonging to the sodium-potassium ATPase inhibitors, ouabain and digoxin, could significantly increase the phagocytic effect of CD47 antibodies (Table 1), while other drugs in the compound library that have anti-tumor effects, such as irinotecan and trametinib, could not increase the phagocytic effect of CD47 antibodies (Table 1 and Figure 1 ), it can be seen that the drug combination formed by the sodium-potassium ATPase inhibitor ouabain or digoxin and the CD47 antibody has a unique property that other anti-tumor drugs do not have, that is, it significantly improves the phagocytic ability of the CD47 antibody.

[0049] Table 1 Statistical table of phagocytic rates of drugs reported to have anti-tumor effects combined with CD47 antibodies

[0050]

[0051] Example 3: Sodium-potassium ATPase inhibitors promote CD47 antibody phagocytosis

[0052] This example uses an in vitro co-culture test to verify the effect of the combined use of ouabain or digoxin and CD47 antibody on tumors, as follows:

[0053] H1975 or A549 were inoculated with 200,000 per well in a 6-well plate overnight. After 12 hours, the original medium was removed and 25, 50 or 100 nM ouabain prepared in 1640 complete medium was added. A549 was inoculated with 200,000 per well in a 6-well plate overnight. After 12 hours, the original medium was removed and 100 or 200 nM digoxin prepared in fresh 1640 complete medium was added. After 24 hours of treatment. After digestion, the cells were stained with CFSE cell tracer and then added to the macrophages isolated in Example 1, and 1 μg / mL CD47 antibody (B6H12, #BE0019-1, InVivoMAb) was added. After co-culture for 3 hours, the cells were digested and added to the flow tube, washed with PBS, and anti-mouse CD16 / 32antibody (clone 93, #101301, Biolegend) was added for blocking. After blocking, APCanti-mouse F4 / 80Antibody (clone BM8, #123115, Biolegend) was added to mark the macrophages. Phagocytosis rate calculation: CFSE + F4 / 80 + Double positive cells accounted for 80% of total F4 + The percentage of positive cells.

[0054] In the in vitro co-culture system, it was found that ouabain at 50 nM significantly increased the phagocytic rate of CD47 antibody (Table 2); digoxin at 200 nM significantly promoted the phagocytic effect of CD47 antibody (Table 3), indicating that sodium-potassium ATPase inhibitors can increase the phagocytic rate of CD47 antibody.

[0055] Table 2 Statistical table of phagocytic rate of ouabain combined with CD47 antibody at different drug ratios

[0056]

[0057] Table 3 Statistical table of phagocytic rate of digoxin combined with CD47 antibody at different drug ratios

[0058]

[0059]

[0060] Example 4 Sodium-potassium ATPase inhibitor promotes the anti-tumor effect of CD47 antibody

[0061] Nude mice (six weeks old) were purchased from the Animal Center of the University of Macau, and the experiment was completed in the SPF-level laboratory of the center. The relevant research will be approved by the center's Animal Ethics Committee. The specific animal experiment plan is as follows: Select mice with basically the same weight, vitality, etc. for the experiment. Take H1975 cells and A549 cells in the logarithmic growth phase, resuspend them in PBS to a suitable concentration, and inject 3 million / mouse (H1975) and 10 million / mouse (A549) under the skin on the right back of nude mice to construct subcutaneous tumors. Measure the tumor (volume = length * width * height / 2) and take the tumor with a relatively uniform volume (≈100mm 3 ) mice were administered, and all animals were weighed before administration, and the tumor volume was measured. According to the tumor size and mouse weight, they were randomly divided into a control group, an ouabain group, a CD47 antibody group, and an ouabain combined with CD47 antibody group, with 8 mice in each group. After grouping, the corresponding dosage of ouabain was 1 mg / kg, and the administration time was once every three days, for a total of six times. The control group was given an equal volume of PBS in the same manner. The dosage of CD47 antibody was 200 μg / mouse, intraperitoneally, and each administration was 24 hours after ouabain administration, for a total of six times. The control group was given an equal mass of isotype control in the same manner. The weight, food and water intake, defecation, fur color, activity and mental state of the experimental animals were regularly tested, and the tumor volume and mouse weight were observed and recorded every other day. The formula for calculating tumor volume is: volume = length * width * height / 2.

[0062] The results showed that the combined administration of ouabain and CD47 antibody significantly enhanced the inhibitory effect of CD47 antibody on tumors. Compared with the group where CD47 antibody was administered alone, the combined administration had a significant anti-tumor effect and had no effect on the body weight of mice ( Figure 2 ).

[0063] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of sodium potassium ATPase inhibitors in the preparation of products that promote the phagocytic effect of CD47 antibodies.

2. The use according to claim 1, characterized in that: The sodium potassium ATPase inhibitor includes at least one of ouabain, digoxin, bufalin and cinobucini.

3. A drug combination comprising a CD47 antibody and a sodium-potassium ATPase inhibitor.

4. The pharmaceutical combination according to claim 3, characterized in that The sodium potassium ATPase inhibitor includes at least one of ouabain, digoxin, bufalin and cinobucini.

5. The pharmaceutical combination according to claim 4, characterized in that The drug combination comprises CD47 antibody and ouabain; or, the drug combination comprises CD47 antibody and digoxin.

6. The pharmaceutical combination according to claim 5, characterized in that The drug combination contains 25-100 nM ouabain; or, the drug combination contains 100-300 nM digoxin.

7. A preparation comprising the drug combination according to any one of claims 3 to 6.

8. The preparation according to claim 7, characterized in that The preparation also includes pharmaceutically acceptable excipients.

9. The preparation according to claim 8, characterized in that The pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

10. Use of the pharmaceutical combination according to any one of claims 3 to 6 or the preparation according to any one of claims 7 to 9 in the preparation of a product for preventing and / or treating tumors and / or inhibiting tumor growth; preferably, the product contains 25 to 100 nM ouabain; and / or, the product contains 100 to 300 nM digoxin.

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