Use of a composition comprising isovaleric acid and lenvatinib for the manufacture of an anticancer drug

Abstract

The application discloses application of a composition containing isovaleric acid and lenvatinib in preparation of anticancer drugs and belongs to the technical field of biological medicines.It is found for the first time that isovaleric acid and lenvatinib have a synergistic effect after being combined, and the anticancer activity of the composition containing isovaleric acid and lenvatinib can be significantly improved, especially the inhibitory activity on liver cancer cells in vitro is better.Then, through animal experiments, it is further verified that the combination of isovaleric acid and lenvatinib can synergistically inhibit the growth of liver cancer cells in mice.Therefore, the composition containing isovaleric acid and lenvatinib has a good application prospect in the prevention and treatment of hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a composition containing isovaleric acid and lenvatinib in preparation of an anticancer drug. BACKGROUND

[0002] According to the latest statistical data released in 2024, more than 830,000 new cases of primary liver cancer are added worldwide each year, ranking third in tumor mortality. Hepatocellular carcinoma has high malignancy and unclear target, and the five-year survival rate of patients with advanced hepatocellular carcinoma is only 16%, which seriously threatens human life safety. Lenvatinib is the first-line drug approved by FDA in 2018 for the treatment of hepatocellular carcinoma, and plays an important role in surgery, local and systemic treatment. However, the clinical research data published in Lancet shows that the effective rate of lenvatinib is still only 24.1%, which seriously limits the clinical application of lenvatinib. At present, there is still a lack of effective means to improve the clinical effective rate of lenvatinib. Therefore, it is particularly important to reveal the potential mechanism of low clinical effective rate of lenvatinib and explore the potential synergistic intervention strategy.

[0003] Intestinal flora is an important influencing factor of the efficacy of antitumor drugs. Intestinal flora can receive signals from the external environment and food, convert them into related metabolic product signal molecules, and realize signal transduction with the host through the circulatory system, playing an important role in human immunity, metabolism, nutrition and nervous system, and being an indispensable important component of the body. A large number of studies have confirmed that intestinal flora disorder is closely related to many cancers such as hepatocellular carcinoma, colorectal cancer, lung cancer and pancreatic cancer. It has been confirmed that intestinal flora can ferment undigested and absorbed carbohydrates of dietary origin into short-chain fatty acids composed of 1-6 carbon atoms, mainly composed of acetic acid, propionic acid, butyric acid and valeric acid, which play an important role in immune balance and physiological and pathological processes of the body.

[0004] At present, there is no research report on the combination of isovaleric acid and lenvatinib for anti-hepatocellular carcinoma activity. SUMMARY

[0005] The purpose of the present application is to provide the application of the composition containing isovaleric acid and lenvatinib in preparation of an anticancer drug. To solve the problem of low anti-hepatocellular carcinoma activity of the existing lenvatinib.

[0006] In a first aspect, the present application provides the application of the composition containing isovaleric acid and lenvatinib in preparation of an anticancer drug.

[0007] In the present application, the inventors have found that the combination of isovaleric acid and lenvatinib has a synergistic effect, which can significantly improve the anticancer activity of the composition comprising isovaleric acid and lenvatinib. Therefore, the composition comprising isovaleric acid and lenvatinib has a good application prospect in the prevention and treatment of cancer.

[0008] In some embodiments, the anticancer drug comprises an anti-hepatocellular carcinoma drug.

[0009] In the present application, the inventors have further found that the combination of isovaleric acid and lenvatinib has a better inhibitory activity, especially for hepatocellular carcinoma.

[0010] It can be understood that the anticancer drug can have a certain preventive and therapeutic effect on the conventional cancer in the prior art, and the anticancer drug in the present application preferably comprises an anti-hepatocellular carcinoma drug.

[0011] In some embodiments, the molar ratio of isovaleric acid to lenvatinib is (200:1)-(10:1), which can be 200:1, 150:1, 100:1, 50:1, 14:1, 10:1 or other values within the range.

[0012] In some embodiments, the molar ratio of isovaleric acid to lenvatinib is any one of 200:1, 100:1, 14:1.

[0013] In some embodiments, the composition comprising isovaleric acid and lenvatinib activates macrophages, thereby inhibiting the proliferation of hepatocarcinoma cells, inducing apoptosis of hepatocarcinoma cells, and ultimately exerting anti-hepatocellular carcinoma activity.

[0014] In a second aspect, the present application provides a pharmaceutical composition for preventing and / or treating hepatocellular carcinoma, comprising a composition of isovaleric acid and lenvatinib.

[0015] In some embodiments, the molar ratio of isovaleric acid to lenvatinib is (200:1)-(10:1), which can be 200:1, 150:1, 100:1, 50:1, 14:1, 10:1 or other values within the range.

[0016] In some embodiments, the molar ratio of isovaleric acid to lenvatinib is any one of 200:1, 100:1, 14:1.

[0017] In some embodiments, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0018] In the present application, the term "pharmaceutically acceptable carrier" refers to an excipient that is widely employed in the pharmaceutical production field. The excipient is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method for allowing the active ingredient to be dissolved at a desired rate after the subject receives the administration, or to facilitate the active ingredient to be effectively absorbed after the subject receives the administration of the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: a binder, a suspending agent, an emulsifying agent, a diluent, a filler, a granulating agent, a gum, a disintegrating agent, a lubricant, an anti-adhesion agent, a flow aid, a wetting agent, a gelling agent, an absorption delaying agent, a dissolution inhibitor, an enhancer, an adsorbent, a buffer, a chelating agent, a preservative, a coloring agent, a flavoring agent, and a sweetening agent.

[0019] The pharmaceutical composition provided by the present application can be prepared according to the disclosed content using any method known to those skilled in the art. For example, including but not limited to conventional mixing, dissolving, granulating, emulsifying, micronizing, encapsulating, entrapping, or lyophilizing processes.

[0020] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semi-solid preparation, a liquid preparation.

[0021] The pharmaceutical composition provided by the present application can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present application can also be a controlled or sustained release dosage form (such as a liposome or a microsphere). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serums. Examples of preparations for parenteral administration include, but are not limited to, injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions, and injection emulsions. Examples of other suitable preparations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0022] The beneficial effects of the present application are: different from the prior art, the present application first discovers that after the combination of isovaleric acid and lenvatinib, the two have a synergistic effect, which can significantly improve the anti-cancer activity of the composition containing isovaleric acid and lenvatinib, especially better inhibitory activity on in vitro hepatocellular carcinoma cells. Then through animal experiments, it is further verified that the combination of isovaleric acid and lenvatinib can synergistically inhibit the growth of hepatocellular carcinoma cells in mice in vivo. Therefore, the composition containing isovaleric acid and lenvatinib has a good application prospect in the prevention and treatment of hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1A is the cell proliferation inhibition result graph of sodium acetate combined with lenvatinib in the embodiment 1 of the present application;

[0024] Figure 1B is the cell proliferation inhibition result graph of sodium propionate combined with lenvatinib in the embodiment 1 of the present application;

[0025] Figure 1C is the cell proliferation inhibition result graph of sodium butyrate combined with lenvatinib in the embodiment 1 of the present application;

[0026] Figure 1D is the cell proliferation inhibition result graph of isobutyric acid combined with lenvatinib in the embodiment 1 of the present application;

[0027] Figure 1E is the cell proliferation inhibition result graph of sodium valerate combined with lenvatinib in the embodiment 1 of the present application;

[0028] Figure 1F is the cell proliferation inhibition result graph of isovaleric acid combined with lenvatinib in the embodiment 1 of the present application;

[0029] Figure 2 is the flow cytometry detection result graph of promoting Hepa1-6 cell apoptosis after isovaleric acid combined with lenvatinib in the embodiment 1 of the present application;

[0030] Figure 3 is the weight change result graph of mice in different treatment and administration groups in the embodiment 2 of the present application;

[0031] Figure 4 is the fluorescence intensity statistical result graph of tumor site of mice in different treatment and administration groups in the embodiment 2 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0033] The experimental methods not specified in the embodiments are generally carried out according to the conventional conditions and the conditions described in the manuals, or according to the conditions suggested by the manufacturers. The general equipment, materials, reagents, etc. used are commercially available, unless otherwise specified.

[0034] In the present application, the hepatocellular carcinoma Hepa1-6 cell line is purchased from Shanghai Tongpai Biotechnology Co., Ltd.; SPF male C57BL / 6J mice, 6 weeks old, weighing 18-22 g, are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK (Jing) 2021-0006).

[0035] Example 1 Cell proliferation test after combination of short-chain fatty acids and lenvatinib

[0036] This example studies the inhibitory effect of different concentrations of short-chain fatty acids: sodium acetate (NAAc, 5 mM, 10 mM), sodium propionate (SP, 0.5 mM, 1 mM), sodium butyrate (SB, 0.5 mM, 1 mM), isobutyric acid (IBA, 1 mM, 2 mM), sodium valerate (SV, 1 mM, 2 mM), and isovaleric acid (IVA, 1 mM, 2 mM) combined with lenvatinib (Len, 10 μM) on the proliferation of hepatocellular carcinoma cells Hepa1-6.

[0037] Specifically, first, Hepa1-6 cell culture and subculture are carried out. The Hepa1-6 cells frozen in liquid nitrogen are taken to a 37℃ constant temperature water bath, shaken to quickly warm up and melt, quickly transferred to a centrifuge tube containing 1 mL complete culture medium (containing 10% FBS and 90% DMEM high-sugar culture medium), centrifuged at 1000 rpm for 5 min, the supernatant is discarded, 1 mL complete culture medium is added to resuspend the cells, and the cells are transferred to a 100 mm culture dish containing 9 mL of culture medium, shaken and then placed in a 37℃, 5% CO2 constant temperature cell incubator for culture; when the confluence rate of Hepa1-6 cells is 80-90%, the culture medium is discarded, washed twice with PBS, 1 mL of 0.25% trypsin solution (containing EDTA) is added, and the cells are digested at 37℃ for 1 min; when the cells show sand-like slippage, 1 mL of complete culture medium is quickly added to terminate the digestion, and the cells are gently blown and collected into a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, and the subculture cells are obtained.

[0038] Then, Hepa1-6 cells in the logarithmic growth phase were collected, digested with trypsin, and seeded in 96-well plates at a density of 800 cells / well. In the lenvatinib group, lenvatinib (10 μM) was administered. In the short-chain fatty acid group, RAW264.7 cells were treated with different concentrations of short-chain fatty acids for 24 h (serum-free medium). After 24 h, the supernatant was collected and centrifuged. The supernatant was then filtered through a 0.22 μm microporous membrane to obtain conditioned medium, which was stored at -80°C and used with 10% FBS and 90% conditioned medium. In the lenvatinib and short-chain fatty acid combination group, the drug concentrations were set as pairwise crossovers of the drug concentrations mentioned above. After cell culture in 96-well plates at 5% CO2 and 37°C overnight, the culture medium was replaced with fresh medium for the blank and control groups, fresh drug-containing medium for the lenvatinib group, conditioned medium for the short-chain fatty acid group, and both the lenvatinib-containing medium and conditioned medium for the lenvatinib and short-chain fatty acid combined group. The cells were then incubated at 5% CO2 and 37°C for 72 hours. Afterward, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 5% CO2 and 37°C for another 4 hours. The solution was then aspirated from the wells, reconstituted with 150 mL of DMSO, and shaken at 300 rpm for 10 min on a microplate shaker. The absorbance of each well was measured at 570 nm. Cell growth inhibition rate (%) = [1 - (OD-treated group - OD blank group) / (OD control group - OD blank group)] × 100%.

[0039] The cell proliferation inhibition results obtained by combining short-chain fatty acids and lenvatinib are as follows: Figures 1A-1F As shown.

[0040] from Figures 1A-1F As can be seen, compared with lenvatinib administration, the combined administration of sodium acetate, sodium propionate, sodium butyrate, isobutyric acid, sodium valerate, and isovaleric acid with lenvatinib significantly inhibited the proliferation of Hepa1-6 liver cancer cells, and the inhibitory effect was better when isovaleric acid was combined with lenvatinib.

[0041] Further, in order to study whether sodium acetate, sodium propionate, sodium butyrate, isobutyric acid, sodium valerate, isovaleric acid has synergistic effect when combined with lenvatinib. The gold Q value method (Kim, J. Synergism of drugs combined. Acta Pharmacol Sin 1980) was used, which is Q = MAB / (MA+Mp-MAxMg). The numerator represents the "measured combined effect", and the denominator is the "expected combined effect", wherein MA, Mp and MAB represent the inhibition rate of drug A, the inhibition rate of drug B and the inhibition rate of the combination of the two drugs at the current dose, respectively. The drug synergy index Q is defined as follows: when Q value is less than 0.85, it is considered that the two drugs have antagonistic effect; when Q value is between 0.85 and 1.15, it is considered that the two drugs are independent of each other, which is additive effect; when Q value is greater than 1.15, it is considered that the two drugs have synergistic effect.

[0042] According to the above measured short-chain fatty acid and lenvatinib after cell proliferation inhibition results, the gold Q value of short-chain fatty acid and lenvatinib after combination was calculated, and the results are shown in Table 1.

[0043] Table 1 Gold Q value of short-chain fatty acid and lenvatinib after combination

[0044]

[0045] As can be seen from Table 1, isobutyric acid (IBA) and isovaleric acid (IVA) have obvious synergistic effect on the inhibition of Hepa1-6 cell proliferation after combined with lenvatinib (Len), and the synergistic inhibition effect of isovaleric acid and lenvatinib is stronger (Q>1.15).

[0046] Further, the cell culture medium after 72h of the above administration treatment (control group, lenvatinib administration group (10μM), isovaleric acid administration group (2mM), isovaleric acid (2mM) and lenvatinib (10μM) combined administration group) was collected in a 2mL EP tube, and centrifuged at 1000rpm for 5min, and the supernatant was discarded. Then 500μL trypsin solution was added to the 6-well plate, and after digestion at room temperature for 2min, an equal amount of DMEM medium was added to terminate the digestion, and then the cell suspension was added to the above 2mL EP tube, and centrifuged at 1000rpm for 5min, and the supernatant was discarded. Then 500μL pre-cooled PBS was used for centrifugal rinsing twice, and the supernatant was discarded. 500μL 1xBingingBuffer was added to resuspend the cells, 5μL Annexin V-FITC and 10μL PI were added to each tube, mixed well, and incubated at room temperature for 5min in the dark, and then flow cytometry detection was performed, and the results are shown in Figure 2

[0047] From Figure 2 ​As can be seen, after administration of isovaleric acid (IVA) and lenvatinib (Len) in combination, the apoptosis of Hepa1-6 cells can be significantly promoted.

[0048] The above results show that, after administration of isovaleric acid (IVA) and lenvatinib (Len) in combination, the proliferation of liver cancer cells is inhibited by activating macrophages, thereby inducing the apoptosis of liver cancer cells, and finally exerting the activity of anti-hepatocellular carcinoma.

[0049] Example 2 Inhibition test of liver cancer cells in mice after isovaleric acid and lenvatinib are used in combination

[0050] This example studies the inhibition of liver cancer cells in mice after isovaleric acid and lenvatinib are used in combination.

[0051] Specifically, first, animal quarantine and routine feeding are performed, which includes: the animal experiment is designed and implemented in accordance with the standard operating procedures of Jianghan University Experimental Animal Management Committee (animal use license number: SYXK (E) 2021-0042). C57BL / 6J mice are raised in a SPF level animal room, the animal room environment temperature is 24±2℃, 12h / 12h day-night cycle, the animals are free to drink water and eat, and the litter is wood shavings litter.

[0052] Then, Luci-Hepa1-6 mouse liver cancer orthotopic tumor modeling is performed: the hair between the chest and abdomen of the mouse is removed using a hair clipper. The Hepa1-6 cells with fluorescent label (Hepa1-6 cells are transferred into firefly luciferase gene-LV16-NC, so that they can produce bioluminescence when the firefly luciferin substrate is injected into the animal body, which is used to detect tumor development) are washed with PBS, then digested with trypsin solution (containing EDTA), centrifuged, and the supernatant is discarded. The cells are washed with PBS twice, the inoculation amount is calculated, and finally the cells are resuspended with PBS and placed on ice for standby. The hair-removed mouse is anesthetized, and after anesthesia, the mouse is fixed. The cell suspension is evenly popped with hands before use, 40μL / dish of cell suspension is extracted with a syringe for standby, the mouse skin and peritoneum are gently cut open with scissors, the mouse liver is exposed, then the needle is inserted at a 30° angle to the epidermis at a distance of 1cm from the liver, the cell solution is injected, the needle position is fixed for 30s, then the needle is slowly removed and hemostasis is performed, the mouse skin is sutured with suture thread, and then the mouse is placed in an incubator to wait for recovery.

[0053] Next, the above-mentioned 32 mice treated were randomly divided into a model group (Model), a lenvatinib administration group (Len), an isovalerate administration group (IVA), an isovalerate combined with lenvatinib administration group (IVA+Len); the administration mode of Len: 30mg / kg by gavage, once a day; the administration mode of isovalerate: 100mg / kg by gavage, once a day, in the combined administration group, lenvatinib and isovalerate were administered at the same time; continuous treatment for 14 days, the body weight of mice in each treatment group was recorded, on the 14th day, the fluorescence intensity of the tumor site of mice in each treatment group was detected, and the results are shown in Figure 3 and 4 .

[0054] From Figure 3 and 4 , compared with mice in the isovalerate (IVA) administration group and the lenvatinib (Len) single administration group, after combined administration, it does not significantly affect the body weight of mice, at the same time, it can significantly inhibit the growth of hepatocellular carcinoma in mice (low fluorescence intensity), the above results show that isovalerate and lenvatinib can significantly inhibit the growth of hepatocellular carcinoma by synergistic effect.

[0055] In summary, the present application first found that after isovalerate and lenvatinib were combined, the two had synergistic effect, and had better inhibitory activity on liver cancer cells in vitro; and the combined administration of isovalerate and lenvatinib can synergistically inhibit the growth of hepatocellular carcinoma in mice.

[0056] It should be noted that each of the above embodiments belongs to the same inventive concept, and the description of each embodiment has its own emphasis, and the description of individual embodiments is not exhaustive. Reference can be made to the description in other embodiments.

[0057] The above-described embodiments only express the implementation of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the inventive concept, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of a composition comprising isovaleric acid and lenvatinib in the manufacture of an anticancer drug, characterized in that, The anticancer drug is an anti-hepatocellular carcinoma drug.

2. Use according to claim 1, characterized in that, The molar ratio of the isovaleric acid to the lenvatinib is any one of (200:1) - (10:1).

3. Use according to claim 2, characterized in that, The molar ratio of the isovaleric acid to the lenvatinib is any one of 200:1, 100:1, 14:

1.

4. Use according to claim 1, characterized in that, The composition comprising the isovaleric acid and the lenvatinib exerts an anti-hepatocellular carcinoma activity by activating macrophages, thereby inhibiting proliferation of hepatoma cells, and further inducing apoptosis of the hepatoma cells.

5. A pharmaceutical composition for preventing and / or treating hepatocellular carcinoma, characterized by, A composition comprising isovaleric acid and lenvatinib.

6. The pharmaceutical composition of claim 5, wherein, The molar ratio of the isovaleric acid to the lenvatinib is any one of (200:1) - (10:1).

7. The pharmaceutical composition of claim 6, wherein, The molar ratio of the isovaleric acid to the lenvatinib is any one of 200:1, 100:1, 14:

1.

8. The pharmaceutical composition of claim 5, wherein, Also included is a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 5, wherein, The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semi-solid preparation, and a liquid preparation.

Images