Application of composition containing isovaleric acid and lenvatinib in preparation of anti-cancer drugs

Abstract

The invention discloses application of a composition containing isovaleric acid and lenvatinib in preparation of anti-cancer drugs, and belongs to the technical field of biological medicines. It is found for the first time that after the isovaleric acid and the lenvatinib are combined for use, the isovaleric acid and the lenvatinib have a synergistic effect, the anti-cancer activity of the composition containing the isovaleric acid and the lenvatinib can be remarkably improved, and especially the composition has better inhibitory activity on in-vitro liver cancer cells. Animal experiments further verify that the combination of isovaleric acid and lenvatinib can synergistically inhibit the growth of liver cancer cells in mice. Therefore, the composition containing the isovaleric acid and the lenvatinib has a relatively good application prospect in the prevention and / or treatment of the hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the use of a composition comprising isovaleric acid and lenvatinib in the preparation of anticancer drugs. Background Art

[0002] According to the latest statistics released in 2024, there are more than 830,000 new cases of primary liver cancer worldwide each year, making it the third leading cause of death from cancer. Hepatocellular carcinoma is highly malignant and its targets are unclear. The five-year survival rate of patients with advanced hepatocellular carcinoma is only 16%, which seriously threatens human life safety. Lenvatinib was first approved by the FDA in 2018 as a first-line drug for the treatment of hepatocellular carcinoma, and plays an important role in surgical, local and systemic treatments. However, clinical research data published in the Lancet showed that the efficacy of lenvatinib is still only 24.1%, which seriously limits the clinical application of lenvatinib. There is currently a lack of effective means to improve the clinical efficacy of lenvatinib. Therefore, it is particularly important to reveal the potential mechanism of the low clinical efficacy of lenvatinib and explore potential synergistic intervention strategies.

[0003] The intestinal flora is an important factor affecting the efficacy of anti-tumor drugs. The intestinal flora can receive signals from the external environment and food, convert them into related metabolite signal molecules, and realize signal transduction between the host and the circulatory system. It plays an important role in the human immune, metabolic, nutritional and nervous systems, and is an indispensable component of the body. A large number of studies have confirmed that intestinal flora disorders are closely related to a variety of cancers such as hepatocellular carcinoma, colorectal cancer, lung cancer, pancreatic cancer, etc. It has been confirmed that the intestinal flora can ferment undigested carbohydrates from dietary sources to produce short-chain fatty acids composed of 1 to 6 carbon atoms, mainly composed of acetic acid, propionic acid, butyric acid, and valeric acid, which play an important role in the body's immune balance and physiological and pathological processes.

[0004] Currently, there are no reports on the combined use of isovaleric acid and lenvatinib for anti-hepatocellular carcinoma activity. Summary of the invention

[0005] The object of the present invention is to provide an application of a composition comprising isovaleric acid and lenvatinib in the preparation of an anticancer drug, so as to solve the problem that the existing lenvatinib has low anti-hepatocellular carcinoma activity.

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

[0007] In the present invention, the inventors have found that after isovaleric acid and lenvatinib are used together, the two have a synergistic effect and can significantly enhance 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 / or treatment of cancer.

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

[0009] In the present invention, the inventors further discovered that the combination of isovaleric acid and lenvatinib has better inhibitory activity, especially against hepatocellular carcinoma.

[0010] It is understandable that anticancer drugs can have certain preventive and / or therapeutic effects on conventional cancers in the prior art, and the anticancer drugs in the present invention preferably include anti-hepatocellular carcinoma drugs.

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

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

[0013] In some embodiments, the composition comprising isovaleric acid and lenvatinib inhibits the proliferation of liver cancer cells by activating macrophages, thereby inducing apoptosis of liver cancer cells, and ultimately exerts anti-hepatocellular carcinoma activity.

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

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

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

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

[0018] In the present invention, the term "pharmaceutically acceptable carrier" refers to an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. Pharmaceutical adjuvants can be inert fillers, 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. Pharmaceutical adjuvants can include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

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

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

[0021] The pharmaceutical composition provided by the present invention 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, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (such as liposomes or microspheres). 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 serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection and emulsions for injection. Examples of other suitable formulations of pharmaceutical compositions include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0022] The beneficial effect of the present invention is that, different from the prior art, the present invention first discovered that after isovaleric acid and lenvatinib are used in combination, the two have a synergistic effect, which can significantly enhance the anticancer activity of the composition comprising isovaleric acid and lenvatinib, especially having better inhibitory activity against liver cancer cells in vitro. 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 comprising isovaleric acid and lenvatinib has a good application prospect in the prevention and / or treatment of hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A This is a graph showing the cell proliferation inhibition results after sodium acetate and lenvatinib were used in combination in Example 1 of the present invention; Figure 1B This is a graph showing the cell proliferation inhibition results after sodium propionate and lenvatinib were used in combination in Example 1 of the present invention; Figure 1C This is a graph showing the cell proliferation inhibition results after sodium butyrate and lenvatinib were used in combination in Example 1 of the present invention; Figure 1D This is a graph showing the cell proliferation inhibition results after isobutyric acid and lenvatinib were used in combination in Example 1 of the present invention; Figure 1E This is a graph showing the cell proliferation inhibition results after sodium valerate and lenvatinib were used in combination in Example 1 of the present invention; Figure 1F This is a graph showing the cell proliferation inhibition results after isovaleric acid and lenvatinib were used in combination in Example 1 of the present invention; Figure 2 This is a flow cytometry result diagram of promoting apoptosis of Hepa1-6 cells after the combination of isovaleric acid and lenvatinib in Example 1 of the present invention; Figure 3 This is a graph showing the weight changes of mice in different treatment and administration groups in Example 2 of the present invention; Figure 4 This is a statistical result diagram of the fluorescence intensity of the tumor site of mice in different treatment and drug administration groups in Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0026] In the present invention, the hepatocellular carcinoma Hepa1-6 cell line was purchased from Shanghai Tongpai Biotechnology Co., Ltd.; SPF-grade male C57BL / 6J mice, 6 weeks old, weighing 18-22 g, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (License No.: SCXK (Jing) 2021-0006).

[0027] Example 1 Cell proliferation assay after combination of short-chain fatty acids and lenvatinib This example studies the inhibitory effect of different concentrations of short-chain fatty acids: sodium acetate (NAAc, 5mM, 10mM), sodium propionate (SP, 0.5mM, 1mM), sodium butyrate (SB, 0.5mM, 1mM), isobutyric acid (IBA, 1mM, 2mM), sodium valerate (SV, 1mM, 2mM), isovaleric acid (IVA, 1mM, 2mM) combined with lenvatinib (Len, 10μM) on the proliferation of liver cancer cells Hepa1-6.

[0028] Specifically, Hepa1-6 cells were first cultured and passaged. Hepa1-6 cells frozen in liquid nitrogen were placed in a 37°C constant temperature water bath, shaken to quickly warm and melt, and quickly transferred to a centrifuge tube containing 1 mL of complete culture medium (containing 10% FBS and 90% DMEM high glucose medium), centrifuged at 1000 rpm for 5 min, discarded the supernatant, added 1 mL of complete culture medium to resuspend the cells, transferred to a 100 mm culture dish containing 9 mL of culture medium, shaken well, and placed at 37°C, 5% CO 2 Culture in a constant temperature cell culture incubator; when the healing rate of Hepa1-6 cells is 80-90%, discard the culture medium, wash twice with PBS, add 1 mL of 0.25% trypsin solution (containing EDTA), and digest for 1 min at 37°C; when the cells appear to slide down in a sandy state, quickly add 1 mL of complete culture medium to terminate digestion, and gently blow to collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min to obtain passage cells.

[0029] Then, the Hepa1-6 cells in the logarithmic growth phase were collected after trypsin digestion and inoculated into 96-well plates at a density of 800 cells / well. In the lenvatinib administration group, lenvatinib (10 μM) was administered; in the short-chain fatty acid administration group, different concentrations of short-chain fatty acids were applied to RAW264.7 cells for 24 hours (serum-free medium), and the supernatant was collected and centrifuged after 24 hours. After centrifugation, the supernatant was collected and filtered using a 0.22 μm microporous filter membrane to obtain a conditioned medium, which was stored at -80°C and used at 10% FBS and 90% conditioned medium; in the lenvatinib and short-chain fatty acid combined administration group, the concentration of each drug was set to be a pairwise cross of the above drug concentrations. After the cells were inoculated in a 96-well plate, the cells were incubated at 5% CO 2 After overnight culture at 37°C, the blank group and the control group were replaced with fresh culture medium, the lenvatinib group was replaced with fresh drug-containing culture medium, the short-chain fatty acid group was replaced with conditioned culture medium, and the lenvatinib and short-chain fatty acid combined treatment group was replaced with lenvatinib drug-containing culture medium and conditioned culture medium at the same time; the cells were kept in 5% CO 2 , incubated in an incubator at 37°C for 72 hours. Then, 20 μL of 5 mg / mL MTT solution was added to each well and the well was kept in a 5% CO 2 , incubate in an incubator at 37°C for 4 hours, then aspirate the solution in the wells, re-dissolve with 150 mL DMSO, and place on a microplate oscillator at 300 rpm for 10 minutes. Measure the absorbance of each well at a wavelength of 570 nm. Cell growth inhibition rate (%) = [1-(OD treatment group-OD blank group) / (OD control group-OD blank group)] × 100%.

[0030] The results of cell proliferation inhibition after the combination of short-chain fatty acids and lenvatinib were as follows Figures 1A-1F shown.

[0031] from Figures 1A-1F It can be seen that compared with the administration of lenvatinib, the combined administration of sodium acetate, sodium propionate, sodium butyrate, isobutyric acid, sodium valerate and isovaleric acid with lenvatinib can significantly inhibit the proliferation of liver cancer cells Hepa1-6, and the inhibitory effect of isovaleric acid combined with lenvatinib is better.

[0032] Furthermore, in order to study whether sodium acetate, sodium propionate, sodium butyrate, isobutyric acid, sodium valerate, and isovaleric acid are combined with lenvatinib to have a synergistic effect. The Q value method recognized in the field was adopted by Jin Zhengjun (Jin Zhengjun's Additive in Combined Use of Drugs [J]. Acta Pharmacologica Sinica, 1980), that is, Q=MAB / (MA+Mp-MA×Mg). In this formula, the numerator represents the "measured combined effect" and the denominator is the "expected combined effect", where 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 the Q value is less than 0.85, the two drugs are considered to have an antagonistic effect; when the Q value is between 0.85 and 1.15, the two drugs are considered to be independent of each other and have an additive effect; when the Q value is greater than 1.15, the two drugs are considered to have a synergistic effect.

[0033] Based on the above-measured cell proliferation inhibition results after the combination of short-chain fatty acids and lenvatinib, the Jin Zhengjun Q value after the combination of short-chain fatty acids and lenvatinib was calculated, and the results are shown in Table 1 below.

[0034] Table 1 Results of Jin Zhengjun Q value after the combination of short-chain fatty acids and lenvatinib

[0035] As can be seen from Table 1, isobutyric acid (IBA) and isovaleric acid (IVA) have a significant synergistic effect on the inhibitory activity of Hepa1-6 cell proliferation when used in combination with lenvatinib (Len), and the synergistic inhibitory effect of isovaleric acid and lenvatinib is stronger (Q>1.15).

[0036] Furthermore, the cell culture medium after 72 hours of the above-mentioned drug treatment (control group, lenvatinib drug group (10μM), isovaleric acid drug group (2mM), isovaleric acid (2mM) and lenvatinib (10μM) combined drug group) was collected in a 2mL EP tube and centrifuged at 1000rpm for 5min, and the supernatant was discarded. Then 500μL of trypsin solution was added to the 6-well plate. 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-mentioned 2mL EP tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded. Rinse twice with 500μL pre-cooled PBS by centrifugation, and the supernatant was discarded. 500μL 1× BingingBuffer was added to each tube to resuspend the cells, and 5μL Annexin V-FITC and 10μL PI were added to each tube respectively. After mixing, incubate at room temperature in the dark for 5min, and then perform flow cytometry analysis. The results are as follows. Figure 2 shown.

[0037] from Figure 2It can be seen that after the combined administration of isovaleric acid (IVA) and lenvatinib (Len), the apoptosis of Hepa1-6 cells can be significantly promoted.

[0038] The above results indicate that after the combined administration of isovaleric acid (IVA) and lenvatinib (Len), by activating macrophages, the proliferation of liver cancer cells is inhibited, and then the apoptosis of liver cancer cells is induced, ultimately exerting the activity against hepatocellular carcinoma.

[0039] Example 2 Inhibitory test of the combination of isovaleric acid and lenvatinib on liver cancer cells in mice This example studied the inhibitory effect of the combination of isovaleric acid and lenvatinib on liver cancer cells in mice.

[0040] Specifically, first, animal quarantine and routine feeding were carried out, which included: The animal experiment was designed and implemented in accordance with the standard operating procedures of the Experimental Animal Management Committee of Jianghan University (Animal Use License No.: SYXK(E)2021-0042). C57BL / 6J mice were housed in a SPF-class animal room, the environmental temperature of the animal room was 24±2°C, with a 12h / 12h day-night cycle, the animals had free access to water and food, and the bedding was wood chip bedding.

[0041] Then, a model of orthotopic transplantation tumor of Luci-Hepa1-6 mouse liver cancer was established: The hair of the mouse between the chest and abdomen was removed using a hair clipper. The Hepa1-6 cells with fluorescent labels (transferring Hepa1-6 cells into the firefly luciferase gene - LV16-NC, so that bioluminescence is generated when the firefly luciferin substrate is injected into the animal body for detecting the development of tumors) were washed with PBS and then digested with trypsin solution (containing EDTA), the supernatant was discarded after centrifugation, the cells were washed with PBS 2 times, the inoculation amount was calculated, and finally the cells were resuspended with PBS and placed on ice for standby. The hair-removed mouse was anesthetized, and after anesthesia, the mouse was fixed. The cell suspension was gently flicked evenly by hand before use, and 40 μL / dish of the cell suspension was drawn into a syringe for standby. The outer skin and peritoneum of the mouse were gently cut open with scissors to expose the liver of the mouse, then a needle was inserted at a 30° angle to the epidermis at a position 1 cm away from the liver, the cell solution was injected, after all the cell solution was slowly pushed into the liver of the mouse, the needle position was fixed for 30 s and then the needle was slowly removed and hemostasis was performed, the outer skin of the mouse was sutured with a suture, and then the mouse was placed in an incubator to wait for recovery.

[0042] Then, the 32 treated mice were randomly divided into a model group (Model), a lenvatinib group (Len), an isovaleric acid group (IVA), and an isovaleric acid combined with lenvatinib group (IVA+Len); Len administration method: 30 mg / kg by intragastric administration, once a day; isovaleric acid administration method: 100 mg / kg by intragastric administration, once a day. In the combined administration group, lenvatinib and isovaleric acid were administered simultaneously; the treatment was continued for 14 days, and the weight of the mice in each treatment group was recorded. On the 14th day, the fluorescence intensity of the tumor site of the mice in each treatment group was detected, and the results were as follows: Figure 3 and 4 shown.

[0043] from Figure 3 and 4 It can be seen that compared with the mice in the isovaleric acid (IVA) administration group and the lenvatinib (Len) single administration group, the combined administration did not significantly affect the weight of the mice. At the same time, it could significantly inhibit the growth of hepatocellular carcinoma in mice (low fluorescence intensity). The above results indicate that isovaleric acid and lenvatinib significantly inhibit the growth of hepatocellular carcinoma through synergistic effects.

[0044] In summary, the present invention discovered for the first time that the combination of isovaleric acid and lenvatinib has a synergistic effect and has better inhibitory activity against liver cancer cells in vitro; and the combination of isovaleric acid and lenvatinib can synergistically inhibit the growth of liver cancer cells in mice.

[0045] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0046] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a composition comprising isovaleric acid and lenvatinib in the preparation of anticancer drugs.

2. The use according to claim 1, characterized in that: The anticancer drugs include anti-hepatocellular carcinoma drugs.

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

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

1.

5. The use according to claim 1, characterized in that: The composition comprising isovaleric acid and lenvatinib inhibits the proliferation of liver cancer cells by activating macrophages, thereby inducing apoptosis of liver cancer cells, and ultimately exerts anti-hepatocellular carcinoma activity.

6. A pharmaceutical composition for preventing and / or treating hepatocellular carcinoma, characterized in that: A composition comprising isovaleric acid and lenvatinib.

7. The pharmaceutical composition according to claim 6, characterized in that The molar ratio of the isovaleric acid to the lenvatinib is (200:1)-(10:1).

8. The pharmaceutical composition according to claim 6, characterized in that The molar ratio of the isovaleric acid to the lenvatinib is any one of 200:1, 100:1, and 14:

1.

9. The pharmaceutical composition according to claim 6, characterized in that Also included are pharmaceutically acceptable carriers.

10. The pharmaceutical composition according to claim 6, characterized in that The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

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