Application of combined drugs in the preparation of drugs for the prevention and treatment of liver cancer

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the application of a combined drug in the preparation of a drug for preventing and treating liver cancer. Experimental results show that the combined use of Torin1 and JQ1 at a molar ratio of 0.1-0.5:3-30 can significantly improve the therapeutic effect of liver cancer. At the same time, JQ1 can reduce the drug resistance of liver cancer cells to Torin1, increase the drug sensitivity of Torin1, and improve the quality of life of patients, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of a combined drug in the preparation of a drug for preventing and treating liver cancer. Background Art

[0002] Primary liver cancer, including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), causes approximately 700,000 deaths each year. Hepatocellular carcinoma is the most common pathological type of liver cancer, accounting for more than 90%. It is characterized by high invasiveness, high metastasis and recurrence rates, and atypical early clinical manifestations. Most hepatocellular carcinoma patients are already in the advanced stage when diagnosed, missing the best time for treatment. Due to tumor recurrence and metastasis, the 5-year survival rate of patients is still only 7%. Currently, the treatment for patients with advanced liver cancer is mainly targeted therapy, but the clinical drugs used, such as sorafenib and lenvatinib, are prone to drug resistance, and most patients cannot achieve the expected treatment effect. There is a lack of treatment options for patients with advanced liver cancer, and exploring new and feasible treatment options is an urgent problem to be solved.

[0003] The chemical name of JQ1 is (S)-(+)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-F][1,2,4]triazolo[4,3-A][1,4]diazepine-6-yl)acetate, which can inhibit the growth of multiple myeloma, brain glioma, ovarian cancer, breast cancer, glioblastoma, midline cancer and other tumors. Its structural formula is shown below:

[0004] .

[0005] JQ1 has certain potential in the treatment of liver cancer, but its effect when used alone is limited. Drug combination is an important treatment strategy in modern medicine. By rationally combining multiple drugs, it can enhance the therapeutic effect, reduce side effects, delay the development of drug resistance, and provide more comprehensive treatment options for complex diseases. However, there are still few drug combination programs for liver cancer. Exploring more drug combination programs for liver cancer treatment plays an important role in tumor treatment. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art and provide an application of a combined drug in the preparation of a drug for preventing and treating liver cancer.

[0007] The second object of the present invention is to provide the use of JQ1 in preparing a drug for improving the drug sensitivity of Torin1.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides use of a combined drug in preparing a drug for preventing and treating liver cancer. The active ingredients of the combined drug include Torin1 and JQ1, and the molar ratio thereof is 0.1-0.5:3-30.

[0010] The inventors found through research that the combined use of JQ1 and Torin1 can increase the drug sensitivity of Torin1, significantly inhibit the growth and proliferation of liver cancer cells, and the effect is better than using JQ1 or Torin1 alone. The combined drug experiment confirmed that the synergy index of JQ1 and Torin1 for lung cancer is higher than 10. Therefore, the combined use of Torin1 and JQ1 for lung cancer has a synergistic effect.

[0011] Specifically, the chemical name of Torin 1 is 1-[4-[4-(1-oxopropyl)-1-piperazinyl]-3-(trifluoromethyl)phenyl]-9-(3-quinolyl)benzo[H]-1,6-naphthyridin-2(1H)-one, and its structural formula is shown below:

[0012] .

[0013] Preferably, the molar ratio of Torin1 to JQ1 is 0.1-0.5:10-30. Most preferably, the molar ratio of Torin1 to JQ1 is 0.5:10. The combined use within this molar ratio range has a more obvious inhibitory effect on the growth and proliferation of liver cancer cells, that is, the combined use within this molar ratio range has a better effect.

[0014] Preferably, the combination drug contains a Torin1 liquid preparation with Torin1 as the active ingredient and a JQ1 liquid preparation with JQ1 as the active ingredient; the concentration ratio of Torin1 to JQ1 in the combination drug is 0.1-0.5 μM: 3-30 μM. Further preferably, the concentration ratio of Torin1 to JQ1 in the combination drug is 0.1-0.5 μM: 10-30 μM.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0016] Preferably, the pharmaceutically acceptable excipient is at least one of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrator, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickener, a inclusion agent, a humectant, an absorbent, a diluent, a flocculant, a deflocculating agent, a filter aid or a release retardant.

[0017] Preferably, the dosage form of the drug is powder, tablet, granule, capsule, solution, emulsion, suspension, aerosol, ointment, suppository, lotion or injection.

[0018] Preferably, the prevention and treatment of liver cancer includes increasing the sensitivity of liver cancer cells to Torin1.

[0019] Preferably, the prevention and treatment of liver cancer includes inhibiting the growth and / or proliferation of liver cancer cells.

[0020] Furthermore, the present invention also provides the use of JQ1 in preparing a drug for improving the drug sensitivity of Torin1.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides the use of Torin1 and JQ1 in combination for preparing drugs for preventing and treating liver cancer. Experimental results show that the combination of Torin1 and JQ1 at a molar ratio of 0.1-0.5:3-30 has a synergistic effect, and the synergistic index of the combination is higher than 10, which can significantly improve the therapeutic effect of liver cancer and inhibit the growth and / or reproduction of liver cancer cells. The use of JQ1 can significantly reduce the drug resistance of liver cancer cells to Torin1, increase the drug sensitivity of Torin1, and improve the quality of life of patients, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The heat maps and 3D graphs corresponding to the three synergistic models (ZIP, HSA, and Bliss) of Torin1 and JQ1 combined therapy for liver cancer.

[0024] Figure 2 The drug dose-response curves and drug dose-response matrix diagrams are for Torin1 and JQ1 alone and in combination.

[0025] Figure 3 The heat map and 3D graph corresponding to the synergistic model (ZIP) of Torin1 and JQ1 combined therapy for colorectal cancer.

[0026] Figure 4 This is a growth curve of the effect of Torin1 and JQ1 combined therapy on the proliferation of liver cancer cells. Figure 4 A in the figure is the growth curve of Huh7 liver cancer cell proliferation. Figure 4 B in the figure is a growth curve of SUN423 liver cancer cell proliferation.

[0027] Figure 5 This is a picture of the staining of liver cancer cell clone formation by the combination of Torin1 and JQ1. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0029] Example 1 Torin1 and JQ1 have a synergistic effect in combination in liver cancer cells

[0030] (1) Preparation of JQ1-containing culture medium: JQ1 was first dissolved in DMSO, and then DMEM complete culture medium (containing 10% serum) was added to prepare JQ1-containing culture medium with final JQ1 concentrations of 30 μM, 10 μM, 3 μM, and 0 μM. No JQ1 was added to the JQ1-containing culture medium with a final concentration of 0 μM, but the amount of DMSO added was consistent with that of the JQ1-containing culture medium with other concentrations.

[0031] Preparation of Torin1-containing culture medium: Torin1 was first dissolved in DMSO, and then added to the culture medium to prepare Torin1-containing culture medium with a final concentration of 500 nM, 300 nM, 100 nM, 30 nM, and 0 nM. Torin1 was not added to the Torin1-containing culture medium with a final concentration of 0 μM, but the amount of DMSO added was consistent with that of Torin1-containing culture medium with other concentrations.

[0032] Preparation of JQ1+ Torin1 drug-containing culture medium: Torin1 and JQ1 were first dissolved in DMSO, and then the aforementioned different JQ1 concentrations were combined with different Torin1 concentrations in sequence, for a total of 20 drug concentration combinations.

[0033] Experimental method: Digest Huh7 liver cancer cells to make cell suspension, add 100 μL of cell suspension to each well of 96-well plate, place in 37℃, 5% CO2 cell culture incubator to continue culturing to allow cells to adhere to the wall. When the cells in the 96-well plate grow to about 50%, remove the old culture medium and divide the cells into 3 groups:

[0034] JQ1 group: JQ1-containing culture medium was added at final concentrations of 30 μM, 10 μM, 3 μM, and 0 μM, respectively.

[0035] Torin1 group: Torin1-containing culture medium was added at final concentrations of 500 nM, 300 nM, 100 nM, 30 nM, and 0 nM, respectively.

[0036] JQ1+ Torin1 group: different final concentrations of JQ1+ Torin1-containing culture medium were added.

[0037] After the above drug addition, continue incubation for 24 h, then remove the old culture medium, add 100 μL of serum-free DMEM complete culture medium containing 10% CCK-8, and incubate in an incubator for 2 h. Measure the OD450 value with an enzyme reader, calculate the inhibition rate of cells = 1-cell survival rate, and calculate the drug synergy score through the SynergyFinder website.

[0038] By using the current more mature pharmacological models (HSA, Bliss and ZIP) to systematically evaluate various types of drug interaction patterns that may occur in high-throughput drug combination screening, the synergistic effect of Torin1 and JQ1 was calculated using the "inhibition index"; a synergy score (Synergy scores) > 10 indicates that the two drugs have a synergistic effect ( Figure 1 The synergy score of -10 < 10 indicates that the effects of the two drugs are additive, which is the additive effect of the drugs; the ZIP synergy score of -10 indicates that there is an antagonistic effect between the two drugs.

[0039] (2) Experimental results:

[0040] The experimental results are shown in Table 1. Figure 1 and Figure 2 As shown, Table 1 is a scoring table of three synergistic models. It can be seen from Table 1 that the synergistic scores of the three synergistic models are all greater than 10, which fully demonstrates that the combination of Torin1 and JQ1 has a synergistic effect on liver cancer. Figure 1 The heat maps and 3D graphs corresponding to the three synergistic models (ZIP, HSA, Bliss) of Torin1 and JQ1 combined therapy for liver cancer show that the synergistic scores of Torin1 and JQ1 combined therapy (HSA, Bliss and ZIP) are all greater than 10. The white box in the figure indicates that the synergistic effect of Torin1 and JQ1 combined therapy is better when the concentration ratio of Torin1 to JQ1 is in the range of 100~500 nM:3~30 μM.

[0041] Table 1 Scoring table of different synergy models of Torin1 and JQ1

[0042]

[0043] Figure 2 The drug dose-response curves and drug dose-response matrix diagrams are for Torin1 and JQ1 alone and in combination. Figure 2The left figure shows that the use of Torin1 and JQ1 alone has a certain inhibitory effect on liver cancer cells, but the effect is limited. The highest concentration of a single drug (Torin1: 500nM, JQ1: 30 μM) only has a 40% inhibitory effect. In the range of drugs with the best synergistic effect, when the lowest concentration of Torin1: 100nM is added to the lowest concentration of JQ1: 3 μM, the cell proliferation inhibition rate reaches 58.33%, which is significantly higher than the inhibitory effect of a single drug. Figure 2 The right figure shows that when Torin1 and JQ1 are used in combination, and the concentration ratio of Torin1 to JQ1 is 100~500 nM: 3~30 μM, that is, within a specific concentration range, the combination of Torin1 and JQ1 will produce a synergistic effect and significantly inhibit the proliferation of liver cancer cells. At Torin1: 500 nM and JQ1: 10 μM, the inhibition rate of the drug combination on cell proliferation reached 74.31%, and the synergistic effect was the best. Therefore, this concentration was applied to the cell growth curve experiment and the plate clone formation experiment for verification.

[0044] (3) Referring to the experimental method in (1) above, the drug combination was also used in combination in colorectal cancer cells LOVO. Figure 3 The heat map and 3D map corresponding to the synergistic model (ZIP) of Torin1 and JQ1 for colorectal cancer are shown in Figure 1. Figure 3 The results showed that in the colorectal cancer cells LOVO, the average synergistic score (ZIP) of the combination of Torin1 and JQ1 was 4.019, between -10 and 10, which was an additive effect. This shows that the combination of Torin1 and JQ1 drugs did not have a synergistic effect in colorectal cancer, but only a simple additive effect, indicating that the combination of Torin1 and JQ1 is specific in liver cancer applications.

[0045] Example 2 Cell growth curve experiment

[0046] Preparation of JQ1-containing medium: JQ1 was first dissolved in DMSO and then added to the culture medium to prepare a JQ1-containing medium with a final JQ1 concentration of 10 μM.

[0047] Preparation of Torin1-containing medium: Torin1 was first dissolved in DMSO, and then added to the culture medium to prepare a Torin1-containing medium with a final Torin1 concentration of 500 nM.

[0048] Preparation of JQ1+ Torin1 drug-containing culture medium: Torin1 and JQ1 were first dissolved in DMSO, and then added to the culture medium to prepare a JQ1+ Torin1 drug-containing culture medium with a final concentration of 500 nM Torin1 and a final concentration of 10 μM JQ1.

[0049] Experimental method: Huh7 and SUN423 liver cancer cells were seeded in 96-well plates at a density of 1000 cells / well, placed in a cell culture incubator at 37°C with 5% CO2 to allow the cells to adhere to the wall, and then the cells were divided into 4 groups.

[0050] Control group (DMSO group): medium without drugs was added. The only difference between the medium of the control group and the drug-added groups (Torin1 single-drug group, JQ1 single-drug group, Torin1 and JQ1 combined drug group) was that no drugs were added to the medium.

[0051] Torin1 single-drug group: Torin1-containing medium was added with a final concentration of 500 nM Torin1.

[0052] JQ1 single-drug group: JQ1-containing medium was added to a final concentration of 10 μM JQ1.

[0053] Torin1 and JQ1 combined treatment group: Add JQ1+Torin1 drug-containing culture medium with a final concentration of 500 nM Torin1 and 10 μM JQ1.

[0054] There were three replicate wells in each group. The volume of drug-free culture medium or drug-containing culture medium added to all groups was the same. No cells were added to the edge wells of the 96-well plate. PBS was added to reduce the effect of liquid evaporation on cell proliferation. The plates were cultured in a cell culture incubator, and cell proliferation was detected on the 1st, 2nd, 3rd, and 4th days of culture.

[0055] Cell proliferation detection method: remove the old culture medium, add 100 μL of serum-free culture medium containing 10% CCK-8, and perform the same operation on the blank control group (no cell group), and incubate in the incubator for 2 h. Use a multifunctional microplate reader to detect the OD450 value, and draw a cell growth curve based on the cell OD value.

[0056] Figure 4 This is a growth curve of the effect of Torin1 and JQ1 combination therapy on liver cancer cell proliferation. Figure 4 A in the figure is the growth curve of Huh7 liver cancer cell proliferation. Figure 4 B in Figure 1 is a growth curve of SUN423 liver cancer cell proliferation. Figure 4 As shown, compared with the control group and the single-drug group, the combination of Torin1 and JQ1 had a significant synergistic inhibitory effect on the proliferation of liver cancer cells, and the cell growth rate in the combination group was the slowest.

[0057] Example 3 Plate clone formation experiment

[0058] Preparation of JQ1-containing medium: JQ1 was first dissolved in DMSO and then added to the culture medium to prepare a JQ1-containing medium with a final JQ1 concentration of 10 μM.

[0059] Preparation of Torin1-containing medium: Torin1 was first dissolved in DMSO, and then added to the culture medium to prepare a Torin1-containing medium with a final Torin1 concentration of 500 nM.

[0060] Preparation of JQ1+ Torin1 drug-containing culture medium: Torin1 and JQ1 were first dissolved in DMSO, and then added to the culture medium to prepare a JQ1+ Torin1 drug-containing culture medium with a final concentration of 500 nM Torin1 and a final concentration of 10 μM JQ1.

[0061] Experimental method: Huh7 and SUN423 liver cancer cells were seeded in 24-well plates at a density of 3000 cells / well, placed in a cell culture incubator at 37°C with 5% CO2 to allow the cells to adhere to the wall, and then the cells were divided into 4 groups.

[0062] Control group (DMSO group): medium without drugs was added. The only difference between the medium of the control group and the drug-added groups (Torin1 single-drug group, JQ1 single-drug group, and Torin1 and JQ1 combined group) was that no drugs were added to the medium.

[0063] Torin1 single-drug group: Torin1-containing medium was added with a final concentration of 500 nM Torin1.

[0064] JQ1 single-drug group: JQ1-containing medium was added to a final concentration of 10 μM JQ1.

[0065] Torin1 and JQ1 combined treatment group: Add JQ1+Torin1 drug-containing culture medium with a final concentration of 500 nM Torin1 and 10 μM JQ1.

[0066] Each group had three replicate wells, and the volume of drug-free medium or drug-containing medium added to all groups was the same. After adding different culture media to the above 4 groups, they were cultured for 2 weeks, and the clone formation was observed every day. The medium was changed every two days. After the culture, the cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 30 minutes, stained with 0.1% crystal violet for 30 minutes, and then dried and photographed.

[0067] Experimental results: The results are as follows Figure 5 The results showed that compared with the control group and the single-drug group, the combination drug group had a significant synergistic inhibitory effect on cell clone formation, and the number of clones formed in the combination drug group was the least.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of a combined drug in the preparation of a drug for preventing and treating liver cancer, characterized in that: The active ingredients of the combined drug include Torin1 and JQ1, and the molar ratio thereof is 0.1-0.5:3-30.

2. The application according to claim 1, characterized in that: The molar ratio of Torin1 to JQ1 is 0.1-0.5:10-30.

3. The application according to claim 2, characterized in that: The molar ratio of Torin1 to JQ1 is 0.5:

10.

4. The use according to claim 1, characterized in that: The combined drug contains a Torin1 liquid preparation with Torin1 as an active ingredient and a JQ1 liquid preparation with JQ1 as an active ingredient; the concentration ratio of Torin1 to JQ1 in the combined drug is 0.1-0.5 μM: 3-30 μM.

5. The application according to claim 4, characterized in that: The concentration ratio of Torin1 and JQ1 in the combined drug is 0.1~0.5 μM:10~30 μM.

6. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 1, characterized in that: The dosage form of the drug is powder, tablet, granule, capsule, solution, emulsion, suspension, aerosol, ointment, suppository, lotion or injection.

8. The use according to claim 1, characterized in that: The method for preventing and treating liver cancer includes increasing the sensitivity of liver cancer cells to Torin1.

9. The use according to claim 1, characterized in that: The prevention and treatment of liver cancer includes inhibiting the growth and / or proliferation of liver cancer cells.

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