Application of JX06 and lenvatinib in preparation of medicine for treating liver cancer

Abstract

The invention relates to the technical field of tumor treatment, in particular to application of JX06 and lenvatinib in preparation of a medicine for treating liver cancer. As a novel pyruvate dehydrogenase kinase 1 specific inhibitor, JX06 can cooperate with a tyrosine kinase inhibitor, namely, lenvatinib to inhibit hepatocellular carcinoma in vitro and in vivo, the median inhibitory concentration (IC50) of the lenvatinib can be reduced by combining JX06 in a cell experiment, and growth of subcutaneous transplantation tumors can be better inhibited by combining JX06 in an animal experiment.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and particularly to the application of JX06 in combination with lenvatinib in the preparation of a medicament for treating liver cancer. Background Art

[0002] Natural or acquired resistance of tumors is one of the difficulties and pain points in clinical treatment. Applications of reversing tumor resistance by inhibiting the pyruvate dehydrogenase kinase family or enhancing efficacy through combination therapy have been reported in some cancer types: in non-small cell lung cancer, targeting pyruvate dehydrogenase kinase 1 (PDK1) can overcome osimertinib resistance driven by the EGFR C797S mutation; in glioblastoma multiforme, targeting PDK1 can enhance the efficacy of anti-EGFR drugs by inhibiting the Warburg effect. Meanwhile, in the clinical practice of treating hepatocellular carcinoma (hereinafter referred to as liver cancer), the problem of resistance to the first-line tyrosine kinase inhibitor lenvatinib also exists. Such drugs can induce upregulation of HIF1α while inhibiting angiogenesis. One of the potential molecular mechanisms for resistance in some patients is that HIF1α-mediated upregulation of OCT4 and SOX2 enhances tumor stemness and thus promotes lenvatinib resistance. Summary of the Invention

[0003] To solve the above problems, the present invention provides the application of JX06 in combination with lenvatinib in the preparation of a medicament for treating liver cancer. The effectiveness of the novel PDK1-specific inhibitor JX06 in liver cancer is not very clear. Therefore, the present invention verifies the effectiveness and safety of JX06 combined with lenvatinib in synergistically inhibiting liver cancer in vitro and in vivo, and seeks possible strategies to reverse patient resistance and improve efficacy.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides the application of JX06 in combination with lenvatinib in the preparation of a medicament for treating liver cancer.

[0006] Preferably, when the combination of JX06 and lenvatinib is used in in vitro experiments, the molar ratio of JX06 to lenvatinib is 1:2;

[0007] When the combination of JX06 and lenvatinib is used in in vivo experiments, the molar ratio of JX06 to lenvatinib is 2:1.

[0008] Preferably, the CAS number of JX06 is: CAS: 729-46-4, and the molecular formula is as shown in Formula I:

[0009]

[0010] Preferably, the CAS number of lenvatinib is: CAS: 417716-92-8, and the molecular formula is shown in Formula II:

[0011]

[0012] The present invention also provides a drug for treating liver cancer, and the drug comprises JX06 and lenvatinib.

[0013] Preferably, when JX06 is used in combination with lenvatinib in vitro experiments, the molar ratio of JX06 to lenvatinib is 1:2;

[0014] When JX06 is used in combination with lenvatinib in in vivo experiments, the molar ratio of JX06 to lenvatinib is 2:1.

[0015] Beneficial effects:

[0016] As a novel PDK1 specific inhibitor, JX06 can synergistically inhibit hepatocellular carcinoma with the first-line tyrosine kinase inhibitor lenvatinib in vitro and in vivo. Combining JX06 can reduce the IC50 of lenvatinib in cell experiments, and the combination of drugs can better inhibit the growth of subcutaneous transplanted tumors in animal experiments. Description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 is the IC50 of JX06 in human hepatocellular carcinoma cell lines Huh7 and SNU449 cells;

[0019] Figure 2 is the IC50 of lenvatinib in Huh7 and SNU449 cells when synergistically using high and low doses of JX06 (Ctrl: blank control group, Len: lenvatinib, the same below);

[0020] Figure 3 is the apoptosis ratio and quantitative analysis of Huh7 and SNU449 cells in the blank control group, lenvatinib group, JX06 group and combined drug group;

[0021] Figure 4 is the diameter change of 3D tumor spheres in the blank control group, lenvatinib group, JX06 group and combined drug group (scale = 100 μm);

[0022] Figure 5 is the gross photograph of subcutaneous tumors in mice (scale = 10 mm), subcutaneous tumor weight and subcutaneous tumor volume change curves in the blank control group, lenvatinib group, JX06 group and combined drug group;

[0023] Figure 6 Ki67 immunohistochemical staining of subcutaneous tumors in mice of the blank control group, lenvatinib group, JX06 group and combination drug group (scale bar for low magnification field = 50 μm, scale bar for high magnification field = 25 μm). Specific implementation manners

[0024] The present invention provides the use of JX06 in combination with lenvatinib in the preparation of a medicament for treating liver cancer. In the present invention, when JX06 is used in combination with lenvatinib for in vitro experiments, the molar ratio of JX06 to lenvatinib is 1:2; when JX06 is used in combination with lenvatinib for in vivo experiments, the molar ratio of JX06 to lenvatinib is 2:1. The present invention has no special limitation on the sources of JX06 and lenvatinib, and conventional commercially available products can be used. In the present invention, the CAS number of JX06 is: CAS: 729-46-4, and the molecular formula is as shown in Formula I:

[0025]

[0026] In the present invention, the CAS number of lenvatinib is: CAS: 417716-92-8, and the molecular formula is as shown in Formula II:

[0027]

[0028] The present invention also provides a medicament for treating liver cancer, and the medicament comprises JX06 and lenvatinib. In the present invention, when JX06 is used in combination with lenvatinib for in vitro experiments, the molar ratio of JX06 to lenvatinib is 1:2; when JX06 is used in combination with lenvatinib for in vivo experiments, the molar ratio of JX06 to lenvatinib is 2:1. The present invention has no special limitation on the dosage form and preparation method of the medicament, and those skilled in the art can use the dosage forms and preparation methods of JX06 and lenvatinib that are medically acceptable.

[0029] In order to further illustrate the present invention, the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1

[0031] 1. Measurement of drug IC50

[0032] (1) Add 2×10 3 liver cancer cells to each well of a 96-well plate, set up a drug concentration gradient and reserve blank wells:

[0033] When measuring the IC50 of JX06, the concentration gradient of JX06 is set to 0 - 0.312 - 0.625 - 1.25 - 2.5 - 5 - 10 - 20 - 40 - 80 μmol / L;

[0034] When measuring the lenvatinib IC50 of Huh7, the concentration gradient was set as 0 - 0.078 - 0.156 - 0.312 - 0.625 - 1.25 - 2.5 - 5 - 10 - 20 μmol / L;

[0035] When measuring the lenvatinib IC50 of SNU449, the concentration gradient was set as 0 - 0.312 - 0.625 - 1.25 - 2.5 - 5 - 10 - 20 - 40 - 80 μmol / L.

[0036] After culturing for 48 h, the culture medium in the wells was replaced with 100 μL of fresh complete medium containing 10% CCK-8, and after culturing in the dark at 37 °C for 2 h, the optical density (OD) value at 450 nm was measured.

[0037] (2) Subtract the OD value of the blank well from the OD value measured in the experimental well, calculate the survival rate of each group with the control group cells as a reference, and calculate the drug IC50 by software fitting the curve.

[0038] 2. Detection of cell apoptosis by flow cytometry

[0039] (1) Huh7 and SNU449 cells were seeded in six-well plates in advance so that the cell density was about 50% during the drug treatment.

[0040] (2) Different cells were grouped and treated according to the results of the preliminary experiment and the drug IC50, as shown in Table 1:

[0041] Table 1 Drug concentrations for different grouped treatments

[0042]

[0043] (3) After 48 h of drug treatment, the cells were digested into centrifuge tubes, centrifuged at 1000 rpm for 5 min, resuspended with PBS and centrifuged again to obtain cell pellets.

[0044] (4) Resuspend the control group cells with 400 μL of the accompanying binding buffer, divide it into two equal parts, one tube as the blank tube without adding reagents later, and resuspend the cell pellets of the remaining groups with 200 μL of the binding buffer.

[0045] (5) Add 5 μL of Annexin V-FITC reagent and 5 μL of propidium iodide (concentration 50 μg / mL) to each tube in the tubes to be tested, gently vortex and mix well, and incubate at room temperature in the dark for 15 - 20 min.

[0046] (6) After incubation, place it on ice and immediately perform on-machine detection. Use the FITC channel for AnnexinV-FITC, and preferably choose the PerCP / Cy5.5 channel for PI.

[0047] (7)Quantify by Flowjo plotting and statistically analyze the differences between groups.

[0048] 3. Drug treatment of 3D tumor spheres

[0049] (1) Use a low-attachment U-bottom 96-well plate for 3D tumor sphere culture. Add 1×10 4 Huh7 cells to each well and place it in an incubator at 37°C and 5% CO2 for 48 h. Observe the formation of tumor spheres under the microscope.

[0050] (2) After the formation of tumor spheres, according to the experimental protocol, replace the medium with complete medium containing 2 μmol / L lenvatinib or 1 μmol / L JX06 or 2 μmol / L lenvatinib + 1 μmol / L JX06. The blank control group only adds an equal amount of complete medium. After continuing to culture for 48 h, observe and photograph under the microscope to record the diameter and morphology of the tumor spheres.

[0051] 4. Drug intervention on subcutaneous tumors in mice

[0052] (1) Expand a sufficient number of Huh7 cells in advance. After digestion and counting, resuspend and dilute the cell pellet with Matrigel and PBS.

[0053] (2) Thoroughly mix the cell suspension using a Pasteur pipette. Inject 200 μL (containing 5×10 6 cells) of the Matrigel suspension subcutaneously into the right posterior neck of each BALB / c-nude mouse, paying attention to the injection depth. A distinct subcutaneous bleb can be seen.

[0054] (3) When the volume of the subcutaneous tumor approaches 100 - 150 mm 3 , randomly divide the tumor-bearing BALB / c-nude mice into 4 groups, with 6 mice in each group, namely the blank control group / lenvatinib group / JX06 group / combination drug group. Measure the baseline diameter of the subcutaneous tumor; the drugs used in the experiment are prepared freshly:

[0055] Preparation of the lenvatinib gavage system: Dissolve lenvatinib powder in 5% DMSO and dilute the lenvatinib stock solution to 1 μg / μL with 0.5% sodium carboxymethylcellulose;

[0056] Preparation of the JX06 intraperitoneal injection system: Dissolve JX06 powder in 5% DMSO and dilute the JX06 stock solution to 2 μg / μL with 40% PEG300, 5% Tween 80, and normal saline;

[0057] Give the following interventions according to the experimental grouping:

[0058] Blank control group: Gavage and intraperitoneal injection with the same amount of drug solvent as the treatment group;

[0059] Lenvatinib group: Gavage with 10 mg / kg lenvatinib;

[0060] Group JX06: Intraperitoneal injection of 20 mg / kg JX06;

[0061] Combined medication group: Gavage of 10 mg / kg lenvatinib + intraperitoneal injection of 20 mg / kg JX06;

[0062] Administer the drugs every other day for a total of 3 weeks. Measure the long and short diameters of the subcutaneous tumors every 4 days, and calculate the subcutaneous tumor volume according to the formula V = (L × W 2 ) / 2, and draw a growth curve.

[0063] (4) Sacrifice the mice after 3 weeks, expose the positions of the subcutaneous tumors and take gross photos in an orderly manner; carefully dissect the subcutaneous tumors, arrange them in the same order and take photos, measure the tumor weights, and then fix the subcutaneous tumors with 4% paraformaldehyde and send them for paraffin-embedded sections. Immunohistochemical staining is used to quantitatively reflect Ki67, which reflects the cell proliferation ability.

[0064] Results:

[0065] In vitro experiments (cell experiments):

[0066] JX06 can inhibit the growth of liver cancer cells. The IC50 in Huh7 cells is 0.51 μmol / L, and the IC50 in SNU449 cells is 1.67 μmol / L ( Figure 1 ).

[0067] The Huh7 cell line is naturally sensitive to lenvatinib treatment, while the SNU449 cell line is relatively naturally resistant. Through combined drug treatment, it was found that JX06 can reduce the IC50 of lenvatinib in these two cell lines:

[0068] The IC50 of lenvatinib alone in Huh7 cells is 2.34 μmol / L. After adding 0.25 μmol / L JX06, the IC50 of lenvatinib is 1.61 μmol / L, a decrease of 31.2%; after adding 0.5 μmol / L JX06, the IC50 of lenvatinib drops to 1.13 μmol / L, a decrease of 51.7%.

[0069] The IC50 of lenvatinib alone in SNU449 cells is 55.37 μmol / L. After adding 1 μmol / L JX06, the IC50 of lenvatinib is 10.06 μmol / L, a decrease of 81.8%; after adding 2 μmol / L JX06, the IC50 of lenvatinib is 4.17 μmol / L, a decrease of 92.5% ( Figure 2 ).

[0070] Flow cytometry detection of liver cancer cell apoptosis found that:

[0071] After treatment with 2 μmol / L lenvatinib for 48 h, the apoptosis rate of Huh7 cells was 9.38%, while the apoptosis rate of cells after combined use of 2 μmol / L lenvatinib and 1 μmol / L JX06 was 30.36%;

[0072] After treatment with 4 μmol / L lenvatinib for 48 h, the apoptosis rate of SNU449 cells was only 4.31%, and the apoptosis rate of cells after combined use of 4 μmol / L lenvatinib and 2 μmol / L JX06 increased to 20.21%.

[0073] It can be seen that the killing effect of the combination drug group on liver cancer is significantly stronger than that of the lenvatinib single drug group ( Figure 3 ).

[0074] Consistent with the above results, the combination drug also has stronger growth inhibition on tumor spheres cultured in 3D than the single drug group ( Figure 4 ).

[0075] In vivo experiment (animal experiment):

[0076] The efficacy of JX06 in synergizing with lenvatinib to inhibit liver cancer in vivo was verified by the method of drug intervention after subcutaneous tumor implantation in BALB / c-nude mice. The results were consistent with those of in vitro experiments. The combination drug could better inhibit the growth of subcutaneous tumors ( Figure 5 ), and immunohistochemical staining indicated that the Ki67 expression in the JX06 and lenvatinib combination group was the lowest ( Figure 6 ).

[0077] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of JX06 in combination with lenvatinib in the preparation of drugs for the treatment of liver cancer.

2. The use according to claim 1, characterized in that: When the JX06 is used in conjunction with lenvatinib in an in vitro experiment, the molar ratio of JX06 to lenvatinib is 1:2; When the JX06 is used in conjunction with lenvatinib in an in vivo experiment, the molar ratio of JX06 to lenvatinib is 2:

1.

3. The use according to claim 1, characterized in that: The CAS number of the JX06 is CAS: 729-46-4, and the molecular formula is shown in Formula I:

4. The use according to claim 1, characterized in that: The CAS number of lenvatinib is: CAS: 417716-92-8, and the molecular formula is shown in Formula II:

5. A drug for treating liver cancer, characterized in that: The drugs include JX06 and lenvatinib.

6. The drug according to claim 5, characterized in that When the JX06 is used in conjunction with lenvatinib in an in vitro experiment, the molar ratio of JX06 to lenvatinib is 1:2; When the JX06 is used in conjunction with lenvatinib in an in vivo experiment, the molar ratio of JX06 to lenvatinib is 2:1.

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