CN111001004A - A drug combination for treating liver cancer and its application - Google Patents

Abstract

The invention discloses the application of the combined use of regorafenib and a PD-1/PD-L1 inhibitor in preparing a medicine for treating liver cancer. The drug combination (regorafenib and PD-1/PD-L1 inhibitor) of the present invention can produce a synergistic anti-liver cancer effect, and the combined administration scheme of the two can not only improve the sensitivity of liver cancer cells to regorafenib, The inhibitory effect on liver cancer cells can be increased, the dosage of regorafenib can also be reduced, the treatment cost of each liver cancer patient can be directly reduced, and more liver cancer patients can benefit; the drug combination of the present invention is low in price and high in safety. The curative effect is affirmative; the pharmaceutical combination of the present invention is convenient to produce, convenient for large-scale production, good curative effect and low cost are also beneficial to social public interests.

Description

Pharmaceutical composition for treating liver cancer and application thereof

Technical Field

The invention relates to the technical field of liver cancer treatment, in particular to a pharmaceutical composition for treating liver cancer and application thereof, and especially relates to application of regorafenib in preparation of a medicament for treating liver cancer in combination with a PD-1/PD-L1 inhibitor BMS-1.

Background

Primary Hepatocellular carcinoma (HCC) is the most common malignant tumor of liver, and develops covertly and rapidly, and the global cancer statistical result in 2018 shows that the incidence of the liver cancer is 6 th of malignant tumor, and the death rate is 4 th of death related to the malignant tumor^[1]. China is a high-incidence area of liver cancer, and the latest data shows that the incidence rate of liver cancer in 2015 is on the 4 th site of tumor-related diseases, the mortality rate is on the 3 rd site of malignant tumors, the incidence rate and the mortality rate are more than half of the total number of the whole world, and the life health of people in China is seriously threatened^[2]. Treatment of early stage liver cancer follows well established guidelines, surgical resection, liver transplantation and local radiofrequency ablation are the primary treatment options for early stage disease, and chemoembolization treatment is suggested for patients with limited liver function and no involvement of vascular invasion of the lesion^[3,4]. Clinically, most liver cancer patients are diagnosed at middle and advanced stages, the treatment opportunity is lost, and the patients can only prolong the life cycle and improve the life quality through a non-radical treatment mode, mainly comprising chemotherapy, medicament treatment, immunotherapy and the like^[5]. For liver cancer patients who are not or no longer eligible for local treatment, the oral drug multi-kinase inhibitor sorafenib is the first systemic treatment approved by the FDA to significantly improve overall survival^[6,7]. However, since the 10-year release of sorafenib treatment results, all phase 3 trials evaluating new systemic drugs failed to improve sorafenib on the first line^[8-11]Or the outcome of second line therapy after sorafenib^[12-15]In a second-line trial of patients with failure of sorafenib treatment, the overall survival of the placebo group was approximately 8 months^[6]. Therefore, the effective systemic therapeutic effect of the patients on the liver cancer is not satisfiedIn particular, the emergence of new drugs or new adjuvant drug combination treatment regimens is urgently desired, especially for patients who have poor efficacy after receiving sorafenib treatment.

Regorafenib (Regorafenib) is a novel oral multi-kinase inhibitor, has a unique molecular target, and has stronger pharmacological activity than sorafenib shown by preclinical research results^[16,17]Can block angiogenesis, tumorigenesis, metastasis and tumor immunoregulation^[17,18]. Research results show that regorafenib can prolong the survival time of HCC patients after failure of sorafenib treatment, and is approved by FDA in 2017 for second-line treatment of patients who cannot resect hepatocellular carcinoma^[19,20]. Median overall survival after regorafenib treatment increased from 7.8 months to 10.6 months in the placebo group, with limited efficacy, regorafenib produced response and survival benefit even in sorafenib-resistant patients^[19]. Nevertheless, drug toxicity, drug resistance and subsequent disease progression remain problematic issues in liver cancer treatment^[21,22]Meanwhile, due to the extremely high selling price of regorafenib, a plurality of liver cancer patients are forbidden, so that the patients urgently need a new auxiliary drug combination treatment scheme, the regorafenib drug dosage is reduced, and the drug treatment effect is improved^[20,23-25]。

Based on the recent success in inhibiting immune checkpoint anti-cancer therapies in different solid tumors, nobel congress awarded the nobel prize in Tasuko Honjo and James p. Compared with traditional chemotherapy or molecular therapy directly aiming at cancer cells, the immunotherapy uses an immune checkpoint inhibitor to block an immune escape mechanism of tumors, inhibit tumor signal transmission and continuously induce strong immune response mainly comprising T cells, thereby realizing the anti-tumor effect^[26]. The most successful form of immunotherapy to date has been the blockade of the immune checkpoint programmed cell death protein 1/programmed death ligand 1(PD-1/PD-L1 receptor) and cytotoxic T lymphocyte protein 4(CTLA-4)^[27-31]. Among these, the main reason for the active development of cancer immunotherapy by blocking the PD-1/PD-L1 pathway is the discovery that PD-L1 is the major ligand expressed in many solid tumors, whereas PD-1 is expressed in tumorsHigh-level expression on tumor-infiltrated lymphocytes^[32-35]. The medicament for blocking PD-1/PD-L1 can block the combination of PD-L1 of tumors and PD-1 of immune cells, thereby avoiding the immune escape of the tumors and maintaining the continuous activation of the immune cells, thus showing great prospect in the aspect of treating various cancers, further promoting the deep research of more and more unique check point molecules as potential treatment targets, and providing a new treatment scheme for patients. However, antibody drugs have many disadvantages in terms of high production cost, low stability, immunogenicity, and the like^[36,37]While novel small molecule inhibitors may provide inherent advantages in terms of pharmacokinetics and pharmacology, resulting in better therapeutic efficacy for cancer treatment^[38]. It was reported that Bristol-Myers Squibb filed a series of compounds having the activity of inhibiting the interaction between PD-1 and PD-L1 in 2015 (patent WO 2015/034820A 1), and BMS-1 is a small molecule PD-1/PD-L1 inhibitor thereof^[39-41]. According to analysis, BMS-1 can be highly overlapped with the surface of PD-1/PD-L1 protein-protein interaction, so that the BMS-1 has the potential effects of preventing PD-1 and PD-L1 from playing normal interaction and blocking signal paths, and can be applied to the treatment of tumor progression^[42]. Because the compound is a small molecular compound, the compound has the advantages of low price, high stability, low immunogenicity, high safety and the like.

Chinese patent document CN104994876A discloses a pharmaceutical composition and combination comprising regorafenib and acetylsalicylic acid, or hydrates, solvates, metabolites or pharmaceutically acceptable salts thereof, or polymorphs thereof, for the treatment, prevention or control of diseases and conditions in humans and other mammals, including hyperproliferative disorders such as cancer chinese patent document CN109069509A discloses that the combination treatment of sorafenib or regorafenib with an phosphoramidate prodrug of troxacitabine having the formula or a pharmaceutically acceptable salt thereof shows unexpected utility in the treatment of liver cancer or liver metastasis chinese patent document CN105879049A discloses an inclusion compound formed with regorafenib as a pharmaceutically active ingredient, β -cyclodextrin as a host molecule (host), molar ratio 1:1, bioavailability is greatly improved, preparation method, conditions are mild, suitable for industrial production, prepared inclusion compound has excellent pertinence to targeted delivery of colon cancer, and safety is good, and can effectively overcome the disadvantages of poor water solubility and low bioavailability of regorafenib itself.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the defects of the prior art and provide a pharmaceutical composition for treating liver cancer, which not only can produce a synergistic effect of inhibiting the growth of liver cancer cells, but also can improve the sensitivity of liver cancer cells to regorafenib, increase the inhibition effect on liver cancer cells, reduce the dosage of regorafenib, directly reduce the treatment cost of each liver cancer patient, and enable more liver cancer patients to benefit.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following two aspects:

in a first aspect, the invention provides the use of regorafenib in combination with a PD-1/PD-L1 inhibitor for the manufacture of a medicament for the treatment of liver cancer.

Preferably, the PD-1/PD-L1 inhibitor is BMS-1.

In another aspect, the invention provides a pharmaceutical combination for treating liver cancer, the pharmaceutical combination comprising regorafenib and a PD-1/PD-L1 inhibitor.

Preferably, the PD-1/PD-L1 inhibitor is BMS-1.

Preferably, the molar ratio of the regorafenib to the BMS-1 is 2-6: 30-40.

Preferably, the regorafenib concentration is 2 μ M and the BMS-1 concentration is 30 μ M.

Preferably, the regorafenib concentration is 4 μ M and the BMS-1 concentration is 30 μ M.

Preferably, the regorafenib concentration is 6 μ M and the BMS-1 concentration is 30 μ M.

Preferably, the regorafenib concentration is 2 μ M and the BMS-1 concentration is 40 μ M.

Preferably, the regorafenib concentration is 4 μ M or 6 μ M and the BMS-1 concentration is 40 μ M.

In conclusion, the beneficial effects of the invention are as follows:

1) the pharmaceutical composition (regorafenib and a PD-1/PD-L1 inhibitor) can generate a synergistic anti-liver cancer effect, and the scheme of combined administration of the regorafenib and the PD-1/PD-L1 not only can improve the sensitivity of liver cancer cells to regorafenib and increase the inhibition effect on the liver cancer cells, but also can reduce the dosage of regorafenib, directly reduce the treatment cost of each liver cancer patient and benefit more liver cancer patients;

2) the medicine composition has low price, high safety and definite curative effect;

3) the medicine composition of the invention has the advantages of convenient production, convenient large-scale production, good curative effect, low cost and benefit to social and public benefits.

Drawings

FIG. 1 is a growth inhibition rate curve of regorafenib single drug on hepatoma cell strains SMMC-7721, Hep3B and SK-Hep 1;

FIG. 2 is a growth inhibition rate curve of BMS-1 single drug on liver cancer cell strains SMMC-7721, Hep3B and SK-Hep 1;

FIG. 3 is a comparison graph of the inhibition effect of regorafenib single drug, BMS-1 single drug and regorafenib combined BMS-1 on liver cancer cell strains SMMC-7721, Hep3B and SK-Hep 1;

FIG. 4 is a graph of the CI values of the combination index of regorafenib single drug, BMS-1 single drug, regorafenib combination BMS-1 to hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 5 is a comparison graph of the inhibition effect of regorafenib single drug, BMS-1 single drug and regorafenib in combination with BMS-1 on liver cancer cell strains SMMC-7721, Hep3B and SK-Hep1 at different action times;

FIG. 6 is an EdU staining pattern and histogram of regorafenib single drug, BMS-1 single drug, regorafenib in combination with BMS-1 on hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 7 is TUNEL staining and histogram of regorafenib single drug, BMS-1 single drug, regorafenib in combination with BMS-1 for hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 8 is a scatter plot and a histogram of apoptosis of regorafenib single drug, BMS-1 single drug, regorafenib in combination with BMS-1 on hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 9 is a graph showing the results of apoptosis analysis of regorafenib single drug, BMS-1 single drug, regorafenib in combination with BMS-1 on hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 10 is a graph showing the results of cell cycle analysis of the individual drugs regorafenib, BMS-1, and regorafenib in combination with BMS-1 on the hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 11 is a graph showing the results of cell migration of regorafenib prodrug, BMS-1 prodrug, regorafenib in combination with BMS-1 against hepatoma cell lines SMMC-7721, Hep3B and SK-Hep 1;

FIG. 12 is a graph showing the results of cell invasion of the hepatoma cell lines SMMC-7721, Hep3B and SK-Hep1 by regorafenib single drug, BMS-1 single drug and regorafenib combined BMS-1.

Detailed Description

According to the invention, regorafenib and BMS-1 are jointly acted on liver cancer cells, and researches show that the two medicines are jointly administrated to generate a synergistic effect of inhibiting the growth of the liver cancer cells. Research shows that the molecular targeted drug can inhibit the signal path for maintaining the immunosuppressive environment, and the combination of the checkpoint inhibitor and the molecular targeted drug can cause the change of the tumor microenvironment, so as to promote the cytotoxic T lymphocyte to enter the tumor tissue and enable the cytotoxic T lymphocyte to permeate the tumor tissue^[43]. Therefore, the combined application of immunotherapy and targeted therapy may be a new idea for liver cancer treatment. The scheme of the combined administration of the two medicines can not only improve the sensitivity of the liver cancer cells to regorafenib and increase the inhibition effect on the liver cancer cells, but also reduce the dosage of regorafenib, directly reduce the treatment cost of each liver cancer patient and benefit more liver cancer patients, and the market prospect and the economic and social benefits brought by the medicine cannot be estimated.

In some embodiments, the invention provides a pharmaceutical combination for treating liver cancer, wherein the molar concentration of the components of the pharmaceutical combination is regorafenib 2 μ M and BMS-130 μ M, regorafenib 4 μ M and BMS-130 μ M, regorafenib 6 μ M and BMS-130 μ M, regorafenib 2 μ M and BMS-140 μ M, regorafenib 4 μ M and BMS-140 μ M, or regorafenib 6 μ M and BMS-140 μ M.

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the concentrations of the reagents in the present invention are mass concentrations. Unless otherwise specified, the reagents, materials or cell lines of the present invention are commercially available or otherwise publicly available.

Example 1

1. Purpose of the experiment: the hepatoma cell strains SMMC-7721, Hep3B and SK-Hep1 are selected to detect the inhibition effect of regorafenib and BMS-1 on hepatoma cells alone.

2. Spreading hepatoma cell strains SMMC-7721, Hep3B and SK-Hep1 in a 96-well plate, respectively adding regorafenib (0.75, 1.5, 3, 6, 12, 24, 48 mu M) and BMS-1(3.25, 7.5, 15, 30, 60, 120, 240 mu M) with different concentrations after 24h, detecting the influence of regorafenib and BMS-1 on the activity of three hepatoma cell strains by using a CCK-8 experimental method after culturing for 36h, drawing a growth inhibition rate curve according to OD values and calculating IC (integrated circuit) of the two drugs corresponding to the three hepatoma cell strains respectively₅₀。

3. The experimental method comprises the following steps: CCK8 test for detecting activity of drug on cells

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 5 × 10^ 3/hole with cell counting plate, homogenizing in 96-well plate, setting 4 multiple holes/group, and placing in 37 deg.C incubator overnight.

② cells are divided into several groups according to the drug concentration gradient or different drug combination design after adhering to the wall, and 200 μ L of drug with corresponding concentration is given to act on the liver cancer cells for a certain time.

③ discarding the culture solution containing the drug from the cells in the biosafety cabinet, preparing CCK8 reagent, namely preparing a serum-free DMEM culture medium and a CCK8 stock solution at a ratio of 10:1 at room temperature in a dark place, adding 110 mu L/hole of CCK8 mixed solution into a plurality of holes, arranging 4 cell-free blank control holes, adding 110 mu L/hole of CCK8 mixed solution, wrapping by using tin foil, and placing in a cell incubator at 37 ℃ in a dark place for incubation for 2 hours.

④ after the incubation time is completed, shaking the mixture on a horizontal shaker at room temperature for 1min at 300rpm, removing air bubbles in the wells, and detecting the OD value at 450nm using an enzyme-linked immunosorbent detector.

⑤ percent cell activity was calculated as (experimental OD value-blank OD value)/(control OD value-blank OD value) × 100%, statistical analysis and plotted using GraphPad Prism7₅₀) The inhibition rate of cell growth according to different drug concentrations was calculated by using GraphPad Prism7 software.

4. The experimental results are as follows: referring to FIGS. 1 and 2, regorafenib dose-dependently inhibits the activity of three hepatoma cell lines, IC of SMMC-7721, Hep3B and SK-Hep1₅₀Are respectively as₁₃50. mu.M, 13.20. mu.M, 9.05. mu.M. As shown in FIG. 2B, the stronger BMS-1 inhibited the activity of hepatoma cells with increasing concentration, the IC of SMMC-7721, Hep3B and SK-Hep1₅₀Respectively at 74.11. mu.M, 73.08. mu.M and 53.76. mu.M.

Example 2

1. Purpose of the experiment: the inhibition effect of the regorafenib combination BMS-1 on liver cancer cells is detected by selecting liver cancer cell strains SMMC-7721, Hep3B and SK-Hep 1.

2. Liver cancer cell strains SMMC-7721, Hep3B and SK-Hep1 are adopted and spread in a 96-well plate, regorafenib (2, 4 and 6 mu M) combined BMS-1(30 and 40 mu M) with different concentrations are respectively added after 24 hours, and after 36 hours of culture, the effect of combined treatment of regorafenib and BMS-1 and single treatment on the activity of three liver cancer cells is detected by adopting a CCK-8 experimental method.

3. The experimental method comprises the following steps: CCK8 test for detecting activity of drug on cells

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 5 × 10^ 3/hole with cell counting plate, homogenizing in 96-well plate, setting 4 multiple holes/group, and placing in 37 deg.C incubator overnight.

② cells are divided into several groups according to the drug concentration gradient or different drug combination design after adhering to the wall, and 200 μ L of drug with corresponding concentration is given to act on the liver cancer cells for a certain time.

③ discarding the culture solution containing the drug from the cells in the biosafety cabinet, preparing CCK8 reagent, namely preparing a serum-free DMEM culture medium and a CCK8 stock solution at a ratio of 10:1 at room temperature in a dark place, adding 110 mu L/hole of CCK8 mixed solution into a plurality of holes, arranging 4 cell-free blank control holes, adding 110 mu L/hole of CCK8 mixed solution, wrapping by using tin foil, and placing in a cell incubator at 37 ℃ in a dark place for incubation for 2 hours.

④ after the incubation time is completed, shaking the mixture on a horizontal shaker at room temperature for 1min at 300rpm, removing air bubbles in the wells, and detecting the OD value at 450nm using an enzyme-linked immunosorbent detector.

⑤ percent cell activity was calculated as (experimental OD value-blank OD value)/(control OD value-blank OD value) × 100%, statistical analysis and plotted using GraphPad Prism7₅₀) The inhibition rate of cell growth according to different drug concentrations was calculated by using GraphPad Prism7 software.

4. The experimental results are as follows: referring to fig. 3, 4 and 5, regorafenib in combination with BMS-1 can produce a synergistic anti-liver cancer effect, and regorafenib and BMS-1 in combination have a stronger anti-liver cancer effect than a regorafenib single-drug group and a BMS-1 single-drug group.

Example 3

1. Purpose of the experiment: the hepatoma cell strains SMMC-7721, Hep3B and SK-Hep1 are selected to detect the inhibition and proliferation effects of the regorafenib combination BMS-1 on hepatoma cells.

2. And adopting EdU staining to detect the staining condition of the cells in the interphase S phase after different treatment groups act on the liver cancer cells for 36 h.

3. The experimental method comprises the following steps: EdU staining detection cell proliferation assay

The cells treated with different drugs were subjected to EdU staining according to the staining method of an EdU kit (purchased from Ruibo Biotech Co., Ltd., Guangzhou, cat # C10310-1) to observe the proliferation of the cells.

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 1 × 10^ 4/hole with cell counting plate, homogenizing in 96-well plate, setting 4 multiple holes/group, and placing in 37 deg.C incubator overnight.

② cells are divided into several groups according to different drug combination designs after being attached to the wall, and 200 mu L of drug with corresponding concentration is given to act on the liver cancer cells for 36 h.

③ EdU labelling (sterile operation in BioSafty Cabinet). A.EdU solution (reagent A) was diluted with fresh medium at a ratio of 1000:1 to prepare a suitable high concentration 50. mu.M EdU medium, b.discarding old medium, adding 100. mu.L/well of 50. mu.M EdU medium, incubating at 37 ℃ for 2 hours in a cell incubator, and c.washing the cells 2 times for 5 min/time with sterile PBS.

④ cell immobilization comprises a removing PBS, adding 100 μ L/well 4% paraformaldehyde to immobilize cells at room temperature for 15min, b removing fixative, adding 50 μ L/well 2mg/mL glycine, incubating on low speed decolorizing shaking table for 5min, c removing glycine, adding 100 μ L/well PBS, washing on low speed decolorizing shaking table for 5min, d removing PBS, adding 100 μ L/well penetrant (0.5% TritonX-100 PBS), incubating on low speed decolorizing shaking table for 10min, e removing penetrant, adding 100 μ L/well PBS, and washing on low speed decolorizing shaking table for 5 min.

⑤ Apollo staining, a.1 XApollo staining reaction liquid (500 μ L dosage) is prepared at room temperature in a dark place, deionized water is 469 μ L + Apollo reaction buffer solution (reagent B) is 25 μ L + Apollo catalyst solution (reagent C) is 5 μ L + Apollo fluorescent dye solution (reagent D) is 1.5 μ L + Apollo buffer additive (reagent E) is 5mg, b.100 μ L/well of 1 XApollo staining reaction liquid is added to the PBS in a dark place and incubated for 30min in a shaking table with low speed and at room temperature in a dark place, c.100 μ L/well of penetrating agent (0.5% TritonX-100 PBS) is added to the staining reaction liquid in a shaking table with low speed and washed for 2-3 times and 10 min/time, d.100 μ L/well of methanol in a washing table for 1-2 times and 5 min/time, e.100 μ L/well of PBS is added to the formaldehyde in a shaking table with low speed and washed for 5 min.

⑥ DNA staining, a, diluting a DAPI reagent with PBS according to the ratio of 20000:1 at room temperature in a dark place to prepare a proper amount of 1 XPAPI reaction liquid, b, removing the PBS, adding 100 mu L/hole of 1 XPAPI reaction liquid, incubating for 1-2 min on a low-speed decolorizing shaking bed at room temperature in a dark place, removing the staining reaction liquid, c, adding 100 mu L/hole of PBS, and washing for 3 times and 5 min/time on the low-speed decolorizing shaking bed.

⑦ fluorescent inverted microscope image acquisition, photographing in a short time after dyeing is completed (the image can be stored for 72h under the conditions of light shielding at 4 ℃ and PBS soaking, but the brightness is reduced to some extent), photographing 3 images/hole under a 100 x-fold mirror (each image contains red light EdU and blue light DAPI), and photographing 5 images/hole under a 200 x-fold mirror.

⑧ statistical analysis and mapping (selected) experiments were repeated 3 times independently, Excel and Photoshop counted 200 Xtimes the number of red light (EdU) and blue light (DAPI) per image, and from the counts for each group, the cell proliferation rate was calculated as EdU/DAPI × 100%, statistical analysis and mapping using GraphPad Prism 7.

4. The experimental results are as follows: referring to fig. 6, regorafenib in combination with BMS-1 produces a synergistic effect of inhibiting liver cancer proliferation, reducing the number of cells in the S phase of the cell interphase.

Example 4

1. Purpose of the experiment: the hepatoma cell strains SMMC-7721, Hep3B and SK-Hep1 are selected to detect the inhibition and proliferation effects of the regorafenib combination BMS-1 on hepatoma cells.

2. The influence of regorafenib in combination with BMS-1 on the apoptosis of the liver cancer cells is detected by a TUNEL staining method and a flow cytometry.

3. The experimental method comprises the following steps: TUNEL staining for detection of cell proliferation apoptosis

The apoptosis of the hepatoma carcinoma cells after the treatment of different drug combinations was detected according to the TUNEL kit instruction (purchased from Promega, USA, Cat: G3250) in a biosafety cabinet in a sterile manner:

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 1 × 10^ 4/hole with cell counting plate, homogenizing in 96-well plate, setting 4 multiple holes/group, and placing in 37 deg.C incubator overnight;

② cells are divided into a plurality of groups according to different drug combination designs after being attached to the wall, and 200 mu L of drug with corresponding concentration is given to act on the liver cancer cells for 36 h;

③ discarding old culture medium, adding 100 μ L/hole 4% paraformaldehyde to fix cells at room temperature for 15min, discarding fixing solution, adding 100 μ L/hole PBS to wash cells for 2 times and 5 min/time;

④ discarding PBS, adding 100 μ L/well penetrant (0.2% TritonX-100 PBS), incubating at room temperature in a dark place with a low-speed decolorizing shaking bed for 5min, discarding penetrant, adding 100 μ L/well PBS, and washing on a low-speed decolorizing shaking bed for 3 times (5 min/time);

⑤ discard PBS, thaw the reagent in TUNEL kit on ice, add 30. mu.L/well of Equilibration Buffer and incubate for 10min at room temperature;

⑥ preparing appropriate amount of TdT reaction mixture (Equilibration Buffer: Nucleotide Mix: rTdT Enzyme: 45:5:1) at room temperature in dark, adding 25 μ L/well mixture, incubating for 1h in a cell incubator at 37 ℃, and properly prolonging the incubation time according to experimental differences, wherein the incubation time should not exceed 2h generally;

⑦ adding 100 μ L/well 2 XSSC liquid (20 XSSC diluted with ultrapure water 10: 1) under dark condition, incubating for 15min, discarding SSC liquid, adding 100 μ L/well PBS, and washing cells for 3 times and 5 min/time;

⑧ discard PBS, DAPI staining stock solution is diluted with PBS at 20000:1 and added to 100. mu.L/well, DAPI is discarded, 100. mu.L/well PBS is added, and washing is carried out 3 times for 5 min/time on a low speed decolorizing shaker.

⑨ fluorescent inverted microscope image acquisition, photographing in a short time after dyeing is completed (the image can be stored for 72h under the conditions of light shielding at 4 ℃ and PBS soaking, but the brightness is reduced to some extent), photographing 3 images/hole under a 100 x-fold microscope (each image contains green light TUNEL and blue light DAPI), and photographing 5 images/hole under a 200 x-fold microscope.

⑩ statistical analysis and mapping (selected) experiments were repeated 3 times independently, Excel and Photoshop were counted 200 × the number of green (TUNEL) and blue (DAPI) light per image taken under the mirror, and from the counts of each group, the apoptosis rate was calculated as TUNEL/DAPI × 100%, statistical analysis was performed and plotted using GraphPad Prism 7.

Flow cytometry to detect levels of apoptosis

Detecting the expression of apoptosis by flow cytometry according to eBioscience^TMAnnexin VApoptosis Detection Kit eFluor ^TM450 and eBioscience^TMPropidium Iodide (both available from Thermo Fisher Scientific Inc. eBioscience, USA)^TMAnnexin V Apoptosis DetectionKit eFluor ^TM450 cargo number: 88-8006-74, eBioscience^TMPropidium Iodide cargo number: BMS500PI) instructions were run on a flow cytometer to measure the apoptosis rate of each group after treatment with different drug combinations.

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 2 × 10^ 5/hole with cell counting plate, homogenizing in 6-hole plate, setting 2 multiple holes/group, and placing in 37 deg.C incubator overnight;

② cells are divided into a plurality of groups according to different drug combination designs after being attached to the wall, and 2mL of drug with corresponding concentration is given to act on the liver cancer cells for 36 h;

③ after the treatment time is over, collecting the old culture medium in the hole, placing in the flow tube marked with the group name, adding the adherent cells in the pancreatin digestion hole into the corresponding flow tube, centrifuging for 5min at the normal temperature at the speed of 1000rpm by a horizontal centrifuge, discarding the supernatant, adding PBS of 3 mL/tube, slightly shaking and mixing for 5s by an oscillator, centrifuging for 5min at the normal temperature at the speed of 1000rpm by the horizontal centrifuge, discarding the PBS, adding PBS of 3 mL/tube, slightly shaking and mixing for 5s by the oscillator, and centrifuging for 5min at the normal temperature at the speed of 1000rpm by the horizontal centrifuge;

④ abandoning PBS, adding 400 μ L Binding buffer into each tube, then lightly shaking and mixing for 5s by using an oscillator, sucking 200 μ L/tube into a new centrifuge tube with a pipette gun to serve as a reagent group, adding 5 μ L Annexin V-450 into each tube of the reagent group, lightly shaking and mixing for 5s by using the oscillator, incubating at normal temperature and in a dark place for 20min, after the incubation time is over, adding 10 μ L PI into each tube of the reagent group, lightly shaking and mixing for 5s by using the oscillator, setting a P1 gate (single cell group) and a cross gate (Annexin V-450(-), PI (-) as a living cell group, Annexin V-450(+), PI (-) as an early apoptosis cell group, Annexin V-450(+), and PI (+) as a late apoptosis cell group, Annexin V-450(-), and PI (+) as a necrotic cell group, and recording the proportion of each cell group;

⑤ experiments were repeated 3 times independently, with statistics based on the proportion of each quadrant population, the sum of the proportion of early apoptotic and late apoptotic populations in each group, statistical analysis and mapping using GraphPad Prism 7.

4. The experimental results are as follows: referring to fig. 7 and 8, regorafenib in combination with BMS-1 groups promotes the apoptosis of hepatoma cells more than the regorafenib monotherapy group and BMS-1 monotherapy group.

Example 5

1. Purpose of the experiment: the protein expression of the hepatoma cells is influenced by the combined administration of regorafenib and BMS-1.

And 2, detecting the expression of related proteins after the liver cancer cells are acted by each group of medicaments for 36 hours by using a Western Blot method.

3. The experimental method comprises the following steps:

western Blot experiment

① protein sample preparation:

selecting liver cancer cells which are in good state and proliferate to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing uniformly with fresh culture medium, counting 2 x 10^ 5/hole by adopting a cell counting plate, homogenizing the plates in a 6-hole plate, grouping according to different drug combination designs, and placing in a 37 ℃ incubator overnight. After the cells are attached to the wall, the cells are divided into a plurality of groups according to different drug combination designs, and 2mL of drugs with corresponding concentrations are given to act on the liver cancer cells for 36 hours. After the treatment time was over, cells in old medium were collected, adherent cells in wells were washed gently 1 time with PBS, and PBS was discarded after tilting 6-well plates for 5 min. An appropriate amount of a fresh cell lysate mixture (PMSF: phosphatase inhibitor: protease inhibitor: RIPA lysate: 1: 1:100) was prepared as needed, and the mixture was uniformly mixed by shaking using a shaker. Adding 80 mu L/hole of the mixture of the added cell lysate, fully scraping cells on an ice box by using a cell shovel after cracking for 10min on ice, inclining a culture plate for 5min, sucking cell cracking products into a 1.5mL EP tube, and using a 30% strength ultrasonic protein sample for 10s in an ice water environment, wherein the interval is 1s every time, and an ultrasonic head needs to be cleaned by using ultrapure water and wiped dry before each ultrasonic. Centrifuging at 12000g speed for 15min in 4 deg.C precooled ultracentrifuge, lightly taking EP tube, and collecting protein sample supernatant in newly labeled EP tube to avoid absorbing precipitate. Samples can be stored for short periods at-80 ℃ and it is recommended that proteins be denatured and stored to prevent protein degradation. Preparing a standard curve (BCA protein gradient concentration is in positive correlation with an OD value with a wavelength of 562 nm) according to an operation instruction of a BCA kit (purchased from Thermo Fisher company, goods number: 23228), detecting the concentration of a protein sample, sucking 2 mu L of the protein sample, 2 multiple holes/group, adding 50:1 of solution A and solution B into AB mixed solution with 200 mu L/hole, placing in an incubator at 37 ℃ for 30min, detecting the OD value with the wavelength of 562nm by using an enzyme-linked immunosorbent assay (ELISA), calculating the concentration of the protein sample according to a standard curve, preparing the protein sample stock solution into a protein sample (generally 2 ug/mu L) containing 1 × loading buffer by using RIPA cell lysis mixed solution and 5 × loading buffer, marking the group, concentration and date of the protein sample, oscillating and uniformly mixing by using an oscillator, centrifuging at low speed, carrying out metal bath at 100 ℃ for 10min, immediately cooling on ice, centrifuging at low speed for 30s, and storing at-80 ℃ for later use.

② SDS-PAGE gel electrophoresis:

a. preparing main instruments and reagents: SDS-PAGE kit, 1.5mm thick glass plate, thin glass plate, sponge sealing gasket, 15-hole comb, glass fixing clamp, glue pouring frame, double distilled water, electrophoresis frame, electrophoresis tank, electrophoresis liquid, ice box, liquid transferring gun, gun head and the like;

b. preparing glue: brushing the thin and thick glass plate by using a detergent, cleaning the thin and thick glass plate by using double distilled water, washing the surface by using ultrapure water, and drying the glass plate in a drying box for later use; the glass fixing clamp is used for fixing the glass plate with the thickness of 1.5mm and the thin glass plate, and the lower edges of the two glass plates are as far as possible on the same horizontal plane. Pressing the glass fixing clamp on the glue filling frame, and tightly pressing the sponge sealing pad on the lower edge of the glass plate; selecting the concentration of the separation gel according to the molecular weight of the target protein, preparing the separation gel according to a fixed ratio (sequentially adding required reagents, fully mixing uniformly, adding to a position (about 6.3mL) of 0.5-1.0cm below a green frame along the middle point of a thick glass plate), slowly adding double distilled water along the middle point of the thick glass plate by using a 1mL gun head, sealing, standing at room temperature for about 45min, and solidifying at a lower temperature for proper extension. When the separation gel is solidified to form a clear boundary with the double distilled water, the double distilled water is poured off and is sucked dry by filter paper, and the gel surface cannot be contacted. Preparing concentrated glue according to a fixed proportion, slowly filling the concentrated glue along the middle point of the thick glass plate until the thick glass plate is full, vertically inserting a 15-hole comb to avoid generating bubbles, and standing at room temperature for about 30min to solidify;

c. preparation before sample loading: when the concentrated glue is solidified and has no bubble or defect, the prepared glue is taken down, two glue blocks are arranged on an electrophoresis frame, and the thin glass plate faces inwards. The electrophoresis frame is placed in the electrophoresis tank after corresponding to the positive electrode and the negative electrode. Preparing an electrophoretic solution: 1000mL of 1 × electrophoresis solution was mixed with 100mL of 10 × electrophoresis solution and 900mL of double distilled water, and the mixture was stored at room temperature. Adding electrophoresis liquid into the inner groove, enabling the electrophoresis liquid to be submerged by more than 2cm of the thin glass plate and not to have bubbles, and taking out the comb vertically after paying attention to no liquid leakage in the groove;

d. loading: taking a protein sample at the temperature of minus 80 ℃, carrying out metal bath for 10min, then carrying out low-speed centrifugation for 30s, and then placing on ice; 3.5. mu.L of marker PM5100 and 20-40. mu.g of the mixed protein sample (no more than 30. mu.L of a 1.5mm 15-well glass plate) are added sequentially from left to right in the order of group by using a 10. mu.L lance tip or a 20. mu.L lance tip. And adding a proper amount of 1 × loadingbuffer into redundant lanes on two sides as required to prevent protein deviation.

e. Electrophoresis: adding 1X electrophoresis liquid into an outer groove of an electrophoresis frame to a corresponding scale mark, covering a power supply cover, correctly connecting an anode and a cathode, connecting a power supply, setting a constant voltage of 80V, starting electrophoresis, adjusting the voltage to 120V after a protein sample is completely electrophoresed from concentrated gel to separation gel (about 30-45min), separating until marker bands with all molecular weights clearly appear, and stopping electrophoresis when a marker with the minimum molecular weight approaches the lower edge of the gel.

③ film transfer:

a. main apparatus and reagents: fresh membrane transferring liquid, a power supply, an electrophoresis tank, a transfer tank, a membrane transferring clamp, black closed sponge, a tray, a glass prying sheet, a roller, a glass rod, an ice bag, a pencil, a PVDF membrane, filter paper, tweezers, scissors and the like.

b. Preparing before film transfer: preparing a membrane transfer solution (1 × 10 × membrane transfer solution: 100% methanol: double distilled water: 1:2:7) in advance, and precooling at 4 ℃. The left upper side of the membrane with 4.5cm × 5.5cm PVDF is cut for marking date, separating gel concentration, protein sample group, etc., and is soaked in methanol for 5min (positive charges on the membrane are activated to facilitate the combination with protein). The sponge, the filter paper and the membrane transferring clamp are completely soaked in the recovered membrane transferring liquid at 4 ℃ for standby.

c. Film transfer: after electrophoresis, the glass plate is taken out, the left hand four fingers support the glass plate, the thick glass plate faces upwards, the thin glass plate faces downwards, the thick glass plate is carefully pried by a prying glass sheet in a wet state, and the concentrated glue is vertically cut off. The rotary membrane clamp, the black sealing sponge and the filter paper which are soaked for recovering the rotary membrane liquid are sequentially placed in the tray, the glue is placed on the filter paper downwards, the PVDF membrane is placed next, the label surface is downward, the filter paper and the black sealing sponge are sequentially placed, the air bubbles are discharged by the back and forth gentle rolling of the roller, the glue can not be excessively deformed by force, and the rotary membrane effect is influenced. Turning on the transfer printing film clamp, vertically pushing into the transfer printing groove, putting the transfer printing groove into the electrophoresis tank according to the directions of black to black (cathode) and white to red (anode), simultaneously putting into an ice box, adding the freshly prepared transfer printing film liquid to the corresponding scale, covering a power supply cover, and putting the electrophoresis tank into an ice basin. The electrodes and the power supply are correctly connected, a 280mA constant current is set for 100min, the initial and final voltages are recorded, and interference factors are eliminated.

④ sealing:

an appropriate amount of 5% milk blocking solution (20mL 5% blocking solution ═ 1g skim milk powder +20mL 1 × TBST) was prepared, and the mixture was shaken and mixed. After the time for membrane transfer, the PVDF membrane was placed in a closed box with the labeled surface facing upward, washed once with 1 XTBST, discarded the TBST, added with 5% blocking solution, and then placed in a horizontal shaker at 50rpm and blocked at room temperature for 1 h. The blocking solution was recovered, washed 1 times with 1 × TBST, and the TBST was discarded.

⑤ antibody incubation:

preparing a proper amount of primary antibody with proper concentration by using a primary antibody diluent, placing the primary antibody on a horizontal shaking table at 40rpm, and incubating for 12-24h at 4 ℃. After recovery of the primary antibody, the primary antibody was washed 3 times for 10 min/time using a1 XTSSL 100rpm shaker. Preparing a proper amount of secondary antibody by using the recovered confining liquid according to the required concentration and the corresponding one-antibody species, placing the secondary antibody on a horizontal shaking table at 50rpm, and incubating for 1h at normal temperature. After discarding the secondary antibody, the secondary antibody was washed 3 times for 10 min/time in a horizontal shaker at 100 rpm.

⑥ exposure and development:

a. preparing main instruments and reagents: a developing machine, an exposure box, a film, an HRP luminescent substrate, absorbent paper, a centrifuge tube, tin foil paper, a timer, a liquid-transfering gun and a gun head, tweezers and the like; checking the state of the developing machine, and starting up for preheating. Taking out HRP luminescent substrate for rewarming 5-10min in advance;

b. in a dark room, the red light is turned on for operation. And clamping the PVDF film, absorbing residual liquid on the dry film by using absorbent paper, and placing the film on a preservative film of an exposure box. Preparing a proper amount of luminescent liquid from HRP luminescent substrate A liquid and B liquid in a ratio of 1:1 in a light-proof centrifugal tube, uniformly dropwise adding luminescent working liquid on a PVDF film by using a liquid transfer gun, covering a preservative film, and observing the luminescent effect. And taking 1-3 films, folding the upper right corner, putting the films along the lower left of the cassette in order, quickly closing the exposure box, and setting exposure time according to the brightness. And taking out the film after the preset time, putting the film into a film developing machine, and adjusting the exposure time according to the exposure result until the target proteins have bands with different strength, clearness and no impurity band.

c. Marking information such as marker molecular weight, antibody, experiment name, exposure time, exposure date and the like, and then acquiring an electronic scanning picture by using a scanner.

d. After independent repeated experiments, the gray value of the strip is calculated, the required strip is intercepted, and the results of the original film and the electronic version film are kept.

4. The experimental results are as follows: referring to fig. 9 and 10, regorafenib, BMS-1 single drug slightly up-regulated the expression of the apoptotic protein clearedparp, but had no significant effect on the proliferative protein PCNA. When the two medicines are combined, the level of the cleaned PARP protein is obviously higher than that of a control group and single medicines of regorafenib and BMS-1, and the expression level of PCNA can be reduced. The regorafenib combined BMS-1 group can down-regulate the expression of cyclins including Cyclin A2, Cyclin B1, Cyclin D1 and Cyclin D3, and influence the proliferation cycle of liver cancer cells. Compared with a single medicine group, the combined group has the most obvious effect of reducing the expression of three hepatoma cell Cyclin A2 and Cyclin B1, and mainly influences the progression of G1/S phase and G2/M phase of cells. Regorafenib is combined with BMS-1 to inhibit the proliferation of liver cancer cells by regulating cell cycle progression.

Example 6

1. Purpose of the experiment: regorafenib combined with BMS-1 can inhibit migration and invasion capacity of liver cancer cells

2. Transwell test was used to observe the effect of 6 μ M regorafenib in combination with 30 μ M BMS-1 on the migratory capacity of hepatoma cells.

3. The experimental method comprises the following steps:

hepatoma cell Transwell cell invasion assay

The principle is that polycarbonate membranes are arranged between the upper layer and the lower layer of the Transwell chamber and are separated, a layer of Matrigel (simulating in-vivo extracellular matrix) is paved on the upper chamber side, and cells can penetrate through the polycarbonate membranes to enter the lower chamber after secreting metalloproteases (MMPs) to degrade the Matrigel in the Matrigel environment, so that the invasion and movement of tumor cells in the body are simulated. The method comprises the steps of inoculating heavy suspension tumor cells in an FBS-free culture medium into an upper chamber, adding a culture medium containing 5% of FBS into a lower chamber, inducing the tumor cells in the upper chamber to secrete metalloproteases to degrade matrigel by utilizing the permeability of a polycarbonate membrane, then penetrating the matrigel into the lower chamber through the polycarbonate membrane, and measuring the invasion capacity of the tumor cells according to the number of the cells entering the lower chamber.

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 2 × 10^ 5/hole with cell counting plate in 6-hole plate, homogenizing, and placing in 37 deg.C incubator overnight;

② cells are divided into a plurality of groups according to different drug combination designs after being attached to the wall, and 2mL of drug with corresponding concentration is given to act on the liver cancer cells for 36 h;

③ preparation before experiment, matrix gel of Matrigel, gun head, Transwell chamber and 24 holes are put into a refrigerator for precooling at 4 ℃;

④ coating basement membrane, taking care that all the operations should be done on ice to avoid the solidification of Matrigel 40. mu.L of Matrigel was put on top of Transwell chamber polycarbonate membrane with 8 μm pore size to make Matrigel evenly cover the membrane and placed in 37 ℃ cell culture box for drying and solidification;

⑤ after the treatment time, using pancreatin to digest adherent cells in the hole, centrifuging for 5min at normal temperature with a horizontal centrifuge at 1000rpm, discarding the supernatant, resuspending with serum-free culture medium, adding 1 × 10^5 cells counted by a cell counting plate into the upper layer of the small chamber, wherein the volume of the culture medium in the upper chamber is no more than 300 μ L, adding 600 μ L of culture medium containing 10% FBS into the lower chamber, and culturing in a cell culture box at 37 ℃ for 24 h;

⑥ after the culture time is over, taking out the Transwell chamber, fixing with 4% paraformaldehyde for 10min, wiping the Matrigel stroma glue on the upper chamber with a cotton swab gently, washing with PBS gently for 2 times, dyeing with 1% crystal violet for 10min, and washing with PBS gently for 2 times;

⑦ after the chamber is naturally air-dried, using an OLYMPUS white light microscope to photograph and record the staining condition of different groups of cells, photographing 3/hole under a 100 times microscope, photographing 5/hole under a 200 times microscope, wherein the parameters of the photographed pictures of all groups in one experiment are kept consistent, and the parameters can not be modified after being adjusted in the whole process;

⑧ statistical analysis and mapping (selected) experiments were repeated 3 times independently and Excel and Photoshop counted the number of cells per image taken under a 200 Xmagnification microscope.

Transwell cell migration assay for hepatoma cells

The principle is that the upper layer and the lower layer of the Transwell chamber are separated by a polycarbonate membrane, and cells penetrate through the polycarbonate membrane to enter the lower chamber, so that the movement capability of tumor cells in a body is simulated. The method comprises the steps of inoculating the resuspended tumor cells in the FBS-free culture medium into an upper chamber, adding a culture medium containing 5% of FBS into a lower chamber, enabling the FBS in the lower chamber to have a tendency effect on the tumor cells in the upper chamber, inducing the tumor cells in the upper chamber to penetrate through a polycarbonate membrane to enter the lower chamber by utilizing the permeability of the polycarbonate membrane, and measuring the movement capacity of the tumor cells according to the number of the cells entering the lower chamber.

① selecting liver cancer cells with good state and proliferation growth to 70% -85% concentration, digesting with pancreatin, horizontally centrifuging, suspending and mixing with fresh culture medium, counting 2 × 10^ 5/hole with cell counting plate in 6-hole plate, homogenizing, and placing in 37 deg.C incubator overnight;

② cells are divided into a plurality of groups according to different drug combination designs after being attached to the wall, and 2mL of drug with corresponding concentration is given to act on the liver cancer cells for 36 h;

③ after the treatment time, using pancreatin to digest adherent cells in the hole, centrifuging for 5min at normal temperature with a horizontal centrifuge at 1000rpm, discarding the supernatant, resuspending with serum-free culture medium, adding 1 × 10^5 cells counted by a cell counting plate into the upper layer of the small chamber, wherein the volume of the culture medium in the upper chamber is no more than 300 μ L, adding 600 μ L of culture medium containing 10% FBS into the lower chamber, and culturing in a cell culture box at 37 ℃ for 12 h;

④ after the culture time is over, taking out the Transwell chamber, fixing with 4% paraformaldehyde for 10min, gently wiping the residual liver cancer cells in the upper layer which do not pass through the polycarbonate membrane with a cotton swab, gently washing with PBS for 2 times, staining with 1% crystal violet for 10min, and gently washing with PBS for 2 times;

⑤ after the chamber is naturally air-dried, using an OLYMPUS white light microscope to photograph and record the staining condition of different groups of cells, photographing 3/hole under a 100 times microscope, photographing 5/hole under a 200 times microscope, wherein the parameters of the photographed pictures of all groups in one experiment are kept consistent, and the parameters can not be modified after being adjusted in the whole process;

⑥ statistical analysis and mapping (selected) experiments were repeated 3 times independently and Excel and Photoshop counted the number of cells per image taken under a 200 Xmagnification microscope.

4. According to the experimental results, referring to fig. 11 and fig. 12, regorafenib combined with BMS-1 can synergistically inhibit invasion and migration of liver cancer cells.

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finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. Application of combination of regorafenib and a PD-1/PD-L1 inhibitor in preparation of a medicine for treating liver cancer.

2. The use of claim 1, wherein the PD-1/PD-L1 inhibitor is BMS-1.

3. A pharmaceutical composition for treating liver cancer, which comprises regorafenib and a PD-1/PD-L1 inhibitor.

4. The pharmaceutical combination of claim 3, wherein the PD-1/PD-L1 inhibitor is BMS-1.

5. The pharmaceutical combination according to claim 4, wherein the molar ratio of regorafenib to BMS-1 is 2-6: 30-40.

6. The pharmaceutical combination according to claim 5, wherein the regorafenib concentration is 2 μ M and the BMS-1 concentration is 30 μ M.

7. The pharmaceutical combination according to claim 5, wherein the regorafenib concentration is 4 μ M and the BMS-1 concentration is 30 μ M.

8. The pharmaceutical combination according to claim 5, wherein the regorafenib concentration is 6 μ M and the BMS-1 concentration is 30 μ M.

9. The pharmaceutical combination according to claim 5, wherein the regorafenib concentration is 2 μ M and the BMS-1 concentration is 40 μ M.

10. The pharmaceutical combination according to claim 5, wherein the regorafenib concentration is 4 μ M or 6 μ M and BMS-1 concentration is 40 μ M.

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