Uses of Trinistra

Abstract

The present invention provides the use of tranilast, in particular the use of tranilast in preparing a sorafenib resistance reversal agent or a drug for preventing and / or treating liver cancer in combination with sorafenib, belonging to the pharmaceutical field. The present invention obtains tranilast, a drug capable of improving sorafenib resistance, through drug screening, and verifies it in vivo and in vitro. The combination of tranilast and sorafenib of the present invention significantly improves the resistance of sorafenib, exhibits a good synergistic anti-liver tumor effect, and provides a new strategy for the clinical treatment of liver cancer. The combination of tranilast and sorafenib provided by the present invention has broad application prospects in the preparation of drugs for reversing sorafenib resistance and preventing and / or treating liver cancer.

Description

Technical Field

[0001] The invention belongs to the pharmaceutical field, and specifically relates to the use of tronilast, in particular to the use of tronilast in preparing a sorafenib resistance reversal agent or an anti-liver cancer drug used in combination with sorafenib. Background Art

[0002] Liver cancer is divided into primary liver cancer and secondary liver cancer. Primary liver cancer is mainly divided into hepatocellular carcinoma (HCC) (75%-85%) and intrahepatic cholangiocarcinoma (ICC) (10%-15%). The occurrence of liver cancer goes through the disease process of liver damage, liver fibrosis, cirrhosis, etc., which is a complex process with multiple genes and multiple steps. There are many pathogenic factors of liver cancer, such as chronic hepatitis virus infection, aflatoxin poisoning, long-term excessive drinking, tumor suppressor gene mutation, type 2 diabetes, obesity, fatty liver, etc. (EL-SERAG 2011). According to the data released by the Global Cancer Information Network (GLOBOCAN) in 2022, there are 865,269 new cases of primary liver cancer and 757,948 deaths worldwide, with an incidence-to-mortality ratio of 1:0.87. The incidence of liver cancer ranks fifth among all cancers, but its mortality rate ranks third among malignant tumors. These data show that liver cancer has become a disease that seriously threatens people's lives and health, and its treatment and diagnosis are very limited.

[0003] The treatment of hepatocellular carcinoma (HCC) is not as precise as other cancers, which can be treated based on their classification. Due to the high heterogeneity and complexity of liver cancer, the treatment of liver cancer can only be based on its staging. In addition, the liver capsule lacks nerves, and the tumor growth process rarely causes serious discomfort. Many patients are diagnosed in the late stage. Patients with advanced HCC often have multiple metastases of cancer cells, and surgical resection of the lesions is no longer realistic. Therefore, non-surgical treatment is the choice of more advanced HCC patients. Non-surgical treatment of HCC mainly includes multi-kinase inhibitor systemic treatment and immunotherapy. Sorafenib is one of the first-line drugs for the treatment of advanced hepatocellular carcinoma. Its chemical name is 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]aminoaminophenoxy]-n-methylpyridine-2-carboxamide, which is an oral multi-kinase inhibitor. It can block tumor cell proliferation by inhibiting the activity of Raf-1, B-Raf and kinases in the Ras / Raf / MEK / ERK signaling pathway. In addition, sorafenib can inhibit tumor proliferation and angiogenesis by targeting hepatocyte cytokine receptor (c-Kit), Fms-like tyrosine kinase (FLT-3), vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor (PDGFR-β) and other tyrosine kinases, and can also promote tumor cell apoptosis to play a therapeutic role. Phase III clinical data showed that sorafenib can significantly prolong the survival time of HCC patients compared with the placebo group, but some patients develop drug resistance in the short term. At present, although the FDA has also approved some other drugs for the treatment of advanced HCC, sorafenib was the only drug approved by the FDA for advanced HCC before 2018. It has a wealth of clinical drug use experience. Whether before or after surgery, sorafenib can often effectively improve the survival rate of HCC patients. It can be said that it is currently a more mature first-line drug for the treatment of advanced HCC. However, sorafenib monotherapy is not suitable for all HCC patients, and drug resistance often occurs in the short term of medication, which leads to patients being insensitive to sorafenib treatment or even treatment failure. Previous phase III clinical trial data showed that sorafenib can prolong the median survival of HCC patients by 3 months, but only about 35% to 43% of patients respond to sorafenib, and the rest of the patients are insensitive to sorafenib or develop resistance within a short period of time after taking it. At present, sorafenib resistance has become the main obstacle to its clinical treatment in patients with advanced HCC, limiting the efficacy and clinical use of sorafenib. Therefore, further exploring the mechanism of sorafenib resistance, screening and identifying the key factors of sorafenib resistance, and screening potential drugs to improve sorafenib resistance on this basis have a positive clinical effect and significance for improving resistance through strategies such as combination therapy and enhancing the efficacy of sorafenib in the clinical treatment of HCC patients.

[0004] How to further reduce the drug resistance of sorafenib against liver cancer is undoubtedly an urgent problem to be solved in current clinical trials. Tranilast, also known as tranilast, has a chemical formula of N-(3,4-dimethoxycinnamoyl) anthranilic acid and is an organic compound with a chemical formula of C 18 H 17 NO5 is mainly used to treat keloids and hypertrophic scars and anti-allergy, and is used as a drug for the skin and respiratory system. Studies have also found that TCR-Ts and quinistad can be used in combination to treat esophageal cancer. After searching, there is no report in the prior art that quinistad can reduce the resistance of sorafenib or that the two can be used in combination to treat hepatocellular carcinoma. Summary of the invention

[0005] In view of the above technical problems, the present invention intends to provide the application of tronilast, in particular, the use of tronilast in the preparation of sorafenib resistance reversal agents or the preparation of drugs for the prevention and / or treatment of liver cancer.

[0006] The present invention obtains the drug tronilast, which can improve the drug resistance of sorafenib, through drug screening, and verifies it in vivo and in vitro. The results show that tronilast can significantly improve the drug resistance of sorafenib, and the combination of the two shows a good synergistic anti-liver tumor effect.

[0007] The invention protects the use of tronilast in preparing a drug that is a sorafenib resistance reversal agent.

[0008] The present invention also protects the use of tronilast combined with sorafenib in preparing a drug for preventing and / or treating liver cancer.

[0009] Furthermore, the mass ratio of sorafenib to tronilast is 1:1-10.

[0010] Preferably, the mass ratio of sorafenib to tronilast is 1:3-5.

[0011] Furthermore, the drug dosage form includes injection or oral dosage, and the oral dosage includes tablets, capsules or pills.

[0012] Compared with the prior art, the present invention has the following technical effects:

[0013] (1) The present invention obtains the drug trenilast that can improve sorafenib resistance through drug screening, which provides a new idea for solving the problem of sorafenib resistance.

[0014] (2) The combined use of quinolones and sorafenib significantly improved the drug resistance of sorafenib and showed a good synergistic anti-liver tumor effect, providing a new strategy for the clinical treatment of liver cancer and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a flow chart for screening drugs combined with sorafenib;

[0016] Figure 2 The IC of the drug in three liver cancer cells 50 The result graph, where Figure 2 AC is the IC of tranistat, sorafenib and their combination in HCCLM3 cells 50 Graphs; Figure 2 DF is the IC of tranilast, sorafenib and their combination in HepG2 cells 50 Graphs; Figure 2 GI is the IC of tranilast, sorafenib and combination therapy in Hepa1-6 cells 50 Curve graph (drug concentrations are in μM);

[0017] Figure 3 To prove in vitro that tranistat improves sorafenib resistance, Figure 3 A is the flow cytometry and quantification diagram of Huh7 cells after 48 hours of treatment with 10 μM sorafenib, 50 μM quinacridone, or both. Figure 3 B is the flow cytometry and quantification diagram of HCCLM3 cells after 48 hours of addition of 15 μM sorafenib, 75 μM tranista, or 15 μM sorafenib and 75 μM tranista at the same time; Figure 3 C is the expression of PARP spliceosomes detected by western blot 48 hours after adding 10 μM sorafenib, 5 μM tranistat or both in Huh7 cells; Figure 3 D is the expression of PARP detected 48 hours after adding 25 μM sorafenib, 100 μM tranista, or 25 μM sorafenib and 100 μM tranista to HCCLM3 cells;

[0018] Figure 4 The results of the combined use of two drugs at different concentrations in three different types of liver cancer cells are shown in Figure 2. Figure 4 A: 20 μM sorafenib and different concentrations of trenilast were added to HCCLM3 cells to detect cell proliferation; Figure 4 B is the detection of cell proliferation by adding 10 μM sorafenib and different concentrations of trenilast to Huh7 cells; Figure 4 C is the detection of cell proliferation in Hepa1-6 cells after adding 20 μM sorafenib and different concentrations of trenilast; Figure 4 D is the detection of cell proliferation in HCCLM3 cells after adding 100 μM tranistat and different concentrations of sorafenib; Figure 4E is the detection of cell proliferation in Huh7 cells after adding 50 μM tranistat and different concentrations of sorafenib; Figure 4 F is the detection of cell proliferation in Hepa1-6 cells after adding 75 μM tranistat and different concentrations of sorafenib;

[0019] Figure 5 This is an in vivo experiment to prove the effect of combining quinistad with sorafenib. Figure 5 A is a schematic diagram of the technical route of the in vitro experiment in nude mice; Figure 5 B is a schematic diagram of tumors in naked mice in each group after drug administration; Figure 5 C is a graph showing the change in tumor volume over time after administration of sorafenib alone; Figure 5 D is a graph showing the changes in tumor volume over time after single and combined administration; Figure 5 E is the statistical graph of the final tumor volume of each group after drug administration; Figure 5 F is the statistical graph of the final tumor weight of each group after drug administration; Figure 5 G is a graph showing the change trend of body weight of mice in each group over time after drug administration. DETAILED DESCRIPTION

[0020] The exemplary embodiments of the present invention will be described in more detail below in conjunction with the accompanying drawings. Unless otherwise specified, the reagents and consumables used in the implementation process can be obtained through commercial channels.

[0021] Example 1 Screening of Trinilast

[0022] In order to find candidate drugs that have synergistic effects with sorafenib in the treatment of liver cancer, the inventors first tested the single-drug IC values ​​of 80 drugs in human liver cancer cells HCCLM3. 50 , uniformly take the same multiple of IC 50 The drug concentration was combined with different concentrations of sorafenib, and the synergy coefficient (CI) of the drug alone and in combination with sorafenib was calculated and compared, where CI < 1 means that the two drugs have a synergistic effect, CI = 1 means that the two drugs have an additive effect, and CI > 1 means that the two drugs have an antagonistic effect. Finally, the compound with the smaller synergy coefficient ( Figure 1 ). Through screening, it was found that the synergy coefficients of many drugs with sorafenib were less than 1, such as CXW39, CXW37, and CXW9. Among them, a compound tranilast was screened, which had the smallest synergy coefficient with sorafenib (Table 1).

[0023] Table 1 Single-drug IC of some compounds in human hepatoma cell line HCCLM3 50 And combined use of IC 50 Results Table

[0024]

[0025] To verify the effect, the inventors also conducted verification in two other liver cancer cells (HepG2 and Huh7) and obtained consistent results (Table 2).

[0026] Table 2 shows the IC values ​​of drugs in three types of liver cancer cells 50 Statistics of synergy coefficient

[0027]

[0028] In order to further study whether the combination of trenilast and sorafenib has a synergistic effect, the inventors detected the cell viability of trenilast, sorafenib and the combination drug in three types of liver cancer cells using CCK-8, and calculated and mapped the drug IC 50 The results showed that the combination of quinistad and sorafenib can significantly inhibit the proliferation of liver cancer cells, and the combination of quinistad and sorafenib can play a synergistic role in anti-liver cancer (such as Figure 2 ).

[0029] Example 2 Combination of tranilast and sorafenib promotes apoptosis of liver cancer cells (in vitro)

[0030] The mechanism of action of sorafenib against liver cancer is mainly to inhibit cancer cell proliferation and induce cell apoptosis. In addition to detecting cell proliferation, the present invention detected the effect of quinistad on sorafenib-induced cell apoptosis. PARP (poly ADP ribose polymerase) is a class of enzymes that play a key role in the DNA repair process. They play an important role in the DNA damage repair pathway, especially in the repair of single-strand breaks. Members of the PARP family participate in the recognition, repair and regulation of DNA damage and apoptosis by adding ADP ribose groups to their substrate proteins. During apoptosis, PARP is activated at the DNA break and is rapidly cleaved, causing it to lose activity. This cleavage can be detected by PARP antibodies, so PARP cleavage is often used as a biomarker for apoptosis.

[0031] The present invention uses sorafenib, tranilast at different concentrations and in combination to treat Huh7 and HCCLM3 cells, and observes the apoptosis of liver cancer cells, the proliferation of liver cancer cells and the PARP splicing state when the drugs are used alone or in combination.

[0032] The results showed that in Huh7 and HCCLM3 cells, by adding different concentrations of sorafenib and quinistad, the researchers observed that the number of apoptotic cancer cells increased significantly when the combination was used compared with sorafenib or quinistad alone (Figure 3A-B). This suggests that quinistad may improve sorafenib resistance by synergizing with sorafenib. In Huh7 cells and HCCLM3 cells, the number of PARP splicing bodies increased after combined use compared with single drug use, suggesting that the combination of drugs increased liver cancer cell apoptosis. The results of WB further proved that the combination of the two drugs improved sorafenib resistance and enhanced the anti-liver cancer effect ( Figure 3 CD).

[0033] like Figure 4 As shown in the figure, specific concentrations of sorafenib and different concentrations of tronista were added to three types of liver cancer cells and cell proliferation was detected. As the concentration of tronista increased, cell proliferation gradually slowed down ( Figure 4 AC), similarly, when a specific concentration of tranistat and different concentrations of sorafenib were added, cell proliferation was slowed down ( Figure 4 DF). These results indicate that troniest can significantly improve sorafenib resistance.

[0034] In summary, tranistat may improve sorafenib resistance in the treatment of liver cancer by synergizing with sorafenib, and may achieve this effect by promoting cell apoptosis and inhibiting cell proliferation.

[0035] Example 3: Trinista improves sorafenib resistance and its combined anti-liver cancer effect (in vivo nude mouse validation)

[0036] The inventors further verified the anti-liver cancer effect of the combination of troniest and sorafenib through animal experiments. Liver cancer cells were inoculated into nude mice to form a tumor model, and then different groups of mice were given corresponding drug treatment. Male immunodeficient mice aged 4 weeks and weighing 20 g were selected and injected with 5x10 6 When the subcutaneous tumor volume reaches 80 mm 3 The nude mice were randomly divided into 8 groups, namely the placebo group, sorafenib group, quinistrazol group, and sorafenib + quinistrazol group. The drugs were administered once every three days, and the mice were killed after four weeks of continuous administration. Figure 5In A, the dosing regimen and experimental timeline are shown. The dosing regimen includes different doses such as placebo, sorafenib 15 mg / kg, sorafenib 30 mg / kg, and sorafenib 60 mg / kg. Subcutaneous tumor tissue is obtained and its volume and weight are measured. Tumor volume change curve of each group (placebo, sorafenib 15 mg / kg, sorafenib 30 mg / kg, sorafenib 60 mg / kg) after administration (measured once a week); Tumor volume change curve of each group (placebo, 60 mg / kg of quinistad, sorafenib 30 mg / kg, sorafenib 30 mg / kg, sorafenib 30 mg / kg + quinistad 30 mg / kg, sorafenib 30 mg / kg + 60 mg / kg of quinistad, sorafenib 30 mg / kg + 120 mg / kg of quinistad) after administration (measured once a week).

[0037] The experimental results showed that as the concentration of sorafenib increased, the tumor inhibition effect gradually increased, but as the treatment time prolonged, the tumor inhibition rate decreased, reflecting the drug resistance of sorafenib ( Figure 5 C) reflects the effect of different doses of sorafenib on tumor growth. Figure 5 D It can be seen that quinistad alone has a certain tumor inhibitory effect, but it is not significant; the combination of sorafenib and quinistad significantly inhibits tumor growth, and as the concentration of quinistad increases, the inhibitory effect gradually increases ( Figure 5 D) reflects the effects of different doses of sorafenib and tranistat and their combination on tumor growth. Figure 5 EF shows that the combination of sorafenib and quinolones reduced tumor weight and volume, and the combination of the two drugs did not cause other side effects, and the weight of the mice did not decrease ( Figure 5 G). The results showed that the combination of quinistad and sorafenib can significantly inhibit tumor growth. The combination of quinistad and sorafenib showed good synergistic anti-liver tumor effect and enhanced anti-tumor effect.

[0038] In summary, the combination of quinistad and sorafenib can significantly improve the drug resistance of sorafenib, and may exert its anti-tumor effect by inhibiting tumor growth. The combination of the two drugs does not cause other side effects, and the mice do not lose weight. The combination of quinistad and sorafenib has a good anti-tumor effect and is an effective drug combination for the treatment of liver cancer.

Claims

1. The use of tranilast combined with sorafenib for preparing a drug for treating liver cancer, characterized in that: The mass ratio of sorafenib to tronilast is 1:1-5.

2. The use according to claim 1, characterized in that: The mass ratio of sorafenib to tronilast is 1:3-5.

3. The use according to claim 1, characterized in that: The dosage form of the medicine includes injection or oral preparation.

4. The use according to claim 3, characterized in that: The oral dosage form includes tablets, capsules or pills.

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