Application of Aurora-A kinase inhibitor combined with dorafenib in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses application of an Aurora-A kinase inhibitor combined with dorafenib in preparation of a medicine for treating hepatocellular carcinoma, and relates to the technical field of medicines. The Aurora-A kinase inhibitor is Alisertib, and the Aurora-A kinase inhibitor is Alisertib. The dorafenib pharmaceutical composition has the beneficial effects that the dorafenib and the Aurora-A inhibitor Alisertib are jointly applied to form the pharmaceutical composition, so that a remarkable synergistic anti-tumor effect is achieved; to a certain extent, the problem of drug resistance during single-drug treatment is effectively solved; moreover, the pharmaceutical composition can achieve a remarkable anti-tumor effect at a relatively low dosage, and the dosage demand when a single drug is used is reduced, so that the potential toxic and side effects are reduced, and the pharmaceutical composition has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and specifically to the use of an Aurora-A kinase inhibitor combined with donafenib in the preparation of a drug for treating hepatocellular carcinoma. Background Art

[0002] Primary liver cancer is one of the most common digestive system malignancies in the world, and the number of new patients in China accounts for about 45% of the world's total. Among them, hepatocellular carcinoma (HCC) is the most common type of liver cancer. Most of the early symptoms of HCC are not obvious, and many patients are already in the middle and late stages when diagnosed. Without effective treatment, the natural survival period is only 3 to 4 months, which seriously threatens human health. For patients who have lost the opportunity for local treatment such as surgery, systemic treatment becomes the only option to effectively delay the progression of the disease, and targeted therapy is an important part of systemic treatment and a research hotspot for HCC treatment.

[0003] Tyrosine kinase inhibitors (TKIs) are a class of small molecule targeted anti-tumor drugs that can act as competitive inhibitors of adenosine triphosphate binding to tyrosine kinases, or as tyrosine analogs, blocking the activity of tyrosine kinases, thereby inhibiting cell proliferation. Currently, TKIs have been used for systemic treatment of HCC, mainly including sorafenib, lenvatinib and donafenib. Donafenib, a deuterated derivative of sorafenib, is an innovative drug for the treatment of targeted hepatocellular carcinoma independently developed in my country. It has been approved as a standard treatment for advanced hepatocellular carcinoma due to its higher stability and better efficacy. However, the patient's survival can only be extended by 1.8 months. Although these targeted drugs provide a variety of options for the treatment of HCC, they often fail to achieve the expected clinical effects due to treatment failure or drug resistance. Therefore, there is an urgent need to find new treatment options for liver cancer.

[0004] Aurora-A kinase (AURKA) is a serine / threonine kinase that regulates the separation, maturation and spindle assembly processes of mitotic centrosomes. Its abnormal activation is closely related to the occurrence and development of various tumors. Accordingly, abnormal expression of the AURKA gene promotes the malignant biological behaviors of cancer, such as cell proliferation, survival, migration, invasion and increased stemness. Therefore, AURKA is considered to be a promising therapeutic target. Currently, a variety of inhibitors targeting Aurora-A kinase are in the preclinical and clinical evaluation stages. MLN8237 (also known as Alisertib) is a second-generation highly selective small molecule inhibitor of Aurora-A kinase developed by Millennium Pharmaceuticals, which can prevent the proliferation of tumor cells and induce apoptosis by inhibiting Aurora-A activity. In different xenograft tumor models, the inhibitor has shown significant anti-tumor activity. In recent years, combination drug strategies have shown great potential in tumor treatment, especially in improving efficacy and reducing drug resistance. At present, there is no basic research to explore whether Aurora-A kinase inhibitors can enhance the therapeutic effect and safety of Donafenib. Summary of the invention

[0005] The object of the present invention is to provide an application of an Aurora-A kinase inhibitor combined with dorafenib in the preparation of a drug for treating hepatocellular carcinoma, so as to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides an application of an Aurora-A kinase inhibitor combined with donafenib in the preparation of a drug for treating hepatocellular carcinoma.

[0008] More optimally, the Aurora-A kinase inhibitor is Alisertib.

[0009] More optimally, the dosage of Alisertib is 30-35 mg / kg, and the administration frequency is once every other day; the dosage of Donafenib is 30-35 mg / kg, and the administration frequency is once every other day.

[0010] More optimally, when the Aurora-A kinase inhibitor is used in combination with Donafenib, the ratio of the two is (1.25-40 μM): (0.625-20 μM).

[0011] More optimally, when the Aurora-A kinase inhibitor is used in combination with Donafenib, the ratio of the two is 2.5 μM:

[0012] (2.5-10μM).

[0013] More optimally, the drug for treating hepatocellular carcinoma further comprises a pharmaceutically acceptable carrier or excipient.

[0014] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0015] The present invention studies the inhibitory effect of Donafenib combined with Aurora-A inhibitor Alisertib on hepatocellular carcinoma. First, the drug synergy is evaluated in HCCLM3 and Huh7 cell lines, and the inhibitory effect of combined treatment on the short-term and long-term proliferation of the two cells is determined. The in vivo efficacy and toxic side effects of the combined treatment are further systematically evaluated in a xenograft tumor model derived from HCCLM3 cells. The results show that the combined use of Donafenib and Alisertib has a significant synergistic anti-tumor effect, which not only effectively overcomes the drug resistance problem during monotherapy, but also achieves significant anti-tumor effects at a lower dose, reducing the dosage requirement when using a single drug, thereby reducing potential toxic side effects, showing a good prospect for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a heat map of cell viability of HCCLM3 cells treated with different concentrations of Alisertib and Donafenib;

[0017] Figure 2 This is a heat map of cell viability of Huh7 cells treated with different concentrations of Alisertib and Donafenib;

[0018] Figure 3 This is the Fa-synergy index (CI) graph of Alisertib and Donafenib in HCCLM3 cells;

[0019] Figure 4 This is the Fa-synergy index (CI) graph of Alisertib and Donafenib in Huh7 cells;

[0020] Figure 5 The heat map of ZIP synergy scores of Alisertib and Donafenib in HCCLM3 cells;

[0021] Figure 6 Heat map of ZIP synergy scores of Alisertib and Donafenib in Huh7 cells;

[0022] Figure 7 The cell viability of HCCLM3 cells treated with different concentrations of donafenib combined with 2.5μM Alisertib;

[0023] Figure 8 The cell viability of Huh7 cells treated with different concentrations of donafenib combined with 2.5μM Alisertib;

[0024] Fig. 9 The clone formation images of HCCLM3 cells treated with different concentrations of Donafenib combined with 2.5μM Alisertib;

[0025] Fig.10 The clone formation images of Huh7 cells treated with different concentrations of Donafenib combined with 2.5μM Alisertib;

[0026] Fig.11 The cloning ability of HCCLM3 cells treated with different concentrations of donafenib combined with 2.5μM Alisertib;

[0027] Fig.12 The cloning ability of Huh7 cells treated with different concentrations of donafenib combined with 2.5 μM Alisertib;

[0028] Fig.13 Schematic diagram of the dosing regimen for the subcutaneous tumor-bearing model of hepatocellular carcinoma;

[0029] Fig.14 These are the tumors isolated from different groups after treatment.

[0030] Fig.15 Comparison of tumor mass in mice of different treatment groups;

[0031] Fig.16 The body weight change curves of mice in different treatment groups;

[0032] Fig.17 The curves of tumor volume changes in mice in different treatment groups;

[0033] Fig.18 Comparison of the indexes of heart, liver, spleen, lung and kidney of mice in different treatment groups;

[0034] Fig.19 Comparison of liver and kidney function indicators in mice in different treatment groups. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] It should be noted that there are no special restrictions on the purchase manufacturers of all raw materials involved in the present invention, which exemplarily include: human liver cancer cell lines (HCCLM3 and Huh7 cells) were purchased from the Cell Bank of the Chinese Academy of Sciences; the experimental mice were female BALB / c mice (5-6 weeks old) purchased from Jicui Yaokang Biological Co., Ltd.; Donafenib and Alisertib used in the present invention were purchased from MCE.

[0037] Example 1: Donafenib combined with Aurora-A inhibitor Alisertib synergistically inhibits liver cancer cells in vitro:

[0038] Hepatocellular carcinoma cell lines: HCCLM3 cells and Huh7 cells

[0039] Drug groups: Donafenib, Aurora-A inhibitor Alisertib, and blank control (PBS)

[0040] Experimental method: A series of concentration gradients were set according to the IC50 values ​​of Donafenib and Alisertib reported in the drug instructions and related literature. Cells were inoculated in 96-well plates according to the pre-set dosing matrix 7×7 (Donafenib concentration: 0uM, 0.625uM, 1.25uM, 2.5uM, 5uM, 10uM, 20uM; Alisertib concentration: 0uM, 1.25uM, 2.5uM, 5uM, 10uM, 20uM, 40uM). After the cells adhered to the wall, the corresponding concentration of drug-containing culture medium was replaced and cultured for 48 hours. The cell viability was determined using the CCK8 test (such as Figure 1 and Figure 2 As shown). Compusyn software and SynergyFinder software were then used to calculate the drug synergy score, respectively. Note: Compusyn software calculates the synergy index (CI) based on the Chou-Talalay method. A CI value of <1 indicates synergy, and the smaller the value, the stronger the synergy; a CI value of >1 indicates antagonism, and the larger the value, the stronger the antagonism; CI = 1 indicates additive effect. The synergy matrix heat map of the combination of two drugs is used to visualize the strength of the combination of two drugs. The ZIPsynergyscores calculated by SynergyFinder are calculated using the built-in zerointeractionpotency (ZIP) method (ZIPscore greater than 0 indicates synergy, and greater than 10 indicates strong synergy).

[0041] Experimental results: Donafenib and Alisertib were combined in a non-fixed ratio to treat HCCLM3 and Huh7 cell lines for 48 hours. The CI values ​​calculated by CompuSyn software based on the Chou-Talalay method showed that the CI values ​​were <1 in most concentration ranges, indicating that the combination of the two drugs showed an ideal synergistic killing effect in the above two hepatocellular carcinoma cell lines (such as Figure 3 and Figure 4 The drug synergy (zero interaction potency, ZIP) score was then calculated using SynergyFinder software, as shown in Figure 5 Figure 6 As shown, the average scores of the anti-tumor response caused by the drug interaction between HCCLM3 cells and Huh7 cells were 14.15 for HCCLM cells and 12.83 for Huh7 cells, and the strongest synergistic region scores were 26.09 and 30.37, respectively, which also demonstrated the synergistic anti-tumor effect of the two drugs (ZIP score >0 indicates synergy, and >10 indicates strong synergy).

[0042] Example 2: Donafenib combined with Alisertib synergistically inhibits short-term and long-term proliferation of hepatocellular carcinoma cells

[0043] Experimental method: Based on the optimal synergistic region confirmed in Example 1, we confirmed that the final dosing concentration of Alisertib is 2.5uM. The optimal concentration of Donafenib needs to be further confirmed, the optimal concentrations of HCCLM3 include 5uM and 10uM, and the optimal concentrations of Huh7 include 2.5uM and 5uM. Therefore, we first set the concentration gradient of the Donafenib monotherapy group: 0.625uM, 1.25uM, 2.5uM, 5uM, 10uM, and then combined with 2.5uM Alisertib to treat HCCLM3 cells and Huh7 cells respectively; after 48 hours of drug treatment, CCK8 was used to determine the number of cells, and the corresponding cell viability was calculated to evaluate the inhibitory effect of the combination of the two drugs on the short-term proliferation of cells; in order to confirm the effect of the drug combination treatment group on the long-term proliferation ability of cells, the inoculation density of HCCLM3 or Huh7 cells was 600 or 800 per six-well plate, respectively, and the medium was changed once every 2-3 days until the clones formed by single cells exceeded 50 cells under the microscope, and then the clones were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (BL802A, Biosharp, China), and then photographed for quantification.

[0044] Experimental results: The combination of the two drugs synergistically inhibited the short-term and long-term proliferation of cells, and showed a concentration-dependent result, with a statistically significant difference (p<0.05). Figure 7 and Figure 8 As shown, Donafenib combined with Alisertib has a synergistic inhibition of the short-term and long-term proliferation effects of hepatocellular carcinoma cells. Given an Alisertib concentration of 2.5uM, HCCLM3 and Huh7 cells were treated with Donafenib at different concentrations in combination or alone. Within the predicted optimal dosing concentration range, the cell viability of the combined treatment group was significantly inhibited, and both showed significant differences in cell viability (p<0.05); within the optimal concentration range, the clone formation ability showed concentration-dependent inhibition, and with the increase in the concentration of Donafenib, the cell proliferation ability showed significant proliferation inhibition in the combination drug group.

[0045] Example 3: Dorafenib combined with Alisertib inhibits the growth of hepatocellular carcinoma in vivo

[0046] Drug groups: control group, Donafenib group, Alisertib group, combined group

[0047] Experimental method: After in vitro amplification culture, the hepatocellular carcinoma (HCC) cell line HCCLM3 with good growth in the logarithmic growth phase was obtained. 6 HCCLM3 cells were injected subcutaneously into the right anterior armpit of each mouse to establish a tumor model. The formula (V = 1 / 2ab 2 ) to calculate the volume of the subcutaneous tumor, where (a) is the longest diameter and (b) is the shortest diameter. Once the volume (V) of the subcutaneous tumor reaches approximately 100 mm 3 , treatment began, and the corresponding drugs were given by subcutaneous injection. The specific dosing regimen included 30 mg / kg / day of Donafenib and 30 mg / kg / day of Alisertib, once every other day. Throughout the study, the tumor volume and body weight of the mice were carefully monitored to assess tumor progression and overall health. After completing seven doses, the mice were anesthetized for orbital bleeding. Subsequently, the tumor and major organs were removed for comprehensive ex vivo analysis and imaging.

[0048] Experimental results: In this study, we investigated the inhibitory effect of Donafenib combined with Alisertib on subcutaneous tumorigenesis of hepatocellular carcinoma. First, a cell-derived subcutaneous tumor model (CDX) was used. After the dosing regimen (such as Fig.13 As shown in Figure 2, it was observed that the tumor mass in the combination group was significantly reduced compared with the single-drug group (p<0.05) (as shown in Figure 2 Figure 14-15 Second, during the entire treatment process, there was no significant difference in the weight changes of mice in different groups, indicating that the combined drug has good safety for mice within the dose range used (as shown in Figure 2A). Fig.16In addition, over time, the subcutaneous tumor volume of mice in the combination group was significantly lower than that in the single-drug group, further confirming the significant inhibitory effect of the combination of Donafenib and Alisertib on tumor growth (as shown in Figure 2). Fig.17 Together, these results support the potential application value of this combination therapy in the treatment of hepatocellular carcinoma. Systemic toxicity in different groups was analyzed by monitoring the body weight of nude mice and assessing damage to major organs. As tumors enlarged, the body weight of mice in each treatment group increased over time. In our study, the use of donafenib alone and its combination with alisertib did not lead to weight loss (as shown in Figure 2). Fig.16 As shown in the figure, the difference between the two groups did not reach statistical significance, which indicates that the doses of Donafenib and Alisertib we used were safe. In addition, we dissected and weighed the main organs of the mice and took blood from their orbits for biochemical tests to evaluate their potential toxic and side effects. Fig.18 As shown in the figure, we measured the indexes of major organs such as heart, liver, spleen, lung and kidney by calculating organ index (%) = [(organ weight) ÷ (mouse weight)] × 100%. The results showed that no significant statistical differences were found between the groups, and the indexes of all organs were within the normal reference range. In addition, the liver and kidney functions of mice were analyzed by biochemical tests, including the levels of creatinine, urea nitrogen, alanine aminotransferase and aspartate aminotransferase, such as Fig.19 As shown in the results, these indicators were also within the normal reference range. This shows that the combined use of drugs did not cause obvious functional damage, proving that it is safe.

[0049] In summary, the combination therapy of Donafenib and Alisertib showed good efficacy and in vivo safety in the hepatocellular carcinoma model, providing a new idea for the treatment of hepatocellular carcinoma.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. Application of Aurora-A kinase inhibitor combined with dorafenib in the preparation of drugs for the treatment of hepatocellular carcinoma.

2. The use according to claim 1, characterized in that: The Aurora-A kinase inhibitor is Alisertib.

3. The use according to claim 1, characterized in that: The dosage of Alisertib is 30-35 mg / kg, and the administration frequency is once every other day; the dosage of Donafenib is 30-35 mg / kg, and the administration frequency is once every other day.

4. The use according to claim 1, characterized in that: When the Aurora-A kinase inhibitor is used in combination with Donafenib, the ratio of the two is (1.25-40 μM): (0.625-20 μM).

5. The use according to claim 4, characterized in that: When the Aurora-A kinase inhibitor is used in combination with Donafenib, the ratio between the two is 2.5 μM: (2.5-10 μM).

6. The use according to any one of claims 1 to 5, characterized in that: The drug for treating hepatocellular carcinoma also includes a pharmaceutically acceptable carrier or excipient.

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