Combination drug of gossypol acetic acid and cdk4 / 6 inhibitor

Abstract

The application discloses a combined medicine of acacetin acetate and a CDK4 / 6 inhibitor, and relates to the technical field of medicines. The medicine composition comprises a CDK6 negative regulator and a CDK4 / 6 inhibitor; the CDK6 negative regulator comprises acacetin acetate or a baicalein derivative. The application proposes a treatment scheme of the combined medicine of acacetin acetate and the CDK4 / 6 inhibitor or a treatment scheme of the combined medicine of the baicalein derivative and the CDK4 / 6 inhibitor, which exhibits excellent synergistic effect and can effectively improve the treatment effect on patients who are not sensitive to the CDK4 / 6 inhibitor.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a combined drug of gossypol acetic acid and a CDK4 / 6 inhibitor. BACKGROUND

[0002] Cancer is a major cause of human death, and there are about 7 million deaths worldwide each year due to cancer. At the present stage, due to the lack of early diagnosis and screening technology for cancer, a large part of cancer patients have already reached the advanced stage of tumor at the first visit, and have lost the opportunity for surgical resection. Chemotherapy and radiotherapy are still the main treatment methods for advanced tumors at the present stage. However, this treatment method has limited efficacy, and most patients will eventually relapse. In addition, this method has a very large side effect, which greatly reduces the quality of life of patients.

[0003] Tumor targeted therapy is to target the functional molecules specifically expressed by tumors, and to design and develop targeted inhibitors including antibodies, small molecules, biological carriers, etc. Such targeted molecules can specifically inhibit the growth, movement and chemotherapy resistance of tumor cells, and have no destructive effect on normal tissue cells. Therefore, targeted therapy can greatly prolong the survival time and quality of life of patients. Cyclin-dependent kinase 4 / 6 (CDK4 / 6) is a tumor treatment target that has become a research hotspot in recent years. At the present stage, the FDA has approved three CDK4 / 6 inhibitors for tumor treatment, namely Palbociclib (Pabociclib), Ribociclib (Ribociclib) and Abemaciclib, and the total sales of the three drugs in 2017 will be nearly 3.3 billion US dollars. CDK4 / 6 is a key factor for regulating the cell cycle, and can trigger the transition of the cell cycle from the growth phase (G1 phase) to the DNA replication phase (S phase). Such inhibitors have strong selectivity for CDK4 / 6, inhibit the phosphorylation of Rb protein, block the CDK4 / 6-Rb pathway, and thus induce tumor cells to arrest in the G1 phase, thereby inhibiting the proliferation of tumor cells.

[0004] However, such CDK4 / 6 inhibitors have the following disadvantages: first, such kinase inhibitors only inhibit the kinase activity of CDK4 / 6 protein, while CDK6 protein also has strong transcriptional activity and can promote cell cycle progression and angiogenesis in a kinase-independent manner; second, clinical trial results show that a large number of patients are insensitive to such kinase inhibitors, and high expression of CDK6 protein is a major reason for clinical insensitivity. Therefore, the use of CDK4 / 6 inhibitors alone is insufficient to completely inhibit the pro-cancer function of CDK4 / 6.

[0005] Due to the above-mentioned deficiencies of the CDK4 / 6 inhibitors, the field has been committed to finding a combination drug that can have a synergistic effect with the CDK4 / 6 inhibitors, so as to further improve the treatment effect of cancer, especially the treatment effect of patients who are not sensitive to the CDK4 / 6 inhibitors. SUMMARY

[0006] The purpose of the present application is to provide a combination drug of baicalein derivatives and CDK4 / 6 inhibitors, and a treatment scheme of the combination drug of baicalein derivatives and CDK4 / 6 inhibitors or a treatment scheme of the combination drug of baicalein derivatives and CDK4 / 6 inhibitors, which shows excellent synergistic effect and can effectively improve the treatment effect of patients who are not sensitive to the CDK4 / 6 inhibitors.

[0007] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0008] The pharmaceutical composition comprises a CDK6 negative regulator and a CDK4 / 6 inhibitor. Based on a large number of experiments, the present application finds that LRPPRC (leucine-rich pentatricopeptide repeat-containing protein) is a key regulatory protein of CDK6 protein expression; LRPPRC directly binds and stabilizes the mRNA of CDK6, resulting in high expression of CDK6 protein. At the same time, the present application screens a LRPPRC specific small molecule inhibitor GAA (gossypol acetic acid), and finds that GAA can directly inhibit the binding of LRPPRC protein to the mRNA of CDK6, thereby reducing the stability of the latter, and can also directly degrade the LRPPRC protein, thereby leading to inhibition of the expression of CDK6 protein and cell cycle inhibition. Based on this, the present application proposes that the combination of the LRPPRC inhibitor GAA and the CDK4 / 6 inhibitor can have a stronger tumor inhibition effect. At the same time, the present application further proposes that the combination of baicalein derivatives, GAA and CKD4 / 6 kinase inhibitors can significantly inhibit the growth and clonogenicity of cancer cells, and show a stronger inhibition effect than the use of single drug, and has a synergistic enhancement.

[0009] Specifically, the CDK6 negative regulator includes at least one of an active substance that directly or indirectly reduces the stability of CDK6 mRNA, an active substance that promotes the degradation of CDK6 mRNA, and an active substance that reduces the level of CD6K protein.

[0010] Specifically, the CDK6 negative regulator includes gossypol acetic acid and / or baicalein derivatives.

[0011] Specifically, the CDK4 / 6 inhibitor includes Palbociclib, Ribociclib or Abemaciclib.

[0012] Further, the above-mentioned pharmaceutical composition also comprises a pharmaceutically acceptable carrier or excipient.

[0013] The application discloses use of baicalein derivatives in preparation of LRPPRC and CDK6 mRNA binding inhibitors.

[0014] The application also discloses use of baicalein derivatives in preparation of preparations for reducing CDK6 mRNA stability and / or promoting CDK6 mRNA degradation and / or reducing CD6K protein level.

[0015] The application further discloses use of baicalein derivatives and gossypol acetic acid in preparation of CDK4 / 6 inhibitor antitumor activity synergists.

[0016] The application further discloses use of baicalein derivatives, gossypol acetic acid and CDK4 / 6 inhibitors in preparation of drugs for preventing or treating tumor diseases.

[0017] The application further discloses use of baicalein derivatives in preparation of CDK4 / 6 inhibitor antitumor activity synergists.

[0018] The application further discloses use of baicalein derivatives and CDK4 / 6 inhibitors in preparation of drugs for preventing or treating tumor diseases.

[0019] Specifically, the above-mentioned baicalein derivatives comprise a product of 4-(4-pyridyloxy)benzenesulfonyl chloride hydrochloride sulfonated baicalein. The application uses 4-(4-pyridyloxy)benzenesulfonyl chloride hydrochloride to chemically modify baicalein to prepare baicalein derivatives, which exhibit better biological activity and improved antitumor ability; the baicalein derivatives are combined with CDK4 / 6 inhibitors to exhibit good synergistic effect and show stronger inhibitory effect than single drug use. It is speculated that the reason may lie in that synthesis of the baicalein derivatives can improve the problem of low solubility, improve the pharmacological activity and expand the clinical application range; and the baicalein derivatives can cause inhibition of CDK6 protein expression and cell cycle inhibition, and the combination of the baicalein derivatives and CDK4 / 6 inhibitors can achieve stronger tumor inhibitory effect. Meanwhile, the combination of the baicalein derivatives, GAA and CDK4 / 6 inhibitors achieves better tumor inhibitory effect.

[0020] Further, the chemical structure of the baicalein derivatives is shown in formula I:

[0021] I.

[0022] The preparation method of the above-mentioned baicalein derivatives comprises the following steps: mixing baicalein and 4-(4-pyridyloxy)benzenesulfonyl chloride hydrochloride, and preparing the baicalein derivatives under a catalytic system.

[0023] Further specifically, the preparation method of the baicalein derivative comprises the following steps:

[0024] Under nitrogen protection, baicalein and 4-(4-pyridyloxy) benzene sulfonyl chloride hydrochloride are mixed, anhydrous acetone is added, then pyridine is added, and the mixture is stirred under ice bath condition for 0.5-1.5 h, the reaction process is monitored by thin layer chromatography (TLC), after the reaction is completed, ice water is added and the mixture is left to precipitate, then filtration is performed, ice ethanol is used for washing, and then the baicalein derivative is obtained by using silica gel column chromatography.

[0025] Specifically, the molar ratio of baicalein and 4-(4-pyridyloxy) benzene sulfonyl chloride hydrochloride is 1:2.1-2.6; the solid-liquid ratio of baicalein and anhydrous acetone is 0.02-0.03 g:1 mL; and the volume ratio of pyridine and anhydrous acetone is 0.1-0.3:1.

[0026] Another purpose of the present application is to provide the use of the above-mentioned pharmaceutical composition in the preparation of a eukaryotic tumor cell proliferation inhibitor.

[0027] Specifically, the eukaryote includes a human or a mammal; and the tumor cell includes at least one of lung cancer cells, breast cancer cells, breast ductal carcinoma cells, liver cancer cells, esophageal cancer cells, colorectal cancer cells and prostate cancer cells, preferably at least one of lung adenocarcinoma cells, lung squamous carcinoma cells, breast cancer cells or breast ductal carcinoma cells.

[0028] Another purpose of the present application is to disclose the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a tumor disease.

[0029] Specifically, the tumor disease includes at least one of lung cancer, breast cancer, breast ductal carcinoma, liver cancer, esophageal cancer, colorectal cancer and prostate cancer, preferably at least one of lung adenocarcinoma, lung squamous carcinoma, breast cancer or breast ductal carcinoma.

[0030] It should be noted that Palbociclib (Pabociclib, trade name: Ibrance) is developed by Pfizer, which is the first CDK4 / 6 inhibitor on the market, and has been approved for the treatment of hormone receptor positive (HR+) and human epidermal growth factor receptor 2 negative (HER2-) locally advanced or metastatic breast cancer, and is currently undergoing a number of clinical trials of single drug or combined with other drugs for various indications. Ribociclib (Ribociclib, trade name: Kisqali) is developed by Novartis, which is a highly selective CDK4 / 6 inhibitor, and has been approved by FDA for the first-line treatment of HR+ and HER2- breast cancer. Abemaciclib (no unified Chinese name, some scholars translate it as: Boluomaxilin, trade name: Verzenio) is developed by Lilly, which has been approved by FDA for the treatment of HR+ and HER2- advanced or metastatic breast cancer.

[0031] The drug names or trade names used herein only represent the active ingredients thereof and should not be regarded as a limitation on the source of the drugs.

[0032] The term "carrier" or "excipient" can be any conventional carrier and excipient in the pharmaceutical field. The selection of specific carriers and excipients will depend on the administration mode or disease type and state for treating a specific patient. The preparation method of suitable pharmaceutical compositions for a specific administration mode is well within the knowledge of those skilled in the pharmaceutical field. For example, the pharmaceutically acceptable carriers or excipients can include carriers, excipients, diluents, fillers, solvents, support agents, binding agents, humectants, disintegrating agents, absorption promoters, surfactants, adsorption carriers and lubricants, etc. which are conventional in the pharmaceutical field. If necessary, flavoring agents, preservatives and sweeteners, etc. can also be included.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application provides a combination drug of GAA and CDK4 / 6 inhibitor, which has a significant effect on inhibiting tumor growth and is significantly better than the use of the two alone, indicating that the combination of the two can play a synergistic effect. At the same time, the present application proposes the mechanism of action of GAA, which can inhibit the expression of CDK6 protein, and the combination of GAA and CDK4 / 6 inhibitor can effectively improve the treatment effect of patients who are not sensitive to CDK4 / 6 inhibitor. In addition, GAA and CDK4 / 6 inhibitor have been proved to be used for clinical treatment, and have high safety. At the same time, the present application discloses a combination drug of baicalein derivative and / or acetyl gossypol combined with CDK4 / 6 inhibitor, which shows stronger inhibition effect, provides a new and more effective treatment scheme for clinical treatment of tumor diseases, and has good clinical application prospect.

[0035] Therefore, the application provides a combined medicine of baicalein derivatives and CDK4 / 6 inhibitors, proposes a combined medicine treatment scheme of baicalein derivatives and CDK4 / 6 inhibitors or a treatment scheme of baicalein derivatives, gossypol acetic acid and CDK4 / 6 inhibitors, which shows excellent synergistic effect and can effectively improve the treatment effect of patients who are not sensitive to CDK4 / 6 inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The enrichment degree detection result of mRNA of CDK6;

[0037] Figure 2 The western blot detection diagram of CDK6 and LRPPRC proteins in lung adenocarcinoma cell line A549, in which the hybridization signal of Actin protein is used as an internal reference for measuring the loading amount;

[0038] Figure 3 The growth rate curve of breast cancer cell line MCF7;

[0039] Figure 4 The survival rate detection diagram of lung adenocarcinoma cell line A549;

[0040] Figure 5 The survival rate detection diagram of lung cancer cell line H1299;

[0041] Figure 6 The microscope imaging diagram of breast cancer cell line BT474 and breast cancer cell line MCF7. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, each embodiment of the application will be described in detail below in combination with examples. However, those skilled in the art can understand that in each embodiment of the application, many technical details are proposed in order to make the reader better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed by the present application can be realized.

[0043] Example 1:

[0044] GAA inhibitor LRPPRC interacts with CDK6

[0045] Through RNA immunoprecipitation, it is verified that GAA can inhibit the binding of mRNA of CDK6 and LRPPRC protein. The experimental steps are as follows:

[0046] (1) Pancreatic enzyme digestion of lung adenocarcinoma cell line A549, lysis of cell extraction of cell lysate, respectively, to the cell lysate added final concentration of 10 µM GAA, with the addition of DMSO sample as a blank control group (NC group).

[0047] (2) Add 2 µg LRPPRC antibody (abcam company, ab97505) and 50 µL protein A / G magnetic beads to each tube, incubate overnight on a four-degree turntable. After incubation, wash with PBS buffer, extract the enriched mRNA on the protein A / G magnetic beads, and perform real-time fluorescent PCR after reverse transcription to detect the enrichment degree of CDK6 mRNA. The enrichment amount of the DMSO treated sample is normalized for statistical analysis. The test results are shown in Figure 1 (Compared with the NC group, the significance is represented by *, P-value <0.05 is represented by *, and P-value <0.01 is represented by **).

[0048] (3) Lung adenocarcinoma cell line A549 was cultured in a φ100mm culture dish. When the cell concentration reached about 60%, different concentrations of GAA (0 µM, 5 µM, 10 µM, 20 µM) were added for cell culture, and the cells were collected after 48h. Extract the cell protein, and after protein quantification, take 40 µg total protein for Western blot experiment, detect the protein expression level of CDK6 and LRPPRC, and take the hybridization signal of Actin protein as the internal reference for measuring the loading amount. The Western blot detection results are shown in Figure 2 .

[0049] From Figure 1 it can be seen that the addition of GAA significantly reduces the binding of CDK6 mRNA and LRPPRC. According to Figure 2 analysis, after GAA treatment, the protein level of LRPRPC and CDK6 is significantly reduced. It shows that GAA can affect the expression of CDK6 protein by blocking the binding of LRPPRC and CDK6 mRNA.

[0050] Example 2:

[0051] GAA combined with CDK4 / 6 inhibitor inhibits tumor growth rate

[0052] In breast cancer cell line MCF7, CDK4 / 6 inhibitor Palbociclib, GAA and their combination were used for treatment (with only PBS buffer as control group), and the cell growth rate curve was detected.

[0053] The test results are shown in Figure 3As shown, the use of Palbociclib alone, GAA alone can slow the growth rate of cells, but can not completely inhibit the growth of cells, when the two are used together, the cell growth rate is completely inhibited.

[0054] Example 3:

[0055] GAA has a synergistic effect with CDK4 / 6 inhibitors

[0056] The lung adenocarcinoma cell line A549 and lung cancer cell line H1299 in the logarithmic growth phase were trypsinized and counted, and were seeded in a 96-well plate at a density of 2000 cells / well, and were cultured for 24 h to adhere, and then the following reagents were added: four groups were set, and GAA (5 µM), Palbociclib (40 µM), and the combination of GAA (5 µM) + Palbociclib (40 µM) were added, and a PBS buffer was added as a blank control group. After 48 h of continuous culture, the cell survival rate was determined by MTS method, and the inhibition rate of each group was calculated.

[0057] The cell survival rate detection results are shown in Table 1. Figure 4 , and the cell survival rate of the blank control group was taken as 1.0 to characterize the cell survival rate of each group.

[0058] The synergistic effect of GAA and Palbociclib was calculated by using the standard synergistic index calculation formula Q = E(A+B) / (E(A)+E(B)-E(A)×E(B)), (E(A+B) is the inhibition rate of the combination of two drugs, E(A) is the inhibition rate of A drug alone, and E(B) is the inhibition rate of B drug alone). When the Q value is greater than 1.15, it is a synergistic effect. In lung adenocarcinoma cell line A549, E(GAA) = 0.36, E(Palbociclib) = 0.39, E(GAA+Palbociclib) = 0.73, Q = 1.20, showing synergistic effect. In lung cancer cell line H1299, E(GAA) = 0.35, E(Palbociclib) = 0.09, E(GAA+Palbociclib) = 0.66, Q = 1.66, showing synergistic effect.

[0059] Example 4:

[0060] GAA and CDK4 / 6 inhibitors inhibit tumor colony formation

[0061] The breast cancer cell line BT474 and breast cancer cell line MCF7 in the logarithmic growth phase were counted by trypsin digestion, respectively, and inoculated in ordinary 96-well plates at a density of 2000 cells per well, and cultured for 24 h to adhere, then the following reagents were added respectively: set four groups, respectively, add GAA (5 µM), Palbociclib (40 µM) and GAA (5 µM) + Palbociclib (40 µM) combination group, and add PBS buffer group as blank control group. Continue to culture for 48 h, then wash the cells with PBS buffer, methanol fixation for 5 min, add 0.5% crystal violet staining solution, stain for 10 min, then wash thoroughly, collect the imaging images of each group of cells under microscope.

[0062] The image acquisition results are shown in Figure 5 . According to Figure 5 analysis, the use of Palbociclib and GAA alone can reduce the number of cell clones, and when the two are used in combination, the number of clones can be inhibited by more than 95%, further illustrating that GAA and CDK4 / 6 inhibitor combination has good synergistic effect.

[0063] Example 5:

[0064] Baicalein derivative and CDK4 / 6 inhibitor combination has synergistic effect

[0065] Preparation of baicalein derivative:

[0066] Under nitrogen protection conditions, baicalein and 4-(4-pyridyloxy) benzene sulfonyl chloride hydrochloride (molar ratio of 1:2.5) were mixed, anhydrous acetone (solid-liquid ratio of baicalein to anhydrous acetone was 0.026 g:1 mL) was added, then pyridine (volume ratio of 0.2:1) was added, and the mixture was stirred in ice bath for 1 h, and the reaction progress was monitored by thin layer chromatography (TLC). After the reaction was completed, ice water was added and precipitated, filtered, ice ethanol was added, then silica gel column chromatography was used for purification (eluent was dichloromethane) to obtain the baicalein derivative, and the chemical structure is as follows:

[0067]

[0068] 1 H NMR (400 MHz, CDCl3): δ: 8.41, 7.06 (8H, Py-H), 7.45~7.80 (13H, Ar-H), 6.69 (s, 1H, C=CH), 5.87 (s, 1H, Ar-H).

[0069] Synergistic effect exploration

[0070] The lung adenocarcinoma cell line A549 and lung cancer cell line H1299 in the logarithmic growth phase were trypsinized and counted, and were seeded in a 96-well plate at a density of 2000 cells / well, and were cultured for 24 h to adhere, and then the following reagents were added: four groups were set, and the groups added with baicalein derivatives (5 µM), palbociclib (40 µM), and baicalein derivatives (5 µM) + palbociclib (40 µM) were added, and the group added with PBS buffer was used as a blank control group. After being continuously cultured for 48 h, the cell survival rate was determined by the MTS method, and the inhibition rate of each group was calculated. The cell survival rate detection result was characterized by taking the cell survival rate of the blank control group as 1.0, and the cell survival rate of each group was represented.

[0071] The synergistic effect of baicalein derivatives and palbociclib was statistically analyzed by using the standard synergistic index calculation formula Q = E(A+B) / (E(A)+E(B)-E(A)×E(B)), (E(A+B) is the inhibition rate of the combination of two drugs, E(A) is the inhibition rate of the single use of A drug, and E(B) is the inhibition rate of the single use of B drug). When the Q value is greater than 1.15, it is a synergistic effect. In the lung adenocarcinoma cell line A549, E(baicalein derivatives) = 0.31, E(palbociclib) = 0.39, E(baicalein derivatives + palbociclib) = 0.82, and Q = 1.42, showing a synergistic effect. In the lung cancer cell line H1299, E(baicalein derivatives) = 0.27, E(palbociclib) = 0.09, E(baicalein derivatives + palbociclib) = 0.73, and Q = 2.17, showing a synergistic effect.

[0072] Example 6:

[0073] Baicalein derivatives, GAA and CDK4 / 6 inhibitor combination have synergistic effect

[0074] The preparation of baicalein derivatives is the same as that of Example 5.

[0075] The lung adenocarcinoma cell line A549 and lung cancer cell line H1299 in the logarithmic growth phase were trypsin-digested and counted, and were seeded in a 96-well plate at a density of 2000 cells per well, and were cultured for 24 h to adhere to the wall, and then the following reagents were added: four groups were set, respectively, a combination group of GAA (2.5 µM) + Palbociclib (30 µM), a combination group of baicalein derivative (2.5 µM) + Palbociclib (30 µM), and a combination group of GAA (2.5 µM) + baicalein derivative (2.5 µM) + Palbociclib (30 µM), and a group added with PBS buffer was taken as a blank control group. After continuing to culture for 48 h, the cell survival rate was determined by MTS method, and the inhibition rate of each group was calculated. The cell survival rate detection result was characterized by taking the cell survival rate of the blank control group as 1.0.

[0076] The synergistic effect of baicalein derivative, GAA and Palbociclib was statistically analyzed by using a standard synergistic index calculation formula Q = E(A+B) / (E(A) + E(B) - E(A) x E(B)), (E(A+B) is the inhibition rate of the combination of three drugs, E(A) is the inhibition rate of the combination of GAA and Palbociclib, and E(B) is the inhibition rate of the combination of baicalein derivative and Palbociclib). When the Q value is greater than 1.15, it is a synergistic effect. In the lung adenocarcinoma cell line A549, E(GAA+Palbociclib) = 0.33, E(baicalein derivative+Palbociclib) = 0.39, E(baicalein derivative+GAA+Palbociclib) = 0.94, and Q = 1.59, showing a synergistic effect. In the lung cancer cell line H1299, E(GAA+Palbociclib) = 0.24, E(baicalein derivative+Palbociclib) = 0.31, E(baicalein derivative+GAA+Palbociclib) = 0.91, and Q = 1.91, showing a synergistic effect.

[0077] It should be noted that the examples are verified by taking lung adenocarcinoma, lung cancer, breast cancer cells as examples. Of course, colon cancer, rectal cancer, esophageal cancer, lung squamous cell carcinoma, pancreatic cancer, gastric cancer and the like can also be taken, and the present embodiment is not limited thereto.

[0078] The conventional techniques in the above examples are prior art known to those skilled in the art, and therefore will not be described in detail here.

[0079] The above description is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A pharmaceutical composition comprising a CDK6 negative regulator and a CDK4 / 6 inhibitor, wherein the CDK6 negative regulator is a baicalein derivative and the CDK4 / 6 inhibitor is palbociclib, and the baicalein derivative is prepared by chemically modifying baicalein with 4-(4-pyridoxy)benzenesulfonyl chloride hydrochloride.

2. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for the prevention and / or treatment of tumor diseases, wherein the tumor is non-small cell lung cancer.

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