Avatrombopag and new therapeutic use thereof

Abstract

The present disclosure relates to avatrombopag and new therapeutic use thereof. Specifically, the present disclosure relates to use of avatrombopag and a pharmaceutically acceptable salt thereof in the preparation of a drug, wherein the drug is used for treating breast cancer, and the pharmaceutically acceptable salt is selected from: maleate, tosylate, hydrochloride, succinate, sulfate, fumarate, acetate, phosphate, and citrate. The avatrombopag and the pharmaceutically acceptable salt thereof can effectively inhibit the level and activity of MET.

Description

Avatrombopag and its new therapeutic uses Technical Field

[0001] This disclosure relates to the fields of biology and medicine. Specifically, it relates to avatrombopag or its pharmaceutically acceptable salts and their clinical applications. Background Technology

[0002] Avatrombopag, also known as avatrombopag tablets, is an oral second-generation non-peptide thrombopoietin (TPO) receptor agonist approved by drug regulatory authorities for use in patients with ITP. 50 =3.3 nM), which can mimic the biological activity of TPO. TPO is a major regulator of normal platelet production.

[0003] Avatrombopag is primarily used to treat primary chronic immune thrombocytopenic purpura (ITP) in adults. It increases platelet production by activating intracellular signaling systems and promotes the production of platelets and megakaryocytes from hematopoietic progenitor cells. Its main therapeutic effects include: increasing platelet count by stimulating platelet production in the bone marrow, promoting platelet growth and maturation, and thus increasing the platelet count; and improving hemostasis by enhancing the patient's ability to stop bleeding and reducing the risk of bleeding.

[0004] Avatrombopag has demonstrated excellent efficacy and high safety in treating related diseases. Thrombocytopenia is a group of diseases characterized by platelet levels below normal, increasing the risk of bleeding. Avatrombopag has attracted widespread attention in this field, with its mechanism of action and clinical performance becoming a focus of research. Patients receiving avatrombopag treatment often exhibit an increase in platelet count, thereby reducing the risk of bleeding due to thrombocytopenia. Avatrombopag is commonly used to treat thrombocytopenia caused by thrombocytopenia in patients with chronic liver disease. Patients can improve platelet levels, alleviate related symptoms, and reduce the risk of complications caused by thrombocytopenia by taking avatrombopag orally.

[0005] However, the application of avatrombopag in cancer treatment is not mentioned in the existing technology. Summary of the Invention

[0006] According to some embodiments of this disclosure, the use of avatrombopag and its pharmaceutically acceptable salts in the preparation of medicines is provided.

[0007] Avatrombopag maleate, chemically named 1-[3-chloro-5-[[[4-(4-chloro-2-thienyl)-5-(4-cyclohexyl-1-piperazinyl)-2-thiazolyl]amino]carbonyl]-2-pyridyl]-4-piperidinecarboxylic acid maleate, is an oral thrombopoietin receptor agonist. Avatrombopag promotes platelet production by stimulating the proliferation and differentiation of megakaryocytes from bone marrow progenitor cells. Thrombopoietin induces a thrombopoietin receptor-dependent immune response that mediates megakaryocyte differentiation and proliferation, leading to an increase in platelet count. In May 2018, avatrombopag was approved by the FDA under the brand name Doptelet for the treatment of thrombocytopenia caused by chronic liver disease (CLD) in adults; it was the first drug approved by the FDA for this purpose.

[0008] In some embodiments of this disclosure, the use of avatrombopag and pharmaceutically acceptable salts thereof in the preparation of medicaments for treating breast cancer is provided.

[0009] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0010] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobroms, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetate, trifluoroacetate, propionates, hexanoates, octanoates, decanoates, undecenoates, glycolates, gluconates, lactates, sebates, adipates, glutarate, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbic acid salts, salicylates, 4-aminosalicylic acid salts, and naphthalene disulfonates.

[0011] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. In some embodiments, the inorganic salt is an ammonium, sodium, potassium, calcium, or magnesium salt. In other embodiments, it is a sodium salt. Salts derived from organic bases include, but are not limited to, the following: primary amines, secondary amines, and tertiary amines; substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Some embodiments include organic bases such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0012] In some embodiments of this disclosure, the pharmaceutically acceptable salt is selected from: maleate, toluenesulfonate, hydrochloride, succinate, sulfate, fumarate, acetate, phosphate, and citrate.

[0013] In particular, the use of avatrombopag maleate in the preparation of medicaments for the treatment of breast cancer is discussed in the specific implementation plan.

[0014] In some embodiments of this disclosure, avatrombopag is as shown in Formula I:

[0015] In some embodiments of this disclosure, avatrombopag and its pharmaceutically acceptable salts exhibit effects (any one or a combination thereof) on breast cancer cells or tissues (in vivo, in vitro, or ex vivo):

[0016] -Inhibits the growth of breast cancer cells or tissues;

[0017] -Inhibits the proliferation of breast cancer cells or tissues;

[0018] -Induces apoptosis in breast cancer cells or tissues;

[0019] - Reduce MET and p-MET protein levels.

[0020] In some embodiments of this disclosure, the breast cancer that is ER-positive is particularly suitable for treatment with avatrombopag and its pharmaceutically acceptable salts.

[0021] In a specific embodiment of this disclosure, the breast cancer suitable for treatment with avatrombopag and its pharmaceutically acceptable salts is ER-positive, PR-positive, HER2-negative breast cancer.

[0022] According to some embodiments of this disclosure, a method for reducing MET or p-MET protein levels in vitro is provided, comprising the steps of: exposing cells to an effective amount of avatrombopag and a pharmaceutically acceptable salt thereof; said pharmaceutically acceptable salt is selected from: maleate, toluenesulfonate, hydrochloride, succinate, sulfate, fumarate, acetate, phosphate, citrate.

[0023] In some embodiments, a method for reducing MET or p-MET protein levels in vitro is provided, comprising the steps of: exposing cells to an effective amount of avatrombopag maleate; wherein the cells are ER-positive breast cancer cells. In some specific embodiments, the breast cancer cells are ER-positive, PR-positive, and HER2-negative.

[0024] In the context of this disclosure, the “method for reducing MET or p-MET protein levels in vitro” does not relate to the diagnosis or treatment of a disease. Attached Figure Description

[0025] Figure 1. Effect of avatrombopag on the viability of MCF-7 cells (*P<0.05, **P<0.01, ***P<0.001 vs. the lowest concentration groups).

[0026] Figure 2. Effect of avatrombopag on apoptosis in MCF-7 cells (log bar is 40 μm).

[0027] Figure 3. Proportion of MCF-7 cells apoptotic due to avatrombopag (**P<0.01, ***P<0.001 vs. DMSO group).

[0028] Figures 4A to 4C. Effects of avatrombopag on the expression of MET and p-MET proteins in MCF-7 cells. Figures 4A to 4C show chemiluminescence imaging and statistical graphs of MET and p-MET protein expression levels, respectively. *P<0.05, ****P<0.0001.

[0029] Figure 5. Changes in transcriptional levels in MCF-7 cells after avatrombopag treatment. (A) PCA plot based on transcriptome sequencing data of MCF-7 cells after avatrombopag treatment; (B) Volcano plot of differentially expressed genes; (C) Heatmap of differentially expressed gene clusters; n=3.

[0030] Figure 6. Bubble diagram of transcriptional functional enrichment in MCF-7 cells after avatrombopag treatment.

[0031] Figure 7. Bubble diagram of transcriptional signaling pathway enrichment in MCF-7 cells after avatrombopag treatment.

[0032] Figure 8. Gene expression levels in MCF-7 cells after avatrombopag treatment. IFI6, IFI27, DDIT3, and DDIT4 are genes annotated in "Apoptosis Process," and CCL5 and STAT1 are genes annotated in "Regulation of Cell Population Proliferation." *P<0.01, ***P<0.001 vs. Control. Detailed Implementation

[0033] Example 1. Surface Plasmon Resonance

[0034] 1. Experimental Procedure

[0035] Table 1. Sample Information

[0036] (1) Reagent preparation

[0037] Prepare running buffer 1 (containing 2 mM potassium dihydrogen phosphate (KH2PO4), 137 mM sodium chloride (NaCl), 10 mM disodium hydrogen phosphate (Na2HPO4), and 2.7 mM potassium chloride (KCl)) by diluting PBS (10×) pH 7.4 10 times with deionized water. Filter the buffer through a 0.22 μM filter membrane before use.

[0038] Prepare Run Buffer 2 (containing 2 mM potassium dihydrogen phosphate (KH2PO4), 137 mM sodium chloride (NaCl), 10 mM disodium hydrogen phosphate (Na2HPO4), 2.7 mM potassium chloride (KCl), and 5% dimethyl sulfoxide (DMSO)) by diluting PBS (10×) with deionized water and adding DMSO. Filter the solution through a 0.22 μM filter before use.

[0039] (2) Chip preparation

[0040] The ligand (MET) was diluted 20-fold with a fixation reagent (10 mM sodium acetate, pH 4.5). First, the surface of the CM5 chip was activated for 900 s with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min. Next, the diluted ligand was injected into the experimental channels (Fc2 / 4) at a flow rate of 30 μL / min, with a fixation volume of approximately 18000 RU. Finally, the chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 900 s. The reference channel (Fc1 / 3) was only activated and blocked, without ligand coupling.

[0041] (3) Combined detection

[0042] a. Prepare solvent calibrators with different DMSO concentrations.

[0043] b. First, dissolve avatrombopag in DMSO to prepare a 10 mM stock solution. Then, prepare a 500 μM avatrombopag solution using PBS (10×) and ultrapure water, keeping the buffer composition consistent with running buffer 2. Dilute avatrombopag to 50 μM using running buffer 2. Inject the diluted avatrombopag sequentially into the experimental channel (Fc2 / 4) and the reference channel (Fc1 / 3) at a flow rate of 30 μL / min. Binding time is 60 s, and dissociation time is 60 s. Both binding and dissociation steps are performed in running buffer 2.

[0044] Table 2. Preparation of calibrators with different DMSO concentrations

[0045] Table 3. Combined with test conditions

[0046] 2. Experimental Results

[0047] The initial concentration of MET was 600 μg / ml, the working concentration was 30 μg / ml, the pH of the coupling buffer was 4, the flow rate was 30 μl / min, the time was 300*2s, the coupling volume was approximately 14000 RU, and the coupling channel was FC2.

[0048] Table 4 shows the binding signal intensity values ​​of MET and its corresponding avatrombopag, where the binding signal intensity is the response value of the absorbed light detected by the instrument.

[0049] The abnormally high binding signal between avatrombopag and the protein MET suggests that it is a relatively stable aggregate.

[0050] Table 4. Signal Intensity of MET Binding with Avatrombopag

[0051] Example 2. Effects of avatrombopag on the proliferation and cytotoxicity of MCF-7 cells.

[0052] 1. MCF-7 cell culture

[0053] MCF-7 breast cancer cells were cultured in α-MEM complete medium containing 10% FBS, 10 μg / mL insulin and 1% penicillin / streptomycin.

[0054] 2. Cell proliferation and toxicity assays

[0055] The effect of different concentrations of avatrombopag on the viability of MCF-7 cells was detected by CCK8 assay, and the IC50 of avatrombopag was determined. The specific steps are as follows:

[0056] (S1) Cell seeding: Prepare a single-cell suspension of well-grown MCF-7 cells and seed them into 96-well plates, approximately 4 × 10⁶ cells per well.3 One per hole;

[0057] (S2) The next day, after all the cells have adhered to the wall, add culture medium containing different concentrations of avatrombopag (see Table 5 for avatrombopag concentrations). Add 100 μL of culture medium to each well, and set up wells without cells and only culture medium as blank control (Blank group). At the same time, add PBS around the measurement wells to keep them moist and prevent liquid evaporation.

[0058] (S3) After culturing for another 24 hours, add 10 μL of CCK8 detection solution to each well and incubate at 37°C for 1 hour. After incubation, measure the absorbance (OD) at 450 nm using a microplate reader. Calculate the effect of avatrombopag on cell viability based on the OD value and calculate the half-maximal inhibitory concentration (IC50).

[0059] IC50 is the concentration of avatrombopag that corresponds to an inhibition rate of 50%.

[0060] Table 5. Concentration gradient settings for detecting avatrombopag in MCF-7 cells

[0061] 3. Experimental Results

[0062] After treating MCF-7 cells with different concentrations of avatrombopag for 24 h, the effects of different concentrations of avatrombopag on cell proliferation were detected, and the IC50 values ​​of the drug were calculated. Figure 1 shows the relationship between the logarithm of avatrombopag at different concentrations and its corresponding inhibition rate. The inhibition rate of avatrombopag at different concentration gradients and its upper and lower bounds were calculated.

[0063] The results showed that different concentrations of avatrombopag inhibited the proliferation of MCF-7 cells, with an IC50 concentration of 1,000 mg / L. 50 The concentration was 0.51 μM. The inhibitory effect of avatrombopag on cell proliferation was significantly different at each concentration compared with the lowest concentration (**P<0.01, ***P<0.001).

[0064] Example 3. Effect of avatrombopag on apoptosis in MCF-7 cells

[0065] 1. Specific experimental procedures for TUNEL staining

[0066] The concentration of avatrombopag in the treatment groups was determined based on the CCK8 results. The effect of avatrombopag on MCF-7 cell apoptosis was detected using the TUNEL apoptosis detection kit. The cells were divided into a control group, a solvent group (DMSO), and an avatrombopag treatment group. Cell apoptosis was observed in each group using a fluorescence microscope, and the proportion of apoptotic cells was counted. The specific steps for TUNEL staining were as follows:

[0067] (1) Cell treatment

[0068] MCF-7 cells were loaded at 2×10 7 Resuspend the cells at a concentration of 1 cell / mL in complete culture medium. Take 50-100 μL of the cell suspension and drop it onto a poly-L-lysine-coated glass slide. Incubate overnight. After the cells adhere to the wall, add an appropriate concentration of avatrombopag for 24 h.

[0069] Cell fixation: Immerse glass slides in freshly prepared 4% paraformaldehyde containing PBS and fix at 4°C for 25 min; wash twice with PBS for 5 min each time; add 0.2% Triton X-100 and incubate at room temperature for 5 min for permeation treatment; wash twice with PBS for 5 min each time.

[0070] (2) Labeling and Detection

[0071] Dilute 5× equilibrium buffer with deionized water at a ratio of 1:5;

[0072] Add 1× equilibration buffer to each sample to completely cover the sample area and incubate at room temperature for 10-30 min. While equilibrating the cells, thaw the FITC-12-dUTP labeled mixture on ice and prepare a sufficient amount of TdT incubation buffer according to Table 6. Negative control system: Prepare a control incubation buffer without TdT enzyme (use ddH2O instead of TdT enzyme).

[0073] Table 6. TdT Incubation Buffer

[0074] After equilibration, blot away most of the 1× equilibration buffer around the area with absorbent paper, then at a depth of 5 cm. 2 Add 50 μL of TdT incubation buffer to the cells (the slide should be protected from light during subsequent operations); cover the cells with a plastic coverslip to ensure even distribution of the reagent, place the slide in a humidified chamber, incubate at 37°C in the dark for 60 min, wash twice with PBS for 5 min each time; stain with 2 μg / mL DAPI solution (freshly prepared and diluted with PBS), stain at room temperature in the dark for 5 min, wash twice with PBS for 5 min each time; immediately take a picture under a fluorescence microscope.

[0075] 2. Experimental Results

[0076] Avatrombopag was used in a TUNEL assay to detect its effect on apoptosis in MCF-7 cells. Figure 2 shows the TUNEL staining results of MCF-7 cells after avatrombopag treatment; all images were taken at 200x magnification. The blue fluorescence indicated by DAPI represents the cell nucleus, and the green fluorescence indicated by TUNEL staining represents apoptotic cells.

[0077] The number of apoptotic cells in avatrombopag and the control group was obtained by TUNEL staining, and the corresponding proportion of positive cells was calculated (Table 7). The results showed that avatrombopag induced apoptosis in MCF-7 cells (Figure 3).

[0078] Table 7. Percentage of positive cells in avatrombopag and the control group (%)

[0079] Example 4. Effect of avatrombopag on MET protein expression in MCF-7 cells

[0080] 1. Specific experimental steps for protein extraction and Western blotting:

[0081] Based on the CCK8 results, an appropriate concentration of avatrombopag was selected as the treatment group. After treating MCF-7 cells for 24 hours, cells were collected, total protein was extracted, and the expression of relevant proteins was detected. The specific operational steps are as follows:

[0082] (1) Cell treatment

[0083] MCF-7 cells were seeded in 6-well plates, with a cell count of approximately 5 × 10⁶ cells. 5 One per hole;

[0084] The next day, after all the cells had adhered to the wall, 1 mL of fresh culture medium containing different concentrations of avatrombopag was added to each well, and the cells were incubated at 37°C for 24 hours.

[0085] (2) Protein extraction

[0086] Cells from each group were scraped off using a cell scraper, resuspended in PBS, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0087] Add protein lysis buffer containing RIPA:PMSF = 100:1 and incubate on ice for 30 minutes;

[0088] Cells were disrupted by sonication and incubated on ice for 10 minutes; then centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was collected as total cell protein.

[0089] (3) Protein quantification

[0090] Protein concentration was determined using an aminomelin quantitative kit, and the specific steps are as follows:

[0091] Add an appropriate amount of loading buffer to the protein supernatant, denature in a 95°C water bath for 5 min, and then quickly place on ice to cool.

[0092] Take 5 μL of each sample and drop it vertically onto the NC membrane. After the liquid dries, rinse the membrane in 50% methanol. After the color of the sample spot disappears, immerse the membrane in 0.1% amino black (30 ml methanol, 10 ml glacial acetic acid, 60 ml water, and 0.1 g amino black mixed thoroughly) for 10-20 min. After the color of the sample spot stabilizes, remove the membrane and rinse it in 50% methanol.

[0093] After the membrane surface is clean and the sample spots no longer lose color, let the membrane dry. Use a punch to remove all the blue parts from the sample spots and place them in 1 mL of 1 mol / L NaOH solution. Shake the membrane up and down until all the color on the NC membrane is eluted into the liquid. Take a 96-well plate and add 200 μL of the eluted liquid to the wells. Set up 3 replicate wells and use NaOH solution as a control. Measure the absorbance value at 650 nm.

[0094] Substitute the numerical values ​​into the standard curve plotted using BSA to calculate the concentration of the protein sample; determine the loading volume based on the measured sample concentration.

[0095] (4) Western imprint

[0096] Gel electrophoresis: Prepare the lower separating gel (10%) and the upper stacking gel (5%) in sequence. After the stacking gel solidifies, take 20 μg of total protein and load it onto the gel. The voltage is 80 V in the stacking gel and 120 V in the separating gel. Stop electrophoresis when the sample reaches 0.5 cm from the bottom edge of the gel plate.

[0097] Transfer: PVDF membrane was selected, and the wet transfer method was used for the transfer. The transfer current was 220mA and the transfer time was 1.5h.

[0098] Sealing: After sealing the PVDF membrane with 5% skim milk powder (prepared by TBST) for 1 hour, wash it three times with a shaker for 10 minutes each time.

[0099] Incubation of primary antibody: Dilute the primary antibody with 5% BSA (prepared with TBST) and incubate overnight at 4°C.

[0100] Secondary antibody incubation: Elute the PVDF membrane three times on a shaker for 10 minutes each time. Dilute the secondary antibody according to the instructions, immerse the membrane in the secondary antibody, incubate at room temperature on a shaker for 2 hours, and then elute three times as before.

[0101] Development: Chemiluminescence was used to develop the protein using a protein gel imaging system. ImageJ software was used to calculate the gray values ​​of the bands and analyze the expression of the target protein.

[0102] 2. Experimental Results

[0103] Changes in MET and p-MET protein levels were detected after avatrombopag treatment of MCF-7 cells.

[0104] After treating MCF-7 cells with avatrombopag for 24 h, total protein was extracted and Western blotted to detect changes in MET and p-MET protein levels. The results are shown in Figures 4A to 4C. After treating cells with 0.51 μM avatrombopag, the levels of MET and p-MET proteins were significantly reduced (P<0.0001).

[0105] Table 8. Gray value ratio of MET and p-MET bands after MCF-7 treatment with avatrombopag

[0106] Table 9. P-values ​​of independent t-tests for relative protein expression levels between the avatrombopag and DMSO groups (normalized relative to the DMSO group).

[0107] Example 5. Transcriptome sequencing of MCF-7 cells after avatrombopag treatment

[0108] Based on the CCK8 results, an appropriate concentration was selected as the avatrombopag concentration for the treatment group. After treating MCF-7 cells for 24 hours, the cells were collected and sent for sequencing.

[0109] 1. The specific operating steps are as follows:

[0110] (1) Transcriptome sequencing

[0111] MCF-7 cells were seeded in 6-well plates, with a cell count of approximately 5 × 10⁶ cells. 4 One per hole;

[0112] The next day, after all the cells had adhered to the wall, 1 mL of fresh culture medium containing avatrombopag was added to each well, and the cells were incubated at 37°C for 24 hours.

[0113] After 24 hours of treatment, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. 1 ml of TRIzol reagent was added to each well, and the cell lysate was collected into 1.5 ml of RNase-free EP tubes to extract total RNA.

[0114] Total RNA quantification and purity analysis were performed using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit. Samples with high-quality RNA (A260 / A280 range of 1.9 to 2.0) and RIN number >7.0 were used for subsequent library construction.

[0115] mRNA was purified from total RNA using Dynabeads Oligo(dT), and then fragmented using divalent cations at 94°C.

[0116] Using mRNA fragments as templates, cDNA first-strand synthesis was performed by PCR amplification using SuperScript™ II reverse transcriptase and random hexamer primers. Second-strand cDNA synthesis was performed using DNA polymerase I and RNase H.

[0117] The 3' end of the double-stranded cDNA is purified and repaired by adding an A, and then ligated with a sequencing adapter;

[0118] Enrichment and purification were performed using PCR amplification technology, and library size was detected using an Agilent 2100 instrument.

[0119] Paired-end sequencing of 2×150bp was performed using an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Hangzhou, China).

[0120] (2) Transcriptome bioinformatics analysis:

[0121] Raw data obtained from transcriptome sequencing was cleaned and read aligned to obtain clean data. Gene expression level analysis was performed based on the clean data and the results of comparison with human reference genome and gene model annotation files. Differentially expressed genes were screened (|log2FC|>1, P<0.05), and differentially expressed genes were enriched for GO function and KEGG pathway based on differential metabolites.

[0122] 2. Sequencing Experiment Results

[0123] To further clarify the major gene changes in MCF-7 cells after avatrombopag treatment, transcriptome sequencing was performed on MCF-7 cells after avatrombopag treatment.

[0124] Figure 5 shows the APCA results, which indicate that there are significant differences between the control group and the avatrombopag group, with clear distinctions in the confidence circles of the two groups. Meanwhile, there are small differences within the control group and the avatrombopag group, with relatively tight clustering of samples within both groups.

[0125] Figure 5 shows the results of the B volcano plot, which indicates that, compared with the control group, a total of 483 genes were differentially expressed after avatrombopag treatment (|log2FC|>1, P<0.05), with 202 significantly upregulated and 281 significantly downregulated. The MET gene showed no significant change.

[0126] Cluster analysis of differentially expressed genes showed significant up- or down-regulation of mRNA levels in the control group and the avatrombopag group. Up-regulated genes included DDIT3, which regulates apoptosis, while down-regulated genes included IFI6, which regulates apoptosis, and CCL5, which regulates cell proliferation.

[0127] To clarify the main functional changes in MCF-7 cells after avatrombopag treatment, transcriptome sequencing results of avatrombopag-treated MCF-7 cells were analyzed using Gene Ontology (GO) (on the Lianchuan Bio Cloud Platform (omicstudio.cn)). Enrichment was achieved in three functional categories: Biological Process, Cellular Components, and Molecular Functions. The top 25, top 15, and top 10 differentially expressed genes were identified and their corresponding cellular biological functions. The results showed that the differentially expressed genes mainly involved biological processes such as "innate immune response," "immune system processes," "immune response," "apoptosis," "protein ubiquitination," and "regulation of cell population proliferation"; cellular components such as "cytoplasm," "cell membrane," "extracellular space," "mitochondria," and "endoplasmic reticulum"; and molecular functions such as "ATP binding," "hydrolytic enzyme activity," "RNA binding," and "DNA binding."

[0128] Figure 6 shows the functional enrichment results of MCF-7 cells after avatrombopag treatment. GO analysis results indicate that differentially expressed genes after avatrombopag treatment were mainly enriched in "regulation of cell population proliferation", "apoptosis", "DNA damage response", "intrinsic apoptosis signaling pathway", and "protein ubiquitination". Therefore, avatrombopag treatment does indeed affect the proliferation and apoptosis levels of MCF-7 cells, and also involves inflammatory responses, cellular energy metabolism, and protein ubiquitination functions.

[0129] Figure 7 shows the results of pathway enrichment analysis of MCF-7 cells after avatrombopag treatment, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). The signaling pathways with the most enriched genes include the NOD-like receptor signaling pathway, JAK-STAT signaling pathway, cytokine-cytokine receptor interaction, RIG-1-like receptor signaling pathway, cytoplasmic DNA sensing pathway, Toll-like receptor signaling pathway, ubiquitin-mediated proteolysis, and nucleocytoplasmic transport. These results indicate that avatrombopag treatment of MCF-7 cells may induce changes in cell function through these corresponding signaling pathways.

[0130] Figure 8 shows the expression levels of key genes regulating apoptosis and cell population proliferation in MCF-7 cells after avatrombopag treatment. After avatrombopag treatment, the gene expression levels of Interferon Alpha Inducible 6 (IFI6), Interferon Alpha Inducible 27 (IFI27), DNA damage inducible transcript 3 (DDIT3), DNA damage inducible transcript 4 (DDIT4), CC motifchemokine ligand 5 (CCL5), and Signal Transducer And Activator Of Transcription 1 (STAT1) changed significantly in MCF-7 cells. Among them, the gene expression levels of IFI6 and IFI27 enriched in the "apoptosis process" (P<0.001) were significantly decreased, while the gene expression levels of DDIT3 (P<0.01) and DDIT4 (P<0.001) were significantly increased. On the other hand, the gene expression levels of CCL5 and STAT1 enriched in the "regulation of cell proliferation" (P<0.001) were significantly decreased.

[0131] Studies have shown that in breast cancer or other disease models, inhibiting the expression of IFI6 and IFI27 genes induces intracellular mitochondrial and DNA damage pathways, promoting apoptosis and reversing G1P3-induced migration and invasion, thereby improving the clinical manifestations of breast cancer patients. Increased expression of DDIT3 and DDIT4 genes triggers cell cycle arrest, leading to apoptosis. CCL5 enhances the proliferation and survival of MCF-7 breast cancer cells by activating mTOR, while STAT1, the gene encoding STAT protein, inhibits ErbB2 / Neu-mediated tumorigenesis. Therefore, significantly reduced expression levels of CCL5 and STAT1 genes can inhibit cell proliferation. In conclusion, avatrombopag treatment affects the apoptosis and proliferation processes of MCF-7 cells.

[0132] Example 6. In vivo pharmacodynamic experiment of antibody in mouse HCC827 model

[0133] 1. MCF-7 human breast cancer cells (purchased from ATCC) (5×10⁻⁶) 6 One xenograft was injected subcutaneously into the right hind limb of female BALB / c mice. The xenograft tumor in the nude mice was approximately 45 mm in size. 3 Animals were randomly divided into groups of 10 each, based on tumor size. The experimental groups were as follows:

[0134] 1) Model group (negative control, equal volume of avatrombopag solvent),

[0135] 2) Avatrombopag-1 group (high-dose experimental group, dosage was 30 mg / kg),

[0136] 3) Avatrombopag-2 group (medium-dose experimental group, dosage of 10 mg / kg) and

[0137] 4) Avatrombopag-3 group (low-dose experimental group, dosage was 3 mg / kg).

[0138] 2. Starting from day 0, the animals were administered the drug via gavage once daily for 2 to 3 weeks. The tumor volume was measured weekly using calipers, and the mice were weighed. After 4 weeks of drug treatment, the mice were euthanized by cervical dislocation under anesthesia, and the tumor tissue was harvested, weighed, and the data were recorded.

[0139] 3. Experimental results show that the avatrombopag disclosed in this invention can significantly inhibit the growth of tumor cells.

[0140] A portion of tumor tissue was fixed by immersion in 4% paraformaldehyde solution for subsequent TUNEL staining, and data were recorded. Experimental results showed that the avatrombopag disclosed in this paper significantly induced apoptosis in tumor cells.

[0141] Partial tumor tissue was used for Western blot analysis, and data were recorded. Experimental results showed that, according to this disclosure, avatrombopag treatment significantly reduced the levels of MET and p-MET proteins in cells.

Claims

1. The use of avatrombopag and its pharmaceutically acceptable salts in the preparation of medicines, wherein: The drug is used to treat breast cancer; The pharmaceutically acceptable salts are selected from: maleate, toluenesulfonate, hydrochloride, succinate, sulfate, fumarate, acetate, phosphate, and citrate.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt is a maleate.

3. The use according to claim 1, wherein the breast cancer is ER-positive breast cancer.

4. The use according to claim 1, wherein avatrombopag is as shown in Formula I:

5. The use according to claim 1 or 3, wherein the breast cancer is ER-positive, PR-positive, HER2-negative breast cancer.

6. The use according to claim 1 or 3, wherein avatrombopag and its pharmaceutically acceptable salts bind to human MET and statistically significantly reduce MET and p-MET protein levels.

7. A method for reducing MET or p-MET protein levels in vitro, comprising the steps of: To expose cells to an effective amount of avatrombopag and its pharmaceutically acceptable salts; The pharmaceutically acceptable salts are selected from: maleate, toluenesulfonate, hydrochloride, succinate, sulfate, fumarate, acetate, phosphate, and citrate; Preferably, the cells are breast cancer cells; More preferably, the breast cancer cells are ER positive; More preferably, the breast cancer cells are ER-positive, PR-positive, and HER2-negative.

8. The method of claim 7, which does not involve the diagnosis or treatment of a disease.

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