Application of Avasimibe in preparation of medicine for treating nasopharynx cancer or oral cancer

Abstract

The invention relates to an application of Avasimibe in preparation of a medicine for treating nasopharynx cancer or oral cancer, and belongs to the field of tumor targeted therapy. An in-vitro cell experiment and an in-vivo animal model experiment verify the anti-tumor effect of the compound. Experiments show that Avasimibe can significantly inhibit the activity, proliferation ability, healing ability and cloning ability of nasopharynx cancer cells and oral cancer cells, can regulate cell cycle arrest in a dose-dependent manner, and induces cell apoptosis. In vivo experiments, the tumor volume of nude mice in an Avasimibe treatment group is remarkably reduced, serum cholesterol is remarkably reduced, and serum biochemical analysis shows that compared with a control group, the Avasimibe treatment group has no statistical difference in indexes such as aspartate transaminase (AST) / glutamic-pyruvic transaminase (ALT) and creatinine (Cr). The invention provides a new indication for resisting nasopharynx cancer and oral cancer for the existing compound Avassimibe, has the clinical advantages of low toxicity and capability of overcoming drug resistance, and provides a new treatment choice for patients with nasopharynx cancer and oral cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeted therapy, and specifically relates to the use of Avasimibe (Chinese name: Avasimibe) in the preparation of a medicament for treating nasopharyngeal carcinoma or oral cancer. Background Art

[0002] Nasopharyngeal carcinoma (NPC) and oral squamous cell carcinoma (OSCC) are common malignancies with high morbidity and mortality. Despite recent advances in cancer treatment, the treatment of these two cancers still faces numerous challenges. Traditional treatments, including radiotherapy, chemotherapy, and surgery, can control tumor growth to a certain extent, but are often associated with severe adverse reactions, such as nausea, vomiting, hair loss, and bone marrow suppression, which severely impact patients' quality of life. Furthermore, targeted drugs are scarce, and cancers are prone to developing drug resistance and resistance to chemotherapy drugs. Due to the hidden anatomical location of the nasopharynx and atypical early symptoms, patients with advanced NPC often fail treatment, primarily due to recurrence and metastasis, and the five-year survival rate for patients with recurrence and metastasis is low. Furthermore, in the case of oral cancer, suboptimal treatment efficacy and toxic side effects remain major challenges in clinical treatment. Safe and effective treatment strategies are limited, primarily due to the complex tumor immune microenvironment. To alleviate the scarcity of existing therapeutic agents, the development of new, low-toxic, targeted drugs is necessary.

[0003] Metabolic reprogramming is considered a key hallmark of cancer, and activated de novo lipid synthesis is considered an emerging hallmark of cancer. The key enzyme mediating cholesterol synthesis is sterol O-acyltransferase (SOAT), also known as acyl-CoA:cholesterol acyltransferase. Avasimibe is a bioavailable acetyl-CoA acetyltransferase inhibitor that can reduce lipid droplet accumulation in tumor cells, cut off energy supply, and achieve tumor cell inhibition. Avasimibe is currently only used as an investigational compound in preclinical and early clinical studies for atherosclerosis, and there is no published evidence supporting its clinical use in the treatment of nasopharyngeal and oral cancers. Summary of the Invention

[0004] In response to the problems existing in the above-mentioned prior art, the present invention provides an application of Avasimibe in drugs for treating nasopharyngeal carcinoma or oral cancer, with the purpose of providing a new use of Avasimibe in drugs for treating nasopharyngeal carcinoma and oral cancer, solving the limitations of existing treatments, such as high toxicity and easy drug resistance, and verifying the clinical transformation potential of Avasimibe through the PDX model (Patient-DerivedXenograft, patient-derived xenograft model). The experimental results show that Avasimibe can significantly inhibit the viability, proliferation ability, migration ability and clone formation ability of nasopharyngeal carcinoma cells and oral cancer cells, and can regulate cell cycle arrest and induce cell apoptosis in a dose-dependent manner. Specifically, Avasimibe has a half inhibitory concentration (IC50) in inhibiting the proliferation of nasopharyngeal carcinoma (NPC) and oral cancer (OSCC) cells. 50 ) ranged from 49.95 to 62.65 μM.

[0005] In order to achieve the above object, the specific scheme of the present invention is as follows:

[0006] Use of Avasimibe in preparing a medicine for treating nasopharyngeal carcinoma or oral cancer, wherein Avasimibe is the sole active ingredient in the medicine for treating nasopharyngeal carcinoma or oral cancer.

[0007] Avasimibe can significantly inhibit the vitality, proliferation ability, migration ability and clone formation ability of nasopharyngeal carcinoma cells and oral cancer cells, and can regulate cell cycle arrest and induce cell apoptosis in a dose-dependent manner.

[0008] The drug for treating nasopharyngeal cancer or oral cancer comprises Avasimibe and a pharmaceutically acceptable carrier, wherein the carrier is dimethyl sulfoxide.

[0009] The dosage form of the drug for treating nasopharyngeal carcinoma or oral cancer is selected from oral preparations, injections, suppositories or local external preparations.

[0010] Avasimibe is used at a concentration range of 10 μM to 160 μM.

[0011] The dosage range of Avasimibe for treating nasopharyngeal carcinoma or oral cancer in animal models is 15 mg / kg per day.

[0012] The invention relates to the use of a composition containing Avasimibe in preparing a medicine for treating nasopharyngeal carcinoma or oral cancer. The composition comprises Avasimibe and pharmaceutically acceptable excipients.

[0013] Advantages of the present invention

[0014] 1. Providing a new treatment strategy: This invention provides the use of Avasimibe in the treatment of nasopharyngeal or oral cancer, particularly for patients with recurrent or metastatic disease and those resistant to conventional chemotherapy and radiotherapy. This provides a new option for clinical treatment and is expected to improve patient outcomes.

[0015] 2. Low toxicity and targeted efficacy: Avasimibe, an acetyl-CoA acetyltransferase inhibitor, can inhibit lipid synthesis in tumor cells, thereby cutting off energy supply and inhibiting tumor cell growth. This mechanism of action is highly targeted, reducing damage to normal cells and lowering the toxic side effects of treatment.

[0016] 3. Overcoming Drug Resistance: This study experimentally validated the inhibitory effects of Avasimibe on nasopharyngeal and oral cancer cells, including inhibition of cell viability, proliferation, migration, and colony formation. These results suggest that Avasimibe may have the potential to overcome drug resistance to traditional therapies, offering new hope for the treatment of drug-resistant patients.

[0017] 4. Extensive Experimental Validation: The present invention not only validates the anti-tumor effects of Avasimibe in in vitro cell studies but also in in vivo animal models. These experimental results provide a solid scientific basis for the clinical application of Avasimibe and enhance its potential for clinical translation.

[0018] 5. Avasimibe, a lipid-lowering drug of the present invention, can affect the malignant biological behavior of nasopharyngeal carcinoma and oral cancer. In in vitro experiments, it significantly inhibited the viability, proliferation, healing, and cloning abilities of nasopharyngeal and oral cancer cells, with no toxic effects on human embryonic kidney epithelial (HEK293T) cells. Avasimibe also induced cell cycle arrest in oral and nasopharyngeal cancer cells in a dose-dependent manner, with significant apoptosis observed after low-dose avasimibe treatment of tumor cells. Animal studies on nasopharyngeal carcinoma PDXs showed a significant reduction in tumor volume (vs. control group, p<0.05) and a significant decrease in serum cholesterol (p<0.001). Serum biochemical analysis showed no statistically significant differences in aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (Cr) levels between the avasimibe-treated and control groups.

[0019] In summary, the use of Avasimibe in the treatment of nasopharyngeal carcinoma or oral cancer, as demonstrated by comprehensive in vitro and in vivo validation, provides strong support for the clinical translation of Avasimibe. These findings not only help promote the use of Avasimibe in the treatment of nasopharyngeal carcinoma and oral cancer but also may offer new insights into the treatment of other related tumors. This approach not only addresses the limitations of existing treatments but also demonstrates multiple advantages, including low toxicity and targeting, overcoming drug resistance, and extensive experimental validation. These advantages make this invention highly innovative and clinically valuable, potentially offering a more effective treatment option for patients with nasopharyngeal carcinoma and oral cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the molecular structural formula of Avasimibe.

[0021] Figure 2 The figure shows the analysis of cell growth after 24 h of treatment with different concentrations of Avasimibe using CCK8 assay.

[0022] Figure 3 The figure shows the analysis of cell proliferation ability after treatment with different concentrations of Avasimibe for different time periods using CCK8 assay.

[0023] Figure 4 Avasimibe inhibits the migration of nasopharyngeal carcinoma cells and oral cancer cells, wherein (a) shows the wound healing assay after nasopharyngeal carcinoma cells and oral cancer cells were treated with Avasimibe for 7 hours and 6 hours, and (b) shows the quantitative results of migration rate.

[0024] Figure 5 This figure is a test of the clone-forming ability of cells treated with Avasimibe.

[0025] Figure 6 Figure 3 Flow cytometric analysis of the cell cycle distribution of nasopharyngeal carcinoma and oral cancer cells after avasimibe treatment.

[0026] Figure 7 The figure shows the flow cytometric analysis of apoptosis in nasopharyngeal carcinoma and oral cancer cell lines after 24 hours of Avasimibe treatment.

[0027] Figure 8Avasimibe inhibits the growth and metastasis of nasopharyngeal carcinoma in vivo, where (a) is a xenograft model established, with the DMSO control group on the upper side and the Avasimibe-treated group on the lower side; (b) to (d) are tumor size and growth curves indicating that Avasimibe can inhibit the proliferation of nasopharyngeal carcinoma cells in vivo; (e) The total cholesterol level in the serum of the Avasimibe-treated group was lower than that of the control group (p<0.01); (f) There was no significant difference in serum creatinine (Cr) between the Avasimibe-treated group and the control group; (g) There was no significant difference in serum aspartate aminotransferase (AST) / alanine aminotransferase (ALT) between the Avasimibe-treated group and the control group. DETAILED DESCRIPTION

[0028] The present invention will be further explained and described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that this specific embodiment is not intended to limit the scope of the present invention. The reagents and materials involved in this specific embodiment are all common commercially available products unless otherwise specified.

[0029] 1. Avasimibe in this example is a known SOAT1 inhibitor (CAS No. 166518-60-1). The molecular formula of Avasimibe is C29H43NO4S, with a molecular weight of 501.72. Its molecular structure is as follows: Figure 1 The chemical synthesis method of Avasimibe and its use in treating atherosclerosis are known in the prior art.

[0030] 2. How to use the drug

[0031] 1. Dosage range

[0032] The dose range of avasimibe for the treatment of nasopharyngeal carcinoma or oral cancer in animal models is 15 mg / kg body weight per day.

[0033] 2. The drug for treating nasopharyngeal carcinoma or oral cancer can be prepared in a variety of dosage forms, including but not limited to oral preparations, injections, suppositories, or topical preparations. The drug of the present invention can be prepared in a variety of dosage forms, including but not limited to oral preparations, injections, suppositories, and topical preparations. The preparation methods of these dosage forms are conventional methods well known to those skilled in the art.

[0034] 3. Concentration range

[0035] The Avasimibe drug powder used in this example was produced and provided by MedChemExpress (Cat. No. HY-13215). In in vitro experiments, the Avasimibe drug powder was prepared into a 190 μM stock solution using dimethyl sulfoxide (DMSO), and the working solution of Avasimibe was used at a concentration range of 10 μM to 160 μM. Avasimibe also exhibited significant inhibitory effects against nasopharyngeal carcinoma and oral cancer cell lines within a concentration range of 10 μM to 160 μM. In in vivo experiments in nude mice, the Avasimibe drug powder was prepared into a 100 mg / ml stock solution using DMSO. The dosage was diluted to 15 mg / kg per mouse body weight and prepared into a uniform suspension for administration, resulting in drug concentrations in the nude mice that effectively inhibited tumor cell growth.

[0036] The nasopharyngeal carcinoma cell lines (TW03, 5-8F) and oral cancer cell lines (SAS, CAL27) used were long-term laboratory-maintained. The nude mice used were provided by the Animal Experiment Center of Guangxi Medical University. No human samples were required for this example.

[0037] Cell Culture: Nasopharyngeal carcinoma and oral cancer cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were maintained in a 37°C, 5% CO2, and humidified incubator. For passaging, when cells reached 80%-90% confluency, the old medium was discarded and the cells were gently rinsed twice with PBS. The cells were then digested with 0.25% trypsin (containing EDTA) for 1-2 minutes. To terminate the digestion, complete serum-supplemented medium (≥3 times the volume of trypsin) was added and single-cell suspensions were obtained by pipetting. After centrifugation (1000 rpm, 5 minutes), the supernatant was discarded and the cells were passaged into new culture flasks at a 1:3 ratio. Fresh medium was replaced every 48-72 hours, and mycoplasma contamination was regularly monitored.

[0038] Statistical methods: SPSS 23.0 and GraphPad Prism 8.0 software were used for statistical analysis and plotting of the results. Two independent sample t-tests were used to compare the data between the two groups. When P < 0.05, it was considered statistically significant.

[0039] Experiment 1: IC50 detection by CCK-8 method

[0040] 1. Methods: Cell viability: CCK8 method was used to determine cell viability 24 h after adding 410 drugs from lipid metabolism compound library and screened. 3Nasopharyngeal carcinoma cell lines (TW03, 5-8F) and oral cancer cell lines (SAS, CAL27) were seeded into 96-well plates at a density of 100 μg / well and cultured for 24 h. Lipid metabolism compound library drugs (10 μmol / L) were added and incubated for another 24 h. The supernatant was discarded, and 110 μL of CCK8 working solution was added to each well. The cells were incubated at 37°C for 30 min, and the optical density (D) at 450 nm was measured using a microplate reader.

[0041] 50% inhibitory concentration: Nasopharyngeal carcinoma cell lines (TW03, 5-8F) and oral cancer cell lines (SAS, CAL27) were seeded in 96-well plates as described above. After 24 h of culture, the cancer cell lines (TW03, 5-8F, SAS, CAL27) were treated with Avasimibe (10, 20, 40, 80, and 160 μM dose groups) and dimethyl sulfoxide (DMSO), respectively. The cells were cultured for another 24 h. Cell viability was measured, and the 50% inhibition concentration (IC50) of Avasimibe was calculated using GraphPad Prism 8.0.

[0042] 2. Results: Avasimibe has an anti-tumor effect on nasopharyngeal carcinoma cells and oral cancer cells

[0043] After treating nasopharyngeal carcinoma cells (TW03, 5-8F) and oral cancer cells (CAL27, SAS) with varying concentrations of avasimibe for 24 hours, the OD values ​​and inhibition rates were measured using the CCK8 assay. The formula for calculating cell viability in the CCK8 assay is as follows: Cell viability (%) = (measured value - blank value) / (control value - blank value) × 100%. The IC50 values ​​of avasimibe for nasopharyngeal carcinoma cells TW03 and 5-8F were 62.65 μM and 49.95 μM, respectively. The IC50 values ​​of avasimibe for oral cancer cells SAS were 56.99 μM and 50.73 μM, respectively. Figure 2 These results fully confirm that Avasimibe can effectively inhibit the proliferation of nasopharyngeal carcinoma and oral cancer cells in vitro. At the same time, it was observed in the experiment that with the increase of drug concentration, the cell morphology gradually changed from polygonal to round and the ability to adhere to the wall was weakened, suggesting that Avasimibe may exert its anti-tumor effect by affecting the cell adhesion properties. Avasimibe showed significant selective toxicity to cancer cells: its IC in human embryonic kidney epithelial cells (HEK293T) was 50 was 729.4 μM, while the IC values ​​for oral cancer cells (CAL27: 50.73 μM; SAS: 56.99 μM) and nasopharyngeal cancer cells (TW03: 62.65 μM; 5-8F: 49.95 μM) were 50The value was reduced by approximately 15-fold (*p* < 0.001, one-way ANOVA).

[0044] Experiment 2: In vitro cell proliferation assay

[0045] 1. Methods: Cell proliferation activity was assessed using the CCK-8 assay. The principle of this assay is that dehydrogenases within living cells reduce the water-soluble tetrazolium salt (WST-8) in CCK-8 to an orange-yellow formazan compound. The amount of formazan produced is positively correlated with cell proliferation activity. Formazan content can be quantified by measuring absorbance at 450 nm (OD), indirectly reflecting viable cell number and proliferation capacity. Nasopharyngeal carcinoma cell lines (TW03, 5-8F) and oral cancer cell lines (SAS, CAL27) were seeded into 96-well plates and cultured for 24 hours. The cells were then treated with varying concentrations of avasimibe and dimethyl sulfoxide (DMSO). CCK-8 was then added at the same time for five consecutive days, and absorbance at 450 nm was measured at the same time. After acquiring data for 120 consecutive hours, proliferation curves were plotted for each group, with the number of days in culture as the horizontal axis and the OD value as the vertical axis.

[0046] 2. Results: Avasimibe inhibits the proliferation of nasopharyngeal carcinoma cells and oral cancer cells

[0047] The cell proliferation assay was performed using the CCK-8 method to compare the proliferation capacity of untreated cells and cells treated with Avasimibe. By calculating the percentage of cell viability in groups treated with different drug concentrations, Avasimibe showed significant proliferation inhibition on all four cancer cell lines, and this inhibition showed obvious dose-dependent and time-dependent characteristics, such as Figure 3 shown.

[0048] Experiment 3: Cell migration assay

[0049] 1. Method: Take cells in logarithmic growth phase, digest and centrifuge them, and then use complete culture medium to prepare single cell suspension. After counting, dilute the cell suspension to 5×10 5 / ml. Use sterile tweezers to place the Ibidi cell culture plug in the center of the well plate, and gently press the plug so that the bottom of the plug is close to the culture plate. Rinse the plug with 1XPBS to reduce the liquid force at the edge of the gap, and check whether there is PBS leakage from the bottom of the plug to prevent the inoculated cell suspension from leaking. Plant 100μl of cell suspension into the culture wells on both sides of the plug. After culturing at 37℃ overnight, pull out the plug, gently rinse the cells with 1XPBS, and then add culture medium containing 20μM Avasimibe. The control group is DMSO group. Continue to culture the cells in the incubator, observe the degree of cell migration under a microscope, and observe the cell migration after 7h of treatment for nasopharyngeal carcinoma cells and 6h of treatment for oral cancer cells and take pictures. Finally, use ImageJ to calculate the changes in cell migration area and calculate the cell migration rate.

[0050] 2. Results: Avasimibe inhibits the migration ability of nasopharyngeal carcinoma cells and oral cancer cells

[0051] The effect of Avassimibe on the migration ability of nasopharyngeal carcinoma cells and oral cancer cells was evaluated by cell scratch test. Compared with the control group, the percentage of wound healing in the experimental group treated with Avasimibe was significantly reduced, and the difference was statistically significant (P < 0.01, such as Figure 4 (a) and (b) in Figure 3) show that Avasimibe can inhibit the wound healing ability of nasopharyngeal carcinoma cells and oral cancer cells. This confirms that Avasimibe has a new function of inhibiting the migration of nasopharyngeal carcinoma and oral cancer cells, providing an important experimental basis for its development as an anti-metastatic drug.

[0052] Experiment 4: Clone formation survival test

[0053] 1. Method: 2 ml of culture medium containing 200 cells was inoculated into each well of a 6-well plate and cultured until colonies of 10 or more cells were present. A 20 μM Avasimibe working solution was then added for 24 hours, followed by replacement with normal culture medium. A DMSO negative control was established. Cells were cultured for 10 to 15 days, until colonies appeared and reached an appropriate size. After removing the culture medium, the cells were fixed and fixed with 4% tissue cell fixative for 15 minutes. After rinsing once with PBS, 1 ml of 0.1% crystal violet solution was added to each well and stained for 30 minutes. The stain was discarded, and the 6-well plate was rinsed with running water. Cell viability was determined by the number of colonies in each well.

[0054] 2. Results: Avasimibe inhibits the cell colony formation ability of nasopharyngeal carcinoma cells and oral cancer cells

[0055] The clone formation assay was used to detect the clone formation ability of nasopharyngeal carcinoma cells (5-8F) and oral cancer cells (CAL27, SAS) after Avasimibe treatment. The results showed that compared with the control group, the number of cell clones in the experimental group was significantly reduced after two weeks of culture ( Figure 5 ), and the number of colonies in the avasimibe-treated group was significantly reduced, and the size of the remaining colonies was significantly smaller, with cells loosely arranged. These results suggest that avasimibe can significantly inhibit the clonogenicity of nasopharyngeal and oral cancer cells. This experiment provides direct evidence for avasimibe's ability to inhibit the long-term survival and proliferation of tumor cells in vitro, further supporting its development as a potential anti-tumor drug.

[0056] Experiment 5: Flow cytometry detection of cell cycle distribution

[0057] 1. Methods: DNA was stained with propidium iodide (PI) and the cell cycle distribution (G0 / G1 phase, S phase, G2 / M phase) was detected by flow cytometry. 5 Cells were seeded / well in a 6-well plate and cultured until the logarithmic growth phase. A 20 μM Avasimibe working solution was added for 24 hours. A DMSO negative control group was established. Cell pellets were harvested according to the normal protocol and fixed overnight with pre-chilled 70% ethanol at 4°C. Ethanol was removed by centrifugation and the cells were washed twice with PBS. Then, 500 μl of DNA staining solution was added and incubated in a dark tube for 30 minutes. Cell cycle distribution was then analyzed by flow cytometry, and data were analyzed using FlowJo software.

[0058] 2. Results: Avasimibe induces cell cycle arrest in oral cancer cells and nasopharyngeal cancer cells

[0059] To explore the cause of the decreased cell proliferation, the cell cycle of nasopharyngeal carcinoma cells (5-8F) and oral cancer cells (CAL27, SAS) was analyzed by flow cytometry. The results showed that Avasimibe treatment for 24 hours could trigger G1 phase arrest in oral cancer cells and S phase cell cycle arrest in nasopharyngeal cancer cells, as shown in Figure 3. Figure 6 shown.

[0060] Experiment 6: Detection of cell apoptosis rate by flow cytometry

[0061] 1. Methods: To determine the rate of cell apoptosis, apoptotic cells were analyzed by flow cytometry using the Annexin V-APC / 7-AAD Apoptosis Detection Kit (Lianke Biotechnology, AP105) according to the manufacturer's instructions. Cells were cultured in 100 mm dishes containing 20 μM Avasimibe for 24 hours. A DMSO negative control was established. After harvest, cells were incubated with Annexin V-APC and 7-AAD at room temperature for 15 minutes. Flow cytometry was then performed to analyze the rate of cell apoptosis. Data were analyzed using FlowJo software.

[0062] 2. Results: Avasimibe-induced apoptosis in nasopharyngeal carcinoma cells and oral cancer cells

[0063] To verify the effect of Avasimibe on apoptosis in nasopharyngeal carcinoma cells (5-8F) and oral cancer cells (CAL27, SAS), Annexin V-APC / 7-AAD double staining was used to determine whether Avasimibe induces apoptosis. The results showed that Avasimibe can induce apoptosis in 5-8F, CAL27 and SAS cells. Figure 7 shown.

[0064] Experiment 7: Establishment of a Nude Mouse Nasopharyngeal Carcinoma Xenograft Model

[0065] 1. Methods: This experiment used male nude mice, aged 4-6 weeks and weighing 14-16 grams, housed under specific pathogen-free (SPF) conditions, obtained from the Guangxi Medical University Experimental Center. Tumor-bearing and non-tumor-bearing mouse models (xenograft) were established. PDX (Patient-Derived Xenograft) models are highly clinically relevant tumor models established by directly transplanting fresh patient tumor tissue into immunodeficient animals (such as nude mice). Their core value lies in their ability to maximize the preservation of the biological characteristics of the original tumor and have become the gold standard tool for oncology research and drug development. The PDX models used in this experiment were proprietary to our laboratory.

[0066] (1) Tumor sample source: Nude mice stably cultured in the laboratory for NPC PDX models were sacrificed by cervical dislocation, and tumor tissue was carefully dissected. The tumor capsule and central necrotic area were carefully removed, leaving only the tumor tissue with a firm texture and adequate blood supply. The tumor tissue was cut into approximately 1 mm3 pieces using scissors and immediately placed in pre-cooled RPMI-1640 medium (containing 10% FBS + 1% double antibody) or tissue preservation medium (such as DMEM + 10% DMSO) and transported on ice (≤ 2 hours).

[0067] (2) Experimental groups: Balb / c nude mice (male, 4-5 weeks, 8 mice) were divided into 2 groups, 4 mice in each group, and subcutaneously inoculated near the back of the right hind limb of the nude mice.

[0068] (3) After anesthetizing the nude mouse, disinfect the skin on the back of the nude mouse near the hind limb with iodine, and then make a wound about 0.5 cm deep subcutaneously in the area. Use ophthalmic forceps to create a 1 to 1.5 cm long "tunnel" between the skin and muscle toward the right hind limb. Subsequently, implant the previously thinned tissue along the "tunnel" into the right hind limb, taking care to avoid the tissue being taken out during the implantation process. After the implantation is completed, suture the wound with sutures and disinfect the skin again with iodine. Ensure that the entire operation is completed within 3 hours after the tumor tissue is removed.

[0069] (4) After the inoculation, pay attention to the condition of the nude mice, check whether the body temperature has recovered, whether they have awakened, and their mental state after awakening. Weigh the nude mice every other day, and use a vernier caliper to measure the long and short diameters of the tumor. After about 10 days, the tumor formed under the skin of the right hind limb can be clearly observed. The formula for calculating the tumor volume is: V (mm³) = long diameter (mm) × short diameter (mm) × short diameter (mm) × 0.5.

[0070] (5) When the tumor volume reaches 25 mm³, the drug is administered every other day. The experimental group is injected with Avasimibe solution at a dose of 15 mg / kg based on the mouse body weight, and the control group is injected with DMSO solution of the same concentration by intraperitoneal injection. If the tumor diameter exceeds 15 mm or the weight loss exceeds 20% of the normal animal weight, or ulcers or infections appear at the tumor growth site, the nude mouse should be killed immediately.

[0071] (6) Tumor acquisition: After 15 days of drug treatment, blood was collected from the eyeballs and the nude mice were killed by cervical dislocation. The tumor was completely dissected and photographed. The heart, liver, spleen, lung and kidney tissues were also dissected and measured and recorded. Blood samples were collected for subsequent colorimetric testing of total cholesterol (TC), aspartate aminotransferase (AST / GOT), alanine aminotransferase (ALT / GPT), and creatinine (Cr). The following are the experimental steps for the colorimetric determination of total cholesterol, aspartate aminotransferase, alanine aminotransferase, and creatinine:

[0072] 1) Serum Collection: Collect blood from the orbital cavity of nude mice using a sterile, enzyme-free EP tube. Allow to clot naturally at room temperature for 10-20 minutes, then centrifuge at 2500-3500 rpm for 15 minutes at 4°C. Carefully collect the supernatant. If precipitation occurs during storage, re-centrifuge. Remove the reagent kit in advance and allow it to equilibrate to room temperature. Prepare the appropriate standards according to the instructions and preheat the reagents.

[0073] 2) Set up standard wells, sample control wells, and sample assay wells. After performing the experimental procedures according to the kit instructions, plot a linear regression curve for the standard in an Excel spreadsheet, with the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis. Substitute the OD value of the sample to be tested into the regression equation to calculate the sample concentration. Multiply this by the dilution factor to obtain the actual sample concentration.

[0074] 2. Results: Avasimibe inhibited the proliferation and metastasis of nasopharyngeal carcinoma in vivo, lowered serum total cholesterol, and had no hepatotoxicity or renal toxicity.

[0075] A nasopharyngeal carcinoma nude mouse PDX model was established to further evaluate the tumor suppressive ability of Avasimibe on nasopharyngeal carcinoma. The results showed that Avasimibe can inhibit the growth of nasopharyngeal carcinoma ( Figure 8 As shown in Figure (a), 15 days after administration, all tumors were removed from the mice. The results showed that the tumor volume and weight of nude mice in the Avasimibe treatment group were reduced (p<0.05, Figure 8 (b) to (d) in the figure), indicating that the proliferation of nasopharyngeal carcinoma cells was strongly inhibited. At the same time, the total cholesterol (TC) in the serum was detected by colorimetry and it was found that the TC content in the serum of nude mice was significantly reduced after administration (p<0.01, Figure 8 The results (e) suggest that the lipid-lowering drug avasimibe can regulate lipid metabolism in nude mice. The standardized PDX model operation process and comprehensive evaluation system established in this experiment also provide a reliable technical platform for subsequent anti-tumor drug development.

[0076] Serum biochemical analysis showed no statistically significant differences in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cr) between the avasimibe-treated and control groups, confirming that avasimibe at the experimental dose (15 mg / kg / day) did not cause liver or kidney damage. The lack of significant differences in Cr compared to the control group confirmed normal renal excretion (f). Furthermore, the absence of elevated AST / ALT values ​​indicated intact liver cell membrane integrity, precluding increased liver metabolic burden and myocardial toxicity (g). Finally, while effectively inhibiting tumor growth, avasimibe exhibited no observable liver or kidney toxicity, providing critical safety support for its clinical development.

Claims

1. The use of Avasimibe in a drug for treating nasopharyngeal carcinoma or oral cancer, characterized in that: Avasimibe is the only active ingredient in the drug for treating nasopharyngeal carcinoma or oral cancer.

2. The use according to claim 1, characterized in that Avasimibe can significantly inhibit the vitality, proliferation ability, migration ability and clone formation ability of nasopharyngeal carcinoma cells and oral cancer cells, and can regulate cell cycle arrest and induce cell apoptosis in a dose-dependent manner.

3. The use according to claim 1, characterized in that The drug for treating nasopharyngeal cancer or oral cancer comprises Avasimibe and a pharmaceutically acceptable carrier, wherein the carrier is dimethyl sulfoxide.

4. The use according to claim 1, characterized in that The dosage form of the drug for treating nasopharyngeal carcinoma or oral cancer is selected from oral preparations, injections, suppositories or local external preparations.

5. The use according to claim 1, characterized in that Avasimibe is used at a concentration range of 10 μM to 160 μM.

6. The use according to claim 1, characterized in that The dosage range of Avasimibe for treating nasopharyngeal carcinoma or oral cancer in animal models is 15 mg / kg per day.

7. Use of a composition containing Avasimibe in the preparation of a medicament for treating nasopharyngeal carcinoma or oral cancer, characterized in that: The composition comprises Avasimibe and pharmaceutically acceptable excipients.

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