Application of mirtazapine in preparation of medicine for enhancing tumor treatment effect of adriamycin

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of mirtazapine in preparation of a medicine for enhancing the tumor treatment effect of adriamycin. Research results of the invention show that MALS3 up-regulating agent mirtazapine can enhance the therapeutic effect of chemotherapeutic drug adriamycin in inhibiting tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of drug and disease treatment technology, specifically relating to the application of mirtazapine in the preparation of drugs for enhancing the therapeutic effect of doxorubicin on tumors. Background Technology

[0002] Malignant tumors are among the major diseases that seriously threaten human health, and their incidence is increasing year by year. Doxorubicin is an anthracycline broad-spectrum antitumor antibiotic that exerts its antitumor effects through mechanisms such as embedding into the DNA double helix, inhibiting topoisomerase II activity, and generating reactive oxygen species. It is widely used in chemotherapy regimens for various solid tumors, including breast cancer, liver cancer, and lung cancer. However, the clinical efficacy of doxorubicin is limited by its severe cardiotoxicity, myelosuppression, and acquired resistance in tumor cells. Furthermore, the objective response rate of monotherapy remains unsatisfactory. Therefore, there is an urgent need to develop combination strategies that can enhance its efficacy and reduce its toxicity. Summary of the Invention

[0003] To develop combination strategies that enhance the efficacy and reduce the toxicity of doxorubicin, addressing the limitations of doxorubicin's clinical efficacy due to its severe cardiotoxicity, myelosuppression, and acquired resistance in tumor cells, as well as the unsatisfactory objective response rate of monotherapy, this invention provides the application of mirtazapine in the preparation of a drug for enhancing the therapeutic effect of doxorubicin on tumors. To achieve the above objectives, this invention employs the following technical solution.

[0004] To develop combination strategies that enhance the efficacy of doxorubicin and reduce its toxicity, this invention provides the use of mirtazapine in the preparation of medicaments for enhancing the therapeutic effect of doxorubicin on tumors. The structure of mirtazapine is shown below:

[0005] .

[0006] This invention addresses the core shortcomings of doxorubicin's clinical application through the combined use of mirtazapine and doxorubicin. As a 5-HT receptor modulator, mirtazapine antagonizes the 5-HT2C receptor, increasing the expression of its downstream molecule MALS3. This inhibits the expression of TAZ, a downstream molecule of MALS3, ultimately weakening the tumor stem cell characteristics regulated by TAZ, thereby reversing acquired resistance in tumors, enhancing doxorubicin-induced tumor cell apoptosis, and significantly improving the objective response rate of monotherapy. This synergistic effect and toxicity reduction mechanism of mirtazapine and doxorubicin not only overcomes the dose-limiting toxicity bottleneck of doxorubicin but also restores its anti-tumor activity by targeting resistance mechanisms. Ultimately, it achieves the combined therapeutic goal of improved efficacy and reduced toxicity, thus solving the problem of doxorubicin's clinical efficacy being limited by its severe cardiotoxicity, myelosuppression, and acquired resistance in tumor cells, and overcoming the still unsatisfactory objective response rate of doxorubicin monotherapy.

[0007] Preferably, the drug uses mirtazapine as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

[0008] Preferably, the drug is selected from any one of tablets, capsules, granules, oral solutions, injections, lyophilized powder for injection, sustained-release agents, controlled-release agents, and transdermal patches.

[0009] Preferably, the excipients are selected from any one or more of fillers, disintegrants, lubricants, binders, flow aids, antioxidants, preservatives, and film coating materials.

[0010] Preferably, mirtazapine is used to enhance the sensitivity of tumor cells to doxorubicin.

[0011] Preferably, the tumor cells are selected from at least one of breast cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells, colorectal cancer cells, ovarian cancer cells, prostate cancer cells, and melanoma cells.

[0012] The present invention also provides a pharmaceutical composition for treating tumors, comprising mirtazapine and doxorubicin; wherein the mass ratio of mirtazapine to doxorubicin is 9-11:1.

[0013] More preferably, the mass ratio of mirtazapine to doxorubicin is 10:1.

[0014] Preferably, the pharmaceutical composition is used for the treatment of solid tumors.

[0015] Preferably, the solid tumor is selected from at least one of breast cancer, liver cancer, lung cancer, stomach cancer, colorectal cancer, ovarian cancer, prostate cancer, and melanoma.

[0016] Preferably, the pharmaceutical composition is an intravenous injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects: To develop combination strategies that enhance the efficacy and reduce the toxicity of doxorubicin, this invention provides the application of mirtazapine in the preparation of drugs for enhancing the therapeutic effect of doxorubicin on tumors, thereby establishing a new method for enhancing the therapeutic effect of doxorubicin in inhibiting the growth of solid tumors. This invention, by combining clinically safe and usable mirtazapine with doxorubicin, significantly improves the tumor inhibition rate against solid tumors such as breast cancer, liver cancer, lung cancer, gastric cancer, colorectal cancer, ovarian cancer, prostate cancer, and melanoma without increasing the dosage of doxorubicin, improves the tumor microenvironment, and reverses drug resistance. This overcomes the shortcomings of doxorubicin monotherapy, including limited efficacy, significant cardiotoxicity, and poor patient tolerance leading to treatment failure.

[0018] This invention addresses the core shortcomings of doxorubicin's clinical application through the combined use of mirtazapine and doxorubicin. As a 5-HT receptor modulator, mirtazapine antagonizes the 5-HT2C receptor, increasing the expression of its downstream molecule MALS3. This inhibits the expression of TAZ, a downstream molecule of MALS3, ultimately weakening the tumor stem cell characteristics regulated by TAZ, thereby reversing acquired resistance in tumors, enhancing doxorubicin-induced tumor cell apoptosis, and significantly improving the objective response rate of monotherapy. This synergistic effect and toxicity reduction mechanism of mirtazapine and doxorubicin not only overcomes the dose-limiting toxicity bottleneck of doxorubicin but also restores its anti-tumor activity by targeting resistance mechanisms. Ultimately, it achieves the combined therapeutic goal of improved efficacy and reduced toxicity, thus solving the problem of doxorubicin's clinical efficacy being limited by its severe cardiotoxicity, myelosuppression, and acquired resistance in tumor cells, and overcoming the still unsatisfactory objective response rate of doxorubicin monotherapy. Attached Figure Description

[0019] Figure 1 This refers to the cell experiment and animal experiment portions of the present invention; wherein: A: Proteins were collected from 4T1 cells after adding different concentrations of mirtazapine for Western blotting. B represents the collection of proteins for Western blotting after adding mirtazapine (10 μM) to control and MALS3-overexpressing 4T1 cells; the four samples in each row from left to right are the control group, control + mirtazapine group, MALS3 overexpression group, and MALS3 overexpression + mirtazapine group. C and D represent the flow cytometry analysis of CD44 in control and MALS3-overexpressing 4T1 cells, respectively, after the addition of mirtazapine. + CD24 - The proportion of subgroups was determined and quantified. E and F represent the results of flow cytometry detection of CD44 in 4T1 cells after the addition of mirtazapine, doxorubicin, or a combination of mirtazapine and doxorubicin, respectively. + CD24 - The proportion of subgroups was determined and quantified. G is a schematic diagram of the animal experimental design for drug treatment; H is a schematic diagram of tumors in mice during drug treatment in animal experiments (n=5 per group); the four groups in each column from top to bottom are solvent group, solvent + mirtazapine group, solvent + doxorubicin group, and solvent + dual drug combination group. I is a statistical analysis graph of the volume and weight of mouse tumor tissue in animal experiments of drug treatment; JO was used to perform immunohistochemical experiments on MALS3, ALDH1A1, c-MYC, NANOG, OCT4 and TAZ in tumor tissues of mice in different groups, and then scored and quantified the results.

[0020] Figure 2 These are representative images of the immunohistochemical experiments of MALS3, ALDH1A1, c-MYC, NANOG, OCT4, and TAZ performed on mouse tumor tissues in animal experiments of drug treatment in this invention. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0022] Example 1 I. Methods 1. Cell Culture and Animal Feeding (1) Cell culture: Cell name: Mouse breast cancer 4T1 cells.

[0023] Cell culture conditions: RPMI-1640 medium supplemented with 10% fetal bovine serum; 37°C, 5% CO2, saturated humidity incubator; trypsin digestion; passaged every 3 days at a passage ratio of 1:4.

[0024] The mouse breast cancer 4T1 cells were sourced from the Peking Union Medical College Cell Resource Center.

[0025] (2) Animal husbandry: SPF-grade Balb / c mice, female, 6-8 weeks old, weighing 22.5g±2.5g; Environment: SPF / clean animal room, temperature 22.5℃±2.5℃, humidity 50%±10%, light cycle 12h / 12h (alternating light and dark); fed standard mouse food, with free access to sterilized purified water.

[0026] The SPF-grade Balb / c mice were sourced from the Experimental Animal Center of the School of Medicine, Xi'an Jiaotong University, and were approved for use by the Medical Ethics Committee of Xi'an Jiaotong University.

[0027] All animal experiments in the examples below were conducted in accordance with the guidelines provided by the Animal Ethics Committee of Xi'an Jiaotong University.

[0028] 2. Construction of a stable 4T1-overexpressing MALS3 cell line (1) Construction of mouse overexpression lvMALS3 lentivirus vector and lentivirus packaging: The construction and packaging of the mouse overexpression lentivirus lvMALS3 vector were commissioned to V-Bio Biotechnology Co., Ltd.

[0029] (2) Lentiviral infection and screening of stable cell lines: 1) After digesting healthy wild-type 4T1 cells (mouse breast cancer 4T1 cells), the cells were seeded into 6cm cell culture dishes. The blank group, control group and MALS3 overexpression group were pre-set. The next experiment was carried out after the cell density reached 40% the next day.

[0030] 2) The next day, after changing the medium for the above 4T1 cells, keep the dish containing 1.5 mL of complete culture medium.

[0031] 3) In an Eppendorf tube containing 0.5 mL of complete culture medium, calculate the MOI (20) required for 4T1 cells, and then add the corresponding viral stock solution, i.e., the control group (add control lentivirus stock solution, titer 9.84 × 10⁻⁶). 8 IU / mL) and MALS3 overexpression group (with added overexpression lentivirus stock solution, titer 6.08×10) 8 (IU / mL). The control lentivirus stock solution was purchased from Shandong Weizhen Biotechnology Co., Ltd.; the control lentivirus was pLent-CMV-P2A-Puro, whose core backbone elements were derived from modifications of the HIV-1 genome. The overexpression lentivirus stock solution was purchased from Shandong Weizhen Biotechnology Co., Ltd.; the overexpression lentivirus was the mouse overexpression lentivirus lvMALS3 vector.

[0032] 4) Continue adding 6 μg / mL of the co-infectant ADV-HR to the Eppendorf tube, gently mix with a pipette, and incubate at 37°C for 0.5 hours. Then add the virus to the corresponding culture dishes, shake the dishes after adding the virus, and place them in an incubator. After 12 hours, add 2 mL of complete culture medium and continue culturing.

[0033] 5) After 24 hours, discard the culture medium containing the virus solution, replace it with fresh complete culture medium and continue culturing.

[0034] 6) After 48 hours, add 3 μg / mL of puromycin to screen for 3 generations or more. After all 4T1 cells in the blank group die and the 4T1 cells in the other two groups no longer die, extract cell proteins to verify the overexpression efficiency.

[0035] 7) After successfully verifying the MALS3 overexpression efficiency, the cell volume was increased and the cells were cryopreserved.

[0036] 3. Western blot 1) DMSO, 5 μM mirtazapine solution and 10 μM mirtazapine solution were added to wild-type 4T1 cells to set up a blank group (no special treatment), a solvent group (DMSO added), and different concentration drug groups (5 μM mirtazapine solution and 10 μM mirtazapine solution added); 10 μM mirtazapine solution was added to the above control group and MALS3 overexpression group 4T1 cell lines to set up a control blank group (no special treatment), a control drug group (10 μM mirtazapine solution added), an overexpression blank group (no special treatment), and an overexpression drug group (10 μM mirtazapine solution added).

[0037] The preparation method for mirtazapine solution is as follows: Accurately weigh mirtazapine powder, use DMSO as solvent, shake to fully dissolve, and prepare a stock solution with a concentration of 1 mg / mL. mirtazapine was purchased from Sigma-Aldrich, catalog number Y0000721.

[0038] 2) 72 hours after drug administration, cells from different groups (wild-type cells with different concentrations of mirtazapine and stable cells with or without mirtazapine) were collected and lysed using Western lysis buffer and IP cell lysis buffer (Beyotime) containing protease inhibitors (MCE) and phosphatase inhibitors (MCE) to obtain lysis products.

[0039] 3) After centrifuging the obtained lysis products at 12,000×g for 20 minutes at 4°C, the supernatant was collected and quantified using the QuickStart™ Bradford protein assay dye reagent (Bio-Rad).

[0040] 4) Subsequently, the proteins from each group were mixed with 5×SDS loading buffer and boiled for 10 minutes to obtain protein samples. Proteins were separated by electrophoresis gel and transferred to a PVDF membrane. The membrane was blocked with 5% (v / v) milk at room temperature for 1 hour, and then incubated with primary antibody overnight at 4°C.

[0041] 5) The next day, after washing the membrane three times with 1×TBST, the membrane was incubated with the secondary antibody at room temperature for 1 hour. The chemiluminescent signals of MALS3, c-MYC, OCT4, NANOG, TAZ and GAPDH were detected using ECL protein blot substrate (MCE).

[0042] The loading buffer consisted of 250 mM Tris-HCl at pH 6.8, 10% SDS (w / v), 25 mM β-mercaptoethanol, 30% glycerol (v / v), and 0.05% bromophenol blue (v / v).

[0043] Overnight stays refer to stays of 12 hours or more.

[0044] TBST: 20mM Tris, 100mM NaCl and 0.1% Tween-20 (v / v).

[0045] 4. Flow cytometry 1) Add 10 μM mirtazapine solution to the control group and MALS3 overexpression group 4T1 cell lines respectively, and set up a control blank group (no special treatment), a control drug group (added 10 μM mirtazapine solution), an overexpression blank group (no special treatment), and an overexpression drug group (added 10 μM mirtazapine solution); add 10 μM mirtazapine solution and 0.5 μM doxorubicin to wild-type 4T1 cells respectively or in combination, and set up a control group (no special treatment), a mirtazapine group (added 10 μM mirtazapine solution), a doxorubicin group (added 0.5 μM doxorubicin solution), and a dual-drug combination group (added 10 μM mirtazapine solution and 0.5 μM doxorubicin solution).

[0046] The preparation method for mirtazapine solution is as follows: Accurately weigh mirtazapine powder, use DMSO as solvent, shake to fully dissolve, and prepare a stock solution with a concentration of 1 mg / mL. mirtazapine was purchased from Sigma-Aldrich, catalog number Y0000721.

[0047] The preparation method for doxorubicin solution is as follows: Accurately weigh doxorubicin powder, use DMSO as solvent, gently mix by pipetting to prepare a 2 mg / mL stock solution. The entire process should be carried out in the dark. After aliquoting, the stock solution should be stored at -80°C. Doxorubicin was purchased from MCE, catalog number HY-15142A.

[0048] 2) After 72 hours of drug treatment, cells from each group were collected, washed twice with PBS, and resuspended to obtain 500 μl of cell suspension. The amount of antibody recommended by the Biolegend manufacturer was added to the cell suspension, and the cells were incubated at 37°C in the dark for 30 minutes. After washing twice again with PBS and resuspending, the cell suspension was collected through a 70 μm cell sieve and analyzed by flow cytometry. CD44 was detected using APC-conjugated CD44 antibody (Biolegend) and PE or FITC-conjugated CD24 antibody (Biolegend). + CD24 − Cell subpopulations.

[0049] The data was analyzed using FlowJo 10.6.2 software.

[0050] The PBS (buffer solution) was purchased from Beyotime Biotechnology Co., Ltd., with a concentration of 0.01 mol / L and a pH of 7.3.

[0051] 5. Animal experiments 1) In the treatment experiment, four groups were set up in the following order: solvent group (adding solvent (corn oil)), solvent + mirtazapine group (adding mirtazapine solution prepared with corn oil at 10 mg / kg), solvent + doxorubicin group (adding doxorubicin solution prepared with corn oil at 1 mg / kg), and solvent + dual drug combination group (adding mirtazapine solution prepared with corn oil at 10 mg / kg and doxorubicin solution prepared with corn oil at 1 mg / kg).

[0052] 2) 5×10 4 Cells from each group were resuspended in 100 μL of serum-free RPMI-1640 medium, mixed with matrix gel at a volume ratio of 1:1, and then inoculated into the right fourth pair of mammary fat pads of 6-week-old female Balb / c mice.

[0053] 3) One week after inoculation, the mice were treated. The control group was given the solvent control via intraperitoneal injection. The treatment groups were given the solvent control mirtazapine solution (10 mg / kg), doxorubicin solution (1 mg / kg), and a combination of mirtazapine solution (10 mg / kg) and doxorubicin solution (1 mg / kg) via intraperitoneal injection.

[0054] 4) After treatment for 21 days and observation for 30 days, tumor development in each mouse of each group was examined by palpation and breast surgical incision. Tumor volume was calculated using the following formula:

[0055] Tumor volume = (major axis × minor axis²) / 2; In the above formula, the major axis refers to the length of the longest diameter of the tumor tissue, and the minor axis refers to the length of the shortest diameter of the tumor tissue in the direction perpendicular to the major axis (both units are mm, and the measuring tool is a vernier caliper).

[0056] Meanwhile, the weight of intact tumors dissected after cervical dislocation in the above-mentioned different treatment groups was measured.

[0057] The solvent used was corn oil, which was purchased from Beyotime Biotechnology Co., Ltd.

[0058] The preparation method for mirtazapine solution is as follows: Accurately weigh mirtazapine powder, use DMSO as solvent, shake to fully dissolve, and prepare a stock solution with a concentration of 1 mg / mL. mirtazapine was purchased from Sigma-Aldrich, catalog number Y0000721.

[0059] The preparation method for doxorubicin solution is as follows: Accurately weigh doxorubicin powder, use DMSO as solvent, gently mix by pipetting to prepare a 2 mg / mL stock solution. The entire process should be carried out in the dark. After aliquoting, the stock solution should be stored at -80°C. Doxorubicin was purchased from MCE, catalog number HY-15142A.

[0060] The combined use of mirtazapine and doxorubicin refers to preparing mirtazapine and doxorubicin into their respective working concentrations according to a preset dosage ratio (mirtazapine 10 mg / kg, doxorubicin 1 mg / kg), and then administering them via divided intraperitoneal injection (mirtazapine is injected first, followed by doxorubicin after a 2-hour interval). The frequency of administration of mirtazapine is once a day, and the frequency of administration of doxorubicin is once every two days.

[0061] 6. Immunohistochemical staining (1) Dehydration, embedding, and sectioning of tumor tissue: After the animal treatment experiments were completed, tumor tissues from mice in each group were collected and dehydrated for embedding. The tumor tissues were placed in embedding cassettes and stored at -20°C. Sections were prepared using a paraffin microtome, and the sections were adhered to glass slides and stored at room temperature for use.

[0062] (2) Pretreatment of glass slides: Slide baking: Place the slides in a constant temperature drying oven at 60°C for 3 hours to ensure that the tissue sections adhere firmly.

[0063] (3) Dewaxing and rehydration treatment: 1) Xylene dewaxing: Immerse the pretreated glass slides in xylene I and II solutions for 10 minutes each to remove paraffin.

[0064] 2) Gradient rehydration: The glass slides dewaxed with xylene were sequentially subjected to hydration treatment with 100% (v / v), 95% (v / v), 85% (v / v) and 75% (v / v) ethanol solutions for 5 min each, and finally rinsed with three-stage distilled water (5 min each time) to obtain the corresponding sections for different groups.

[0065] (4) Antigen epitope repair: 1) Thermal induced repair: The sections were completely immersed in 0.01M citrate buffer (pH 6.0) and microwave thermal repair was performed (heated to boiling at medium-high power and then maintained at a gentle boil for 15 minutes).

[0066] 2) After natural cooling, rinse three times with phosphate-buffered saline (PBS) (5 min / cycle).

[0067] (5) Endogenous peroxidase blocking: 1) Cover the tissue area with 3% (v / v) hydrogen peroxide methanol solution and block at room temperature for 15 min.

[0068] 2) Rinse three times with PBS buffer (5 min / cycle).

[0069] (6) Blocking of non-specific binding sites: 1) Add normal goat serum blocking solution and incubate at room temperature for 30 minutes.

[0070] 2) Pour off the sealing liquid; no washing is required.

[0071] (7) Specific antibody incubation: 1) Primary antibody reaction: The tissue area was evenly covered with the monoclonal primary antibodies MALS3, ALDH1A1, c-MYC, NANOG, OCT4 and TAZ (1:200, diluted with PBS), and incubated in a humidified chamber at 4°C for 16 h.

[0072] 2) Secondary antibody reaction: After warming to room temperature, wash three times with PBS, add biotinylated secondary antibody working solution (SP kit), and incubate at 37°C for 60 min in a humidified chamber.

[0073] (8) Signal amplification system: After rinsing three times with PBS, the streptavidin-HRP complex was added dropwise and reacted at room temperature for 15 min.

[0074] (9) Color development and counterstaining: 1) DAB color development: After thorough rinsing with PBS, add freshly prepared DAB color development solution, monitor the color development under a microscope, and rinse with running water to stop the reaction.

[0075] 2) Hematoxylin counterstaining: stain the nucleus with conventional staining for 5 minutes, then rinse with running water for 10 minutes to remove excess stain.

[0076] (10) Differentiation and Blue Reversion Treatment: 1) Differentiate with 1% (v / v) hydrochloric acid ethanol solution for 15 seconds.

[0077] 2) 0.2% (v / v) ammonia solution turns blue in 5 seconds.

[0078] (11) Dehydration sealing: 1) Gradient dehydration: Dehydration with increasing concentrations of ethanol (75% (v / v), 85% (v / v), 95% (v / v), 100% (v / v)) for 5 min each.

[0079] 2) Xylene clearing: Xylene I and Xylene II were each cleared for 10 minutes.

[0080] 3) Seal with neutral resin.

[0081] (12) Result interpretation criteria: A double-blind evaluation method was used. Ten high-power fields (400×) were randomly selected from each group for scoring using a two-parameter integral method. 1) Positive cell percentage score: 0 points (positive cell percentage <10%), 1 point (positive cell percentage 10%~25%), 2 points (positive cell percentage 25%~50%), 3 points (positive cell percentage 50%~75%), 4 points (positive cell percentage >75%).

[0082] 2) Color intensity grading: 1 point (light yellow), 2 points (brownish yellow), 3 points (dark brown).

[0083] 3) Overall score = percentage score × intensity score, and the average of 10 fields of view is taken as the final score.

[0084] II. Results 1. Mirtazapine, as a MALS3 upregulator, significantly increases MALS3 expression levels. Previous studies have shown that the antidepressant mirtazapine can target and inhibit the 5-Hydroxytryptamine 2C Receptor (HTR2C), thereby upregulating its downstream molecule MALS3. Therefore, this invention investigated the upregulation effect of mirtazapine on MALS3 and its inhibitory effect on the downstream molecule TAZ by adding different concentrations of mirtazapine. The results showed that a mirtazapine concentration of 10 μM had a significant effect. Figure 1 (A in the middle).

[0085] 2. Mirtazapine, as a MALS3 upregulator, significantly inhibits the characteristics of tumor stem cells. Consistently, after the addition of mirtazapine to the control group and MALS3 overexpressing cells, the protein levels of the target gene TAZ and its downstream stemness-related molecules decreased to varying degrees. Figure 1 (B in the text). Flow cytometry results further indicated that mirtazapine can effectively reduce CD44. + CD24 - The proportion of cell subsets ( Figure 1 C and Figure 1 (D in the middle).

[0086] 3. Mirtazapine, as a MALS3 upregulator, significantly enhances the therapeutic effect of chemotherapy drugs in vivo. Subsequently, the therapeutic efficacy of this combination therapy strategy was evaluated in vivo and in vitro. Compared with cells treated with doxorubicin alone, the combination of mirtazapine and doxorubicin effectively reduced CD44 levels. + CD24 - The proportion of cell subsets ( Figure 1 E and Figure 1 (F in the text). Similar to in vitro results, in in vivo experiments, the addition of mirtazapine significantly enhanced the efficacy of doxorubicin in inhibiting tumor growth compared to doxorubicin alone. Figure 1 G~ Figure 1 Immunohistochemical results of mouse tumor tissues showed that the addition of mirtazapine effectively enhanced the expression of MALS3, while significantly reducing the expression of stemness-related molecules ALDH1A1, c-MYC, NANOG, OCT4, and TAZ. Figure 1 J~ Figure 1 The O in Figure 2 In summary, these results indicate that MALS3 upregulation of mirtazapine can enhance the therapeutic effect of the chemotherapy drug doxorubicin in inhibiting tumor growth.

[0087] The above experimental results show that mirtazapine, as a MALS3 upregulator, can effectively increase the protein expression level of MALS3, thereby inhibiting the expression of its downstream TAZ, and ultimately suppressing the tumor stem cell characteristics regulated by TAZ. In vivo experiments further demonstrate that mirtazapine combined with the chemotherapy drug doxorubicin can effectively increase the tumor's sensitivity to doxorubicin treatment, achieving an anti-cancer effect.

[0088] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0089] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The use of mirtazapine in the preparation of drugs for enhancing the therapeutic effect of doxorubicin on tumors, characterized in that, The structure of mirtazapine is shown below: 。 2. The use according to claim 1, characterized in that, The drug uses mirtazapine as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that, The drug is selected from any one of tablets, capsules, granules, oral solutions, injections, lyophilized powder for injection, sustained-release agents, controlled-release agents, and transdermal patches.

4. The use according to claim 3, characterized in that, The excipients are selected from any one or more of fillers, disintegrants, lubricants, binders, flow aids, antioxidants, preservatives, and film coating materials.

5. The use according to claim 1, characterized in that, Mirtazapine is used to increase the sensitivity of tumor cells to doxorubicin.

6. The use according to claim 5, characterized in that, The tumor cells are selected from at least one of breast cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells, colorectal cancer cells, ovarian cancer cells, prostate cancer cells, and melanoma cells.

7. A pharmaceutical composition for treating tumors, characterized in that, It includes mirtazapine and doxorubicin as described in claim 1; the mass ratio of mirtazapine to doxorubicin is 9-11:

1.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an intravenous injection.

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