A pharmaceutical composition for treating breast cancer

Abstract

The present invention discloses a pharmaceutical composition for treating breast cancer, wherein the active ingredient in the pharmaceutical composition is cycloserine, and the cycloserine is one of L-cycloserine, D-cycloserine, racemic cycloserine, or a pharmaceutically acceptable salt thereof. The present invention finds that cycloserine, particularly L-cycloserine, exerts a pharmacological effect of inhibiting breast cancer cells by inhibiting the expression of GPX4 and regulating the Ferroptosis pathway, and cycloserine has not been reported as a new potential drug for the clinical treatment of breast cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition for treating breast cancer. Background Art

[0002] The World Health Organization's International Agency for Research on Cancer (IARC) released global cancer data for 2020, showing that new cases of breast cancer surpassed lung cancer for the first time, becoming the most common cancer in the world, accounting for 11.7%. Currently, most anti-cancer treatments mainly work by activating cell death pathways, including cancer cell apoptosis. However, the generation of acquired drug resistance in cancer cells and the high recurrence rate of tumors are important issues facing treatment. Therefore, in-depth research on new treatment strategies is imperative, and new therapeutic drugs and targets are urgently needed to improve the treatment efficiency of breast cancer.

[0003] Ferroptosis was first proposed by Dr. Brent R. Stockwell of Columbia University in 2012. It is an iron-dependent, new type of programmed cell death that is different from apoptosis, necrosis, and autophagy. The essence of ferroptosis is the depletion of glutathione, the decrease in the activity of glutathione peroxidase (GPX4), and the inability of lipid oxides to be metabolized by the glutathione reductase reaction catalyzed by GPX4. Then, divalent iron ions oxidize lipids to produce reactive oxygen species, thereby promoting the occurrence of ferroptosis.

[0004] More and more studies have shown that iron is involved in the occurrence and metastasis of cancer. 2+ Ions generate hydroxyl radicals through the Fenton reaction, inducing ferroptosis in cancer cells. Therefore, inducing ferroptosis in cancer cells has become a new approach for tumor research and treatment.

[0005] Ma et al. showed that siramesin and lapatinib induced ferroptosis in breast cancer cells by reducing ferritin expression and increasing transferrin expression. Timmerman et al. reported that salazosulfapyridine induced ferroptosis in nutrient-deficient triple-negative breast cancer cells by inhibiting SLC7A11. The above studies show that the Ferroptosis pathway is related to breast cancer, and targeting the Ferroptosis pathway can be used as a new and effective method to promote cancer cell death and prevent tumor recurrence. Summary of the invention

[0006] At present, although the research on the ferroptosis pathway is gradually increasing, there are few reports on drugs that treat tumors by inducing ferroptosis in cells through the ferroptosis pathway. The inventor combined network pharmacology and bioinformatics methods to verify that L-cycloserine can exert anti-tumor activity by regulating the ferroptosis pathway, and predicted that L-cycloserine can be used as a new small molecule drug for the clinical treatment of breast cancer, and verified that cycloserine does have the activity of promoting the ferroptosis pathway, and the activity of L-cycloserine is significantly stronger than that of D-cycloserine. The above discovery provides a novel potential drug for the treatment of breast cancer, and the present invention proposes the following technical solution based on this.

[0007] One object of the present invention is to provide a pharmaceutical composition for treating breast cancer with cycloserine as an active ingredient. Another object of the present invention is to provide a use of cycloserine or a pharmaceutical composition containing cycloserine in preparing a drug for preventing and / or treating breast cancer.

[0008] In a first aspect, the present invention provides a pharmaceutical composition for treating breast cancer, wherein the active ingredient in the pharmaceutical composition is cycloserine.

[0009] Preferably, the cycloserine is one of L-cycloserine, D-cycloserine, racemic cycloserine, or a pharmaceutically acceptable salt thereof;

[0010] More preferably, the active ingredient is L-cycloserine.

[0011] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, which include one or a combination of two or more of a diluent, a binder, a lubricant, and a wetting agent.

[0012] The diluent is selected from one or a combination of two or more of starch, lactose, sucrose, mannitol, glucose, sorbitol, cellulose (such as ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylate, calcium hydrogen phosphate, and calcium sulfate.

[0013] The adhesive is selected from one or a combination of two or more of magnesium aluminum silicate, starch paste, gelatin, tragacanth gum, gum arabic, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, and hydroxypropyl methyl cellulose.

[0014] The lubricant is selected from one or a combination of two or more of silicon dioxide, talc, magnesium stearate or calcium salt, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate, and polyethylene glycol.

[0015] The wetting agent is selected from starch slurry, syrup, sodium lauryl sulfate, or a combination of two or more thereof.

[0016] Preferably, the pharmaceutical composition further comprises a combined drug, and the combined drug is selected from at least one of a cancer cell proliferation inhibitor, an anticancer agent, an immunosuppressant, and a pain reliever.

[0017] The cancer cell proliferation inhibitor, anticancer agent, and immunosuppressant are selected from one or a combination of two or more of platinum agents, angiogenesis inhibitors, cyclin-dependent kinase (CDK) inhibitors, BRAF inhibitors, EGFR inhibitors, and human epidermal growth factor receptor 2 (HER2) inhibitors.

[0018] The pain reliever is selected from at least one of aspirin, ibuprofen, paracetamol, celecoxib and tramadol.

[0019] The pharmaceutical composition of the present invention can be prepared into a pharmaceutical preparation administered by any suitable route, including transdermal, nasal, oral, rectal or inhalation administration. In certain embodiments, the pharmaceutical composition is administered by intradermal, subcutaneous, intravenous, intramuscular, intranasal or intratumoral injection via a syringe or other device.

[0020] The pharmaceutical composition can be prepared into pharmaceutical preparations in the following forms: tablets, capsules, syrups, suspensions, powders, granules, lozenges, creams, ointments, gels, emulsions, and aerosols.

[0021] In a specific embodiment of the present invention, the pharmaceutical preparation is preferably a unit dose preparation, which contains 0.01-1500 mg of the L-cycloserine; preferably, the unit dose preparation contains 0.1-500 mg of the L-cycloserine.

[0022] The unit dose preparation described in the present invention refers to a preparation containing an appropriate amount of active ingredient, which can be a capsule, a tablet or any dosage form, for example, a tablet or capsule packaged in aluminum foil, or a powder packaged in an ampoule, etc.

[0023] In a second aspect, the present invention provides use of cycloserine or a pharmaceutical composition comprising cycloserine as described above in preparing a drug for preventing and / or treating breast cancer.

[0024] Preferably, the cycloserine or the pharmaceutical composition comprising cycloserine as described above can be used alone or in combination with other kinds of pharmaceutical preparations and / or therapeutic methods.

[0025] The other types of pharmaceutical preparations and / or treatment methods include, but are not limited to: cancer cell proliferation inhibitors, anticancer agents, immunosuppressants, non-steroidal anti-inflammatory drugs, anti-tumor vaccines, adoptive cellular immunotherapy or radiotherapy.

[0026] In a third aspect, the present invention provides a method for inhibiting GPX4 in a cell, the method comprising incubating the cell with an effective amount of cycloserine, wherein the cell is a cancer cell.

[0027] Preferably, the cancer cells are breast cancer cells, and the cycloserine is L-cycloserine at a concentration of 50-200 μmol / L.

[0028] More preferably, the concentration of L-cycloserine is 150-200 μmol / L.

[0029] The advantages of the present invention are:

[0030] The present invention discloses a new use of cycloserine for the treatment of breast cancer by targeting ferroptosis. L-cycloserine and D-cycloserine are two structures of cycloserine. The inventors tested the viability of MDA-MB-231 cells under the incubation conditions of L-cycloserine through CCK8 experiments, and found that the activity of L-cycloserine was significantly stronger than that of D-cycloserine. After determining the efficacy of L-cycloserine, in order to further verify the safety of the drug, the inventors used normal cells MCF-10A and BEAS-2B cells for CCK-8 experiments. The results showed that under the condition of a maximum concentration of 200 μmol / L, the inhibition rates of MCF-10A and BEAS-2B cells were 4.33% and 18.8%. This result shows that the drug provided by the present invention has greater safety in the treatment of breast cancer. Then the inventors detected the cell survival rate, reactive oxygen species (ROS) and iron ion levels of two breast cancer cells MDA-MB-231 and MCF-7 under the conditions of ferroptosis inhibitor (Fer-1), and the expression level of ferroptosis key protein GPX4. The results showed that in the presence of ferroptosis inhibitor Fer-1, the inhibitory effect of L-cycloserine on MDA-MB-231 and MCF-7 breast cancer cells was eliminated. At the same time, the accumulation of reactive oxygen species (ROS), Fe3+ and GPX4 expression in the control group and the L-cycloserine intervention group were compared. The ROS and Fe3+ of MDA-MB-231 and MCF-7 cells intervened by L-cycloserine increased significantly, and GPX4 expression was significantly downregulated. This result shows that L-cycloserine exerts a pharmacological effect against breast cancer by targeting and regulating the ferroptosis pathway. Based on this result, the inventors believe that L-cycloserine has good drug potential in the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The viability curves of triple-negative breast cancer cells (MDA-MB-231), MCF-7 cells, normal breast epithelial cells (MCF-10A) and normal lung epithelial cells (BEAS-2B) incubated with L-cycloserine for 72 hours;

[0032] Figure 2Viability curve of MDA-MB-231 cells incubated with D-cycloserine for 72 h;

[0033] Figure 3 L-cycloserine inhibits the proliferation of MDA-MB-231 and MCF-7 cells by activating the Ferroptosis pathway;

[0034] Figure 4 Expression levels of GPX4, a key protein in ferroptosis, in MDA-MB-231 and MCF-7 cells incubated with L-cycloserine for 72 h. DETAILED DESCRIPTION

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

[0036] Example 1 Cycloserine exerts biological anti-breast cancer effects by regulating the Ferroptosis pathway

[0037] (1) Inhibitory effect of cycloserine on proliferation of MDA-MB-231 and MCF-7 breast cancer cells

[0038] Cell culture and viability assays

[0039] MDA-MB-231, BEAS-2B and MCF-7 cells were cultured in high-glucose DMEM containing 10% fetal bovine serum. MCF-10A was cultured in DMEM / F12 and supplemented with 5% horse serum, 20ng / mL epidermal growth factor (EGF), 0.5μg / mL Hydrocortisone, 10μg / mL insulin, 1% MEM non-essential amino acid solution (NEAA) and 1% penicillin-streptomycin solution (double antibody, P / S). All cells were incubated at 37°C in a 5% carbon dioxide incubator for 2-3 days for passage. The culture medium was replaced twice a week.

[0040] After cell counting, MDA-MB-231, BEAS-2B, MCF-10A and MCF-7 cells were seeded in 96-well plates at a density of 5000 cells / well. After the cells adhered to the wall, L-cycloserine at concentrations of 50 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmo1 / L was added to the 96-well plate for incubation for 72 h. The cell activity was detected using the CCK-8 kit. CCK-8 reagent (10 μL) was added to each well for 3.5 h of incubation. The OD value at 450 nm was measured using an enzyme reader. The following formula was used to calculate cell viability:

[0041] Cell survival rate (%) = [A(treated) - A(blank)] / [A(control) - A(blank)]

[0042] Effects of L-cycloserine on the viability of MDA-MB-231 and MCF-7 cells are shown in Figure 1 A and 1C, the effects of L-cycloserine on the viability of BEAS-2B and MCF-10A cells are shown in Figure 1 B and 1D. Figure 1 As shown in A and 1C, L-cycloserine significantly inhibited the viability of MDA-MB-231 and MCF-7 breast cancer cells in a concentration-dependent manner, and the inhibitory effect on MDA-MB-231 cells was stronger than that on MCF7 cells. This may be because triple-negative breast cancer is more sensitive to ferroptosis than MCF-7 (ER-positive breast cancer). Figure 1 As shown in B and 1D, L-cycloserine had no significant inhibitory effect on the cell viability of normal cells BEAS-2B and MCF-10A, and this result provided evidence for the safety of the drug.

[0043] We also tested the inhibitory effect of D-cycloserine on the viability of MDA-MB-231 breast cancer cells. The experimental method was similar to that of L-cycloserine, except that D-cycloserine was added to the 96-well plate at concentrations of 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L, respectively. The results are shown in Table 1. Figure 2 shown.

[0044] pass Figure 1 , Figure 2We tested the activity of two isomers of cycloserine on MDA-MB-231 breast cancer cells and found that the inhibition rates of L-cycloserine at 50μm, 100μm, 150μm, and 200μm were 24.25%, 32.97%, 60.35%, and 80.98%, respectively. The inhibition rates of D-cycloserine at 100μm, 200μm, 400μm, and 800μm were 2.11%, 13.44%, 32.05%, and 51.55%, respectively. This shows that the inhibitory activity of L-cycloserine on breast cancer is significantly higher than that of D-cycloserine.

[0045] (2) L-cycloserine induces ferroptosis and inhibits proliferation of MDA-MB-231 and MCF-7 cells

[0046] ① Cell activity assay

[0047] When MDA-MB-231 and MCF-7 cells grew to 60%, the cells were counted and plated in 96-well plates at 5000 / well. After the cells adhered to the wall, 5 groups of experiments (excluding the control) were set up, including 50 μmol / L, 100 μmol / L L-cycloserine, 50 μmol / L L-cycloserine + Ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and 100 μmol / L L-cycloserine + Ferrostatin-1. Ferrostatin-1 (2uM) was incubated for 24h in advance, and L-cycloserine was incubated for 72h. The cell activity was detected using the CCK-8 kit, and CCK-8 reagent (10 μL) was added to each well. The cells were incubated for 3.5h, and the OD value at 450nm was measured using an enzyme reader. The cell viability was calculated according to the method described above.

[0048] ②Fe 3+ analyze

[0049] When MDA-MB-231 and MCF-7 cells grow to 60%, count the cells and plate them in 6-well plates at 1×10^6. After the cells adhere to the wall, incubate with L-cycloserine (50μmol / L, 100μmol / L, 150μmol / L, 200μmol / L) for 72h, and use the Intracellular Iron Colorimetric Assay Kit (from Prile) to detect. Wash the DMEM-cultured cells twice with cold PBS 2ml, discard the PBS, lyse the cells with 200ul / well lysis buffer, and place them on a shaker for 2h. Strictly follow the instructions for standard dilution. Finally, incubate at 60℃ for 1h. When the temperature cools to room temperature, centrifuge the droplets on the tube cap and tube wall into the bottom of the tube. Add 30ul of iron ion detector, mix well, incubate at room temperature for 30min, gently blow with a pipette to dissolve the precipitate, add 200ul / well to a 96-well plate, measure the absorbance at 550nm, draw a standard curve and calculate the iron ion concentration.

[0050] ③Active oxygen determination

[0051] When MDA-MB-231 and MCF-7 cells grew to 60%, the cells were counted and plated in 6-well plates at 1×10^6. After the cells adhered to the wall, a negative control group (cells without DCFH-DA probe, only PBS) and a positive control group (cell suspension with DCFH-DA fluorescent probe and reactive oxygen donor) were set up. L-cycloserine (50μmol / L, 100μmol / L, 150μmol / L, 200μmo1 / L) was incubated for 72h, DCFH-DA (20uM) probe working solution was added, and the cells were placed in a 37℃ cell culture incubator for 45min, centrifuged at 1000r for 5min, washed twice with PBS, and the cell number was adjusted to 1*10 5 200ul of cell suspension was added to each well of a 96-well ELISA plate with a black bottom, and the fluorescence intensity was detected using a bioket ELISA reader at an excitation wavelength of 500nm and an emission wavelength of 525nm.

[0052] The results of the above cell activity experiments are as follows Figure 3As shown in A and B, MDA-MB-231 and MCF-7 cells were co-incubated with L-cycloserine in the presence or absence of Ferrostatin-1 (Fer-1, a ferroptosis inhibitor). It can be seen that in the absence of Ferrostatin-1, the viability of MDA-MB-231 and MCF-7 cells treated with 50 μmol / L and 100 μmol / L L-cycloserine was significantly decreased compared with the control group; when Ferrostatin-1 was added to L-cycloserine, the inhibitory effect of L-cycloserine on cell viability was eliminated. Since Ferrostatin-1 is a ferroptosis inhibitor, it can be proved that L-cycloserine inhibits cell viability by activating the Ferroptosis pathway.

[0053] L-cycloserine affects the expression of Fe in MDA-MB-231 and MCF-7 cells 3+ The results of the effect of concentration are shown in Figure 3 As shown in Figures C and D, in MDA-MB-231 and MCF-7 cells treated with different concentrations of L-cycloserine, the corresponding iron ion levels increased significantly in an L-cycloserine concentration-dependent manner.

[0054] The results of ROS level determination in MDA-MD-231 and MCF-7 cells treated with L-cycloserine are shown in Figure 3 As shown in Figures E and F, compared with the control group, the intracellular ROS levels of MDA-MD-231 and MCF-7 cells incubated with L-cycloserine increased significantly, and the ROS levels increased in an L-cycloserine concentration-dependent manner. The above results indicate that L-cycloserine induces cell ferroptosis by promoting the accumulation of reactive oxygen species and iron ions in MDA-MB-231 and MCF-7 breast cancer cells.

[0055] Effect of L-cycloserine on GPX4 expression in breast cancer cells

[0056] Furthermore, the inventors evaluated the effect of L-cycloserine on breast cancer cells through the GPX4-mediated Ferroptosis pathway. GPX4 is a key indicator of the Ferroptosis pathway, and the inventors used Western blotting to detect the protein expression level of GPX4.

[0057] Western blot analysis method: An appropriate amount of MDA-MB-231 and MCF-7 cells were seeded in a 60 mm culture dish and incubated with L-cycloserine at a concentration of 100 μmol / L and 150 μmol / L for 72 hours, and a control group was set up. Total protein was extracted with lysis buffer. Subsequently, 40 μg of cell lysate from each sample was separated by 10% SDS-PAGE, and the protein was transferred to a polyvinylidene fluoride membrane. The membrane was first incubated with the primary antibody (GPX4) at 4 ° C overnight, and then incubated with the corresponding secondary antibody at room temperature for 2 hours. Finally, the target protein band was analyzed to determine the expression level of GPX4.

[0058] The results are as follows Figure 4 As shown in A and B, the expression of GPX4 was significantly reduced in MDA-MB-231 and MCF-7 cells incubated with L-cycloserine, and the expression level of GPX4 was significantly reduced when the administration concentration of L-cycloserine increased. The above results indicate that L-cycloserine activates the Ferroptosis pathway by downregulating the expression level of GPX4, thereby exerting an anti-breast cancer effect.

[0059] The present invention uses SPSS 26.0, Prism 8.0, and Image J to perform statistical analysis on the results. Independent sample t test and nonparametric independent sample test were used to evaluate the differences between all experimental groups, and the results are expressed as mean ± SD. P < 0.05 is the threshold for statistically significant differences.

[0060] In summary, L-cycloserine significantly inhibited the cell activity of MD-MBA-231 and MCF-7, and the ROS and Fe ions of MD-MBA-231 and MCF7 cells after drug intervention were significantly higher than those of the control group. Western blot results also showed that GPX4 expression was downregulated. This research result suggests that L-cycloserine can achieve the purpose of treating breast cancer by targeting ferroptosis.

[0061] Example 2 Preparation of a pharmaceutical composition containing L-cycloserine

[0062] Example 2-1 Tablet

[0063] S1: 50 g of L-cycloserine was added with 300 g of lactose in an incremental manner, the mixture was mixed and ground for 50 min, 35 g of microcrystalline cellulose and 10 g of sorbitol were added, and the mixture was ground again for 30 min, and the ground mixture was cooled to room temperature to obtain a drug-lactose mixture;

[0064] S2: the drug-lactose mixture obtained in step S1 is put into a wet granulator and mixed for 5 minutes, Tween-80 is dissolved in 95% ethanol, 4 ml of Tween-80 is added per kg of the preparation weight, and then mixed with 0.5% povidone K30 aqueous solution at a ratio of 1:20 as a binder and added to the wet granulator mixture for granulation;

[0065] S4: Magnesium stearate was added to the granules obtained in S2, and the hardness of the tablet press was controlled within 4-5 kgf, and 1000 tablets were obtained by tableting.

[0066] Example 2-2 Capsules

[0067] S1: Dissolve 4.0 g of sodium hydroxide in a small amount of 50% ethanol aqueous solution, add 50 g of L-cycloserine, mix and grind to obtain a mixture;

[0068] S2: Add 300 g of lactose to the mixture, mix, dry at 55°C for 50 min, crush, and pass through a 40-mesh sieve to obtain mixed fine powder;

[0069] S3: adding 30 ml of 5% ethanol solution of povidone K30 to the mixed fine powder obtained in step S2 to prepare a soft material, and passing through a 20-mesh sieve to prepare wet granules;

[0070] S4: drying the wet granules obtained in step S3 at 50° C. for 6 h, and then passing through an 18-mesh sieve for granulation;

[0071] S5: Determine the content of L-cycloserine in the granules, calculate the filling amount, encapsulate, and package after inspection to obtain 1000 capsules.

[0072] Example 2-3 Injection

[0073] S1: Prepare a solution by dissolving 20 mg of L-cycloserine in 1000 mL of phosphate buffer solution to obtain a solution with a drug concentration of 0.02 mg / mL;

[0074] S2: Activated carbon treatment, add 0.2% of the total solution of the needle to the drug solution for adsorption treatment with activated carbon for 0.5 hours;

[0075] S3: Primary filtration, filtering the liquid medicine with filter paper;

[0076] S4: Filtration sterilization, using a microporous filter membrane with a pore size of 0.22 μm to sterilize the drug solution and remove the heat source;

[0077] S5: Divide and seal the liquid medicine into 2 mL units to obtain an injection solution.

[0078] D-cycloserine and L-cycloserine are two structures of cycloserine, and their application range is relatively wide. D-cycloserine is currently one of the combined drugs for the clinical treatment of tuberculosis, and studies have shown that D-cycloserine has considerable efficacy in the treatment of Alzheimer's disease, schizophrenia and pain; L-cycloserine plays an important role in the treatment of epilepsy and mental illness. Clinical studies have shown that the side effects of cycloserine can be eliminated by the combined use of vitamin B6. Our research found that cycloserine has considerable efficacy in the treatment of breast cancer and has certain safety. Therefore, the application of cycloserine in clinical research has good prospects. The patent of cycloserine compounds has expired, and it will be a very meaningful work to obtain independent intellectual property rights of cycloserine in the treatment of breast cancer.

[0079] In conclusion, the results of this study indicate that GPX4 is a key protein in the Ferroptosis pathway, and L-cycloserine exerts its pharmacological effect in inhibiting breast cancer cells by inhibiting the expression of GPX4 and regulating the Ferroptosis pathway. This suggests that cycloserine, especially L-cycloserine, can be used as a new potential drug for the clinical treatment of breast cancer.

Claims

1. Use of L-cycloserine in preparing medicine for treating breast cancer.

2. The use according to claim 1, characterized in that: The administration routes of L-cycloserine include transdermal, nasal, oral, rectal or inhalation administration.

3. The use according to claim 1, characterized in that: The drug is in one of the following dosage forms: tablets, capsules, syrups, suspensions, powders, granules, lozenges, creams, ointments, gels, emulsions, and aerosols.

4. The use according to claim 1, characterized in that: The medicine is a unit dose preparation, which contains 0.01-1500 mg of the L-cycloserine.

5. The use according to claim 1, characterized in that: The medicine is a unit dose preparation, which contains 0.1-500 mg of the L-cycloserine.

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