Application of pharmaceutical composition targeting Beclin 1 in preparation of antitumor drugs

Abstract

The invention discloses an application of a Beclin 1 targeting pharmaceutical composition in preparation of antitumor drugs. The pharmaceutical composition comprises stapled peptides targeting Beclin 1 and platinum chemotherapeutic drugs. The treatment effect of the pharmaceutical composition on ovarian cancer is verified through an in-vitro cell model and an in-vivo mouse model, the combined use of the stapled peptide i7-01s-31 targeting Beclin 1 and cis-platinum can further amplify the anti-tumor effect of the stapled peptide i7-01s-31 and cis-platinum, the synergistic interaction effect is achieved, and the pharmaceutical composition can be used for treating ovarian cancer. The problem that drug resistance is easily generated due to single use of cis-platinum in actual clinical application is avoided; meanwhile, the dosage of chemotherapeutic drugs can be reduced through drug combination, so that the treatment safety is improved, and the toxicity is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to the application of a drug composition targeting Beclin 1 in the preparation of anti-tumor drugs. Background Art

[0002] Cell autophagy is a lysosome-dependent metabolic process that maintains homeostasis by degrading intracellular components. Research has shown that autophagy plays an important role in clearing abnormal proteins and regulating metabolism, and its dysfunction is closely related to tumors, neurodegenerative diseases, etc.

[0003] As a key protein of autophagy, Beclin 1 has an anti-cancer effect in various cancers. It has been found that 75% of ovarian cancers have monoallelic deletion of Beclin 1, resulting in reduced protein expression. Beclin 1 can also be ubiquitinated and degraded by CUL3, inhibiting autophagy and promoting tumor growth. Clinical data show that the expression levels of Beclin 1, LC3, and HMGB-1 are related to the prognosis of patients and can be used as predictive markers for chemotherapy resistance.

[0004] The fatality rate of ovarian cancer ranks first among gynecological tumors, and about 1 / 78 women will be affected. Due to the lack of early screening methods, most patients are diagnosed at an advanced stage, and the main symptoms include abdominal distension, abdominal pain, etc. The standard treatment plan is tumor debulking surgery combined with platinum-based / paclitaxel chemotherapy. However, the problem of cisplatin resistance is serious, and the recurrence rate at 18 months is more than 75%. The resistance mechanism is complex and involves the interaction of multiple pathways such as multidrug resistance (MDR), oxidative stress, and DNA repair, which is the main obstacle to treatment. Drug-resistant cancer cells can enhance drug efflux and detoxification ability by changing the expression of transporters and other ways. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. For this purpose, the present invention proposes the application of a drug composition targeting Beclin 1 in the preparation of anti-tumor drugs, and this drug composition exerts a synergistic anti-tumor effect in multiple ovarian cancer cell lines and a mouse ovarian cancer model.

[0006] The present invention also proposes a drug composition.

[0007] According to the first aspect of the present invention, there is provided the application of a drug composition targeting Beclin 1 in the preparation of anti-tumor drugs, and the drug composition includes a stapled peptide targeting Beclin 1 and a platinum-based chemotherapeutic drug.

[0008] In some embodiments of the present invention, the amino acid sequence of the stapled peptide targeting Beclin 1 includes SEQ ID NO: 1.

[0009] In some embodiments of the present invention, the platinum-based chemotherapeutic drug includes at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0010] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.1 - 5).

[0011] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.4 - 4).

[0012] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.5 - 2).

[0013] In some embodiments of the present invention, the dosages of the stapled peptide targeting Beclin 1 and the platinum-based chemotherapeutic drug in a mouse subcutaneous tumorigenesis model are 5 - 15 mg / kg of the stapled peptide per day and 1 - 2 mg / kg of the platinum-based chemotherapeutic drug per week, respectively.

[0014] In some embodiments of the present invention, the tumor includes at least one of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer, and bladder cancer.

[0015] According to the second aspect of the present invention, a pharmaceutical composition is provided, which includes a stapled peptide targeting Beclin 1 and a platinum-based chemotherapeutic drug.

[0016] In some embodiments of the present invention, the amino acid sequence of the stapled peptide targeting Beclin 1 includes SEQ ID NO:1.

[0017] In some embodiments of the present invention, the platinum-based chemotherapeutic drug includes at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0018] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.1 - 5).

[0019] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.4 - 4).

[0020] In some embodiments of the present invention, the molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic drug is 1:(0.5 - 2).

[0021] In some embodiments of the present invention, the dosages of the stapled peptide targeting Beclin 1 and the platinum-based chemotherapeutic drug in the mouse subcutaneous tumorigenesis model are 5-15 mg / kg of the stapled peptide per day and 1-2 mg / kg of the platinum-based chemotherapeutic drug per week, respectively.

[0022] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable excipient comprises at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent, and a surfactant.

[0024] In some embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose, or polyvinylpyrrolidone.

[0025] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, crospovidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethyl cellulose, carboxymethyl cellulose calcium, or alginic acid.

[0026] In some embodiments of the present invention, the lubricant is selected from at least one of colloidal silicon dioxide, magnesium stearate, calcium stearate, stearic acid, talc, or anhydrous silicon dioxide.

[0027] In some embodiments of the present invention, the coating agent comprises at least one of hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, polyvinyl alcohol phthalate, ethyl cellulose, or cellulose acetate.

[0028] In some embodiments of the present invention, the suspending agent comprises at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose, or aluminum stearate gel.

[0029] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate, or glyceryl monostearate.

[0030] The present invention has at least the following beneficial effects:

[0031] The present invention provides a drug composition targeting Beclin 1, which can be used to prepare anti-tumor drugs; moreover, the present invention verifies the therapeutic effect of this drug composition on ovarian cancer through in vitro cell models and in vivo mouse models. The combined use of the stapled peptide i7-01s-31 targeting Beclin 1 and cisplatin can further amplify their anti-tumor effects, exert a synergistic effect, and avoid the problem of easy drug resistance caused by single use of cisplatin in actual clinical applications; meanwhile, the combined use of drugs can reduce the dosage of chemotherapeutic drugs, thereby improving the safety of treatment and reducing toxicity.

[0032] Additional aspects and advantages of the present invention will be given in part in the following description and will become apparent in part from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described below in conjunction with the drawings and embodiments, where:

[0034] Figure 1 It is a schematic diagram of the chemical structure of the all-carbon hydrogen chain in Example 1 of the present invention. Two amino acids containing olefin side chains are separated by 6 amino acid residues on the stapled peptide sequence; the two side chains are connected through a ruthenium-catalyzed ring-closing metathesis reaction to obtain an 11-carbon long chain as shown in the figure;

[0035] Figure 2 It is a schematic diagram of the binding mode in Example 1 of the present invention; where a is a schematic diagram of the binding mode of the Tat-SP4 stapled peptide to the coiled-coil domain of Beclin 1, and b is a schematic diagram of the binding mode of the I7-01s scaffold to the coiled-coil domain of Beclin 1;

[0036] Figure 3 It is a graph of the cell activity detection results in Example 2 of the present invention; where A is ovarian cancer cells, B is normal cells, ns indicates no significant difference, ***P < 0.001, ****P < 0.0001;

[0037] Figure 4 It is a graph of the results of the cell colony formation experiment in Example 2 of the present invention; where A is OVCAR3 cells, B is SK-OV3 cells, and C is ID8 cells;

[0038] Figure 5 It is for the IC 50 value statistical chart in Example 2 of the present invention; where A is OVCAR3 cells and B is SK-OV3 cells;

[0039] Figure 6 It is a graph of the dose-effect curve and the relationship between the effect fraction (Fa) - CI in Example 2 of the present invention; where A and B are OVCAR3 cells, and C and D are SK-OV3 cells;

[0040] Figure 7 This is the Western blot detection result graph in Example 2 of the present invention;

[0041] Figure 8 This is the flow cytometry result graph in Example 2 of the present invention; wherein, A is OVCAR3 cells, B is SK-OV3 cells, and C is ID8 cells;

[0042] Figure 9 This is the result graph of the efficacy and safety experiments of the ID8 ovarian cancer mouse model in Example 2 of the present invention; wherein, A is the tumor growth curve, B is the tumor weight analysis, C is the actual tumor photo, D is the mouse body weight curve, and E is the photos of the main organs of the mouse. Detailed implementation manners

[0043] The following will clearly and completely describe the concept of the present invention and the technical effects generated in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0044] The reagents, methods and equipment used in the present invention, unless otherwise specified, are all conventional reagents, methods and equipment in the technical field.

[0045] Example 1 A drug composition targeting Beclin 1

[0046] This example provides a drug composition targeting Beclin 1, and the drug composition includes a stapled peptide targeting Beclin 1 and cisplatin.

[0047] The above stapled peptide targeting Beclin 1 is obtained by taking the amino acid residues 191-205 (Native-P1 peptide) on the coiled-coil domain of Beclin 1 as a template and performing amino acid mutation and modification on it.

[0048] The fragment of Native-P1 peptide was selected as the template because previous studies have found that this fragment is part of the Beclin 1 homodimer interface but does not overlap with the binding site of UVRAG. Therefore, amino acid residues 191-205 (Native-P1 peptide) on the coiled-coil domain of Beclin 1 are expected to bind to the coiled-coil domain of Beclin 1, reduce its homodimerization, and promote the formation of Beclin 1-Atg14l / UVRAG heterodimers, subsequently promoting the upregulation of autophagy and endosome-lysosome degradation. When the Native-P1 peptide binds to the coiled-coil domain of Beclin 1, an α-helical structure is formed. In this example, a fully hydrocarbon chain was used to connect two amino acid residues (i.e., E195 amino acid residue and N202 amino acid residue) separated by 6 residues on the Native-P1 sequence, so that the fully hydrocarbon chain can pass through two turns of the helix, thereby stabilizing the α-helical structure of the stapled peptide.

[0049] The present invention selects the i7-01s scaffold to insert the fully hydrocarbon chain. As Figure 1 shown, the E195 amino acid residue and N202 amino acid residue on the Native-P1 sequence were mutated and connected by a fully hydrocarbon chain. The E195 amino acid residue, N202 amino acid residue, and the fully hydrocarbon chain are all near the hydrophobic region on the surface of the coiled-coil domain, as Figure 2 shown. Therefore, the mutation of the E195 amino acid residue and N202 amino acid residue may reduce the hydrophobic-hydrophilic mismatch between the coiled-coil domain and the stapled peptide. In addition, according to the predicted model, the fully hydrocarbon chain on i7-01s is actually stacked on the surface of the coiled-coil domain, which may further enhance the binding of the stapled peptide to Beclin 1. Based on the i7-01s scaffold, the present invention carried out systematic single-point mutations on the Native-P1 sequence, and combined and screened to obtain a multi-point mutant with higher binding affinity, named i7-01s-31, and its amino acid sequence is as follows: Ace-VLFNR8LVDVIKS5RKV-Nme (SEQ ID NO:1).

[0050] Application of the drug composition targeting Beclin 1 in the preparation of anti-tumor drugs in Example 2

[0051] In this example, the anti-tumor effect of the drug composition targeting Beclin 1 provided in Example 1 was verified through in vitro and in vivo experiments. The specific experimental methods and experimental results are as follows:

[0052] 1. Cell viability detection

[0053] 1) Cell culture:

[0054] OVCAR-3 (human ovarian cancer cells, cisplatin-resistant cell line), SK-OV3 (human ovarian cancer cells, cisplatin-resistant cell line), L-O2 (human normal liver cells) and HEK293 cell line (human embryonic kidney cells) were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Life Technologies), 1% penicillin G (100 U / mL) and streptomycin (100 mg / mL). HEY-T30 (human ovarian cancer cells) and ID8 cell line (mouse ovarian epithelial cancer cells) were cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (Life Technologies), 1% penicillin G (100 U / mL) and streptomycin (100 mg / mL). Cells were cultured in an incubator at 37 °C with 5% CO2. All cell lines used in the experiments were tested using a mycoplasma detection kit (Lonza), and the results were all negative.

[0055] 2) Cell viability assay:

[0056] The standard trypan blue staining method was used to detect cell viability. This method distinguishes live / dead cells based on cell membrane integrity. Live cells can exclude trypan blue dye and maintain transparent cytoplasm, while dead cells are stained blue due to membrane damage. The cells to be tested were seeded in 96-well plates at the optimal growth density (final density 80%-90%) and pre-cultured overnight. After 24 hours of drug treatment, 100 μL of trypsin was added to each well to obtain a cell suspension. Trypan blue staining solution was added to the cell suspension, and cell counting was performed using a Z1 particle counter (Beckman Coulter). The results are as Figure 3 shown.

[0057] As Figure 3 shown in A, after simultaneous administration of cisplatin and i7-01s-31, significant differences in cell viability were observed in each ovarian cancer cell line. In OVCAR3 cells, the cell death rate in the combination treatment group (i7-01s-31 + cisplatin) reached 47.2%, significantly higher than that in the cisplatin alone group (30.1%) and the i7-01s-31 alone group (3.5%), indicating that the inhibitory effect of combination treatment is not only better than single drug treatment, but also exceeds the simple addition of the effects of the two drugs. Similar phenomena were also observed in the SK-OV3 and ID8 cell lines, suggesting that i7-01s-31 and cisplatin may have a synergistic effect in ovarian cancer cells.

[0058] As Figure 3 shown in B, when the same combination treatment was given to normal cell lines L-O2 and HEK293 cells, there was no significant difference in the cell death rate between the combination treatment group and the single drug group, indicating that in normal cells, i7-01s-31 cannot amplify the anti-tumor effect of cisplatin.

[0059] 2. Cell colony formation assay

[0060] The colony formation assay is an in vitro cell survival detection method based on the proliferative ability of single cells (the gold standard for in vitro evaluation of cytotoxicity), used to verify the effect of combination therapy. The specific operation steps are as follows: Ovarian cancer cells are inoculated in a 6-well plate and allowed to adhere overnight. After treatment with the corresponding drugs, they are cultured for 6 - 7 days according to the cell proliferation rate. Subsequently, the culture medium is discarded, and the cells are washed twice with PBS. 500 μL of 4% paraformaldehyde is added to each well and fixed at room temperature for 15 minutes. After staining with 0.5% crystal violet solution at room temperature for 30 minutes, the cells are washed twice with PBS again. Finally, the plate is scanned and imaged using a ChemiDoc imaging system (Bio-Rad), and the results are as Figure 4 shown.

[0061] As Figure 4 can be seen, the inhibitory effect of the combination of different concentrations of i7-01s-31 and cisplatin on the colony formation of ovarian cancer cells is significantly stronger than that of single drug treatment, and it is dose-dependent, confirming the synergistic effect of cisplatin and i7-01s-31 in ovarian cancer cells.

[0062] 3. IC 50 value (half-inhibitory concentration) determination

[0063] The standard trypan blue staining method is used to determine IC 50 . For the staining method, refer to the trypan blue staining method given in the first part of this example. The cells to be tested are inoculated in a 96-well plate at the optimal growth density (final density 80% - 90%) and pre-cultured overnight. After treatment with 2.5 μM cisplatin and different gradient concentrations of i7-01s-31 for 48 hours, 100 μL of trypsin is added to each well to obtain a cell suspension. Trypan blue staining solution is added to the cell suspension, and a Z1 particle counter (Beckman Coulter) is used for cell counting, and IC 50 is calculated. The results are as Figure 5 shown.

[0064] As Figure 5 can be seen, the IC 50 values of i7-01s-31 in OVCAR3 and SK-OV3 cells are 7.109 μM and 6.621 μM respectively. When 2.5 μM cisplatin is combined with i7-01s-31, the IC 50 values of i7-01s-31 decrease to 1.78 μM and 2.81 μM respectively.

[0065] 4. Analysis of the combination index (CI) of combined drug use

[0066] To explore the synergistic effect of polypeptides and cisplatin in ovarian cancer cells, the Chou-Talalay method was used in this example, and combination drug analysis was performed using CalcuSyn software. The specific experimental method is as follows:

[0067] According to the optimal growth rate of each cell line, cells were seeded in 96-well plates at a final confluence of 80%-90% and cultured overnight. Then, different concentration gradients of polypeptides, cisplatin, or their combination (using a fixed molar ratio of 1:1 for combined drug administration) were added, and the concentration gradients were 0.5 μM, 1 μM, 2 μM, 4 μM, and 6 μM. After incubation for 24 hours, cell viability was detected by trypan blue staining. The experimental data were analyzed by CalcuSyn software to calculate the CI. A CI < 1 indicates a synergistic effect. The resulting dose-effect curve and the effect fraction (Fa)-CI relationship diagram are as Figure 6 shown; based on the standard trypan blue exclusion assay data, the CI values at different effect levels calculated by CalcuSyn software are shown in Table 1, and the dose reduction indices at different effect fractions are shown in Tables 2 and 3.

[0068] Table 1 Combination index at different effect levels

[0069]

[0070] Among them, Dm (median effect dose) is a parameter characterizing drug efficacy; m is an index characterizing the "S" shape (shape feature) of the dose-effect curve; r is the linear correlation coefficient.

[0071] As can be seen from Table 1, in the two ovarian cancer cell lines OVCAR3 and SK-OV3, the average CI values at the ED 50 , ED 75 and ED 90 levels are all < 1, confirming that i7-01s-31 and cisplatin exerted a synergistic inhibitory effect in ovarian cancer cell lines.

[0072] The dose reduction index (DRI) quantitatively characterizes the degree to which the dose of each drug can be reduced compared to single-drug treatment when combined drugs are used, while still maintaining the same efficacy. This indicator is particularly important in clinical practice because it can reduce host toxicity while ensuring efficacy. A DRI > 1 indicates that the combined drug use has more advantages and can reduce the drug dosage on the premise of achieving the expected therapeutic effect, which is of great significance for reducing the toxic and side effects brought by high doses. The higher the DRI value, the more significant the degree of synergy.

[0073] Table 2 DRI values of combined drugs in OVCAR3 cells

[0074]

[0075] Table 3 DRI values of combined drugs in SK-OV3 cells

[0076]

[0077]

[0078] As can be seen from Table 2 and Table 3, except for the case where Fa < 0.2 in the OVCAR3 cell line, the DRI values of the combination of i7-01s-31 and cisplatin at each effect fraction were > 1. The CI and DRI data obtained by analyzing with CalcuSyn software jointly verified the in vitro synergistic effect of i7-01s-31 and cisplatin in ovarian cancer cells.

[0079] 5. Detection of cell apoptosis by Western blot

[0080] To explore the mechanism of the synergistic antitumor effect of stapled peptide and cisplatin, in this example, the cleavage activation of Caspase was detected by Western blot. The specific method is as follows:

[0081] Inoculate the SK-OV3 cells to be tested in a 6-well plate at a density of 70%. After the cells adhere overnight, treat them with the corresponding drugs. After 48 h of incubation, first collect the culture medium, and wash the well plate twice with PBS (the washing solution and the culture medium are collected together), and obtain the suspended cells after centrifugation. Subsequently, add freshly prepared lysis buffer (containing 2% SDS, 25% glycerol, 5% β-mercaptoethanol, and add 1% protease inhibitor mixture before use), collect the cells with a cell scraper, transfer the lysis buffer system to a 1.5 mL centrifuge tube, and place it in a dry bath at 100 °C for 10 minutes to obtain protein samples. Before loading, mix the protein samples with 5×SDS loading buffer and boil at 100 °C for 5 minutes. After SDS-PAGE electrophoresis, use a Mini-Trans-Blot electrotransfer apparatus (Bio-Rad) to transfer the proteins to a 0.45 μm PVDF membrane (Bio-Rad). After the transfer is completed, block the non-specific binding sites on the membrane with TBST buffer (137 mM NaCl, 20 mM Tris, 0.1% Tween 20) containing 5% skim milk powder. Incubate the blocked membrane with the specific primary antibody overnight at 4 °C. Finally, wash the membrane 3 times with TBST buffer, 10 minutes each time, and then incubate with the HRP-labeled secondary antibody (diluted 1:2000 in TBST containing 5% skim milk powder) for 1 hour, and repeat the washing step. Use Western HRP substrate (Millipore) and ChemiDoc imaging system (Bio-Rad) for signal detection, and the obtained results are as Figure 7 shown.

[0082] As a key executor of apoptosis, Caspase3 can play a role by cleaving a variety of key proteins including the ribozyme PARP. As Figure 7 shown, both the i7-01s-31 and cisplatin monotherapy groups produced slight Caspase3 and PARP cleavage bands, while the combined treatment group had significantly increased Caspase3 and PARP cleavage bands, indicating that the combined treatment significantly induced apoptosis of ovarian cancer cells.

[0083] 6. Detection of cell apoptosis and necrosis by flow cytometry

[0084] To explore the mechanism of the synergistic antitumor effect of stapled peptide and cisplatin, in this example, cell apoptosis and necrosis were detected by flow cytometry. The specific method is as follows:

[0085] After collecting cells by trypsin digestion, they were labeled with the Annexin V-FITC / PI double staining reagent from Invitrogen and detected and analyzed by a BD Accuri C6 flow cytometer. According to the fluorescence signal characteristics, cells can be divided into four subpopulations: Annexin V- / PI- are live cells (lower left quadrant), Annexin V+ / PI- indicate early apoptotic cells (lower right quadrant), Annexin V+ / PI+ are late apoptotic cells (upper right quadrant), and Annexin V- / PI+ are necrotic cells (upper left quadrant). All experimental steps were strictly in accordance with the operating specifications of the reagent instructions, and data analysis was completed using the BD Accuri C6 software supporting the instrument. The results obtained are as Figure 8 shown.

[0086] As Figure 8 shown, compared with the monotherapy, the combined use of i7-01s-31 and cisplatin can significantly increase the number of cells in the upper left quadrant, upper right quadrant and lower right quadrant, indicating that the combined treatment induced apoptosis and necrotic death of ovarian cancer cells.

[0087] 7. Verification of the efficacy of combined treatment using a mouse subcutaneous tumorigenesis model

[0088] To explore the mechanism of the synergistic antitumor effect of stapled peptide and cisplatin, in this example, cell apoptosis and necrosis were detected by flow cytometry. The specific method is as follows:

[0089] Female C57BL / 6J mice at 4 - 6 weeks of age were selected, and each mouse was subcutaneously injected with a mixed suspension (volume ratio 1:1, total volume 100 μL) of PBS containing 5×10 6 ID8 cells and Matrigel (Corning, 356237) to establish a transplanted tumor model. When the tumor volume reached approximately 100 mm 3When it was time, treatment was initiated by intraperitoneal injection of stapled peptide, cisplatin, or their combination. The experimental groups were respectively administered 10 mg / kg i7-01s-31 daily, 0.75 mg / kg cisplatin twice a week, and their combination (10 mg / kg i7-01s-31 + 0.75 mg / kg cisplatin), while the control group was injected with PBS. Thereafter, the tumor volume (calculation formula: 1 / 2 × long diameter × short diameter²) and animal body weight were measured regularly. After 19 days of drug administration, the mice were euthanized, the subcutaneous tumors were removed for weighing analysis, and the main organs were dissected to observe their morphology. The results are as Figure 9 shown.

[0090] As can be seen from Figure 9 A- Figure 9 C, all treatment groups could effectively inhibit tumor growth, and the combination treatment group showed the best tumor inhibitory effect.

[0091] As can be seen from Figure 9 D, compared with the control group, cisplatin, i7-01s-31, and the combination treatment group had no significant effect on the body weight of mice.

[0092] As can be seen from Figure 9 E, the morphological observation of the main organs showed no significant difference between the cisplatin, i7-01s-31, and combination treatment groups and the control group, comprehensively indicating that the combination treatment regimen has good safety.

[0093] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. Use of a drug composition targeting Beclin 1 in the preparation of an anti-tumor drug, characterized in that, The pharmaceutical composition comprises a stapled peptide targeting Beclin 1 and a platinum-based chemotherapeutic agent.

2. The application according to claim 1, wherein The amino acid sequence of the stapled peptide targeting Beclin 1 comprises SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The platinum-based chemotherapeutic agent comprises at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin and lobaplatin.

4. The application according to claim 1, wherein The molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic agent is 1:(0.1 - 5).

5. The application according to claim 1, wherein The tumor comprises at least one of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer and bladder cancer.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a stapled peptide targeting Beclin 1 and a platinum-based chemotherapeutic agent.

7. The pharmaceutical composition according to claim 6, wherein, The amino acid sequence of the stapled peptide targeting Beclin 1 comprises SEQ ID NO:

1.

8. The pharmaceutical composition according to claim 6, wherein The platinum-based chemotherapeutic agent comprises at least one of cisplatin, carboplatin, nedaplatin, oxaliplatin and lobaplatin.

9. The pharmaceutical composition according to claim 6, wherein The molar ratio of the stapled peptide targeting Beclin 1 to the platinum-based chemotherapeutic agent is 1:(0.1 - 5).

10. The pharmaceutical composition according to claim 6, wherein The tumor comprises at least one of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, cervical cancer, endometrial cancer, prostate cancer, ovarian cancer and bladder cancer.

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