A small molecule peptide having inhibitory activity against breast cancer cells, formulations and use thereof in the treatment of breast cancer

Abstract

The application discloses a small-molecule peptide with inhibitory activity on breast cancer cells, a preparation and application thereof in treating breast cancer, and belongs to the biomedical technical field. The amino acid sequence of the small-molecule peptide is shown in SEQ ID NO. 1, in-vitro cell experiments prove that the small-molecule peptide can significantly inhibit the proliferation of various breast cancer cells, and has no cytotoxicity and good safety. The application further provides a pharmaceutical preparation containing the small-molecule peptide. The small-molecule peptide and the preparation can significantly inhibit the growth rate of human breast cancer cell MDA-MB-231 transplanted tumor in nude mice, improve the levels of IL-6, IFN-gamma and TNF-alpha cytokines in serum, and enhance the immune function of the nude mice, and can be effectively used for treating breast cancer, thereby providing a new candidate drug and treatment strategy for the clinical treatment of breast cancer, and having important clinical application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule peptide, preparation, and its use in the treatment of breast cancer that has inhibitory activity against breast cancer cells. Background Technology

[0002] Breast cancer is one of the most common malignant tumors among women worldwide, and its incidence rate is increasing year by year, with the age of onset gradually becoming younger. It has become a major disease burden threatening women's lives and health. With the advancement of medical technology, the treatment of breast cancer has formed a comprehensive treatment system with surgery as the core, combined with chemotherapy, radiotherapy, endocrine therapy, targeted therapy, and other methods, which has significantly improved the prognosis of some patients.

[0003] However, current breast cancer treatments still have many problems that urgently need to be solved. Among them, chemotherapy drugs, as an important means of clinical treatment, can inhibit tumor progression to a certain extent, but they lack targeted specificity. While killing tumor cells, they can easily cause serious damage to normal tissue cells such as bone marrow hematopoietic cells and gastrointestinal mucosal cells, leading to a series of serious adverse reactions such as bone marrow suppression, nausea and vomiting, and hair loss. These reactions greatly reduce the quality of life of patients and even cause some patients to discontinue treatment because they cannot tolerate the adverse reactions.

[0004] The emergence of targeted therapy drugs has brought a new breakthrough in breast cancer treatment. By specifically targeting key sites on tumor cells, they can reduce damage to normal cells and improve treatment safety. However, currently marketed targeted drugs for breast cancer still have problems such as limited applicable populations and the easy development of drug resistance. For example, targeted drugs for HER2-positive breast cancer are only effective for patients with HER2 gene amplification or overexpression. For subtypes such as triple-negative breast cancer, due to the lack of clear effective targets, targeted therapy options are scarce, and clinical treatment still relies mainly on chemotherapy, which has poor efficacy and significant side effects.

[0005] Endocrine therapy is mainly suitable for patients with hormone receptor-positive breast cancer. Although it has relatively fewer side effects, it still faces the problem of drug resistance and is ineffective for patients with hormone receptor-negative breast cancer. Radiotherapy is mainly used for postoperative adjuvant therapy or palliative treatment for advanced patients. It is prone to causing complications such as local skin damage and radiation pneumonitis, and its efficacy against metastatic tumors is limited.

[0006] Therefore, the development of novel anti-breast cancer drugs with strong targeting, significant efficacy, and low toxicity, especially for refractory breast cancer subtypes, has become a research hotspot and urgent need in the biomedical field. Small molecule peptides, due to their unique advantages such as small molecular weight, strong penetration, easily regulated targeting, and low toxicity, have shown great application potential in the field of tumor targeted therapy. Compared with traditional large molecule antibody drugs, small molecule peptides have relatively simple preparation processes, lower costs, and are less likely to induce immune responses, making them more suitable for large-scale clinical applications. Based on this, developing a small molecule peptide with highly efficient inhibitory activity against breast cancer cells and low toxicity to normal cells is of great significance for enriching the variety of breast cancer treatment drugs, improving treatment efficacy, and enhancing patients' quality of life, providing a new and effective strategy for the clinical treatment of breast cancer. Summary of the Invention

[0007] To address the aforementioned technical issues, this invention provides a small molecule peptide that can inhibit the proliferation of breast cancer cells and enhance immune function, and can be widely used in the clinical treatment of breast cancer.

[0008] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0009] This invention provides a small molecule peptide with inhibitory activity against breast cancer cells, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] Furthermore, the amino acid sequence of the small molecule peptide is Met-Leu-Tyr-Gly-Asp-Phe-Val-Ala-Ala-Ile-Leu-Asn-Pro (MLYGDFVAAILNP).

[0011] Furthermore, the molecular weight of the small molecule peptide is 1.42 kDa (1423.67 Da), and the theoretical isoelectric point PI is 9.36.

[0012] Furthermore, the small molecule peptide is synthesized using a solid-phase synthesis method.

[0013] The present invention also provides a pharmaceutical preparation, characterized in that the pharmaceutical preparation contains the aforementioned small molecule peptide.

[0014] Furthermore, the small molecule peptide includes its pharmaceutically acceptable salt.

[0015] Furthermore, the pharmaceutically acceptable salt includes at least one of hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, and organic amine salt.

[0016] Furthermore, the pharmaceutical preparation also contains pharmaceutically acceptable excipients.

[0017] Furthermore, the pharmaceutical formulation can be formulated into any pharmacologically acceptable dosage form.

[0018] Furthermore, the dosage form of the pharmaceutical preparation includes liquid, solid, or semi-solid formulations.

[0019] The present invention also provides the use of the small molecule peptide or the pharmaceutical preparation in the preparation of a drug for treating tumors.

[0020] Furthermore, the tumor is breast cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The amino acid sequence of the small molecule peptide of this invention is shown in SEQ ID NO.1. In vitro cell experiments have confirmed that this small molecule peptide can significantly inhibit the proliferation of various breast cancer cells, and has no cytotoxicity and good safety. This invention also provides pharmaceutical formulations containing the above-mentioned small molecule peptide. These formulations use the small molecule peptide as the active ingredient, supplemented with pharmaceutically acceptable excipients, and can be prepared into various clinical dosage forms. The small molecule peptide and formulation of this invention can significantly inhibit the growth rate of human breast cancer cell MDA-MB-231 xenograft tumors in nude mice, increase the serum levels of IL-6, IFN-γ, and TNF-α cytokines, and enhance the immune function of nude mice. They can be effectively used for the treatment of breast cancer, providing new candidate drugs and treatment strategies for the clinical treatment of breast cancer, and have significant clinical application value and market prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the molecular structure of the small peptide of the present invention;

[0025] Figure 2 This is a predicted three-dimensional structure diagram of the small molecule peptide of the present invention (A represents the main chain backbone of the small molecule peptide, and B represents the amino acid residues of the side chain further shown on the basis of the main chain backbone).

[0026] Figure 3 The results of the cytotoxicity test of the small molecule peptides of this invention;

[0027] Figure 4 This invention investigates the effects of different concentrations of small molecule peptides on the proliferative activity of breast cancer cells. Detailed Implementation

[0028] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods.

[0029] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and components may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention may comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0030] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available, conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0031] The experimental data of this invention were analyzed using SPSS 20.0 software. All data were tested for normality and homogeneity of variance. The experimental data are expressed as x±s. One-way ANOVA was used to analyze the results. ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0032] Example 1: Solid-phase synthesis of small molecule peptides

[0033] Raw materials: Rink Amide MBHA resin as the solid-phase carrier was purchased from Beijing Bailingwei Technology Co., Ltd., product number: 910548; other amino acids protected by Fmoc, such as Fmoc-Met-OH, were purchased from Shanghai Jier Biochemical, with a purity ≥99%; the activators were O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt); the organic base was N,N-diisopropylethylamine (DIEA); the deprotecting agent was 20% piperidine / N,N-dimethylformamide (DMF) solution; the cleavage reagent was TFA:EDT: benzyl sulfide:phenol:water = 86:5:5:2:2 (volume ratio); the remaining reagents (DMF, dichloromethane (DCM), etc.) were all analytical grade and purchased from Shanghai Jier Biochemical.

[0034] This embodiment uses solid-phase synthesis to prepare the target small molecule peptide MLYGDFVAAILNP (SEQ ID NO:1). The specific preparation steps are as follows:

[0035] (1) Resin swelling: Accurately weigh 1.0g of Rink Amide MBHA resin and place it in a solid-phase synthesis reaction column. Add 10mL of DMF solution and soak at room temperature for 30min.

[0036] (2) Ligation of the first amino acid (Pro): The swollen resin was washed three times with DMF (5 mL each time, 3 min each time) to remove impurities from the resin surface; then 10 mL of 20% piperidine / DMF solution was added, and the mixture was shaken at room temperature for 20 min to remove the Fmoc protecting group on the resin; after deprotection, the resin was washed five times with DMF (5 mL each time) and three times with DCM to completely remove residual piperidine. 1.5 mmol Fmoc-Pro-OH, 1.5 mmol HBTU and 1.5 mmol HOBt were weighed and placed in a dry reaction flask, dissolved in 5 mL of anhydrous DMF, and then 3 mmol DIEA was added. The mixture was stirred and activated at room temperature for 5 min; the activated amino acid mixture was added to the reaction column and the reaction was shaken at room temperature for 2 h to allow the carboxyl group of Fmoc-Pro-OH to form an amide bond with the amino group of the resin. After the reaction was completed, the resin was washed three times with DMF. A small amount of resin was taken for ninhydrin detection (Kaiser test): if the resin was colorless and transparent, it indicated that the amino acid linkage was successful; if the resin was blue, 1.0 mmol of Fmoc-Pro-OH and the corresponding proportion of activator and organic base were added, and the activation and linkage reaction was repeated for 1 hour until the test was qualified.

[0037] (3) Stepwise ligation of subsequent amino acids: Following the C-terminus to N-terminus sequence of the target polypeptide sequence MLYGDFVAAILNP (Pro→Asn→Leu→Ile→Ala→Ala→Val→Phe→Asp→Gly→Tyr→Leu→Met), repeat the "deprotection-washing-amino acid activation-ligation-detection" operation in step 2. In each reaction, the molar ratio of Fmoc protected amino acid, HBTU, HOBt to resin substitution degree is 3:3:3:1, the activation time is 5 min, and the ligation reaction time is 2 h. After each ligation step, ninhydrin detection is required to ensure that each amino acid is ligated efficiently and accurately, avoiding mismatches or deletions.

[0038] (4) Peptide chain cleavage and deprotection: After all amino acids were linked, the resin was washed 5 times with DMF and 5 times with DCM. Then the resin was transferred to a dry round-bottom flask, 10 mL of pre-cooled cleavage reagent was added, and the mixture was stirred at room temperature for 2.5 h to break the peptide chain from the resin and remove all protecting groups. After cleavage, the mixture was filtered through a sintered glass funnel and the filtrate was collected. The resin was washed 3 times with a small amount of TFA, and the washings and filtrate were combined. The combined filtrate was slowly added dropwise to 50 mL of pre-cooled anhydrous diethyl ether, and the mixture was stirred vigorously to precipitate the peptide. After standing at 4 °C for 30 min, the precipitate was collected by centrifugation (5000 r / min for 10 min). The precipitate was washed 3 times with anhydrous diethyl ether and dried under vacuum to obtain crude small molecule peptide.

[0039] (5) Crude product purification: The crude small molecule peptide was purified by high performance liquid chromatography (HPLC) using an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm). Mobile phase A was 0.1% TFA / water solution, and mobile phase B was 0.1% TFA / acetonitrile solution. The gradient elution program was: 0-5 min, 5% B; 5-20 min, 5%-60% B; 20-32 min, 60%-95% B; flow rate 1.0 mL / min; detection wavelength 220 nm. The eluent corresponding to the target peak was collected and freeze-dried to obtain the pure small molecule peptide MLYGDFVAAILNP (structural formula as shown in the figure). Figure 1 As shown, the molecular weight is 1.42 kDa. The purity was determined by HPLC to be ≥98%. It should be stored at low temperature for future use.

[0040] (6) Structure prediction: The tertiary structure prediction tool PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ) was used to predict that the small peptide of SEQ ID NO:1 was helical. The prediction results are as follows: Figure 2 As shown.

[0041] Example 2 Safety evaluation of small molecule peptides

[0042] Fully adherent HeLa cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0101) were digested with 0.25% trypsin and cultured in high-glucose DMEM containing 5% FBS to achieve a cell density of 1×10⁻⁶ cells / cells. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity for 24 h. The complete culture medium was then aspirated. The experimental groups were added to high-glucose DMEM medium diluted to concentrations of 0.1 mg / ml, 1 mg / ml, 5 mg / ml, and 10 mg / ml, respectively, to the peptide solution prepared in Example 1. The control group consisted of cells cultured in DMEM medium, and the blank group consisted of cell-free DMEM medium. The cells were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent (GLPbio) was added to each group, and the cells were incubated for 1–4 h. The absorbance (OD value) of each well was measured using an ELISA reader at 450 nm. Cell viability was calculated based on the mean absorbance of each group using the following formula:

[0043]

[0044] Experimental results are as follows Figure 3 As shown, the cell survival rates of the small molecule peptides of the present invention at different concentrations and the control group were all above 100%, and the activity of the small molecule peptides was improved compared with the blank group, indicating that the small molecule peptides of the present invention have no cytotoxicity and good safety.

[0045] Example 3: Effects of small molecule peptides on breast cancer cell proliferation

[0046] Breast cancer cell sources: MCF-7 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0149), MDA-MB-231 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0150), and SK-BR-3 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0211).

[0047] Experimental groups: Experimental group: breast cancer cells + 10% FBS medium (Sigma) + small molecule peptide of the present invention; Control group: breast cancer cells + 10% FBS medium, no small molecule peptide; Blank group: no cells, no small molecule peptide, 10% FBS medium.

[0048] Experimental method: Three types of breast cancer cells with appropriate densities were inoculated into 96-well plates according to the need, with a volume of 100 μL / well. After the cells adhered to the wall, small molecule peptides were added to a final concentration of 0.5 mg / mL, 2 mg / mL, and 4 mg / mL respectively. The control group did not add small molecule peptides. After co-culturing the cells with the small molecule peptides for 36 h, the 96-well plates were transferred to the ultra-clean bench in the cell room. The culture medium was discarded, and the residual liquid was aspirated clean. Under light avoidance conditions, 100 μL of serum-free medium and 10 μL of CCK-8 were added to each well, and PBS was added to the periphery of the 96-well plates to prevent the volatilization of reagents. The 96-well plates were placed in an incubator at 37 °C for light avoidance culture for 1 hour to measure the absorbance value OD450nm, and repeated measurements were taken 3 times and the mean value was taken. Calculate according to the formula:

[0049] .

[0050] The experimental results are as Figure 4 shown. The small molecule peptide of the present invention can inhibit the proliferation activity of breast cancer cells. After grouped culture, compared with the control group, the proliferation activities of the three types of breast cancer cells treated with the small molecule peptide were significantly reduced, indicating that the small molecule peptide has an inhibitory effect on the proliferation ability of breast cancer cells.

[0051] Example 4 Anti-breast cancer cell xenograft tumor test of small molecule peptide

[0052] Animal source: SPF-grade BALB / c-nu female nude mice, 6 weeks old, with a body weight of 15 - 18 g, purchased from Zhuhai BestBio Technology Co., Ltd., with the use license number SYXK(Yue)2025 - 0229. After 1 week of adaptive feeding, the experiment was started.

[0053] Breast cancer xenograft tumor mouse model: Human breast cancer cells MDA-MB-231 in the logarithmic growth phase were made into a cell suspension with a cell density of 3.0×10 7 cells / mL with normal saline. 0.2 mL of the cell suspension was slowly injected subcutaneously into the axilla of the nude mice. After inoculation, the inoculated area was gently pressed with a sterilized cotton swab for a moment to establish a human breast cancer xenograft tumor model. After inoculation, the body weight of the tumor-bearing nude mice was weighed every 3 days, and the tumor size was measured at the same time. When the tumor volume reached 100 mm 3 , it indicated that the MDA-MB-231 breast cancer tumor-bearing mouse model was successfully established, and relevant experiments could be carried out. 6 normal mice were randomly selected and 0.2 mL of normal saline was injected at the corresponding site as the normal control group.

[0054] Grouping and Drug Administration: After successful establishment of the MDA-MB-23 breast cancer xenograft mouse model, mice were randomly divided into a model group, a cyclophosphamide (CTX, 25 mg / kg) group, and low- and high-dose (50 and 500 mg / kg) small molecule peptide groups, with 6 mice in each group. The small molecule peptide group was administered the corresponding concentration of peptide solution by gavage, while the normal group and the model group were administered an equal volume of sterile water for injection by gavage, once daily. The CTX group was administered the drug intraperitoneally once a week for 21 consecutive days. During the drug administration period, the mice's weight, mental state, coat, behavior, and other general indicators were observed.

[0055] Tumor growth volume and tumor inhibition rate were measured: Starting from day 1 of drug administration, the major diameter (L) and minor diameter (d) of the tumors in tumor-bearing mice were measured every 3 days using vernier calipers. The tumor volume was recorded and calculated, and a tumor growth curve was plotted. Tumor volume V = (L × d) 2 ) / 2. Twenty-four hours after the last administration, nude mice with breast cancer were euthanized by injecting phenobarbital. The tumors and spleens were dissected, and the residual blood was blotted with filter paper before weighing. The tumor inhibition rate was calculated. Tumor inhibition rate = (mean tumor weight in the model group - mean tumor weight in the experimental group) / mean tumor weight in the model group × 100%.

[0056] ELISA was used to detect serum cytokine levels: 24 hours after the last administration, blood was collected from the fundus venous plexus of mice and allowed to stand for 1 hour before being analyzed by ELISA at 3500 rpm. -1 Centrifuge for 15 min, separate serum, and measure the levels of IL-6, TNF-α and IFN-γ in mouse serum according to the instructions of the ELISA kits (all purchased from Wuhan Beinlai Biotechnology Co., Ltd.).

[0057] Experimental results:

[0058] (1) Effects of small molecule peptides on growth status, body weight, and tumors in nude mice with xenografts

[0059] The experimental results are shown in Table 1. Compared with the normal group, the nude mice in the model group were lethargic, sluggish, and had dry, yellow, and fluffy fur. Each drug-treated group showed varying degrees of relief after drug intervention. There were no significant differences in body weight among the normal group, model group, CTX group, and different dosage groups of the small molecule peptide (P > 0.05). Compared with the model group, the tumor weight of nude mice in the CTX group and the high and low dosage groups of the small molecule peptide was inhibited to varying degrees (P < 0.01), and the inhibitory effect of the small molecule peptide on tumor growth in breast cancer xenograft mice showed a significant dose-dependent effect.

[0060] Table 1. Effects of small molecule peptides on body weight and tumor inhibition rate in nude mice with transplanted tumors (x±s, n=6)

[0061]

[0062] Compared with the model group, *p < 0.05, **p < 0.01; compared with the CTX group, #p < 0.05, ##p < 0.01.

[0063] (2) Effects of small molecule peptides on serum cytokine levels in nude mice with xenografts

[0064] The experimental results are shown in Table 2. Compared with the control group, the serum levels of IL-6, IFN-γ and TNF-α in the model group mice were significantly reduced (p < 0.01), indicating that the modeling was successful. Compared with the model group and the CTX group, the serum levels of IL-6, IFN-γ and TNF-α in the small molecule peptide groups were significantly increased (p < 0.05, 0.01).

[0065] Table 2 Effects of small molecule peptides on serum cytokine levels in x±s, n=6 in nude mice with xenografts

[0066]

[0067] Compared with the normal group, Δp < 0.05, ΔΔp < 0.01; compared with the model group, *p < 0.05, **p < 0.01; compared with the CTX group, #p < 0.05, ##p < 0.01.

[0068] The small molecule peptides of this invention can significantly inhibit the growth rate of human breast cancer cell MDA-MB-231 xenograft tumors in nude mice and reduce tumor mass. The small molecule peptides can significantly regulate abnormal leukocyte levels in the peripheral blood of xenograft tumor-bearing nude mice and increase serum levels of IL-6, IFN-γ, and TNF-α cytokines, indicating that the small molecule peptides of this invention can improve the disordered immune system and enhance immune function in xenograft tumor-bearing nude mice.

[0069] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A small molecule peptide with inhibitory activity against breast cancer cells, characterized in that, The amino acid sequence of the small molecule peptide is shown in SEQ ID NO:

1.

2. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the small molecule peptide as described in claim 1.

3. The pharmaceutical preparation according to claim 2, characterized in that, The small molecule peptides include their pharmaceutically acceptable salts.

4. The pharmaceutical preparation according to claim 3, characterized in that, The pharmaceutically acceptable salts include at least one of the following: hydrochloride, sulfate, acetate, methanesulfonate, succinate, fumarate, citrate, malate, and organic amine salts.

5. The pharmaceutical preparation according to any one of claims 2-4, characterized in that, The pharmaceutical preparation also contains pharmaceutically acceptable excipients.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation can be formulated into any pharmacologically acceptable dosage form.

7. The pharmaceutical preparation according to claim 6, characterized in that, The dosage forms of the pharmaceutical preparations include liquid preparations, solid preparations, or semi-solid preparations.

8. Use of the small molecule peptide of claim 1 or the pharmaceutical preparation of any one of claims 2-7 in the preparation of a medicament for treating breast cancer.

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