Application of alpha spiral polypeptide and tamoxifen in preparation of anti-breast cancer pharmaceutical composition

Abstract

The invention discloses application of alpha spiral polypeptide combined with tamoxifen to preparation of an anti-breast cancer pharmaceutical composition, and belongs to the technical field of biological medicine. A large number of experimental studies show that the alpha spiral polypeptide 6w and tamoxifen are combined for medication, the killing ability of the ERalpha high-expression breast cancer cell line can be obviously improved, the phenomena of apoptosis, cell cycle arrest in the G0 / G1 phase and the like of the ERalpha high-expression breast cancer cell line can be obviously improved, an estrogen receptor signal channel can be inhibited, and the ERalpha high-expression breast cancer cell line can be used for treating the ERalpha high-expression breast cancer cell line. The expression level of related genes such as pS2 at the downstream of a signal is obviously reduced, and the proliferation of tamoxifen drug-resistant cells can be better inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the use of an alpha helical polypeptide combined with tamoxifen to prepare an anti-breast cancer pharmaceutical composition. Background Art

[0002] Breast cancer is one of the most common cancers among women worldwide, seriously endangering their health. According to the latest global cancer burden data released in 2022 by the World Health Organization's International Agency for Research on Cancer (IARC), breast cancer is the most common cancer in women.

[0003] Estrogen receptor (ERα)-positive breast cancer refers to breast cancer that is estrogen receptor (ER)-positive. It is a specific and common subtype of breast cancer, accounting for approximately 70% of all cases. Estrogen promotes the proliferation of estrogen-dependent ERα-positive cancer cells. Its incidence is significantly age-dependent, rising from 65% in women under 50 years old to 75% in those over 50 years old. Estrogen primarily acts through three types of receptors: nuclear ERα, ERβ, and membrane G protein-coupled ER (GPER, also known as GPR30). ERα has been shown to be a core molecule driving breast cancer development and progression, making it a promising therapeutic target. While endocrine therapy is effective and well-tolerated as the mainstay of treatment for hormone receptor-positive breast cancer, it also carries significant side effects. When estrogen binds to ERα, it activates downstream signaling pathways, promoting tumor cell growth and proliferation. ERα regulates the transcription of target genes by binding to estrogen response elements (EREs) on DNA, thereby influencing cell proliferation, differentiation, epithelial-mesenchymal transition (EMT), and invasion and metastasis. ER-positive breast cancer is often linked to factors such as genetics and hormone changes. When detected early, prompt surgical resection can yield excellent results. Following surgery, patients should follow their doctor's advice for comprehensive treatment options, including radiotherapy and chemotherapy.

[0004] Tamoxifen is an important endocrine therapy drug that has been widely used in the treatment of ERα-positive breast cancer. It competes with estrogen for binding to estrogen receptors, thereby inhibiting estrogen-activated gene transcription and cell proliferation. Taking tamoxifen may reduce the risk of estrogen receptor-positive breast cancer patients developing further invasive breast cancer. However, after long-term treatment with these endocrine therapy drugs, patients often develop drug resistance and increase the risk of uterine cancer. The development of drug resistance has brought great difficulties to the fields of drug development and disease treatment. The problem of tamoxifen resistance in breast cancer patients has become one of the important issues that need to be addressed in the field of breast cancer treatment. Therefore, it is particularly important to develop new therapies or drug combination therapies to address the problem of tamoxifen resistance. Summary of the Invention

[0005] In view of the above problems, the technical problem to be solved by the present invention is to provide an application of an α-helical polypeptide combined with tamoxifen to prepare an anti-breast cancer pharmaceutical composition, so as to better inhibit the proliferation of tamoxifen-resistant cells.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows: An application of an α-helical polypeptide combined with tamoxifen to prepare an anti-breast cancer pharmaceutical composition; The structural formula of α-helical polypeptide is: ; The structural formula of tamoxifen: .

[0007] As a preferred embodiment, the α-helical polypeptide can inhibit the proliferation of tamoxifen-resistant cells MCF-7 and / or T47D.

[0008] As a preferred embodiment, the concentration of the α-helical polypeptide is 5-20 μM.

[0009] The pharmaceutical composition for inhibiting the proliferation of tamoxifen-resistant breast cancer cells comprises alpha helical polypeptide 6w and tamoxifen.

[0010] In cell experiments and animal experiments, the pharmaceutical composition of the present invention has a drug concentration of 10 μM for the α-helical polypeptide and a drug concentration of 5 μM for tamoxifen.

[0011] The pharmaceutical composition of the present invention is in the form of tablets, granules, pills, capsules, solutions or injections.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The α-helical peptide 6w of the present invention, combined with tamoxifen, significantly enhanced apoptosis and cell cycle arrest in the G0 / G1 phase of ERα breast cancer cell lines. It also inhibited the estrogen receptor signaling pathway, significantly downregulated the expression levels of downstream genes such as pS2, and significantly inhibited the cell proliferation activity of tamoxifen-resistant breast cancer cells. Therefore, α-helical peptide 6w has promising application prospects in improving tamoxifen resistance in breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Fluorescence polarization experiments were used to detect the binding of peptides to ERα protein in the presence of tamoxifen (TAM); Figure 2 Fluorescence polarization experiments were used to detect the binding of peptides to ERα protein in the presence of 4-hydroxytamoxifen (OHT); Figure 3This is an in vitro serum stability test for peptides; Figure 4 It is a hemolytic test of polypeptide; Figure 5 For pull-down experiments of peptides modified with streptavidin-biotin; Figure 6 For in vitro heat transfer experiments; Figure 7 Peptide 6w enhances the thermal stability of ERα protein at the cellular level; Figure 8 To analyze the killing ability of peptide 1w, 6w, TAM and 6w+TAM combination on T47D, MCF7, MDA-MB-231 and MCF10A cells using MTT assay; Figure 9 To analyze the killing ability of peptides at specific concentrations at 1 week, 6 weeks, TAM and 6 weeks + TAM combination on T47D cells using MTT assay; Figure 10 Statistical graph showing the apoptosis of T47D cells induced by peptide or TAM; Figure 11 This is a statistical diagram of the apoptosis of T47D cells induced by 6 weeks of peptide; Figure 12 This is a statistical diagram of cell cycle arrest in T47D cells; Figure 13 For RT-qPCR experiments, the effects of the peptides on the pS2 gene in T47D cells were examined; Figure 14 Western blot experiments were used to detect the effects of peptides on ERα, PR, VDR and other proteins; Figure 15 The changes in plasma concentration of the peptide over time in mice after different administration methods for 6 weeks; Figure 16 To stabilize the effect of peptide on the body weight of mice transplanted with MCF7 human breast cancer; Figure 17 To test the in vivo distribution of stable peptides labeled with Cy5 fluorescent group for infrared imaging after intravenous and intratumoral injection; Figure 18 Infrared imaging of tumor, kidney, liver, spleen, heart, lung, and brain tissues in tumor-bearing mice 24 hours after intravenous and intratumoral injection of stable peptides labeled with Cy5 fluorescent groups; Figure 19 Hematoxylin-eosin staining was used to detect the effects of stable peptides on mouse heart, liver, spleen, lung and kidney tissues. DETAILED DESCRIPTION

[0014] The specific implementation of the present invention is further described below with reference to the examples.

[0015] Example 1

[0016] 1.1 Fluorescence polarization (FP) assay to determine the binding affinity of peptide 6-FITC to ERα In order to verify the binding affinity of peptide 6w to ERα protein and the effect of tamoxifen (TAM) on the binding of peptide 6w to ERα protein, peptide 6w was labeled with FITC (named peptide 6-FITC), and the protein was expressed after the target plasmid was introduced into E. coli. After purification, the pure ERα-LBD protein was obtained. The binding affinity of peptide 6-FITC to ERα was then determined using the fluorescence polarization (FP) method. The experiment was conducted at a concentration of 10 nM peptide 6-FITC. As the concentration of TAM gradually increased, the effect of TAM on the binding affinity of 6-FITC to ERα was tested. The experimental results are shown in Figure 2. Figure 1 shown. Figure 1 Middle (A) shows the binding of 6-FITC peptide to ERα protein with increasing tamoxifen concentration in fluorescence polarization experiment. Figure 1 (B) Summary of the binding dissociation constant K between 6-FITC peptide and ERα protein D value.

[0017] Experimental results showed that low TAM concentrations had little effect on the binding of the 6-FITC peptide to ERα. However, when TAM concentrations were significantly higher than 6-FITC by 500 or 1000 times, it did affect the binding of the 6-FITC peptide to ERα, manifesting as a gradual decrease in the binding affinity of the 6-FITC peptide to ERα, ultimately leading to a lack of binding or a trend of negative competition. Therefore, in subsequent cell experiments, lower TAM concentrations were used, which theoretically should not affect the binding of the 6w peptide to ERα.

[0018] At the same time, the experimental results also suggest that if the ligand binding pocket on the ERα protein that binds to estrogen such as estradiol molecules is competitively bound by tamoxifen, the ERα protein may undergo an allosteric effect, resulting in ERα being unable to bind to the coactivator, and the peptide targeting the binding pocket of the coactivator and ERα is also difficult to bind to ERα. However, in the presence of low concentrations of tamoxifen, the binding affinity between the peptide and ERα will not be significantly affected, indicating that the two can reach an equilibrium point in concentration, and the ERα protein may be stabilized in a certain state at the same time. Therefore, when using peptide 6w in combination with tamoxifen in the study of breast cancer, it is necessary to pay attention to the dosage concentration of both.

[0019] 1.2 Fluorescence polarization (FP) assay to determine the effect of 4-hydroxytamoxifen (OHT) on the binding affinity of 6-FITC peptide to ERα The metabolite of tamoxifen in the human body is 4-hydroxytamoxifen (OHT). The structural formula of 4-hydroxytamoxifen (OHT) is:

[0020] Fluorescence polarization experiments were used to detect the binding of peptides to ERα protein in the presence of 4-hydroxytamoxifen (OHT). The experimental results are as follows: Figure 2 This experiment was conducted at a concentration of 10 nM peptide 6-FITC, with increasing concentrations of OHT, to test the effect of OHT on the binding affinity of 6-FITC to ERα. Figure 2 (A) shows the binding of 6-FITC peptide to ERα protein with increasing concentration of 4-hydroxytamoxifen in fluorescence polarization experiment. Figure 2 (B) Summary of the binding dissociation constant K between 6-FITC peptide and ERα protein D value.

[0021] The experimental results also showed that the presence of low concentrations of 4-hydroxytamoxifen did not significantly affect the binding affinity between the peptide and ERα. Higher concentrations of 4-hydroxytamoxifen affected the binding of the 6-FITC peptide to the ERα protein, which was the same experimental phenomenon as the above-mentioned tamoxifen. The results indicate that the peptide 6w can be used in combination with tamoxifen (TAM) or 4-hydroxytamoxifen (OHT) for the treatment of breast cancer.

[0022] 1.3 In vitro serum stability test of peptides Experimental method: 1 week and 6 week peptides were co-incubated in 25% mouse serum for a specific time point, and then the samples were taken out for serum precipitation. After high-speed centrifugation, the supernatant samples were taken for LCMS analysis. The relative percentage of peptide content was obtained by comparing the peak areas of the corresponding peptide peaks on the liquid chromatography spectrum. The experimental results are as follows: Figure 3 shown.

[0023] Experimental results showed that the ring-closed α-helical peptide 6w exhibited improved serum stability compared to the unringed linear peptide 1w (peptide control group). Peptide 6w also exhibited a low degradation rate in 25% mouse serum, with approximately 70% of the peptide remaining after 24 hours of incubation. This indicates that the amide bond cyclization strategy on the peptide side chain can improve its serum stability. This higher serum stability facilitates its biological effects within cells, preventing rapid degradation by serum or other peptidases.

[0024] 1.4 Hemolytic test In order to detect the hemolytic activity of the peptide and the combination of the peptide and tamoxifen, and to determine whether the peptide can be safely used in cells, the peptide was co-incubated with fresh mouse red blood cells to conduct a hemolytic experiment to detect the hemolytic activity of the peptide on mouse red blood cells at 1 week, 6 weeks, TAM, and 6 weeks + TAM combination. The experimental results are as follows Figure 4 shown.

[0025] The experimental results showed that at specific dosing concentrations of the polypeptide and the concentrations of the polypeptide-tamoxifen combination, the hemolysis caused by cell membrane rupture in mouse red blood cells in each dosing group was weak, indicating that the polypeptide or polypeptide-tamoxifen combination did not cause significant damage to the cell membrane of mouse red blood cells at specific cell dosing concentrations, and each dosing group could be safely used in subsequent cell experiments.

[0026] 1.5 Streptavidin-biotin-modified peptide pull-down experiment The aforementioned FR experiments have demonstrated that the peptides can interact with E. coli-expressed ERα protein with high binding affinity. To determine whether the peptides can interact with endogenous estrogen receptor ERα, this experiment attempted to react cell lysates with biotin-labeled peptides (1w-biotin and 6w-biotin). Pull-down experiments were then performed to verify whether the peptides can interact with ERα protein derived from cells.

[0027] Experimental method: Cell lysate was obtained by lysing T47D cells, and the supernatant of the cell lysate was obtained by high-speed centrifugation. The biotin-modified polypeptide was first incubated with the cell lysate at 4°C for more than 12 hours to allow the polypeptide to fully bind to the target protein in the cell lysate. Then, streptavidin-modified beads were incubated with the above-mentioned biotin-polypeptide-target protein complex at 4°C for 4 hours. Streptavidin and biotin have very high binding affinity and can form a streptavidin-biotin-polypeptide-target protein complex. Western blot experiments were then performed to detect whether the addition of 1w-biotin or 6w-biotin polypeptide can interact with the endogenous ERα protein in the cell. 400 μL of cell lysate was taken and incubated with 1w-biotin or 6w-biotin polypeptide. The group without polypeptide was used as the control group. In the experiment, equal amounts of cell lysate were taken for western blot experiments to detect the amount of ERα and GAPDH as internal reference for the sample amount. The experimental results are shown in the figure. Figure 5 shown.

[0028] The experimental results showed that the linear peptide 1w-biotin had a weak interaction effect with ERα, which may be due to its low binding affinity with ERα in the cell lysate, and therefore no ERα band could be detected; 6w-biotin could clearly detect the ERα band, which may be due to its strong binding affinity with ERα protein and its ability to interact with endogenous estrogen receptor ERα in cells. This result strongly demonstrated that the 6w-biotin peptide can bind to endogenous ERα protein from cells.

[0029] 1.6 In vitro thermal shift assay (TSA) In vitro thermal shift experiments are used to characterize the effect of adding small molecules or peptides to target proteins on the increase or decrease of protein stability. m Generally speaking, the melting temperature T m The value becomes larger, indicating that the target protein can be more stable after adding the peptide; on the contrary, if the melting temperature T m The value decreases, indicating that the addition of polypeptides reduces the stability of the protein, making it more susceptible to degradation and denaturation.

[0030] Experimental method: In vitro thermal shift assay (TSA) was performed using ERα-LBD protein expressed in E. coli. After incubating the peptide with ERα-LBD protein for 2 h, SYBRO Orange dye was added and mixed. The TSA experimental program with temperature gradient setting was used in a PCR instrument to collect data. The experimental results are shown in Figure 2. Figure 6 shown.

[0031] The experimental results showed that the experimental groups of 10 μM peptide 6w and 10 μM 6w+5 μM TAM could increase the melting temperature of ERα m The T values ​​of 5 μM TAM and the control group with PBS increased by about 4-5 ℃. m There was no significant change compared with the values, indicating that the addition of 10 μM 6w can slightly stabilize the ERα protein. A higher temperature is required to denature and precipitate the ERα protein during heating. This result indirectly suggests that the combined administration of polypeptide 6w or 6w+TAM may stabilize the ERα protein in a certain state.

[0032] 1.7 Cellular thermal shift assay (CETSA) In vitro thermal shift experiments verified that the addition of polypeptide 6w would make ERα expressed in Escherichia coli more stable. Western blot experiments were then used to study the effect of polypeptide 6w on the stability of endogenous ERα protein in cells.

[0033] Experimental Method: First, incubate with T47D cell lysate for 5 hours with or without 10 μM peptide 6w. Then, take 30 μl of each complex and heat it for 2 minutes at the following temperatures. Unstable proteins may precipitate. Then, place on ice for 2 minutes, centrifuge at high speed, and take equal amounts of supernatant (avoiding the precipitate). After adding SDS loading buffer, inactivate at 100°C for 10 minutes. Then, take equal amounts of the solution for western blot analysis to detect the relative content of ERα protein. The experimental results are as follows: Figure 7 shown. Figure 7 (A) Equal volumes of T47D cell lysate were incubated with or without peptide for 6 weeks, and the soluble ERα protein content at different temperatures was analyzed by western blot analysis. Figure 7 (B) Semi-quantitative analysis of the relative content of ERα in the cell heat shift experiment, with the experimental group containing only ERα protein at 50°C as the reference (set to 1.0).

[0034] The experimental results showed that the addition of 10 μM peptide for 6 weeks may further stabilize the ERα protein. Under temperature gradient heating, the ERα content was slightly higher than that of the control group without peptide addition, which was consistent with the results of the above in vitro heat transfer experiment.

[0035] 1.8 MTT assay to detect the viability of related cells After confirming that the peptide 6w can interact with the endogenous ERα protein, in order to further study the effect of the peptide or the peptide combined with tamoxifen on breast cancer cell lines with high ERα expression such as T47D and MCF7, and triple-negative breast cancer cell lines with low ERα expression such as MDA-MB-231 cell line, and normal breast epithelial cells such as MCF10A, this experiment used MTT assay to analyze the killing ability of peptide 1w, 6w, TAM and 6w+TAM combination on T47D, MCF7, MDA-MB-231 and MCF10A cells. The experimental results are as follows Figure 8 shown.

[0036] Experiments showed that peptides 6w, TAM, and 6w+TAM had strong killing abilities against breast cancer cell lines with high ERα expression, such as T47D and MCF7. This killing ability increased with increasing drug concentration. The effect of the unclosed linear peptide 1w was weaker. In contrast, peptides 1w, 6w, TAM, and 6w+TAM had weaker killing abilities against triple-negative breast cancer cell lines with low ERα expression, such as MDA-MB-231. Furthermore, peptides 1w, 6w, TAM, and 6w+TAM had lower toxicity against normal breast epithelial cells, such as MCF10A.

[0037] The experiments showed that the peptide 6w, TAM, and 6w+TAM had high selectivity for breast cancer cell lines with high ERα expression, and the 6w+TAM combination group had a higher killing ability against breast cancer cell lines with high ERα expression. From the MTT assay results of the T47D cell line, it can be seen that 10 μM 6w+10 μM TAM can kill approximately 50% of the cells. Combined with the above FP assay results, low concentrations of TAM do not interfere with the binding of the peptide to the ERα protein. Therefore, TAM was added at a lower concentration than the 6w concentration, and the combination drug concentration ratio was set to 10 μM 6w+5 μM TAM to reasonably characterize the effect of the combination drug.

[0038] At the same time, the MTT experiment was used to characterize the killing ability of 10 μM 6w, 5 μM TAM, and 10 μM 6w+5 μM TAM on T47D cells at the drug concentrations used in the cell experiment. The experimental results are as follows: Figure 9 As shown, the experimental results show that combined use can enhance the effect of inhibiting the growth and proliferation of cancer cells with high ERα expression.

[0039] 1.9 Cell apoptosis assay In order to further study whether the peptide can induce apoptosis in breast cancer cells, the Annexin V / PI double staining kit (Genstar) was used to detect whether the peptide can induce apoptosis in T47D cells. The cell apoptosis experiment first incubated the peptide and T47D cells for 12 hours, then collected the cells in the culture supernatant of the cell plate and the cells at the bottom of the cell plate, centrifuged, added FITC Annexin V and PI dyes, and tested the cell apoptosis using flow cytometry. The results of the cell apoptosis experiment are shown in Figure 2. Figure 10 As shown, A in the figure is a statistical diagram of T47D cells induced by polypeptide or TAM to produce cell apoptosis, the flow cytometry graph and Q3 in the statistical diagram represent early apoptotic cells, and Q2 represent mid-to-late apoptotic cells.

[0040] The experimental results showed that 1 week of peptide, 6 weeks of peptide, TAM, and 6 weeks + TAM could induce early apoptosis (increased proportion of cells in the Q3 region of the flow cytometer) and mid-to-late stage apoptosis (increased proportion of cells in the Q2 region of the flow cytometer) in T47D cells, indicating that peptide and TAM can inhibit the growth and proliferation of breast cancer cells by inducing apoptosis.

[0041] This experiment further determined the effect of increasing the concentration of peptide 6w on the apoptosis of T47D cells. After the peptide and T47D cells were co-incubated for 12 hours, the cells at the bottom of the cell plate were uniformly collected to test the apoptosis of T47D cells induced by peptide 6w. The experimental results are as follows: Figure 11It can be clearly found that peptide 6w can induce concentration-dependent apoptosis in T47D cells, that is, the higher the concentration of peptide 6w, the more the proportion of mid-to-late stage apoptosis (Q2 region) of T47D cells will increase significantly.

[0042] 1.10 Cell cycle assay Studies have shown that estrogen receptor inhibitors can cause cell cycle arrest in breast cancer cells, and tamoxifen can arrest cancer cells in the G0 / G1 phase. In order to detect the effect of peptides on the cell cycle, this experiment tested the effect of peptides on the cell cycle. The cell cycle arrest statistics of T47D cells are shown in the figure below. Figure 12 Data are shown as mean ± SD, based on at least three independent experiments. Standard deviations are shown as error bars in the figures.

[0043] The experimental results showed that the effect of linear peptide for 1 week was not obvious, and there was little difference compared with the blank control group; peptide for 6 weeks could arrest the cell cycle in the G0 / G1 phase; tamoxifen arrested cells in the G0 / G1 phase, which was consistent with literature reports; the effect of the combination of peptide for 6 weeks and tamoxifen was more significant, and a higher proportion of T47D cells could be arrested in the G0 / G1 phase.

[0044] 1.11 RT-qPCR assay to detect the effect of peptides on the pS2 gene In the estrogen receptor signaling pathway, activated estrogen receptors interact with estrogen response elements (EREs), mediating the transcription of downstream target genes. Literature indicates that pS2 is a target gene in the estrogen receptor signaling pathway. To examine the effect of peptides on pS2 transcription, RT-qPCR experiments were performed.

[0045] Experimental method: After adding peptides or TAM to T47D cells and incubating for 24 hours, all cells were collected and RNA was extracted for RT-qPCR experiment. Figure 13 shown.

[0046] RT-qPCR experiments showed that peptide 6w, TAM, and 6w+TAM downregulated pS2 mRNA expression, indicating that peptide 6w, TAM, and the combination of 6w+TAM can mediate and downregulate pS2 gene levels in the ERα downstream signaling pathway, with the 6w+TAM combination showing a greater effect. The linear peptide 1w may have a limited effect on pS2 gene expression due to its weak cell membrane penetration ability or low binding affinity to ERα.

[0047] 1.12 Western blot assay to detect the effects of peptides on ERα and other proteins Nuclear receptor family proteins include estrogen receptor (ER), progesterone receptor (PR), vitamin D receptor (VDR), etc. These nuclear receptor proteins all contain LXXLL sequence modules, which have homology in nuclear receptor proteins. In order to determine the effect of polypeptides on nuclear receptor proteins such as ERα, PR, and VDR in T47D cells, western blot experiments were performed. After T47D cells were cultured in six-well plates overnight, the old culture medium was removed, the cells were washed once with PBS, and 10 μM polypeptide was added respectively. After incubation for 24 hours, the supernatant cells in the six-well plates and the cell lysate after the bottom cells were combined were extracted for western blot experiments. The antibody dilution ratio was 1:1000. The results are shown in the figure. Figure 14 shown.

[0048] The experimental results showed that adding the peptide for 1 week or 6 weeks and incubating for 24 hours had no significant effect on the content of ERα, PR, VDR and other proteins in T47D cells, and the peptide could not downregulate the expression levels of ERα, PR, VDR and other proteins.

[0049] Example 2

[0050] 2.1 Pharmacokinetics of Stable Peptide 6w in Mice after Different Administration Methods To verify the in vivo anti-tumor effect of the most potent stable peptide 6w, its pharmacokinetic profile in mice was first determined. The plasma drug concentrations of female Balb / c mice following intravenous, intraperitoneal, or subcutaneous administration for 6 weeks are shown in Tables 1-3, the corresponding pharmacokinetic parameters are shown in Table 4, and the corresponding concentration-time curves are shown in Table 5. Figure 15 .

[0051] After intravenous administration of 3 mg / kg for 6 weeks, the blood concentration at 5 minutes was 30000±4101 ng / mL. The area under the blood concentration curve (AUC 0-24h, plasma ) is 20964 ng∙h / mL, and the elimination half-life is 0.35 hours. The clearance rate after 6 weeks is 0.14 L·h -1 kg -1 , the apparent volume of distribution is 0.10 L / kg.

[0052] After intraperitoneal administration of 10 mg / kg for 6 weeks, the peak concentration reached 2 hours after administration, with a peak concentration of 22550±212 ng / mL. The area under the plasma concentration curve (AUC 0-24h, plasma ) was 60057 ng∙h / mL. The bioavailability of intraperitoneal administration relative to intravenous injection was 85.9%.

[0053] After subcutaneous administration of 10 mg / kg for 6 weeks, the peak concentration was 7835±983 ng / mL at 1 hour after administration. The area under the plasma concentration curve (AUC 0-24h, plasma ) is 25239 ng∙h / mL. The bioavailability of subcutaneous administration relative to intravenous injection is 36.1%.

[0054] Table 1 Plasma concentrations of 6w (ng / mL) after 6w intravenous administration (3 mg / kg)

[0055] BLOQ: below the limit of quantification (1 ng / mL) Table 2 Plasma concentrations of 6w (ng / mL) after intraperitoneal administration of 6w (10 mg / kg)

[0056] BLOQ: below the limit of quantification (1 ng / mL) Table 3 Plasma concentrations of 6w (ng / mL) after subcutaneous administration of 6w (10 mg / kg)

[0057] BLOQ: below the limit of quantification (1 ng / mL) Table 4 Pharmacokinetic parameters after 6 weeks of administration by different routes

[0058] Table 4 Pharmacokinetic parameters after 6 weeks of administration by different routes

[0059] The plasma concentration of peptide 6w in mice after different administration methods is shown in the following curves: Figure 15 As shown in the figure, blue, red, and green represent intravenous, intraperitoneal, and subcutaneous injections, respectively. Peptide 6w has the highest bioavailability when injected intraperitoneally, and maintains effective concentrations for a longer period of time. Therefore, the efficacy experiments later in this article will use intraperitoneal injection as the administration route.

[0060] 2.2 Effect of stabilized peptides on body weight in mice bearing MCF7 human breast cancer xenografts This study tested the effect of stable peptide administration on the body weight of MCF7 human breast cancer Balb / c nude mouse xenograft tumor model mice. Figure 16 The data showed that whether the stable peptide was administered alone or in combination with tamoxifen, the effect on the body weight of mice was very small, indicating that the stable peptide was not significantly toxic to mice when administered in vivo.

[0061] 2.3 Infrared imaging test of peptide distribution in nude mouse human breast cancer transplant model This experiment used infrared imaging to test the distribution of Cy5 infrared group-labeled stable peptides in living tumor-bearing mice. In vivo infrared imaging was performed using the IVISSpectrum in vivo imaging system (Perkin Elmer) before drug injection and 10 min, 30 min, 1 h, 2 h, 4 h, and 24 h after drug injection. The ventral surface of the tumor was imaged. The results are shown below. Figure 17 As shown, the tumor site is circled. The results demonstrate that the peptide 6-Cy5 accumulates at the tumor site when injected intravenously. Furthermore, when injected intratumorally, the stable peptide primarily accumulates at the tumor site, rarely disseminating to other organs, and maintains high fluorescence intensity even 24 hours later.

[0062] After the whole-body imaging of the mice was completed at 24 hours, the tumor, kidney, liver, spleen, heart, lung, and brain tissues of the mice were removed and infrared imaging was performed using the IVIS Spectrum in vivo imaging system. The infrared fluorescence intensity of each tissue and organ was calculated using the corresponding infrared imaging software. The results are as follows: Figure 18 The results show that 6-Cy5 is still abundant in tumor tissue 24 hours after intravenous injection, with some distribution in the kidney, liver, and lungs, but virtually absent in other tissues. Twenty-four hours after intratumoral injection, 6-Cy5 is still concentrated in tumor tissue with high intensity, but absent in other tissues. This suggests that the stabilized peptide can accumulate at the tumor site after injection, exerting its anti-tumor effect.

[0063] 2.4 Hematoxylin-eosin staining to detect the effects of peptides on various body tissues of mice After the administration of the stabilized peptide against the growth of MCF7 human breast cancer transplanted tumor mice, the mice were treated. The tumors and kidney, liver, spleen, heart, lung, and brain tissues of the mice were removed. Some samples were immediately placed in formalin for preservation. The effects of the peptide on the various body tissues of the mice were detected by hematoxylin-eosin staining. The results are as follows: Figure 19 As shown in the figure, it can be seen that the administration of the peptide alone for 6 weeks and the combination with tamoxifen had no significant effect on the tissues and organs of the mice compared with the control group, indicating that the stable peptide drug had no obvious toxic effects on the tissues and organs of the mice.

[0064] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should be covered by the patent of the present invention.

Claims

1. An application of an α-helical polypeptide combined with tamoxifen to prepare an anti-breast cancer pharmaceutical composition; The structural formula of α-helical polypeptide is: ; The structural formula of tamoxifen: .

2. The use according to claim 1, wherein the α-helical polypeptide can inhibit the proliferation of tamoxifen-resistant cells MCF-7 and / or T47D.

3. The use according to claim 1, wherein the α-helical polypeptide is used at a concentration of 5-20 μM.

4. A pharmaceutical composition for inhibiting the proliferation of tamoxifen-resistant breast cancer cells, comprising α-helical polypeptide 6w and tamoxifen.

5. The pharmaceutical composition according to claim 4, wherein in cell experiments and animal experiments, the concentration of the α-helical polypeptide is 10 μM, and the concentration of tamoxifen is 5 μM. The pharmaceutical composition according to claim 4 , which is in the form of tablets, granules, pills, capsules, solutions or injections.

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