A polypeptide suspension and its use in the preparation of an antitumor medicament

Abstract

The application provides a polypeptide suspension and application thereof in preparation of an anti-tumor medicine, and belongs to the technical field of biological medicines. D F D F D The self-assembled polypeptide suspension with Y.D configuration not only has good anti-degradation, higher biocompatibility and stability, but also diffuses to the whole body after oral absorption, reduces the expression of COX-2 at the tumor site, improves the tumor immune microenvironment, and thus inhibits the growth of tumor cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide suspension and its application in the preparation of antitumor drugs. Background Technology

[0002] Compared to traditional chemotherapy, radiotherapy, and surgery, tumor immunotherapy, with its advantages of high specificity, long duration of action, and fewer side effects, can effectively improve problems such as tumor resistance and recurrence caused by tumor cells evading the body's immune surveillance. It has long been considered the ultimate means of curing cancer. Prospective studies have shown that cyclooxygenase-2 (COX-2) is a protein highly expressed in tumor tissues. On the one hand, it can directly or through the synthesis of prostaglandin E2 (PGE2) promote cancer cell proliferation and metastasis, and inhibit cancer cell apoptosis. On the other hand, it can inhibit interferon and T cell function, mediating tumor immune escape, and is one of the important indicators of tumor occurrence and development. Therefore, COX-2 is particularly important in specifically addressing the problems of tumor immune escape and immune tolerance, and is a favorable way to turn "cold" tumors into "hot" ones, considered a promising anti-tumor therapeutic target.

[0003] As is well known, among the available routes of administration, oral administration is the most convenient, simple, and of greatest concern to patients, offering high patient compliance, wide applicability, and ease of administration. Oral medications are primarily available in solid dosage forms, such as granules, capsules, and sustained-release tablets, and liquid dosage forms, such as solutions, suspensions, and emulsions. Suspensions are not only easy to swallow and have a rapid onset of action, but also allow for precise dosage control, making them an invaluable option for medication administration.

[0004] Currently, there is no oral peptide nanomedicine suspension that can effectively and selectively degrade COX-2 protein, improve the tumor tissue microenvironment, and achieve tumor treatment. Summary of the Invention

[0005] The purpose of this invention is to provide an oral polypeptide suspension with anti-tumor effects, which inhibits tumor cell growth by degrading COX-2 in tumor cells, improving the immune microenvironment at the tumor site, and thus inhibiting tumor cell growth. Furthermore, the drug suspension has advantages in terms of stability.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of a polypeptide suspension in the preparation of an antitumor drug, wherein the active ingredient of the polypeptide suspension is Npx-G. D F D F D Y.

[0008] Preferably, the Npx-GD F D F D The structural formula of Y is shown in equation (I):

[0009]

[0010] Preferably, the solvent for the polypeptide suspension is PBS buffer.

[0011] Preferably, the active ingredient Npx-G D F D F D The mass-to-volume ratio of Y to PBS buffer is 0.5–2 mg: 1–4 mL.

[0012] Preferably, the polypeptide suspension achieves its anti-tumor effect by degrading COX-2 protein.

[0013] Preferably, the tumor includes breast cancer and colon cancer.

[0014] The present invention also provides a polypeptide suspension for the aforementioned application.

[0015] Preferably, the pH of the polypeptide suspension is 7.2 to 7.4.

[0016] Preferably, the polypeptide suspension is an oral preparation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The active ingredient in the polypeptide suspension provided by this invention is Npx-G. D F D F D Y, a self-assembled polypeptide G with Npx naproxen as the D configuration. D F D F D The Y-terminated group was dissolved in buffer to obtain a peptide suspension. The D-configuration self-assembled peptide suspension not only has good anti-degradation properties, high biocompatibility and stability, but also diffuses throughout the body after oral absorption, reducing COX-2 expression at the tumor site, improving the tumor immune microenvironment, and thus inhibiting tumor cell growth. Attached Figure Description

[0019] Figure 1 The polypeptide Npx-G prepared in Example 1 D F D F D High-resolution mass spectrum of Y;

[0020] Figure 2 High-resolution mass spectrum of the peptide Npx-GFFY prepared for Comparative Example 1;

[0021] Figure 3 Optical photographs of the polypeptide suspension and polypeptide hydrogel prepared for Experiment Example 1;

[0022] Figure 4 The peptide Npx-G was measured in Experimental Example 2. D F D F D Microscopic morphology diagram of Y;

[0023] Figure 5 The graph shows the anti-degradation ability of peptide D-suspension under the action of proteinase K as determined in Experiment Example 3.

[0024] Figure 6 The tumor growth trend of mice measured in Experiment Example 4 is shown in the graph.

[0025] Figure 7 This is a graph showing the changes in mouse body weight as measured in Experiment Example 4;

[0026] Figure 8 The expression and statistical graph of COX-2 in 4T1 cells after treatment with the peptide in Experiment Example 5;

[0027] Figure 9 The expression and statistical graph of COX-1 in 4T1 cells after treatment with the peptide in Experiment Example 5. Detailed Implementation

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] The peptide Npx-G was synthesized using the classic Fmoc-short peptide solid-phase synthesis method. D F D F D Y, the specific steps are as follows:

[0031] 1) Weigh 0.5 mmol of 2-chlorotriphenylmethyl chloride resin (purchased from Jier Biochemical (Shanghai) Co., Ltd., substitution rate 1.158 mmol / g) into a solid phase synthesis tube, add 15 mL of dichloromethane (DCM, purchased from Tianjin Chemical Reagent Co., Ltd.), place on a shaker and shake for 15 min to allow the 2-chlorotriphenylmethyl chloride resin to swell completely.

[0032] 2) Use a syringe to completely squeeze the DCM out of the solid-phase synthesis tube, and wash repeatedly with 10 mL of DCM 3 times, 1 min each time;

[0033] 3) Weigh out 1 mmol of 9-fluorenylmethoxycarbonyl protecting group (Fmoc) protected by... DY(Fmoc-Tyr(tBu)-OH) was dissolved in 15 mL of DCM, and 2 mmol of N,N-diisopropylethylamine (DIEPA, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added. After mixing, the mixture was added to a solid-phase synthesis tube and placed on a shaker to react at room temperature for 1 h.

[0034] 4) Remove the reaction solution from the solid-phase synthesis tube with a rubber bulb, wash it three times with 10 mL of DCM for 1 min each time, and add 15 mL of solution containing DCM, DIEPA, and methanol (purchased from Tianjin Concord Technology Co., Ltd.) (the volume ratio of DCM:DIEPA:methanol is 17:1:2) to the solid-phase synthesis tube after washing with DCM. React at room temperature for 15 min.

[0035] 5) Remove the reaction solution from the solid-phase synthesis tube with a rubber bulb, wash three times with 10 ml of DCM for 1 min each time, then wash three times with 10 ml of N,N-dimethylformamide (DMF, purchased from Tianjin Chemical Reagent Company) for 1 min each time. Add 15 mL of 20% piperidine (purchased from Tianjin Chemical Reagent Company) to the solid-phase synthesis tube, using DMF as the diluent. React for 20 min, then wash five times with 10 ml of DMF for 1 min each time.

[0036] 6) Add 1 mmol D F(Fmoc-Phe-OH), 1 mmol HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, purchased from Tianjin Xiens Biochemical Technology Co., Ltd., purity 98%) and 2 mmol DIEPA were mixed and dissolved in 10 mL DMF. After dissolution, the solution was placed in the solid-phase synthesis tube that had been washed 5 times with DMF in step 5). The reaction time was 1 h.

[0037] 7) Repeat steps 5) and 6), wherein, in repeating step 6), 1 mmol is used sequentially. D F(Fmoc-Phe-OH), 1 mmol G(Fmoc-Gly-OH), 1 mmol of the end-capping group Npx (naproxen), after attaching the end-capping group, it is not necessary to remove Fmoc;

[0038] 8) Discard the reaction solution, wash 3 times with DMF for 1 minute each time, and then wash 5 times with DCM for 1 minute each time.

[0039] 9) Prepare 20 mL of cutting solution (95% TFA (trifluoroacetic acid, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., purity 99%), 2.5% TIS (triisopropylsilane, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity 99%), 2.5% H2O (volume ratio)). Add the cutting solution to a solid-phase synthesis tube and react at room temperature for 45 min. Cut the product off from 2-chlorotriphenylmethyl chlororesin, remove the solvent using a rotary evaporator, and obtain the crude product. Then separate and purify using HPLC (high performance liquid chromatograph, purchased from Lumtech, Germany) to obtain the antitumor oral peptide, Npx-G. D F D F D Y.

[0040] The antitumor oral peptides prepared by LC-MS (brand: Shimadzu, Japan, model LC-MS2020) showed the following results: Figure 1 As shown, the structural formula of the polypeptide is shown in Formula (I).

[0041]

[0042] Comparative Example 1

[0043] The polypeptide Npx-GFFY was prepared according to the solid-phase synthesis method of Example 1, wherein... D Y、 D F, D Replacing F with Y, F, and F respectively, Npx-GFFY was synthesized. The synthesized peptide was analyzed by LC-MS, and the results are as follows. Figure 2 As shown, the structural formula is as shown in formula (Ⅱ).

[0044]

[0045] Experimental Example 1

[0046] Preparation of L-hydrogel, D-hydrogel and D-suspension

[0047] The obtained Npx-G D F D F D Y was resuspended in PBS, and the pH was adjusted to 7.4 with sodium carbonate. The suspension was then vortexed to obtain a 1 mM polypeptide suspension, abbreviated as D-suspension.

[0048] The obtained Npx-G D F D F D Y was resuspended in PBS, the pH was adjusted to 7.4 with sodium carbonate, heated to 100°C and cooled to room temperature (25°C) to obtain a 1mM polypeptide hydrogel, abbreviated as D-hydrogel.

[0049] Resuspend the Npx-GFFY pure product in PBS, adjust the pH to 7.4 with sodium carbonate, heat to 100°C and cool to room temperature (25°C) to obtain a 1mM polypeptide hydrogel, abbreviated as L-hydrogel.

[0050] Finally, optical photographs were obtained, such as Figure 3 As shown. From Figure 3 Optical photographs show that after heating and cooling, L-hydrogel and D-hydrogel appear as clear and transparent hydrogels on a macroscopic scale, while D-suspension appears as a suspension with a certain degree of fluidity. Suspension has better stability and oral compliance compared to hydrogel.

[0051] Experimental Example 2

[0052] Use a toothpick to apply a small amount of dry, uncontaminated polypeptide Npx-G. D F D F D Y (prepared in Example 1) was evenly applied to the sample stage on which conductive tape had already been adhered, and then compacted. The sample, along with the sample stage, was placed in a container for gold sputtering. Subsequently, scanning electron microscopy (SEM: JEOL JSM-7900F) was used for imaging, and the surface microstructure was observed using a low-voltage, secondary electron detector. The results are as follows. Figure 4 As shown. By Figure 4 As can be seen, the polypeptide powder in Example 1 has a slender rod-shaped structure.

[0053] Experimental Example 3

[0054] Degradation resistance assay

[0055] Following the method in Example 1, prepare 500 μL of 500 μM L-hydrogel, D-hydrogel, and D-suspension in 1.5 mL EP tubes, and add proteinase K (purchased from Beyotime) to achieve a final concentration of 1 mg / mL.

[0056] After mixing the resulting solution, it was placed in a 37℃ incubator for 24 hours. At six time points (0, 2, 4, 8, 12, and 24 hours after the start of the reaction), 50 μL of sample was dissolved in 200 μL of methanol to prepare a dilution. The content of the compound in the dilution was determined by LC-MS, and the percentage of peptide degradation under the action of proteinase K was calculated based on the peak area. The results are as follows: Figure 5 As shown.

[0057] Figure 5The results showed that within 24 hours of proteinase K treatment, L-hydrogel was almost completely degraded, D-hydrogel was degraded by 27.80%, while D-suspension was degraded by only 13.02%, less than half the degradation of D-hydrogel. These results demonstrate that D-configured peptides have stronger anti-degradation capabilities compared to L-configured peptides, with D-suspension showing the best effect. This also proves the significant advantage of peptide suspensions in resisting degradation in vivo.

[0058] Experiment Example 4

[0059] Mouse test

[0060] An orthotopic tumor model was constructed using COX-2-positive mouse breast cancer cells (4T1). Six-week-old female BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. 4T1 tumor cells were prepared in advance and inoculated into the lower part of the second pair of mammary fat pads in the mice, with 1 million tumor cells injected into each mouse. When the tumor cells reached a size of 70 mm², the tumor cells were inoculated. 3 Mice were randomly divided into five groups (5 mice per group): PBS group, Npx (naproxen) group, L-hydrogel group, D-hydrogel group, and D-suspension group. Administered via gavage every two days at the following concentrations: Npx group 9.2 mg / kg, L-hydrogel group 29.8 mg / kg, D-hydrogel group 29.8 mg / kg, and D-suspension group 29.8 mg / kg, for a total of five administrations. Tumor volume and body weight were monitored for 14 consecutive days, starting from day 0 of administration. Mice were then euthanized. The tumor volume was calculated as length × width. 2 / 2, the result is as follows Figure 6 and Figure 7 As shown.

[0061] Depend on Figure 6 It was found that the D-suspension group had the smallest average tumor volume and an inhibition rate of 67.24%, indicating that D-suspension can significantly inhibit tumor cell growth. In contrast, Npx, L-hydrogel, and D-hydrogel only inhibited tumor growth by 25.62%, 30.90%, and 22.03%, respectively, which is mainly related to the high stability of D-suspension. Meanwhile, Figure 7The results showed that the body weight of mice in all groups maintained a slow growth trend, indicating that the peptides prepared in this invention have good biocompatibility. These results demonstrate that, at the animal level, D-suspension can inhibit tumor cell proliferation with oral administration alone, making it a very promising oral formulation.

[0062] Experimental Example 5

[0063] Protein expression level determination

[0064] Five groups were set up: Control, NPX, L-hydrogel, D-hydrogel, and D-suspension. 4T1 cells were evenly seeded into six-well plates at 300,000 cells per well. After cell attachment, the cells were treated with 1.5 mL of empty culture followed by 400 μM NPX, L-hydrogel, D-hydrogel, and D-suspension for 24 hours, respectively, before protein extraction. Cells were washed three times with PBS, scraped off with a cell scraper, and lysed with RIPA lysis buffer for 20 min. The cells were then centrifuged at 12,000 rpm, 4°C for 10 min, and the supernatant was collected. Loading buffer was added to the sample, and the mixture was boiled at 95°C for 8 min to fully denature the proteins. A 10% gel (purchased from Beyotime, catalog number: P0456S) was used, and protein markers and samples were added sequentially. Electrophoresis was performed at a constant voltage of 160V for 35 min to separate the proteins. The PVDF membrane was activated with methanol for 30 s and then equilibrated with membrane equilibration buffer for 10 min. The membrane and gel were placed in a transfer apparatus for transfer. After transfer, the membrane was blocked with 5% skim milk powder at room temperature for 1 hour. It was then washed four times with TBST for 8 minutes each time. The membrane was cut to the target band size, and the band was incubated with primary antibody (COX-2 or COX-1) diluted with 3% BSA overnight on a shaker at 4°C. The membrane was washed three times with TBST for 10 minutes each time to remove unbound primary antibody. The band was then co-incubated with HRP-labeled secondary antibody diluted with 3% BSA at room temperature on a shaker for 1 hour. The membrane was washed three times with TBST for 10 minutes each time. Subsequently, ECL chemiluminescent substrate reaction solution was added to expose the band. The grayscale values ​​of the bands were measured using ImageJ software and normalized. The results are shown below. Figure 8 and Figure 9 As shown.

[0065] like Figure 8 As shown, cells treated with D-suspension exhibited the lowest COX-2 expression level, with a COX-2 degradation rate of 62.06% after normalization, while COX-1 expression remained almost unchanged. Figure 9 This indicates that D-suspension can selectively degrade cyclooxygenase, which is crucial for resisting tumor immune escape.

[0066] In summary, this invention provides an orally administered polypeptide suspension, specifically polypeptide Npx-G. D F D F D Y exhibits a rod-shaped nanostructure with excellent resistance to proteinase K degradation. It can selectively degrade COX-2 protein, which is highly expressed in tumor cells, thereby reducing toxic side effects. Furthermore, animal experiments have shown that it has good anti-tumor effects and can inhibit the growth of tumor cells.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a polypeptide suspension in the preparation of antitumor drugs, characterized in that, The active ingredient in the polypeptide suspension is Npx-G. D F D F D Y; The Npx-G D F D F D The structural formula of Y is shown in equation (I): （I）； The tumor is breast cancer.

2. The application as described in claim 1, characterized in that, The solvent for the polypeptide suspension is PBS buffer.

3. The application as described in claim 2, characterized in that, The active ingredient Npx-G D F D F D The mass-to-volume ratio of Y to PBS buffer is 0.5~2 mg: 1~4 mL.

4. A polypeptide suspension used in any one of claims 1 to 3.

5. The polypeptide suspension as described in claim 4, characterized in that, The pH of the polypeptide suspension is 7.2 to 7.

4.

6. The polypeptide suspension as described in claim 5, characterized in that, The polypeptide suspension is an oral preparation.

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