Polypeptide suspension and application thereof in preparation of antitumor drugs

Abstract

The invention provides a polypeptide suspension and application thereof in preparation of antitumor drugs, and belongs to the technical field of biological medicines. The active component of the polypeptide suspension provided by the invention is Npx-GDFDFDY (N, N '-GDFDY). The D-configuration self-assembled polypeptide suspension not only has a good anti-degradation effect and relatively high biocompatibility and stability, but also diffuses to the whole body after being orally taken and absorbed, so that the expression of COX-2 at a tumor part is reduced, and a tumor immune microenvironment is improved, thereby inhibiting the growth of tumor cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide suspension and application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Compared with traditional chemotherapy, radiotherapy and surgical treatment, tumor immunotherapy, with its advantages of strong specificity, long duration of action and few side effects, can effectively improve the problems of tumor resistance and easy recurrence caused by tumor cells escaping the body's immune surveillance, and has always been considered the ultimate means to cure tumors. 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) to promote cancer cell proliferation, metastasis, inhibit cancer cell apoptosis, and on the other hand, it can inhibit interferon and T cell function, mediate tumor immune escape, and is one of the important indicators of tumor occurrence and development. Therefore, COX-2 is particularly important in solving the problems of tumor immune escape and immune tolerance in a targeted manner. It is a favorable way to make "cold" tumors "hot" and is considered to be a promising anti-tumor therapeutic target.

[0003] As we all know, among the available routes of administration, the most convenient and simple one that is most concerned by patients is the oral route, which has high patient compliance, a wide range of applications, and is easy to take. The dosage forms of oral drugs mainly include solid dosage forms represented by granules, capsules, and sustained-release tablets, and liquid dosage forms represented by solutions, suspensions, and emulsions. The suspension method is not only easy to swallow and has a fast onset of action, but also can achieve precise control of dosage, which is one of the rare options for drugs.

[0004] However, there is currently no oral polypeptide nanodrug 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 the present invention is to provide an oral polypeptide suspension with anti-tumor effect, which can degrade COX-2 of tumor cells and improve the immune microenvironment of tumor sites, thereby inhibiting the growth of tumor cells. The drug suspension has more advantages in terms of stability.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a polypeptide suspension in the preparation of an anti-tumor 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 formula (I):

[0009]

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

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

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

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

[0014] The invention also provides a polypeptide suspension for use.

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

[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 of the polypeptide suspension provided by the present invention is Npx-G D F D F D Y, Npx naproxen as a self-assembling peptide G in D configuration D F D F D The end-capping group of Y is dissolved in a buffer to obtain a polypeptide suspension. The self-assembled polypeptide suspension of D configuration not only has good anti-degradation effect, high biocompatibility and stability, but also diffuses throughout the 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. BRIEF DESCRIPTION OF THE DRAWINGS

[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 This is a high-resolution mass spectrum of the polypeptide Npx-GFFY prepared in Comparative Example 1;

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

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

[0023] Figure 5 This is a graph showing the anti-degradation ability of the polypeptide D-suspension under the action of proteinase K determined in Experimental Example 3;

[0024] Figure 6 This is a graph showing the tumor growth trend of mice measured in Experimental Example 4;

[0025] Figure 7 This is a graph showing the weight changes of mice measured in Experimental Example 4;

[0026] Figure 8 The expression and statistical graph of COX-2 after the polypeptide in Experimental Example 5 was used to treat 4T1 cells;

[0027] Fig. 9 This is the expression and statistical graph of COX-1 after 4T1 cells were treated with the polypeptide of Experimental Example 5. DETAILED DESCRIPTION

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

[0029] Example 1

[0030] The peptide Npx-G was synthesized by 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-chlorotrityl 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 Company), and shake on a shaker for 15 min to completely swell the 2-chlorotrityl chloride resin;

[0032] 2) Use an ear bulb to completely squeeze out the DCM from the solid phase synthesis tube, and wash repeatedly with 10 mL of DCM three times, each time for 1 min;

[0033] 3) Weigh 1 mmol of 9-fluorenylmethoxycarbonyl protecting group (Fmoc) protected 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, it was added to the solid phase synthesis tube and placed on a shaker for reaction at room temperature for 1 h.

[0034] 4) Remove the reaction solution in the solid phase synthesis tube with an ear bulb, wash with 10 mL DCM three times, 1 min each time, add 15 ml of a 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 DCM washing, and react at room temperature for 15 min;

[0035] 5) Remove the reaction solution in the solid phase synthesis tube with an ear bulb, wash with 10 ml of DCM 3 times, 1 min each time, then wash with 10 ml of N,N-dimethylformamide (DMF, purchased from Tianjin Chemical Reagent Company) 3 times, 1 min each time, add 15 mL of 20% piperidine (purchased from Tianjin Chemical Reagent Company) to the solid phase synthesis tube, use DMF as the diluent, react for 20 min, and wash with 10 ml of DMF 5 times, 1 min each time;

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

[0037] 7) Repeating steps 5) and 6), wherein when repeating step 6), 1 mmol D F (Fmoc-Phe-OH), 1mmol G (Fmoc-Gly-OH), 1mmol end-capping group Npx (naproxen), no need to remove Fmoc after connecting the end-capping group;

[0038] 8) Squeeze out the reaction solution, wash with DMF 3 times, 1 min each time, and then wash with DCM 5 times, 1 min 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 the solid phase synthesis tube, react at room temperature for 45 minutes, cut the product from the 2-chlorotrityl chloride resin, remove the solvent with a rotary evaporator, and obtain a crude product. Then use HPLC (high performance liquid chromatography, purchased from Lumtech, Germany) to separate and purify, and the obtained anti-tumor oral peptide, Npx-G D F D F D Y.

[0040] The obtained oral anti-tumor peptide was detected by LC-MS (brand: Shimadzu, Japan, model: LC-MS2020). 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 F was replaced with Y, F, and F to synthesize Npx-GFFY. The obtained peptide was detected by LC-MS. The results are as follows Figure 2 As shown, the structural formula is 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, the pH was adjusted to 7.4 with sodium carbonate, and vortexed to obtain a 1 mM polypeptide suspension, referred to 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 1 mM polypeptide hydrogel, referred to as D-hydrogel.

[0049] PBS was added to the pure Npx-GFFY product for resuspending, the pH was adjusted to 7.4 with sodium carbonate, heated to 100°C and cooled to room temperature (25°C) to obtain 1 mM polypeptide hydrogel, referred to as L-hydrogel.

[0050] Finally, the optical photograph is obtained as Figure 3 As shown. Figure 3 It can be seen from the optical photos that after heating and cooling, L-hydrogel and D-hydrogel present the appearance of clear and transparent hydrogels on a macroscopic scale, while D-suspension presents the appearance of a suspension with a certain fluidity. Compared with hydrogels, suspensions have better stability and oral compliance.

[0051] Experimental Example 2

[0052] Use a toothpick to pick up a small amount of dry, uncontaminated peptide Npx-G D F D F D Y (prepared in Example 1) was evenly applied on the sample table with the conductive tape attached and compacted. The sample and the sample table were placed in a container for gold spraying. Subsequently, a scanning electron microscope (SEM, JEOL, model JSM-7900F) was used to take photos. The surface microstructure was observed using a low voltage, secondary electron detector. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the polypeptide powder in Example 1 is in the form of a slender rod.

[0053] Experimental Example 3

[0054] Anti-degradation assay

[0055] According to the method of Experimental Example 1, 500 μL of 500 μM L-hydrogel, D-hydrogel and D-suspension were respectively prepared in 1.5 mL EP tubes, and proteinase K (purchased from Beyotime) was added to make the final concentration 1 mg / mL.

[0056] After the obtained solution is mixed, it is placed in a 37°C thermostat for 24 hours. At 0, 2, 4, 8, 12, and 24 hours after the start of the reaction, 50 μL of sample is taken and dissolved in 200 μL of methanol to prepare a dilution solution. The content of the compound in the dilution solution is determined by LC-MS, and the degradation percentage of the peptide under the action of proteinase K is calculated based on the peak area. The results are as follows: Figure 5 shown.

[0057] Figure 5The results showed that within 24 hours under the action of proteinase K, L-hydrogel was almost completely degraded, D-hydrogel was degraded by 27.80%, and D-suspension was only degraded by 13.02%, which was less than half of D-hydrogel. The above results show that compared with L-configuration peptides, D-configuration peptides have stronger anti-degradation ability, among which D-suspension has the best effect, which also proves the great advantage of peptide suspension in anti-degradation in vivo.

[0058] Experimental Example 4

[0059] Mouse test

[0060] The orthotopic tumor model was established using COX-2-positive mouse breast cancer cell 4T1. The mice used were 6-week-old female BALB / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). 4T1 tumor cells were prepared in advance and inoculated into the lower part of the second pair of mammary fat pads of mice. One million tumor cells were injected into each mouse. When the tumor cell volume grew to 70 mm 3 When the mice were about 24 hours old, they were randomly divided into 5 groups, including PBS group, Npx (naproxen) group, L-hydrogel group, D-hydrogel group, and D-suspension group, with 5 mice in each group. The mice were administered by gavage once every two days, and the concentrations were: 9.2 mg / kg for Npx group, 29.8 mg / kg for L-hydrogel group, 29.8 mg / kg for D-hydrogel group, and 29.8 mg / kg for D-suspension group, for a total of five times. The tumor volume and body weight changes of the mice were monitored, with the day of administration as the 0th day of monitoring, and the monitoring was continued for 14 days, and the mice were euthanized. The calculation formula for the tumor volume is: length × width 2 / 2, the result is Figure 6 and Figure 7 shown.

[0061] Depend on Figure 6 It can be seen that the average tumor volume of the D-suspension group was the smallest, and the tumor inhibition rate was 67.24%, indicating that D-suspension can significantly inhibit tumor cell growth, while Npx, L-hydrogel, and D-hydrogel can only inhibit 25.62%, 30.90%, and 22.03%, respectively. This is mainly related to the high stability of D-suspension. Figure 7The results showed that the weight of mice in all groups maintained a slow growth trend, indicating that the polypeptide prepared by the present invention has good biocompatibility. The above results show that at the animal level, D-suspension can inhibit tumor cell proliferation by oral administration alone, and is a very promising oral administration preparation.

[0062] Experimental Example 5

[0063] Protein expression determination

[0064] Five groups were set up, namely Control, Npx, L-hydrogel, D-hydrogel, and D-suspension. 4T1 cells were evenly spread in a six-well plate with 300,000 cells per well. After the cells adhered to the wall, the cells were treated with 1.5mL of empty culture, 400μM of Npx, L-hydrogel, D-hydrogel, and D-suspension for 24 hours, and then they could be used for protein extraction. After washing the cells three times with PBS, the cells were scraped off with a cell scraper, and the cells were lysed with RIPA lysis buffer for 20 minutes, centrifuged at 12000rpm and 4℃ for 10 minutes, and the supernatant was aspirated. The sample was added with loading buffer and boiled at 95℃ for 8 minutes to fully denature the protein. 10% gel (purchased from Biyuntian, item number: P0456S) was selected, protein marker and sample were added in turn, and electrophoresis was performed at a constant voltage of 160V to separate the protein for 35 minutes. The PVDF membrane was activated with methanol for 30 seconds, and the membrane balance solution was added for 10 minutes. Place the membrane and gel in a transfer apparatus for transfer. After transfer, block with 5% skim milk powder at room temperature for 1 hour. Wash with TBST 4 times, 8 minutes each time. Cut the membrane according to the size of the target band, and incubate the band with the primary antibody (COX-2 or COX-1) diluted with 3% BSA on a shaker at 4°C overnight. Wash the membrane with TBST for 10 minutes each time, wash 3 times, and remove the unbound primary antibody. Incubate the band with the HRP-labeled secondary antibody diluted with 3% BSA, shake at room temperature for 1 hour, wash the membrane with TBST for 10 minutes each time, wash 3 times, then add the ECL chemiluminescent substrate reaction luminescent liquid to expose the band. Use ImageJ software to measure the grayscale value of the band and perform normalization. The results are shown as follows. Figure 8 and Fig. 9 shown.

[0065] like Figure 8 As shown in the figure, cells treated with D-suspension showed the lowest COX-2 expression. After normalization, the degradation of COX-2 reached 62.06%, while the expression of COX-1 remained almost unchanged ( Fig. 9 ), indicating that D-suspension can achieve selective degradation of cyclooxygenase, which is very important for resisting tumor immune escape.

[0066] In summary, the present invention provides an orally administrable polypeptide suspension, the polypeptide Npx-G D F D F D Y is a rod-shaped nanostructure with excellent resistance to (proteinase K) degradation. It can selectively degrade COX-2 protein highly expressed in tumor cells and reduce toxic side effects. Experiments at the animal level have found that it has good anti-tumor effects and can inhibit the growth of tumor cells.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a polypeptide suspension in the preparation of an anti-tumor drug, characterized in that: The active ingredient of the polypeptide suspension is Npx-G D F D F D Y.

2. The use according to claim 1, characterized in that The Npx-G D F D F D The structural formula of Y is shown in formula (I):

3. The use according to claim 1, characterized in that The solvent of the polypeptide suspension is PBS buffer.

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

5. The use according to claim 4, characterized in that The polypeptide suspension achieves the anti-tumor effect by degrading COX-2 protein.

6. The use according to claim 5, characterized in that The tumors include breast cancer and colon cancer.

7. A polypeptide suspension for use according to any one of claims 1 to 6.

8. The polypeptide suspension according to claim 7, characterized in that The pH of the polypeptide suspension is 7.2-7.

4.

9. The polypeptide suspension according to claim 8, characterized in that The polypeptide suspension is an oral preparation.

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