Polypeptide PT-09014 and application thereof in antitumor drugs

Abstract

The invention relates to a novel polypeptide drug PT-09014 with antitumor activity, which has an amino acid sequence as shown in SEQ ID NO: 1, and belongs to the field of polypeptide drugs. The polypeptide has a good killing effect on ovarian cancer cells and is suitable for ovarian cancer treatment. The invention also provides a preparation method for microwave-promoted Fmoc protection solid-phase synthesis of the polypeptide, and an application of the polypeptide in preparation of antitumor drugs. Experimental results show that the IC50 of the polypeptide PT-09014 on the SKOV3 ovarian cancer cells is 79.84 mu g / mL, and the polypeptide PT-09014 shows obvious killing efficacy. The polypeptide PT-09014 provides a new treatment strategy for ovarian cancer patients, and is expected to improve the treatment effect and the life quality of the patients.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide drugs, and particularly relates to the preparation of polypeptide PT-09014 and its application in anti-tumor drugs. Background Art

[0002] Peptide drugs have shown great potential in the field of anti-tumor and have become a hot topic of research in recent years. By binding to specific targets, peptide drugs can regulate cell signal transduction, induce tumor cell apoptosis or inhibit their proliferation. Compared with traditional small molecule chemical drugs, peptide drugs have higher specificity and targeting, can significantly reduce damage to normal cells and reduce side effects. In addition, compared with biological macromolecule drugs such as monoclonal antibodies, peptide drugs have small molecular weight, simple structure, easy synthesis and modification, low production cost and good stability. Peptide drugs can also enter the body through a variety of delivery methods (such as injection, oral, transdermal, etc.), further improving the flexibility and practicality of their clinical application. Therefore, peptide drugs have unique advantages in anti-tumor treatment and provide cancer patients with safer and more efficient treatment options.

[0003] Ovarian cancer has the worst prognosis among common gynecological malignancies, with a 5-year survival rate of only 30-40%, which seriously endangers women's health. Since the early symptoms of ovarian cancer are not obvious, patients are often in the late stage when diagnosed, which makes treatment more difficult. Traditional treatment methods include surgery, chemotherapy and other treatment methods, but these methods have many limitations. For example, surgery cannot completely eliminate all cancer cells, and chemotherapy drugs often have strong toxic side effects, and long-term use is prone to drug resistance.

[0004] For example, the current standard treatment is tumor reduction surgery combined with platinum-based chemotherapy, but 70% of patients will relapse within 2 years after initial treatment. After multiple relapses and repeated chemotherapy, they eventually develop platinum resistance. The sensitivity of ovarian cancer patients to platinum chemotherapy regimens is a decisive factor affecting their survival, and patients with platinum resistance have a very poor prognosis. Therefore, there is an urgent need to discover new targeted drugs based on resistance mechanisms to provide new treatment strategies for platinum-resistant patients.

[0005] In the treatment of ovarian cancer, peptide drugs can effectively identify and target cancer cells, reduce damage to normal cells, and thus reduce the side effects of treatment. This feature makes peptide drugs superior to traditional chemotherapy drugs in terms of efficacy and safety. In addition, peptide drugs are highly modifiable. By modifying the structure of peptide molecules, their stability and half-life can be significantly improved, and the efficacy of the drug can be enhanced. On the other hand, peptide drugs can also be used in combination with other treatment methods, such as radiotherapy, chemotherapy, and immunotherapy, to achieve a synergistic effect and further improve the treatment effect.

[0006] With the continuous progress of biotechnology and synthetic chemistry, the research and development and application prospects of peptide drugs are broader. The development of modern biotechnology has made the synthesis, modification and screening of peptides more efficient and accurate, greatly promoting the clinical transformation of peptide drugs. In addition, with the in-depth study of the molecular mechanism of ovarian cancer, more and more targets have been discovered, providing more possibilities for the design of peptide drugs. In the future, the application of peptide drugs in the treatment of ovarian cancer will continue to expand and deepen. By combining multidisciplinary technical means such as genomics and proteomics, personalized peptide drugs for different subtypes of ovarian cancer are developed to provide more accurate and efficient treatment options, ultimately bringing greater hope of survival and higher quality of life to ovarian cancer patients.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide an anti-tumor polypeptide which has high anti-tumor activity and low cytotoxicity and is suitable for the preparation of anti-tumor products.

[0009] The present invention provides an anti-tumor polypeptide and use of a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof in the preparation of an anti-tumor drug, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. Each amino acid in the sequence independently has a D-configuration or an L-configuration, or, in a specific embodiment, each amino acid of the polypeptide has a D-configuration.

[0010] The polypeptide of the present invention is a polypeptide chain independently developed and designed by the applicant. It is found that it has mild toxicity to normal cells and can produce good killing effects on a variety of tumor cells. It can be used as a drug active ingredient to prepare anti-tumor drugs.

[0011] The invention provides a method for preparing the polypeptide, which comprises preparing a polypeptide-resin complex by adopting a microwave-promoted Fmoc protection solid phase synthesis method, cleaving the polypeptide-resin complex with a cleavage agent to obtain a crude product, and purifying the crude product to obtain the polypeptide.

[0012] The present invention provides an application of the polypeptide or the polypeptide obtained by the preparation method in the preparation of anti-tumor drugs.

[0013] The present invention provides a polypeptide or a pharmaceutically acceptable salt, hydrate, mixture or prodrug of the polypeptide obtained by the preparation method in the preparation of an anti-tumor drug.

[0014] Preferably, the anti-tumor drug is a drug that promotes tumor cell apoptosis. Experiments have shown that the polypeptide can achieve anti-tumor effects by promoting tumor cell apoptosis.

[0015] Preferably, the tumor is ovarian cancer.

[0016] The present invention provides a pharmaceutical composition, which comprises the polypeptide and / or the polypeptide and a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof.

[0017] The present invention provides a pharmaceutical composition, which comprises the polypeptide and a pharmaceutically acceptable salt, hydrate, mixture or prodrug-acceptable carrier and / or excipient thereof.

[0018] The beneficial effects of the present invention are as follows:

[0019] The polypeptide of the present invention has good inhibitory and killing effects on various tumor cells including ovarian cancer cells, and can be used to prepare corresponding anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A graph showing the results of the peptide PT-09014 cytotoxicity test on SKOV3 tumor cells;

[0021] Figure 2 The fitting curve diagram showing the half inhibition concentration IC50 of peptide PT-09014;

[0022] Figure 3 A diagram showing the mass spectrometry detection results of the polypeptide PT-09014 obtained in Example 1 of the present invention;

[0023] Figure 4 The HPLC chromatogram of the polypeptide PT-09014 obtained in Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0024] The present invention provides an anti-tumor polypeptide, the amino acid sequence is shown in SEQ ID NO: 1, specifically: LVWKQSCSQTSSTR, wherein each amino acid in the sequence independently has a D-configuration or an L-configuration, or, in a specific embodiment, each amino acid of the polypeptide has a D-configuration. In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following examples are listed for illustration. It should be pointed out that the following examples do not limit the scope of protection claimed by the present invention.

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0027] Example 1: Preparation of polypeptide PT-09014

[0028] This embodiment provides a method for synthesizing a polypeptide PT-09014, whose amino acid sequence is: LVWKQSCSQTSSTR, and each amino acid in the sequence is in D-configuration, which can be used to prepare anti-tumor drugs.

[0029] Main materials and instruments

[0030] Synthesis of polypeptide: The polypeptide PT-09014 of the present invention is synthesized by the polypeptide synthesis laboratory of our company by solid phase synthesis method, and the purity is above 95%.

[0031] The main experimental reagents (source) of the present invention are: Fmoc-D-Arg (pbf) -Wang resin (Gill Biochemical), Fmoc-D-Ser-OH (ACMEC), Fmoc-D-Thr (tBu) -OH (ACMEC), Fmoc-D-Leu-OH (ACMEC), Fmoc-D-Val-OH (ACMEC), Fmoc-D-Trp (Boc) -OH (ACMEC), Fmoc-D-Gln(trt)-OH(ACMEC), Fmoc-D-Cys(trt)-OH(ACMEC), Fmoc-D-Lys(Boc)-OH(ACMEC), nitrogen, nitrogen-dimethylformamide (DMF) (Sinopharm), dichloromethane (Sinopharm), trifluoroacetic acid (McLean), triisopropylsilane (Aladdin), acetonitrile (Aladdin), nitrogen, nitrogen-diisopropylcarbodiimide (DIC) (Aladdin), ethyl 2-oxime cyanoacetate (McLean), 4-methylpiperidine (McLean), methyl tert-butyl ether (McLean).

[0032] The main experimental instruments (sources) of the present invention are: Agilent HPLC-1260 series high performance liquid chromatographs (HPLC-1260 vial sampler, Quat Pump, MCT and DAD HS), Agilent HPLC / MSD iQ liquid chromatography tandem mass spectrometer (HPLC-1260 vial sampler, Quat Pump, MCT, DAD HS and / MSD iQ), and CEM Liberty blue 2.0 fully automatic peptide synthesizer.

[0033] The specific synthesis steps are as follows:

[0034] Synthesis of peptide resin: Weigh 0.6g of solid phase synthesis carrier Fmoc-D-Arg (pbf)-Wang resin resin and place it in a peptide synthesis solid phase reaction tube, add DCM (10mL) to swell the resin for 10 minutes, vacuum remove DCM, add DMF (10mL) to wash the resin, and repeat washing twice. The peptide resin synthesis was completed using Liberty blue 2.0 fully automatic peptide synthesizer, in which amide bond condensation used 5 times excess of the corresponding Fmoc-protected amino acid, and the amino acid condensation was carried out using nitrogen, nitrogen-diisopropylcarbodiimide / 2-oxime ethyl cyanoacetate at 90°C microwave reaction for 2 minutes. Fmoc group deprotection used a DMF solution containing 20% ​​4-methylpiperidine at 90°C microwave reaction for 1 minute.

[0035] Cleavage of resin peptide: The resin peptide obtained in the above step was washed with DCM three times and then vacuum dried, and then 10 mL of cleavage solution (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was added and shaken for 2.5 hours at room temperature. After the reaction was completed, it was filtered, and the resin was washed with trifluoroacetic acid twice. After the filtrate was combined, a large amount of frozen methyl tert-butyl ether was added to precipitate solids, and the supernatant was removed after centrifugation to obtain the crude product of polypeptide PT-09014.

[0036] Purification of crude peptide: The crude peptide was dissolved in a mixed solvent containing 20% ​​acetonitrile / water, filtered through a 0.45 μm membrane, and separated using a high-pressure preparation system. The mobile phases were A (0.1% trifluoroacetic acid, 10% acetonitrile / water solution, v / v) and B (0.1% trifluoroacetic acid, 90% acetonitrile / water solution, v / v). During the purification process, the chromatograph detection wavelength was set to 220 nm and the flow rate was 20 mL / min. The product-related fractions were collected and lyophilized to obtain a pure polypeptide numbered PT-09014. The purity and molecular weight of the pure polypeptide were determined by analytical high performance liquid chromatography and liquid chromatography tandem mass spectrometry, where the purity = 98.28%, and the molecular weight was correct.

[0037] Example 2: Trypan blue detection of the effect of polypeptide PT-09014 on tumor cell survival

[0038] Main materials and instruments

[0039] The main experimental reagents (sources) of the present invention: SKOV3 (ovarian cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the required experimental materials were: DMEM basal culture medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; trypan blue.

[0040] The main experimental instruments (sources) of the present invention are: fully automatic cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.), inverted microscope (OLYMPUS), and BIOBASE carbon dioxide incubator (Jinan Xinbeisi Biotechnology Co., Ltd.).

[0041] Cell recovery: Take the cryopreserved tube of SKOV3 cell line out of the liquid nitrogen tank and place it in a 37℃ water bath to shake until it melts. Pipette the thawed cell line into a 15mL sterile centrifuge tube in a biosafety cabinet, centrifuge at 1000r / min for 5min, and remove the supernatant; add DMEM complete medium to SKOV3. Finally, transfer to a 25cm 2 The cells were cultured in sterile culture bottles at 5% CO2 and 37°C.

[0042] Cell passaging: Observe the cells under a microscope. When the cell morphology is normal and the growth density reaches about 90%, cell passaging is performed. First, take out the culture bottle containing cells and place it in the clean bench, aspirate the original culture medium, wash the cells once with PBS buffer, aspirate the PBS and add 2mL of trypsin so that it can completely cover the bottom of the culture bottle, tighten the bottle cap and place it in a 37℃ incubator for digestion. After digestion, blow the cells off the bottle wall with fresh complete culture medium, then aspirate the cell suspension into a 15mL sterile centrifuge tube and centrifuge it at 1000r / min for 5min, remove the supernatant, add fresh complete culture medium to resuspend the cells and perform cell passaging at a ratio of 1:3.

[0043] Cell cryopreservation: After centrifugation and removal of the supernatant according to the above cell passage process, add freezing solution (fetal bovine serum: DMSO = 9:1) to resuspend the cells, and add the suspension to 1.5 mL cell cryopreservation tubes (each tube of cell suspension contains approximately 2×10 6 After labeling, they were cryopreserved by gradient cooling method.

[0044] In this experiment, SKOV3 tumor cells were treated with different concentrations of peptide PT-09014 for 24 hours, and then trypan blue in situ staining and cell viability detection were performed.

[0045] Trypan blue in situ staining: After discarding the culture medium, add trypan blue dye solution with a final concentration of 0.4%, incubate at room temperature for 8 minutes, then discard the dye solution, wash once with PBS buffer, and observe and take pictures under a microscope.

[0046] Cell viability test: Digest the cells into a cell suspension, then mix the cell suspension with 0.2% trypan blue dye at a ratio of 1:1, and take 20 μL of the mixture for counting using a cell counter. The experimental results showed that the IC50 of peptide PT-09014 against SKOV3 was 79.84 μg / mL.

[0047] Table 1 Toxicity of peptide PT-09014 to SKOV3 tumor cells

[0048] Dosing concentration 800 μg / mL 600 μg / mL 300 μg / mL 100 μg / mL 50 μg / mL Cell death rate 100％ 100％ 100％ 66.47％ 19.26％

[0049] Combined with the above experiments, it can be shown that the peptide PT-09014 exhibits significant killing effect on SKOV3 tumor cells.

Claims

1. A polypeptide PT-09014 and a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof, characterized in that: The amino acid sequence of the polypeptide PT-09014 is shown in SEQ ID NO: 1, wherein each amino acid in the sequence independently has a D-configuration or an L-configuration, or, in a specific embodiment, each amino acid of the polypeptide has a D-configuration.

2. A method for preparing the polypeptide according to claim 1, characterized in that: The polypeptide-resin complex was prepared by microwave-promoted Fmoc protection solid phase synthesis method, and the polypeptide-resin complex was cleaved by a cleavage agent to obtain a crude product, and the crude product was purified to obtain polypeptide PT-09014.

3. Use of the polypeptide according to claim 1 or the polypeptide obtained by the preparation method according to claim 2 in the preparation of anti-tumor drugs.

4. Use of the polypeptide according to claim 1 or the polypeptide obtained by the preparation method according to claim 2 in a pharmaceutically acceptable salt, hydrate, mixture or prodrug in the preparation of anti-tumor drugs.

5. The use according to claim 1, characterized in that: The anti-tumor drug is a drug that promotes tumor cell apoptosis.

6. The use according to any one of claims 1 or 3, 4, characterized in that: The tumor is ovarian cancer.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 and / or a pharmaceutically acceptable salt, hydrate, mixture or prodrug of the polypeptide.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide according to claim 1 and / or a pharmaceutically acceptable salt, hydrate, mixture or prodrug-acceptable carrier and / or excipient of the polypeptide.

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