Polypeptide pt-1203 and application thereof in antitumor drugs

Abstract

The present application relates to a kind of polypeptide drugs PT-1203 with antitumor activity, and its amino acid sequence is LVWKQSCSQTSK, belongs to polypeptide drug field.The drug has innovation significance in the field of polypeptide drug research.By using microwave promoted Fmoc protection solid-phase synthesis method, the drug can be quickly and efficiently prepared, which not only improves the synthesis efficiency, but also guarantees the stability of synthesis process.Due to its excellent antitumor effect and high specificity, the drug shows great potential and application prospect in the field of antitumor therapy.In addition, its good practicability indicates that in the preparation of antitumor drugs, the drug will have wide application and significant market value.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drugs, and specifically relates to the preparation of a polypeptide PT-1203 and its application in antitumor drugs. Background Technology

[0002] Ovarian cancer, one of the most common malignant tumors of the female reproductive system, suffers from poor prognosis due to difficulties in early diagnosis and limited treatment options in later stages. Currently, treatment for ovarian cancer mainly relies on traditional methods such as surgery, chemotherapy, and radiotherapy. However, these methods have many shortcomings, including low early diagnosis rates, limitations of chemotherapy drugs, radiotherapy side effects, surgical risks, and a lack of targeted therapies. Therefore, new treatment strategies are urgently needed.

[0003] Peptide drugs can achieve highly specific recognition of tumor cell surface markers by designing specific amino acid sequences. Compared with traditional chemotherapy drugs, peptide drugs have lower toxicity and side effects, reducing damage to normal cells. Peptide drugs are easily enzymatically digested in vivo, reducing the potential toxicity caused by long-term accumulation. They are easy to synthesize and modify: peptide drugs can be efficiently synthesized through methods such as solid-phase synthesis, and their pharmacokinetic properties can be improved through chemical modification. Peptide drugs can achieve targeted delivery to tumor tissues by conjugating specific tumor homing peptides. Some peptide drugs have the potential to activate the body's immune system and enhance anti-tumor immune responses. Peptide drugs can inhibit tumor angiogenesis, cut off the tumor's nutrient supply, and inhibit tumor growth and metastasis. Peptides can self-assemble into nanostructures, serving as drug carriers and improving the stability of drug delivery systems.

[0004] Given the shortcomings of existing technologies for treating ovarian cancer, this patent aims to develop a novel targeted peptide drug for ovarian cancer, achieving precision treatment. The design of this patented drug fully utilizes the advantages of peptide drugs, such as high specificity, low toxicity, and easy biodegradability. Simultaneously, innovative synthesis and modification strategies enhance the drug's stability and therapeutic efficacy. Furthermore, this invention will explore the potential of peptide drugs in reducing the toxicity of ovarian cancer cells, providing ovarian cancer patients with a safer and more effective treatment option.

[0005] With the development of biotechnology, peptide drugs have broad application prospects in the treatment of ovarian cancer, and are expected to provide patients with safer and more effective treatment options. Therefore, this invention is proposed. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the application of polypeptide PT-1203 in the preparation of antitumor drugs, wherein the polypeptide PT-1203 has low toxicity to normal cells but exhibits good killing effect on ovarian cancer cells.

[0007] This invention provides a polypeptide PT-1203 with antitumor activity and the application of its pharmaceutically acceptable salt in the preparation of antitumor drugs. The amino acid sequence of the polypeptide PT-1203 is: LVWKQSCSQTSK. The polypeptide PT-1203 contains 12 amino acid residues and has a molecular weight of 1393.70.

[0008] The polypeptide PT-1203 is a polypeptide chain independently developed and designed by the applicant. It has been found that the polypeptide PT-1203 has mild toxicity to normal cells and can produce good killing effects on a variety of tumor cells. It can be used as a pharmaceutical active ingredient to prepare anti-tumor drugs.

[0009] The polypeptide PT-1203 described in this invention has a relatively short peptide chain length and can be synthesized using a solid-phase synthesis method, which is convenient and rapid.

[0010] This invention provides a method for preparing the polypeptide PT-1203, which uses microwave-promoted Fmoc protected solid-phase synthesis to prepare a polypeptide-resin complex, cleaving the polypeptide-resin complex with a cleaving agent to obtain a crude product, and purifying the crude product to obtain polypeptide PT-1203.

[0011] This invention provides the application of the polypeptide PT-1203 described above or the polypeptide PT-1203 obtained by the preparation method described above in the preparation of antitumor drugs.

[0012] This invention provides the use of a pharmaceutically acceptable salt of polypeptide PT-1203 or polypeptide PT-1203 obtained by the preparation method described herein in the preparation of antitumor drugs.

[0013] Preferably, the antitumor drug is a tumor cell apoptosis-promoting drug. Experiments have shown that the polypeptide PT-1203 can achieve an antitumor effect by promoting tumor cell apoptosis.

[0014] Preferably, the tumor is ovarian cancer.

[0015] The present invention provides a pharmaceutical composition comprising the polypeptide PT-1203 as described above and / or a pharmaceutically acceptable salt of the polypeptide PT-1203.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) The polypeptide PT-1203 described in this invention has low toxicity to normal cells and high safety.

[0018] (2) The polypeptide PT-1203 described in this invention has good inhibitory and killing effects on a variety of tumor cells, including ovarian cancer cells, and can be used to prepare corresponding anti-tumor drugs. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the background art, the accompanying drawings used in the embodiments of the present invention or the background art will be described below.

[0020] Figure 1 The graph shows the results of the cytotoxicity test of peptide PT-1203 against SKOV3 tumor cells.

[0021] Figure 2 The graph shows the results of the cytotoxicity test of peptide PT-1203 against A2780 tumor cells.

[0022] Figure 3 This image shows the mass spectrometry results of the polypeptide PT-1203 obtained in Example 1 of this invention.

[0023] Figure 4 The image shows a high-performance liquid chromatogram of the polypeptide PT-1203 obtained in Example 1 of this invention. Detailed Implementation

[0024] This invention provides an antitumor polypeptide PT-1203, with the amino acid sequence shown as LVWKQSCSQTSK. To enable those skilled in the art to more clearly understand the technical solution described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] Unless otherwise defined, 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 is described.

[0027] Example 1: Preparation of polypeptide PT-1203

[0028] This embodiment provides a method for synthesizing the polypeptide PT-1203, whose amino acid sequence is LVWKQSCSQTSK, which can be used to prepare anti-tumor drugs.

[0029] Main materials and instruments

[0030] Synthesis of peptides: The peptide PT-1203 used in the experiment was synthesized by our company's peptide synthesis laboratory using solid-phase synthesis method, with a purity of over 95%.

[0031] The main experimental reagents disclosed in this paper (sources) are: Fmoc-Lys(Boc)-Wang resin (Gil Biochemical), Fmoc-Ser-OH (Gil Biochemical), Fmoc-Thr(tBu)-OH (Gil Biochemical), Fmoc-Leu-OH (Gil Biochemical), Fmoc-Val-OH (Gil Biochemical), Fmoc-Trp(Boc)-OH (Gil Biochemical), Fmoc-Gln(trt)-OH (Gil Biochemical), F... moc-Cys(trt)-OH (Jier Biochemical), Fmoc-Lys(Boc)-OH (Jier Biochemical), Nitrogen, Nitrogen-Dimethylformamide (DMF) (China National Pharmaceutical Group), Dichloromethane (China National Pharmaceutical Group), Trifluoroacetic acid (Maclean), Triisopropylsilane (Aladdin), Acetonitrile (Aladdin), Nitrogen, Nitrogen-Diisopropylcarbodiimide (DIC) (Aladdin), Ethyl 2-oxime cyanoacetate (Maclean), 4-Methylpiperidine (Maclean), Methyl tert-butyl ether (Maclean).

[0032] The main experimental instruments disclosed in this report (source): 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 spectrometry (HPLC-1260 Vial sampler, Quat Pump, MCT, DAD HS, and / or MSD iQ), and CEM Liberty Blue 2.0 fully automated peptide synthesizer.

[0033] The specific synthesis steps are as follows:

[0034] Synthesis of peptide resin: 0.1 mmol of Fmoc-Lys(Boc)-Wang resin, the solid-phase synthesis support, was weighed and placed in a solid-phase reaction tube for peptide synthesis. DMF (10 mL) was added to swell the resin for 10 minutes. The DMF was removed under vacuum, and the resin was washed with DMF (10 mL) twice. Peptide resin synthesis was performed using a Liberty Blue 2.0 fully automated peptide synthesizer. Amide bond condensation was achieved using a 5-fold excess of the corresponding Fmoc-protected amino acid, reacted with ethyl N,N-diisopropylcarbodiimide / 2-oxime cyanoacetate at 90°C for 2 minutes via microwave induction. Deprotection of the Fmoc group was performed using a DMF solution containing 20% ​​4-methylpiperidine at 90°C via microwave induction for 1 minute.

[0035] Cleavage of the resin peptide: The resin peptide obtained in the above steps was washed three times with DCM and then vacuum dried. 10 mL of lysis buffer (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) was added, and the mixture was shaken and reacted at room temperature for 2.5 hours. After the reaction, the mixture was filtered, and the resin was washed twice with trifluoroacetic acid. The filtrates were combined, and a large amount of frozen methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the supernatant was removed, and crude peptide PT-1203 was obtained.

[0036] Purification of crude peptides: The crude peptides were dissolved in a mixed solvent containing 20% ​​acetonitrile / water, filtered through a 0.45 μm membrane, and then 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 purification, the chromatographic detection wavelength was set to 220 nm, and the flow rate was 20 mL / min. The relevant fractions of the product were collected and lyophilized to obtain the pure peptide, designated PT-1203. The purity and molecular weight of the pure peptide were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), with a purity of 98.31% and a correct molecular weight.

[0037] Example 2: Trypan Blue assay for the killing efficiency of PT-1203 on SKOV3 tumor cells.

[0038] Main materials and instruments

[0039] The main experimental reagents disclosed in this paper (source): SKOV3 (human ovarian cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. The required experimental materials are: DMEM basal culture medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; trypan blue.

[0040] The main experimental instruments disclosed in this paper (source): fully automated cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.), inverted microscope (OLYMPUS), BIOBASE CO2 incubator (Jinan Xinbeixi Biotechnology Co., Ltd.)

[0041] Cell thawing: Remove the SKOV3 cell line cryopreservation tubes from the liquid nitrogen container and place them in a 37°C water bath with shaking until thawed. Transfer the thawed cell line to 15mL sterile centrifuge tubes in a biosafety cabinet, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add DMEM complete medium to the SKOV3 solution. Finally, transfer to 25cm... 2 The sterile culture flasks were incubated at 37°C with 5% CO2.

[0042] Cell passage: Cells were observed under a microscope and, when their morphology was normal and their growth density reached approximately 90%, were passaged. First, the culture flask containing the cells was removed and placed in a clean bench. The original culture medium was aspirated, and the cells were washed once with PBS buffer. After aspirating the PBS, 2 mL of trypsin was added, ensuring it completely covered the bottom of the flask. The cap was tightened, and the flask was placed in a 37°C incubator for digestion. After digestion, the cells were blown down the flask wall with fresh complete culture medium. The cell suspension was then transferred to a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in fresh complete culture medium and passaged at a ratio of 1:3.

[0043] Cell cryopreservation: After centrifuging to remove the supernatant following the cell passage process described above, resuspend the cells in cryopreservation buffer (fetal bovine serum:DMSO = 9:1) and transfer the suspension to 1.5 mL cell cryopreservation tubes (approximately 3 × 10⁶ cells per tube). 6 (Each cell) was labeled and then cryopreserved using a gradient cooling method.

[0044] In this experiment, SKOV3 tumor cells were treated with different concentrations of the peptide PT-1203 for 24 hours. Then, trypan blue in situ staining and cell counting were performed.

[0045] Trypan blue in situ staining: After aspirating the culture medium, add trypan blue staining solution to a final concentration of 0.4%, incubate at room temperature for 8 min, then aspirate the staining solution, rinse once with PBS buffer, and observe and photograph under a microscope. In situ counting was performed on cells treated with different concentrations after staining to calculate the number of cells stained with trypan blue, determine the cell death rate, and thus determine the killing efficiency of peptide PT-1203 against SKOV3 tumor cells.

[0046] Table 1 Killing efficiency of peptide PT-1203 against SKOV3 tumor cells

[0047] Cell death rate 100.00％ 83％ 56％ 20％

[0048] The counting results, combined with trypan blue in situ staining, indicate that the peptide PT-1203 has a killing effect on SKOV3 tumor cells.

[0049] Example 3: Trypan Blue assay for the killing efficiency of PT-1203 on A2780 tumor cells

[0050] Main materials and instruments

[0051] The main experimental reagents disclosed in this paper (source): A2780 (ovarian cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd. The required experimental materials are: DMEM basal culture medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; trypan blue.

[0052] The main experimental instruments disclosed in this paper (source): fully automated cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.), inverted microscope (OLYMPUS), BIOBASE CO2 incubator (Jinan Xinbeixi Biotechnology Co., Ltd.)

[0053] Cell thawing: Remove the A2780 cell line cryopreservation tubes from the liquid nitrogen container and place them in a 37°C water bath with gentle shaking until thawed. Transfer the thawed cell line to 15mL sterile centrifuge tubes in a biosafety cabinet, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add DMEM complete medium to the A2780. Finally, transfer to 25cm... 2 The sterile culture flasks were incubated at 37°C with 5% CO2.

[0054] Cell passage: Cells were observed under a microscope and, when their morphology was normal and their growth density reached approximately 90%, were passaged. First, the culture flask containing the cells was removed and placed in a clean bench. The original culture medium was aspirated, and the cells were washed once with PBS buffer. After aspirating the PBS, 2 mL of trypsin was added, ensuring it completely covered the bottom of the flask. The cap was tightened, and the flask was placed in a 37°C incubator for digestion. After digestion, the cells were blown down the flask wall with fresh complete culture medium. The cell suspension was then transferred to a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in fresh complete culture medium and passaged at a ratio of 1:3.

[0055] Cell cryopreservation: After centrifuging to remove the supernatant following the cell passage process described above, resuspend the cells in cryopreservation buffer (fetal bovine serum:DMSO = 9:1) and transfer the suspension to 1.5 mL cell cryopreservation tubes (approximately 3 × 10⁶ cells per tube). 6 (Each cell) was labeled and then cryopreserved using a gradient cooling method.

[0056] In this experiment, A2780 tumor cells were treated with different concentrations of the peptide PT-1203 for 24 hours. Then, trypan blue in situ staining and cell counting were performed.

[0057] Trypan blue in situ staining: After aspirating the culture medium, add trypan blue staining solution to a final concentration of 0.4%, incubate at room temperature for 8 min, then aspirate the staining solution, rinse once with PBS buffer, and observe and photograph under a microscope. In situ counting was performed on cells treated with different concentrations after staining to calculate the number of cells stained with trypan blue, determine the cell death rate, and thus determine the killing efficiency of peptide PT-1203 against A2780 tumor cells.

[0058] Table 2 Killing efficiency of peptide PT-1203 against A2780 tumor cells

[0059] Cell death rate 100％ 100％ 97.10％ 98％

[0060] Combined with trypan blue in situ staining and counting, it can be shown that the peptide PT-1203 exhibits significant killing efficiency against A2780 tumor cells.

Claims

1. A polypeptide PT-1203, characterized in that, The amino acid sequence of the polypeptide is: LVWKQSCSQTSK.

2. The use of a polypeptide PT-1203 with antitumor activity and its pharmaceutically acceptable salt in the preparation of an anti-ovarian cancer drug, characterized in that, The amino acid sequence of the polypeptide PT-1203 is: LVWKQSCSQTSK.

3. A method for preparing the polypeptide PT-1203 as described in claim 1, characterized in that: A peptide-resin complex was prepared by microwave-promoted Fmoc protected solid-phase synthesis. The peptide-resin complex was cleaved with a cleavage agent to obtain a crude product, which was then purified to obtain peptide PT-1203.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide PT-1203 as described in claim 1 and / or a pharmaceutically acceptable salt of the polypeptide PT-1203.

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