Cyclic peptide PT-C07 and application thereof in antitumor drugs

Abstract

The invention relates to a novel cyclopeptide drug PT-C07 with antitumor activity. The amino acid sequence of the novel cyclopeptide drug PT-C07 is shown as SEQ ID NO: 1. The cyclic peptide has a good killing effect on renal cell carcinoma cells and is suitable for treating renal cell carcinoma. The invention also provides a preparation method of the cyclopeptide through microwave-promoted Fmoc protection solid-phase synthesis, and an application of the cyclopeptide in preparation of antitumor drugs. Experimental results show that the IC50 of the cyclopeptide PT-C07 to the 786-O kidney cancer cells is 77.53 mu M, and the IC50 of the cyclopeptide PT-C07 to the A498 kidney cancer cells is 67.06 mu M, so that the cyclopeptide PT-C07 can obviously reduce the cell viability of the 786-O and A498 and shows obvious killing efficacy. The cyclic peptide PT-C07 provides a new treatment strategy for patients with renal cell carcinoma, 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 technical field of cyclic peptide drugs, and particularly relates to the preparation of a cyclic peptide PT-C07 and its application in anti-tumor drugs. Background Art

[0002] Renal cell carcinoma (RCC) is a malignant tumor that originates from the renal tubular epithelium and accounts for 80% to 90% of renal malignant tumors. Among its histopathological types, clear cell carcinoma is the most common, followed by papillary renal cell carcinoma and chromophobe renal cell carcinoma. The incidence of renal cell carcinoma is increasing year by year worldwide, and it has a high mortality rate among urinary system tumors. Early renal cell carcinoma may have no obvious symptoms. As the disease progresses, symptoms such as hematuria, low back pain, and abdominal masses may appear. In addition, renal cell carcinoma is highly invasive and has a tendency to metastasize. Approximately 20% to 30% of patients have distant metastases at the time of diagnosis.

[0003] Surgery is the standard treatment for localized renal cell carcinoma, including radical nephrectomy and nephron-sparing surgery. However, about one-third of patients who receive radical treatment will experience recurrence and metastasis. For metastatic renal cell carcinoma, treatment options have been continuously enriched in recent years, including targeted therapy, immunotherapy, etc. Targeted therapy drugs such as sunitinib and pazopanib work by inhibiting tumor angiogenesis and tumor cell proliferation. Immunotherapy mainly activates the patient's own immune system to attack tumor cells, such as PD-1 / PD-L1 inhibitors. Despite this, the treatment of renal cell carcinoma still faces many challenges, such as drug resistance and treatment side effects, and there is an urgent need to explore more effective treatment strategies.

[0004] Cyclic peptides are cyclic structures formed by amino acids connected by peptide bonds. They have the characteristics of small molecular weight, high stability, and diverse biological activities. In recent years, the research on cyclic peptide drugs has made significant progress, and many cyclic peptide drugs have entered the clinical research stage. In the treatment of renal cell carcinoma, cyclic peptide drugs have shown unique advantages. First, cyclic peptide drugs can specifically target receptors or proteins on the surface of renal cell cancer cells, thereby accurately exerting anti-tumor effects. Secondly, the molecular weight of cyclic peptide drugs is relatively small, which can better penetrate tumor tissue and increase the concentration of drugs at the tumor site. In addition, cyclic peptide drugs are highly stable and can maintain activity in the body for a long time, thereby prolonging the duration of drug action. These advantages make cyclic peptide drugs a promising new option for the treatment of renal cell carcinoma, bringing more hope to patients.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide an anti-tumor cyclic hexapeptide that has high anti-tumor activity and low cytotoxicity and is suitable for use as an anti-tumor drug.

[0007] The present invention provides an anti-tumor cyclic peptide and its use in the preparation of anti-tumor drugs in the form of a pharmaceutically acceptable salt, hydrate, mixture or prodrug, wherein the amino acid sequence of the cyclic peptide is shown in SEQ ID NO: 1. Each amino acid in the sequence independently has a D-configuration or an L-configuration.

[0008] The cyclic peptide described in the present invention is a cyclic peptide independently developed by the applicant. It has been found to have mild toxicity to normal cells and can produce good killing effects on various tumor cells. It can be used as an active pharmaceutical ingredient to prepare anti-tumor drugs.

[0009] The present invention provides a method for preparing the cyclic peptide, comprising the steps of: preparing a fully protected linear peptide-resin complex by adopting a microwave-promoted Fmoc protection solid-phase synthesis method; cleaving the fully protected linear peptide-resin complex with a cleavage agent to obtain a crude fully protected linear peptide; cyclizing the crude fully protected linear peptide to obtain a crude fully protected cyclic peptide; cleaving the crude fully protected cyclic peptide with a cleavage agent to obtain a crude cyclic peptide; and purifying the crude product to obtain the cyclic peptide PT-C07.

[0010] The present invention provides use of the cyclic peptide or the cyclic peptide obtained by the preparation method in preparing anti-tumor drugs.

[0011] The present invention provides a cyclic peptide or the use of a pharmaceutically acceptable salt, hydrate, mixture or prodrug of the cyclic peptide obtained by the preparation method in the preparation of an anti-tumor drug.

[0012] Preferably, the anti-tumor drug is a drug that promotes tumor cell apoptosis. Experiments have shown that the cyclic peptide can achieve anti-tumor effects by promoting tumor cell apoptosis.

[0013] Preferably, the tumor is renal cell carcinoma.

[0014] The present invention provides a pharmaceutical composition, which comprises the cyclic peptide and / or the cyclic peptide and a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof.

[0015] The present invention provides a pharmaceutical composition comprising the cyclic peptide and a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof, and a carrier and / or excipient acceptable thereto.

[0016] The beneficial effects of the present invention are as follows:

[0017] The cyclic peptide of the present invention has good inhibitory and killing effects on various tumor cells including renal cell carcinoma cells, and can be used to prepare corresponding anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Represents the cell status after different concentrations of PT-C07 acted on 786-O cells for 72 h;

[0019] Figure 2 Represents the cell status when different concentrations of PT-C07 acted on A498 cells for 72 h;

[0020] Figure 3 The fitting curve diagram represents the half-maximal inhibitory concentration IC50 of cyclic peptide PT-C07 in 786-O cells;

[0021] Figure 4 The fitting curve diagram represents the half-maximal inhibitory concentration IC50 of cyclic peptide PT-C07 in A498 cells;

[0022] Figure 5 Graph showing the mass spectrometry results of the cyclic peptide PT-C07 obtained in Example 1 of the present invention;

[0023] Figure 6 The figure shows the high performance liquid chromatogram of the cyclic peptide PT-C07 obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] The present invention provides an anti-tumor cyclic hexapeptide having an amino acid sequence as shown in SEQ ID NO: 1, specifically: CRLIIX, i.e., Cys-Arg-Leu-Ile-Ile-X, wherein X is cyclopentylglycine. The anti-tumor cyclic peptide is obtained by cyclization of the above amino acids, and each amino acid in the sequence independently has a D-configuration or an L-configuration. To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the following examples are provided for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present 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 only used to explain the present invention and are not intended 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 cyclic peptide PT-C07

[0028] This embodiment provides a method for synthesizing a cyclic peptide PT-C07, whose amino acid sequence is: CRLIIX, i.e., Cys-Arg-Leu-Ile-Ile-X, where X is cyclopentylglycine. The anti-tumor cyclic peptide is obtained by cyclization of the above amino acids. Each amino acid in the sequence independently has a D-configuration or an L-configuration and can be used to prepare anti-tumor drugs.

[0029] Main materials and instruments

[0030] Synthesis of cyclic peptide: The cyclic peptide PT-C07 of the present invention is synthesized by the peptide synthesis laboratory of our company using solid phase synthesis method, and the purity is above 95%.

[0031] The main experimental reagents of the present invention (source): 2-CTC Resin (2-chlorotrityl resin) (Gill Biochemical), Fmoc-L-Arg(pbf)-OH (Fmoc-Pbf-arginine) (ACMEC), Fmoc-L-Ile-OH (Fmoc-L-isoleucine) (McLean), Fmoc-L-Leu-OH (Fmoc-L-leucine) (McLean), Fmoc-L-Cys(trt)-OH (Fmoc-S-trityl-L- Cysteine) (Biden Pharmaceuticals), Fmoc-cyclopentylglycine (Aicon), nitrogen, nitrogen-dimethylformamide (DMF) (Sinopharm), dichloromethane (Sinopharm), trifluoroacetic acid (McLean), triisopropylsilane (Aladdin), acetonitrile (Aladdin), nitrogen, nitrogen-diisopropylcarbodiimide (DIC) (Aladdin), ethyl 2-oximecyanoacetate (McLean), 4-methylpiperidine (McLean), methyl tert-butyl ether (McLean), trifluoroethanol (ACMEC).

[0032] The main experimental instruments (sources) of the present invention are: Agilent HPLC-1260 series high performance liquid chromatograph (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 Libertyblue 2.0 fully automatic peptide synthesizer.

[0033] The specific synthesis steps are as follows:

[0034] Peptide resin synthesis: 3 g of solid-phase synthesis support 2-CTC resin was weighed and placed in a peptide synthesis solid-phase reaction tube. DCM (10 mL) was added to swell the resin for 10 minutes. The DCM was removed under vacuum, and the resin was washed with DMF (10 mL). Washing was repeated twice. 3 mmol of Fmoc-cyclopentylglycine was weighed and added to the modified resin with DIEA. Peptide resin synthesis was completed using a Libertyblue 2.0 fully automated peptide synthesizer. Amide bond condensation used a 5-fold excess of the corresponding Fmoc-protected amino acid. Amino acid condensation was performed using nitrogen-diisopropylcarbodiimide / ethyl 2-oxime cyanoacetate in a microwave oven at 90°C for 2 minutes. Fmoc group deprotection was performed using a DMF solution containing 20% 4-methylpiperidine in a microwave oven at 90°C for 1 minute.

[0035] Cleavage of the resin peptide: The resin peptide obtained in the above step was washed three times with DCM and then vacuum dried. 10 mL of cleavage buffer (trifluoroethanol:DCM = 20:80, v:v) was added and the reaction was shaken at room temperature for 1 hour. This reaction was repeated three times. After the reaction was completed, the filtrate was filtered, and the combined filtrates were spin-dried to obtain the corresponding fully protected peptide.

[0036] Cyclization of fully protected peptide: PYBOP and DIEA were used for cyclization reaction for 6 hours and then dried to obtain fully protected cyclic peptide.

[0037] Cleavage of the fully protected cyclic peptide: Add 10 mL of cleavage buffer (trifluoroacetic acid: triisopropylsilane: water = 90:5:5, v:v:v) and shake at room temperature for 2.5 hours. Add a large amount of chilled methyl tert-butyl ether to precipitate a solid. After centrifugation, remove the supernatant and obtain the crude cyclic peptide PT-C07.

[0038] Purification of cyclic 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 cyclic peptide numbered PT-C07. The purity and molecular weight of the pure cyclic peptide were determined by analytical high performance liquid chromatography and liquid chromatography tandem mass spectrometry, where the purity = 95.25% and the molecular weight was correct.

[0039] Example 2: Cell viability of 786-O (renal cancer cells) after addition of cyclic peptide PT-C07 was detected by microscopic observation and CCK-8 kit

[0040] Main materials and instruments

[0041] The main experimental reagents (sources) of the present invention: 786-O (renal cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the required experimental materials were: DMEM high-glucose medium; RPMI 1640 medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; Cell Counting Kit (CCK-8); CCK-8 kit (Yisheng Biological).

[0042] 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.).

[0043] Cell recovery: Remove the cryopreserved tube of 786-O cell line from the liquid nitrogen tank and place it in a 37°C water bath to shake until thawed. Pipette the thawed cell line into a 15mL sterile centrifuge tube in a biosafety cabinet, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Add RPMI 1640 complete medium to the 786-O tube and transfer it to a 25cm 2 The cells were cultured in sterile culture bottles at 5% CO2 and 37°C.

[0044] Cell passaging: Observe the cells under a microscope. Passage the cells when the cell morphology is normal and the growth density reaches about 90%. First, take out the culture flask containing the cells and place it in the clean bench. Aspirate the original culture medium and wash the cells once with PBS buffer. After absorbing the PBS, add 2mL of trypsin so that it can completely cover the bottom of the culture flask. After tightening the bottle cap, place it in a 37℃ incubator for digestion. After digestion, blow the cells off the wall of the flask with fresh complete culture medium. Then, aspirate the cell suspension into a 15mL sterile centrifuge tube and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, add fresh complete culture medium to resuspend the cells and perform cell passaging at a ratio of 1:3.

[0045] Cell cryopreservation: After centrifugation and removal of the supernatant according to the above cell passage process, add freezing solution (complete culture medium: DMSO = 9:1) to resuspend the cells, and add the suspension to 1.5 mL cell cryopreservation tubes (3 × 10 6 After labeling, cryopreservation was performed using the gradient cooling method.

[0046] In this experiment, 786-O cells were treated with different concentrations of cyclic peptide PT-C07 for 72 hours. During this period, the cell status after treatment was observed every day and photos were taken. At 72 hours, cell viability and IC were detected using CCK-8 reagent. 50 Drawing of fitting curve graph.

[0047] Cell status observation:

[0048] After treating 786-O cells with different concentrations of cyclic peptide PT-C07, the cell status after treatment was observed every day and photos were collected. Figure 1 shown.

[0049] Killing efficacy test:

[0050] 1. Prepare 100 μL of cell suspension in a 96-well plate and place the plate in an incubator for 24 hours (37°C, 5% CO2).

[0051] 2. Add different concentrations of PT-C07 to the culture plate.

[0052] 3. Incubate the culture plate in an incubator for 72 hours.

[0053] 4. Add 10 μL of CCK-8 solution to each well.

[0054] 5. Incubate the culture plate in the incubator for 1.5 hours.

[0055] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0056] Data processing was performed according to the CCK-8 reagent instructions. Cell viability after treatment is shown in Table 1.

[0057] Table 1 Cell viability after adding cyclic peptide PT-C07 to 786-O

[0058]

[0059] IC 50 Plotting the fitting curve:

[0060] Perform IC in GraphPad Pirsm 50 Drawing of fitting curve. Figure 3 As shown. 786-O IC 50 =77.53μM.

[0061] Example 3: Cell viability of A498 (renal cancer cells) after addition of cyclic peptide PT-C07 was detected by microscopic observation and CCK-8 kit

[0062] Main materials and instruments

[0063] The main experimental reagents (sources) of the present invention: A498 (renal cancer cells) were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and the required experimental materials were: DMEM high-glucose medium; RPMI 1640 medium; fetal bovine serum; ampicillin and streptomycin; DMSO; trypsin; PBS; Cell Counting Kit (CCK-8) CCK-8 kit (Yisheng Biological).

[0064] 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.).

[0065] Cell recovery: Take the A498 cell line cryotube out of the liquid nitrogen tank and place it in a 37°C water bath to shake until thawed. Pipette the thawed cell line into a 15mL sterile centrifuge tube in a biosafety cabinet, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant. Add DMEM complete medium to the A498. Finally, transfer to a 25cm 2 The cells were cultured in sterile culture bottles at 5% CO2 and 37°C.

[0066] Cell passaging: Observe the cells under a microscope. Passage the cells when the cell morphology is normal and the growth density reaches about 90%. First, take out the culture flask containing the cells and place it in the clean bench. Aspirate the original culture medium and wash the cells once with PBS buffer. After absorbing the PBS, add 2mL of trypsin so that it can completely cover the bottom of the culture flask. After tightening the bottle cap, place it in a 37℃ incubator for digestion. After digestion, blow the cells off the wall of the flask with fresh complete culture medium. Then, aspirate the cell suspension into a 15mL sterile centrifuge tube and centrifuge at 1000r / min for 5 minutes. Remove the supernatant, add fresh complete culture medium to resuspend the cells and perform cell passaging at a ratio of 1:3.

[0067] Cell cryopreservation: After centrifugation and removal of the supernatant according to the above cell passage process, add freezing solution (complete culture medium: DMSO = 9:1) to resuspend the cells, and add the suspension to 1.5 mL cell cryopreservation tubes (3 × 10 6 After labeling, cryopreservation was performed using the gradient cooling method.

[0068] In this experiment, A498 cells were treated with different concentrations of cyclic peptide PT-C07 for 72 hours. During this period, the cell status after treatment was observed every day and photos were collected. At 72 hours, cell viability and IC were detected using CCK-8 reagent. 50 Drawing of fitting curve graph.

[0069] Cell status observation:

[0070] After A498 cells were treated with different concentrations of cyclic peptide PT-C07, the cell status after treatment was observed every day and photos were collected. Figure 2 shown.

[0071] Killing efficacy test:

[0072] 1. Prepare 100 μL of cell suspension in a 96-well plate and place the plate in an incubator for 24 hours (37°C, 5% CO2).

[0073] 2. Add different concentrations of PT-C07 to the culture plate.

[0074] 3. Incubate the culture plate in an incubator for 72 hours.

[0075] 4. Add 10 μL of CCK-8 solution to each well.

[0076] 5. Incubate the culture plate in the incubator for 1.5 hours.

[0077] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0078] Data processing was performed according to the CCK-8 reagent instructions. The killing efficacy after treatment is shown in Table 2.

[0079] Table 2 Cell viability after adding cyclic peptide PT-C07 to A498

[0080]

[0081] IC 50 Plotting the fitting curve:

[0082] Perform IC in GraphPad Pirsm 50 Drawing of fitting curve. Figure 4 As shown. A498 IC 50 =67.06μM.

[0083] Combined with the above experiments, it can be shown that the cyclic peptide PT-C07 can significantly reduce the cell viability of 786-O and A498.

Claims

1. A cyclic hexapeptide PT-C07 and a pharmaceutically acceptable salt, hydrate, mixture or prodrug thereof, characterized in that: The amino acid sequence of the cyclic peptide PT-C07 is shown in SEQ ID NO: 1, wherein each amino acid in the sequence independently has a D-configuration or an L-configuration.

2. A method for preparing the cyclic peptide according to claim 1, characterized in that: A fully protected linear peptide-resin complex was prepared by microwave-promoted Fmoc protection solid-phase synthesis. The fully protected linear peptide-resin complex was cleaved by a cleavage agent to obtain a crude fully protected linear peptide. The crude fully protected linear peptide was cyclized to obtain a crude fully protected cyclic peptide. The crude fully protected cyclic peptide was cleaved by a cleavage agent to obtain a crude cyclic peptide. The crude product was purified to obtain the cyclic peptide PT-C07.

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

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

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 renal cell carcinoma.

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

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

Images