Polypeptide conjugate, hydrogel, use, and Anti-tumor drug

Abstract

The present invention pertains to the technical field of biology. Disclosed is a polypeptide conjugate comprising a polypeptide and a chemotherapeutic drug. The polypeptide and the chemotherapeutic drug are connected by means of a covalent bond. The polypeptide has at least one D-amino acid. The polypeptide is formed by connecting a self-assembled polypeptide and a transmembrane peptide. Research has found that when D-amino acids are used in place of the corresponding amino acids in a polypeptide having transmembrane and self-assembly functions, the water solubility and anti-tumor activity of a chemotherapeutic drug having poor water solubility can be effectively improved. In addition, the present invention provides a hydrogel, use of the conjugate, and a drug.

Description

Polypeptide conjugate, hydrogel, use and antitumor drug TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide conjugate, hydrogel, use and antitumor drug. BACKGROUND

[0002] According to the International Agency for Research on Cancer of the World Health Organization, 1 out of 5 people in the world will suffer from cancer in their lifetime, and the prevention and treatment of cancer has become one of the most important public health challenges in the 21st century. The main advantage of chemotherapy is fast-acting, and chemotherapy drugs can kill a large number of tumor cells, but they have poor specificity and often cause serious toxic side effects, which is called palliative chemotherapy. Therefore, patients are afraid of chemotherapy, which reduces patient compliance and is not conducive to recovery. Therefore, it is urgent to develop new tumor treatment strategies to efficiently and specifically target tumor cells, thereby improving tumor treatment effects and reducing toxicity to normal tissues.

[0003] CN102552929A discloses a method for improving the targeting selectivity of a drug delivery system by modifying a cell-penetrating peptide, which is described as follows: the drug delivery system is composed of an activatable cell-penetrating peptide capable of improving the targeting selectivity and an anti-cancer and / or tracer and / or biologically active substance and / or carrier. The activatable cell-penetrating peptide includes a shielding peptide sequence, an enzymatic substrate peptide sequence, and a cell-penetrating peptide sequence, and the enzymatic substrate peptide sequence is between the shielding peptide sequence and the cell-penetrating peptide sequence. It is further described that the anti-cancer drugs include doxorubicin, paclitaxel, docetaxel, mitomycin, daunorubicin, cisplatin, carboplatin, camptothecin, hydroxycamptothecin, vincristine, bleomycin, 5-fluorouracil, cyclophosphamide, gemcitabine, methotrexate, capecitabine, lomustine, and etoposide, the tracers include fluorescent substances, radioisotopes, and quantum dots, and the biologically active substances include DNA, RNA, proteins, or peptide substances.

[0004] The scheme discloses that the cell-penetrating peptide is combined with paclitaxel to achieve targeted delivery to target cells.

[0005] In Example 5, Example 6, and Example 7 of the case, the liposome and the micelle form are used to encapsulate the liposome, and the essential reason is that the case only finds that the cell-penetrating peptide has a cell delivery effect on paclitaxel, and does not solve the problem of water solubility optimization before delivery, so it must rely on the liposome or the micelle to achieve the purpose of the scheme.

[0006] Moreover, the polypeptide of the case has good water solubility, which further proves that the polypeptide of the case has no substantial contribution to the water solubility optimization of paclitaxel.

[0007] The present applicant found through further research that some chemotherapy drugs such as paclitaxel and camptothecin have poor water solubility, resulting in low cell uptake rate and insufficient anti-tumor activity of the chemotherapy drugs.

[0008] Therefore, the technical problem solved by the present application is how to improve the cell uptake rate and anti-tumor activity of chemotherapy drugs with poor water solubility. SUMMARY

[0009] In view of the deficiencies in the prior art, the present application aims to provide a polypeptide conjugate. The present application has found that when D-type amino acids are used to replace corresponding amino acids in a polypeptide having a membrane-penetrating and self-assembling function, the water solubility and anti-tumor activity of chemotherapy drugs with poor water solubility can be effectively improved.

[0010] Meanwhile, the present application also provides a hydrogel, application of the conjugate and a medicament.

[0011] To achieve the object of the present application, the present application adopts the following technical solution: a polypeptide conjugate comprising a polypeptide and a chemotherapy drug; the polypeptide and the chemotherapy drug are connected by a covalent bond; the polypeptide has at least one D-type amino acid; and the polypeptide is formed by connecting a self-assembling polypeptide and a membrane-penetrating peptide.

[0012] In the polypeptide conjugate described above, the amino acid sequence of the polypeptide is as shown in SEQ ID No. 1.

[0013] The polypeptide as shown in SEQ ID No. 1 described in the present application is discussed in detail in the prior application CN116253805B of the present applicant.

[0014] The prior application comprehensively discusses the polypeptide from the aspects of characterization, stability, toxicity, cell uptake rate, anti-tumor mechanism and the like. It has been found through research that the cell uptake rate of polypeptide WDL is 158.3 times that of polypeptide WLL, and the cell uptake rate of polypeptide WDD is 541.9 times that of polypeptide WLL after 24 hours. The prior application can prove that D-type amino acids have a large contribution to the cell uptake rate.

[0015] In the present application, the polypeptide and the chemotherapy drug are connected in the form of a covalent bond, and it has been surprisingly found that this not only significantly optimizes the cell uptake rate, but also significantly improves the water solubility of the chemotherapy drug with poor water solubility, thereby achieving the purpose of improving the anti-tumor activity of the chemotherapy drug.

[0016] Preferably, all the amino acids in the self-assembling polypeptide and / or the membrane-penetrating peptide are D-type amino acids.

[0017] Preferably, the chemotherapy drug is paclitaxel, camptothecin, navelbine, SN38 or exatecan.

[0018] Meanwhile, the application also discloses a hydrogel containing the polypeptide conjugate.

[0019] In the use of the polypeptide conjugate in the preparation of an antitumor drug.

[0020] Finally, the application also discloses an antitumor drug containing the polypeptide conjugate.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application adds D-type amino acids into the synthesis of the polypeptide, which can significantly enhance the stability and improve the cell uptake rate, and covalently connects the polypeptide containing D-type amino acids with camptothecin to form PDC, which has significantly enhanced water solubility and cell uptake rate compared with camptothecin and the peptide alone, and exhibits significantly enhanced antitumor activity in vivo.

[0023] The polypeptide conjugate drug group of the application can significantly slow down the growth of tumors, has better curative effect than the chemotherapy drug camptothecin, and has important research value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a molecular structure of the polypeptide conjugate provided in Embodiment 1 of the application;

[0025] Fig. 2 is a serum stability test result of the polypeptide conjugate synthesized by full D-type amino acids;

[0026] Fig. 3 is a test result of the lipophilicity of the polypeptide conjugate;

[0027] Fig. 4 is a test result of the water solubility of the polypeptide conjugate;

[0028] Fig. 5 is a test result of the cell uptake rate of the polypeptide conjugate;

[0029] Fig. 6 is a test result of the cell uptake rate of the polypeptide conjugate;

[0030] Fig. 7 is a test result of the cytotoxicity of the polypeptide conjugate;

[0031] Fig. 8 is a test result of the mouse antitumor of the polypeptide conjugate. DETAILED DESCRIPTION

[0032] The technical solutions of the application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application and should not be regarded as specific limitations on the application.

[0033] Unless otherwise indicated, the specific experimental procedures or conditions in the examples were carried out according to the conventional experimental procedures described in the literature. The reagents or instruments used were not specified by the manufacturer, and were all conventional reagents available on the market.

[0034] Example 1

[0035] Synthesis of drugs

[0036] The synthesis was carried out using dichloro resin as the carrier. The required amino acid, small molecule drug, HBTU and DIEA were weighed and dissolved separately, and then placed in different synthesis bottles. The synthesis was set up according to the operation of the automatic synthesis instrument. The resin was cut and precipitated to obtain the crude polypeptide. The corresponding polypeptide conjugated drug lyophilized powder was obtained by purification and lyophilization using a high-performance liquid chromatograph.

[0037] The structure of the synthesized drug is shown in Figure 1.

[0038] Among them, CPT1-DL represents: camptothecin and polypeptide 1-DL are connected by a covalent bond, in which FFKLV is a D-type amino acid; RRVR is an L-type amino acid.

[0039] CPT1-DD represents: camptothecin and polypeptide DL are connected by a covalent bond, in which FFKLVRRVR is all D-type amino acid.

[0040] CPTFF1-DD represents: camptothecin and polypeptide FF1-DD are connected by a covalent bond, in which FFFFFKLVRRVR is all D-type amino acid.

[0041] Cbl-1LL represents: leucovorin and polypeptide LL are connected by a covalent bond, in which FFKLVRRVR is all L-type amino acid.

[0042] 1DD represents: FFKLVRRVR is all D-type, and the N-terminal is covalently connected with naphthalene acetic acid.

[0043] 1DL represents: FFKLV is a D-type amino acid; RRVR is an L-type amino acid, and the N-terminal is covalently connected with naphthalene acetic acid.

[0044] Example 2

[0045] Stability of polypeptide conjugated drugs

[0046] The polypeptide conjugated drug was prepared into a 20 mM stock solution, diluted to 300 μM using PBS buffer containing 10% FBS, and then sampled at different time points to detect the degradation of the polypeptide by analytical HPLC, and the degradation curve was plotted by calculation.

[0047] Results refer to Figure 2; the polypeptide conjugated drug of all D-form amino acids has higher serum stability.

[0048] Since CPT1-LL has poor stability, subsequent tests were performed using 1-DL, 1-DD, CPT1-DL, CPT1-DD, and CPTFF1-DD.

[0049] Example 3

[0050] Lipophilicity of polypeptide conjugated drugs

[0051] The molecular formulas of camptothecin (CPT), polypeptides (1-DL and 1-DD), and polypeptide conjugated drugs (CPT1-DL and CPT1-DD) were drawn by ChemDraw 18.0, and then each molecular formula was selected, and the values could be directly read in the Chemical Properties Window in ChemDraw 18.0.

[0052] Results refer to Figure 3; the lipophilicity of camptothecin (CPT), polypeptides (1-DL and 1-DD), and polypeptide conjugated drugs (CPT1-DL and CPT1-DD) was simulated and calculated by ChemDraw 18.0, and the results showed that the hydrophilicity of the polypeptide conjugated drugs was significantly stronger than that of camptothecin and polypeptides, proving that the polypeptide conjugated drugs can enhance the drugability of camptothecin.

[0053] Example 4

[0054] Solubility test of polypeptide conjugated drugs in PBS buffer

[0055] 2 mg of sample was added to 100 μL of water, and then ultrasonic-assisted dissolution was performed, followed by the addition of 100 μL of 2xPBS, and then incubation was performed at 37°C in a constant temperature incubator for 12 h.

[0056] Figure 4 shows that polypeptides 1-DL and 1-DD both form hydrogels, camptothecin CPT forms a turbid liquid, and polypeptide conjugated drugs CPT1-DL and CPT1-DD are both clear and transparent solutions, and after changing the sequence, CPTFF1-DD still forms a hydrogel, indicating that the polypeptide sequence-specific conjugated drugs CPT1-DL and CPT1-DD can significantly improve the water solubility of camptothecin, and thus enhance its drugability.

[0057] Example 5

[0058] Cellular uptake rate of polypeptide conjugated drugs

[0059] The qualitative analysis of cell uptake was carried out by fluorescence confocal microscopy. The polypeptide CPT1-DD, CPT1-DL, CPT and cells were incubated for different time. The fluorescence confocal imaging showed that the cell uptake rate of polypeptide CPT1-DD was the highest, while the uptake efficiency of CPT was the lowest. With the increase of time, the content of polypeptide CPT1-DDL in cells increased, and the uptake efficiency of CPT was far lower than that of polypeptide conjugated drug CPT1-DD within 12h (Figure 5). The qualitative analysis by flow cytometry showed that the cell uptake rate of polypeptide CPT1-DD was 6 times that of CPT after 4h (Figure 6), which further confirmed that polypeptide conjugated drug could affect the cell uptake rate.

[0060] Referring to Figure 5, the HCT-116 cells were treated with different compounds, and it was found that the polypeptide conjugated drug could significantly improve the internalization of CPT in cells, and the D-type amino acid had the best cell uptake rate with the polypeptide conjugated drug CPT1-DD, which indicated that the D-type polypeptide not only improved the water solubility of CPT, but also promoted the internalization of polypeptide.

[0061] Referring to Figure 6, the flow cytometry was used to quantitatively analyze the cells after drug treatment. The experimental results were the same as those of fluorescence confocal microscopy. The polypeptide conjugated drug could improve the internalization of CPT in cells, and the D-type polypeptide conjugated drug significantly improved the internalization efficiency of CPT in cells.

[0062] Example 6

[0063] Cytotoxicity of polypeptide conjugated drug

[0064] The polypeptide conjugated drug and CPT were configured into a 20mM stock solution, which was gradiently diluted and added to the 96-well plate with HCT116 cells pre-plated, 100μL per well, three replicate wells for each concentration, the treatment time was 3 days, and then the OD value was measured at 572nm by MTT, and the obtained value was calculated.

[0065] Referring to Figure 7, the cytotoxicity of CPT and polypeptide conjugated drug of CPT was tested, and it was found that the polypeptide conjugated drug did not reduce the toxicity of CPT, but the polypeptide conjugated drug greatly changed the solubility of CPT, which could enhance the possibility of clinical use of CPT.

[0066] Example 7

[0067] Effect of polypeptide conjugated drug on mice

[0068] HCT-116 tumor cells were injected subcutaneously into nude mice, and after the tumors grew to a certain size, different solutions were injected around the tumors of the nude mice. The solutions were divided into polypeptide drug 1-DD group (20 mg / kg), CPT1-DD (20 mg / kg), anticancer drug CPT group, PBS group, and the mice were given the drugs once every two days, 100 μL each time, 6 mice in each group, the body weight of the nude mice was measured twice a day, and the tumor volume of the nude mice was measured using a vernier caliper, and the nude mice were sacrificed after 10 days (Figure 7, the first row is the PBS group, the second row is the 20 mg / kg 1-DD group; the third row is the 20 mg / kg CPT1-DD group, and the fourth row is the camptothecin).

[0069] Referring to Figure 8, the polypeptide conjugated drug group can significantly slow down the growth of the tumor, and has better efficacy than the chemotherapy drug camptothecin, and has important research value.

Claims

1. A polypeptide conjugate, characterized in that, It includes a polypeptide and a chemotherapeutic drug; the polypeptide and the chemotherapeutic drug are linked by covalent bonds; the polypeptide contains at least one D-type amino acid; the polypeptide is formed by linking a self-assembled polypeptide and a membrane-penetrating peptide.

2. The polypeptide conjugate according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

3. The polypeptide conjugate according to claim 1, characterized in that, All amino acids in the self-assembled polypeptides and / or membrane-penetrating peptides are D-type amino acids.

4. The polypeptide conjugate according to claim 1, characterized in that, The chemotherapy drugs mentioned are paclitaxel, camptothecin, leukoxetine, SN38, or ethnetam.

5. A hydrogel, characterized in that, Contains a polypeptide conjugate as described in any one of claims 1 to 4.

6. Use of the polypeptide conjugate according to any one of claims 1 to 4 in the preparation of antitumor drugs.

7. An antitumor drug, characterized in that, Contains a polypeptide conjugate as described in any one of claims 1 to 4.

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