Polypeptide for targeted hepatic fibrosis treatment, preparation method and application

Abstract

The invention discloses a polypeptide for targeted hepatic fibrosis treatment as well as a preparation method and application thereof. The amino acid sequence of the polypeptide for targeting hepatic fibrosis is as shown in the specification: PEG3-YVNKEIQNAVNGVKQIKTLI. The anti-fibrosis polypeptide provided by the invention can specifically promote endocytosis of extracellular collagen to enter cells, and decomposition is carried out through intracellular lysosome, so that a large number of extracellular matrixes in fibrotic tissues are degraded, and the anti-fibrosis effect is achieved. Therefore, the compound can be used for preparing the anti-hepatic fibrosis medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a polypeptide for treating targeted liver fibrosis, a preparation method and an application thereof. Background Art

[0002] Liver fibrosis is a pathological process caused by various chronic liver injuries, and is usually an important pathological basis for liver diseases such as chronic hepatitis, cirrhosis and liver cancer. The main feature of liver fibrosis is the excessive accumulation of extracellular matrix (ECM), which leads to liver tissue structure destruction and liver function decline. At present, the clinical methods used to treat liver fibrosis include antiviral therapy, antioxidant therapy, inhibition of hepatic stellate cell activation, etc., but these methods often have problems such as poor therapeutic effects or obvious side effects. Therefore, the development of highly effective and low-toxic drugs for the treatment of liver fibrosis is of great clinical significance.

[0003] In recent years, peptide-based drug development has become an emerging means of treating liver fibrosis due to its good targeting, low toxicity and structural adjustability. Peptides have gradually attracted the attention of researchers due to their small molecular weight, easy penetration of tissues and cells, and good stability. Peptides can significantly reduce the progression of liver fibrosis by regulating the activation of hepatic stellate cells and inhibiting the activation of inflammatory factors and fibrosis signaling pathways. Summary of the invention

[0004] The present invention aims to provide a polypeptide with strong targeting and significant therapeutic effect for effectively treating liver fibrosis, especially showing excellent therapeutic potential in inhibiting excessive deposition of fibrosis-related extracellular matrix.

[0005] The main purpose of the present invention is to provide a polypeptide for the treatment of liver fibrosis. Through preparation and activity verification, the polypeptide can effectively target cells in the liver fibrosis area, inhibit extracellular matrix deposition, and alleviate symptoms related to liver fibrosis.

[0006] Specifically, the polypeptide involved in the present invention can target and bind to mesothelial cells in the liver, and reduce the excessive deposition of extracellular matrix by promoting mesothelial cells to internalize collagen secreted by surrounding fibroblasts, thereby slowing down the progression of liver fibrosis and promoting liver tissue repair.

[0007] The technical solution of the present invention is as follows:

[0008] A polypeptide for targeted treatment of liver fibrosis, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO.3, SEQ ID NO.6 or SEQ ID NO.19.

[0009] Preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.3.

[0010] Preferably, the last amino acid in the amino acid sequence of the polypeptide is deprotected and then acetylated or pegylated.

[0011] Preferably, the last amino acid in the amino acid sequence of the polypeptide is deprotected and then pegylated.

[0012] Preferably, the last amino acid in the amino acid sequence of the polypeptide is deprotected and then trimerized, and its specific structure is PEG3-YVNKEIQNAVNGVKQIKTLI.

[0013] A method for preparing a polypeptide for targeted treatment of liver fibrosis, comprising the following steps:

[0014] a. Under the action of a condensing agent, the first amino acid at the C-terminus is coupled to a solid phase carrier; a deprotection agent is used to remove the Fmoc protecting group on the amino acid; the next amino acid is connected under the action of a condensing agent; the deprotection-coupling operation is repeated to synthesize a peptide chain according to the amino acid sequence.

[0015] Preferably, the method comprises the following steps:

[0016] After step a, proceed to step b.

[0017] b. The last amino acid is deprotected and then acetylated or PEGylated.

[0018] Preferably, the method comprises the following steps:

[0019] After step b, proceed to step c,

[0020] c. The peptide was purified by reverse HPLC under the following conditions: chromatographic column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A was 0.1% TFA / water, mobile phase B was 0.1% TFA / acetonitrile; gradient elution program: 25% B for 0-5 min, 25% B-45% B for 5-60 min; flow rate was 15 mL / min, injection volume was 5 mL, and detection wavelength was 214 nm.

[0021] An application in the preparation of a drug for treating liver fibrosis, comprising any of the above-mentioned polypeptides.

[0022] Preferably, an application in the preparation of a drug for treating liver fibrosis is characterized in that the injection dose of the drug is ≤50 mg / kg.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0024] The polypeptide of the present application specifically targets the lesion site of liver fibrosis and inhibits the excessive deposition of fibrosis-related extracellular matrix, thereby achieving the effect of treating liver fibrosis. The advantages of the present application are:

[0025] 1. Strong targeting: The peptide specifically recognizes mesothelial cells, activates the endocytosis function of mesothelial cells, improves the targeting of treatment, and reduces side effects on normal tissues.

[0026] 2. Good stability: The in vivo stability of the peptide is improved by modification with polyethylene glycol, which prolongs the half-life of the drug in the body and enhances its efficacy.

[0027] 3. Low toxicity and side effects: Compared with traditional anti-fibrosis drugs, the polypeptide of the present invention has significantly reduced toxicity and is suitable for long-term treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 This is the fluorescence intensity effect diagram of the screened peptide flow analysis after mesothelial cells internalize collagen;

[0030] Figure 2 This is the fluorescence intensity effect diagram of the three peptides screened out by flow cytometry analysis of endocytic collagen;

[0031] Figure 3 This is the mass spectrum of peptide 3 without polyethylene glycol attached to the mass spectrometry analysis to identify the peptide;

[0032] Figure 4 This is the mass spectrum of peptide 3 linked to trimerized polyethylene glycol mass spectrometry analysis to identify the peptide;

[0033] Figure 5 This is a diagram showing the results of peptide treatment of liver fibrosis;

[0034] Figure 6 This is a diagram showing the results of the in vivo safety evaluation of small molecule peptides. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Experimental reagents and materials:

[0037] Human mesothelial cell line MET-5A (manufacturer: SUNNCELL, catalog number SNL-364);

[0038] MET-5A special culture medium (manufacturer: Shanghai Jinyuan Biotechnology Co., Ltd., catalog number: JY-H079);

[0039] Oregon Green TM 488 conjugate (Manufacturer: Thermo, Catalog No. G13186).

[0040] 1. Design and screening of peptides

[0041] The present invention combines in vitro screening with computer simulation, and designs 21 polypeptides Peptide1 to Peptide121 (sequences as follows) composed of several amino acids according to the helical folding fragments in the secondary structure of the protein, which have high intrahepatic mesothelial cell targeting and cell endocytosis-promoting abilities.

[0042] Peptide1 MMKTLLLFVGLLLTWESGQV(SEQ ID NO.1)

[0043] Peptide2 LGDQTVSDNELQEMSNQGSK(SEQ ID NO.2)

[0044] Peptide3 YVNKEIQNAVNGVKQIKTLI(SEQ ID NO.3)

[0045] Peptide4 EKTNEERKTLLSNLEEAKKK(SEQ ID NO.4)

[0046] Peptide5 KEDALNETRESETKLKELPG(SEQ ID NO.5)

[0047] Peptide6 VCNETMMALWEECKPCLKQTCMKFYARVCR(SEQ ID NO.6)

[0048] Peptide7 SGSGLVGRQLEEFLNQSSPFYFWMN(SEQ ID NO.7)

[0049] Peptide8 GDRIDSLLENDRQQTHMLDV(SEQ ID NO.8)

[0050] Peptide9 MQDHFSRASSIIDELFQDRF(SEQ ID NO.9)

[0051] Peptide10 FTREPQDTYHYLPFSLPHRR(SEQ ID NO.10)

[0052] Peptide11 PHFFFPKSRIVRSLMPFSPY(SEQ ID NO.11)

[0053] Peptide12 EPLNFHAMFQPFLEMIHEAQQAMDI(SEQ ID NO.12)

[0054] Peptide13HFHSPAFQHPPTEFI(SEQ ID NO.13)

[0055] Peptide14 REGDDDRTVCREIRHNSTGCLRMKD(SEQ ID NO.14)

[0056] Peptide15 QCDKCREILSVDCSTNNPSQAKLRR(SEQ ID NO.15)

[0057] Peptide16 ELDESLQVAERLTRKYNELLKSYQW(SEQ ID NO.16)

[0058] Peptide17 KMLNTSSLLE(SEQ ID NO.17)

[0059] Peptide18 QLNEQFNWVSRLANLTQGED(SEQ ID NO.18)

[0060] Peptide19 QYYLRVTTVASHTSDSDVPS(SEQ ID NO.19)

[0061] Peptide20 GVTEVVVKLFDSDPITVTVP(SEQ ID NO.20)

[0062] Peptide21 VEVSRKNPKFMETVAEKALQEYRKKHREE(SEQ ID NO.21)

[0063] After sequence optimization, the optimal sequence is Peptide3: YVNKEIQNAVNGVKQIKTLI. Compared with other sequences, this sequence can promote the endocytosis of extracellular collagen at a minimum dose, and has better targeting and affinity.

[0064] The specific experimental steps are as follows:

[0065] 1) Dissolve the candidate peptide powder in DMSO solution to prepare a 10 mM concentration stock solution.

[0066] 2) Human medullary cell line MET-5A was cultured at 1×10 5 pc / mL were inoculated in a 24-well plate, and the cells were allowed to adhere to the wall and continue to grow until the density was greater than 70% for peptide incubation;

[0067] 3) Before peptide incubation, replace the MET-5A special medium with a serum-free medium, add 300 μL of medium and 100 μM small molecule peptide to each cell sample, have three replicate wells for each peptide, and incubate in a 37°C incubator for 40 minutes;

[0068] 4) After the peptide incubation, continue to add pig skin gelatin and Oregon Green to the sample TM 488 conjugate 0.5 mg / mL, incubated in a 37°C incubator for 15 minutes;

[0069] 5) discarding the culture medium, washing twice with PBS buffer, digesting the mesothelial cells with trypsin, digesting them into single cells, and performing flow cytometry analysis;

[0070] 6) The effect of the peptide on promoting mesothelial cell endocytosis was determined based on the intracellular FITC fluorescence intensity. Figure 1 Based on the results, we screened out the candidate peptide3, peptide6 and peptide19 as potential effective peptides, and further analyzed the specific effects of different peptide concentrations on mesothelial cells.

[0071] 7) The three candidate peptides were treated with 50 and 100 μM concentrations, and the above experimental steps were repeated. Finally, flow cytometry analysis was performed. The results are as follows: Figure 2 As shown, the polypeptide sequence is Peptide3:

[0072] YVNKEIQNAVNGVKQIKTLI works best.

[0073] 2. Modification and optimization of peptides

[0074] In order to enhance the in vivo stability and bioavailability of the peptide, the small molecule peptide was chemically modified with polyethylene glycol at the N-terminus. The modified peptide has a longer in vivo half-life and enhanced targeting in the liver. The resulting polyethylene glycol-modified peptide sequence is PEG3-YVNKEIQNAVNGVKQIKTLI.

[0075] 3. Preparation method of polypeptide

[0076] (1) Solid phase peptide synthesis (SPPS): The peptide is synthesized step by step by using the Fmoc protecting group solid phase synthesis method. The steps are as follows: the first amino acid at the C-terminus is coupled to the solid phase support under the action of a condensing agent; the Fmoc protecting group on the amino acid is removed using a deprotecting agent; the next amino acid is connected under the action of a condensing agent; the deprotection-coupling operation is repeated to synthesize a peptide chain according to the amino acid sequence; the last amino acid is deprotected and then acetylated or pegylated;

[0077] (2) After synthesis, high performance liquid chromatography (HPLC) purification was used to ensure that the purity of the peptide reached the pharmaceutical standard. Steps: The purification method adopted was reverse HPLC, and the conditions were as follows: chromatographic column: YMC-Pack ODS-AQ column; mobile phase: mobile phase A was 0.1% TFA / water, mobile phase B was 0.1% TFA / acetonitrile; gradient elution program: 25% B elution 0-5min, 25% B-45% B, 5-60min; flow rate was 15mL / min, injection volume was 5mL, and detection wavelength was 214nm.

[0078] (3) Identify the molecular weight and sequence integrity of the polypeptide by mass spectrometry analysis.

[0079] The mass spectrum of peptide 3 without polyethylene glycol mass spectrometry analysis is shown in Figure 3 As shown;

[0080] HR-Q-TOF-MS and HPLC of Peptide3 YVNKEIQNAVNGVKQIKTLI.

[0081] HR-Q-TOF-MS m / z calcd for C 102 H 174 N 28 O 30 2272.68;

[0082] found[M+2H] 2+ =1136.34, [M+3H] 3+ =757.56, [M+4H] 4+ =568.17.

[0083] Peptide3 has been linked to polyethylene glycol. The mass spectrum of the peptide identified by mass spectrometry is shown below. Figure 4 As shown;

[0084] HR-Q-TOF-MS and HPLC of Ppeptide3:PEG3-YVNKEIQNAVNGVKQIKTLI.

[0085] HR-Q-TOF-MS m / z calcd for C 112 H 194 N 28 O 36 2490.94;

[0086] found[M+2H] 2+ =1245.47, [M+3H] 3+ =830.31.

[0087] In summary, it has been verified that there is no problem with the molecular weight and sequence integrity of PEG3-YVNKEIQNAVNGVKQIKTLI.

[0088] 5. The effect of polypeptide on inhibiting liver fibrosis in vivo

[0089] The therapeutic effect of the peptide drug was evaluated in a CCl4-induced liver fibrosis mouse model. The reduction in fibrosis was evaluated by biochemical analysis and histopathological examination. The experimental results showed that the peptide significantly reduced the progression of fibrosis.

[0090] The experimental steps are as follows:

[0091] 1) Establishment of mouse liver fibrosis model: intraperitoneal injection of CCl4 reagent (CCl4: olive oil ratio of 1:4), 2 μL / g CCl4 reagent per mouse, twice a week;

[0092] 2) Two weeks after the mice were modeled for liver fibrosis, they were randomly divided into two groups;

[0093] 3) Two weeks after CCl4 modeling, 100 μL PBS solution and small molecule peptide (5 mg / kg, dissolved in PBS solution) were injected intraperitoneally from the third week, once a day for 2 weeks;

[0094] 4) After the model was established for 2 weeks, liver tissues were collected and the therapeutic effect of small molecule peptides on liver fibrosis was evaluated by Sirius red, MASSON staining and collagen expression level.

[0095] The results are as follows Figure 5 As shown:

[0096] A: Hematoxylin eosin, Sirius red and MASSON staining showed that liver fibrosis was significantly alleviated after small molecule peptide injection. B: Collagen area statistics after Sirius red and MASSON staining. C: Immunoprotein blot showed the expression level of collagen in the control and small molecule peptide injection groups. D: Statistics of the relative expression level of collagen. ** indicates p<0.01.

[0097] 6. Peptide safety evaluation

[0098] The toxicity of small molecule peptides in vivo was evaluated by injecting them into mice at high doses, and liver function indicators and liver tissue morphology were tested. The results showed that even if the injection dose was increased by 10 times, there was no effect on liver function and tissue morphology, indicating that it is relatively safe.

[0099] The specific experimental steps are as follows:

[0100] 1) Six normal wild-type mice were randomly divided into two groups, with three mice in each group. One group was intraperitoneally injected with 100 μL PBS solution, and the other group was intraperitoneally injected with 100 μL of a small molecule peptide at a dose of 50 mg / kg.

[0101] 2) 24 hours after intraperitoneal injection, liver tissue and mouse serum were collected, and the safety of small molecule peptides in vivo was evaluated by observing liver function biochemical indicators and liver tissue morphology.

[0102] The results are as follows Figure 6 As shown:

[0103] A: The concentrations of ALT, AST and ALP in the serum of the two groups of mice showed no significant difference; B: Hematoxylin and eosin staining showed no significant changes in the liver tissue structure of the two groups of mice. ns indicates no statistical difference.

[0104] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0105] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide for treating liver fibrosis, characterized in that: The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO.3, SEQ ID NO.6 or SEQ ID NO.

19.

2. A polypeptide for treating liver fibrosis according to claim 1, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

3.

3. A polypeptide for treating liver fibrosis according to claim 2, characterized in that: The last amino acid in the amino acid sequence of the polypeptide is deprotected and then acetylated or pegylated.

4. A polypeptide for treating liver fibrosis according to claim 3, characterized in that: The last amino acid in the amino acid sequence of the polypeptide is deprotected and then pegylated.

5. A fluorescent probe for determining nuclease activity according to claim 4, characterized in that: The last amino acid in the amino acid sequence of the polypeptide is deprotected and then trimerized, and its specific structure is PEG3-YVNKEIQNAVNGVKQIKTLI.

6. A method for preparing a polypeptide for targeted treatment of liver fibrosis, characterized in that: The following steps are involved: a. Under the action of a condensing agent, the first amino acid at the C-terminus is coupled to a solid phase carrier; Use a deprotection reagent to remove the Fmoc protecting group on the amino acid; connect the next amino acid under the action of a condensing agent; repeat the deprotection-coupling operation to synthesize a peptide chain according to the amino acid sequence.

7. The method for preparing a polypeptide for treating liver fibrosis according to claim 6, characterized in that: The following steps are involved: After step a, proceed to step b. b. The last amino acid is deprotected and then acetylated or PEGylated.

8. The method for preparing a polypeptide for treating liver fibrosis according to claim 7, characterized in that: The following steps are involved: After step b, proceed to step c, c. Peptide purification was performed by reverse HPLC under the following conditions: Chromatographic column: YMC-Pack ODS-AQ column; Mobile phase: Mobile phase A was 0.1% TFA / water, mobile phase B was 0.1% TFA / acetonitrile; gradient elution program: 25% B elution for 0-5 min, 25% B-45% B, 5-60 min; flow rate was 15 mL / min, injection volume was 5 mL, and detection wavelength was 214 nm.

9. An application in the preparation of a drug for treating liver fibrosis, characterized in that: The method comprises the polypeptide according to any one of claims 1 to 5.

10. The use of claim 9 in preparing a drug for treating liver fibrosis, characterized in that: The injection dose of the drug is ≤50 mg / kg.

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