A polypeptide for targeted treatment of liver fibrosis, preparation method and application

Abstract

The present invention discloses a polypeptide for targeted treatment of liver fibrosis, a preparation method and an application thereof. The amino acid sequence of the polypeptide for targeted liver fibrosis is shown as follows: PEG3-YVNKEIQNAVNGVKQIKTLI. The anti-fibrotic polypeptide in the present invention can specifically promote the endocytosis of extracellular collagen into cells and decompose it through intracellular lysosomes, thereby causing the degradation of a large amount of extracellular matrix in fibrotic tissues, so as to achieve the anti-fibrotic effect. Therefore, it can be used to prepare drugs for anti-liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a polypeptide for targeted treatment of liver fibrosis, a preparation method thereof, 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, liver cirrhosis, and liver cancer. The main feature of liver fibrosis is the excessive accumulation of extracellular matrix (ECM), leading to the destruction of liver tissue structure and the decline of liver function. At present, the methods for treating liver fibrosis clinically include antiviral treatment, antioxidant treatment, inhibition of hepatic stellate cell activation, etc., but these methods mostly have problems such as poor treatment effect or obvious side effects. Therefore, the development of liver fibrosis treatment drugs with high efficiency and low toxicity has important clinical significance.

[0003] In recent years, the development of polypeptide-based drugs has become an emerging means for treating liver fibrosis due to their good targeting, low toxicity, and adjustable structure. Polypeptides have gradually attracted the attention of researchers because of their small molecular weight, easy penetration into tissues and cells, and good stability. Polypeptides 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 curative effect for effectively treating liver fibrosis, especially showing excellent treatment potential in inhibiting the excessive deposition of fibrosis-related extracellular matrix.

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

[0006] Specifically, the polypeptide involved in the present invention can target and bind to mesothelial cells in the liver, and by promoting the endocytosis of collagen secreted by surrounding fibroblasts by mesothelial cells, reduce the excessive deposition of extracellular matrix, 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, the amino acid sequence of the polypeptide is shown as 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 shown as SEQ ID NO.3.

[0010] Preferably, the last amino acid of the amino acid sequence of the polypeptide is acetylated or polyethylene glycolated after deprotection.

[0011] Preferably, the last amino acid of the amino acid sequence of the polypeptide is polyethylene glycolated after deprotection.

[0012] Preferably, the last amino acid of the amino acid sequence of the polypeptide is triethylene glycolated after deprotection, and its specific structure is PEG3-YVNKEIQNAVNGVKQIKTLI.

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

[0014] a. Under the action of a condensing agent, the first amino acid at the C-terminus is coupled with a solid-phase carrier respectively; a deprotecting agent is used to remove the Fmoc protecting group on the amino acid; the next amino acid is linked 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, it comprises the following steps:

[0016] After the step a above, step b is carried out.

[0017] b. After the last amino acid is deprotected, it is acetylated or polyethylene glycolated.

[0018] Preferably, it comprises the following steps:

[0019] After the step b above, step c is carried out.

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

[0021] An application in the preparation of a drug for treating liver fibrosis, comprising the polypeptide described in any one of the foregoing.

[0022] Preferably, an application in the preparation of a drug for treating liver fibrosis, characterized in that the injection dose of the drug ≤ 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 liver fibrosis lesion site, inhibits the excessive deposition of fibrosis-related extracellular matrix, and thus achieves the effect of treating liver fibrosis. The advantages of the present application are:

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

[0026] 2. Good stability: The stability of the polypeptide in vivo is improved by polyethylene glycol modification, the half-life of the drug in vivo is prolonged, and the curative effect is enhanced.

[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 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, but do not constitute a limitation to the present invention. In the drawings:

[0029] Figure 1 is the fluorescence intensity effect diagram of screening polypeptide flow cytometry for mesothelial cells to endocytose collagen;

[0030] Figure 2 is the fluorescence intensity effect diagram of three screened polypeptides for flow cytometry to endocytose collagen;

[0031] Figure 3 is the mass spectrometry diagram of Peptide3 without polyethylene glycol modification for identifying polypeptides;

[0032] Figure 4 is the mass spectrometry diagram of Peptide3 with polyethylene glycol modification for identifying polypeptides;

[0033] Figure 5 is the effect diagram of the polypeptide in the treatment of liver fibrosis;

[0034] Figure 6 is the result diagram of the in vivo safety evaluation of small molecule polypeptides. DETAILED DESCRIPTION OF THE INVENTION

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] Experimental reagents and materials:

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

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

[0039] Oregon Green TM 488 conjugate (manufacturer: Thermo, product number G13186).

[0040] 1. Design and screening of polypeptides

[0041] In the present invention, through the combination of in vitro screening and computer simulation, according to the helical folding fragments in the protein secondary structure, 21 polypeptides Peptide1 - Peptide121 composed of several amino acids were designed (sequences are as follows), which have high liver endothelial cell targeting and the ability to promote endocytosis.

[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] Peptide13 HFHSPAFQHPPTEFI(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 in cells with the smallest dose and has good targeting and affinity.

[0064] The specific experimental steps are as follows:

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

[0066] 2) The human mesothelial cell line MET-5A was inoculated at 1×10 5 cells / mL into a 24-well plate. After the cells adhered to the wall, they continued to grow until the density was greater than 70% for polypeptide incubation;

[0067] 3) Before polypeptide incubation, the special medium for MET-5A was replaced with a serum-free medium. 300 μL of medium and 100 μM of small molecule polypeptide were added to each cell sample. Each polypeptide had three replicates and was incubated in a 37 °C incubator for 40 minutes;

[0068] 4) After polypeptide incubation, porcine skin gelatin and Oregon Green TM 488 conjugate at 0.5 mg / mL were added to the samples and incubated in a 37 °C incubator for 15 minutes;

[0069] 5) The medium was discarded, and the mesothelial cells were washed twice with PBS buffer. After being digested with trypsin into single cells, flow cytometry analysis was performed;

[0070] 6) The ability of the polypeptide to promote mesothelial cell endocytosis was judged according to the FITC fluorescence intensity in the cells. According to Figure 1 the results, we screened out the candidate peptides peptide3, peptide6 and peptide19, these potentially effective polypeptides, and further analyzed the specific effects of different polypeptide concentrations on mesothelial cells.

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

[0072] YVNKEIQNAVNGVKQIKTLI had the best effect.

[0073] 2. Modification and optimization of polypeptides

[0074] To enhance the in vivo stability and bioavailability of the polypeptide, the small molecule polypeptide was chemically modified with N-terminal polyethylene glycol. The modified polypeptide had a longer in vivo half-life and enhanced targeting in the liver. The obtained polyethylene glycol-modified polypeptide sequence was PEG3-YVNKEIQNAVNGVKQIKTLI.

[0075] 3. Preparation method of polypeptides

[0076] (1) Solid-phase peptide synthesis (SPPS): The peptide was synthesized step by step by the Fmoc protection-based solid-phase synthesis method. The steps are as follows: Under the action of a condensing agent, the first amino acid at the C-terminus was coupled to the solid-phase carrier respectively; the Fmoc protecting group on the amino acid was removed using a deprotecting reagent; the next amino acid was linked under the action of a condensing agent; the deprotection-coupling operation was repeated to synthesize the peptide chain according to the amino acid sequence; after the last amino acid was deprotected, it was acetylated or polyethylene glycolated;

[0077] (2) After synthesis, it was purified by high-performance liquid chromatography (HPLC) to ensure that the purity of the peptide reached the drug standard. Steps: The purification method used was reverse-phase high-performance liquid chromatography, and the conditions were as follows: Chromatographic column: YMC-Pack ODS-AQ column; Mobile phase: Mobile phase A was 0.1% TFA / water, and mobile phase B was 0.1% TFA / acetonitrile; Gradient elution program: 25% B was eluted 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.

[0078] (3) The molecular weight and sequence integrity of the peptide were identified by mass spectrometry analysis.

[0079] The mass spectrometry analysis identification of the peptide without the attachment of triethylene glycol for Peptide3 is shown as Figure 3 follows;

[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] The mass spectrometry analysis identification of the peptide with the attachment of triethylene glycol for Peptide3 is shown as Figure 4 follows;

[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, after verification, it can be determined that there are no problems with the molecular weight and sequence integrity of PEG3 - YVNKEIQNAVNGVKQIKTLI.

[0088] 5. Effect of the polypeptide on inhibiting liver fibrosis in vivo

[0089] In a mouse model of CCl4 - induced liver fibrosis, the therapeutic effect of the polypeptide drug was evaluated. The degree of fibrosis reduction was evaluated through biochemical analysis and histopathological detection. The experimental results showed that the polypeptide significantly reduced the fibrosis progression.

[0090] The experimental steps are as follows:

[0091] 1) Establish a mouse liver fibrosis model: Inject CCl4 reagent (CCl4: olive oil ratio is 1:4) intraperitoneally, and inject 2 μL / g of CCl4 reagent into each mouse, twice a week;

[0092] 2) Two weeks after establishing the mouse liver fibrosis model, randomly divide the mice into two groups;

[0093] 3) Two weeks after CCl4 modeling, starting from the 3rd week, inject 100 μL of PBS solution and small - molecule polypeptide (5 mg / kg, dissolved in PBS solution) intraperitoneally once a day for 2 weeks;

[0094] 4) Two weeks after continuing the modeling, collect liver tissues, and evaluate the therapeutic effect of the small - molecule polypeptide on liver fibrosis through Sirius red, MASSON staining, and the expression level of collagen.

[0095] The results are as Figure 5 shown:

[0096] A: Hematoxylin - eosin, Sirius red, and MASSON staining showed that after injection of the small - molecule polypeptide, liver fibrosis was significantly alleviated. B: Statistical analysis of the collagen area after Sirius red and MASSON staining. C: Immunoblot showed the expression levels of collagen in the control and small - molecule polypeptide injection groups. D: Statistical analysis of the relative expression levels of collagen. ** indicates p < 0.01.

[0097] 6. Safety evaluation of the polypeptide

[0098] Inject it into mice at a high dose, and evaluate the toxicity of the small molecule polypeptide in vivo by detecting liver function indicators and liver tissue morphology. The results show that even when the injection dose is increased by 10 times, there is no impact on liver function and tissue morphology, indicating its high safety.

[0099] The specific experimental steps are as follows:

[0100] 1) Randomly divide 6 normal wild-type mice into two groups, with 3 mice in each group. One group is intraperitoneally injected with 100 μL of PBS solution, and the other group of mice is intraperitoneally injected with 100 μL of a small molecule polypeptide with a dose of 50 mg / kg.

[0101] 2) After 24 hours of intraperitoneal injection, collect liver tissue and mouse serum, and evaluate the safety of the small molecule polypeptide in vivo by observing liver function biochemical indicators and liver tissue morphology.

[0102] The results are as Figure 6 shown:

[0103] A: The concentrations of ALT, AST, and ALP in the sera of the two groups of mice show that there is no significant difference; B: Hematoxylin and eosin staining shows that there is no obvious change in the liver tissue structure of the two groups of mice. n.s. indicates no statistical difference.

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

[0105] Finally, it should be noted that: The above are only preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

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

19.

2. The polypeptide for targeted liver fibrosis treatment according to claim 1, wherein, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

3.

3. The polypeptide for targeted liver fibrosis treatment according to claim 2, wherein The last amino acid of the amino acid sequence of the polypeptide is acetylated or polyethylene glycolated after deprotection.

4. The polypeptide for targeted liver fibrosis treatment according to claim 3, wherein The last amino acid of the amino acid sequence of the polypeptide is polyethylene glycolated after deprotection.

5. A polypeptide for targeted liver fibrosis treatment according to claim 4, characterized in that, The last amino acid of the amino acid sequence of the polypeptide is triethylene glycolated after deprotection, and its specific structure is PEG3 - YVNKEIQNAVNGVKQIKTLI.

6. A method for preparing a polypeptide for targeted liver fibrosis treatment according to any one of claims 1 to 5, characterized in that, Comprising the following steps: a. Under the action of a condensing agent, the first amino acid at the C - terminus is coupled with a solid support respectively; Use a deprotecting 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 preparation method according to claim 6, characterized in that, Comprising the following steps: After the step a described above, step b is carried out. b. The last amino acid is acetylated or polyethylene glycolated after deprotection.

8. The preparation method according to claim 7, wherein Comprising the following steps: After the step b described above, step c is carried out. c. The polypeptide is purified by reverse high - performance liquid chromatography under the following conditions: chromatographic column: YMC - Pack ODS - AQ column; Mobile phase: mobile phase A is 0.1% TFA / water, mobile phase B is 0.1% TFA / acetonitrile; gradient elution program: 25% B elutes for 0 - 5 min, 25% B - 45% B, 5 - 60 min; flow rate is 15 mL / min, injection volume is 5 mL, detection wavelength is 214 nm.

9. Use of the polypeptide according to any one of claims 1 - 5 in the preparation of a drug for treating liver fibrosis.

10. The application according to claim 9, wherein The injection dose of the drug is ≤50 mg / kg.

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