Polypeptide for treating liver fibrosis as well as synthesis method and application thereof

Abstract

The invention discloses a polypeptide for treating liver fibrosis as well as a synthesis method and application thereof. The amino acid sequence of the polypeptide is as shown in the specification: YGRKKRRQRRRFSLDKIYLIGGDLGPFNPGLPVEV PLWLAI, and the amino acid sequence of the polypeptide is as shown in the specification. The synthesis method of the polypeptide can be an Fmoc solid-phase synthesis method. The polypeptide can effectively inhibit the liver fibrosis process and relieve liver cell injury, and can be used for preparing medicines for treating or preventing liver fibrosis diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polypeptide for treating liver fibrosis and a synthesis method and application thereof. BACKGROUND

[0002] Liver fibrosis is a common pathological process of various chronic liver diseases (such as viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, etc.), and its essence is the excessive deposition of extracellular matrix (ECM) in the liver. Eventually, it can progress to cirrhosis, liver failure and even liver cancer. At present, there are various drugs for liver disease treatment in clinical practice, but there is a lack of effective anti-fibrosis drugs directly targeting the activation of hepatic stellate cells (HSC) and collagen deposition. Existing therapies such as obeticholic acid and pirfenidone have defects such as slow onset, large side effects or inability to reverse the formed fibrosis. Therefore, there is an urgent need in the art for a new type of safe, efficient and targeted anti-liver fibrosis treatment. SUMMARY

[0003] The purpose of the present application is to provide a polypeptide for treating liver fibrosis and a synthesis method and application thereof, so as to at least partially solve the technical problems pointed out in the background.

[0004] In a first aspect, the present application provides a polypeptide for treating liver fibrosis, and the amino acid sequence thereof is as follows:

[0005] YGRKKRRQRRRFSLDKIYLIGGDLGPFNPGLPVEVPLWLAI.

[0006] In a second aspect, the present application provides a synthesis method of the above-mentioned polypeptide, and the method is Fmoc solid-phase synthesis.

[0007] The Fmoc solid-phase synthesis method comprises the following steps: using swelled resin as a carrier, loading C-terminal amino acid onto the resin, and then removing fluorenylmethyloxycarbonyl protection; from the second amino acid, coupling amino acids one by one through Fmoc-SPPS coupling cycles in turn, and finally cutting the polypeptide from the resin.

[0008] In at least one embodiment, the resin is swelled with DCM for 10 min, then washed with DCM twice and DMF three times;

[0009] The removal of fluorenylmethyloxycarbonyl protection is as follows: using 20% piperidine / DMF solution for reaction twice, 5 min and 10 min respectively, and then washing with DMF three times and DCM twice;

[0010] The coupling method of the amino acid is: adding fluorenylmethyloxycarbonyl-protected amino acid, HATU, DIPEA, dissolving in DMF, reacting at room temperature for 45-60 min, washing the resin with DMF for 3 times and DCM for 2 times after the reaction;

[0011] The cutting method is: after the last defluorination, washing the resin thoroughly, adding TFA / TIS / H2O cutting solution, reacting at room temperature for 2-3 h, collecting the filtrate by filtration, washing the resin with TFA and combining; precipitating the combined filtrate in cold ether, centrifuging or standing, discarding the supernatant, washing 2-3 times with cold ether, and blowing dry or air-drying the precipitate.

[0012] In at least one embodiment, the precipitate after blowing dry or air-drying is dissolved in 30%-50% acetonitrile / 0.1% trifluoroacetic acid solution, and then purified by reverse-phase high-performance liquid chromatography, with mobile phase A being 0.1% trifluoroacetic acid aqueous solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution, the gradient change of mobile phase B being 5% to 60%, and the time being 30-45 min; and the target peak is collected and freeze-dried.

[0013] In a third aspect, the present application provides use of the above-mentioned polypeptide in the preparation of a medicament for treating liver fibrosis.

[0014] Specifically, the medicament comprises the polypeptide and a pharmaceutically acceptable excipient. The dosage form of the medicament includes injection preparations and oral preparations.

[0015] In a fourth aspect, the present application provides a medicament for treating liver fibrosis, which comprises the above-mentioned polypeptide and a pharmaceutically acceptable excipient.

[0016] Specifically, the dosage form of the medicament includes injection preparations and oral preparations.

[0017] The polypeptide provided by the present application exhibits significant anti-liver fibrosis activity, overcoming the deficiencies of the prior art drugs, mainly embodied in:

[0018] 1. Good cell compatibility: in the in vitro hepatic stellate cell experiment, the polypeptide can significantly improve the cell viability, indicating that the polypeptide has good biological safety and no toxicity to cells.

[0019] 2. Significant reduction of liver cell damage: in the liver fibrosis animal model, after treatment with the polypeptide of the present application, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are markers of liver cell damage in the serum of the test animals, are significantly reduced, indicating that the polypeptide has a definite protective effect on the liver.

[0020] 3. Effective mitigation of liver fibrosis: liver tissue HE staining results show that the fibrous interval and collagen deposition in the liver tissue of the polypeptide treatment group are significantly reduced, and the liver cells are arranged more regularly, indicating that the polypeptide can effectively block or reverse the progression of liver fibrosis.

[0021] 4. Good biocompatibility: multi-organ tissue HE staining results show that the polypeptide treatment group has no obvious pathological changes in the heart, spleen, kidney and lung, indicating that the polypeptide has no toxicity to the main organs at the treatment dose and is highly safe. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1: Effect of polypeptide (SEQ ID NO: 1) on serum ALT levels of liver fibrosis mice.

[0023] Figure 2 Figure 2: Effect of polypeptide (SEQ ID NO: 1) on serum AST levels of liver fibrosis mice.

[0024] Figure 3 Figure 3: HE staining results of liver tissue of mice in each group.

[0025] Figure 4 Figure 4: Polypeptide cell compatibility test results.

[0026] Figure 5 Figure 5: HE staining results of multi-organ tissue of mice in each group (heart, spleen, lung, kidney). DETAILED DESCRIPTION

[0027] The present application provides a polypeptide for treating liver fibrosis, which is specifically referred to the nucleotide sequence table (SEQ ID NO: 1), and the amino acid sequence is as follows: YGRKKRRQRRRFSLDKIYLIGGDLGPFNPGLPVEVPLWLAI. The polypeptide can be synthesized by referring to the conventional preparation method in the prior art, such as the commonly used Fmoc solid-phase synthesis method. The polypeptide can be used for preparing a medicine for treating liver fibrosis, and the medicine comprises the polypeptide and a pharmaceutically acceptable excipient, and the optional medicine dosage form includes but is not limited to injection preparations and oral preparations. The preparation and properties of the polypeptide will be specifically described hereinafter through specific examples.

[0028] Example 1: Preparation and identification of polypeptide

[0029] The target polypeptide (SEQ ID NO: 1) is prepared by using the solid-phase synthesis method, and the detailed synthesis steps are as follows:

[0030] 1) Resin swelling

[0031] Weigh 200 mg of resin, add 5 mL of dichloromethane (DCM) to swell for 10 min, and discard the solution. Repeat the washing with dichloromethane (DCM) twice and N,N-dimethylformamide (DMF) three times to ensure the resin is fully swelled.

[0032] 2) Removal of fluorene methoxycarbonyl (Fmoc) protection

[0033] Add 5 mL of 20% piperidine / N,N-dimethylformamide (DMF) solution and react for 5 min. Discard the solution and add another 5 mL of the same solution, reacting for 10 min. Wash 3 times with N,N-dimethylformamide (DMF) and 2 times with dichloromethane (DCM). Confirm the free amine using the Kaiser Test.

[0034] 3) Amino acid coupling

[0035] Add fluorenemethyloxycarbonyl-protected amino acid (Fmoc-AA) (4 eq, 0.4 mmol), condensing agent (HATU) (4 eq, 0.4 mmol), and N,N-diisopropylethylamine (DIPEA) (8 eq, 0.8 mmol) to each step. Dissolve in 3-4 mL of N,N-dimethylformamide (DMF). React at room temperature for 45-60 min, gently shaking. After the reaction, wash 3 times with N,N-dimethylformamide (DMF) and 2 times with dichloromethane (DCM). Detect with Kaiser's reagent; if positive (blue), repeat the coupling step.

[0036] The Fmoc-AA used corresponds to the sequence of the target polypeptide, including: Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, etc.

[0037] 4) Repeat steps 2) and 3) until the last amino acid at the N-terminus is complete.

[0038] 5) After the final removal of the fluorene methyloxycarbonyl (Fmoc) group, thoroughly clean the resin and prepare for cutting.

[0039] 6) Cutting and removing protection

[0040] Add a trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water (H2O) (95 / 2.5 / 2.5, 5 mL) cutting reagent and shake at room temperature for 2–3 hours. Filter and collect the filtrate, wash the resin with a small amount of trifluoroacetic acid (TFA) and combine the filtrate. Slowly pour the filtrate into 40 mL of cold diethyl ether to precipitate the crude peptide. Centrifuge or allow to stand, discard the supernatant, and wash 2–3 times with cold diethyl ether. Dry the precipitate under nitrogen (nitrogen blowing) or air drying for later use.

[0041] 7) Crude peptide purification

[0042] The crude peptide was dissolved in a 30–50% acetonitrile (ACN) / 0.1% trifluoroacetic acid (TFA) solution. Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC): mobile phase A = water + 0.1% trifluoroacetic acid (TFA), mobile phase B = acetonitrile (ACN) + 0.1% trifluoroacetic acid (TFA). Gradient: 5% → 60% B, 30–45 min. The target peak was collected and lyophilized.

[0043] 8) Analysis and identification

[0044] Molecular weight was determined using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) or matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF). Purity was assessed by analytical high-performance liquid chromatography (analytical HPLC) (target ≥95%).

[0045] Example 2: Evaluation of peptide biocompatibility

[0046] 2.1 Cell compatibility evaluation

[0047] The effect of peptides on cell viability was detected using the CCK8 assay. The experimental results showed that ( Figure 4 The cell viability of the normal group, drug group, peptide treatment group (-50μM) and peptide treatment group (-100μM) was around 100%, and there was no significant difference among the groups (P>0.05), indicating that the peptide of the present invention has good cell compatibility and no significant inhibitory effect on cell survival.

[0048] 2.2 In vivo biocompatibility evaluation

[0049] The in vivo biocompatibility of the peptides was assessed by observing HE staining of multiple organ tissues in mice. The results showed ( Figure 5 Heart: Myocardial fibers in all groups were relatively well-arranged, with no obvious degeneration or necrosis. Spleen: Splenic corpuscles were clearly structured in all groups, with a clear boundary between the red and white pulp. Lungs: The alveolar structure in the polypeptide treatment group was improved compared to the drug group, and the inflammatory response was reduced. Kidneys: The glomeruli and renal tubules in all groups were intact, with no obvious swelling or necrosis. The results indicate that the polypeptides of this invention have no obvious toxic effects on major organs and have good biocompatibility.

[0050] Example 3: In vivo pharmacodynamic study of the anti-liver fibrosis effect of polypeptides

[0051] 3.1 Laboratory Animals and Grouping

[0052] Forty male C57BL / 6 mice (6-8 weeks old, weighing 20-25g) were selected. The animals were randomly divided into four groups: a blank control group, a model group, a peptide treatment group (50μM), and a peptide treatment group (10 mice per group). The blank control group received saline, the model group underwent induced fibrosis without treatment, and the peptide treatment group received peptides (50μM or 100μM) via intraperitoneal injection.

[0053] 3.2 Establishment of animal models and drug administration

[0054] Animal model establishment and drug administration: A liver fibrosis model was induced using CCl4 (0.5 mL / kg, dissolved in olive oil, twice a week, intraperitoneal injection) for 8 weeks. The peptide treatment group received the drug three times a week for 4 weeks, starting from week 4 of model establishment. The blank control group and the model group received an equal volume of physiological saline.

[0055] 3.3 Indicator Testing and Results

[0056] Mouse serum was collected, and ALT and AST levels were measured using a fully automated biochemical analyzer. Results are as follows: Figure 1 and Figure 2 As shown in the figure, serum ALT and AST levels in the model group were significantly higher than those in the blank control group (P<0.01), indicating that the liver fibrosis model was successfully established. Serum ALT and AST levels in the peptide treatment groups (50 μM and 100 μM) were significantly lower than those in the model group (P<0.05), with the 100 μM peptide group showing a more significant effect, and ALT and AST levels approaching those of the blank control group. This indicates that the peptide can effectively protect hepatocytes and reduce liver damage.

[0057] Liver histopathological examination results ( Figure 3 The morphological changes in liver tissue were observed using HE staining. Results showed that in the normal group, hepatocytes were neatly arranged, structurally intact, and showed no necrosis or inflammation. In the model group, hepatocytes exhibited significant vacuolar degeneration, necrotic foci, and inflammatory cell infiltration. In the polypeptide treatment group (50 μM), hepatocyte damage was less severe than in the model group, vacuolar degeneration was reduced, and the area of ​​necrosis was smaller. In the polypeptide treatment group (100 μM), the hepatocyte structure was closer to that of the normal group, vacuolar degeneration was further reduced, and there was no significant large-scale necrosis, suggesting that polypeptides can effectively block or reverse the process of liver fibrosis.

Claims

1. A polypeptide for treating liver fibrosis, characterized in that, The amino acid sequence of the polypeptide is as follows: YGRKKRRQRRRFSLDKIYLIGGDLGPFNPGLPVEVPLWLAI.

2. A method for synthesizing the polypeptide of claim 1, comprising the following steps: using Fmoc solid-phase synthesis to prepare the polypeptide, wherein the C-terminal amino acid is loaded onto a resin, and then the fluorenylmethyloxycarbonyl protection is removed; from the second amino acid, the amino acids are coupled one by one through Fmoc-SPPS coupling cycles, and finally the polypeptide is obtained by cleaving from the resin.

3. The method of claim 2, wherein: the resin is swelled in DCM for 10 min, and then washed twice with DCM and three times with DMF; the fluorenylmethyloxycarbonyl protection is removed by using 20% piperidine / DMF solution for 2 times, 5 min and 10 min respectively, and then washing three times with DMF and twice with DCM; the coupling method of the amino acid is as follows: after adding the fluorenylmethyloxycarbonyl-protected amino acid, HATU and DIPEA, dissolving in DMF, reacting at room temperature for 45-60 min, and then washing three times with DMF and twice with DCM; and the cleavage method is as follows: after the last fluorenylmethyloxycarbonyl protection, the resin is washed thoroughly, TFA / TIS / H2O cleavage solution is added, and the reaction is carried out at room temperature for 2-3 h, the filtrate is collected by filtration, the resin is washed with TFA, and the filtrate is combined; the combined filtrate is precipitated in cold ether, centrifuged or left to stand, the supernatant is discarded, and the precipitate is washed twice or three times with cold ether, and the precipitate is dried under nitrogen or air.

4. The method of claim 2 or 3, wherein: after cleavage, the precipitate is purified by dissolving the precipitate in 30%-50% acetonitrile / 0.1% trifluoroacetic acid solution, and then using reverse-phase high-performance liquid chromatography for purification, wherein the mobile phase A is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution, the gradient change of the mobile phase B is 5% to 60%, and the time is 30-45 min; and after collecting the target peak, the product is freeze-dried.

5. Use of the polypeptide of claim 1 in the preparation of a medicament for treating liver fibrosis. The medicament comprises the polypeptide and pharmaceutically acceptable excipients. The dosage forms of the medicament include injection preparations and oral preparations. The polypeptide of claim 1 and pharmaceutically acceptable excipients. The dosage forms of the medicament include injection preparations and oral preparations. ​ ​ 6. Use according to claim 5, characterized in that: ​ 7. Use according to claim 5, characterized in that: ​ 8. A medicament for treating liver fibrosis, characterized by: ​ 9. The medicament according to claim 8, characterized in that: ​

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