Use of a polypeptide in the preparation of a medicament for treating or preventing non-alcoholic fatty liver

Abstract

The application provides application of a polypeptide in preparation of a medicine for treating or preventing non-alcoholic fatty liver, wherein the polypeptide comprises 10-22 polypeptide fragments in an amino acid sequence shown in SEQ ID NO. 1, or the polypeptide comprises opposite direction polypeptide fragments consisting of 10-22 consecutive amino acids in the amino acid sequence shown in SEQ ID NO. 1. The polypeptide of the application is derived from a pannexin 1 protein amino acid sequence, has high selectivity to the pannexin 1 protein, can reduce off-target effects, thereby improving the treatment effect on non-alcoholic fatty liver, and can also work in the case of low dosage and has low side effects; the polypeptide of the application only contains 20 amino acid residues, can be chemically synthesized, has no animal source impurities in the production process, and has no substances causing immune reactions, and therefore has high safety; in terms of production cost and therapeutic administration, the polypeptide of the application has more advantages than macromolecular drugs such as proteins or antibodies.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of a polypeptide in the preparation of drugs for the treatment or prevention of non-alcoholic fatty liver disease. Background Technology

[0002] The main characteristic of non-alcoholic fatty liver disease (NAFLD) is the excessive accumulation of lipids, such as triglycerides and cholesterol, in hepatocytes. It is estimated that NAFLD affects approximately 25% of the global population. The prevalence of NAFLD is around 50% in obese individuals, approximately 23% in those with type 2 diabetes, approximately 70% in those with hyperlipidemia, approximately 40% in those with hypertension, and approximately 41% in those with hypertriglyceridemia. NAFLD can easily progress to other liver diseases, such as non-alcoholic steatohepatitis (NASH), hepatocellular carcinoma (HCC), and cirrhosis. Because the pathogenesis of NAFLD is unclear, regulatory agencies have not yet approved specific drugs for its treatment. Therefore, the main treatment currently involves lifestyle modifications, such as dietary adjustments, exercise, and weight loss. Medical guidelines suggest that a weight loss of 3% to 5% can improve NAFLD, while greater weight loss (7% to 10%) is beneficial in improving the histopathological features of NAFLD, including liver fibrosis. Because the risk of death from cardiovascular disease and related illnesses in patients with non-alcoholic fatty liver disease (NAFLD) outweighs the side effects of statins (used to treat atherosclerosis), statins are also used to treat NAFLD when no other effective treatment options are available. However, lipid-lowering drugs are not specific treatments for NAFLD and may cause liver, kidney, and muscle damage. Therefore, it is essential to seek alternative medications to control and treat NAFLD.

[0003] The inventors applied for Chinese patent CN115785228 A in 2022, entitled "Peptide Antagonist Targeting Pannexin1 Channel Protein," which relates to the amino acid sequence of a peptide, its preparation method, and its application in the preparation of drugs for treating stroke, epilepsy, inflammation, chronic pain, cancer, Parkinson's disease, Crohn's disease, ischemia, glaucoma, ulcerative colitis, sepsis, and organ explantation. Building upon these existing achievements, the inventors have obtained new results through further experimental research; this peptide can also be used to treat or prevent non-alcoholic fatty liver disease. Summary of the Invention

[0004] This invention provides the application of a polypeptide in the preparation of drugs for the treatment or prevention of non-alcoholic fatty liver disease. The polypeptide is derived from the amino acid sequence of the pannexin1 protein, has high selectivity for the pannexin1 protein, can reduce off-target effects, thereby improving the therapeutic effect on non-alcoholic fatty liver disease, and is effective even at low doses with low side effects.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned objective is: the application of a polypeptide in the preparation of a drug for treating or preventing non-alcoholic fatty liver disease, wherein the polypeptide comprises a polypeptide fragment consisting of 10-22, preferably 10-20, and most preferably 20 consecutive amino acids from the amino acid sequence shown in SEQ ID NO.1, wherein the amino acid sequence of SEQ ID NO.1 is as follows:

[0006] XFKHLWLPLNGSESQLSNKXXX

[0007] In SEQ ID NO.1, X in the first position is F or does not exist; X in the 20th position is Q or E; X in the 21st position is Q or E; X in the 22nd position is V or does not exist.

[0008] Further, the polypeptide comprises a peptide segment consisting of an amino acid sequence selected from any of the sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0009] Further, the polypeptide consists of the amino acid sequence shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0010] Furthermore, one or more amino acids in the polypeptide may be in D-enantiomer form; preferably, all amino acids in the polypeptide are in D-enantiomer form.

[0011] Furthermore, the polypeptide is further modified.

[0012] Furthermore, the modifications include N-terminal modifications, C-terminal modifications, side chain modifications, amino acid modifications, peptide backbone modifications, and binding to other peptides or proteins.

[0013] Further, the modification includes modification by linking stearic acid, and / or amination modification, preferably, the modification includes amination modification by linking stearic acid to the end of the peptide (e.g., N-terminus), and / or C-terminus.

[0014] Further, the polypeptide comprises a reverse polypeptide fragment consisting of 10-22 consecutive amino acids in the amino acid sequence shown in SEQ ID NO:1, preferably a reverse polypeptide fragment consisting of 10-20 consecutive amino acids, and most preferably a reverse polypeptide fragment consisting of 20 consecutive amino acids.

[0015] This invention discloses a pharmaceutical composition for treating or preventing non-alcoholic fatty liver disease, comprising the aforementioned polypeptide and statin.

[0016] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least:

[0017] First, the polypeptide of this invention is derived from the amino acid sequence of the pannexin1 protein and has high selectivity for the pannexin1 protein, which can reduce off-target effects and minimize adverse effects and side effects on non-target tissues or cells, thereby improving the therapeutic effect on non-alcoholic fatty liver disease. This is demonstrated by its effectiveness even at low doses and low side effects. In addition, the polypeptide of this invention has a different mechanism of action than statins, and can be used in combination with a certain dose of statins to minimize the adverse effects of statins while achieving a synergistic effect.

[0018] Secondly, the polypeptide of the present invention is a short peptide containing only 20 amino acid residues, which can be chemically synthesized. The production process is free of animal-derived impurities and does not contain substances that cause immune responses, thus ensuring high safety. As it is a short peptide, it is easy to modify or alter it through chemical and biological methods to improve its stability and further enhance its drug efficacy. In terms of production cost and therapeutic administration, the polypeptide of the present invention has advantages over large molecule drugs such as proteins or antibodies. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0020] Figure 1 The image shows the serum detection results of the polypeptide of the present invention on mice with non-alcoholic fatty liver disease, where (A) alanine aminotransferase (ALT); (B) aspartate aminotransferase (AST); (C) cholesterol (CHO); (D) triglycerides (TG); and (E) low-density lipoprotein (LDL).

[0021] Figure 2 This is a schematic diagram of the liver of a mouse with non-alcoholic fatty liver disease, where (A) is the blank control group and (B) is the polypeptide treatment group.

[0022] Figure 3 The figure shows the effect of the polypeptide of the present invention on the liver / body weight ratio in mice with non-alcoholic fatty liver disease, where (A) is the body weight of the mouse; and (B) is the liver weight of the mouse.

[0023] Figure 4The images show liver sections stained by non-alcoholic fatty liver disease in mice. (A) HE staining in the blank control group; (B) O-red staining in the blank control group; (C) HE staining in the polypeptide treatment group; and (D) O-red staining in the polypeptide treatment group. Detailed Implementation

[0024] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0025] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0027] Example 1 Synthesis of polypeptides

[0028] The peptide was synthesized by the peptide synthesis company (CASLO ApS, c / o Techbical University Denmark, Diplomvej 381, DK-2800, Kongens Lyngby, Denmark) via solid-phase synthesis. The amino acid sequences of SEQ ID NO.2-SEQ ID NO.8 are as follows:

[0029] SEQ ID NO.2: FFKHLWLPLNGSESQLSNKQQV;

[0030] SEQ ID NO.3: FKHLWLPLNGSESQLSNKQQV;

[0031] SEQ ID NO.4: FKHLWLPLNGSESQLSNKQQ;

[0032] SEQ ID NO.5: FKHLWLPLNGSESQLSNKQE;

[0033] SEQ ID NO.6: FKHLWLPLNGSESQLSNKEE;

[0034] SEQ ID NO.7: FKHLWLPLNG;

[0035] SEQ ID NO.8: WRQAAFVDSY.

[0036] Stearic acid was conjugated to the N-terminus of peptide SEQ ID NO. 5 (FKHLWLPLNGSESQLSNKQE) and then amination was applied to the C-terminus to obtain peptide QE20. The specific steps are as follows: First, the amination peptide was synthesized by solid-phase method. Then, stearic acid was conjugated to the N-terminus of the peptide through chemical reaction. The stearic acid peptide was released from the solid phase by chemical reaction. The synthesized peptide was purified to a purity >95% by reversed-phase chromatography. The purity of the peptide was determined by high-performance liquid chromatography (HPLC), and the molecular weight of the peptide was determined by mass spectrometry (MALDI-TOF).

[0037] Example 2: Effects of peptide QE20 on a mouse model of non-alcoholic fatty liver disease

[0038] Male C57BL / 6J mice aged 6-8 weeks were selected as the research subjects and fed a high-fat, high-cholesterol, high-fructose diet (D09100310, Research Diets) for 12 weeks to establish a non-alcoholic fatty liver disease (NAFLD) mouse model. The NAFLD mice were randomly divided into a blank control group and a peptide treatment group. The blank control group received an intraperitoneal injection of 100 μL of 0.01 M phosphate-buffered saline every 2 days; the peptide treatment group received an intraperitoneal injection of 200 μg of peptide QE20 every 2 days for 12 weeks. After fasting for 12 hours, blood was collected from the orbital sinus to detect serum ALT (alanine aminotransferase), AST (aspartate aminotransferase), CH3O (cholesterol), TG (triglycerides), and LDL (low-density lipoprotein). The liver was quickly removed and weighed after blood collection to calculate the liver / body weight ratio. Hematoxylin and eosin (HE) staining and O-red staining were used to observe liver morphology and the degree of lipid accumulation in the liver, assessing the effect of the peptide on the liver.

[0039] Depend on Figure 1It was found that the levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), CHO (cholesterol), TG (triglycerides), and LDL (low-density lipoprotein) in the serum of mice treated with peptides were significantly lower than those in the blank control group, showing significant differences. As shown in Figure 3, peptide intervention significantly reduced the liver / body weight ratio in mice with non-alcoholic fatty liver disease, suggesting a reduction in lipid accumulation in the liver. Figure 4 HE staining images showed that the number of vacuolated structures in the liver tissue of mice treated with peptides was reduced, indicating that there were fewer lipid droplets inside the cells. Figure 4 O-red staining revealed a significant reduction in both the number and size of red lipid droplets in the mouse liver. Based on the above analysis, the polypeptide of this invention has a significant effect in treating non-alcoholic fatty liver disease.

[0040] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of a polypeptide in the preparation of a drug for treating non-alcoholic fatty liver disease, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

5.

2. The application as described in claim 1, characterized in that, The polypeptide is further modified, including modification via stearic acid linking to the N-terminus of the polypeptide and / or C-terminal amination modification.

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