Therapeutic uses of triple agonist having activity for all of glucagon, GLP-1 and GIP receptors, or conjugates thereof, for liver disease

Abstract

To provide a pharmaceutical composition for the prevention or treatment of liver disease, which contains a peptide that has activity for a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor and a glucose-dependent insulinotropic polypeptide (GIP) receptor, or a conjugate thereof.SOLUTION: The present invention relates to therapeutic uses of a triple agonist having activity for all of glucagon, GLP-1 and GIP receptors, and long-acting conjugates thereof, for liver disease.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the therapeutic use of a triple active agent or a conjugate thereof having activity at all of the glucagon, GLP-1 and GIP receptors for treating liver disease. [Background technology]

[0002] The liver is one of the main organs of the body in animals, and representative liver-related diseases include non-alcoholic fatty liver, hepatitis, hepatic fibrosis, cholestatic liver disease, cirrhosis, liver failure, liver cancer, etc. It is known that inflammation of the liver occurs due to viruses, alcohol, drugs, immune disorders, metabolic diseases, etc., and the progression and chronicization of liver inflammation leads to diseases such as liver fibrosis, cirrhosis, and liver cancer.

[0003] In general, hepatitis, which causes inflammation of the liver, accounts for the majority of liver diseases, and it is known that as liver inflammation progresses, various liver diseases (liver fibrosis, cirrhosis, etc.) that are accompanied by or caused by liver inflammation develop. Depending on the state of liver inflammation, it is classified as acute hepatitis or chronic hepatitis, and depending on the cause, it is classified as viral hepatitis, alcoholic hepatitis, drug-induced hepatitis, etc. Cholestasis liver disease is also presumed to be caused by inflammatory diseases.

[0004] Representative examples of liver diseases include metabolic liver diseases such as fatty liver, non-alcoholic fatty liver disease, and steatohepatitis, as well as liver fibrosis, liver cirrhosis, liver failure, and liver cancer. These liver diseases are not noticeable in the early stages and are only discovered after they have progressed significantly, making them the leading cause of death not only in Korea but also around the world, and there is a strong demand for the development of drugs to treat them.

[0005] Since liver fibrosis is the result of the damage recovery process against repeated liver damage, it will recover normally when the cause of liver damage disappears, but if the liver fibrosis process continues repeatedly and liver fibrosis progresses, liver cirrhosis occurs. Pathologically, liver cirrhosis is a chronic disease accompanied by necrosis, inflammation, and fibrosis of liver cells, and ultimately progresses to liver cirrhosis complications such as liver failure (liver decompensation), liver cancer, and other diseases, leading to death. In particular, since there are no subjective symptoms in the early stages and it is discovered after the disease has progressed considerably, research is being actively conducted to develop a method for quickly treating liver fibrosis, which is a state before it progresses to liver cirrhosis. In recent years, Dr. Knos' team has reported a drug that is a chemical improvement of ibipinapant, a type of cannabinoid type 1 (CB1) receptor antagonist that penetrates the brain, but its actual effect as a drug is still unknown (Non-Patent Document 1). Therefore, there is still a need to develop drugs that can treat fibrosis of various tissues or liver fibrosis while ensuring patient convenience and without side effects.

[0006] Nonalcoholic fatty liver disease (NAFLD), a metabolic liver disease, is a type of disease that shows tissue findings similar to alcoholic hepatitis, even though it is not related to alcohol intake, and includes simple steatosis, nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), etc. NAFLD tends to increase as the population of obesity and diabetes increases, and the annual incidence rate in Korea has reached about 16%.

[0007] In order to prevent and / or treat such non-alcoholic fatty liver disease, many efforts have been made to improve insulin resistance. For example, clinical trials of TZDs (thiazolidinedinones), which are a type of insulin sensitizer, and metformin are currently being actively conducted (Non-Patent Document 2).

[0008] However, it is known that the treatment using TZD drugs has the disadvantages of causing a large weight gain and slowing down the flow of body fluids, and therefore cannot be applied to patients with heart disease. As can be seen from these results, it is known in the art that drugs known to be effective in the treatment of diabetes, such as insulin sensitizers, can cause problems such as side effects when used as a treatment for non-alcoholic fatty liver disease.

[0009] On the other hand, macrophages are known to play an important role in immune responses in the liver and to be involved in non-alcoholic fatty liver disease, including non-alcoholic fatty liver disease (Non-Patent Document 3). Specifically, macrophages are activated in patients with non-alcoholic fatty liver disease, and it is known that drugs targeting macrophages suppress inflammation and fibrosis in the liver and are effective in treating non-alcoholic fatty liver disease.

[0010] GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insuliontropic polypeptide) are representative gastrointestinal hormones and, as neurohormones, are substances involved in the regulation of blood sugar levels through food intake. Glucagon is a peptide hormone secreted from the pancreas, and, together with the two aforementioned substances, is involved in the regulation of blood sugar levels.

[0011] GLP-1 is a hormone secreted from the small intestine in response to food intake, and promotes insulin secretion from the pancreas in a blood glucose concentration-dependent manner and inhibits glucagon secretion, thereby lowering blood glucose concentration. It also acts as a satiety factor, slowing gastrointestinal digestion and slowing the gastrointestinal transit time of food digests, thereby reducing food intake. It has also been reported that administration of GLP-1 to mice suppresses food intake and reduces body weight, and it has been confirmed that these effects are similar in both normal and obese states, suggesting its potential as a therapeutic agent for obesity.

[0012] GIP, one of the gastrointestinal hormones that is secreted in response to food intake along with GLP-1, is a hormone composed of 42 amino acids secreted from K cells in the small intestine. It lowers blood glucose levels by promoting insulin secretion from the pancreas in a blood glucose concentration-dependent manner, and has also been reported to have the effect of increasing GLP-1 activity.

[0013] Glucagon is produced by the pancreas when blood glucose levels begin to drop due to drug treatment, disease, hormone or enzyme deficiency, etc. Glucagon acts to raise blood glucose levels to normal levels by sending a signal to the liver to break down glycogen and release glucose. In addition to its blood glucose-raising effect, glucagon has been reported to have anti-obesity effects in animals and humans by suppressing appetite, activating hormone sensitive lipase in adipocytes to promote lipolysis, and promoting energy expenditure. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 97 / 034631 [Patent Document 2] International Publication No. 96 / 032478 [Non-patent literature]

[0015] [Non-Patent Document 1] JCI Insight. 2016;1(11):e87336.doi:10.1172 / jci.insight.87336 [Non-Patent Document 2] Hepatology(2003) 38:1008-17, J Clin Invest. (2001) 108:1167-74 [Non-Patent Document 3] Nat Rev Gastroenterol Hepatol. 2019 Mar;16(3):145-159. [Non-Patent Document 4] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 [Non-Patent Document 5] GJ Webb et al, J. Autoimmunity, 2015 Nov; 64: 42-52 Summary of the Invention [Problem to be solved by the invention]

[0016] An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of liver diseases, which comprises a peptide having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insuliontropic polypeptide (GIP) receptor, or a conjugate thereof.

[0017] Another object of the present invention is to provide a method for preventing or treating liver disease, comprising the step of administering the peptide or a composition containing it to an individual in need thereof.

[0018] A further object of the present invention is to provide use of the peptide or a composition containing the same in the manufacture of a medicament for the prevention or treatment of liver disease. [Means for solving the problem]

[0019] One embodiment of the present invention is a pharmaceutical composition for preventing or treating liver diseases, comprising a peptide or a conjugate thereof having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insuliontropic polypeptide (GIP) receptor.

[0020] In one specific example, the present invention provides a pharmaceutical composition for preventing or treating a liver disease, comprising a pharma- ceutical acceptable excipient and a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 102 in a pharma- ceutical effective amount.

[0021] In another embodiment, the peptide is in the form of a persistent conjugate, the persistent conjugate being characterized by being represented by chemical formula (1).

[0022] XLF···(1)

[0023] Here, X is a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 102, L is a linker containing a repeating ethylene glycol unit, F is an immunoglobulin Fc fragment or a derivative thereof, and - indicates a covalent bond between X and L and between L and F.

[0024] In a composition according to any of the previous embodiments, the peptide is characterized in that its C-terminus is amidated.

[0025] The composition according to any of the previous embodiments, wherein the liver disease is liver inflammation.

[0026] In a composition according to any of the above-mentioned embodiments, the pharmaceutical composition is characterized in that, upon administration, it reduces the expression of at least one of TNF-α, MCP-1, and IL-6 in liver tissue.

[0027] The composition according to any of the previous embodiments, wherein the liver disease is a metabolic liver disease.

[0028] A composition according to any of the previous embodiments, wherein the pharmaceutical composition is characterized in that, upon administration, it reduces triglyceride and / or cholesterol levels in liver tissue.

[0029] In the composition according to any of the above-mentioned specific examples, the liver disease is characterized in that it is at least one disease selected from the group consisting of simple fatty liver, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer.

[0030] In the composition according to any of the above-mentioned embodiments, the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

[0031] In a composition according to any of the previous embodiments, the liver disease is characterized as being non-alcoholic steatohepatitis accompanied by fatty liver, liver fibrosis or cirrhosis.

[0032] In the composition according to any of the above embodiments, the liver disease is liver cancer due to non-alcoholic steatohepatitis.

[0033] In the composition according to any of the above-mentioned embodiments, the liver disease is characterized in that it is at least one disease selected from the group consisting of simple fatty liver, non-alcoholic fatty liver and cirrhosis of the liver.

[0034] In the composition according to any of the above embodiments, the liver disease is characterized in that it is at least one disease selected from the group consisting of liver inflammation, non-alcoholic steatohepatitis and liver fibrosis.

[0035] In a composition according to any of the above-mentioned embodiments, the liver disease is liver fibrosis, and the pharmaceutical composition is characterized in that, upon administration, it reduces blood levels of TIMP-1 and / or hyaluronic acid in the individual to which it is administered.

[0036] In a composition according to any of the previous embodiments, the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

[0037] In a composition according to any of the previous embodiments, the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97 and 100.

[0038] In a composition according to any of the previous embodiments, the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96.

[0039] In the composition according to any of the above-mentioned specific examples, the chemical formula weight of the repeating unit portion of ethylene glycol in L is in the range of 1 to 100 kDa.

[0040] Another embodiment of the invention is a method for preventing or treating liver disease, comprising the step of administering said peptide or a composition comprising it to an individual in need thereof.

[0041] Yet another embodiment of the invention is the use of said peptide or a composition comprising it in the manufacture of a medicament for the prevention or treatment of liver diseases.

[0042] Yet another embodiment of the present invention is the use of said peptide or a composition comprising it for the prevention or treatment of liver diseases. Effect of the Invention

[0043] The triple activity entity or its conjugate according to the present invention has application in the prevention or treatment of liver diseases. [Brief description of the drawings]

[0044] [Figure 1] FIG. 1 shows the change in NAS score in mice administered a long-acting conjugate of SEQ ID NO: 42 once every two days for 28 days in a NASH mouse model induced by feeding an MCD diet (p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Diagram 2] FIG. 1 shows the results of confirming the fatty liver improving effect of a long-acting conjugate of SEQ ID NO: 42 in mice with AMLN diet-induced steatohepatitis. [Diagram 3] FIG. 1 shows the results of confirming the effect of a long-acting conjugate of SEQ ID NO: 42 in reducing the steatosis score in mice with AMLN diet-induced steatohepatitis. [Figure 4] FIG. 1 shows the change in ELF score by administration of a long-acting conjugate of SEQ ID NO: 42 in a mouse model of liver fibrosis induced by TAA administration (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Diagram 5]FIG. 1 shows changes in the positive area of ​​sirius red staining in liver tissue by administration of a long-acting conjugate of sequence number 42 in a mouse model of hepatic fibrosis induced by TAA administration (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 6] FIG. 1 shows changes in blood concentrations of liver fibrosis markers following administration of a long-acting conjugate of SEQ ID NO:42 in a mouse model of liver fibrosis induced by BDL (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA, †††p<0.01 vs. obeticholic acid by unpaired t-test). [Figure 7a] FIG. 1 shows the results of sirius red staining following administration of a long-acting conjugate of SEQ ID NO: 42 in a mouse model of liver fibrosis induced by BDL. [Figure 7b] FIG. 1 shows the fibrosis scores of liver tissue following administration of a long-acting conjugate of SEQ ID NO: 42 in a mouse model of liver fibrosis induced by BDL (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 8] FIG. 1 shows changes in H&E staining and inflammation score of liver tissue following administration of a long-acting conjugate of sequence number 42 in a PBC mouse model (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 9] FIG. 1 shows changes in H&E staining and parenchmal necrosis score of liver tissue following administration of a long-acting conjugate of SEQ ID NO:42 in a PSC mouse model (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 10]FIG. 1 shows changes in bile duct hyperplasia score following administration of a long-acting conjugate of SEQ ID NO: 42 in a PSC mouse model (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 11] FIG. 1 shows changes in expression of inflammation-related cytokines in liver tissue following administration of a long-acting conjugate of SEQ ID NO: 42 (*p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA). [Figure 12] FIG. 1 shows the results of confirming the effect of triple activators of SEQ ID NOs: 42, 66, 67, 97 and 100 in reducing human tumor necrosis factor α (TNF-α) in a human macrophage line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention will now be described in more detail.

[0046] In addition, each description and embodiment disclosed in the present invention is applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0047] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid Asp,D Cysteine ​​Cys,C Glutamic acid Glu,E Glutamine Gln,Q Glycine Gly,G Histidine Isoleucine Ile,I Leucine Leu,L Lysine Lys,K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin,V

[0048] One embodiment of the present invention is a pharmaceutical composition for preventing or treating a liver disease, comprising a peptide having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insuliontropic polypeptide (GIP) receptor, specifically a peptide having an amino acid sequence set forth in any of SEQ ID NOs: 1 to 102.

[0049] The above-mentioned "peptides having activity on the glucagon receptor, the GLP-1 receptor and the GIP receptor" are used interchangeably with triple active substances in the present invention.

[0050] Such peptides include various substances, eg, various peptides, that have significant levels of activity at the glucagon, GLP-1 and GIP receptors.

[0051] The triple active substance having a significant level of activity against glucagon, GLP-1 and GIP receptors has in vitro activity against one or more of glucagon, GLP-1 and GIP receptors, specifically two or more of the receptors, more specifically all three receptors, which is about 0.001% or more, about 0.01% or more, about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, but is not particularly limited thereto as long as it is significantly increased, compared to the natural ligands of the receptors (native glucagon, native GLP-1 and native GIP).

[0052] Here, examples of the activity against the receptor include, but are not limited to, in vitro activity against the receptor of 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or about 200% or more, compared to the natural receptor.

[0053] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0054] A method for measuring the in vitro activity of such a triple-active substance is shown in Example 1 of the present specification, but is not particularly limited thereto.

[0055] On the other hand, the peptide is characterized in that it has one or more, two or more, particularly three activities of the following i) to iii), specifically, that it has significant activities. i) Activation of GLP-1 receptors ii) Activation of glucagon receptors iii) Activation of GIP receptors

[0056] Here, activating a receptor means increasing the in vitro activity of the receptor to about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more, compared to the natural receptor, but is not limited thereto.

[0057] Furthermore, the peptide has a longer half-life in the body than any one of native GLP-1, native glucagon, and native GIP, but is not particularly limited thereto.

[0058] Such peptides may be non-naturally occurring, but are not particularly limited thereto.

[0059] The peptide may be, but is not limited to, an analog of native glucagon. Specifically, the analog of native glucagon includes a peptide having at least one difference in amino acid sequence compared to native glucagon, a peptide modified by modifying the native glucagon sequence, and a mimic of native glucagon.

[0060] On the other hand, native glucagon has the following amino acid sequence, but is not particularly limited thereto. His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 118)

[0061] Specifically, the peptide may be, but is not limited to, an analog of native glucagon in which an alteration selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in the native glucagon sequence has been made.

[0062] Furthermore, the amino acid substitution includes all substitutions with amino acids and substitutions with non-natural compounds.

[0063] Furthermore, the addition may be at the N-terminus and / or C-terminus of the peptide. The length of the added amino acids may be, but is not limited to, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids, and in a broad sense includes, but is not limited to, the addition of a polypeptide.

[0064] More specifically, the peptide is selected from the group consisting of positions 1, 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 of the amino acid sequence of native glucagon, It may have 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 amino acids substituted with other amino acids, or may have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more amino acids added independently or in addition to the C-terminus, but is not limited to these.

[0065] More specifically, the peptide is selected from the group consisting of 1, 2, 3, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 amino acids in the amino acid sequence of native glucagon. , 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, or 19 amino acids may be substituted with other amino acids, or 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids may be added independently or additionally to the C-terminus, but is not limited to these.

[0066] More specifically, the peptide may be one in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or 17 amino acids selected from the group consisting of 1, 2, 3, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 28, and 29 in the amino acid sequence of native glucagon have been substituted with other amino acids, or one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids have been added to the C-terminus, independently or additionally, but is not limited to these.

[0067] More specifically, the peptide may be one in which 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or 14 amino acids selected from the group consisting of 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the amino acid sequence of native glucagon have been substituted with other amino acids, or one or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acids have been added independently or additionally to the C-terminus, but is not limited thereto.

[0068] As described above, the amino acid to be introduced may be selected from the group consisting of tyrosine, α-methylglutamic acid, Aib, methionine, glutamic acid, histidine, lysine, leucine, isoleucine, glutamine, valine, glycine, alanine, cysteine, serine, alanine, aspartic acid, and arginine, but is not limited thereto.

[0069] For example, as described above, the added amino acid sequence may be one or more amino acid sequences derived from the amino acid sequence of native GLP-1, native GIP, or native exendin-4.

[0070] Such a peptide may contain an intramolecular bridge (e.g., a covalent or non-covalent bridge), and specifically may have a ring-containing form, for example, a ring formed between the 16th and 20th amino acids of the peptide, but is not limited thereto.

[0071] Examples of such rings include, but are not limited to, lactam bridges (or lactam rings).

[0072] The peptides also include all those that have been modified to contain a ring by including amino acids that form a ring at a desired position.

[0073] For example, the 16th and 20th amino acid pairs of the peptide may be substituted with glutamic acid or lysine, respectively, which form a ring, but the present invention is not limited thereto.

[0074] Such a ring may be formed between the side chains of amino acids in the peptide, for example, in the form of a lactam ring formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0075] Examples of peptides produced by combining the above-mentioned methods include, but are not limited to, peptides that have at least one amino acid sequence different from that of native glucagon, have the alpha carbon of the N-terminal amino acid residue removed, and have activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor. Peptides used in the present invention can be produced by combining various methods for producing analogs.

[0076] In addition, in the peptide of the present invention, some of the amino acids are substituted with other amino acids or non-natural compounds in order to avoid the recognition action of active form degrading enzymes and extend the half-life in the body, but this is not particularly limited to these.

[0077] Specifically, the peptide may have an extended half-life in the body due to a substitution of the second amino acid in the amino acid sequence of the peptide, thereby avoiding the recognition action of degradative enzymes, but any amino acid substitution or modification for avoiding the recognition action of degradative enzymes in the body may be used.

[0078] Furthermore, such modifications for the production of peptides include modifications using L- or D-amino acids and / or non-natural amino acids, and / or modifications of the native sequence, such as modifications of side chain functional groups, covalent bonds within the molecule, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, and other modifications.

[0079] Further included are all those in which at least one amino acid has been added to the N- and / or C-terminus of native glucagon.

[0080] The substituted or added amino acids can be any of the 20 amino acids commonly found in human proteins, as well as unusual or non-naturally occurring amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides and typical peptide sequences containing these amino acids can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0081] Amino acid derivatives can also be obtained in the same manner, one example being 4-imidazoacetic acid.

[0082] In addition, the peptide according to the present invention may be in a form in which the N-terminus and / or C-terminus, etc. are chemically modified, protected with an organic group, or modified by adding an amino acid to the peptide terminus, etc., in order to protect it from in vivo protease and improve its stability.

[0083] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, so that in order to remove the charge, acetylation of the N-terminus and / or amidation of the C-terminus may be performed, but is not limited to these.

[0084] The peptide according to the present invention includes the peptide itself, a salt thereof (e.g., a pharma- ceutically acceptable salt of the peptide), or a solvate thereof. Furthermore, the peptide may be in any form as long as it is pharma- ceutically acceptable.

[0085] The type of the salt is not particularly limited, although it is preferable that the salt is in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0086] The term "pharmacologically acceptable" refers to a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reactions, and the like, within the scope of medical judgment.

[0087] The "pharmaceutically acceptable salt" in the present invention includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Suitable salts derived from bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0088] In addition, the term "solvate" in the present invention means a complex formed between the peptide or a salt thereof according to the present invention and a solvent molecule.

[0089] Another example of the peptide is a peptide having an amino acid sequence represented by general formula 1.

[0090] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103)

[0091] In the general formula 1, Xaa1 is histidine, 4-imidazoacetyl or tyrosine, Xaa2 is glycine, α-methylglutamic acid or Aib, Xaa3 is glutamic acid or glutamine, Xaa7 is threonine or isoleucine, Xaa10 is leucine, tyrosine, lysine, cysteine ​​or valine, Xaa12 is lysine, serine or isoleucine, Xaa13 is glutamine, tyrosine, alanine or cysteine, and Xaa14 is leucine. Xaa15 is cysteine, aspartic acid, glutamic acid, or leucine; Xaa16 is glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine; Xaa18 is alanine, glutamine, arginine, or histidine; Xaa19 is alanine, glutamine, cysteine, or valine; and Xaa20 is lysine, glutamine, or arginine. Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid, Xaa23 is isoleucine or valine, Xaa24 is alanine, glutamine, cysteine, asparagine, aspartic acid, or glutamic acid, Xaa27 is valine, leucine, or lysine, Xaa28 is cysteine, lysine, alanine, asparagine, or aspartic acid, and Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or hyaluronan. and Xaa30 is cysteine, glycine, lysine or histidine or is absent, and R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107) or m-SSGQPPPS-n (SEQ ID NO: 108) or is absent, where m is -Cys-, -Pro- or -Gly-Pro- and n is -Cys-, -Gly-, -Ser- or -His-Gly- or is absent.

[0092] Examples of the triple activity include those containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 13 to 102, and those consisting of an amino acid sequence (essential) selected from the group consisting of SEQ ID NOs: 1 to 11, 13 to 102, but are not limited thereto.

[0093] Furthermore, even if the present invention describes a peptide as being "represented" by a particular sequence number, this does not exclude meaningless sequence additions, naturally occurring mutations, or silent mutations before or after the amino acid sequence of the sequence number, so long as the peptide has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, and it goes without saying that those having such sequence additions or mutations are also included in the present invention.

[0094] The above also applies to other embodiments or aspects of the present invention, but is not limited thereto.

[0095] Specifically, in general formula 1, Xaa14 is leucine or methionine, and Xaa15 is cysteine, aspartic acid, or leucine.

[0096] Examples of such peptides include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 11, 14 to 17, and 21 to 102, and peptides consisting of said amino acid sequences (essential), but are not particularly limited to these.

[0097] Such a peptide can significantly activate at least one of glucagon receptor, GLP-1 receptor, and GIP receptor, but is not particularly limited thereto. Specifically, it is a peptide that significantly activates GLP-1, or further, a peptide that significantly activates glucagon receptor and / or GIP receptor, but is not particularly limited thereto.

[0098] More specifically, in the general formula 1, Xaa2 is glycine, α-methylglutamic acid, or Aib, Xaa7 is threonine, Xaa10 is tyrosine, cysteine, or valine, Xaa12 is lysine or isoleucine, Xaa13 is tyrosine, alanine, glutamine, or cysteine, Xaa14 is leucine, cysteine, or methionine, Xaa15 is cysteine, leucine, glutamic acid, or aspartic acid, Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic acid, or lysine, and Xaa18 is glycine, arginine, isoleucine, cysteine, glutamic acid, or lysine. The peptide may be, but is not limited to, a peptide in which aa18 is alanine, glutamine, arginine, or histidine, Xaa19 is alanine, glutamine, valine, or cysteine, Xaa20 is lysine, arginine, or glutamine, Xaa21 is glutamic acid, glutamine, leucine, cysteine, or aspartic acid, Xaa23 is isoleucine or valine, Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic acid, or aspartic acid, and Xaa27 is leucine or lysine.

[0099] More specifically, in the general formula 1, Xaa2 is glycine, α-methylglutamic acid, or Aib, Xaa7 is threonine, Xaa10 is tyrosine, cysteine, or valine, Xaa12 is lysine or isoleucine, Xaa13 is tyrosine, alanine, or cysteine, Xaa14 is leucine or methionine, Xaa15 is cysteine ​​or aspartic acid, and Xaa17 is glutamine, arginine, isoleucine, cysteine, or is lysine, Xaa18 is alanine, arginine or histidine, Xaa19 is alanine, glutamine or cysteine, Xaa20 is lysine or glutamine, Xaa21 is glutamic acid, cysteine ​​or aspartic acid, Xaa23 is valine, Xaa24 is alanine, glutamine, cysteine, asparagine or aspartic acid, and Xaa27 may be leucine or lysine, but is not limited to these.

[0100] More specifically, in general formula 1, Xaa2 is α-methylglutamic acid or Aib, Xaa7 is threonine, Xaa10 is tyrosine or cysteine, Xaa12 is lysine or isoleucine, Xaa13 is tyrosine, alanine or cysteine, Xaa14 is leucine or methionine, Xaa15 is cysteine ​​or aspartic acid, Xaa16 is glutamic acid, and Xaa17 is arginine, isoleucine, cysteine, ... Xaa21 may be a glutamic acid or aspartic acid, Xaa23 is valine, Xaa24 is glutamine, asparagine or aspartic acid, Xaa27 is leucine, and Xaa28 is cysteine, alanine, asparagine, or aspartic acid.

[0101] Specifically, in general formula 1, Xaa1 is histidine or 4-imidazoacetyl, Xaa2 is α-methylglutamic acid or Aib, Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, Xaa12 is isoleucine, Xaa13 is alanine or cysteine, Xaa14 is methionine, Xaa15 is aspartic acid, Xaa16 is glutamic acid, and Xaa17 is isoleucine. Xaa18 is alanine or histidine, Xaa19 is glutamine or cysteine, Xaa20 is lysine, Xaa21 is aspartic acid, Xaa23 is valine, Xaa24 is asparagine, Xaa27 is leucine, Xaa28 is alanine or asparagine, Xaa29 is glutamine or threonine, and Xaa30 is cysteine ​​or lysine or may be absent.

[0102] More specifically, in general formula 1, Xaa2 is glycine, α-methylglutamic acid, or Aib, Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, cysteine, or valine, Xaa12 is lysine, Xaa13 is tyrosine, Xaa14 is leucine, Xaa15 is aspartic acid, Xaa16 is glycine, glutamic acid, or serine, and Xaa17 is glutamine, arginine, cysteine, or lysine. , Xaa18 is alanine, arginine or histidine, Xaa19 is alanine or glutamine, Xaa20 is lysine or glutamine, Xaa21 is glutamic acid, cysteine ​​or aspartic acid, Xaa23 is valine, Xaa24 is alanine, glutamine or cysteine, Xaa27 is leucine or lysine, and Xaa29 may be glycine, glutamine, threonine or histidine, but is not limited to these.

[0103] Such a peptide may be one that activates the GLP-1 receptor and the glucagon receptor to a significantly higher degree than that of the GIP receptor, one that activates the GLP-1 receptor, the glucagon receptor and the GIP receptor to a significantly higher degree than that of the GLP-1 receptor, the glucagon receptor and the GIP receptor, or one that activates the GLP-1 receptor and the GIP receptor to a significantly higher degree than that of the glucagon receptor, but is not limited thereto.

[0104] Examples of such peptides include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 21 to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88, 89, 91 to 93, and 95 to 102, and peptides consisting of said amino acid sequences (essential), but are not limited to these.

[0105] In a specific embodiment, the peptide may comprise an amino acid sequence represented by general formula 2.

[0106] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa2 1-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO: 104)

[0107] In the above formula, Xaa1 is 4-imidazoacetyl, histidine or tyrosine, Xaa2 is glycine, α-methylglutamic acid or Aib, Xaa10 is tyrosine or cysteine, Xaa13 is alanine, glutamine, tyrosine or cysteine, Xaa14 is leucine, methionine or tyrosine, Xaa15 is aspartic acid, glutamic acid or leucine, Xaa16 is glycine, glutamic acid or serine, Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine ​​or lysine, Xaa18 is alanine, glutamine, arginine or histidine, Xaa19 is alanine, Xaa20 is lysine, glutamine or arginine, Xaa21 is cysteine, glutamic acid, glutamine, leucine or aspartic acid, Xaa23 is isoleucine or valine, Xaa24 is cysteine, alanine, glutamine, asparagine or glutamic acid, Xaa28 is lysine, cysteine, asparagine or aspartic acid, Xaa29 is glycine, glutamine, cysteine ​​or histidine, Xaa30 is cysteine, glycine, lysine or histidine, Xaa31 is proline or cysteine, and Xaa40 is cysteine ​​or absent.

[0108] More specifically, in general formula 2, Xaa13 may be alanine, tyrosine or cysteine, Xaa15 may be aspartic acid or glutamic acid, Xaa17 may be glutamine, arginine, cysteine ​​or lysine, Xaa18 may be alanine, arginine or histidine, Xaa21 may be cysteine, glutamic acid, glutamine or aspartic acid, Xaa23 may be isoleucine or valine, Xaa24 may be cysteine, glutamine or asparagine, Xaa28 may be cysteine, asparagine or aspartic acid, Xaa29 may be glutamine, cysteine ​​or histidine, and Xaa30 may be cysteine, lysine or histidine.

[0109] Examples of such peptides include, but are not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 95 to 102, more specifically, a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 96 to 102, and a peptide consisting of said amino acid sequence (essential).

[0110] In a specific embodiment, the peptide may comprise an amino acid sequence of general formula 3.

[0111] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Ph e-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 3, sequence number 105)

[0112] In general formula 3, Xaa1 is histidine or tyrosine, Xaa2 is α-methylglutamic acid or Aib, Xaa13 is alanine, tyrosine or cysteine, Xaa17 is arginine, cysteine ​​or lysine, Xaa18 is alanine or arginine, Xaa19 is alanine or cysteine, Xaa21 is glutamic acid or aspartic acid, Xaa24 is glutamine or asparagine, Xaa28 is cysteine ​​or aspartic acid, Xaa29 is cysteine, histidine or glutamine, Xaa30 is cysteine ​​or histidine, Xaa31 is proline or cysteine, and Xaa40 is cysteine ​​or may be absent.

[0113] Examples of such peptides include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 71, 75 to 77, and 96 to 102, and peptides consisting of said amino acid sequences (essential), but are not particularly limited to these.

[0114] In addition, in general formula 1, R1 is cysteine, GKKNDWKHNIT (sequence number 106), CSSGQPPPS (sequence number 109), GPSSGAPPPS (sequence number 110), GPSSGAPPPSC (sequence number 111), PSSGAPPPS (sequence number 112), PSSGAPPPSG (sequence number 113), PSSGAPPPSHG (sequence number 114), PSSGAPPPSS (sequence number 115), PSSGQPPPS (sequence number 116) or PSSGQPPPSC (sequence number 117), or is absent, but is not limited to these.

[0115] Furthermore, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0116] Specifically, the peptides of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other method known in the art. Thus, the peptides of the present invention can be synthesized in a number of ways, including, for example, (a) synthesizing the peptide stepwise in solid or liquid phase methods or by fragment assembly, and isolating and purifying the final peptide product, (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture, (c) expressing a nucleic acid construct encoding the peptide in a cell-free test tube and recovering the expression product, or recovering fragments of the peptide by any combination of (a), (b), and (c), and then ligating the fragments to obtain the peptide, thereby recovering the peptide.

[0117] Specifically, the composition according to the present invention may be a pharmaceutical composition for preventing or treating a liver disease, which contains a pharma- ceutical composition comprising a pharma- ceutical acceptable excipient and a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102, or a peptide consisting of said amino acid sequence (essential), in a pharma- ceutical effective amount.

[0118] More specifically, the peptide may be, but is not limited to, one that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97, and 100, or one that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97, and 100, or one that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96, or one that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96, but is not limited to these. In addition, in the present invention, the peptide is in the form of a long-acting conjugate, and the long-acting conjugate may be one in which a biocompatible substance for extending the in vivo half-life is bound to a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor. The biocompatible substance of the present invention is mixed with a carrier.

[0119] The peptide conjugate of the present invention has a longer-lasting effect than the peptide not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" in the present invention, which is used interchangeably with "conjugate."

[0120] Furthermore, such conjugates may be non-naturally occurring.

[0121] Specifically, the persistent conjugate is represented by chemical formula (1), but is not limited thereto.

[0122] XLF···(1)

[0123] Here, X is a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102, L is a linker containing a repeating ethylene glycol unit, F is an immunoglobulin Fc fragment or a derivative thereof, and - indicates a covalent bond between X and L and between L and F.

[0124] In the conjugate, F is a substance capable of extending the half-life of X, i.e., a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor, specifically, a peptide having any of the amino acid sequences of SEQ ID NOs: 1 to 102, and is a component constituting a part of the conjugate of the present invention.

[0125] The F's may be bonded to X by a covalent or non-covalent chemical bond, specifically, to X via L by a covalent chemical bond.

[0126] In a specific example, the F is an immunoglobulin Fc fragment or a derivative thereof, and more specifically, the immunoglobulin Fc fragment or a derivative thereof may be derived from IgG, but is not limited thereto.

[0127] In the present invention, the "immunoglobulin Fc fragment" refers to a portion of an immunoglobulin that does not include the heavy and light chain variable regions and that includes the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). The immunoglobulin Fc fragment may be a component that constitutes a part of the conjugate of the present invention.

[0128] In the present invention, the Fc fragment includes not only the native sequence obtained by papain digestion of an immunoglobulin, but also derivatives thereof, such as sequences that differ from the native sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of at least one amino acid residue of the native sequence.

[0129] The F has a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only through a nitrogen atom of one of the two chains, but is not limited thereto. The linkage through the nitrogen atom may be a linkage to the ε-amino atom of lysine or the amino group at the N-terminus by reductive amination.

[0130] Reductive amination refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, which is then reduced to form an amine bond, and is an organic synthesis reaction well known in the art.

[0131] In one embodiment, the F may be linked via the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0132] Such an immunoglobulin Fc fragment may contain, but is not limited to, a hinge portion in the heavy chain constant region.

[0133] The immunoglobulin Fc fragment according to the present invention may contain a specific hinge sequence at the N-terminus.

[0134] The term "hinge sequence" as used herein means a site located in a heavy chain that forms a dimer of immunoglobulin Fc fragments via inter disulfide bonds.

[0135] The hinge sequence in the present invention may be a mutated hinge sequence having the following amino acid sequence in which a portion of the sequence has been deleted to have only one cysteine ​​residue, but is not limited thereto. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 119)

[0136] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 119 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention is one that contains only one cysteine ​​residue and is composed of 3 to 12 amino acids, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 120) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 121) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 122) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 123) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 124) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 125) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 126) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 127) Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 128) Pro-Ser-Cys-Pro (SEQ ID NO: 129) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 130) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 131) Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 132) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 133) Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 134) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 135) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 136) Glu-Pro-Ser-Cys (SEQ ID NO: 137) Ser-Cys-Pro (SEQ ID NO: 138)

[0137] More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 129 (Pro-Ser-Cys-Pro) or SEQ ID NO: 138 (Ser-Cys-Pro), but is not limited thereto.

[0138] The immunoglobulin Fc fragment of the present invention is in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence, and the conjugate of the present invention represented by chemical formula (1) is in a form in which one end of a linker is linked to one chain of the dimeric immunoglobulin Fc fragment, but is not limited thereto.

[0139] In the present invention, the "N-terminus" refers to the amino terminus of a protein or polypeptide, and includes the extreme amino terminus, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc fragment of the present invention may contain a hinge sequence at the N-terminus, but is not limited thereto.

[0140] Furthermore, the immunoglobulin Fc fragment of the present invention may be an extended Fc fragment that includes a part or the whole of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) except for the variable regions of the heavy and light chains of an immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a fragment in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0141] For example, the immunoglobulin Fc fragment of the present invention is, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0142] In one embodiment, the immunoglobulin Fc fragment may be in a dimeric form, and may have one Fc fragment in a dimeric form to which an X1 molecule is covalently linked, and the immunoglobulin Fc and X may be linked to each other via a non-peptidic polymer. Alternatively, the immunoglobulin Fc fragment in a dimeric form may have one X2 molecule symmetrically linked to which an Fc fragment in a dimeric form may have one Fc fragment in a dimeric form to which an X2 molecule is symmetrically linked, and the immunoglobulin Fc and X may be linked to each other via a non-peptidic linker. However, the present invention is not limited thereto.

[0143] Furthermore, the immunoglobulin Fc fragment of the present invention includes not only the naturally occurring amino acid sequence but also its sequence derivatives, which means that at least one amino acid residue of the naturally occurring amino acid sequence is different from that of the naturally occurring amino acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0144] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as sites suitable for modification.

[0145] Various derivatives are also used, such as derivatives in which the site forming disulfide bonds has been removed, derivatives in which some amino acids at the N-terminus of native Fc have been deleted, and derivatives in which a methionine residue has been added to the N-terminus of native Fc. Furthermore, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc fragments are disclosed in Patent Documents 1 and 2, etc.

[0146] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 4). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, the amino acid residues may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0147] The above-mentioned Fc derivative may exhibit biological activity equivalent to that of the Fc fragment of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc fragment.

[0148] Furthermore, such Fc fragments may be obtained from natural sources isolated from the living bodies of animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or may be recombinant products or derivatives thereof obtained from transformed animal cells or microorganisms. Here, the method for obtaining from natural sources may involve isolating the entire immunoglobulin from the living body of humans or animals and then treating it with a protease. Treatment with papain cleaves it into Fab and Fc, and treatment with pepsin cleaves it into pF'c and F(ab) 2 These can be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc fragment is a recombinant immunoglobulin Fc fragment obtained from a microorganism.

[0149] Furthermore, the immunoglobulin Fc fragment may be in a form having a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. For increasing or decreasing or removing such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used. Here, an immunoglobulin Fc fragment from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (c1q) and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, immunoglobulin Fc fragments from which the sugar chains have been removed or which have been deglycosylated are suitable for their original purpose as drug carriers.

[0150] In the present invention, "deglycosylation" refers to an Fc fragment from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc fragment that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0151] On the other hand, the immunoglobulin Fc fragment may be of human origin or of animal origin, such as bovine, goat, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment is of human origin.

[0152] The immunoglobulin Fc fragment may be derived from IgG, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In a more specific embodiment, the immunoglobulin Fc fragment is an IgG4 Fc fragment, and in a most specific embodiment, the immunoglobulin Fc fragment is a non-glycosylated Fc fragment derived from human IgG4, but is not limited thereto.

[0153] In a specific embodiment, the immunoglobulin Fc fragment is a human IgG4 Fc fragment, and may be in the form of a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteines of each monomer, and in this case, each monomer of the homodimer independently has / may have an internal disulfide bond between the 35th and 95th cysteines, and an internal disulfide bond between the 141st and 199th cysteines, i.e., two internal disulfide bonds (intra-chain form). Each monomer is composed of 221 amino acids, and the amino acids forming the homodimer are composed of a total of 442 amino acids, but are not limited thereto. Specifically, the immunoglobulin Fc fragment is a homodimer in which two monomers having the amino acid sequence of SEQ ID NO: 139 (consisting of 221 amino acids) form a disulfide bond between the third amino acid cysteines of each monomer, and each monomer of the homodimer independently forms an internal disulfide bond between the 35th and 95th cysteines, and an internal disulfide bond between the 141st and 199th cysteines, but is not limited to this.

[0154] On the other hand, the term "combination" in the present invention means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc fragment of the same origin is bound to a single-chain polypeptide of a different origin, i.e., a dimer or multimer can be produced from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0155] Alternatively, L may be a non-peptidic linker, such as a linker containing repeating ethylene glycol units.

[0156] The "non-peptidic linker" of the present invention includes a biocompatible polymer having at least two repeating units bonded together. The repeating units are linked to each other by any covalent bond except a peptide bond. The non-peptidic linker is a component that forms a part of the conjugate of the present invention, and corresponds to L in chemical formula (1). The non-peptidic linker used in the present invention may be any polymer that is resistant to in vivo protease. The non-peptidic linker of the present invention is used in combination with a non-peptidic polymer.

[0157] The non-peptide linker is a linker containing a repeating unit of ethylene glycol, such as polyethylene glycol, but is not particularly limited thereto. In addition, the present invention also includes derivatives thereof known in the art and derivatives that can be easily prepared at the technical level in the art.

[0158] The repeating unit of the non-peptidic linker may be a repeating unit of ethylene glycol, and specifically, the non-peptidic linker may contain a repeating unit of ethylene glycol and a functional group used for preparing a conjugate at its end. The long-lasting conjugate according to the present invention is in a form in which X and F are linked via the functional group, but is not limited thereto. The non-peptidic linker according to the present invention contains two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0159] Specifically, the linker is polyethylene glycol (PEG) represented by chemical formula (2), but is not limited thereto.

[0160] JPEG2025085685000002.jpg1528...(2)

[0161] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited to these.

[0162] The PEG portion of the persistent conjugate includes, but is not limited to, the -(CH2CH2O)n- structure as well as the oxygen atom(s) intervening between the linking element and the -(CH2CH2O)n-.

[0163] In a specific embodiment, the conjugate has a structure in which a peptide (X) having an amino acid sequence of any one of SEQ ID NOs: 1 to 102 and an immunoglobulin fragment (F) are covalently linked via a linker containing a repeating unit of ethylene glycol, but is not limited thereto. The polyethylene glycol includes, but is not limited to, ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG).

[0164] The molecular weight of the non-peptidyl polymer is in the range of 1 to 100 kDa, specifically in the range of 1 to 20 kDa or in the range of 1 to 10 kDa, but is not limited thereto. Furthermore, the non-peptidyl linker of the present invention to be bound to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0165] In a specific embodiment, both ends of the non-peptidic linker can be bound to an amino or thiol group of F, for example an immunoglobulin Fc fragment, and an amino or thiol group of X, respectively.

[0166] Specifically, the non-peptidyl polymer contains reactive groups at both ends that are bonded to F (e.g., an immunoglobulin Fc fragment) and X, respectively, more specifically, reactive groups that are bonded to X, or the amino group located at the N-terminus or lysine of F, or the thiol group of cysteine, but are not limited thereto.

[0167] Furthermore, the reactive group of the non-peptidyl polymer bound to F, e.g., an immunoglobulin Fc fragment, and X is selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0168] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0169] In the above, examples of the succinimide derivative include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0170] The non-peptide linker is linked to X and F via such a reactive group, but is not particularly limited thereto.

[0171] Furthermore, the final products generated by reductive amination with aldehyde bonds are much more stable than those linked with amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0172] The reactive groups at both ends of the non-peptidic linker may be the same or different. For example, one end may have a maleimide group and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. However, the non-peptidic linker is not particularly limited to these, as long as F, specifically, an immunoglobulin Fc fragment and X are bound to each end of the non-peptidic linker.

[0173] For example, the non-peptide linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end.

[0174] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the long-acting protein conjugate of the present invention can be produced by activating the hydroxy groups into the above-mentioned reactive groups by a known chemical reaction or by using commercially available polyethylene glycol having modified reactive groups.

[0175] In a specific embodiment, the non-peptidyl polymer is linked to a cysteine ​​residue of X, more specifically, to a -SH group of cysteine, but is not limited thereto.

[0176] Examples of the non-peptide polymer that is linked to the 10th cysteine ​​residue, the 13th cysteine ​​residue, the 15th cysteine ​​residue, the 17th cysteine ​​residue, the 19th cysteine ​​residue, the 21st cysteine ​​residue, the 24th cysteine ​​residue, the 28th cysteine ​​residue, the 29th cysteine ​​residue, the 30th cysteine ​​residue, the 31st cysteine ​​residue, the 40th cysteine ​​residue, or the 41st cysteine ​​residue in the peptide corresponding to X include, but are not limited to, those.

[0177] Specifically, the reactive group of the non-peptidyl polymer may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X by a thioether bond, and the aldehyde group can be linked to F, specifically, the -NH group of immunoglobulin Fc. 2 The linkage can be achieved by, but is not limited to, a reductive amination reaction with the group, which are merely examples.

[0178] In the conjugate, the reactive group of the non-peptidyl polymer is -NH 2 However, this is merely one example.

[0179] Furthermore, the conjugates described above may have improved duration of efficacy over native GLP-1, GIP or glucagon, or over X where F is not modified, and such conjugates include all of the above forms as well as forms encapsulated in biodegradable nanoparticles, etc.

[0180] The peptide or conjugate thereof according to the present invention may have prophylactic or therapeutic applications against liver diseases.

[0181] In the present invention, the term "liver disease" refers to a disease that occurs in the liver, including, but not limited to, metabolic liver disease or liver inflammation. Representative examples of the liver disease include simple fatty liver, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure, liver cancer, etc., and the liver disease in the present invention may be any disease that causes abnormalities in liver tissue or function. It is known that in many cases, inflammation of the liver occurs due to viruses, alcohol, drugs, immune abnormalities, metabolic diseases, etc., and diseases such as liver cirrhosis and liver cancer develop due to the progression and chronicity of liver inflammation. The composition according to the present invention is effective against liver diseases accompanied by or caused by liver inflammation, such as, but not limited to, liver inflammation, non-alcoholic steatohepatitis, or liver fibrosis. On the other hand, the composition according to the present invention also exerts a preventive or therapeutic effect on liver diseases not accompanied by inflammation. Examples of such liver diseases include, but are not limited to, simple fatty liver, non-alcoholic fatty liver, and cirrhosis of the liver.

[0182] The liver diseases for which the peptide or its conjugate of the present invention has a therapeutic effect include, but are not limited to, metabolic liver diseases, which are caused by abnormal chemical reactions in the body that interfere with the body's metabolism, and include simple fatty liver, fatty liver, steatohepatitis, etc.

[0183] The composition according to the present invention exerts a preventive or therapeutic effect on metabolic liver disease by reducing the amount of triglyceride and / or cholesterol in liver tissue when administered, but is not limited thereto. The metabolic liver disease may or may not be accompanied by inflammation. Examples of liver diseases that can be treated by the composition of the present invention include, but are not limited to, simple fatty liver, non-alcoholic fatty liver, non-alcoholic steatohepatitis, etc.

[0184] "Nonalcoholic fatty liver disease (NAFLD)", a representative example of metabolic liver disease, refers to a condition in which fatty liver is present despite no history of alcohol intake or no association with alcohol intake. Fatty liver refers to a phenomenon that occurs when neutral fat is abnormally deposited in liver cells. Approximately 5% of a normal liver is made up of adipose tissue, and neutral fat, fatty acid, phospholipid, cholesterol, and cholesterol ester are the main components of fat. Once fatty liver occurs, most of the components are replaced by neutral fat, and when the amount of neutral fat is 5% or more of the liver weight, fatty liver is diagnosed. Fatty liver is caused by a fat metabolism disorder in liver cells or a defect in the process of transporting excess fat, and is mainly caused by a fat metabolism disorder in the liver. Most of the fat accumulated in the fatty liver is neutral fat (triglyceride).

[0185] Nonalcoholic fatty liver disease refers to a group of diseases including simple steatosis, which is caused only by excessive accumulation of fat in liver cells, nonalcoholic fatty liver, and nonalcoholic steatohepatitis (NASH), which is accompanied by hepatocyte necrosis, inflammation, and fibrosis, but may be any disease that can be treated with the composition of the present invention. Nonalcoholic fatty liver disease according to the present invention is associated with nonalcoholic steatohepatitis, but is not limited thereto.

[0186] In addition, the liver disease for which the peptide or its conjugate of the present invention has a therapeutic effect is liver inflammation, but is not limited thereto. In the present invention, "liver inflammation" refers to a disease that causes inflammation in the liver, which is the most important cause of liver disease, and is divided into acute hepatitis and chronic hepatitis depending on the cause and symptoms. The main causes are viruses, alcohol, drugs, immune disorders, metabolic diseases, etc.

[0187] When administered, the composition according to the present invention can reduce the expression of at least one of TNF-α, MCP-1, and IL-6 in liver tissue, thereby exerting a preventive or therapeutic effect against liver inflammation, but is not limited thereto.

[0188] The peptide and its conjugate according to the present invention not only have the effect of alleviating liver inflammation itself, but also have an effect on diseases that are accompanied by or caused by liver inflammation, such as hepatitis, non-alcoholic steatohepatitis, and hepatic fibrosis.

[0189] "Nonalcoholic steatohepatitis" in the present invention is a type of nonalcoholic fatty liver disease, and is a representative example of liver disease accompanied by hepatocyte necrosis, inflammation and fibrosis. The composition according to the present invention exerts an effect on nonalcoholic steatohepatitis by suppressing liver inflammation and fibrosis, specifically, exerts an effect on nonalcoholic steatohepatitis accompanied by fatty liver, hepatic fibrosis or cirrhosis, or liver cancer caused by nonalcoholic steatohepatitis, but is not limited thereto.

[0190] In the present invention, "liver fibrosis" refers to the result of a recovery process from repeated liver injury, and refers to the formation of excessive fibrous connective tissue in organs and tissues during a reparative or reactive process. Chronicity and aggravation of liver inflammation are known to be one of the causes of onset. Unlike cirrhosis, it is known to be reversible, composed of thin fibrils, and without nodule formation, and will recover to normal when the cause of liver injury disappears. However, if such a liver fibrosis process continues, crosslinking between ECM (extra cellular matrix) increases, progressing to irreversible cirrhosis with nodules. The composition according to the present invention exerts a preventive or therapeutic effect on liver fibrosis, specifically liver fibrosis associated with nonalcoholic steatohepatitis, but is not limited thereto.

[0191] When administered, the composition of the present invention exerts a preventive or therapeutic effect against liver fibrosis by reducing the blood concentration of TIMP-1 and / or hyaluronic acid in the individual to which it is administered, but this is not limited thereto.

[0192] Specifically, the composition containing the peptide according to the present invention or a conjugate thereof may be effective against liver fibrosis, and specifically may prevent or treat liver fibrosis by reducing the ELF score (enhanced liver fibrosis score).

[0193] The ELF score (enhanced liver fibrosis score) is a score that indicates the degree of healing of liver fibrosis, and can be calculated by the following formula. It can be calculated by measuring the concentrations of hyaluronic acid (HA), PIIINP (N-terminal propeptide of procollagen type III), and TIMP-1 (tissue inhibitor of metalloproteinase-1) in blood samples.

[0194]

number

[0195] The reduction in the ELF score is, compared to a group not administered the peptide or its long-acting conjugate according to the present invention, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, or about 14% to about 30%, but is not limited to these.

[0196] The compositions prevent or treat liver fibrosis by reducing the ELF score of an individual to which they are administered to, but not limited to, about 9.8 or less, about 9.7 or less, about 9.6 or less, about 9.5 or less, about 9.4 or less, about 9.3 or less, about 9.2 or less, or about 9.1 or less.

[0197] In the present invention, "cholestasis" refers to a condition in which the flow of bile from the liver to the duodenum is slowed or blocked, and "cholestasis liver disease" refers to a condition in which bile formation in the liver is impeded by conditions such as various diseases, extended jugular feeding, or side effects of certain drugs (e.g., some antibiotics). Common symptoms of cholestasis include fatigue, itchy skin (pruritus), jaundice, and xanthomas (deposition of cholesterol-rich material under the skin). The effects of cholestasis are severe and widespread, which can lead to the deterioration of liver disease into systemic disease, liver failure, and the need for liver transplantation. Causes of cholestatic liver disease include acute hepatitis, inflammation of the bile duct, etc.

[0198] The cholestatic liver diseases include, but are not limited to, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), Alagille syndrome (AS), and the like.

[0199] Primary biliary cholangitis (PBC), also known as primary biliary cholangitis (PBC), is a chronic cholestatic liver disease of unknown etiology. Progressive bile duct damage due to portal and periportal inflammation can lead to progressive fibrosis and ultimately cirrhosis. Immunologic, genetic and environmental factors are currently believed to be potential causes of the disease. Primary biliary cirrhosis is primarily seen in middle-aged women, and symptoms of PBC may include early onset fatigue, pruritus and unexplained dyslipidemia.

[0200] It is now known that primary biliary cirrhosis is an immune-mediated disease, specifically immunohistochemical staining of T lymphocytes in the portal and periportal areas shows CD4 positive and CD8 negative T cells. Also, abnormal suppressor T cell activity has been reported in asymptomatic first-degree relatives of affected individuals. Interleukins have been reported to play a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis (Non-Patent Document 5).

[0201] The treatment for PBC is bile acid therapy with ursodeoxycholic acid (UDSA) and obeticholic acid (OCA). The mechanism of action of both drugs in PBC is related to their ability to activate FXR and TGFR-5 and exert anti-inflammatory effects. However, approximately 40% of patients treated with UDCA did not achieve an adequate biochemical response.

[0202] Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease caused by inflammation and fibrosis of the biliary tract inside and outside the liver, the cause of which is unknown. Specifically, it is an inflammatory disease of the bile duct and biliary tract, and as the disease progresses, fibrosis occurs, causing the bile duct wall to thicken and narrow or become strictured. Although the cause is still unknown, it is suspected to be a complex combination of various factors, including genetic factors, environmental factors, and associated immune responses.

[0203] If blood liver function tests show elevated alkaline phosphatase scores, elevated aminotransferase scores, and gammaglobulinemia, a diagnosis of primary sclerosing cholangitis is made.

[0204] No clear treatment for PSC has yet been reported, and liver transplantation is the only treatment that can provide a fundamental cure.

[0205] Therefore, there remains a need to develop drugs that can treat PBS and PSC while ensuring patient convenience and without side effects.

[0206] In the present invention, "liver cirrhosis" is a chronic disease that develops due to repeated regeneration of liver cells and increase in fibrous tissue, and pathologically involves necrosis, inflammation, and fibrosis. Ultimately, it progresses to diseases such as liver cirrhosis complications such as liver failure, liver cancer, and death. In particular, since there are no subjective symptoms in the early stages and it is discovered after the disease has progressed, it is necessary to quickly treat liver fibrosis, which is a state before it progresses to liver cirrhosis, etc. The composition according to the present invention exhibits a preventive or therapeutic effect against liver cirrhosis, specifically liver cirrhosis accompanied by non-alcoholic steatohepatitis, but is not limited thereto.

[0207] In the present invention, "liver decompensation" refers to a state in which the liver is unable to perform normal physiological functions such as protein synthesis and metabolism due to weakened liver function caused by liver damage or liver disease due to viral hepatitis, cirrhosis, drugs, alcohol, etc. Depending on the rate of progression, it is divided into acute liver failure and chronic liver failure, and is known to cause various complications. The composition according to the present invention exerts effects such as suppression of inflammation and fibrosis, thereby exerting a preventive or therapeutic effect against liver failure.

[0208] In the present invention, "hepatocellular carcinoma" refers to a malignant tumor originating from hepatocytes, and is divided into primary hepatic cancer (hepatocellular carcinoma) occurring in hepatic cells themselves and metastatic hepatic cancer caused by metastasis of cancer of other tissues to the liver, with approximately 90% or more of hepatic cancer being primary hepatic cancer. As main causes, in addition to hepatitis and chronic liver disease, alcohol, smoking, obesity, etc. are known to be included. The composition according to the present invention exerts a preventive or therapeutic effect against hepatic cancer, specifically, hepatic cancer caused by non-alcoholic steatohepatitis, but is not limited thereto.

[0209] The model induced by the MCD diet used in the examples of the present invention is known as a non-alcoholic steatohepatitis model, and the model induced by the AMLN diet is known as a fatty liver and steatohepatitis model. The AMLN / TAA mouse model is also known to be used as a liver fibrosis or non-alcoholic steatohepatitis model. These models are used in various liver disease-related studies, and in the examples of the present invention, the effects of the peptide (triple activity) according to the present invention or its long-acting conjugate were confirmed in each model. This suggests that it is useful for preventing or treating liver diseases such as hepatitis, liver fibrosis, simple fatty liver, non-alcoholic fatty liver, and non-alcoholic steatohepatitis. In the examples of the present invention, the ameliorative effect of the triple activity long-acting conjugate of the present invention was evaluated using a PBC and / or PSC model, and the effect on cholestatic liver disease was also confirmed.

[0210] The composition according to the present invention is characterized in that it does not cause weight gain, a side effect of conventional drugs for treating liver diseases, or the degree of weight gain is relatively low.

[0211] The composition of the present invention has at least one of the following characteristics (a) to (k), and thus can prevent or treat liver diseases, but is not limited thereto. (a) Decrease in NAS score (NAFLD Activity Score) (b) Reduction of triglycerides in the liver (c) Reduction of blood cholesterol (d) Reduction in steatosis score (e) Decreased levels of TNF-α, MCP-1, and IL-6 in liver tissue (f) Reduction in liver inflammation score (g) Reduction in parenchymal necrosis score (h) Decrease in bile duct hyperplasia score (i) Reduction in ELF (enhanced liver fibrosis) score (j) Reduction in blood levels of TIMP-1 and / or hyaluronic acid, which are markers of liver fibrosis (k) Decrease in fibrosis score

[0212] In the present invention, "prevention" means any action of suppressing or delaying the onset of liver disease by administering the peptide or a composition containing it, and "treatment" means any action of improving or favorably changing the symptoms of liver disease by administering the peptide or a composition containing it.

[0213] The pharmaceutical compositions of the present invention may further comprise a pharma- ceutically acceptable excipient, carrier, or diluent. Such pharma- ceutically acceptable excipient, carrier, or diluent may be non-naturally occurring.

[0214] In the present invention, "pharmacologically acceptable" means a sufficient amount to exert a therapeutic effect and to cause no side effects, and can be easily determined by a person skilled in the art based on factors known in the medical field, such as the type of disease, the patient's age, weight, health condition, sex, sensitivity to the drug, administration route, administration method, number of doses, treatment period, combination, and drugs used concomitantly.

[0215] The pharmaceutical composition containing the peptide of the present invention may further contain a pharma- ceutically acceptable excipient. The excipient may be, for oral administration, a binder, a lubricant, a disintegrant, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., for injection, a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, etc., for topical administration, a base, an excipient, a lubricant, a preservative, etc., but is not limited thereto.

[0216] The composition of the present invention can be prepared in various forms by mixing with the pharma- ceutically acceptable excipients as described above.For example, for oral administration, it can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be prepared in the form of disposable ampoules or multiple doses.Other forms include solutions, suspensions, tablets, pills, capsules, sustained release preparations, etc.

[0217] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, a preservative, etc.

[0218] Furthermore, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized agents, and suppositories.

[0219] Furthermore, the composition can be formulated into a single-dose formulation suitable for administration to the body of a patient by a conventional method in the pharmaceutical field, specifically into a formulation form useful for administration of a protein pharmaceutical, and administered orally or via a parenteral administration route, including, but not limited to, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method commonly used in the art.

[0220] Furthermore, the conjugate can be used by mixing it with various medicament-acceptable carriers such as physiological saline or organic solvents, and in order to improve stability and absorbability, carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, other stabilizers, etc. can be used as drugs.

[0221] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, the severity of the disease, etc. Specifically, the composition of the present invention contains a pharmacologic effective amount of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102 or a long-acting conjugate comprising the same, but is not limited thereto.

[0222] The term "containing a pharmacologic effective amount of the peptide or long-acting conjugate" means that the peptide or long-acting conjugate is contained to such an extent that the desired pharmacological activity (e.g., prevention, improvement, or treatment of liver disease) can be obtained, and also means that the peptide or long-acting conjugate is contained at a pharmacologic acceptable level that is free of or has only slight toxicity or side effects on the individual to which it is administered, but is not limited thereto. Such a pharmacologic effective amount is determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0223] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredients depending on the severity of the disease. Specifically, the total dose of the conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the dose of the conjugate is determined by taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these into consideration, a person having ordinary knowledge in the art would be able to determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0224] Since the pharmaceutical composition of the present invention has excellent in vivo durability and potency, the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced.

[0225] Another embodiment of the present invention provides a method for preventing or treating liver disease, comprising the step of administering said peptide or a composition comprising it to an individual in need thereof.

[0226] The peptide or a composition containing the same, liver disease, prevention and treatment are as described above.

[0227] The individual in the present invention is an individual suspected of having a liver disease, and the individual suspected of having a liver disease means a mammal, including humans, mice, livestock, etc., that has developed or is at risk of developing a liver disease, but may be any individual that can be treated with the conjugate of the present invention or the composition containing the same.

[0228] In the present invention, "administration" means introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition can be any common route that can deliver the composition to an in vivo target, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0229] The method of the invention may include administering a pharmaceutical composition comprising said peptide in a pharma- ceutical effective amount. A suitable total daily dose will be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and extent of the response to be achieved, whether or not other formulations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0230] Yet another embodiment of the present invention is the use of said peptide or a composition comprising it for the prevention or treatment of liver diseases.

[0231] Yet another embodiment of the invention is the use of said peptide or a composition comprising it in the manufacture of a medicament for the prevention or treatment of liver diseases.

[0232] The peptide or a composition containing the same, liver disease, prevention and treatment are as described above.

[0233] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. EXAMPLES

[0234] Determination of in vitro activity of the triple-active compound and its long-acting conjugate Example 1-1: Preparation of triple active A triple active compound was prepared that has activity against the GLP-1, GIP and glucagon receptors, the sequence of which is shown in Table 1.

[0235] [Table 1] JPEG2025085685000005.jpg222131 JPEG2025085685000006.jpg222131 JPEG2025085685000007.jpg222130 JPEG2025085685000008.jpg224130 JPEG2025085685000009.jpg147131

[0236] In the sequences shown in Table 1, the amino acid represented by X is the unnatural amino acid Aib (aminoisobutyric acid), and the underlined amino acids indicate that the underlined amino acids form a ring with each other. Also, in Table 1, CA means 4-imidazoacetyl, and Y means tyrosine.

[0237] Example 1-2: Preparation of triple-active long-acting conjugate In order to PEGylate the cysteine ​​residues of the triple-active substance of Example 1 (SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96) with 10 kDa PEG having a maleimide group and an aldehyde group at both ends, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the molar ratio of the triple-active substance to maleimide-PEG-aldehyde was set to 1:1-3, the protein concentration was set to 1-5 mg / ml, and the reaction was carried out at a low temperature for 0.5-3 hours. Here, the reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple-active substance mono-PEGylated to cysteine.

[0238] Next, the purified mono-PEGylated triple active substance and immunoglobulin Fc were reacted at a molar ratio of 1:1-5, a protein concentration of 10-50 mg / ml, and at 4-8°C for 12-18 hours. The reaction was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was added with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-30% isopropanol. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing the triple active substance and immunoglobulin Fc.

[0239] After preparation, the purity was analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography to be 95% or more.

[0240] Here, the conjugate in which the triple activity of SEQ ID NO: 21 and immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 21 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 21." These terms are used interchangeably in the present invention.

[0241] Here, the conjugate in which the triple activity of SEQ ID NO: 22 and immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 22 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 22." These terms are used interchangeably in the present invention.

[0242] Here, the conjugate in which the triple activity form of SEQ ID NO: 42 and the immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 42 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 42." These terms are used interchangeably in the present invention.

[0243] Here, the conjugate in which the triple activity of SEQ ID NO: 43 and the immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 43 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 43." These terms are used interchangeably in the present invention.

[0244] Here, the conjugate in which the triple activity of SEQ ID NO: 50 and the immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 50 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 50." These terms are used interchangeably in the present invention.

[0245] Here, the conjugate in which the triple activity of SEQ ID NO: 77 and immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 77 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 77." These terms are used interchangeably in the present invention.

[0246] Here, the conjugate in which the triple activity of SEQ ID NO: 96 and immunoglobulin Fc are linked via PEG is named a "conjugate comprising SEQ ID NO: 96 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 96." These terms are used interchangeably in the present invention.

[0247] Example 1-3: Measurement of in vitro activity of triple-active complex and its long-acting conjugate In order to measure the activity of the triple-active substance and its long-acting conjugate prepared in Examples 1-1 and 1-2, a method for measuring cell activity in vitro was used using cell lines transformed with GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor, respectively.

[0248] The above cell lines were transformed into CHO (chinese hamster ovary) cells to express human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, and are suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity of each part was measured using each transformed cell line.

[0249] To measure the GLP-1 activity of the triple active substance and its long-acting conjugate prepared in Examples 1-1 and 1-2, human GLP-1 was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple active substance and its long-acting conjugate prepared in Examples 1-1 and 1-2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing human GLP-1 receptor, and 5 μl of each of the serially diluted substances was added to the cells, followed by adding 5 μl of a buffer containing a cAMP antibody, and then culturing at room temperature for 15 minutes. Next, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to extract EC from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative potencies against human GLP-1 are shown in Tables 2 and 3.

[0250] To measure the GCG activity of the triple activity complex and its long-acting conjugate prepared in Examples 1-1 and 1-2, human GCG was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple activity complex and its long-acting conjugate prepared in Examples 1-1 and 1-2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing human GCG receptor, and 5 μl of each of the serially diluted substances was added to the cells, followed by adding 5 μl of a buffer containing a cAMP antibody, and then culturing at room temperature for 15 minutes. Next, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to extract EC from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative potencies against human GCG are shown in Tables 2 and 3.

[0251] To measure the GIP activity of the triple activity complex and its long-acting conjugate prepared in Examples 1-1 and 1-2, human GIP was serially diluted 4-fold from 50 nM to 0.000048 nM, and the triple activity complex and its long-acting conjugate prepared in Examples 1-1 and 1-2 were serially diluted 4-fold from 400 nM to 0.00038 nM. The culture medium was removed from the cultured CHO cells expressing human GIP receptor, and 5 μl of each of the serially diluted substances was added to the cells, followed by adding 5 μl of a buffer containing a cAMP antibody, and then culturing at room temperature for 15 minutes. Next, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to extract EC from the accumulated cAMP. 50 The values ​​were calculated and compared with each other. The relative potencies against human GIP are shown in Tables 2 and 3.

[0252] [Table 2] JPEG2025085685000011.jpg221129 JPEG2025085685000012.jpg220129 JPEG2025085685000013.jpg37129

[0253] [Table 3]

[0254] The novel triple-activator long-acting conjugate prepared as described above has a triple-activator function capable of activating all of the GLP-1 receptor, the GIP receptor and the glucagon receptor, and therefore can be used as a therapeutic agent for a target disease. EXAMPLES

[0255] Confirmation of the therapeutic effect of triple-activator on metabolic liver disease The present inventors attempted to confirm the therapeutic effect of the triple-active compound of the present invention on metabolic liver diseases.

[0256] Example 2-1: NASH treatment effect in mice with NASH induced by MCD diet intake First, the effect of the triple activation long-acting conjugate on nonalcoholic steatohepatitis (NASH) was confirmed as follows.

[0257] C57BL / 6 mice were fed a methionine-choline deficient (MCD) diet for two weeks to induce a non-alcoholic steato hepatitis (NASH) mouse model.

[0258] To confirm the efficacy of the developed substance in treating NASH, mice were divided into normal mice, NASH-induced mice (vehicle control group), and groups administered the long-acting conjugate of SEQ ID NO: 42 (0.36, 0.72, 1.44 nmol / kg, Q2D), and the substance was repeatedly administered subcutaneously over a period of 4 weeks. After 4 weeks of repeated administration, liver tissues from each mouse were collected by autopsy, and then stained with H&E (Haematoxylin and Eosin) to evaluate the degree of progression of NASH using NAS (NAFLD Activity Score).

[0259] As shown in FIG. 1, repeated administration of the long-acting conjugate of SEQ ID NO:42 over a period of 4 weeks resulted in a significant reduction in the NAS score in liver tissue (NASH vehicle control group=3.71, long-acting conjugate of SEQ ID NO:42 0.36 nmol / kg=2.57, long-acting conjugate of SEQ ID NO:42 0.72 nmol / kg=0.43, long-acting conjugate of SEQ ID NO:42 1.44 nmol / kg=0).

[0260] Therefore, the therapeutic effect of the long-acting conjugate of SEQ ID NO: 42, which is a representative triple-activity conjugate according to the present invention, on NASH was confirmed.

[0261] Example 2-2: Improvement of fatty liver in mice induced by AMLN (amylin) diet Furthermore, the present inventors used an AMLN (amylin) mouse model to confirm the fatty liver improving effect of the triple activity form according to the present invention.

[0262] Because the AMLN diet is high in fat, fructose, and cholesterol, it is known that long-term intake of the diet can induce obesity and steatohepatitis, and the AMLN mouse model is used as one of the models for steatohepatitis.

[0263] Mice induced with AMLN diet for 37 weeks were divided into a vehicle control group, an obeticholic acid (30mg / kg, QD, oral) group, and a long-acting conjugate of SEQ ID NO:42 (2.6nmol / kg, Q2D, subcutaneous) group, and were repeatedly administered for 12 weeks. After 12 weeks of repeated administration, liver tissues of each mouse were collected by autopsy, and the fat content in the liver tissue was measured, and the efficacy of improving fatty liver was evaluated by H&E staining.

[0264] As a result, it was confirmed that when the long-acting conjugate of SEQ ID NO: 42 was repeatedly administered for 12 weeks, triglyceride and cholesterol scores were significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared to the vehicle control group and the obeticholic acid group. Therefore, it was confirmed that the long-acting conjugate of SEQ ID NO: 42 according to the present invention reduces intrahepatic fat content (Figure 2).

[0265] In addition, to further confirm the fatty liver improving effect of administration of a long-acting conjugate of SEQ ID NO: 42, the present inventors examined the change in steatosis score after administration of a long-acting conjugate of SEQ ID NO: 42 in the same manner as described above.

[0266] As a result, it was confirmed that the steatosis score, which indicates the level of steatosis, was significantly reduced when the long-acting conjugate of sequence number 42 was administered, compared to the vehicle control group and the obeticholic acid-administered group (Figure 3). EXAMPLES

[0267] Confirmation of the therapeutic effect of triple-active compound on liver fibrosis Example 3-1: Confirmation of the effect of improving liver fibrosis indicators in mice with liver fibrosis induced by TAA administration To confirm the efficacy of the triple activity long-acting conjugate prepared in Example 1 in improving liver fibrosis, an AMLN / TAA (Thioacetamide) mouse model, known as a liver fibrosis model, was used. Briefly, the model was induced in C57BL / 6 mice by feeding the AMLN diet and administering TAA (50-400 mg / kg, TIW-three times a week) for 16 weeks. The induced animals were divided into a vehicle control group and a group administered the long-acting conjugate of SEQ ID NO: 42 (1.3 nmol / kg, Q2D), which was selected as a representative triple activity, and the substance was repeatedly administered subcutaneously for 8 weeks at the end of the induction period. Mice that received only the AMLN diet were used as a negative control group. After 8 weeks of repeated administration, the concentrations of hyaluronic acid, TIMP-1 (tissue inhibitor of metalloproteinase-1), and PIIINP (N-terminal propeptide of procollagen type III) in the blood samples were analyzed, and the ELF (enhanced liver fibrosis) score, known as a non-invasive indicator of liver fibrosis, was then calculated.

[0268] As shown in FIG. 4, it was confirmed that repeated administration of the long-acting conjugate of SEQ ID NO:42 over 8 weeks significantly reduced the ELF score, which was elevated in the AMLN / TAA vehicle control group.

[0269] These results suggest that the triple activity compound or its long-acting conjugate of the present invention dramatically reduces the ELF score and has a preventive or therapeutic effect on liver fibrosis.

[0270] Example 3-2: Confirmation of therapeutic effect on liver fibrosis in mice with liver fibrosis induced by TAA administration Based on the non-invasive liver fibrosis index improving effect confirmed in Example 3-1, an invasive method was used to clearly evaluate the liver fibrosis therapeutic effect of the triple activity sustained conjugate. Briefly, liver tissues of the mice used in Example 3-1 (repeated administration for 8 weeks) were collected by autopsy and then stained with Sirius Red.

[0271] As a result, it was confirmed that the area of ​​positive Sirius Red staining in liver tissue was also significantly reduced by administration of the triple activity long-acting conjugate (Figure 5).

[0272] These results suggest that the triple activity complex or its long-acting conjugate of the present invention has a preventive or therapeutic effect on liver fibrosis.

[0273] Example 3-3: Confirmation of the effect of improving liver fibrosis in mice with liver fibrosis induced by BDL In order to confirm the hepatic fibrosis improving effect of the long-acting conjugate of SEQ ID NO: 42 confirmed in Examples 3-1 and 3-2, a BDL (bile duct ligation) mouse model, known as a hepatic fibrosis model, was used. Briefly, C57BL / 6 mice were anesthetized, and then the bile duct was sutured by surgical therapy to induce cholestasis, thereby inducing hepatic fibrosis. The induced animals were divided into an excipient control group and a group administered with a long-acting conjugate of SEQ ID NO: 42 selected as a representative triple-active substance (1.3 nmol / kg, Q2D, subcutaneous administration). As a control substance, mice administered with obeticholic aicd (30 mg / kg, QD, oral administration), which is the active pharmaceutical ingredient of Ocaliva (registered trademark), were used. Drug administration was repeated over a period of two weeks starting two days after surgery. Sham mice were used as a negative control group. The blood concentrations of TIMP-1 and hyaluronic acid, which are markers of liver fibrosis, were measured in blood samples taken from mice that had been repeatedly administered the drug over a period of two weeks.

[0274] The results confirmed that the concentrations of TIMP-1 and hyaluronan were consistently decreased by administration of the triple activity long-acting conjugate (Figure 6).

[0275] These results again suggest that the triple activity compound or its long-acting conjugate of the present invention has a therapeutic effect on liver fibrosis.

[0276] Example 3-4: Confirmation of the therapeutic effect of liver fibrosis in mice with liver fibrosis induced by BDL Based on the non-invasive liver fibrosis index improving effect confirmed in Example 3-3, an invasive method was used to clearly evaluate the liver fibrosis therapeutic effect of the triple activity sustained conjugate. Briefly, the liver tissues of the mice used in Example 3-3 (repeated administration for 2 weeks) were collected by autopsy, and then stained with Sirius Red, and the fibrosis score was measured based on the results.

[0277] As a result, it was confirmed that repeated administration of the long-acting conjugate of SEQ ID NO: 42 over a period of two weeks significantly reduced the fibrosis score that increased in the BDL vehicle control group (FIGS. 7a and 7b).

[0278] These results suggest that the triple activity complex or its long-acting conjugate of the present invention can be used as an agent for preventing or treating liver fibrosis. EXAMPLES

[0279] Confirmation of the effect of triple-active compound on liver inflammation In order to confirm the therapeutic effect on cholestatic liver disease, which is a type of liver disease, the following experiment was carried out.

[0280] Example 4-1: Confirmation of the effect of improving PBC (primary biliary cirrhosis) in mice with PBC induced by BDL A bile duct ligation (BDL) mouse model, known as a PBC model, was used to confirm the PBC-improving efficacy of the triple activity long-acting conjugate prepared in Example 1. Briefly, C57BL / 6 mice were anesthetized, and then the bile duct was sutured by surgical therapy to induce cholestasis, thereby inducing liver inflammation.

[0281] The induced animals were divided into a vehicle control group and a group administered with a long-acting conjugate of SEQ ID NO: 42 (1.3 nmol / kg, Q2D, subcutaneous administration). As a control substance, mice administered with obeticholic aicd (30 mg / kg, QD, oral administration), which is an active pharmaceutical ingredient of Ocaliva (registered trademark), which is commercially available as a PBC disease treatment agent, were used. Drug administration was repeated over a period of two weeks starting two days after surgery. Sham mice were used as negative controls. After two weeks of repeated administration, liver tissues from each mouse were collected by autopsy, and the effect of improving liver inflammation was evaluated by H&E staining.

[0282] As a result, it was confirmed that repeated administration of the triple activity long-acting conjugate over a period of two weeks significantly reduced the inflammation score in liver tissue, which was elevated in the BDL vehicle control group (Figure 8).

[0283] Therefore, it was confirmed that the triple activity long-acting conjugate has an excellent effect of improving liver inflammation in PBC mice.

[0284] Example 4-2: Confirmation of the PSC improvement effect in PSC (primary sclerosing cholangitis) mice induced by BDL To confirm the PSC improving efficacy of the triple activity long-acting conjugate prepared in Example 1, a bile duct ligation (BDL) mouse model, which is known as a PSC model, was used.

[0285] Specifically, C57BL / 6 mice were anesthetized, and then the bile duct was sutured by surgical therapy to induce cholestasis, thereby inducing liver and bile duct injury. The animals were divided into a vehicle control group and a group administered with a long-acting conjugate of SEQ ID NO: 42 (1.3 nmol / kg, Q2D), and were repeatedly administered subcutaneously for two weeks starting two days after surgery. Sham-treated mice were used as a negative control group. After two weeks of repeated administration, liver tissues from each mouse were collected by necropsy, and the effect of improving liver and bile duct injury was evaluated by H&E staining.

[0286] As a result, it was confirmed that repeated administration of the triple activity long-acting conjugate over a period of 2 weeks significantly reduced the parenchymal necrosis score due to bile reflux, which was elevated in the BDL vehicle control group (Figure 9).

[0287] Furthermore, as shown in FIG. 10, it was confirmed that the bile duct hyperplasia score, which increases due to bile duct injury, was significantly reduced by administration of the triple activity long-acting conjugate.

[0288] Therefore, it was confirmed that the triple activity long-acting conjugate improves liver and bile duct injuries and alleviates liver inflammatory responses in PSC mice.

[0289] Example 4-3: Confirmation of the effect of improving liver inflammation in TAA-administered mice The present inventors used an AMLN / TAA (thioacetamide) mouse model to confirm the efficacy of the triple activity long-acting conjugate prepared in Example 1 in improving inflammation in the liver.

[0290] Specifically, the model was induced in C57BL / 6 mice by feeding them with the AMLN diet and administering TAA (50-400 mg / kg, TIW-three times a week) for 16 weeks. The induced animals were divided into a vehicle control group and a group administered with a long-acting conjugate of SEQ ID NO:42 (1.3 nmol / kg, Q2D), and the substance was repeatedly administered subcutaneously over a period of 8 weeks at the end of the induction period. Mice that only consumed the AMLN diet were used as a negative control group. In addition, the expression levels of cytokines in the liver tissue of each mouse collected by autopsy were measured.

[0291] Specifically, as shown in Figure 11, the relative expression level of MCP-1, when taken as 1.0 for AMLN (vehicle control group), was 1.506 for AMLN / TAA (vehicle control group) and 0.984 for the long-acting conjugate of SEQ ID NO: 42, and the relative expression level of IL-6, when taken as 1.0 for AMLN (vehicle control group), was 1.61 for AMLN / TAA (vehicle control group) and 1.048 for the long-acting conjugate of SEQ ID NO: 42. In the group administered with the triple activity long-acting conjugate, it was confirmed that MCP-1 and IL-6 were reduced by 34.7% and 34.9%, respectively, compared to AMNL / TAA.

[0292] That is, as shown in FIG. 11, it was confirmed that the expression of MCP-1 and / or IL-6 in liver tissue was consistently decreased by administration of the triple activity long-acting conjugate.

[0293] In order to further confirm the hepatic inflammation improving effect confirmed in FIG. 11, the change in the level of human tumor necrosis factor-α (TNF-α) in a human macrophage line (THP-1 cell line) was measured.

[0294] Specifically, human macrophage strains were treated with phorbol 12-myristate 13-acetate (PMA) to differentiate for 72 hours, and then the medium was treated with the triple activator of SEQ ID NO: 42, the triple activator of SEQ ID NO: 66, the triple activator of SEQ ID NO: 67, the triple activator of SEQ ID NO: 97, and the triple activator of SEQ ID NO: 100, each of which was prepared in Example 1, at a concentration of 1 μM, and used as test groups. After treatment with each triple activator for 48 hours, the cells were further treated with lipopolysaccharide (LPS) for 6 hours to activate the inflammatory response.

[0295] After 12 hours, the changes in secreted human tumor necrosis factor α in the medium of the test groups treated with each triple activity, the negative control group not treated with either triple activity or LPS, and the positive control group not treated with the triple activity but only treated with LPS were measured using the Human TNF-α ELISA Kit and compared (Figure 12). Statistical analysis was performed using one-way ANOVA to compare the positive control group, the test group, and the negative control group.

[0296] As a result, it was confirmed that in all test groups in which the human macrophage line was treated with the triple activator, which can activate the GLP-1 receptor, the GIP receptor, and the glucagon receptor without being limited to a specific sequence, the amount of human tumor necrosis factor α secreted in the medium was significantly reduced compared to the positive control group treated with LPS only, and therefore the therapeutic effect against liver diseases accompanied by inflammation was also confirmed.

[0297] From the above examples, the present inventors have confirmed that the triple activity sustained conjugate of the present invention has therapeutic effects on various liver diseases such as non-alcoholic steatohepatitis, fatty liver, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

[0298] In summary, the triple activity compound and its long-acting conjugate of the present invention have therapeutic effects on liver diseases and are therefore useful for the production of drugs.

[0299] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A pharmaceutical composition for preventing or treating a liver disease, comprising: A pharma- ceutically acceptable excipient; A pharmaceutical composition comprising a pharma- ceutical effective amount of a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 102.

2. The pharmaceutical composition of claim 1 , wherein the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following chemical formula (1): 【Chemistry 1】 wherein X is a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 102; L is a linker containing repeating ethylene glycol units; F is an immunoglobulin Fc fragment or a derivative thereof; - indicates a covalent bond link between X and L, and between L and F.

3. The pharmaceutical composition according to claim 1 or 2, wherein the peptide is amidated at its C-terminus.

4. The pharmaceutical composition according to claim 1 or 2, wherein the liver disease is liver inflammation.

5. The pharmaceutical composition according to claim 4, characterized in that, when administered, the pharmaceutical composition reduces the expression of at least one of TNF-α, MCP-1, and IL-6 in liver tissue.

6. The pharmaceutical composition according to claim 1 or 2, wherein the liver disease is a metabolic liver disease.

7. The pharmaceutical composition according to claim 6, characterized in that, when administered, the pharmaceutical composition reduces the amount of triglycerides and / or cholesterol in liver tissue.

8. 3. The pharmaceutical composition according to claim 1 or 2, wherein the liver disease is at least one disease selected from the group consisting of simple fatty liver, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer.

9. 9. The pharmaceutical composition of claim 8, wherein the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

10. The pharmaceutical composition of claim 8, wherein the liver disease is non-alcoholic steatohepatitis accompanied by fatty liver, liver fibrosis or cirrhosis.

11. The pharmaceutical composition according to claim 8, wherein the liver disease is hepatocellular carcinoma caused by non-alcoholic steatohepatitis.

12. The pharmaceutical composition according to claim 1 or 2, wherein the liver disease is at least one disease selected from the group consisting of simple fatty liver, non-alcoholic fatty liver, and cirrhosis.

13. The pharmaceutical composition according to claim 1 or 2, wherein the liver disease is at least one disease selected from the group consisting of hepatic inflammation, non-alcoholic steatohepatitis and hepatic fibrosis.

14. The pharmaceutical composition of claim 13, wherein the liver disease is liver fibrosis, and the pharmaceutical composition, when administered, reduces blood levels of TIMP-1 and / or hyaluronic acid in an individual to which the pharmaceutical composition is administered.

15. 3. The pharmaceutical composition of claim 1 or 2, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

16. 16. The pharmaceutical composition of claim 15, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97 and 100.

17. 17. The pharmaceutical composition of claim 16, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96.

18. The pharmaceutical composition according to claim 2, wherein the formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

Images