EA046413B1 - THERAPEUTIC USE OF TRIPLE GLUCAGON/GLP-1/GIP RECEPTOR AGONIST OR ITS CONJUGATE AGAINST LIVER DISEASE - Google Patents

Description

Field of invention

The present invention relates to therapeutic uses against liver disease of a triple agonist having activities at all of the glucagon receptors, GLP-1 (glucagon-like peptide) and GIP (glucose-dependent insulinotropic peptide) or conjugates thereof.

Prior Art

The liver is one of the major organs in a living animal body, and typical examples of liver-related diseases include non-alcoholic fatty liver (NAFL), hepatitis, liver fibrosis, cholestatic liver disease, cirrhosis, liver failure, liver cancer, etc. It is known that viruses, alcohol, drugs, immune disorders, metabolic diseases, etc. can cause inflammation of the liver, and diseases such as liver fibrosis, cirrhosis, liver cancer, etc. can develop as liver inflammation progresses and becomes chronic.

In general, hepatitis, which is a disease characterized by inflammation of the liver, is the cause of most liver diseases, and it is known that as hepatitis progresses, various liver diseases (such as liver fibrosis, cirrhosis, etc.) may appear, accompanied by inflammation liver or caused by inflammation of the liver. Hepatitis can be divided, according to its characteristics, into acute hepatitis and chronic hepatitis, and it can be divided, according to its causes, into viral hepatitis, alcoholic hepatitis and drug-induced hepatitis. It is also assumed that cholestatic liver disease is caused by an inflammatory disease.

Other typical examples of liver disease include metabolic liver disease (eg fatty liver, non-alcoholic fatty liver disease (NAFLD; non-alcoholic steatohepatitis), steatohepatitis, liver fibrosis, cirrhosis, liver failure, liver cancer, etc.). Since these liver diseases can only be detected after significant development, due to the absence of any symptoms or their recognition in the early stages, they are considered the leading causes of death not only in Korea but also throughout the world, and thus there is there is a great need for the development of therapeutic drugs.

Liver fibrosis is the result of the wound healing process due to repeated liver injury, and normal recovery may be possible when the cause of liver injury is eliminated, but when liver fibrosis recurs, cirrhosis occurs and the fibrosis increases. Cirrhosis is a chronic disease that is pathologically accompanied by necrosis, inflammation and fibrosis of liver cells, and which develops into diseases such as complications of cirrhosis (eg liver failure), liver cancer, etc., ultimately leading to death. In particular, since cirrhosis can only be detected after significant development due to lack of recognition of its symptom in the early stages of the disease, research is being actively carried out to develop a method for quickly treating liver fibrosis, which is a condition preceding its development into cirrhosis, etc. Dr. Kunos' group recently described a drug that is being developed by chemically improving ibipinapant, which is a type of brain-penetrating cannabinoid receptor type 1 (CB-1) antagonist, but it is still unclear whether the drug is effective as a real-life drug (JCI Insight. 2016; 1(11): e87336.doi:10.1172/jci.insight.87336). Accordingly, there is still a need to develop a drug capable of treating fibrosis of various tissues or liver fibrosis, which can provide patient convenience without side effects.

Non-alcoholic steatohepatitis (NAFLD), a metabolic liver disease, is a disease that exhibits tissue abnormalities similar to alcoholic hepatitis despite no association with alcohol consumption, and it includes simple steatosis, non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) and etc. Non-alcoholic steatohepatitis (NAFLD) has shown an increasing trend along with increasing populations with obesity and diabetes, and the annual incidence rate in Korea is approximately 16%.

In order to prevent and/or treat such non-alcoholic steatohepatitis, efforts are being made to improve insulin resistance. For example, clinical trials are still active on thiazolidinediones (TZD) or metformin, which is a type of insulin sensitizer (Hepatology (2003) 38: 1008-17, J Clin Invest. (2001) 108: 1167-74).

However, in the case of treatment using TZD-based drugs, they have the disadvantages that they can cause large weight gain and reduce the flow rate of body fluids. Therefore, it is known that the use of these drugs is not possible for patients with heart disease. Because of these results, etc. The art has learned in various ways that direct use of drugs that are known to be effective in treating diabetes (eg, insulin resistance improving agents) as a therapeutic agent for the treatment of non-alcoholic steatohepatitis (NAFLD) may cause problems such as side effects.

Meanwhile, macrophages are known to be responsible for important immune responses in the liver and are involved in non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH)

- 1 046413 (Nat Rev Gastroenterol Hepatol. 2019 Mar; 16(3): 145-159). In particular, it is known that macrophages are activated in patients with nonalcoholic fatty liver disease (NAFLD), and that drugs targeting such macrophages can inhibit inflammation and fibrosis in the liver and demonstrate therapeutic efficacy against nonalcoholic steatohepatitis (NASH).

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are typical gastrointestinal hormones and neurohormones involved in the regulation of blood glucose components according to food intake. Glucagon is a peptide hormone secreted by the pancreas and is involved in the regulation of blood glucose levels, along with the two substances described above.

GLP-1 is a hormone secreted by the small intestine and is stimulated by food intake, and it stimulates the secretion of insulin in the pancreas in a blood glucose-dependent manner and inhibits the secretion of glucagon, thereby aiding the action of lowering blood glucose levels. In addition, GLP-1 plays a role in slowing digestive activity in the gastrointestinal tract by acting as a satiety factor and reducing food intake by slowing the time it takes digested food to pass through the gastrointestinal tract. In addition, administration of GLP-1 to mice has been reported to have the effect of inhibiting food intake and reducing body weight, and these effects have been confirmed to occur similarly in both normal and obese states, thus demonstrating the potential of GLP-1. 1 as a therapeutic agent for the treatment of obesity.

GIP, one of the gastrointestinal hormones secreted by food stimulation, like GLP-1, is a 42-amino acid hormone secreted by intestinal K cells. It has been reported that GIP has the functions of stimulating insulin secretion in the pancreas in a blood glucose-dependent manner, and helping to reduce blood glucose levels, having the effect of increasing GLP-1 activation, etc.

Glucagon is produced in the pancreas when blood glucose levels fall due to reasons such as drug exposure, illness, hormone or enzyme deficiency, etc. Glucagon sends a signal to break down glycogen in the liver to induce the release of glucose and thereby increase blood glucose levels to normal levels. In addition to the effect of increasing blood glucose levels, glucagon has been reported to suppress appetite in animals and humans, and to activate hormone-sensitive adipocyte lipase so as to stimulate lipolysis and energy expenditure, thereby demonstrating an anti-obesity effect.

Description of the invention

Technical problem.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of liver disease that contains a peptide that has activities against the glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic polypeptide (GIP) receptor, or a conjugate thereof.

Another object of the present invention is to provide a method for preventing or treating liver disease, which includes administering a peptide or a composition containing the peptide to a subject in need thereof.

Another object of the present invention is to provide the use of a peptide or a composition containing a peptide in the preparation of a medicament for the prevention or treatment of liver disease.

Technical solution.

To achieve the above objects, according to one aspect of the present invention there is provided a pharmaceutical composition for the prevention or treatment of liver disease, which contains a peptide having activities against the glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic polypeptide (GIP) receptor, or its conjugate.

In a specific embodiment, the pharmaceutical composition for preventing or treating liver disease comprises a pharmaceutically acceptable excipient; and a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102, in a pharmaceutically effective amount.

In another specific embodiment, the peptide is characterized in that it is in the form of a long-acting conjugate, and the long-acting conjugate is characterized in that it is represented by the following formula 1:

I[Formula 1]:

X-L-F, where in the above formula 1

X is a peptide with the amino acid sequence of any of SEQ ID NO: 1-102;

L is a linker comprising an ethylene glycol repeating unit;

F is an immunoglobulin Fc fragment or a derivative thereof; And

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- represents a covalent bond between X and L, and between L and F.

In the composition according to any of the previous specific embodiments, the peptide is characterized in that the C-terminus of the peptide is amidated.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is an inflammation of the liver.

In the composition according to any of the previous specific embodiments, the pharmaceutical composition is characterized in that it reduces the expression of at least one of TNF-α (tumor necrosis factor-alpha), MCP-1 (monocyte chemotactic protein-1) and IL-6 (interleukin-α). 6) in liver tissue upon administration.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is a metabolic liver disease.

In the composition according to any of the previous specific embodiments, the pharmaceutical composition is characterized in that it reduces the amount of triglycerides and/or cholesterol in liver tissue upon administration.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver (NAFL), liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic disease liver, liver fibrosis, cirrhosis, liver failure and liver cancer.

In the composition according to any of the previous specific embodiments, the cholestatic liver disease is characterized in that it is a disease selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is non-alcoholic steatohepatitis (NASH), which is accompanied by fatty liver, liver fibrosis or cirrhosis.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is liver cancer caused by non-alcoholic steatohepatitis (NASH).

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver (NAFL) and cirrhosis.

In the composition according to any of the previous specific 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 (NASH) and liver fibrosis.

In the composition according to any of the previous specific embodiments, the liver disease is characterized in that it is liver fibrosis, and the pharmaceutical composition is characterized in that it reduces, upon administration, the blood concentration of TIMP-1 (tissue inhibitor of metalloproteinases 1) and/or hyaluronic acid in the subject, to whom this pharmaceutical composition was administered.

In the composition according to any of the previous specific embodiments, the peptide is characterized in that it includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100.

In the composition according to any of the previous specific embodiments, the peptide is characterized in that it includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97 and 100 .

In the composition according to any of the previous specific embodiments, the peptide is characterized in that it includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 77 and 96.

In the composition according to any of the previous specific embodiments, the molecular weight according to the formula of the ethylene glycol repeating unit in L is in the range from 1 to 100 kDa.

According to another aspect of the present invention, there is provided a method of preventing or treating liver disease, which includes administering a peptide or a composition containing the peptide to a subject in need thereof.

According to another aspect of the present invention, there is provided the use of a peptide or a composition containing the peptide in the manufacture of a medicament for the prevention or treatment of liver disease.

According to yet another aspect of the present invention, there is provided the use of a peptide or a composition containing the peptide for the prevention or treatment of liver disease.

Beneficial effects.

The triple agonist or conjugate thereof of the present invention can be used to prevent or treat liver disease.

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Brief description of graphic materials

In fig. 1 is a graph illustrating the results of changes in NAFLD activity score (NAS) in mice when the long-acting conjugate SEQ ID NO: 42 was administered once every 2 days for 28 days in a mouse model of non-alcoholic steatohepatitis (NASH) induced by ingestion of methionine deficiency and choline (MCD) (*p less than 0.05; **p less than 0.01; ***p less than 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 2 is a graph illustrating the results of confirming the effect of improving fatty liver with the long-acting conjugate SEQ ID NO: 42 in mice with AMLN diet-induced steatohepatitis.

In fig. 3 is a graph and images illustrating the results of confirming the steatosis score reduction effect of the long-acting conjugate SEQ ID NO: 42 in mice with AMLN diet-induced steatohepatitis.

In fig. 4 is a graph illustrating the results of changes in ELF score according to the administration of long-acting conjugate SEQ ID NO: 42 in a mouse model of TAA (thioacetamide)-induced liver fibrosis (*p less than 0.05; **p less than 0.01; *** p less than 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 5 is a graph illustrating changes in the Sirius Red positive region of liver tissue following administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of TAA-induced liver fibrosis (*p less than 0.05; **p less 0.01; ***p less than 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 6 is a graph illustrating changes in the blood concentration of a liver fibrosis marker following administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of BDL (bile duct ligation)-induced liver fibrosis (*p less than 0.05; **p less than 0.01 ; ***p less than 0.001 relative to vehicle when determined by one-way ANOVA, fffp less than 0.01 relative to obeticholic acid when determined by unpaired t-test).

In fig. 7a shows images illustrating the results of Sirius red staining according to the administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of BDL-induced liver fibrosis.

In fig. 7b is a graph illustrating the liver tissue fibrosis score according to the administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of BDL-induced liver fibrosis (*p less than 0.05; **p less than 0.01; ***p less than 0.001 vs. carrier when determined by one-way ANOVA).

In fig. 8 shows images illustrating the results of H&E (hematoxylin and eosin) staining and a graph illustrating changes in the liver tissue inflammation score following administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of primary biliary cirrhosis (PBC) (*p less than 0.05; **p less than 0.01; ***p less than 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 9 shows images illustrating the results of H&E staining and a graph illustrating changes in the liver parenchymal necrosis score according to the administration of the long-acting conjugate SEQ ID NO: 42 in a mouse model of primary sclerosing cholangitis (PSC) (*p less than 0.05; **p less 0.01; ***p less than 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 10 is a graph illustrating changes in bile duct hyperplasia scores following administration of long-acting conjugate SEQ ID NO: 42 in a PSC (primary sclerosing cholangitis) mouse model (*p less than 0.05; **p less than 0.01; ***p less 0.001 relative to vehicle as determined by one-way ANOVA).

In fig. 11 is a graph illustrating changes in the expression levels of inflammation-related cytokines in liver tissue following administration of long-acting conjugate SEQ ID NO: 42 (*p less than 0.05; **p less than 0.01; ***p less than 0.001 vs. carrier when determined by one-way ANOVA).

In fig. 12 is a graph illustrating results confirming the effect of reducing human tumor necrosis factor-α (TNF-α) by triple agonists with SEQ ID NOs: 42, 66, 67, 97 and 100 in a human macrophage cell line.

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Detailed Description of the Invention

Hereinafter, the present invention will be described in more detail.

However, each of the explanations and exemplary embodiments disclosed in this specification may be applied to every other explanation and exemplary embodiment. That is, all combinations of the various factors disclosed herein are within the scope of the present invention. Moreover, the scope of the present invention should not be limited by the specific disclosure set forth below in the description of the invention.

Throughout the description of the present invention, not only the traditional one- and three-letter codes for naturally occurring amino acids are used, but also those three-letter codes that are generally accepted for other amino acids, such as α-aminoisobutyric acid (Aib), Sar (N-methylglycine), and α -methylglutamic acid. In addition, the amino acids mentioned here are abbreviated according to the rules of IUPAC-IUB (International Union of Pure and Applied Chemistry - International Biochemical Union) nomenclature as follows:

Alanine (Ala, A) arginine (Arg, R) asparagine (Asn, N) aspartic acid (Asp, D) cysteine (Cys, C) glutamic acid (Glu, E) glutamine (Gin, Q) glycine (Gly, G) histidine (His, H) isoleucine (Pe, I) leucine (Leu, L) lysine (Lys, K) methionine (Met, M) phenylalanine (Phe, F) proline (Pro, P) serine (Ser, S) threonine (Thr, T) tryptophan (Trp, W) tyrosine (Tyr, Y) valine (Vai, V)

To achieve the objects of the present invention, in one aspect of the present invention there is provided a pharmaceutical composition for the prevention or treatment of liver disease, which contains a peptide having activities against the glucagon receptor, glucagon-like peptide-1 (GLP-1) receptor and glucose-dependent insulinotropic polypeptide (GIP) receptor, and, in particular, a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102.

In the present invention, the term peptide having activities at the glucagon receptor, GLP-1 receptor and GIP receptor may be used interchangeably with the term triple agonist.

Such a peptide includes various substances that have significant levels of activity at the glucagon, GLP-1 and GIP receptors (eg various peptides).

A triple agonist having significant levels of activities at the glucagon, GLP-1 and GIP receptors may exhibit in vitro activities that are about 0.001% or greater, about 0.01% or greater, about 0.1% or greater, about 1 % or higher, about 2% or higher, about 3% or higher, about 4% or higher, about 5% or higher, about 6% or higher, about 7% or higher, about 8% or higher, about 9% or higher, about 10% or higher, about 20% or higher, about 30% or higher, about 40% or higher, about 50% or higher, about 60% or higher, about 70% or higher, about 80% or higher, about 90% or higher and about 100% or higher, with respect to one or more receptors, in particular two or more receptors and more particularly all three receptors of the glucagon receptors, GLP-1 and GIP compared to the natural ligands of the corresponding receptors (natural glucagon , natural GLP-1 and natural GIP), but the triple agonist is not specifically limited to these, and significant increase activity intervals are included without limitation.

In particular, receptor activities may include, for example, cases where in vitro activities are 0.1% or higher, 1% or higher, 2% or higher, 3% or higher, 4% or higher, 5% or higher , 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher , 70% or higher, 80% or higher, 90% or higher, 100% or higher, and about 200% or higher compared to, but are not limited to, natural receptors.

The term approximately, when used here, refers to an interval including all of plus/minus 0.5, plus/minus 0.4, plus/minus 0.3, plus/minus 0.2, plus/minus 0.1, etc. further, and it includes, but is not limited to, all meanings equivalent to, but not limited to, the meanings immediately following the term approximately, or meanings in a similar interval.

The method for measuring in vitro activity of a triple agonist may refer to Example 1 of the present invention, but the method is not specifically limited to this.

In this case, the triple agonist is characterized by the presence of one or more activities (1)-(3) described below, and, in particular, by its significant activity:

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1) activation of the GLP-1 receptor;

2) activation of the glucagon receptor and

3) activation of the GIP receptor.

In particular, receptor activation may include, for example, cases where the in vitro activities are about 0.1% or greater, about 1% or greater, about 2% or greater, about 3% or greater, about 4% or greater, about 5% or higher, about 6% or higher, about 7% or higher, about 8% or higher, about 9% or higher, about 10% or higher, about 20% or higher, about 30% or higher, about 40% or higher, about 50% or higher, about 60% or higher, about 70% or higher, about 80% or higher, about 90% or higher, and about 100% or higher compared to, but not limited to, natural receptors .

In addition, the peptide may be a peptide that has an increased in vivo half-life compared to, but is not particularly limited to, any of natural GLP-1, natural glucagon, and natural GIP.

The peptide may be a peptide that does not occur in nature, but is not particularly limited to these.

The peptide may be an analogue of natural glucagon, but is not particularly limited to these.

In particular, a natural glucagon analogue includes peptides that have at least one difference in amino acid sequence compared to the amino acid sequence of natural glucagon; peptides that are modified by modifying the sequence of natural glucagon; and natural glucagon mimetics.

Naturally occurring glucagon may have the following amino acid sequence, but is not particularly limited to:

His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-AlaGln-Asp-Phe-Val-Gln-Trp-Leu- Met-Asn-Thr (SEQ GO NO: 118)

In particular, the peptide may be a natural glucagon analogue in which at least one amino acid of the natural glucagon sequence has undergone a change selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof, but the peptide is not particularly limited to these.

In addition, an amino acid substitution includes both a substitution with an amino acid and a substitution with a non-natural compound.

Additionally, the addition may occur at the N-terminus and/or C-terminus of the peptide. However, the length of the amino acid segment to be attached is not particularly limited, and 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 than, 10 or more, 11 or more amino acids, and in a broad sense, joining may include joining a polypeptide, but the joining is not particularly limited to this.

More specifically, a peptide may be peptides wherein 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, 17 or more, 18 or more, 19 or more, or 20 amino acids selected from the group consisting of amino acids at positions 1 , 2, 3, 7, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28 and 29 in the amino acid sequence of natural glucagon are replaced by other amino acids, and, in addition In addition, may be peptides, wherein 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 are independently or additionally appended to their C-terminus, but the peptide is not specifically limited to them.

Even more specifically, a peptide may be peptides wherein 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, 17 or more, 18 or more, or 19 amino acids selected from the group consisting of amino acids at positions 1, 2, 3 , 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28 and 29 in the amino acid sequence of natural glucagon are replaced by other amino acids, and, in addition, may represent peptides where 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, independently or additionally attached to their C-terminus, but the peptide is not particularly limited to these.

Even more specifically, a given peptide may be peptides wherein 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 than, 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 amino acids at positions 1, 2, 3, 10, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 23, 24, 28 and 29 in the amino acid sequence of natural glucagon are replaced by other amino acids, and may further represent peptides, where 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

- 6,046,413 or more amino acids independently or additionally appended to their C-terminus, but the peptide is not specifically limited to these.

Even more specifically, a given peptide may be peptides wherein 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 than, 11 or more, 12 or more, 13 or more, or 14 amino acids selected from the group consisting of amino acids at positions 1, 2, 13, 16, 17, 18, 19, 20, 21, 23, 24, 27 , 28 and 29 in the amino acid sequence of natural glucagon are replaced by other amino acids, and may further be peptides, wherein 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 are independently or additionally appended to their C-terminus, but the peptide is not particularly limited to these.

The amino acids to be administered may be selected from the group consisting of tyrosine, αmethyl-glutamic acid, Aib, methionine, glutamic acid, histidine, lysine, leucine, isoleucine, glutamine, valine, glycine, alanine, cysteine, serine, alanine, aspartic acid and arginine, but the amino acids to be administered are not particularly limited to them.

For example, the amino acid sequence(s) to be attached may be one or more amino acid sequences derived from a native GLP-1 amino acid sequence, a native GIP amino acid sequence, or a native exendin-4 amino acid sequence.

Such a peptide may include an intramolecular bridge (eg, a covalent cross-link or a non-covalent cross-link) and, in particular, may be in a form including a ring, for example may be in a form where the ring is formed by the 16th amino acid and the 20th amino acid of the peptide, but the peptide is not specifically limited to this.

A non-limiting example of a ring may include a lactam bridge (or lactam ring).

In addition, peptide includes all peptides that are modified to include a ring or to include an amino acid capable of forming a ring at the target position.

For example, the peptide may be a peptide where one of the 16th and 20th amino acids is replaced by glutamic acid and the other by lysine, which may form a ring, but the peptide is not limited to this.

Such a ring may be formed by amino acid side chains within the peptide; for example, the ring may be in a form where a lactam ring is formed between the lysine side chain and the glutamic acid side chain, but the ring is not particularly limited to these.

Examples of a peptide produced by a combination of these methods may include peptides in which their amino acid sequences differ from the amino acid sequence of natural glucagon by at least one amino acid, and the α-carbon at their N-terminus is removed, in the presence of activities at the glucagon receptor, GLP-receptor 1 and GIP receptor, etc., but the peptide is not limited to this, and the peptide applicable to the present invention can be obtained by a combination of different methods for producing analogues.

In addition, with the peptide of the present invention, some of the amino acids may be replaced with other amino acids or non-natural compounds to avoid recognition by a degrading enzyme to increase the half-life of the peptide in vivo, but the peptide is not particularly limited to this.

In particular, the peptide may be a peptide in which the in vivo half-life is increased by avoiding recognition by a degradative enzyme by replacing the 2nd amino acid sequence among the amino acid sequences of the peptide, but any substitution or modification of amino acids to avoid recognition by a degrading enzyme in vivo included without limitation.

Moreover, such modification to produce a peptide includes all modifications using L-type or D-type amino acids and/or unnatural amino acids; and/or modification of the natural sequence, for example modification of a side chain functional group, intramolecular covalent binding (eg formation of a ring between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation, etc.

In addition, this modification also includes all modifications where one or more amino acids are added to the amino and/or carboxy terminus of the natural glucagon.

Not only the 20 amino acids commonly found in human proteins, but also atypical or non-naturally occurring amino acids can be used as amino acids to replace or add. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptides incorporating these amino acids and representative peptide sequences can be synthesized and purchased from commercial peptide synthesis companies (eg American Peptide Company, Bachem (USA) or Anygen (Korea)).

Amino acid derivatives can be prepared in the same way, and 4-imidazoacetic acid, etc. can be used as one such example.

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In addition, the peptide of the present invention may be in the form of a variant wherein the N-terminus and/or C-terminus, etc. the peptide is chemically modified or protected with organic groups, or amino acids may be added to the end of the peptide to protect it from proteases in vivo, increasing its stability.

In particular, in the case of a chemically synthesized peptide, its N- and C-termini are electrically charged. Therefore, in order to remove such charge, the N-terminus of a given peptide may be acetylated and/or the C-terminus of a given peptide may be amidated, but modification of the peptide is not particularly limited to this.

In addition, the peptide of the present invention includes all peptides in the form of the peptide itself, a salt thereof (eg a pharmaceutically acceptable salt thereof) or a solvate thereof. In addition, the peptide may be in any pharmaceutically acceptable form.

The type of salt is not particularly limited. However, the salt is preferably a salt that is in a safe and effective form for a subject (eg, a mammal), but the salt is not particularly limited to this.

The term pharmaceutically acceptable refers to a substance that can be effectively used for the desired use within the scope of a pharmacomedical solution without inducing excessive toxicity, irritation, allergic responses, etc.

The term pharmaceutically acceptable salt as used herein refers to a salt prepared from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids may 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-psulfonic acid, tartaric acid, acetic acid acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metal salts (eg sodium, potassium, etc.); alkaline earth metals (for example magnesium); ammonium, etc.

The term solvate, as used herein, refers to a complex formed by a peptide of the present invention or a salt thereof and a solvent molecule.

In another embodiment of the peptide, it may be a peptide that includes the amino acid sequence represented by the general formula I below:

Xaal-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaal0-Ser-Xaal2-Xaal3-Xaal4Xaa 15 -Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa20-Xaa21 -Phe-Xaa23 -Xaa24 -T rp-Leu-Xaa27Xaa28-Xaa29-Xaa30-Rl (General Formula 1, SEQ Ш NO: 103).

In general formula 1 above,

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;

Xaa14 is leucine, methionine or tyrosine;

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;

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;

Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid or histidine;

Xaa30 is cysteine, glycine, lysine or histidine, or absent; And

- 8 046413

R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107) or m-SSGQPPPS-n (SEQ ID NO: 108), or absent, where:

m is -Cys-, -Pro- or -Gly-Pro-; and n is -Cys-, -Gly-, -Ser- or -His-Gly-, or is absent.

Examples of a triple agonist may include examples that include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11 and SEQ ID NOs: 13-102, and examples that (substantially) consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11 and SEQ ID NO: 13-102, but the triple agonist is not limited to these.

In addition, although described in the present invention as a peptide consisting of a particular SEQ ID NO, such description does not exclude a mutation that may occur by adding a nonsense sequence above or below the amino acid sequence of the corresponding SEQ ID NO, or a mutation that may occur naturally manner, or a silent mutation thereof, provided that the peptide has an activity identical to or corresponding to that of a peptide that consists of the amino acid sequence of the corresponding SEQ ID NO, and it is clearly within the scope of the present invention even when the peptide has such sequence addition or mutation in it.

The above may be applicable to, but is not limited to, other specific embodiments or aspects of the present invention.

Specifically, in General Formula 1 above, Xaa14 may be leucine or methionine, and Xaa15 may be cysteine, aspartic acid or leucine.

Examples of such a peptide may include a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, 14-17 and 21-102; or a peptide that (essentially) consists of it, but the peptide is not particularly limited to these.

The peptide may significantly activate at least one of a glucagon receptor, a GLP-1 receptor, and a GIP receptor, but the peptide is not particularly limited to this. In particular, the peptide may be a peptide that significantly activates the GLP-1 receptor, or additionally significantly activates the glucagon receptor and/or the GIP receptor, but is not particularly limited to this.

More specifically, the peptide may be a peptide wherein in general formula 1 above

Xaa2 is glycine, α-methyl-glutamic 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;

Xaa18 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, but the peptide is not particularly limited to these.

More specifically, the peptide may be a peptide wherein in general formula 1 above

Xaa2 is glycine, α-methyl-glutamic 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;

Xaa17 is glutamine, arginine, isoleucine, cysteine or 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;

- 9 046413

Xaa23 is valine;

Xaa24 is alanine, glutamine, cysteine, asparagine or aspartic acid; And

Xaa27 is leucine or lysine, but the peptide is not particularly limited to this.

More specifically, the peptide may be a peptide wherein in general formula 1 above

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa7 is threonine;

Xaa10 is a 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;

Xaa17 is arginine, isoleucine, cysteine or lysine;

Xaa18 is alanine, arginine or histidine;

Xaa19 is alanine, glutamine or cysteine;

Xaa20 is lysine or glutamine;

Xaa21 is 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.

In particular, the peptide may be a peptide wherein in general formula 1 above

Xaa1 is histidine or 4-imidazoacetyl;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is a tyrosine;

Xaa12 is isoleucine;

Xaa13 is an alanine or cysteine;

Xaa14 is methionine;

Xaa15 is aspartic acid;

Xaa16 is glutamic acid;

Xaa17 is isoleucine or lysine;

Xaa18 is an 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 asparapine;

Xaa29 is glutamine or threonine; And

Xaa30 is cysteine or lysine, or absent.

More specifically, the peptide may be a peptide wherein in general formula 1 above

Xaa2 is glycine, α-methyl-glutamic acid or Aib;

Xaa3 is glutamine;

Xaa7 is threonine;

Xaa10 is tyrosine, cysteine or valine;

Xaa12 is lysine;

Xaa13 is a tyrosine;

Xaa14 is leucine;

Xaa15 is aspartic acid;

Xaa16 is glycine, glutamic acid or serine;

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;

- 10 046413

Xaa27 is leucine or lysine; And

Xaa29 is glycine, glutamine, threonine or histidine, but the peptide is not specifically limited to these.

Such a peptide may correspond to the case where the peptide has significant levels of activation at both the GLP-1 receptor and the glucagon receptor, or higher levels of activation compared to the level of activation at the GIP receptor; the case where a given peptide has significant levels of activation on all of the GLP-1 receptor, glucagon receptor and GIP receptor; or the case where the peptide has significant levels of activation at both the GLP-1 receptor and the GIP receptor and higher levels of activation compared to the level of activation at the glucagon receptor; but these cases are not specifically limited to them.

Examples of the peptide may include a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 9, 21-37, 39, 42, 43, 49-61, 64-83, 85, 86, 88, 89 , 91-93 and 95-102; or a peptide which (essentially) consists of it, but the peptide is not particularly limited to these.

In a specific embodiment, the peptide may include the amino acid sequence represented by the general formula 2 below.

Xaa 1 -Xaa2-Gln-Gly-Thr-Phe-Thr- Ser- Asp-Xaa 10- Ser-Ly s-Xaa 13 -Xaa 14-Xaa 15Xaa 16-Xaa 17-Xaa 18-Xaa 19-Xaa20-Xaa21 - Phe-Xaa23 -Xaa24-T rp-Leu-Leu-Xaa28 -Xaa29Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 2, SEQ Ш

NO: 104).

In General Formula 2 above, the peptide may be a peptide where:

Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;

Xaa2 is glycine, α-methyl-glutamic acid or Aib;

Xaa10 is a 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, glutamine, cysteine or valine;

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;

Xaa30 is proline or cysteine; And

Xaa40 is a cysteine or absent.

More specifically, the peptide may be a peptide wherein in general formula 2:

Xaa13 is alanine, tyrosine or cysteine;

Xaa15 is aspartic acid or glutamic acid;

Xaa17 is glutamine, arginine, cysteine or lysine;

Xaa18 is alanine, arginine or histidine;

Xaa21 is cysteine, glutamic acid, glutamine or aspartic acid;

Xaa23 is isoleucine or valine;

Xaa24 is cysteine, glutamine or asparagine;

Xaa28 is cysteine, asparagine or aspartic acid;

Xaa29 is glutamine, cysteine or histidine; And

Xaa30 is a cysteine, lysine or histidine.

Examples of the peptide may include a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 64-77 and 95-102; more specifically, a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64-77 and 96-102; or a peptide that (essentially) consists of them, but the peptide is not particularly limited to these.

In a specific embodiment, the peptide may include the amino acid sequence represented by the general formula 3 below.

- 11 046413

Xaa 1 -Xaa2-Gln-Gly-Thr-Phe-Thr- Ser- Asp-T yr-S er-Ly s-Xaa 13 -Leu-Asp-Glu-Xaa 17Xaal8-Xaal9-Lys-Xaa21-Phe-Val- Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-SerSer-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (General Formula 3, SEQ Ш NO: 105).

The peptide may be a peptide wherein in General Formula 3 above:

Xaal is histidine or tyrosine;

Xaa2 is α-methyl-glutamic acid or Aib;

Xaa13 is alanine, tyrosine or cysteine;

Xaa17 is arginine, cysteine or lysine;

Xaa18 is alanine or arginine;

Xaa19 is an alanine or cysteine;

Xaa21 is glutamic acid or aspartic acid;

Xaa24 is glutamine or asparagine;

Xaa28 is cysteine or aspartic acid;

Xaa29 is a cysteine, histidine, or glutamine;

Xaa30 is a cysteine or histidine;

Xaa31 is proline or cysteine; And

Xaa40 is a cysteine or absent.

Examples of the peptide may include a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 64-71, 75-77 and 96-102; or a peptide that (essentially) consists of it, but the peptide is not particularly limited to these.

Additionally, the peptide may be a peptide in which, in General Formula 1 above, R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO : 116) or PSSGQPPPSC (SEQ ID NO: 117), or absent, but the peptide is not particularly limited to these.

In addition, the peptide of the present invention can be synthesized according to its length by a method well known in the art (for example, using an automatic peptide synthesizer) and can be obtained through genetic engineering technology.

Specifically, the peptide of the present invention can be obtained using a standard synthesis method, a recombinant expression system, or any other method known in the art. Accordingly, the peptide of the present invention can be synthesized by many methods, including, for example, the methods described below:

(a) a method for synthesizing a peptide by a stepwise solid-phase or liquid-phase method, or by fragment assembly, followed by isolation and purification of the final peptide product; or (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and isolating the expressed product from a culture of the host cell; or (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and isolating the expressed product therefrom; or a method of producing peptide fragments by any combination of methods (a), (b) and (c), obtaining the peptide by coupling the peptide fragments and then isolating the peptide.

In particular, the composition according to the invention is a pharmaceutical composition for preventing or treating liver disease, and it may be a pharmaceutical composition that contains a pharmaceutically acceptable excipient; and a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102, or a peptide (essentially) consisting thereof, in a pharmaceutically effective amount.

In a more specific embodiment, the peptide may be a peptide that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 64, 66, 67, 70, 71, 76, 77, 96, 97 and 100, or (essentially) composed of it; a peptide that includes or (essentially) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 42, 43, 50, 66, 67, 77, 96, 97 and 100; or a peptide that includes or (essentially) consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96; but this peptide is not limited to these. Moreover, in the present invention, the peptide is in the form of a long-acting conjugate, and the long-acting conjugate may be a conjugate in which a biocompatible substance for increasing the half-life of the peptide in vivo is associated with a peptide having activities on the glucagon receptor, GLP-1 receptor and the GIP receptor. In the present description of the invention, the term biocompatible substance may be used interchangeably with the term carrier.

In the present invention, the peptide conjugate may exhibit an increased duration of effectiveness compared to a peptide to which the carrier is not coupled, and in the present invention

- 12 046413 In this paper, such a conjugate is called a long-acting conjugate, which can be used interchangeably with the conjugate.

In this case, the conjugate may be a conjugate that does not occur in nature.

In particular, the long-acting conjugate may be a conjugate represented by Formula I below, but the long-acting conjugate is not limited to this.

([Formula 1]

X-L-F where in formula 1 above

X is a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102;

L is a linker containing an ethylene glycol repeating unit;

F is an immunoglobulin Fc fragment or a derivative thereof; And

- represents a covalent bond between X and L, and between L and F.

In the above conjugate, F is a substance capable of increasing the half-life of X (i.e., a peptide having activities at the glucagon receptor, GLP-1 receptor and GIP receptor; and, in particular, a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102), and it corresponds to the structure of the moiety that constitutes the conjugate of the present invention.

F may be a substance that is linked to X by a covalent chemical bond or a non-covalent chemical bond, and in particular, F and X may be linked to each other through L by a covalent chemical bond.

In a particular embodiment, F may be an immunoglobulin Fc fragment or a derivative thereof, and more specifically, an immunoglobulin Fc fragment or derivative thereof may be derived from IgG, but F is not particularly limited to these.

In the present invention, the term immunoglobulin Fc portion refers to a region that includes portions of the heavy chain constant region 2 (CH2) and/or the heavy chain constant region 3 (CH3), excluding the variable regions of the immunoglobulin heavy and light chains. The Fc portion of an immunoglobulin may be a structure constituting a moiety of the conjugate of the present invention.

In the present invention, the Fc fragment includes not only the natural sequence obtained through papain cleavage of immunoglobulin, but also its derivative (i.e., sequences in which one or more amino acid residues in the natural sequence are modified by deletion, insertion, non-conservative or conservative substitutions or combinations thereof) and thus differs from the sequence of the natural form.

F may have a structure in which two polypeptide chains are linked by a disulfide bond, or a structure in which two polypeptide chains are linked through a nitrogen atom in only one of the two chains, but the structure of F is not limited to these. Bonding through the nitrogen atom can be accomplished with the epsilon N atom or the N-terminal amino group of lysine via reductive amination.

Reductive amination reaction refers to a reaction in which the amine group or amino group of a reactant reacts with the aldehyde of another reactant (ie, a functional group capable of reductive amination) to produce an amine, and then an amine bond is formed through reductive amination. The reductive amination reaction is an organic synthesis reaction widely known in the art.

In one embodiment, F may be a structure that binds through the N-terminal nitrogen of proline, but F is not limited to this.

Such an immunoglobulin Fc fragment may include, but is not limited to, a hinge region in the heavy chain constant region.

In the present invention, the Fc portion of an immunoglobulin may include a particular hinge sequence at the N-terminus.

The term hinge sequence, as used herein, refers to the region that is located in the heavy chain and forms a dimer of immunoglobulin Fc fragments through an intermolecular disulfide bond.

In the present invention, the hinge sequence may be a modified sequence in which part of the hinge sequence having the following amino acid sequence is deleted and thus there is only one cysteine residue in the sequence, but the hinge sequence is not limited to this:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ IDNO: 119).

The hinge sequence may be a sequence in which the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO: 119 is deleted, and thus only one cysteine residue is included in the sequence. The hinge sequence of the present invention may consist of 3-12 amino acids, including only one residue

- 13 046413 cysteine, but the hinge sequence is not limited to this. More specifically, the hinge sequence of the present invention may have the following sequences:

Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ Ш NO: 120), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ Ш NO: 121), Glu-Ser-Lys-Tyr-GlyPro-Pro-Cys-Pro-Ser (SEQ Ш NO: 122), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro -Pro (SEQ Ш NO: 123), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ Ш NO: 124), Glu-Ser-Lys-Tyr-Gly-ProPro-Cys (SEQ Ш NO : 125), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ Ш NO: 126), Glu-Ser-ProSer-Cys-Pro (SEQ Ш NO: 127), Glu-Pro-Ser-Cys- Pro (SEQ Ш NO: 128), Pro-Ser-Cys-Pro (SEQ Ш NO: 129), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ Ш NO: 130) , Lys-TyrGly-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-ProPro-Cys-Pro (SEQ ID NO: 134), Glu-Ser-Lys-Pro-Ser -Cys-Pro (SEQ ID NO: 135), Glu-SerPro-Ser-Cys-Pro (SEQ ID NO: 136), Glu-Pro-Ser-Cys (SEQ ID NO: 137) and Ser-Cys-Pro ( SEQ ID NO: 138).

More specifically, the hinge sequence may be a sequence that includes the amino acid sequence SEQ ID NO: 129 (Pro-Ser-Cys-Pro) or SEQ ID NO: 138 (Ser-Cys-Pro), but the hinge sequence is not limited to this.

The immunoglobulin Fc portion of the present invention may be in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence, and further, the Formula 1 conjugate of the present invention may be in a form in which one end of the linker associated with one chain of dimeric immunoglobulin Fc fragments, but the immunoglobulin Fc fragment and the conjugate of formula 1 are not limited to them.

The term N-terminus, as used herein, refers to the amino terminus of a protein or polypeptide, and it may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids from the most terminal end or terminal end of the amino end. The Fc portion of the immunoglobulin of the present invention may include a hinge sequence at the N-terminus, but the Fc portion of the immunoglobulin is not limited to this.

In addition, the Fc fragment of the immunoglobulin of the present invention may be an extended Fc fragment that includes all or part of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1), excluding the heavy chain and light chain variable regions immunoglobulin chain, provided that it has substantially the same or improved effect compared to its natural type. In addition, the Fc fragment of the immunoglobulin of the present invention may be a fragment in which some rather long amino acid sequences corresponding to CH2 and/or CH3 have been deleted.

For example, the Fc portion of the immunoglobulin of the present invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) CH1 domain and CH2 domain; 3) CH1 domain and CH3 domain; 4) CH2 domain and CH3 domain; 5) a combination of one or two or more domains of a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, and an immunoglobulin hinge region (or part of a hinge region); and 6) a dimer of each heavy chain constant region and light chain constant region domain, but the immunoglobulin Fc portion is not limited to these.

Additionally, in a particular embodiment, the immunoglobulin Fc moiety may be in dimeric form, or one molecule X may be covalently linked to one Fc moiety in dimeric form, and in particular, the immunoglobulin Fc and X may be linked to each other via a non-peptide polymer .

It is also possible that two X molecules are symmetrically bound to one Fc fragment in dimeric form. In particular, the immunoglobulin Fc and X may be linked to each other through a non-peptide linker, but the connection between the immunoglobulin Fc portion and X is not limited to the embodiments described above.

In addition, the Fc portion of the immunoglobulin of the present invention includes natural amino acid sequences as well as derivative sequences thereof. A derivative of an amino acid sequence means that a given amino acid sequence differs from its natural amino acid sequence due to the presence of a deletion, addition, conservative or non-conservative substitution, or a combination thereof at one or more than one amino acid residue in the natural amino acid sequence.

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

- 14 046413

In addition, different types of derivatives are possible, for example a derivative where the site capable of forming an intermolecular disulfide bond is removed; a derivative where several N-terminal amino acids have been removed from the natural Fc; derivative, where a methionine residue is added to the N-terminus of the natural Fc, etc. In addition, complement binding sites (eg C1q binding sites) or antibody-dependent cellular cytotoxicity (ADCC) sites may be deleted to eliminate effector function. Technologies for obtaining derivatives of the sequence of the Fc fragment of immunoglobulin are disclosed in international publications WO 97/34631, WO 96/32478, etc.

Amino acid substitutions in a protein or peptide that do not change the entire activity of the molecule are well known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most common substitutions occur 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, amino acids may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

In addition, the Fc derivatives described above may be derivatives that exhibit the same biological activity as the Fc region of the present invention and have improved structural stability of the Fc region with respect to heat, pH, etc.

In addition, such an Fc fragment may be derived from a naturally occurring type isolated from humans and animals (eg cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc.), or may be a recombinant or their derivatives obtained from transformed animal cells or microorganisms. In particular, the Fc fragment can be obtained from natural Fc by isolating complete immunoglobulins from human or animal organisms and treating them with a protease. Papain processing of the Fc fragment generates Fab and Fc fragments, and pepsin processing of the Fc fragment produces pF'c and F(ab) ₂ fragments. These fragments can be subjected to gel filtration to isolate Fc or pF'c. In a more specific embodiment, the Fc fragment may be a recombinant immunoglobulin Fc fragment, wherein the human Fc fragment is derived from a microorganism.

In addition, the Fc portion of an immunoglobulin may be in a naturally occurring glycan form, an increased glycan content relative to its native type, a reduced glycan content relative to its native type, or a deglycosylated form. Increasing, decreasing or removing immunoglobulin Fc glycans can be achieved by conventional methods such as a chemical method, an enzymatic method and a genetic engineering method using a microorganism. In particular, the Fc portion of an immunoglobulin, where glycans have been removed from the Fc, exhibits a significant reduction in binding affinity for complement (C1q) and a reduction or elimination of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and thus does not induce unnecessary immune responses in vivo. In this regard, an immunoglobulin Fc fragment in deglycosylated or aglycosylated form may be more suitable to fulfill the primary purpose of the present invention as a drug carrier.

The term deglycosylation, as used herein, means the removal of glycans from an Fc moiety using an enzyme, and the term aglycosylation means that the Fc moiety is produced in a non-glycosylated form in prokaryotes, and in a more specific embodiment in E. coli.

The Fc portion of the immunoglobulin may be derived from humans or animals (eg cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc.), and in a more specific embodiment, it may be derived from humans.

In addition, the Fc portion of an immunoglobulin may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, the Fc portion of an immunoglobulin may be derived from IgG or IgM, which are among the most abundant proteins in human blood, and in an even more specific embodiment, it may be derived from IgG, which is known to increase the half-lives of ligand binding proteins . In an even more specific embodiment, the immunoglobulin Fc fragment may be an IgG4 Fc fragment, and in a most specific embodiment, it may be an aglycosylated Fc fragment derived from human IgG4, but the immunoglobulin Fc fragment is not limited to this.

Additionally, in a particular embodiment, the immunoglobulin Fc fragment, which is a fragment of human IgG4, may be in the form of a homodimer in which the two monomers are linked by an internal disulfide bond (interchain form) between cysteines that represent the 3rd amino acid of each monomer. In particular, each monomer of the homodimer independently has/or may have an internal disulfide bond between the cysteines at positions 35 and 95; and an internal disulfide bond between cysteines at positions 141 and 199 (i.e., two internal disulfide bonds (interchain form)). In terms of the number of amino acids, each monomer may consist of 221 amino acids, and the amino acids forming a homodimer may consist of a total of 442 amino acids, but the number of amino acids is not limited thereto.

In particular, the Fc fragment of an immunoglobulin may be a fragment in which two

- 15 046413 monomers having the amino acid sequence SEQ ID NO: 139 (consisting of 221 amino acids) form a homodimer through an internal disulfide bond between cysteines, which represent the 3rd amino acid of each monomer, and in which the monomers of the homodimer independently form an internal disulfide bond between cysteines at positions 35 and 95, and an internal disulfide bond between cysteines at positions 141 and 199, but the Fc portion of immunoglobulin is not limited to this.

The term combination, as used herein, means that polypeptides encoding single chain immunoglobulin Fc fragments of the same origin are linked to a single chain polypeptide of a different origin to form a dimer or multimer. That is, a dimer or multimer can be prepared from two or more fragments selected from the group consisting of Fc fragments of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc.

In this case, L may be a non-peptide linker, for example a linker containing a repeating unit of ethylene glycol.

In the present invention, the term non-peptide linker includes a biocompatible polymer in which two or more than two repeating units are connected. The repeating units are linked to each other by any covalent bond that is not a peptide bond. The non-peptide linker may be one structure that constitutes the moieties of the conjugate of the present invention and corresponds to L in Formula 1 above. As a non-peptide linker that can be used in the present invention, any polymer that has resistance to proteases in vivo can be used, without limitation. In the present invention, the term non-peptide linker can be used interchangeably with non-peptide polymer.

The non-peptide linker, although not particularly limited, may be a linker containing an ethylene glycol repeat unit (for example, polyethylene glycol), and in addition those derivatives already known in the art and derivatives that can be easily prepared in the art. level of those skilled in the art are included within the scope of the present invention.

The non-peptide linker repeat unit may be an ethylene glycol repeat unit, and, in particular, the non-peptide linker may be a linker that includes a functional group used to end the conjugate upon inclusion of an ethylene glycol repeat unit. The long-acting conjugate of the present invention may be in a form in which X and F are linked through a functional group, but the long-acting conjugate is not limited to this. In the present invention, the non-peptide linker may include two, three or more than three functional groups, and each functional group may be the same or different from each other, but the non-peptide linker is not limited to this.

In particular, the linker may be polyethylene glycol (PEG), represented by Formula 2 below, but the linker is not limited to:

[Formula 2]

where n is from 10 to 2400, n is from 10 to 480, or n is from 50 to 250, but the range of n is not limited to this.

In the above long-acting conjugate, the PEG group may include not only the -(CH ₂ CH ₂ O) _n structure, but also an oxygen atom located between the linking element and the -(CH ₂ CH ₂ O) _n structure, but the PEG group is not limited to this .

Additionally, in a particular embodiment, the conjugate may have a structure in which an immunoglobulin fragment (F) is linked to a peptide (X) containing the amino acid sequence of any of SEQ ID NO: 1-102 by a covalent bond through a linker containing an ethylene glycol repeat unit, but the structure of the conjugate is not limited to this. Polyethylene glycol is a general term that includes all forms of ethylene glycol homopolymers, PEG copolymers and monomethyl PEG polymers (mPEG), but polyethylene glycol is not specifically limited to these.

The molecular weight of the non-peptide polymer may range from 1 to 100 kDa, in particular from 1 to 20 kDa, or from 1 to 10 kDa, but the molecular weight of the non-peptide polymer is not limited to this. In addition, the non-peptide linker of the present invention, which is linked to the polypeptide corresponding to F, may include not only one kind of polymer, but also a combination of different types of polymers.

In a particular embodiment, one end of the non-peptide linker may be linked to an amino group or thiol group F (for example, an immunoglobulin Fc moiety), while the other end may be linked to an amino group or thiol group X.

In particular, the non-peptide polymer may include a reactive group that may

- 16 046413 be associated with F (eg the Fc fragment of an immunoglobulin) and X at both ends thereof, respectively, and, more specifically, a reactive group that can be associated with an amino group located at the N-terminus of lysine or a thiol group of cysteine X; or with an amino group located at the N-terminus of lysine, or a thiol group of cysteine F, but the non-peptide polymer is not limited to these.

In addition, the reactive group of the non-peptide polymer, which can be linked to F (eg, the Fc portion of an immunoglobulin) and X, can be selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but the reactive group is not limited to this.

In the above, a propionaldehyde group or a butyraldehyde group can be used as an example of the aldehyde group, but the aldehyde group is not limited to this.

In the above, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate can be used as the succinimidyl derivative, but the succinimidyl derivative is not limited to this.

The non-peptide linker may be linked to X and F through these reactive groups, but the reactive groups are not particularly limited to them.

In addition, the final product obtained by reductive amination via an aldehyde bond is significantly more stable than a product linked by an amide bond. The aldehyde reactive group reacts selectively with the N-terminus at low pH, but it can form a covalent bond with the lysine residue at high pH (eg pH 9.0).

In addition, the reactive groups at each end of the non-peptide linker may be the same or different from each other. For example, a non-peptide linker may have a maleimide reactive group at one end, with an aldehyde group, propionaldehyde group or butyraldehyde group at the other end. However, the reactive groups are not particularly limited to them, provided that F (in particular the Fc portion of an immunoglobulin) may be linked to an X at each end of the non-peptide linker.

For example, the non-peptide linker may include, as a reactive group, a maleimide group at one end while including an aldehyde group, propionaldehyde group or butyraldehyde group at the other end.

When using polyethylene glycol having a reactive hydroxyl group at both ends thereof as a non-peptide polymer, the long-acting protein conjugate of the present invention can be prepared by activating the hydroxyl group to different reactive groups through known chemical reactions or by using commercially available polyethylene glycol having a modified reactive group. group.

In a specific embodiment, the non-peptide polymer may be a polymer that is linked to a cysteine X residue, and more specifically to the -SH group of the cysteine, but the non-peptide polymer is not limited to this.

For example, the non-peptide polymer may be a polymer that is linked to a peptide corresponding to X at the position of the 10th cysteine residue, the 13th cysteine residue, the 15th cysteine residue, the 17th cysteine residue, the 19th cysteine residue, 21 nth cysteine residue, 24th cysteine residue, 28th cysteine residue, 29th cysteine residue, 30th cysteine residue, 31st cysteine residue, 40th cysteine residue or 41st cysteine residue, but non-peptide polymer is not specifically limited to this.

In particular, the reactive group of a given non-peptide polymer may be linked to the SH group of a cysteine residue, and all of the above descriptions may apply to this reactive group. In the case where a maleimide-PEG aldehyde is used, the maleimide group is linked to the -SH group of X through a thioether bond, and the aldehyde group may be linked to F (in particular to the -NH2 Fc group of immunoglobulin) through a reductive amination reaction, but the linkage is not limited to this, and this connection simply represents one embodiment.

In addition, in the above conjugate, the reactive group of the non-peptide polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc portion, but this linkage simply represents one embodiment.

In addition, the conjugate may be a conjugate having an increased duration of effectiveness compared to native GLP-1, GIP or glucagon, or X, which is not modified with F, and such conjugate includes not only the forms described above, but also all forms encapsulated in biodegradable nanoparticles, etc.

The peptide of the present invention or a conjugate thereof may have use in the prevention or treatment of liver disease.

The term liver disease, as used herein, refers to a disease occurring in the liver, and it may include, but is not limited to, metabolic liver disease or liver inflammation. Common examples of liver disease may include simple steatosis, non-alcoholic fatty liver (NAFL), liver inflammation, non-alcoholic steatohepatitis

- 17 046413 (NASH), cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, liver cancer, etc., and provided that the disorder occurs in the tissues and functions of the liver, it may constitute a liver disease according to the present invention. In many cases, liver inflammation can occur due to reasons such as viruses, alcohol, drugs, immune disorders, metabolic diseases, etc., and liver inflammation is known to develop into diseases such as cirrhosis, liver cancer, etc. d. in accordance with the development and chronic nature of liver inflammation. The composition of the present invention may demonstrate an effect on liver disease accompanied or caused by liver inflammation (eg liver inflammation, non-alcoholic steatohepatitis (NASH) or liver fibrosis), but liver disease is not limited to this.

Moreover, the composition of the present invention may show an effect even in preventing or treating liver disease that is not accompanied by inflammation, and examples of such liver disease may include simple steatosis, non-alcoholic fatty liver (NAFL), cirrhosis, etc., but liver disease is not limited to this.

The liver disease for which the peptide of the present invention or a conjugate thereof has a therapeutic effect may be a metabolic liver disease, but the liver disease is not limited to this. Metabolic liver disease is a disease caused by an abnormal chemical reaction of the body that interferes with the body's metabolism, and it includes simple steatosis, fatty liver, steatohepatitis, etc.

The composition of the present invention may be a composition that exhibits a preventive effect or a therapeutic effect on metabolic liver disease by reducing the amount of triglycerides and/or cholesterol in liver tissue upon administration, but the composition is not limited to this. Metabolic liver disease may or may not be accompanied by inflammation, and examples of liver diseases that can be treated by the composition of the present invention may include simple steatosis, non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), etc., but liver diseases are not limited to them.

Non-alcoholic fatty liver disease (NAFLD), which is a typical example of metabolic liver disease, refers to a disease that is accompanied by fatty liver degeneration, even though the subject has no history of alcohol consumption, or is not associated with alcohol consumption. Fatty liver refers to the occurrence of a phenomenon in which triglycerides appear to be deposited abnormally in liver cells, unlike normal cases. Approximately 5% of a normal liver consists of fatty tissue. Although triglycerides, fatty acids, phospholipids, cholesterol and cholesterol esters are the main components of fat, once fatty liver disease occurs, most of the components are replaced by triglycerides, and when the amount of triglycerides is 5% or more of the liver weight, it is diagnosed as fatty liver disease . Fatty liver is caused by a disorder of fat metabolism in the liver cells or a defect in the process of transporting excess fat, etc., and it is mainly caused by a disorder of fat metabolism in the liver. Most of the fat accumulated during fatty liver disease may be triglycerides.

Non-alcoholic steatohepatitis disease (NAFLD) refers to a group of diseases that includes simple steatosis with only excess accumulation of fat in liver cells, non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) accompanied by hepatocellular necrosis, inflammation and fibrosis, etc. etc., but non-alcoholic steatohepatitis (NAFLD) is not limited to this, provided that the disease can be treated with the composition of the present invention. The non-alcoholic steatohepatitis disease (NAFLD) of the present invention may be a disease that is accompanied by non-alcoholic steatohepatitis (NASH), but non-alcoholic steatohepatitis disease (NAFLD) is not limited to this.

In addition, the liver disease for which the peptide of the present invention or a conjugate thereof has a therapeutic effect may be liver inflammation, but the liver disease is not limited to this. As used herein, the term liver inflammation, which is the most common cause of liver disease, refers to a disease that causes inflammation of the liver, and it is divided into acute hepatitis and chronic hepatitis according to causes and symptoms. Viruses, alcohol, drugs, immune disorders, metabolic diseases, etc. are the main reasons.

The composition of the present invention can reduce the expression of at least one of TNF-α, MCP-1 and IL-6 in liver tissue upon administration, and, thereby, the composition can exhibit a preventive effect or a therapeutic effect on liver inflammation, but the effects of this compositions are not limited to this.

The peptide of the present invention or a conjugate thereof may exhibit not only the effect of alleviating inflammation of the liver itself, but also a therapeutic effect on diseases accompanied or caused by liver inflammation (for example, hepatitis, non-alcoholic steatohepatitis (NASH), fibroid

- 18 046413 liver roses, etc.).

As used herein, non-alcoholic steatohepatitis (NASH), which is one of the non-alcoholic steatohepatitis diseases, is a typical example of liver disease accompanied by liver cell necrosis, inflammation and fibrosis. The composition of the present invention can suppress inflammation and fibrosis of the liver and, thereby, demonstrates an effect on non-alcoholic steatohepatitis (NASH), and in particular, can demonstrate an effect on non-alcoholic steatohepatitis (NASH) accompanied by fatty liver, liver fibrosis or cirrhosis; or liver cancer caused by non-alcoholic steatohepatitis (NASH), but the diseases are not limited to this.

The term liver fibrosis, as used herein, refers to the formation of excess fibrous connective tissue in organs or tissues during the reparative or response process as a result of the wound healing process due to repeated injury to the liver. Chronicity and exacerbation of liver inflammation are known to cause liver fibrosis. It is known that liver fibrosis is reversible (unlike cirrhosis), consists of thin fibrils and is not accompanied by the formation of nodules. Once the cause of liver damage stops, restoration of a normal liver may be possible. However, with the constant repetition of the process of liver fibrosis, the formation of cross-links between extracellular matrices (ECM) increases, thereby leading to the progression of irreversible cirrhosis with nodules.

The composition of the present invention may exhibit a preventive effect or a therapeutic effect on liver fibrosis and, in particular, liver fibrosis associated with non-alcoholic steatohepatitis (NASH), but the effects are not limited to this.

The composition of the present invention, when administered to a subject, may exhibit a preventive effect or a therapeutic effect on liver fibrosis in the subject to whom the composition is administered by reducing blood levels of TIMP-1 and/or hyaluronic acid, but the effects are not limited to this.

In particular, the peptide of the present invention or a conjugate thereof may exhibit an effect on liver fibrosis, and in particular, the effect may be to prevent or treat liver fibrosis by reducing the enhanced liver fibrosis (ELF) score.

The ELF score (Enhanced Liver Fibrosis Score) is a score that confirms the degree of healing of liver fibrosis, and it can be calculated according to the following equation. The ELF score can be calculated using the following equation after measuring the concentrations of hyaluronic acid (HA), procollagen type III N-terminal propeptide (PIIINP), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in blood samples:

ELF score = 2.278 + 0.851 In(CHA) + 0.751 In(CPIIINP) + 0.394 In(CTIMP-l)

The above reduction in ELF score may be a reduction from about 10% to about 100%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, or about 14% to about 30% compared to a group not administered a peptide of the present invention or a long-acting conjugate thereof, but the decline in ELF score does not stop there.

The composition may prevent or treat liver fibrosis by reducing the ELF score of a subject administered the composition to about 9.8 or lower, about 9.7 or lower, about 9.6 or lower, about 9.5 or lower, about 9.4 or below, about 9.3 or below, about 9.2 or below, or about 9.1 or below, but this reduction in ELF score is not limited to this.

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 cholestatic liver disease means that the production of bile in the liver is impaired due to conditions such as various diseases, dilated jugular nutrition, or side effects of specific medications (for example, some antibiotics). Common signs of cholestasis include fatigue, pruritis (itching), bile spillage, and xanthoma (subcutaneous deposits of cholesterol-rich substances). The effects of cholestasis are limitless and wide-ranging, causing worsening of liver disease to systemic disease, liver failure, and the need for liver transplantation. Causes of cholestatic liver disease may include acute hepatitis, inflammation of the bile ducts, etc.

Cholestatic liver disease may include primary biliary cholangitis (PBC), primary sclerosing cholangitis (PBC), progressive hereditary intrahepatic cholestasis (PFIC), and Alagille syndrome (AS), but cholestatic liver disease is not limited to these.

Primary biliary cirrhosis, which is also known as primary biliary cholangitis (PBC), is a cryptogenic chronic cholestatic liver disease. Progressive damage to the bile duct due to portal or periportal inflammation can cause progressive fibrosis and ultimately cirrhosis. To date, immunological, genetic and environmental factors are known as potential causes of primary biliary cirrhosis. Primary biliary cirrhosis mainly occurs in women

- 19 046413 middle age, and symptoms such as weakness, itching and hyperlipidemia of unknown origin may also appear with early onset of primary biliary cirrhosis.

It is now understood that primary biliary cirrhosis is an immune-mediated disease, and in particular, immunohistochemical staining of T lymphocytes in the portal and periportal regions demonstrates CD4-positive and CD8-negative T cells.

In addition, abnormal suppressor T cell activity has been described in asymptomatic first-degree relatives of affected subjects. It has been reported that interleukins may play a role in the pathogenesis of PBC by promoting altered immune functions and fibrosis (G. J. Webb et al., J. Autoimmunity, 2015 Nov; 64: 42-52).

The treatment for PBC is bile acid therapy using ursodeoxycholic acid (UDSA) and obeticholic acid (OCA). The mechanism of action of these two drugs in PBC is associated with their ability to activate FXR and TGFR-5 to exert their anti-inflammatory effects. However, a sufficient biochemical response was not achieved in almost 40% of patients treated with UDCA.

Primary sclerosing cholangitis (PSC) is a cryptogenic chronic cholestatic liver disease caused by inflammation and fibrosis of the intrahepatic and extrahepatic bile ducts. Specifically, it is an inflammatory disease of the bile ducts and bile tract, and as the disease progresses, fibrosis occurs and the wall of the bile duct becomes thickened, thereby narrowing the bile ducts.

The causes of this disease have not yet been identified, but a possible cause appears to be a combination of various factors, such as genetic factors, environmental factors and associated immune responses.

A subject is diagnosed as having primary sclerosing cholangitis when results of liver function tests through blood tests demonstrate increased alkaline phosphatase levels, increased aminotransferase levels, and an indication of gammaglobulinemia.

At the moment, the treatment method for PSC has not been clearly described, and liver transplant surgery is the only treatment that can radically cure PSC.

Accordingly, there is still a need to develop a drug capable of treating PBS and PSC without side effects while providing patient convenience.

Liver cirrhosis of the present invention is a chronic disease that occurs with repeated increasing regeneration of liver cells and fibrous tissue, it is pathologically accompanied by necrosis, inflammation and fibrosis and develops into complications of cirrhosis (for example liver failure) and disease (for example liver cancer), ultimately ultimately leading to death. In particular, since liver cirrhosis can only be detected after significant development due to lack of recognition of one's own symptoms in the early stages of this disease, it is necessary to quickly treat liver fibrosis, which is a condition before it develops into cirrhosis, etc. The composition of the present invention may exhibit a preventive effect or a therapeutic effect against liver cirrhosis and in particular non-alcoholic steatohepatitis (NASH) liver cirrhosis, but the effects of the composition are not limited to this.

In the present invention, liver failure refers to a condition in which liver function is weakened and the liver is unable to perform protein synthesis and metabolic functions as normal physiological functions due to viral hepatitis, cirrhosis, liver damage from drugs or alcohol, or liver disease. Liver failure is classified into acute liver failure and chronic liver failure according to the rate of development, and is known to cause various complications. Since the composition of the present invention exhibits effects such as inhibition of inflammation and fibrosis, it may exhibit a preventive effect or a therapeutic effect on liver failure.

In the present invention, liver cancer (hepatocellular carcinoma) refers to a malignant tumor arising from liver cells, and it can be classified into primary liver cancer (hepatocellular carcinoma), which occurs in the liver cells themselves, and metastatic liver cancer, which in which cancers of other tissues have metastasized to the liver, and approximately 90% or more of liver cancers are primary liver cancer. The main causes are alcohol, smoking, obesity, etc., besides hepatitis and chronic liver disease.

The composition of the present invention may exhibit a preventive effect or a therapeutic effect on liver cancer and in particular liver cancer caused by non-alcoholic steatohepatitis (NASH), but the effects of the composition are not limited to this.

The models used in the examples of the present invention are known as: MCD diet-induced non-alcoholic steatohepatitis (NASH) model; and AMLN diet-induced fatty liver and steatohepatitis models. In addition, the AMLN/TAA mouse model is known to be used as a model of liver fibrosis or non-alcoholic steatohepatitis (NASH). The above model is a model used in various disease-related studies

- 20 046413 liver, and in the examples of the present invention, the effect of the peptide of the present invention (triple agonist) or its long-acting conjugate was confirmed in each model, indicating that the peptide of the present invention (triple agonist) or its long-acting conjugate action is useful for the prevention or treatment of liver disease (eg hepatitis, liver fibrosis, simple steatosis, non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), etc.). Moreover, in the examples of the present invention, the improvement effect of the long-acting triple agonist conjugate was confirmed in PBC and/or PSC models, and the effect on cholestatic liver disease was also confirmed.

The composition of the present invention may be characterized in that there is no weight gain or a relatively mild degree of weight gain, which is a side effect of a traditional therapeutic agent for liver disease.

The composition of the present invention can prevent or treat liver disease by performing one or more of the following characteristics (a) to (k), but the characteristics to be performed are not limited to them.

(a) decrease in NAS values (non-alcoholic steatohepatitis disease activity score (NAFLD));

(b) reduction of triglyceride levels in the liver;

(c) reducing blood cholesterol levels;

(d) decrease in steatosis score;

(e) decrease in the levels of TNF-α, MCP-1 and IL-6 in liver tissue;

(e) decrease in liver inflammation score;

(g) decrease in the parenchymal necrosis score;

(h) decrease in the bile duct hyperplasia score; (i) reduction in enhanced liver fibrosis (ELF) score;

(j) a decrease in the blood concentration of TIMP-1 and/or hyaluronic acid (i.e., markers of liver fibrosis); and (k) decrease in fibrosis score.

The term prevention, as used herein, refers to all activities that inhibit or delay the onset of liver disease through administration of the above peptide or a composition containing the peptide, and the term treatment refers to all activities that improve or beneficially modify the symptoms of liver disease through administration the above peptide or a composition containing this peptide.

The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient, carrier or diluent. A pharmaceutically acceptable excipient, carrier or diluent may be a pharmaceutically acceptable excipient, carrier or diluent that does not occur naturally.

The term pharmaceutically acceptable, as used herein, refers to the property of having a sufficient amount to exhibit a therapeutic effect and not cause side effects, and can be readily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, age, body weight, health status, gender, patient sensitivity to the drug, route of administration, route of administration, frequency of administration, duration of treatment, drug(s) to be mixed or administered simultaneously, etc.

The pharmaceutical composition of the present invention containing this peptide may further contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may include, for oral administration, a binder, lubricant, disintegrant, solubilizer, dispersant, stabilizer, suspending agent, coloring agent, flavoring agent, etc.; for injection: buffering agent, preservative, analgesic, solubilizer, isotonic agent, stabilizer, etc., which can be combined for use; and for local administrations: base, excipient, lubricant, preservative, etc.

The formulation type of the composition of the present invention can be prepared in various ways by combining with a pharmaceutically acceptable excipient described above. For example, for oral administration, the composition can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, the composition can be prepared in single-dose ampoules or multi-dose containers. This composition may also be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained release preparations, etc.

Examples of suitable carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone , water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, this composition may additionally contain fillers

- 21 046413 body, anticoagulant, lubricant, humectant, fragrance, preservative, etc.

Moreover, the pharmaceutical composition of the present invention may be any type of preparation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid oral medicine, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized drugs and suppositories.

In addition, the present composition can be formulated into a unit dosage form suitable for administration to a patient, and specifically can be formulated as a drug used for peptide drugs according to a conventional method in the pharmaceutical field, so as to be administered by the oral or parenteral route (including, but not limited to, the dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal route.

In addition, the conjugate can be used mixed with various pharmaceutically acceptable carriers such as saline or organic solvents. To increase stability or absorption capacity, carbohydrates (eg glucose, sucrose or dextrans), antioxidants (eg ascorbic acid or glutathione), chelating agents, low molecular weight proteins or other stabilizers, etc. can be used as pharmaceutical agents.

The dosage and frequency of administration of the pharmaceutical composition of the present invention is determined by the type of active ingredient(s), along with various factors such as the disease being treated, route of administration, age, sex and weight of the patient, severity of the disease, etc. In particular, the composition of the present invention may be a composition that contains a peptide containing the amino acid sequence of any of SEQ ID NO: 1-102, or a long-acting conjugate containing the peptide, in a pharmaceutically effective amount, but the composition of the present invention is not limited to this.

The content of a peptide or a long-acting conjugate thereof in a pharmaceutically effective amount refers to the level at which the desired pharmacological activity (for example, the prevention, amelioration or treatment of liver disease) can be obtained by the peptide or a long-acting conjugate thereof, and may further refer to the level where no toxicity or side effects are observed or are observed at a non-negligible level in the subject to be administered, or may be at, but not limited to, a pharmaceutically acceptable level. The pharmaceutically effective amount as such can be determined by comprehensive consideration of the number of administrations, the patient, the drugs, etc.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose or may be administered over an extended period of time in multiple doses according to a fractionated treatment protocol. In the pharmaceutical composition of the present invention, the content of the active ingredient(s) may vary depending on the severity of the disease. In particular, the total daily dose of the conjugate of the present invention may range from about 0.0001 mg to 500 mg per kg of body weight of the patient. However, the effective dose of the conjugate is determined by taking into account various factors, including the patient's age, body weight, health status, gender, severity of the disease, diet and excretion rate, as well as the route of administration and frequency of treatment with the pharmaceutical composition. In this regard, those skilled in the art can readily determine an effective dose suitable for a particular use of the pharmaceutical composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited to the type of preparation and the route and method of administration, as long as it exhibits the effects of the present invention.

The pharmaceutical composition of the present invention has excellent duration of in vivo efficacy and titer, and thus the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced.

To achieve the objects of the present invention, according to yet another aspect of the present invention, there is provided a method of preventing or treating liver disease, which includes administering a peptide or a composition containing the peptide to a subject in need thereof.

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

In the present invention, the subject refers to a subject suspected of having liver disease, and the subject suspected of having liver disease refers to a mammal, including humans, rats, cattle, etc., that has or is at risk of developing liver disease , but any subject that can be treated with a conjugate of the present invention or a composition containing the conjugate is included without limitation.

The term administration, as used herein, refers to the administration of a specific substance to subjects

- 22 046413 that by any suitable route, and the route of administration of the composition may be any conventional route that provides delivery of the composition to the target (for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.), but the route of administration is not limited to this.

The method of the present invention may include administering a pharmaceutical composition containing the peptide in a pharmaceutically effective amount.

A suitable total daily dosage of the pharmaceutical composition may be determined within the scope of the proper medical judgment of the practitioner, and the pharmaceutical composition may be administered one or more times in divided doses. However, for the purposes of the present invention, it is preferable that the specific therapeutically effective dose of the pharmaceutical composition for any particular patient will vary depending on the type and extent of responses to be achieved, the particular compositions, including the possible use with them of other agents from time to time, the age of the patient, body weight, health status, gender and diet, time of administration, route of administration, release rate of the composition, duration of treatment, other drugs used in combination or concurrently with particular compositions, and similar factors well known in the medical field.

To achieve the objects of the present invention, according to yet another aspect of the present invention, the use of a peptide or a composition containing the peptide for the prevention or treatment of liver disease is provided.

To achieve the objects of the present invention, according to yet another aspect of the present invention, there is provided the use of a peptide or a composition containing the peptide in the preparation of a medicament for the prevention or treatment of liver disease.

The peptide or composition containing the peptide, liver disease, prevention and treatment are as described above.

Below, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Measurement of in vitro activities of triple agonists and their long-acting conjugates.

Example 1-1. Preparation of triple agonists.

Triple agonists showing activity at all of the GLP-1, GIP and glucagon receptors have been prepared and their sequences are shown in Table 1. 1 below.

Table 1

SEQ ID NO Subsequence Information 1 HXQGTFTSDVSSYLDGQAAKEFIAWLVKGC - 2 HXQGTFTSDVSSYLDGQAQKEFIAWLVKGC - 3 HXQGTFTSDVSSYLLGQAAKQFIAWLVKGGG PSSGAPPPSC - 4 HXQGTFTSDVSSYLLGQQQKEFIAWLVKGC - 5 HXQGTFTSDVSSYLLGQQQKEFIAWLVKGGG PSSGAPPPSC - 6 HXQGTFTSDVSSYLDGQAAKEFVAWLLKGC - 7 HXQGTFTSDVSKYLDGQAAKEFVAWLLKGC - 8 HXQGTFTSDVSKYLDGQAAQEFVAWLLKGC - 9 HXQGTFTSDVSKYLDGQAAQEFVAWLLAGC - 10 HXQGTFTSDVSKYLDGQAAQEFVAWLLAGG GP S S GAPPP S C - AND CAGEGTFTSDLSKYLDSRRQQLFVQWLKAGG R S S GAPPP SHG -

- 23 046413

12 CAGEGTFISDLSKYMDEQAVQLFVEWLMAGG P S S GAPPP SHG - 13 CA GE G T F I S D Y S I Q L D E I A V Q D F VE W L L A Q K P SSGAPPPSHG - 14 CAGQGTFTSDYSIQLDEIAVRDFVEWLKNGGP SSGAPPPSHG - 15 CAGQGTFTSDLSKQMDEEAVRLFIEWLKNGG P S S GAPPP SHG - 16 CAGQGTFTSDLSKQMDSEAQQLFIEWLKNGG P S S GAPPP SHG - 17 CAGQGTFTSDLSKQMDEERAREFIEWLLAQK P S S GAPPP SHG - 18 CAGQGTFTSDLSKQMDSERAREFIEWLKNTG P S S GAPPP SHG - 19 CAGQGTFTSDLSIQYDSEHQRDFIEWLKDTGP S S GAPPP SHG - 20 CAGQGTFTSDLSIQYEEEAQQDFVEWLKDTG P S S GAPPP SHG - 21 YXQGTFTSDYSKYLDECRAKEFVQWLLDHHP SSGQPPPS Education rings 22 YXQGTFTSDYSKCLDEKRAKEFVQWLLDHHP SSGQPPPS Education rings 23 YXQGTFTSDYSKYLDECRAKEFVQWLLAQK GKKNDWKHNIT Education rings 24 YXQGTFTSDYSKYLDECRAKEFVQWLKNGG P S S GAPPP S Education rings 25 HXQGTFTSDCSKYLDERAAQDFVQWLLDGG P S S GAPPP S - 26 HXQGTFTSDCSKYLDSRAAQDFVQWLLDGGP SSGAPPPS - 27 HXQGTFTSDYSKYLDERACQDFVQWLLDQG GPS SGAPPPS -

- 24 046413

28 HXQGTFTSDYSKYLDEKRAQEFVCWLLAQK GKKNDWKHNIT - 29 HXQGTFTSDYSKYLDEKAAKEFVQWLLNTC Education rings thirty HXQGTFTSDYSKYLDEKAQKEFVQWLLDTC Education rings 31 HXQGTFTSDYSKYLDEKACKEFVQWLLAQ Education rings 32 HXQGTFTSDYSKYLDEKACKDFVQWLLDGG P S S GAPPP S Education rings 33 HXQGTFTSDYSIAMDEIHQKDFVNWLLAQKC Education rings 34 HXQGTFTSDYSKYLDEKRQKEFVNWLLAQK C Education rings 35 HXQGTFTSDYSIAMDEIHQKDFVNWLLNTKC Education rings 36 HXQGTFTSDYSKYLCEKRQKEFVQWLLNGGP SSGAPPPSG Education rings 37 HXQGTFTSDYSKYLDECRQKEFVQWLLNGGP SSGAPPPSG Education rings 38 CAXQGTFTSDKSSYLDERAAQDFVQWLLDGG P S S GAPPP S S - 39 HXQGTFTSDYSKYLDGQHAQCFVAWLLAGG GPS SGAPPPS - 40 HXQGTFTSDKSKYLDERACQDFVQWLLDGG P S SGAPPPS - 41 HXQGTFTSDKSKYLDECAAQDFVQWLLDGG P S SGAPPPS - 42 YXQGTFTSDYSKYLDEKRAKEFVQWLLDHHP SSGQPPPPSC Education rings 43 YXQGTFTSDYSKYLDEKRAKEFVQWLLDHH CSSGQPPPS Education rings

- 25 046413

44 HGQGTFTSDCSKQLDGQAAQEFVAWLLAGG P S S GAPPP S - 45 HGQGTFTSDCSKYMDGQAAQDFVAWLLAGG P S S GAPPP S - 46 HGQGTFTSDCSKYLDEQHAQEFVAWLLAGGP SSGAPPPS - 47 HGQGTFTSDCSKYLDGQRAQEFVAWLLAGG P S S GAPPP S - 48 HGQGTFTSDCSKYLDGQRAQDFVNWLLAGG P S S GAPPP S - 49 CAXQGTFTSDYSICMDEIHQKDFVNWLLNTK Education rings 50 HXQGTFTSDYSKYLDEKRAKEFVQWLLDHHP SSGQPPPSC Education rings 51 HXQGTFTSDYSKYLDEKRQKEFVQWLLNTC Education rings 52 HXQGTFTSDYSKYLDEKRQKEFVQWLLDTC Education rings 53 HXEGTFTSD YSIAMDEIHQKDFVNWLLAQC Education rings 54 HXEGTFTSD YSIAMDEIHQKDFVDWLLAEC Education rings 55 HXQGTFTSDYSIAMDEIHQKDFVNWLLAQC Education rings 56 HXQGTFTSDYSKYLDEKRQKEFVNWLLAQC Education rings 57 HXQGTFTSDYSIAMDEIHQKDFVNWLLNTC Education rings 58 HXQGTFTSDYSKYLDEKRQKEFVQWLLNTKC Education rings 59 CAXQGTFTSDYSICMDEKHQKDFVNWLLNTK Education rings

- 26 046413

60 C A X Q G T F T S D Y S IA M D E K N S K D F V N W L L N T K Education rings 61 CA X Q G T F T S D Y S I AM D E I A S K D F VN W L LN T K Education rings 62 CAXQGTFTSDKSKYLDERAAQDFVQWLLDGG P S S GAPPP S - 63 CAXQGTFTSDCSKYLDERAAQDFVQWLLDGG P S S GAPPP S - 64 YXQGTFTSDYSKYLDECAAKEFVQWLLDHHP SSGQPPPS Education rings 65 HXQGTFTSDYSKCLDEKRAKEFVQWLLDHHP SSGQPPPS Education rings 66 YXQGTFTSDYSKYLDECRAKDFVQWLLDHH P S S GQPPP S Education rings 67 YXQGTFTSDYSKYLDECAAKDFVQWLLDHH P S S GQPPP S Education rings 68 YXQGTFTSDYSKCLDEKAAKEFVQWLLDHHP SSGQPPPS Education rings 69 YXQGTFTSDYSKCLDERAAKEFVQWLLDHHP SSGQPPPS Education rings 70 YXQGTFTSDYSKCLDEKRAKDFVQWLLDHH P S S GQPPP S Education rings 71 YXQGTFTSDYSKYLDERACKDFVQWLLDHH P S S GQPPP S Education rings 72 YXQGTFTSDCSKYLDERAAKDFVQWLLDHH P S S GQPPP S Education rings 73 CAXQGTFTSDYSKYLDECRAKEFVQWLLDHH P S S GQPPP S Education rings 74 CAXQGTFTSDYSKCLDEKRAKEFVQWLLDHH P S S GQPPP S Education rings 75 YXQGTFTSDYSKYLDEKAAKEFVQWLLDHH PSSGQPPPSC Education rings

- 27 046413

76 YXQGTFTSDYSKYLDEKRAKDFVQWLLDHH PSSGQPPPSC Education rings 77 YXQGTFTSDYSKYLDEKAAKDFVQWLLDHH PSSGQPPPSC Education rings 78 HXQGTFTSDYSKYLDEKRQKEFVQWLLDTKC Education rings 79 HXEGTFTSDYSIAMDEIHQKDFVNWLLAQKC Education rings 80 HXEGTFTSDYSIAMDEIHQKDFVDWLLAEKC Education rings 81 CAXQGTFTSDYSKYLDEKRQKEFVQWLLNTC Education rings 82 CAXQGTFTSDYSKYLDEKRQKEFVQWLLDTC Education rings 83 CAXEGTFTSDYSIAMDEIHQKDFVNWLLAQC Education rings 84 CAXEGTFTSDYSIAMDEIHQKDFVDWLLAEC Education rings 85 CAXQGTFTSDYSIAMDEIHQKDFVNWLLAQC Education rings 86 CAXQGTFTSDYSKYLDEKRQKEFVNWLLAQC Education rings 87 CAXQGTFTSDYSIAMDEIHQKDFVNWLLNTC Education rings 88 CAXQGTFTSDYSKYLDEKRQKEFVQWLLNTK C Education rings 89 CAXQGTFTSDYSKYLDEKRQKEFVQWLLDTK C Education rings 90 CAXEGTFTSDYSIAMDEIHQKDFVNWLLAQK C Education rings 91 CAXEGTFTSDYSIAMDEIHQKDFVDWLLAEK C Education rings

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92 CAXQGTFTSDYSIAMDEIHQKDFVNWLLAQK C Education rings 93 CAXQGTFTSDYSKYLDEKRQKEFVNWLLAQK WITH Education rings 94 CAXQGTFTSDYSIAMDEIHQKDFVNWLLNTK C Education rings 95 YXQGTFTSDYSKYLDEKRAKEFVQWLLCHHP SSGQPPPS Education rings 96 YXQGTFTSDYSKYLDEKRAKEFVQWLLDHCP SSGQPPPS Education rings 97 YXQGTFTSDYSKYLDEKRAKEFVQWLLDCHP SSGQPPPS Education rings 98 YXQGTFTSDYSKALDEKAAKEFVNWLLDHH PSSGQPPPSC Education rings 99 YXQGTFTSDYSKALDEKAAKDFVNWLLDHH PSSGQPPPSC Education rings 100 YXQGTFTSDYSKALDEKAAKEFVQWLLDQH PSSGQPPPSC Education rings 101 YXQGTFTSDYSKALDEKAAKEFVNWLLDQH PSSGQPPPSC Education rings 102 YXQGTFTSDYSKALDEKAAKDFVNWLLDQH PSSGQPPPSC Education rings

In the sequences described in table. 1, the amino acids indicated by X represent aminoisobutyric acid (Aib), which is a non-natural amino acid, and the underlined amino acids represent the formation of a ring between the underlined amino acids. In addition, in table. 1 CA represents 4-imidazoacetyl and Y represents tyrosine.

Example 1-2. Preparation of a long-acting triple agonist conjugate.

To PEGylate the cysteine residue of the triple agonists (SEQ ID NOs: 21, 22, 42, 43, 50, 77 and 96) from Example 1 using PEG (10 kDa) having a maleimide group and an aldehyde group at each end, i.e. maleimide-PEG-aldehyde (10 kDa, NOF, Japan), triple agonists and maleimidePEG-aldehyde were reacted in a molar ratio of 1:1-3 with a protein concentration of 1 mg/ml to 5 mg/ml at low temperature for 0. 5-3 hours. Specifically, this reaction was carried out in a medium in which 20% to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). Upon completion of this reaction, the reagents were applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple agonists, which were mono-PEGylated on cysteine.

The purified monopegylated triple agonists and immunoglobulin Fc were then reacted in a molar ratio of 1:1-5, with protein concentrations of 10 mg/mL to 50 mg/mL at 4°C to 8°C for 12 to 18 hours. This reaction was carried out in a medium in which 10 mM to 50 mM sodium cyanoborohydride (NaCNBH ₃ ; reducing agent) and 10% to 30% isopropanol were added to 100 mM potassium phosphate buffer (pH 6.0). Upon completion of this reaction, the reagents were applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify conjugates involving triple agonists and immunoglobulin Fc.

Once prepared, the purity, analyzed by reverse phase chromatography, gel filtration and ion exchange chromatography, was shown to be 95% or higher.

In particular, a conjugate in which a triple agonist of SEQ ID NO: 21 and an immunoglobulin Fc were linked through a PEG was referred to as a conjugate comprising a triple agonist of SEQ ID NO: 21 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 21, and these terms may be used interchangeably in the present invention.

In particular, the conjugate in which the triple agonist of SEQ ID NO: 22 and an immunoglobulin Fc were linked through PEG was called a conjugate comprising a triple agonist of SEQ ID NO: 22 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 22, and these terms may be used interchangeably in the present invention.

In particular, the conjugate in which the triple agonist of SEQ ID NO: 42 and an immunoglobulin Fc were linked through PEG was called a conjugate comprising a triple agonist of SEQ ID NO: 42 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 42, and data the terms may be used interchangeably in the present invention.

Specifically, a conjugate in which the triple agonist of SEQ ID NO: 43 and the immunoglobulin Fc were

- 29 046413 linked via PEG, a conjugate comprising a triple agonist of SEQ ID NO: 43 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 43 was named, and these terms can be used interchangeably in the present invention.

In particular, a conjugate in which a triple agonist of SEQ ID NO: 50 and an immunoglobulin Fc were linked through PEG was called a conjugate comprising a triple agonist of SEQ ID NO: 50 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 50, and these terms may be used interchangeably in the present invention.

In particular, a conjugate in which a triple agonist of SEQ ID NO: 77 and an immunoglobulin Fc were linked through PEG was called a conjugate comprising a triple agonist of SEQ ID NO: 77 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 77, and these terms may be used interchangeably in the present invention.

In particular, a conjugate in which a triple agonist of SEQ ID NO: 96 and an immunoglobulin Fc were linked through a PEG was called a conjugate comprising a triple agonist of SEQ ID NO: 96 and an immunoglobulin Fc or a long-acting conjugate of SEQ ID NO: 96, and these terms may be used interchangeably in the present invention.

Example 1-3: In vitro measurement of the activities of triple agonists and their long-acting conjugates

The activities of the triple agonists and their long-acting conjugates prepared in Examples 1-1 and 1-2 were measured using an in vitro cellular activity assay using cell lines transformed with the GLP-1 receptor, the glucagon receptor (GCG) and the GIP receptor respectively.

Each of the above cell lines is a cell line in which the genes of human GLP-1 receptor, human GCG receptor and human GIP receptor are transformed into Chinese hamster ovary (CHO), respectively, to be expressed therein and are thus suitable for measurements of GLP-1, GCG and GIP activities. Accordingly, the activity of each part was measured using the corresponding transformed cell line.

To measure the GLP-1 activities of the long-acting triple agonists and their conjugates prepared in Examples 1-1 and 1-2, human GLP-1 was subjected to a 4-fold serial dilution from 50 nM to 0.000048 nM, and the triple agonists and their conjugates long-acting, obtained in examples 1-1 and 1-2, were subjected to 4-fold serial dilution from 400 nM to 0.00038 nM. The culture solution was removed from the cultured CHO cells in which the human GLP1 receptor was expressed, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μl, respectively, and a buffer solution containing anti-cAMP antibody was added therein in an amount of 5 μl and cultured at room temperature. temperature for 15 minutes. Then, a detection mixture containing a cell lysis buffer was added to them in an amount of 10 μl to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After completion of the reaction, cell lysates were applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC ₅₀ value through accumulated cAMP, and these values were compared with each other. Relative titers compared to human GLP-1 are shown in Table. 2 and 3 below.

To measure the GCG activities of the triple agonists and their long-acting conjugates prepared in Examples 1-1 and 1-2, human GCG was subjected to a 4-fold serial dilution from 50 nM to 0.000048 nM, and the triple agonists and their long-acting conjugates obtained in examples 1-1 and 1-2, were subjected to 4-fold serial dilution from 400 nM to 0.00038 nM. The culture solution was removed from the cultured CHO cells in which the human GCG receptor was expressed, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μl, respectively, and a buffer solution containing anti-cAMP antibody was added therein in an amount of 5 μl and cultured at room temperature. temperature for 15 minutes. Then, a detection mixture containing a cell lysis buffer was added to them in an amount of 10 μl to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After completion of the reaction, cell lysates were applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC ₅₀ value through accumulated cAMP, and these values were compared with each other. Relative titers compared to human GCG are shown in Table. 2 and 3 below.

To measure the GIP activities of the triple agonists and their long-acting conjugates prepared in Examples 1-1 and 1-2, human GIP was subjected to a 4-fold serial dilution from 50 nM to 0.000048 nM, and the triple agonists and their long-acting conjugates obtained in examples 1-1 and 1-2, were subjected to 4-fold serial dilution from 400 nM to 0.00038 nM. The culture solution was removed from the cultured CHO cells in which the human GIP receptor was expressed, and each of the serially diluted substances was added to the CHO cells in an amount of 5 μl, respectively, and a buffer solution containing anti-cAMP antibody was added to them in an amount of 5 μl and cultured at room temperature for 15 minutes. Then, a detection mixture containing cell lysis buffer was added to them in an amount of 10 μl to lyse the cells, and the reaction was carried out at room temperature for 90 minutes. After completion of the reaction, cell lysates

- 30 046413 was applied to the LANCE cAMP kit (PerkinElmer, USA) to calculate the EC50 value through accumulated cAMP, and these values were compared with each other. Relative titers compared to human GIP are shown in Table. 2 and 3 below.

Table 2 _____Relative ratio of triple agonist titers_____

In vitro activity compared to natural peptide (%) SEQ Ш NO rel. GLP-1 rel. glucagon rel. GIP 1 3.2 <0.1 <0.1 2 5.9 <0.1 <0.1 3 1.8 <0.1 <0.1 4 8.5 <0.1 <0.1 5 42.1 <0.1 <0.1 6 17.0 <0.1 <0.1 7 13.7 <0.1 <0.1 8 14.2 0.10 <0.1 9 32.1 0.13 <0.1 10 46.0 <0.1 <0.1 AND 1.4 <0.1 <0.1 12 0.4 <0.1 <0.1 13 <0.1 <0.1 <0.1 14 28.0 <o,1 <0.1 15 79.2 <0.1 <0.1

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16 2.1 <0.1 <0.1 17 0.2 <o,l <0.1 18 <θ,ι <0.1 <0.1 19 <0.1 <0.1 <0.1 20 <0.1 <0.1 <0.1 21 17.8 267 22.7 22 20.1 140 59.7 23 4.01 9.3 <0.1 24 41.2 9.3 <0.1 25 82.6 ο,ι <0.1 26 64.5 0.2 <0.1 27 83.1 0.8 0.9 28 17.2 1.6 <0.1 29 38.5 6.0 <0.1 thirty 142 0.7 0.8 31 135 2.2 2.4 32 151 1.7 8.8 33 24.5 <0.1 10.4 34 19.1 0.92 0.6 35 7.5 <0.1 1.3 36 37.4 0.39 0.2 37 236 6.21 2.2 38 2.3 - - 39 13.9 0.53 <0.1 40 75.2 <0.1 <0.1 41 34.3 <0.1 <0.1 42 33.9 205.8 7.8 43 12.6 88.4 3.70 44 1.3 <0.1 <0.1 45 6.6 <0.1 <0.1 46 1.4 <0.1 <0.1 47 2.4 <0.1 <0.1

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48 1.5 <θ,ι <0.1 49 29.8 <0.1 3.3 50 67.4 50.5 2.7 51 14.4 2.0 0.1 52 44.1 7.5 0.3 53 161 8.4 1.3 54 30.6 1.4 0.1 55 27.1 0.7 2.4 56 57.9 4.9 0.8 57 11.7 <0.1 o, s 58 39.1 2.6 0.2 59 40.3 <0.1 4.0 60 106.2 <0.1 8.2 61 59.8 <0.1 2.8 62 5.2 <0.1 <0.1 63 15.3 <0.1 <0.1 64 64.6 60.1 92.9 65 95.4 25.2 11.6 66 15.8 172 17.2 67 28.5 46.2 39.8 68 27.9 8.8 107 69 24.3 9.6 62.8 70 15.1 71.3 64.4 71 90.1 12.7 94.7 72 11.5 ι,ο 1.6 73 22.6 5.4 h,o 74 12.9 0.9 ι,ο 75 35.1 8.5 18.0 76 10.3 47.6 11.7 77 38.7 12.2 35.5 78 51.0 14.0 0.12 79 41.5 4.9 1.4

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80 8.1 0.0 0.1 81 7.8 0.3 <0.1 82 9.5 1.1 <0.1 83 47.3 1.3 0.4 84 4.2 <0.1 <0.1 85 4.3 <0.1 o, s 86 28.4 0.4 0.2 87 0.9 <0.1 <0.1 88 9.6 0.3 <0.1 89 7.1 0.7 <0.1 90 7.4 <0.1 <0.1 91 31.9 16.8 0.3 92 0.8 <0.1 0.4 93 5.7 o, s 0.7 94 0.5 <0.1 <0.1 95 2.1 0.4 <0.1 96 34.4 194.8 5.2 97 10.5 62.8 2.6 98 28.1 8.2 47.1 99 20.9 14.9 57.7 100 42.2 12.7 118.5 101 23.2 13.9 40.1 102 23.3 29.5 58.0

Table 3

Relative titer ratio of long-acting triple agonist conjugates

Long acting conjugate In vitro activity compared to natural peptide (%) rel. GLP-1 rel. glucagon rel. GIP 21 0.1 1.6 0.2 22 0.1 0.9 0.5 42 3.1 23.1 1.2 43 2.1 13.5 0.6 50 15.4 6.9 0.7 77 6.7 1.7 6.6 96 o, s 4.0 o, s

The novel long-acting triple agonist conjugates obtained above have the function of triple agonists that can activate all of GLP-1 receptors, GIP receptors and glucagon receptors, and thus these long-acting triple agonist conjugates can be used as a therapeutic agent for the treatment of target disease.

Example 2: Confirmation of the therapeutic effect of triple agonists on metabolic liver disease.

The present inventors have attempted to confirm the therapeutic effect of the triple agonists of the present invention on metabolic liver disease.

Example 2-1. Effect of NASH treatment in MCD diet-induced NASH mice.

First, the effect of long-acting triple agonist conjugates on non-alcoholic steatohepatitis (NASH) was confirmed as follows.

A mouse model of non-alcoholic steatohepatitis (NASH) was induced by feeding a methionine-choline-deficient (MCD) diet for 2 weeks in C57BL/6 mice.

To confirm the therapeutic effect of the developed substance on the treatment of NASH, mice were divided into normal mice; mice with NASH induction (control group with excipient) and group

- 34 046413 pu, to which the long-acting conjugate SEQ ID NO: 42 was administered (0.36 nmol/kg, 0.72 nmol/kg and 1.44 nmol/kg Q2D (once every two days)), and this substance in repeated regimen was administered subcutaneously for 4 weeks. After repeated administration for 4 weeks, liver tissue was collected from each mouse by autopsy and subjected to hematoxylin and eosin (H&E) staining to assess the extent of NASH development through the non-alcoholic steatohepatitis (NAS) activity score.

As shown in FIG. 1, as a result of repeated administration of the long-acting conjugate SEQ ID NO: 42 for 4 weeks, it was confirmed that the NAS value in liver tissue was significantly reduced (control group NASH with excipient - 3.71; group that was administered the conjugate SEQ ID NO: 42 long-acting (0.36 nmol/kg) - 2.57; group that was administered the conjugate SEQ ID NO: 42 long-acting (0.72 nmol/kg) - 0.43; group that was administered the conjugate SEQ ID NO: 42 long-acting (1.44 nmol/kg) - 0).

From the above results, the therapeutic effect on NASH was confirmed for the long-acting conjugate SEQ ID NO: 42, which is a typical triple agonist conjugate of the present invention.

Example 2-2. Effect of ameliorating fatty liver induced by AMLN diet in mice.

In addition, the present inventors used a mouse model of AMLN so as to confirm the effect of the triple agonists of the present invention on improving fatty liver.

The AMLN diet is known to be high in fat, fructose and cholesterol, and thus is known to induce obesity and steatohepatitis when fed over a long period of time. Therefore, the AMLN mouse model is used as a model of steatohepatitis.

Mice induced by a 37-week AMLN diet were divided into an excipient control group; obeticholic acid group (30 mg/kg, QD (once daily), oral) and long-acting conjugate group SEQ ID NO: 42 (2.6 nmol/kg, Q2D, subcutaneous) ; and they were subjected to repeated administration for 12 weeks. After 12 weeks of repeated administration, liver tissue was collected from each mouse by autopsy, and the effectiveness of improvement in fatty liver was assessed by measuring the fat content of the liver tissue and by H&E staining.

As a result, it was confirmed that with repeated administration for 12 weeks of SEQ ID NO: 42 long-acting conjugate, triglyceride and cholesterol levels were significantly reduced in the SEQ ID NO: 42 long-acting conjugate group compared to the levels of the excipient control group and the excipient control group. which was administered obeticholic acid. From this result, it was confirmed that the liver fat content was reduced by the long-acting conjugate SEQ ID NO: 42 of the present invention (FIG. 2).

In addition, to further confirm the improvement effect on fatty liver according to the administration of the long-acting conjugate SEQ ID NO: 42, the present inventors tested the changes in steatosis score upon administration of the long-acting conjugate SEQ ID NO: 42 in the same manner as described above.

As a result, it was confirmed that upon administration of the long-acting conjugate SEQ ID NO: 42, the steatosis score (i.e., a value indicating the level of steatosis) was significantly reduced compared to the levels of the excipient control group and the obeticholic acid group (FIG. 3). ).

Example 3: Confirmation of the therapeutic effect of triple agonists on liver fibrosis.

Example 3-1. Confirmation of the effect of improving liver fibrosis index in mice with liver fibrosis induced by TAA administration.

To confirm the improvement effect on liver fibrosis by the long-acting triple agonist conjugates obtained in Example 1, an AMLN/TAA (thioacetamide) mouse model known as a model of liver fibrosis was used. Briefly, C57BL/6 mice were exposed to AMLN chow and TAA administration (50 mg/kg to 400 mg/kg, TIW: 3 times per week) for 16 weeks to induce the model. Induced animals were divided into an excipient control group and a long-acting conjugate SEQ ID NO: 42 group (1.3 nmol/kg, Q2D), which was selected as a typical triple agonist, and the corresponding substance was administered subcutaneously in a repeated manner over the course of 8 weeks induction period. Mice fed only the AMLN diet were used as negative controls. After 8 weeks of repeated administration, blood concentrations of hyaluronic acid, tissue inhibitor of metalloproteinase-1 (TIMP-1) and procollagen type III N-terminal propeptide (PIIINP) were analyzed in blood samples obtained by blood collection and, thereby, an enhanced score was calculated. liver fibrosis (ELF), which is known as a non-invasive index of liver fibrosis.

As shown in FIG. 4, it was confirmed that as a result of repeated administration of the conjugate

- 35 046413

SEQ ID NO: 42 long-acting over 8 weeks, the ELF score, which increased in the AMLN/TAA excipient control group, decreased significantly.

These results indicate that the triple agonists of the present invention or a long-acting conjugate thereof can significantly reduce the ELF score and thus have a preventive or therapeutic effect on liver fibrosis.

Example 3-2. Confirmation of the therapeutic effect on liver fibrosis in mice with liver fibrosis induced by TAA administration.

Based on the effect of improving the non-invasive liver fibrosis index confirmed in Example 3-1, an invasive method was used to directly evaluate the therapeutic effect of long-acting triple agonist conjugates on liver fibrosis. Briefly, liver tissue from mice used in Example 3-1 (8-week repeat administration) was collected by autopsy and then subjected to Sirius red staining.

The result confirmed that the positive area in liver tissue detected by Sirius red staining was also significantly reduced by administration of the long-acting triple agonist conjugate (FIG. 5).

These results indicate that the triple agonist of the present invention or a long-acting conjugate thereof has a preventive effect or a therapeutic effect on liver fibrosis.

Example 3-3. Confirmation of the improvement effect on liver fibrosis in mice with BDL-induced liver fibrosis.

In order to confirm the improvement effect on liver fibrosis of the long-acting conjugate SEQ ID NO: 42 confirmed in Examples 3-1 and 3-2, a mouse model of bile duct ligation (BDL), which is known as a model of liver fibrosis, was used. Briefly, C57BL/6 mice were anesthetized and cholestasis was induced in these mice by suturing the bile duct through surgery, and thereby liver fibrosis was induced in the mice. Induced animals were divided into an excipient control group and a long-acting conjugate SEQ ID NO: 42 selected as a representative triple agonist (1.3 nmol/kg, Q2D, subcutaneous administration). Mice treated with obeticholic acid (30 mg/kg, QD, oral administration), which is the active pharmaceutical ingredient of Ocaliva®, were used as a control. The drug administration was repeated for 2 weeks, starting on the 2nd day after surgery. Sham-exposed mice were used as a negative control. Blood concentrations of TIMP-1 and hyaluronic acid, which are typical markers of liver fibrosis, were measured using blood samples obtained by drawing blood from mice that were repetitively dosed with excipient, long-acting conjugate SEQ ID NO: 42, or obeticholic acid for 2 weeks. .

The results confirmed that the concentrations of TIMP-1 and hyaluronic acid were concordantly decreased by administration of the long-acting triple agonist conjugate (Figure 6).

These results again indicate that the triple agonist of the present invention or its long-acting conjugate has a therapeutic effect on liver fibrosis.

Example 3-4. Confirmation of therapeutic effect on liver fibrosis in mice with BDL-induced liver fibrosis.

Based on the effect of improving the non-invasive liver fibrosis index confirmed in Example 3-3, an invasive method was used to directly evaluate the therapeutic effect of long-acting triple agonist conjugates on liver fibrosis. Briefly, liver tissue from mice used in Example 3-3 (2-week boost) was collected by autopsy and then subjected to Sirius red staining. The fibrosis score was measured based on Sirius red staining.

As a result, it was confirmed that with repeated administration for 2 weeks of the long-acting conjugate SEQ ID NO: 42, the fibrosis score, which increased in the BDL excipient control group, was significantly reduced (Figs. 7a and 7b).

The above results indicate that the triple agonist of the present invention or a long-acting conjugate thereof can be used as an agent for the prevention or treatment of liver fibrosis.

Example 4: Confirmation of the effect of a triple agonist on liver inflammation.

In order to confirm the therapeutic effect on cholestatic liver disease, which is a liver disease, experiments were carried out as follows.

Example 4-1. Confirmation of the improvement effect in primary biliary cirrhosis (PBC) in BDL-induced PBC mice.

To confirm the improvement effect in primary biliary cirrhosis (PBC) by the long-acting triple agonist conjugates obtained in Example 1, a bile duct ligation (BDL) mouse model, which is known as the PBS model, was used. Briefly, C57BL/6 mice were anesthetized and cholestasis was induced in these mice by suturing the bile duct through surgery, and thereby liver inflammation was induced in the mice.

- 36 046413

Induced animals were divided into an excipient control group and a long-acting conjugate group SEQ ID NO: 42 (1.3 nmol/kg, Q2D, subcutaneous administration). As a control, mice were treated with obeticholic acid (30 mg/kg, QD, oral administration), which is the active pharmaceutical ingredient of Ocaliva®, commercially available as a therapeutic agent for the treatment of PBC. The drug administration was repeated for 2 weeks, starting on the 2nd day after surgery. Sham-exposed mice were used as a negative control. After 2 weeks of repeated administration, liver tissue was collected from each mouse by autopsy, and the effect of improving liver inflammation was assessed by H&E staining.

The result confirmed that with repeated administration of the long-acting triple agonist conjugate over 2 weeks, the inflammation score, which had increased in the BDL excipient control group, was significantly reduced (Figure 8).

From the above results, it was confirmed that the long-acting triple agonist conjugate had an excellent ameliorating effect on liver inflammation in PBC mice.

Example 4-2. confirmation of the ameliorative effect on primary sclerosing cholangitis (PSC) in BDL-induced PSC mice.

To confirm the ameliorative effect on primary sclerosing cholangitis (PSC) by the long-acting triple agonist conjugates prepared in Example 1, a bile duct ligation (BDL) mouse model, which is known as a PSC model, was used.

Specifically, C57BL/6 mice were anesthetized and cholestasis was induced in the mice by suturing the bile duct through surgical therapy, and thereby liver and bile duct lesions were induced in the mice. Induced animals were divided into an excipient control group and a long-acting conjugate SEQ ID NO: 42 group (1.3 nmol/kg, Q2D). Repeated subcutaneous administration of this conjugate was carried out for 2 weeks, starting on the 2nd day after surgery. After 2 weeks of repeated administration, liver tissue was collected from each mouse by autopsy, and the improvement effect on liver and bile duct injuries was assessed by H&E staining.

As a result, it was confirmed that with repeated administration of the long-acting triple agonist conjugate for 2 weeks, the parenchymal necrosis score measured by bile reflux, which increased in the BDL excipient control group, was significantly reduced (Fig. 9).

Moreover, as shown in FIG. 10, it was confirmed that the increase in bile duct hyperplasia score due to bile duct injury was significantly reduced by administration of a long-acting triple agonist conjugate.

From the above results, it was confirmed that the long-acting triple agonist conjugate could improve liver and bile duct damage and alleviate liver inflammation in PSC mice.

Example 4-3. Confirmation of the improvement effect on liver inflammation in TAA-treated mice.

To confirm the effect of improving liver inflammation by the long-acting triple agonist conjugates obtained in Example 1, the present inventors used an AMLN/TAA (thioacetamide) mouse model.

Specifically, C57BL/6 mice were exposed to an AMLN diet and TAA administration (50 mg/kg to 400 mg/kg, TIW: 3 times per week) for 16 weeks to induce the model. Induced animals were divided into an excipient control group and a SEQ ID NO: 42 long-acting conjugate group (1.3 nmol/kg, Q2D) and the corresponding compound was administered subcutaneously in a repeated manner over the last 8 weeks of the induction period. Mice fed only the AMLN diet were used as negative controls. In addition, the level of cytokine expression in the liver tissue of each mouse collected by autopsy was measured.

In particular, referring to FIG. 11, it was confirmed that when the MCP1 expression level of AMLN (excipient control group) was set to 1.0, the relative expression level of MCP-1 was 1.506 for AMLN/TAA (excipient control group) and 0.984 for AMLN/TAA ( long-acting conjugate SEQ ID NO: 42); and, taking the IL-6 expression level of AMLN (excipient control group) to 1.0, the relative IL-6 expression level was 1.61 for AMLN/TAA (excipient control group) and 1.048 for AMLN/TAA (conjugate SEQ ID NO: 42 long acting). It was confirmed that the expression levels of MCP-1 and IL-6 in the long-acting triple agonist conjugate groups were reduced by 34.7% and 34.9% relative to the level for AMLN/TAA, respectively.

That is, as shown in FIG. 11, it was confirmed that the expression levels of MCP-1 and/or IL-6 in liver tissue were concordantly decreased by administration of a long-acting triple agonist conjugate.

- 37 046413

Moreover, to further confirm the improvement effect on liver inflammation confirmed in FIG. 11, measured changes in human tumor necrosis factor-α (TNFα) levels in a human macrophage cell line (THP-1 cell line).

Specifically, this human macrophage cell line was treated with phorbol 12-myristate 13-acetate (PMA) and allowed to differentiate for 72 hours. The resulting human macrophage cell line was then treated with each of the triple agonists SEQ ID NO: 42, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 97 and SEQ ID NO: 100 in media to a concentration of 1 μM, and each was used as an experimental group. Treatment with each of the triple agonists was carried out for 48 hours and then treated with lipopolysaccharide (LPS) for 6 hours so as to activate the inflammatory response.

After 12 hours, using an ELISA kit for human TNF-α, changes in the amount of human tumor necrosis factor-α (TNFα) that was secreted into each of the media of the experimental groups treated with each of the triple agonists were measured; negative control group not treated with triple agonist and LPS; and a positive control group treated with LPS only without triple agonist treatment, and the results were compared (Fig. 12). For statistical analysis, results were compared between the positive control group, test groups and negative control group using one-way ANOVA.

As a result, it was confirmed that the amount of human tumor necrosis factor-α (TNFα) that was secreted into the media of all experimental groups in which treatment with triple agonists that are capable of activating all of the GLP-1, GIP and glucagon receptors, without limitation to specific sequences, carried out on a human macrophage cell line was significantly reduced compared to the amount in the positive control group treated with LPS only. From this result, the therapeutic effect of triple agonists on liver disease accompanied by inflammation was confirmed.

Through the above Examples, the present inventors have confirmed that the long-acting triple agonist conjugates of the present invention can demonstrate a therapeutic effect on various liver diseases such as non-alcoholic steatohepatitis (NASH), fatty liver, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis ( PSC).

In general, the triple agonists of the present invention and their long-acting conjugates have a therapeutic effect on liver disease, and thus can be effectively used in the manufacture of drugs.

From the foregoing, one skilled in the art to which the present invention relates will appreciate that the present invention may be embodied in other specific forms without modification of the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention. To the contrary, the present invention is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present invention as defined in the appended claims.

List of sequences <110> HANMI PHARM. CO., LTD.

<120> Therapeutic use of Trigonal glucagon/GLP-1/GIP receptor agonist or a conjugate thereof for liver disease <130> OPA20113 <150> KR 10-2019-0077776 <151> 2019-06-28 <150> KR 10- 2020-0004379 <151> 2020-01-13 <150> KR 10-2020-0004386 <151> 2020-01-13 <150> KR 10-2020-0069219 <151> 2020-06-08 <160> 139

- 38 046413 <170> KoPatentIn3.0 <210>1 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<400> His Xaa 1 1 Gln Gly Thr 5 Phe Thr Ser Asp Val 10 Ser Ser Tyr Leu Asp Gly 15 Gln Ala Ala Lys Glu 20 Phe Ile Ala Trp 25 Leu Val Lys Gly Cys thirty <210> <211> <212> <213> 2 thirty PRT Artificial Sequence <220> <223> Triple agonist

<220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15 Gln Ala Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25 thirty <210> 3

<211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <400> 3

His 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Val 10 Ser Ser Tyr Leu Leu 15 Gly Gln Ala Ala Lys 20 Gln Phe Ile Ala Trp 25 Leu Val Lys Gly Gly thirty Gly Pro

- 39 046413

Ser Ser Gly Ala Pro Pro Pro Ser Cys

<210> 4 <211> thirty <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib)

<400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Leu Gly 1 5 10 15 Gln Gln Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25 thirty <210> 5

<211> 41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <400> 5

His 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Val 10 Ser Ser Tyr Leu Leu 15 Gly Gln Gln Gln Lys 20 Glu Phe Ile Ala Trp 25 Leu Val Lys Gly Gly thirty Gly Pro Ser Ser Gly 35 Ala Pro Pro Pro Ser 40 Cys

<210>6 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <400> 6

His

Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly

- 40 046413

Gln Ala Ala Lys Glu Phe Val Ala Trp Leu Leu Lys Gly Cys <210> 7 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <400> 7

His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly

5 10 15

Gln Ala Ala Lys Glu Phe Val Ala Trp Leu Leu Lys Gly Cys <210> 8 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <400> 8

His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly

5 10 15

Gln Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Lys Gly Cys <210> 9 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <400>9

His Xaa Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly

5 1015

- 41 046413

Gln Ala Ala Gln Glu Phe Val Ala

Trp Leu Leu Ala Gly Cys

<210> 10 <211> 41 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<400> His Xaa 1 10 Gln Gly Thr 5 Phe Thr Ser Asp Val 10 Ser Lys Tyr Leu Asp 15 Gly Gln Ala Ala Gln 20 Glu Phe Val Ala Trp 25 Leu Leu Ala Gly Gly thirty Gly Pro Ser Ser Gly 35 Ala Pro Pro Pro Ser 40 Cys

<210>11 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <400> 11

Xaa 1 Gly Glu Gly Thr 5 Phe Thr Ser Asp Leu 10 Ser Lys Tyr Leu Asp 15 Ser Arg Arg Gln Gln Leu Phe Val Gln Trp Leu Lys Ala Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser His Gly

40 <210>12 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA)

- 42 046413 <400>12

Xaa Gly Glu Gly Thr Phe Ile Ser Asp Leu Ser Lys Tyr Met Asp Glu 1 5 1015

Gln Ala Val Gln Leu Phe Val Glu Trp Leu Met Ala Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser His Gly

3540 <210>13 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <400> 13

Xaa 1 Gly Glu Gly Thr 5 Phe Ile Ser Asp Tyr 10 Ser Ile Gln Leu Asp 15 Glu Ile Ala Val Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser His Gly

40

<210> 14 <211> 41 <212> PRT <213> Artificial Sequence

<220>

<223>

Triple agonist

<220> <221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA)

<400> 14

Xaa 1 Gly Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Gln Leu Asp 15 Glu Ile Ala Val Arg Asp Phe Val Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser His Gly

40

<210> 15 <211> 41 <212> PRT <213> Artificial Sequence

- 43 046413 <220>

<223> Triple agonist

<220> <221> <222> <223> MISC_FEATURE (1) Xaa represents 4-imidazoacetyl (CA) <400> Xaa Gly 1 15 Gln Gly Thr Phe Thr Ser 5 Asp Leu 10 Ser Lys Gln Met Asp 15 Glu Ala Val Arg Leu Phe Ile Glu 20 Trp 25 Leu Lys Asn Gly Gly thirty Pro Ser Gly Ala Pro Pro Pro Ser His 35 40 Gly <210> <211> <212> <213> 16 41 PRT Artificial Sequence <220> <223> Triple agonist <220> <221> <222> <223> MISC_FEATURE (1) Xaa represents 4-imidazoacetyl (CA) <400> Xaa Gly 1 16 Gln Gly Thr Phe Thr Ser 5 Asp Leu 10 Ser Lys Gln Met Asp 15 Glu Ala Gln Gln Leu Phe Ile Glu 20 Trp 25 Leu Lys Asn Gly Gly thirty Pro Ser Gly Ala Pro Pro Pro Ser His 35 40 Gly <210> <211> <212> <213> 17 41 PRT Artificial Sequence <220> <223> Triple agonist <220> <221> <222> <223> MISC_FEATURE (1) Xaa represents 4-imidazoacetyl (CA) <400> Xaa Gly 1 17 Gln Gly Thr Phe Thr Ser 5 Asp Leu 10 Ser Lys Gln Met Asp 15 Glu Arg Ala Arg Glu Phe Ile Glu 20 Trp 25 Leu Leu Ala Gln Lys thirty Pro Ser Gly Ala Pro Pro Pro Ser His Gly

Glu

Ser

Ser

Ser

Glu

Ser

- 44 046413

3540 <210>18 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA)

<400> Xaa Gly 1 18 Gln Gly Thr 5 Phe Thr Ser Asp Leu 10 Ser Lys Gln Met Asp 15 Ser Glu Arg Ala Arg 20 Glu Phe Ile Glu Trp 25 Leu Lys Asn Thr Gly thirty Pro Ser Ser Gly Ala 35 Pro Pro Pro Ser His 40 Gly

<210>19 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <400> 19

Xaa 1 Gly Gln Gly Thr 5 Phe Thr Ser Asp Leu 10 Ser Ile Gln Tyr Asp 15 Ser Glu His Gln Arg Asp Phe Ile Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser His Gly

40 <210>20 <211>41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA)

- 45 046413 <400>

Xaa 1 Gly Gln Gly Thr 5 Phe Thr Ser Asp Leu 10 Ser Ile Gln Tyr Glu 15 Glu Glu Ala Gln Gln Asp Phe Val Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser His Gly

<210> 21 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 21 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 CysArg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>22 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223>

amino acids at positions 16 and 20 form a ring

<400> 22 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Cys Leu Asp 15 Glu Tyr 1 Xaa Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty

- 46 046413

Ser Gly Gln Pro Pro Pro Ser 35 <210> 23 <211> 41 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 23 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 CysArg Ala Lys Glu Phe Val Gln Trp Leu Leu Ala Gln Lys Gly Lys 20 25 thirty Lys Asn Asp Trp Lys His Asn Ile Thr 35 40

<210>24 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring <400> 24

Tyr 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Lys Asn Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser

<210> 25

- 47 046413 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist

<220> <221> <222> <223> MISC_FEATURE (2) Xaa represents aminoisobutyric acid (Aib) <400> 25 His Xaa Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser 35 <210> 26 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Triple agonist <220> <221> MISC_FEATURE <222> (2) <223> Xaa represents aminoisobutyric acid (Aib)

<400> 26

His 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Cys 10 Ser Lys Tyr Leu Asp 15 Ser Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser

<210> 27 <211> 40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <400>27

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 1015

- 48 046413

Arg Ala Cys Gln Asp 20 Phe Val Gln Trp Leu Leu Asp 25 Gln Gly Gly Pro 30 Ser Ser Gly Ala Pro 35 Pro Pro Ser 40 <210> <211> <212> <213> 28 41 PRT Artificial Sequence <220> <223> Triple agonist <220> <221> <222> <223> MISC_FEATURE (2) Xaa represents aminoisobutyric acid (Aib)

<400> His Xaa 1 28 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Lys Arg Ala Gln 20 Glu Phe Val Cys Trp 25 Leu Leu Ala Gln Lys thirty Gly Lys Lys Asn Asp 35 Trp Lys His Asn Ile 40 Thr

<210> 29 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 29 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 His 1 Xaa Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 thirty

<210> thirty <211> thirty <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist

- 49 046413 <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222>

<223>

(16)..(20) amino acids at positions 16 and 20 form a ring <400>30

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

5 1015

Lys Ala Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 2530

<210> 31 <211> 29 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 31 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Cys Lys Glu Phe Val Gln Trp Leu Leu Ala Gln 20 25

<210> 32 <211> 39 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 50 046413 <400>32

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 1015

Lys Ala Cys Lys Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 2530

Ser Gly Ala Pro Pro Pro Ser 35 <210>33 <211>31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib)

<220> <221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> His Xaa 1 33 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Ile His Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Ala Gln Lys thirty Cys

<210> 34 <211> 31 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> His Xaa 1 34 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Lys Arg Gln Lys 20 Glu Phe Val Asn Trp 25 Leu Leu Ala Gln Lys thirty Cys

- 51 046413 <210> 35 <211> 31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222>

<223>

(16)..(20) amino acids at positions 16 and 20 form a ring

<400> His Xaa 1 35 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Ile His Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Asn Thr Lys thirty Cys

<210> 36 <211> 40 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222>

<223>

(16)..(20) amino acids at positions 16 and 20 form a ring

<400> His Xaa 1 36 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Cys 15 Glu Lys Arg Gln Lys 20 Glu Phe Val Gln Trp 25 Leu Leu Asn Gly Gly thirty Pro Ser Ser Gly Ala 35 Pro Pro Pro Ser Gly 40

<210> 37 <211> 40 <212> PRT

- 52 046413 <213>

<220>

<223>

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

<400>

His Xaa

CysArg

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring

Ser Gly

37 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Gly Gly Pro Ser 20 25 thirty Ala Pro Pro Pro Ser Gly 35 40

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <400>

Xaa Xaa

Arg Ala

Ser Gly

38 Gln Gly Thr 5 Phe Thr Ser Asp Lys 10 Ser Ser Tyr Leu Asp 15 Glu Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ala Pro Pro Pro Ser Ser 35 40

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist

- 53 046413 <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <400>

His Xaa

Gln His

Ser Ser

39 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Gly Ala Gln 20 Cys Phe Val Ala Trp 25 Leu Leu Ala Gly Gly thirty Gly Pro Gly 35 Ala Pro Pro Pro Ser 40

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

<400> His Xaa

Arg Ala

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib)

Ser Gly

Gln Gly Thr 5 Phe Thr Ser Asp Lys 10 Ser Lys Tyr Leu Asp 15 Glu Cys Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ala Pro Pro Pro Ser

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <400>

His Xaa

Cys Ala

Ser Gly

41 Gln Gly Thr 5 Phe Thr Ser Asp Lys 10 Ser Lys Tyr Leu Asp 15 Glu Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ala Pro Pro Pro Ser

<210>

- 54 046413 <211> 40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 42 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>43 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring <400> 43

Tyr 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Cys Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 44 <211> 39 <212> PRT <213> Artificial Sequence

<223> Triple agonist <220>

- 55 046413 <400> 44

His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Gln Leu Asp Gly

5 10 15

Gln Ala Ala Gln

Glu Phe Val Ala Trp Leu 25

Leu Ala Gly Gly Pro Ser 30

Ser Gly Ala Pro Pro Pro Ser 35

<210> <211> <212> <213> 45 39 PRT Artificial Sequence <220> <223> <400> His Gly Triple agonist 45 Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Met Asp Gly 1 Gln Ala 5 10 15 Ala Gln Asp Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser Ser Gly <210> <211> <212> <213> <220> <223> <400> His Gly 20 25 30 Ala Pro Pro Pro Ser 35 46 39 PRT Artificial Sequence Triple agonist 46 Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Glu 1 Gln His 5 10 15 Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser Ser Gly <210> <211> <212> <213> <220> <223> <400> His Gly 20 25 30 Ala Pro Pro Pro Ser 35 47 39 PRT Artificial Sequence Triple agonist 47 Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Gly 1 Gln Arg 5 10 15 Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser

- 56 046413 <210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <400>

His Gly

Gln Arg

Ser Gly

48 Gln Gly Thr 5 Phe Thr Ser Asp Cys 10 Ser Lys Tyr Leu Asp 15 Gly Ala Gln Asp Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 thirty Ala Pro Pro Pro Ser

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring <400>

Xaa Xaa

Ile His

49 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Cys Met Asp Glu 15 Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Asn Thr Lys thirty

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

MISC_FEATURE (2)

- 57 046413 <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 50 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty

Ser Gly Gln Pro Pro Pro Ser Cys

3540 <210>51 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 51 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 thirty

<210> 52 <211> thirty <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 58 046413

<400> 52 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 His 1 Xaa Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 25 thirty

<210> 53 <211> thirty <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib)

<220> <221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 53 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 His 1 Xaa Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 thirty

<210> 54 <211> thirty <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 54 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 His 1 Xaa Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Cys 20 25 thirty

<210> 55 <211> 30 <212> PRT <213> Artificial Sequence

- 59 046413 <220>

<223>

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring <400>

His Xaa

Ile His

55 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Ala Gln Cys thirty

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring <400>

His Xaa

Lys Arg

Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Gln Lys 20 Glu Phe Val Asn Trp 25 Leu Leu Ala Gln Cys thirty

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

- 60 046413 <221>

<222>

<223>

MISC_FEATURE (16) .. (20) amino acids at positions 16 and 20 form a ring

<400> 57 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 His 1 Xaa Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Cys 20 25 thirty

<210> 58 <211> 31 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 58 His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Lys Cys 20 25 thirty

<210>59 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 61 046413

<400> 59 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Cys Met Asp Glu 15 Xaa 1 Xaa Lys His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 thirty

<210> 60 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 60 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met Asp Glu 1 5 10 15 Lys His Cys Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Lys 20 25 thirty

<210>61 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 62 046413 <400>61

Xaa Xaa Gln Gly Thr Phe Thr Ser 15

Ile Ala Cys Lys Asp Phe Val Asn 20

Asp Tyr Ser Ile Ala Met Asp Glu

1015

Trp Leu Leu Asn Thr Lys

2530 <210> 62 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa represents

4-imidazoacetyl (CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <400> 62

Xaa 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Lys 10 Ser Lys Tyr Leu Asp 15 Glu Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser

<210>63 <211>39 <212> PRT <213> Artificial Sequence <220>

<223>

Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <400> 63

Xaa 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Cys 10 Ser Lys Tyr Leu Asp 15 Glu Arg Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 thirty Ser Gly Ala Pro Pro Pro Ser

- 63 046413 <210> 64 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 64 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Cys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>65 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222>

<223>

(16)..(20) amino acids at positions 16 and 20 form a ring

<400> 65 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Cys Leu Asp 15 Glu His 1 Xaa Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 6 6 <211> 39 <212> PRT

- 64 046413 <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 66 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 CysArg Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>67 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> Tyr Xaa 1 67 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Cys Ala Ala Lys 20 Asp Phe Val Gln Trp 25 Leu Leu Asp His His thirty Pro Ser Ser Gly Gln Pro Pro Pro Ser

<210> 68 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Triple agonist

- 65 046413 <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 68 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>69 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 69 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Arg Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>70 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib)

- 66 046413 <220>

<221> MISC_FEATURE <222> (16) .. (20)

<223> amino acids in provisions 16 and 20 form ring <400> 70 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 71 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 71 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Arg Ala Cys Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>72 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 67 046413 <400> 72

Tyr 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Cys 10 Ser Lys Tyr Leu Asp 15 Glu Arg Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 73 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring <400> 73

Xaa 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 74 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib)

- 68 046413 <220>

<221> MISC_FEATURE <222> (16) .. (20)

<223> amino acids in provisions 16 and 20 form ring <400> 74 Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210> 75 <211> 40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 75 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>76 <211>40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 69 046413

<400> Tyr Xaa 1 76 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Lys Arg Ala Lys 20 Asp Phe Val Gln Trp 25 Leu Leu Asp His His thirty Pro Ser Ser Gly Gln 35 Pro Pro Pro Ser Cys 40

<210> 77 <211> 40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 77 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>78 <211>31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> His Xaa 1 78 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Lys Arg Gln Lys 20 Glu Phe Val Gln Trp 25 Leu Leu Asp Thr Lys thirty Cys

- 70 046413 <210> 79 <211> 31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

MISC_FEATURE (16)..(20) amino acids at positions 16 and form a ring

<400> His Xaa 1 79 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Ile His Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Ala Gln Lys thirty Cys

<210> 80 <211> 31 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring <400>80

His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met AspGlu

5 1015

Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu LysCys

2530 <210>81 <211>30 <212> PRT <213> Artificial Sequence

- 71 046413 <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 81 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Xaa 1 Xaa Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr Cys 20 25 thirty

<210> 82 <211> 30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 82 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Xaa 1 Xaa Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Cys 20 25 thirty

<210> 83 <211> 30 <212> PRT <213> Artificial Sequence

- 72 046413 <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<220> <221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 83 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Xaa 1 Xaa Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 thirty

<210>84 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 84 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Xaa 1 Xaa Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu Cys 20 25 thirty

<210> 85 <211> 30 <212> PRT <213> Artificial Sequence

- 73 046413 <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<220> <221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 85 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Xaa 1 Xaa Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 thirty

<210>86 <211>30 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 86 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Xaa 1 Xaa Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln Cys 20 25 thirty

<210> 87 <211> 30 <212> PRT <213> Artificial Sequence

- 74 046413 <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib)

<220> <221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 87 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Xaa 1 Xaa Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr Cys 20 25 thirty

<210>88 <211>31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> Xaa Xaa 1 88 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Lys Arg Gln Lys 20 Glu Phe Val Gln Trp 25 Leu Leu Asn Thr Lys thirty Cys

<210> 89 <211> 31

- 75 046413 <212>

<213>

<220>

<223>

PRT Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring <400>

Xaa Xaa

Lys Arg

Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Lys Cys

25 30 <210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

<400> Xaa Xaa

Ile His

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring

90 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Ala Gln Lys thirty Cys

- 76 046413 <210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

<400>

Xaa Xaa

Ile His

MISC_FEATURE (16)..(20)

amino acids in provisions 16 and 20 form ring 91 Glu Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Gln Lys 20 Asp Phe Val Asp Trp 25 Leu Leu Ala Glu Lys thirty Cys

<210>

<211>

<212>

<213>

<220>

<223>

PRT

Artificial Sequence

Triple agonist <220>

<221>

<222>

<223>

MISC_FEATURE (1)

Xaa is 4-imidazoacetyl (CA) <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is aminoisobutyric acid (Aib) <220>

<221>

<222>

<223>

<400>

Xaa Xaa

MISC_FEATURE (16)..(20) amino acids at positions 16 and 20 form a ring

Gln Gly Thr Phe Thr Ser Asp Tyr

10

Ser Ile Ala Met Asp Glu

- 77 046413

Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala

Gln Lys Cys 30 <210> 93 <211> 31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> Xaa Xaa 1 93 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Glu 15 Lys Arg Gln Lys 20 Glu Phe Val Asn Trp 25 Leu Leu Ala Gln Lys thirty Cys

<210>94 <211>31 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is 4-imidazoacetyl(CA) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

- 78 046413

<400> Xaa Xaa 1 94 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Ile Ala Met Asp Glu 15 Ile His Gln Lys 20 Asp Phe Val Asn Trp 25 Leu Leu Asn Thr Lys thirty Cys

<210> 95 <211> 39 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 95 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Cys His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>96 <211>39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222>

<223>

(16)..(20) amino acids at positions 16 and 20 form a ring

<400> 96 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp 15 Glu Tyr 1 Xaa Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His Cys Pro Ser 20 25 thirty

- 79 046413

Ser Gly Gln Pro Pro Pro Ser 35 <210> 97 <211> 39 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 97 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu 1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Cys His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser

<210>98 <211>40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring

<400> 98 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Ala Leu Asp 15 Glu Tyr 1 Xaa Lys Ala Ala Lys Glu Phe Val Asn Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys

40 <210> 99 <211> 40

- 80 046413 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 99 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Asn Trp Leu Leu Asp His His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>100 <211>40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20) <223> amino acids at positions 16 and 20 form a ring <400> 100

Tyr 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Ala Leu Asp 15 Glu Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Gln His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys

40

<210> 101 <211> 40 <212> PRT <213> Artificial Sequence

<220>

<223> Triple agonist

- 81 046413 <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 101 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Glu Phe Val Asn Trp Leu Leu Asp Gln His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>102 <211>40 <212> PRT <213> Artificial Sequence <220>

<223> Triple agonist <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is aminoisobutyric acid (Aib) <220>

<221> MISC_FEATURE <222> (16)..(20)

<223> amino acids in provisions 16 and 20 form ring <400> 102 Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu Asp Glu 1 5 10 15 Lys Ala Ala Lys Asp Phe Val Asn Trp Leu Leu Asp Gln His Pro Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Cys 35 40

<210>103 <211>30 <212>PRT <213> Artificial Sequence <220>

<223> General formula1 <220>

<221> MISC_FEATURE <222>(1) <223> Xaa is His (H), 4-imidazoacetyl (CA), or Tyr (Y)

- 82 046413 <220>

<221>

<222>

<223>

MISC_FEATURE (2)

Xaa is Gly (G), alpha-methylglutamic acid or Aib (aminoisobutyric acid) <220>

<221>

<222>

<223>

MISC_FEATURE (3)

Xaa is Glu (E) or Gln (Q) <220>

<221>

<222>

<223>

MISC_FEATURE (7)

Xaa represents Thr(T) or Ile(I) <220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

MISC_FEATURE (10)

Xaa presents yourself Leu (L), Tyr (Y), Lys (K), Cys (C) or Val (V) MISC_FEATURE (12) Xaa presents yourself Lys (K), Ser (S) or Ile (I)

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

MISC_FEATURE (13) Xaa represents Gln (Q), Tyr (Y), Ala (A) or Cys(C) MISC_FEATURE (14) Xaa represents Leu (L), Met (M) or Tyr (Y)

<220>

<221>

<222>

<223>

MISC_FEATURE (15)

Xaa represents Cys(C)

Asp (D), Glu (E) or Leu (L) <220>

<221>

<222>

<223>

MISC_FEATURE (16)

Xaa is Gly (G), Glu (E) or Ser (S) <220>

<221>

<222>

<223> (K)

MISC_FEATURE (17)

Xaa represents Gln(Q)

Arg (R), Ile (I), Glu (E), Cys (C) or Lys <220>

<221>

<222>

<223>

MISC_FEATURE (18)

Xaa represents Ala(A),

Gln (Q), Arg (R) or His (H) <220>

- 83 046413

<221> <222> <223> MISC_FEATURE Ala (19) Xaa represents (A), Gln (Q), Cys (C) or Val(V) <220> <221> <222> <223> <220> <221> <222> MISC_FEATURE (20) Xaa represents MISC_FEATURE (21) Lys (K), Gln (Q) or Arg (R) <223> <220> <221> <222> <223> <220> <221> <222> Xaa represents MISC_FEATURE (23) Xaa represents MISC_FEATURE (24) Glu Ile (E), Gln (Q), Leu (L) (I) or Val (V) , Cys (C) or Asp (D) <223> (E) <220> <221> <222> <223> <220> <221> <222> Xaa represents MISC_FEATURE (27) Xaa represents MISC_FEATURE (28) Ala Val (A), Gln (Q), Cys (C) (V), Leu (L), Lys (K) , Asn (N), Asp (D) or Glu or Met(M) <223> <220> <221> <222> Xaa represents MISC_FEATURE (29) Cys (C), Lys (K), Ala (A) , Asn (N) or Asp (D) <223> (H) <220> <221> <222> Xaa represents MISC_FEATURE (30) Cys (C), Gly (G), Gln (Q) , Thr (T), Glu (E) or His <223> howls; Xaa represents and Cys (C), Gly (G), Lys (K) or His (H), or absent Xaa may be further linked to R1, where R1 is Cys(C), GKKNDWKHNIT, m-SSGAPPPS-n or m-SSGQPPPS-n, or absent, and where m -Gly-, -Ser- represents -Cys-, or -His -Pro - or -Gly-Pro-, and n represents -Cys-,

<400>103

Xaa Xaa Xaa Gly Thr Phe Xaa Ser Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa

5 1015

Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Xaa Xaa Xaa Xaa

2530 <210> 104

- 84 046413 <211>40 <212>PRT <213> Artificial Sequence <220>

<223> General formula2 <220>

<221> MISC_FEATURE <222>(1) <223> Xaa represents His (H),

4-Imidazoacetyl (CA) or Tyr (Y) <220>

<221> MISC_FEATURE <222>(2) <223> Xaa is Gly (G), alpha-methylglutamic acid or Aib (aminoisobutyric acid) <220>

<221> MISC_FEATURE <222>(10) <223> Xaa is Tyr (Y) or Cys (C) <220>

<221> MISC_FEATURE <222>(13) <223> Xaa is Gln (Q), Tyr (Y), Ala (A) or Cys (C) <220>

<221> MISC_FEATURE <222> (14)

<223> Xaa is yourself Leu (L) Met (M) or Tyr (Y) <220> <221> <222> <223> MISC (15) Xaa _FEATURE is yourself Asp (D) Glu (E) or Leu (L)

<220> <221> MISC_FEATURE <222> (16) <223> Xaa is Gly (G), Glu (E) or Ser (S)

<220>

<221> MISC_FEATURE <222>(17) <223> Xaa is Gln (Q), Arg (R), Ile (I), Glu (E), Cys (C) or Lys (K) <220>

<221> MISC_FEATURE <222> (18) <223> Xaa is Ala (A), Gln (Q), Arg (R) or His (H) <220>

<221> MISC_FEATURE <222> (19) <223> Xaa is Ala (A), Gln (Q), Cys (C) or Val (V)

- 85 046413 <220>

<221> MISC_FEATURE <222>(20) <223> Xaa is Lys (K), Gln (Q) or Arg (R) <220>

<221> MISC_FEATURE <222>(21) <223> Xaa is Glu (E), Gln (Q), Leu (L), Cys (C) or Asp (D) <220>

<221> MISC_FEATURE <222>(23) <223> Xaa is Ile (I) or Val (V) <220>

<221> MISC_FEATURE <222> (24)

<223> Xaa presents yourself Ala (A), Gln (Q), Cys (C), Asn (N) or Glu (E) <220> <221> <222> <223> MISC_FEATURE (28) Xaa presents yourself Cys (C), Lys (K), Asn (N) or Asp (D)

<220> <221> MISC_FEATURE <222> (29) <223> Xaa is Cys (C), Gly (G), Gln (Q) or His (H)

<220> <221> MISC_FEATURE <222> (thirty) <223> Xaa is Cys (C), Gly (G), Lys (K) or His (H)

<220>

<221> MISC_FEATURE <222>(31) <223> Xaa is Pro (P) or Cys (C) <220>

<221> MISC_FEATURE <222>(40) <223> Xaa is Cys (C) or missing <400> 104

Xaa 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Xaa 10 Ser Lys Xaa Xaa Xaa 15 Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Xaa Xaa Xaa Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Xaa

40

<210> 105 <211> 40 <212> PRT <213> Artificial Sequence

- 86 046413 <220>

<223> General formula 3 <220>

<221> MISC_FEATURE <222> (1) <223> Xaa is His (H) or Tyr (Y) <220>

<221> MISC_FEATURE <222> (2) <223> Xaa is alpha-methylglutamic acid or Aib (aminoisobutyric acid)

<220> <221> <222> <223> MISC_FEATURE (13) Xaa presents yourself Tyr (Y) , Ala (A) or Cys (C) <220> <221> <222> <223> MISC_FEATURE (17) Xaa presents yourself Arg (R) , Cys (C) or Lys (K) <220> <221> <222> <223> MISC_FEATURE (18) Xaa presents yourself Ala (A) or Arg (R) <220> <221> <222> <223> MISC_FEATURE (19) Xaa presents yourself Ala (A) or Cys (C) <220> <221> <222> <223> MISC_FEATURE (21) Xaa presents yourself Glu (E) or Asp (D) <220> <221> <222> <223> MISC_FEATURE (24) Xaa presents yourself Gln (Q) or Asn (N) <220> <221> <222> <223> MISC_FEATURE (28) Xaa presents yourself Cys (C) or Asp (D) <220> <221> <222> <223> MISC_FEATURE (29) Xaa presents yourself Cys (C) , Gln i(Q) or His (H) <220> <221> <222> <223> MISC_FEATURE (30) Xaa presents yourself Cys (C) or His (H)

- 87 046413 <220>

<221> MISC_FEATURE <222>(31) <223> Xaa is Pro (P) or Cys (C) <220>

<221> MISC_FEATURE <222>(40) <223> Xaa is Cys (C) or missing <400> 105

Xaa 1 Xaa Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Xaa Leu Asp 15 Glu Xaa Xaa Xaa Lys Xaa Phe Val Xaa Trp Leu Leu Xaa Xaa Xaa Xaa Ser 20 25 thirty Ser Gly Gln Pro Pro Pro Ser Xaa

40

<210> 106 <211> eleven <212> PRT <213> Artificial Sequence

<220>

<223>R1

<400> 106 Gly Lys 1 Lys Asn Asp Trp Lys His Asn Ile Thr 5 10

<210> 107 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> R1

<400>107

Ser Ser Gly Ala Pro Pro Pro Ser

<210> 108 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> R1

<400>108

Ser Ser Gly Gln Pro Pro Pro Ser

<210> 109 <211> 9 <212> PRT <213> Artificial Sequence

- 88 046413 <220>

<223>R1

<400> Cys Ser 1 109 Ser Gly Gln 5 Pro Pro Pro Ser <210> <211> <212> <213> 110 10 PRT Artificial Sequence <220> <223> R1 <400> Gly Pro 1 110 Ser Ser Gly 5 Ala Pro Pro Pro Ser 10 <210> <211> <212> <213> 111 eleven PRT Artificial Sequence <220> <223> R1 <400> Gly Pro 1 111 Ser Ser Gly 5 Ala Pro Pro Pro Ser Cys 10 <210> <211> <212> <213> 112 9 PRT Artificial Sequence <220> <223> R1

<400>112

Pro Ser Ser Gly Ala Pro Pro Pro Ser

<210> 113 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> R1

<400>113

Pro Ser Ser Gly Ala Pro Pro Pro

Ser Gly 10

<210> 114 <211> eleven <212> PRT <213> Artificial Sequence

- 89 046413 <220>

<223>R1

<400> Pro Ser 1 114 Ser Gly Ala 5 Pro Pro Pro Ser His 10 Gly <210> <211> <212> <213> 115 10 PRT Artificial Sequence <220> <223> R1 <400> Pro Ser 1 115 Ser Gly Ala 5 Pro Pro Pro Ser Ser 10 <210> <211> <212> <213> 116 9 PRT Artificial Sequence <220> <223> R1 <400> Pro Ser 1 116 Ser Gly Gln 5 Pro Pro Pro Ser <210> <211> <212> <213> 117 10 PRT Artificial Sequence <220> <223> R1 <400> Pro Ser 1 117 Ser Gly Gln 5 Pro Pro Pro Ser Cys 10 <210> <211> <212> <213> 118 29 PRT Homo sapiens <400> His Ser 1 118 Gln Gly Thr 5 Phe Thr Ser Asp Tyr 10 Ser Lys Tyr Leu Asp Ser 15 Arg Arg Ala Gln Asp 20 Phe Val Gln Trp 25 Leu Met Asn Thr

<210> 119 <211> 12 <212> PRT <213> HOMO SAPIENS

- 90 046413 <400>

Glu Ser

119

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro 5 10 <210>

<211>

<212>

<213>

<220>

<223>

120

PRT

Artificial Sequence <400>

Glu Ser

Hinge area option

120

Lys Tyr Gly Pro Pro Pro Ser Cys Pro 5 10 <210>

<211>

<212>

<213>

<220>

<223>

121

PRT

Artificial Sequence <400>

Glu Ser

Hinge area option

121

Lys Tyr Gly Pro Pro Cys Pro Ser Pro 5 10 <210>

<211>

<212>

<213>

<220>

<223>

122

PRT

Artificial Sequence

Hinge area option <400>

Glu Ser

122

Lys Tyr Gly Pro Pro Cys Pro Ser

10 <210>

<211>

<212>

<213>

<220>

<223>

123

PRT

Artificial Sequence

Hinge area option <400>

Glu Ser

123

Lys Tyr Gly Pro Pro Cys Pro Pro

10 <210>

<211>

<212>

<213>

<220>

<223>

124

PRT

Artificial Sequence

Hinge area option

- 91 046413

<400> Lys Tyr 1 124 Gly Pro Pro Cys Pro Ser 5 <210> <211> <212> <213> 125 8 PRT Artificial Sequence

<220> <223> Hinge area option <400> Glu Ser 1 125 Lys Tyr Gly Pro Pro Cys 5 <210> <211> <212> <213> 126 7 PRT Artificial Sequence

<220> <223> Hinge area option <400> Glu Lys 1 126 Tyr Gly Pro Pro Cys 5 <210> <211> <212> <213> 127 6 PRT Artificial Sequence

<220> <223> Hinge area option <400> Glu Ser 1 127 Pro Ser Cys Pro 5 <210> <211> <212> <213> 128 5 PRT Artificial Sequence

<220> <223> Hinge area option <400> Glu Pro 1 128 Ser Cys Pro 5 <210> <211> <212> <213> 129 4 PRT Artificial Sequence

<220> <223> Hinge area option

- 92 046413 <400>129

Pro Ser Cys Pro 1

<210> 130 <211> 10 <212> PRT <213> Artificial Sequence

<220>

<223> Hinge area option <400>130

Glu Ser Lys Tyr Gly Pro Pro Ser Cys Pro

510

<210> <211> <212> <213> 131 9 PRT Artificial Sequence <220> <223> Hinge area option <400> 131 Lys Tyr 1 Gly Pro Pro 5 Pro Ser Cys Pro

<210> 132 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Hinge area option

<400> 132 Glu Ser Lys Tyr Gly Pro Ser Cys Pro 1 5

<210> 133 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Hinge area option

<400> 133 Glu Ser 1 Lys Tyr Gly Pro Pro Cys 5

<210>134 <211>7 <212> PRT <213> Artificial Sequence <220>

<223> Hinge area option

- 93 046413 <400>134

Lys Tyr Gly Pro Pro Cys Pro

<210> 135 <211> 7 <212> PRT <213> Artificial Sequence

<220>

<223> Hinge area option <400>135

Glu Ser Lys Pro Ser Cys Pro

<210> 136 <211> 6 <212> PRT <213> Artificial Sequence

<220>

<223> Hinge area option <400>136

Glu Ser Pro Ser Cys Pro

<210> 137 <211> 4 <212> PRT <213> Artificial Sequence

<220>

<223> Hinge area option <400>137

Glu Pro Ser Cys

<210> 138 <211> 3 <212> PRT <213> Artificial Sequence

<220>

<223> Hinge area option <400>138

Ser Cys Pro 1 <210>139 <211>221 <212>PRT <213> homo sapiens <400>139

Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

5 1015

-

Claims (6)

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 20 2530 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 35 4045 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 50 5560 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser ValLeu 65 70 7580 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys 85 9095 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 100 105110 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 115 120125 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 130 135140 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn GlyGln 145 150 155160 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser AspGly 165 170175 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 180 185190 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 195 200205 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215220 CLAIM 1. The use of a pharmaceutical composition for the prevention or treatment of liver disease accompanied or caused by liver inflammation or steatosis, containing: pharmaceutically acceptable excipient; and a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 31, 32, 37, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101 and 102, in a pharmaceutically effective amount, wherein the liver disease is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver (NAFL), liver inflammation, non-alcoholic steatohepatitis (NASH), metabolic liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure and liver cancer. 2. Use according to claim 1, where the peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 21, 22, 43, 50, 64, 65, 66, 67, 70, 71, 76, 77, 96, 97, 100, 101 and 102. 3. Use according to claim 1, wherein the peptide contains an amino acid sequence selected from the group consisting of SEQ ID NO: 66, 67, 97 and 100. 4. Use according to any one of claims 1 to 3, wherein the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by Formula 1 below: [Formula 1] X-L-F, where in formula 1 above, X is the peptide described above; L is a linker containing an ethylene glycol repeating unit; F is an immunoglobulin Fc fragment or a derivative thereof; And - represents a covalent bond between X and L, and between L and F. 5. Use according to claim 1, wherein the C-terminus of the peptide is amidated. 6. Use according to claim 1, wherein the liver disease is inflammation of the liver. -