Liver-targeted drugs and their uses

Abstract

The present invention relates to a liver-targeted drug and its therapeutic use for diseases requiring drug action in the liver. The present invention also relates to a method for targeting to liver tissue or inducing increased distribution in liver tissue after administration in vivo using a substance active against glucagon.

Description

[Technical field]

[0001] The present invention relates to a liver-targeted drug and its therapeutic use for diseases requiring drug action in the liver. The present invention also relates to a method for targeting a liver-targeted drug to liver tissue or for inducing increased distribution in liver tissue after administration in vivo. [Background technology]

[0002] The liver is one of the largest organs in the human body and plays an important role in various and comprehensive metabolic processes. Many plant and animal substances that humans ingest, and the metabolic products resulting from the performance of biological functions, are beneficial to the body, but many are harmful. The liver performs chemical processes to help the body properly utilize the beneficial substances, and helps the harmful substances to be safely excreted from the body through urine and feces through chemical metabolic processes. The liver plays an essential role in biological functions by synthesizing and secreting several proteins, fats, and carbohydrates that are necessary for the human body. Therefore, when liver function deteriorates, various functional abnormalities can appear. Insufficient production of clotting factors involved in blood clotting often causes bleeding, and frequent bleeding from weak gums, etc., and in patients with cirrhosis of the liver, insulin decomposition does not work well and the liver's glycogen storage is insufficient, resulting in hypoglycemia due to hunger. The liver also plays a central role in defense against bacterial invasion. In particular, Kupffer cells in the liver act as macrophages, consuming foreign substances or bacteria, and expose viruses that have entered the body to the immune system, inducing the body's natural immune function. Bile, the main substance synthesized and secreted by liver cells, is produced at a rate of about 800-1000cc per day, and is mainly composed of water, electrolytes, bile acids, cholesterol, phospholipids, and bilirubin. The main functions of bile are that bile acids in bile play an important role in digesting and absorbing fats and fat-soluble vitamins in the small intestine, and bile itself excretes many waste products produced in the body as feces. Liver diseases are diseases caused by various causes that require drug action in the liver, and there are various diseases such as hepatitis caused by viral infection, primary biliary cirrhosis, non-alcoholic fatty liver disease, liver cirrhosis, liver cancer, etc.

[0003] Primary biliary cirrhosis (PBC) is a rare disease that is a chronic inflammatory disease that shows biliary fibrotic stenosis and chronic cholestasis. It causes partial or total fibrotic stenosis of the extrahepatic and intrahepatic bile ducts, and eventually leads to liver failure due to damage to liver cells through chronic cholestasis and biliary cirrhosis. It is most common in middle-aged women, and biochemical tests show cholestatic biochemical abnormalities such as alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), and elevated bilirubin, and 90-95% of patients are positive for antimitochondrial antibodies. Currently, there is no specific treatment for primary biliary cirrhosis. Drugs such as ursodeoxycholic acid (UDCA), which improves cholestasis in cholestatic diseases and protects and restores liver cells, are used, and obeticholic acid, a drug developed by Intercept Pharmaceuticals that improves fatty liver, was recently approved in the United States for the above indication.

[0004] In particular, nonalcoholic steatohepatitis disease (NAFLD) is a type of disease that shows similar histological findings to alcoholic hepatitis, even though it is not related to alcohol intake, and is a disease that includes nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinomas. Nonalcoholic fatty liver disease is on the rise as the obese and diabetic population increases, and the annual incidence rate in Korea has reached about 16%.

[0005] The pathogenesis of non-alcoholic fatty liver disease is known to be due to various factors such as insulin resistance, obesity, lipotoxicity, inflammatory response, etc. Among these, the most important cause is insulin resistance.

[0006] In order to prevent and / or treat such non-alcoholic fatty liver disease, many efforts have been made to improve insulin resistance. For example, clinical trials of insulin sensitizers, such as TZDs (thiazolidinedinones) and metformin, are currently being actively conducted (Hepatology (2003) 38:1008-17, J Clin Invest. (2001) 108:1167-74).

[0007] Meanwhile, in order to treat the above-mentioned liver diseases, one of the important technical challenges is to deliver and distribute drugs to the liver at high concentrations (Korean Patent Publication No. KR10-2013-0131227).

[0008] Liver-targeted drugs can optimize drug therapy by minimizing side effects of existing drugs, maximizing efficacy and effectiveness, and efficiently delivering the required amount of drugs. This increases the amount of drug that reaches the target site, increasing bioavailability and enabling more effective treatment, prevents delivery to sites other than the target site, reducing side effects, and improves the patient's response to the drug, playing a major role in promoting patient compliance. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent Publication Number KR10-2013-0131227 [Patent Document 2] International Patent Publication No. WO 97 / 34631 [Patent Document 3] International Patent Publication No. 96 / 32478 [Patent Document 4] International Publication No. WO 2020 / 263063

Non-licensed documents

[0010]

Non-patent document 1

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Non-patent document 10

[0011] In order to improve the therapeutic effect of diseases requiring drug action in the liver, there is a need to develop an excellent liver-targeted drug that has a therapeutic effect while having a high distribution rate in the liver among the organs in the body of an individual to which it is administered. [Means for solving the problem]

[0012] One object of the present invention is to provide a pharmaceutical composition containing a liver-targeted drug, specifically, a pharmaceutical composition containing a liver-targeted drug, which has a high distribution rate of the drug in the liver among the internal organs of an individual to which it is administered.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising a liver-targeted drug, specifically a pharmaceutical composition comprising a liver-targeted drug for preventing or treating a disease requiring a drug action in the liver.

[0014] Another object of the present invention is to provide a method for preventing or treating a disease requiring drug action in the liver, comprising the step of administering the pharmaceutical composition or a physiologically active substance targeted to liver tissue to an individual in need thereof.

[0015] Another object of the present invention is to provide a use of the liver-targeted drug, specifically a physiologically active substance targeted to liver tissue, for use in the manufacture of a medicament for the prevention or treatment of a disease requiring a drug action in the liver.

[0016] Another object of the present invention is to provide a method for inducing liver targeting by administering the liver-targeted drug, specifically a physiologically active substance targeted to liver tissue, to an individual in need thereof.

[0017] Another object of the present invention is to provide a method for inducing increased distribution of the physiologically active substance in liver tissue by administering the liver-targeted drug, specifically, a physiologically active substance targeted to liver tissue, to an individual in need thereof. Effect of the Invention

[0018] The drug of the present invention (e.g., a physiologically active protein or peptide derivative) containing a substance that binds to a receptor on hepatocytes can reach liver tissue from blood vessels more effectively and, after administration in vivo, is provided as a physiologically active substance that is distributed relatively more in liver tissue among internal organs, specifically, by having a binding ability to the glucagon receptor. Since the distribution in the liver is high even within about 7 days, the drug can be applied to the treatment of diseases requiring drug action in the liver, such as various liver diseases, while reducing the inconvenience of administering medication to patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] One embodiment of the present invention relates to a pharmaceutical composition comprising a liver-targeted drug, specifically a pharmaceutical composition in which the drug is highly distributed in the liver among the body organs of an individual to which the composition is administered.

[0020] As one specific example, the liver-targeted drug is a physiologically active substance targeted to liver tissue, and is characterized in that it is in the form of a peptide containing the amino acid sequence of the following general formula 1 or a long-acting conjugate containing the same.

[0021] In one embodiment, the liver targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 1:

[0022] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-X aa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (general formula 1, sequence number 103)

[0023] In the general formula 1, Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y); Xaa2 is glycine (Gly, G), alpha-methyl-glutamic acid, or Aib (aminoisobutyric acid); Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q); Xaa7 is threonine (Thr, T) or isoleucine (Ile, I); Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V); Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I); Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C); Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y); Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L); Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S); Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K); Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H); Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V); Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R); Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D); Xaa23 is isoleucine (Ile, I) or valine (Val, V); Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E); Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K) or methionine (Met, M); Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D); Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H); Xaa30 is cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), histidine (His, H), or absent; R1 is cysteine ​​(Cys,C), GKKNDWKHNIT (SEQ ID NO:106), m-SSGAPPPS-n (SEQ ID NO:107), or m-SSGQPPPS-n (SEQ ID NO:108), or is absent; Where: m is Cys, Pro or Gly-Pro; n is Cys, Gly, Ser, His-Gly, or is absent.

[0024] In another embodiment, the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is a pharmaceutical composition represented by the following formula 1:

[0025] [Chemical formula 1] XLF

[0026] Where: X is a peptide comprising the amino acid sequence of general formula 1; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region, - indicates a covalent bond link between X and L, and between L and F.

[0027] In the pharmaceutical composition according to any one of the above-mentioned specific examples, the internal organs are the liver, heart, lungs, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney and brain, and are characterized by the highest distribution in the liver among the above-mentioned organs.

[0028] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the composition is for use in the prevention or treatment of a disease requiring a drug action in the liver.

[0029] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeted drug is characterized in that the T / S ratio (tissue-to-serum ratio) in the liver after administration is one or more selected from the following: (a) a T / S ratio of 25% to 50% 2 to 5 hours after administration; (b) a T / S ratio of 40% to 60% 40 to 50 hours after administration; and (c) a T / S ratio of 45% to 75% 160 to 180 hours after administration.

[0030] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeted drug is characterized in that the T / S ratio (tissue-to-serum ratio) in the liver after administration is one or more selected from the following: (a) a T / S ratio of about 30% to about 50% at about 2 hours to about 5 hours after administration; (b) a T / S ratio of about 45% to about 55% at about 40 hours to about 50 hours after administration; and (c) a T / S ratio of about 50% to about 65% at about 160 hours to about 180 hours after administration.

[0031] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeted drug is characterized in that the T / S ratio in the liver after administration is one or more selected from the following: (a) a T / S ratio of 35% to 45% 4 hours after administration; (b) a T / S ratio of 45% to 55% 2 days after administration; and (c) a T / S ratio of 55% to 70% 7 days after administration.

[0032] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeted drug is characterized in that the T / S ratio in the liver after administration is one or more selected from the following: (a) a T / S ratio of about 41% to about 43% about 4 hours after administration; (b) a T / S ratio of about 48% to about 52% about 2 days after administration; and (c) a T / S ratio of about 60% to about 65% about 7 days after administration.

[0033] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the liver-targeted drug is characterized in that the distribution ratio in the liver compared to the lung tissue after administration is about 1:2 to about 4.

[0034] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the liver-targeted drug is characterized in that the distribution ratio in the liver compared to the lung tissue after administration is about 1:3 to about 4.

[0035] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the liver-targeted drug is characterized in that the distribution ratio in the liver compared to the lung tissue after administration is 1:about 3.5 to about 4.

[0036] The pharmaceutical composition according to any one of the above-mentioned specific examples is characterized in that the distribution ratio in the liver compared to the lung tissue after the administration is a distribution ratio at about 40 hours to about 180 hours after the administration.

[0037] The pharmaceutical composition according to any one of the above-mentioned specific examples is characterized in that the distribution ratio in the liver compared to the lung tissue after the administration is a distribution ratio for about 2 to about 7 days after the administration.

[0038] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeted drug is characterized in that (a) the distribution ratio in the liver compared to the heart is 1:1.5-3.0 2-5 hours after administration; (b) the distribution ratio in the liver compared to the heart is 1:2.0-3.0 40-50 hours after administration; and (c) the distribution ratio in the liver compared to the heart is 1:5.5-7.0 160-180 hours after administration.

[0039] In the pharmaceutical composition according to any one of the above-mentioned specific examples, the liver-targeting drug is characterized in that it is in the form of a peptide or a long-acting conjugate containing the same, which is distributed relatively more in the liver among the organs in the body after administration.

[0040] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the liver-targeted drug has therapeutic activity for a disease requiring drug action in the liver.

[0041] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the disease requiring a drug action in the liver is characterized in that it is a liver disease.

[0042] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the liver disease is characterized in that it is liver cancer.

[0043] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the liver-targeting drug is characterized in that it is a protein or peptide containing a peptide sequence having binding ability to the glucagon receptor.

[0044] The pharmaceutical composition according to any one of the above embodiments, In the general formula 1, Xaa2 is glycine, alpha-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, tyrosine, 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; Xaa27 is characterized as being leucine or lysine.

[0045] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the peptide is characterized in that it is a peptide comprising an amino acid sequence represented by the following general formula 2:

[0046] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa2 1-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO: 104)

[0047] In the general formula 2, Xaa1 is 4-imidazoacetyl, histidine, or tyrosine; Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib; Xaa10 is tyrosine or cysteine; Xaa13 is alanine, glutamine, tyrosine, or cysteine; Xaa14 is leucine, methionine, or tyrosine; Xaa15 is aspartic acid, glutamic acid, or leucine; Xaa16 is glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine; Xaa18 is alanine, glutamine, arginine, or histidine; Xaa19 is alanine, 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; Xaa31 is proline or cysteine; Xaa40 is cysteine ​​or absent. The pharmaceutical composition according to any one of the above embodiments, In the general formula 1, Xaa2 is glycine, alpha-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; Xaa23 is valine, Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; Xaa27 is characterized as being leucine or lysine. The pharmaceutical composition according to any one of the above embodiments, In the 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; Xaa30 is characterized as being cysteine, lysine, or histidine.

[0048] The pharmaceutical composition according to any one of the above embodiments, In the general formula 1, Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa7 is threonine, Xaa10 is tyrosine or cysteine; Xaa12 is lysine or isoleucine; Xaa13 is tyrosine, alanine, or cysteine; Xaa14 is leucine or methionine; Xaa15 is cysteine ​​or aspartic acid; Xaa16 is glutamic acid, 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, Xaa28 is characterized as being cysteine, alanine, asparagine, or aspartic acid.

[0049] The pharmaceutical composition according to any one of the above embodiments, In the general formula 1, Xaa1 is histidine or 4-imidazoacetyl; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, Xaa12 is isoleucine, Xaa13 is alanine or cysteine; Xaa14 is methionine, Xaa15 is aspartic acid, Xaa16 is glutamic acid, Xaa17 is isoleucine or lysine; Xaa18 is alanine or histidine; Xaa19 is glutamine or cysteine; Xaa20 is lysine, Xaa21 is aspartic acid, Xaa23 is valine, Xaa24 is asparagine, Xaa27 is leucine, Xaa28 is alanine or asparagine; Xaa29 is glutamine or threonine; Xaa30 is characterized as being cysteine, lysine, or absent.

[0050] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the peptide is characterized in that it is a peptide comprising an amino acid sequence of the following general formula 3:

[0051] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Ph e-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 3, sequence number 105)

[0052] In the general formula 3, Xaa1 is histidine or tyrosine; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa13 is alanine, tyrosine or cysteine; Xaa17 is arginine, cysteine, or lysine; Xaa18 is alanine or arginine; Xaa19 is alanine or cysteine; Xaa21 is glutamic acid or aspartic acid; Xaa24 is glutamine or asparagine; Xaa28 is cysteine ​​or aspartic acid; Xaa29 is cysteine, histidine, or glutamine; Xaa30 is cysteine ​​or histidine; Xaa31 is proline or cysteine; Xaa40 is cysteine ​​or absent. The pharmaceutical composition according to any one of the above embodiments, The R1 is characterized in that it is cysteine, GKKNDWKHNIT (sequence number 106), CSSGQPPPS (sequence number 109), GPSSGAPPPS (sequence number 110), GPSSGAPPPSC (sequence number 111), PSSGAPPPS (sequence number 112), PSSGAPPPSG (sequence number 113), PSSGAPPPSHG (sequence number 114), PSSGAPPPSS (sequence number 115), PSSGQPPPS (sequence number 116), or PSSGQPPPSC (sequence number 117), or is absent.

[0053] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-102.

[0054] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that in the peptide, the amino acid Xaa16 and the amino acid Xaa20 form a ring together.

[0055] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the peptide is characterized in that its C-terminus is amidated or has a free carboxyl group (-COOH).

[0056] The composition according to any one of the above embodiments is characterized in that the C-terminus of the peptide is amidated.

[0057] The composition according to any one of the above-mentioned embodiments is characterized in that the chemical formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

[0058] In the composition according to any one of the above-mentioned embodiments, the structure of Formula 1 is characterized by being a structure of Formula 3 below:

[0059] [Chemical formula 3] JPEG2025502749000001.jpg3398

[0060] Here, X and F are as defined in Chemical Formula 1.

[0061] The composition according to any one of the above specific examples is characterized in that the ethylene glycol repeating unit is [OCH2CH2]n, where n is a natural number, and the average molecular weight, for example, the number average molecular weight, of the [OCH2CH2]n portion within the peptide bond is determined to be 1 to 100 kDa.

[0062] In the composition according to any one of the above-mentioned specific examples, the value of n is characterized in that the average molecular weight, for example the number average molecular weight, of the [OCH2CH2]n moiety in the peptide conjugate is determined to be 10 kDa.

[0063] In the pharmaceutical composition according to any one of the above embodiments, L is polyethylene glycol.

[0064] In the composition according to any one of the above embodiments, X is linked through a sulfur atom of a cysteine ​​in the peptide.

[0065] The pharmaceutical composition according to any one of the above embodiments is characterized in that F is an IgG Fc region.

[0066] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the immunoglobulin Fc region is derived from IgG4.

[0067] In a composition according to any one of the above-mentioned specific examples, F is a structure in which two polypeptide chains are linked by a disulfide bond, and is characterized in that the two chains are linked only through a nitrogen atom of one of the chains.

[0068] The composition according to any one of the above embodiments, wherein F comprises a monomer having the amino acid sequence of SEQ ID NO:139.

[0069] The composition according to any one of the above embodiments, wherein F is a homodimer of a monomer having the amino acid sequence of SEQ ID NO:139.

[0070] The composition according to any one of the above embodiments, wherein F is a homodimer comprising the amino acid sequence of SEQ ID NO:140.

[0071] The composition according to any one of the above embodiments is characterized in that F is linked through the nitrogen atom of its N-terminal proline.

[0072] The composition according to any one of the above embodiments is characterized in that the immunoglobulin Fc domain, F and X, is non-glycosylated.

[0073] Another aspect of the present invention is a physiologically active substance targeted to liver tissue, specifically, a physiologically active substance targeted to liver tissue, comprising a substance that has binding ability with a receptor present in the liver.

[0074] Another aspect of the present invention is a method for preventing or treating a disease requiring drug action in the liver, comprising administering the pharmaceutical composition or a physiologically active substance targeted to liver tissue to an individual in need thereof.

[0075] In the composition according to any one of the above-mentioned embodiments, the disease requiring a medicinal effect in the liver is characterized in that it is a liver disease.

[0076] The composition according to any one of the above-mentioned embodiments, wherein the liver disease is a metabolic liver disease.

[0077] In the composition according to any one of the above-mentioned specific examples, the liver disease is characterized in that it is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer.

[0078] In the composition according to any one of the above-mentioned embodiments, the cholestatic liver disease is at least one disease selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

[0079] Another aspect of the present invention is the use of the liver-targeted drug, specifically a physiologically active substance targeted to liver tissue, for use in the manufacture of a medicament for the prevention or treatment of a disease requiring drug action in the liver.

[0080] Another aspect of the present invention is a method for inducing liver targeting by administering the liver-targeted drug, specifically a physiologically active substance targeted to liver tissue, to an individual in need thereof.

[0081] Another aspect of the present invention is to provide a method for administering the liver-targeted drug, particularly a physiologically active substance targeted to liver tissue, to an individual in need thereof to induce increased distribution of the physiologically active substance within liver tissue.

[0082] The present invention will now be described in further detail.

[0083] Meanwhile, each description and embodiment disclosed in the present application can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present invention. In addition, the following specific description is not considered to limit the scope of the present invention.

[0084] Moreover, those of ordinary skill in the art will be able to recognize or ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of the invention described herein. Such equivalents are intended to be encompassed by the present invention. The documents described herein may be incorporated by reference in their entirety into this application. Also, numerous papers and patent documents are referenced and citations are provided throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference in their entirety into this specification to more clearly explain the state of the art to which this application pertains and the contents of the present invention.

[0085] Throughout this specification, the usual one-letter and three-letter codes for the naturally occurring amino acids are used, as well as the commonly accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, the amino acids referred to as abbreviations in this specification are described according to the IUPAC-IUB nomenclature system.

[0086] Alanine Ala, A Arginine Arg, R Asparagine Asn, N Aspartic acid Asp, D Cysteine ​​Cys, C Glutamic acid Glu, E Glutamine Gln, Q Glycine Gly, G Histidine, H Isoleucine, Ile 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 (Val), V

[0087] In this specification, "Aib" may be used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid", and 2-aminoisobutyric acid and aminoisobutyric acid may be used interchangeably.

[0088] One embodiment of the present invention provides a composition, e.g., a pharmaceutical composition, containing a liver-targeting drug. Specifically, one embodiment of the present invention relates to a pharmaceutical composition containing a liver-targeting drug, in which the drug is highly distributed in the liver among the organs in the body of an individual to which the composition is administered.

[0089] A high distribution rate in the liver among the internal organs means, but is not limited to, that among the internal organs consisting of the liver, heart, lungs, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney and brain, after the liver-targeted drug is administered into the body, the distribution of the liver-targeted drug is highest in the liver.

[0090] The pharmaceutical composition may be for use in the prevention or treatment of a disease requiring a drug action in the liver.

[0091] In this application, the term "liver-targeted drug" refers to a drug that can be targeted to liver tissue. Targeting to liver tissue means, but is not limited to, that the drug is distributed more in the liver than in other organs after administration. The liver-targeted drug has therapeutic activity against diseases that require drug action in the liver.

[0092] The liver-targeting drug may have a T / S ratio (tissue-to-serum ratio) in the liver after administration selected from one or more of the following, but is not limited thereto: (a) a T / S ratio of about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 45%, about 41% to about 44%, or about 42% to about 43% at about 2 hours to about 5 hours after administration; (b) a T / S ratio of about 40% to about 60%, about 45% to about 55%, about 46% to about 50% at about 40 hours to about 50 hours after administration; and (c) a T / S ratio of about 45% to about 75%, about 50% to about 65%, about 55% to about 65%, about 58% to about 65%, about 59% to about 65%, about 60% to about 65%, about 61% to about 65%, about 62% to about 64%, or about 63% to about 64% at about 160 hours to about 180 hours after administration. The characteristic may be, but is not limited to, one or more, two or more, or all three selected from (a), (b), or (c).

[0093] The liver-targeted drug may have a T / S ratio in the liver after administration that is one or more selected from the following, but is not limited thereto: (a) a T / S ratio of about 35% to about 45%, about 40% to about 45%, about 41% to about 43%, about 42% to about 43%, or about 42.4% at about 4 hours after administration; (b) a T / S ratio of about 45% to about 55%, about 48% to about 52%, about 49% to about 52%, about 49% to about 51%, about 50% to about 51%, or about 50.1% at about 2 days after administration; and (c) a T / S ratio of about 55% to about 70%, about 60% to about 65%, about 61% to about 65%, about 62% to about 64%, about 63% to about 64%, or about 63.7% at about 7 days after administration. The features may be, but are not limited to, one or more, two or more, or all three selected from (a), (b), or (c).

[0094] The T / S ratio is the concentration ratio of tissue to serum, which is converted into a percentage and can be measured by a known method. For example, the T / S ratio (%) is calculated by tissue concentration / serum concentration x 100. The concentration is measured by removing the organ and then measuring the substance concentration by ELISA or the like.

[0095] The higher the T / S ratio, the higher the distribution of the administered substance in the tissue compared to tissues with a lower T / S ratio. In general, when a drug is administered to the body, the organs with the highest distribution of the drug are the lungs and the heart. When the distribution in the lung tissue and the heart tissue is compared to the liver tissue, if the distribution in the liver tissue is higher than that in the lung tissue and the heart tissue, this means that the liver-targeted drug of the present invention is effectively targeted to the liver tissue. In addition, tissues that can be used to measure the T / S ratio to confirm that the liver-targeted drug is effectively targeted to the liver tissue and compare it with the T / S ratio of the liver tissue can be included without limitation.

[0096] The liver-targeted drug may have a distribution ratio in the liver compared to the lung tissue after administration of, but is not limited to, about 1:2 to about 4.2, about 1:3 to about 4.2, about 1:3 to about 4, about 1:3.2 to about 4, about 1:3.3 to about 4, about 1:3.4 to about 4, about 1:3.5 to about 4, about 1:3.5 to about 3.9, about 1:3.5 to about 3.8, about 1:3.6 to about 3.8, about 1:3.6 to about 3.9, or about 1:3.65 to about 3.89.

[0097] The distribution ratio can be measured based on T / S (%), and can be confirmed based on the multiple of T / S (%) in the liver when T / S (%) in the lung is set to 1 as a standard.

[0098] The distribution ratio in the liver compared to the lung tissue after administration may be, but is not limited to, about 40 hours to about 180 hours, about 45 hours to about 170 hours, or about 2 days to about 7 days after administration.

[0099] The distribution ratio in the liver compared to the lung tissue after the administration may be, but is not limited to, about 1: about 3.2 to about 3.9, or 1: about 3.6 to about 3.7 at about 2 days after administration, and about 1: about 3.5 to about 4.2, 1: about 3.7 to 3.9, or 1: about 3.8 to about 3.9 at about 7 days after administration.

[0100] The distribution ratio in the liver compared to the heart after the administration may be, but is not limited to, (a) a distribution ratio in the liver compared to the heart 2 to 5 hours after administration of 1:1.5 to 3.0; (b) a distribution ratio in the liver compared to the heart 40 to 50 hours after administration of 1:2.0 to 3.0; and (b) a distribution ratio in the liver compared to the heart 160 to 180 hours after administration of 1:5.5 to 7.0.

[0101] Regarding the distribution ratio in the liver compared to the heart after the administration, (a) 2 to 5 hours after administration, the distribution ratio in the liver compared to the heart is 1:about 1.5 to about 3.0, 1:about 1.8 to about 3.0, 1:about 2.0 to about 2.8, 1:about 2.1 to about 2.5, or 1:about 2.2 to about 2.8; (b) about 40 to about 50 hours, about 45 to about 50 hours, or about 2 days after administration, the distribution ratio in the liver compared to the heart is 1:about 2.0 to 3.0, 1:about 2.4 to 3.0, 1:about 2.5 to 3.0, 1:about 2.6 to 3.0, 1:about 2.7 to 3.0, 1:about 2.8 to about 3.0, or 1:about 2.8 to 2.9. and (c) the distribution ratio in the liver compared to the heart at about 160 hours to about 180 hours, about 160 hours to about 180 hours, or about 7 days after administration may be, but is not limited to, 1: about 5.5 to about 7.0, 1: about 5.8 to about 7.0, 1: about 5.9 to about 7.0, 1: about 6.0 to about 7.0, 1: about 6.1 to about 7.0, 1: about 6.2 to about 7.0, 1: about 6.3 to about 7.0, 1: about 6.4 to about 7.0, 1: about 6.5 to about 7.0, 1: about 6.5 to about 6.9, 1: about 6.5 to about 6.8, 1: about 6.5 to about 6.7, 1: about 6.5 to about 6.6, or 1: about 6.55 to about 6.6.

[0102] The distribution ratio can be measured based on T / S (%), and can be determined by the multiple of T / S (%) in the liver when T / S (%) in the heart is set as the standard of 1.

[0103] In this application, the term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.05, etc., including all numerical values ​​in a range equal to or similar to the numerical value following the term about.

[0104] The liver targeting drug may include a substance that can be targeted to liver tissue. Specifically, the liver targeting drug may include a peptide sequence that binds to a glucagon receptor that can induce liver targeting and has activity against glucagon receptor, GLP-1 receptor, and GIP receptor. For example, the peptide may be in the form of a peptide containing the amino acid sequence of general formula 1 or a long-acting conjugate containing the same, but is not limited thereto. The peptide may be a peptide that has activity against glucagon receptor, GLP-1 receptor, and GIP receptor, but is not limited thereto.

[0105] Meanwhile, in the present invention, the term "having binding ability to glucagon receptor" refers to having binding ability to glucagon receptor to such an extent that the drug can be targeted from blood to liver when delivered into the blood of an animal. More specifically, when the organ distribution ratio of the drug is confirmed at least 1 hour, at least 4 hours, at least 48 hours, or at least 168 hours after the drug is delivered into the blood of an animal, the ratio of the drug distributed in the liver is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, or about 40% or more higher than at least one other organ such as heart, lung, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney, or brain, but is not particularly limited thereto.

[0106] The above-mentioned "peptide having activity on the glucagon receptor, the GLP-1 receptor, and the GIP receptor" may be used in the present invention as a triple-active peptide or a mixture of triple-active peptides.

[0107] Such peptides include a variety of substances, eg, a variety of peptides, that have significant levels of activity at the glucagon, GLP-1, and GIP receptors.

[0108] In particular, but not limited thereto, the triple active substance having a significant level of activity against the glucagon, GLP-1, and GIP receptors may exhibit in vitro activity against one or more of the glucagon, GLP-1, and GIP receptors, specifically, two or more of the receptors, more specifically, against all three receptors, of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more, compared to the natural ligands of the receptors (natural glucagon, natural GLP-1, and natural GIP).

[0109] More specifically, the triple active substance may exhibit in vitro activity against the glucagon receptor of about 0.1% or more, about 1% or more, about 2% or more, 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more compared to the natural ligand (natural glucagon), but is not limited thereto.

[0110] The method for measuring the in vitro activity of such a triple-active substance can be seen in Experimental Example 1 of the present specification, but is not particularly limited thereto.

[0111] On the other hand, the peptide is characterized in that it has one or more, two or more, specifically three activities, specifically significant activities, among the following i) to iii):

[0112] i) activation of the glucagon receptor; ii) activation of the GLP-1 receptor; and iii) activation of the GIP receptor.

[0113] More specifically, the compound may have activity against the glucagon receptor, activity against the GLP-1 receptor and / or activity against the GIP receptor, but is not particularly limited thereto.

[0114] Here, examples of activating a receptor include, but are not limited to, in vitro activity against the receptor of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more compared to the natural receptor.

[0115] The peptide may have a longer half-life in the body than any one of native glucagon, native GLP-1, and native GIP, but is not particularly limited thereto.

[0116] Although not limited thereto, such peptides may be non-naturally occurring.

[0117] However, without being limited thereto, native glucagon can have the following amino acid sequence:

[0118] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 118)

[0119] Specifically, the peptide may be an analog of native glucagon in which at least one amino acid in the native glucagon sequence has been subjected to a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof, and as long as the peptide has a high distribution in the liver among the organs in the body of an individual to which it is administered, it may fall within the scope of the present invention.

[0120] Furthermore, the amino acid substitution includes all substitutions with amino acids and substitutions with non-natural compounds.

[0121] Examples of glucagon analogs produced by such a combination of methods include, but are not limited to, peptides that have one or more amino acid sequences different from those of native glucagon, have the alpha carbon of the N-terminal amino acid residue removed, and have activity against the glucagon receptor, GLP-1 receptor, and GIP receptor. Native glucagon analogs applicable to the present invention can be produced by combining various methods for producing analogs.

[0122] In addition, although not limited thereto, some amino acids of the peptide of the present invention may be replaced with other amino acids or non-natural compounds to avoid the recognition action of activated decomposition enzymes in order to increase the half-life in the body.

[0123] Specifically, the triple activity peptide may be a peptide that has increased half-life in the body by avoiding the recognition action of degradative enzymes through a substitution of the second amino acid sequence in the amino acid sequence of the triple activity peptide, but any substitution or modification of amino acids to avoid the recognition action of degradative enzymes in the body is included without limitation.

[0124] Such modifications to produce analogs of native glucagon also include modifications using L- or D-amino acids, and / or non-naturally occurring amino acids; and / or modifications of the native sequence, such as modifications of side chain functional groups, intramolecular covalent bonds, e.g., side chain intercyclization, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, and the like.

[0125] It also includes all of the additions of one or more amino acids to the amino and / or carboxy termini of native glucagon.

[0126] The substituted or added amino acids can be atypical or non-naturally occurring amino acids as well as the 20 amino acids commonly observed in human proteins. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing such amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0127] Amino acid derivatives can also be obtained in a similar manner, with 4-imidazoacetic acid being just a few examples.

[0128] In one specific embodiment, the peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor, which is the liver targeting drug, may include an amino acid sequence represented by the following general formula 1:

[0129] Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-X aa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (general formula 1, sequence number 103)

[0130] In the general formula 1, Xaa1 is histidine, 4-imidazoacetyl, or tyrosine; Xaa2 is glycine, alpha-methyl-glutamic 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, lysine, or methionine; Xaa28 is cysteine, lysine, alanine, asparagine, or aspartic acid; Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine; Xaa30 is cysteine, glycine, lysine, histidine, or is absent; R1 is cysteine, GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; Where: m is -Cys-, -Pro-, or -Gly-Pro-; n is -Cys-, -Gly-, -Ser-, -His-Gly-, or absent.

[0131] Examples of the triple activity substance include, but are not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102, or an amino acid sequence (essentially) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102.

[0132] In addition, even if the present application describes a "peptide consisting of a specific sequence number," it does not exclude meaningless additions of sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof, so long as it has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, and it is self-evident that such additions or mutations of sequences also fall within the scope of the present application. In other words, even if there is a partial difference in sequence, if it shows a certain level of homology or identity or higher, and the distribution of the peptide in the liver among the internal organs of an individual to which it is administered is high, it can fall within the scope of the present invention.

[0133] Examples of such peptides include peptides consisting of a specific sequence number, or peptides having at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more sequence identity to a peptide containing a specific sequence number, and are not limited to a specific sequence as long as the peptide has a high distribution in the liver among the organs in the body of an individual to which the peptide is administered.

[0134] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0135] Whether any two peptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters as in, for example, Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, or using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later). (The GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0136] Peptide homology, similarity or identity can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48: 443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines a sequence as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., amino acids). Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, as used herein, the terms "homology" or "identity" indicate the relevance between sequences.

[0137] The above may be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0138] As an example, in general formula 1 above, Xaa14 may be leucine or methionine, and Xaa15 may be cysteine, aspartic acid, or leucine.

[0139] Examples of such peptides include, but are not limited to, peptides that contain or are (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, 14 to 17, and 21 to 102.

[0140] Such a peptide may significantly activate one or more of glucagon receptor, GLP-1 receptor, and GIP receptor, but is not particularly limited thereto. Specifically, the peptide may significantly activate GLP-1, and may also significantly activate glucagon receptor and / or GIP receptor, but is not particularly limited thereto.

[0141] Also, as an example, In the general formula 1, Xaa2 is glycine, alpha-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, tyrosine, 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; Xaa27 is leucine or lysine and may be a peptide, but is not limited thereto.

[0142] Also, as an example, In the general formula 1, Xaa2 is glycine, alpha-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; Xaa23 is valine, Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; Xaa27 may be, but is not limited to, leucine or lysine.

[0143] Also, as an example, In the general formula 1, Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa7 is threonine, Xaa10 is tyrosine or cysteine; Xaa12 is lysine or isoleucine; Xaa13 is tyrosine, alanine, or cysteine; Xaa14 is leucine or methionine; Xaa15 is cysteine ​​or aspartic acid; Xaa16 is glutamic acid, 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, Xaa28 can be cysteine, alanine, asparagine, or aspartic acid.

[0144] Also, as an example, In the general formula 1, Xaa1 is histidine or 4-imidazoacetyl; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, Xaa12 is isoleucine, Xaa13 is alanine or cysteine; Xaa14 is methionine, Xaa15 is aspartic acid, Xaa16 is glutamic acid, Xaa17 is isoleucine or lysine; Xaa18 is alanine or histidine; Xaa19 is glutamine or cysteine; Xaa20 is lysine, Xaa21 is aspartic acid, Xaa23 is valine, Xaa24 is asparagine, Xaa27 is leucine, Xaa28 is alanine or asparagine; Xaa29 is glutamine or threonine; Xaa30 can be cysteine, lysine, or absent.

[0145] Also, as an example, In the general formula 1, Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib; Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, cysteine, or valine; Xaa12 is lysine, Xaa13 is tyrosine, Xaa14 is leucine, Xaa15 is aspartic acid, Xaa16 is glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine, cysteine, or lysine; Xaa18 is alanine, arginine, or histidine; Xaa19 is alanine or glutamine; Xaa20 is lysine or glutamine; Xaa21 is glutamic acid, cysteine, or aspartic acid; Xaa23 is valine, Xaa24 is alanine, glutamine, or cysteine; Xaa27 is leucine or lysine; Xaa29 may be, but is not limited to, glycine, glutamine, threonine, or histidine.

[0146] Such a peptide may be applicable when the activation level of the glucagon receptor and the GLP-1 receptor is significant and higher than the activation level of the GIP receptor; when the activation levels of the GLP-1 receptor, the glucagon receptor and the GIP receptor are all significant; or when the activation levels of the GLP-1 receptor and the GIP receptor are significant and higher than the activation level of the glucagon receptor, but is not particularly limited thereto.

[0147] When the activation level of GLP-1 receptor and GIP receptor is significant and higher than that of glucagon receptor, a peptide with higher blood glucose regulating ability can be provided with weight reduction efficacy, and when the activation levels of GLP-1 receptor, glucagon receptor and GIP receptor are all significant, there is an advantage that the weight reduction effect is maximized. However, it is not particularly limited thereto, and as long as it can be targeted to the liver and optionally has therapeutic activity on the liver, it is all included in the scope of the present invention.

[0148] Examples of such peptides include peptides that contain or are (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 21-37, 39, 42, 43, 49-61, 64-83, 85, 86, 88, 89, 91-93, and 95-102, but are not limited thereto.

[0149] As a specific example, the peptide that is the liver targeting drug may include an amino acid sequence represented by the following general formula 2.

[0150] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa 17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31- Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO: 104)

[0151] In the above formula, Xaa1 is 4-imidazoacetyl, histidine, or tyrosine; Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib; Xaa10 is tyrosine or cysteine Xaa13 is alanine, glutamine, tyrosine, or cysteine; Xaa14 is leucine, methionine, or tyrosine; Xaa15 is aspartic acid, glutamic acid, or leucine; Xaa16 is glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine; Xaa18 is alanine, glutamine, arginine, or histidine; Xaa19 is alanine, 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; Xaa31 is proline or cysteine; Xaa40 is cysteine ​​or absent. More specifically, in the 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; Xaa30 can be cysteine, lysine, or histidine.

[0152] Examples of such peptides include, but are not limited to, an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 95 to 102, more specifically, a peptide that includes or is (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64 to 77, and 96 to 102.

[0153] In one embodiment, the peptide that is the liver targeting drug may include an amino acid sequence of the following general formula 3:

[0154] Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Ph e-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 3, sequence number 105)

[0155] In the general formula 3, Xaa1 is histidine or tyrosine; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa13 is alanine, tyrosine, or cysteine; Xaa17 is arginine, cysteine, or lysine; Xaa18 is alanine or arginine; Xaa19 is alanine or cysteine; Xaa21 is glutamic acid or aspartic acid; Xaa24 is glutamine or asparagine; Xaa28 is cysteine ​​or aspartic acid; Xaa29 is cysteine, histidine, or glutamine; Xaa30 is cysteine ​​or histidine; Xaa31 is proline or cysteine; Xaa40 may be cysteine ​​or absent. Examples of such peptides include, but are not limited to, peptides that contain or are (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 42, 43, 50, 64-71, 75-77, and 96-102.

[0156] In addition, in the general formula 1, R1 may be cysteine, GKKNDWKHNIT (sequence number 106), CSSGQPPPS (sequence number 109), GPSSGAPPPS (sequence number 110), GPSSGAPPPSC (sequence number 111), PSSGAPPPS (sequence number 112), PSSGAPPPSG (sequence number 113), PSSGAPPPSHG (sequence number 114), PSSGAPPPSS (sequence number 115), PSSGQPPPS (sequence number 116), or PSSGQPPPSC (sequence number 117), or may be absent, but is not particularly limited thereto.

[0157] The above-mentioned triple activity peptide may include an intramolecular bridge (e.g., a covalent or non-covalent bridge), and specifically may be in the form of a ring. For example, the triple activity peptide may be in the form of a ring formed between the 16th and 20th amino acids, but is not particularly limited thereto.

[0158] Non-limiting examples of such rings can include lactam bridges (or lactam rings).

[0159] The triple activity peptide also includes all peptides modified to include a ring and to include an amino acid capable of forming a ring at a desired position.

[0160] Such a ring may be formed between the side chains of amino acids in the triple activity peptide, for example, a lactam ring may be formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0161] For example, the peptides containing the amino acid sequences of general formulas 1 to 3 may be, but are not limited to, those in which the amino acids in the amino acid pairs Xaa10 and Xaa14, Xaa12 and Xaa16, Xaa16 and Xaa20, Xaa17 and Xaa21, Xaa20 and Xaa24, and Xaa24 and Xaa28 in general formulas 1 to 3 are substituted with glutamic acid or lysine, respectively. In the Xaan (n is a natural number), n indicates the amino acid position from the N-terminus of the presented amino acid sequence.

[0162] In addition, the peptides containing the amino acid sequences of general formulas 1 to 3 may each be substituted with glutamic acid or lysine capable of forming a ring in the amino acid pair of Xaa12 and Xaa16, the amino acid pair of Xaa16 and Xaa20, or the amino acid pair of Xaa17 and Xaa21, but are not limited thereto.

[0163] Furthermore, in the general formulas 1 to 3, among the amino acid pairs of Xaa10 and Xaa14, Xaa12 and Xaa16, Xaa16 and Xaa20, Xaa17 and Xaa21, Xaa20 and Xaa24, and Xaa24 and Xaa28, at least one of the amino acid pairs may have a ring (e.g., a lactam ring) formed between each amino acid in each amino acid pair, but is not limited thereto.

[0164] Furthermore, in the general formulae 1 to 3, Xaa16 may be glutamic acid, Xaa20 may be lysine, and the side chains of Xaa16 and Xaa20 may form a lactam ring, but is not limited thereto.

[0165] In addition, the peptide according to the present invention may have an unmodified N-terminus and / or C-terminus, but the peptide according to the present invention also includes those modified by chemically modifying the N-terminus and / or C-terminus or protecting them with an organic group to protect them from in vivo protease and increase their stability, or by adding an amino acid to the peptide terminus, etc. When the C-terminus is unmodified, the terminus of the peptide according to the present invention has a carboxyl group, but is not particularly limited thereto.

[0166] Alternatively, in the case of a chemically synthesized peptide, since the N- and C-termini are charged, the N-terminus may be acetylated and / or the C-terminus may be amidated to remove such charges, but is not particularly limited thereto.

[0167] Unless otherwise specified in the present specification, the detailed description and claims of the "peptide" according to the present invention or the "conjugate" in which such a peptide is covalently linked to an immunoglobulin Fc region are applicable to the peptide or conjugate, as well as to a category including all forms of the salt of the peptide or conjugate (e.g., a pharma- ceutically acceptable salt of the peptide), or a solvate thereof. Thus, even if the specification only describes a "peptide" or a "conjugate," the description is equally applicable to a specific salt thereof, a specific solvate thereof, or a specific solvate of a specific salt thereof. Such a salt form may be, for example, a form using any pharma- ceutically acceptable salt.

[0168] The type of the salt is not particularly limited, but is preferably in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0169] The term "pharmaceutical acceptable" means a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reaction, or the like, within the scope of medical judgment.

[0170] In this application, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, potassium, alkaline earth metals such as magnesium, and ammonium, and the like.

[0171] The term "solvate" used in the present invention refers to a complex formed between the peptide, conjugate, or salt thereof according to the present invention and a solvent molecule.

[0172] The C-terminus of the peptide according to the present invention may be amidated or may have a free carboxyl group (-COOH), or may include a peptide with an unmodified C-terminus, but is not limited thereto.

[0173] In one specific example, the peptide may be amidated at the C-terminus, but is not limited thereto.

[0174] In one embodiment, but not limited to, the peptide may be non-glycosylated.

[0175] The peptide of the present invention may be synthesized by solid phase synthesis, may be produced by recombinant methods, or may be commercially manufactured, but is not limited thereto.

[0176] Furthermore, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0177] In particular, the peptides of the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the peptides according to the present invention can be synthesized in a number of ways, including, for example, the following:

[0178] (a) synthesis of peptides stepwise or by fragment assembly by means of solid-phase or solution-phase techniques, followed by isolation and purification of the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method comprising obtaining peptide fragments by any combination of (a), (b) and (c), subsequently ligating the fragments to obtain a peptide, and recovering the peptide.

[0179] In addition, the peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor may be in the form of a long-acting conjugate in which the peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor is conjugated to a biocompatible material portion that increases its in vivo half-life, but is not limited thereto. In the present specification, the biocompatible material portion may be mixed with a carrier.

[0180] The peptide comprising the amino acid sequence of any one of general formulas 1 to 3 in the form of a long-acting conjugate (conjugate form), or the peptide comprising the amino acid sequence of any one of general formulas 1 to 3 itself is a liver-targeted drug, and is an active ingredient of a pharmaceutical composition in which the drug is highly distributed in the liver among the internal organs of an individual to which it is administered.

[0181] In the present application, the term "long-acting conjugate" refers to a form in which a biocompatible substance or carrier is bound to a physiologically active substance (e.g., a triple activity peptide). Specifically, the conjugate includes a peptide moiety and a biocompatible substance moiety covalently linked to the peptide moiety, and the peptide moiety may be the same as or include any one of the amino acid sequences of the general formulas 1 to 3 or any one of the sequences selected from SEQ ID NOs: 1 to 102. In the long-acting conjugate, the biocompatible substance moiety or carrier may be covalently linked to a physiologically active substance, but is not particularly limited thereto. The C-terminus of the peptide having the same sequence as or include any one of the amino acid sequences of the general formulas 1 to 3 or any one of the sequences selected from SEQ ID NOs: 1 to 102 may be amidated, or may have a free carboxyl group (-COOH), or may include a peptide with an unmodified C-terminus, but is not limited thereto.

[0182] In the present invention, the peptide conjugate may exhibit an increased duration of efficacy and / or increased half-life in the blood compared to the peptide not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" in the present invention.

[0183] Alternatively, such conjugates may be non-naturally occurring.

[0184] In a specific embodiment of the present invention, the long-acting conjugate refers to a form in which an immunoglobulin Fc region is linked to a triple activity peptide. Specifically, the conjugate may be a triple activity peptide and an immunoglobulin Fc region covalently linked to each other via a linker, but is not particularly limited thereto.

[0185] In one embodiment, but not limited to, the immunoglobulin Fc region and X may be non-glycosylated.

[0186] In one embodiment of the present invention, the persistent conjugate is a conjugate represented by the following formula 1:

[0187] [Chemical formula 1] XLF

[0188] Where: X is the peptide; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region, - indicates a covalent bond link between X and L, and between L and F.

[0189] X of the persistent conjugate of Formula 1 may be, but is not limited to, the triple activation peptide described above.

[0190] In one specific example, X of the persistent conjugate of formula 1 may be a peptide comprising the amino acid sequence of formula 1.

[0191] In one specific example, X of the persistent conjugate of formula 1 may be a peptide comprising the amino acid sequence of formula 2 or 3.

[0192] Specifically, the X may be a peptide containing an amino acid sequence of any one of SEQ ID NOs: 1 to 102, but is not limited thereto.

[0193] In the conjugate, F is X, i.e., a liver targeting drug, specifically, a drug containing a substance having binding ability to the glucagon receptor, for example, a peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor, which corresponds to one of the moieties constituting the conjugate of the present invention. All of the above applies to the liver targeting drug.

[0194] The F may be bonded to X by a covalent or non-covalent chemical bond, or F and X may be bonded to each other through L by a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0195] In the present invention, the term "long-acting conjugate of Chemical Formula 1" refers to a form in which a triple activity peptide and an immunoglobulin Fc region are linked to each other via a linker, and the conjugate may exhibit increased persistence of efficacy compared to a triple activity peptide to which an immunoglobulin Fc region is not linked.

[0196] In the long-acting conjugate, F is a substance that increases the half-life of X, i.e., the triple activation peptide, and corresponds to one component of the moiety that constitutes the conjugate of the present invention.

[0197] In the long-acting conjugate of Chemical Formula 1, the triple-active peptide X and the immunoglobulin Fc region may be linked by a physical or chemical bond, or a non-covalent or covalent bond, specifically, by a covalent bond, but is not limited thereto.

[0198] In addition, the method of linking the triple activity peptide X of the long-acting conjugate of Chemical Formula 1 to the immunoglobulin Fc region is not particularly limited, and the triple activity peptide and the immunoglobulin Fc region may be linked to each other via a linker.

[0199] In Formula 1, X and F are bonded to each other via L by a covalent bond.

[0200] More specifically, X and L, and L and F may be linked to each other by a covalent bond, and in this case, the conjugate may be a conjugate in which X, L, and F are linked to each other by a covalent bond in the order of Chemical Formula 1.

[0201] Moreover, the X may be linked to F via a linker (L).

[0202] Alternatively, L may be a non-peptidic linker, such as a linker containing repeating ethylene glycol units.

[0203] In the present invention, the "non-peptidic linker" includes a biocompatible polymer in which two or more repeating units are bonded. The repeating units are linked to each other through any covalent bond, not a peptide bond. The non-peptidic linker may be one of the moieties of the conjugate of the present invention, and corresponds to L in Chemical Formula 1. The non-peptidic linker that can be used in the present invention can be any polymer that is resistant to in vivo protease, without any limitations. In the present invention, the non-peptidic linker may be used in combination with a non-peptidic polymer.

[0204] Although not particularly limited thereto, the non-peptidic linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and derivatives thereof already known in the art and derivatives that can be easily prepared at the technical level in the art are also included within the scope of the present invention.

[0205] The repeating unit of the non-peptidic linker may be an ethylene glycol repeating unit, and specifically, the non-peptidic linker may contain an ethylene glycol repeating unit and a functional group used in the preparation of a conjugate at its end before being constructed into a conjugate. The long-lasting conjugate according to the present invention may be in a form in which X and F are linked through the functional group, but is not limited thereto. In the present invention, the non-peptidic linker may contain two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0206] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following formula 2:

[0207] [Chemical formula 2] JPEG2025502749000002.jpg2035

[0208] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

[0209] In the persistent conjugate, the PEG moiety is -(CH2CH2O) n -Not only the structure, but also the linking elements and this -(CH2CH2O) n It may also include, but is not limited to, an oxygen atom between - and -.

[0210] As one specific example, the ethylene glycol repeating unit is represented by, for example, [OCH2CH2]n, where the value of n is a natural number and is determined so that the average molecular weight of the [OCH2CH2]n moiety in the peptide bond, for example, the number average molecular weight, is greater than 0 to about 100 kDa, but is not limited thereto. In another example, the value of n is a natural number and the average molecular weight of the [OCH2CH2]n site in the peptide bond, for example, the number average molecular weight, is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 1 5 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa The molecular weight may be, but is not limited to, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, or about 10 kDa.

[0211] In one specific embodiment, the long-acting conjugate of Chemical Formula 1 may have a structure in which a peptide (X) having an amino acid sequence of any one of General Formulas 1 to 3 and an immunoglobulin domain (F) are covalently linked via a linker containing an ethylene glycol repeating unit, but is not limited thereto.

[0212] The polyethylene glycol is a term that encompasses any of the forms of ethylene glycol homopolymer, PEG copolymer, and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

[0213] The molecular weight of the non-peptidic polymer is in the range of 1 to 100 kDa, specifically in the range of 1 to 20 kDa or in the range of 1 to 10 kDa, but is not limited thereto. In addition, the non-peptidic linker of the present invention to be bonded to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0214] In one specific example, both ends of the linker may be bound to a thiol group, an amino group, or a hydroxyl group of the immunoglobulin Fc region and a thiol group, an amino group, an azide group, or a hydroxyl group of the peptide (X), but is not limited thereto.

[0215] Specifically, the linker may contain reactive groups at both ends capable of binding to an immunoglobulin Fc region and peptide (X), specifically, reactive groups that are bound to a thiol group of cysteine ​​in the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine, and / or histidine; and / or a hydroxyl group located at the C-terminus, and can bind to a thiol group of cysteine; an amino group of lysine, arginine, glutamine, and / or histidine; an azide group of azidolysine; and / or a hydroxyl group of peptide (X), but are not limited thereto.

[0216] More specifically, the reactive group of the linker may be at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0217] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0218] In the above, examples of the succinimide derivative include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0219] The linker is linked to F, which is an immunoglobulin Fc region, and X, which is a peptide (triactivator), through the reactive groups described above, and converted into a linker linker.

[0220] In addition, the final product generated by reductive amination (or reductive alkylation) via an aldehyde bond is much more stable than that linked via an amide bond. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form a covalent bond with lysine residues at high pH conditions, such as, but not limited to, pH 9.0.

[0221] In addition, the reactive groups at both ends of the linker of the present invention may be the same or different from each other. The linker may have an aldehyde reactive group at its terminal, or may have an aldehyde group and a maleimide reactive group at each end, or an aldehyde group and a succinimide reactive group at each end, but is not limited thereto as long as F, specifically, the immunoglobulin Fc region and X are bound to each end of the linker.

[0222] For example, one end may have a maleimide group and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. Also, as an example, one end may have a succinimidyl group and the other end may have a propionaldehyde group or a butyraldehyde group.

[0223] When polyethylene glycol having a hydroxy reactive group at the propionyl end is used as a linker, the hydroxy group can be activated with the various reactive groups by known chemical reactions, or the conjugate of the present invention can be prepared using commercially available polyethylene glycol having a modified reactive group.

[0224] In one specific embodiment, the reactive group of the linker may be linked to a cysteine ​​residue of the peptide (X), more specifically, to the -SH group of cysteine, but is not limited thereto.

[0225] When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of the peptide via a thioether bond, and the aldehyde group can be linked to the -NH2 group of the immunoglobulin Fc via a reductive alkylation reaction, but this is just one example and is not limited thereto.

[0226] Through such reductive alkylation, the oxygen atom at one end of PEG is linked to the N-terminal amino group of the immunoglobulin Fc region through a linker functional group having the structure -CH2CH2CH2- to form a structure similar to -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and a structure in which one end of PEG is linked to a sulfur atom at a cysteine ​​residue of a peptide through a thioether bond can be formed. The above-mentioned thioether bond is It can contain the structure JPEG2025502749000003.jpg4033.

[0227] However, the present invention is not particularly limited to the above example, which is merely an example.

[0228] In the above-mentioned conjugate, the reactive group of the linker may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, which is just one example.

[0229] In the conjugate, the peptide according to the present invention may be linked to a linker having a reactive group through the C-terminus, which is just one example.

[0230] In the present invention, the "C-terminus" refers to the carboxy terminus of a peptide, and for the purposes of the present invention, refers to a position that can be bound to a linker. Examples of the C-terminus include, but are not limited to, not only the most terminal amino acid residue of the C-terminus, but also any amino acid residues around the C-terminus, specifically, the first to 20th amino acid residues from the most terminal, but are not limited thereto.

[0231] As a specific example, the conjugate of Formula 1 may have a structure of Formula 3 below.

[0232] [Chemical formula 3] JPEG2025502749000004.jpg3398

[0233] In the formula 3, X is the peptide (triple active compound) described above; F is a human immunoglobulin Fc region; n may be a natural number, and in this case, the explanation for n is as described above.

[0234] As a specific example, the long-acting conjugate of Chemical Formula 3 may have a structure in which peptide X and human immunoglobulin Fc region F are covalently linked via an ethylene glycol repeat moiety, and X may be linked to a succinimide ring of Chemical Formula 3, and F may be linked to an oxypropylene group of Chemical Formula 3.

[0235] In Chemical Formula 3, the value of n may be determined so that the average molecular weight of the [OCH2CH2]n moiety in the peptide bond, for example, the number average molecular weight, is 1 to 100 kDa, or 1 to 20 kDa, or 10 kDa, but is not limited thereto.

[0236] In one embodiment, the site at which X is linked to the succinimide ring of Formula 3 may be the sulfur atom of the C-terminal cysteine ​​of X.

[0237] The site in F linked to the oxypropylene group is not particularly limited. In one embodiment of the present invention, the site of F linked to the oxypropylene group may be the N-terminal nitrogen or a nitrogen atom of an internal residue of F (e.g., the epsilon nitrogen of lysine). In a specific embodiment of the present invention, the site of F linked to the oxypropylene group may be, but is not limited to, the N-terminal proline of F.

[0238] In the conjugate, the reactive group of the nonpeptidic polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, which is just one example.

[0239] As a specific example, F in the above Chemical Formula 1 is an immunoglobulin Fc region. For example, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.

[0240] In the present invention, the "immunoglobulin Fc region" refers to a portion including heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3) excluding the variable regions of the heavy and light chains of an immunoglobulin. The immunoglobulin Fc region may be one of the components constituting the moiety of the long-acting conjugate of the present invention. The immunoglobulin Fc region may be used interchangeably with "immunoglobulin Fc fragment".

[0241] In the present specification, the term "Fc region" includes not only the native sequence obtained by papain digestion of immunoglobulin, but also its derivatives, such as variants in which one or more amino acid residues in the native sequence are deleted, inserted, non-conservative or conservatively substituted, or a combination thereof to produce a sequence different from the native sequence. The derivatives, substitutions, and variants are premised on having the ability to bind to FcRn. In the present invention, F may be a human immunoglobulin region, but is not limited thereto. F may be a structure in which two polypeptide chains are linked by a disulfide bond, and may be a structure in which only one of the two chains is linked via a nitrogen atom, but is not limited thereto. The linkage via the nitrogen atom may be a linkage to the epsilon amino atom of lysine or the amino group at the N-terminus by reductive amination.

[0242] The reductive amination reaction refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, which is then reduced to form an amine bond, and is an organic synthesis reaction well known in the art.

[0243] In one specific example, the F may be linked through the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0244] Such an immunoglobulin Fc region may include, but is not limited to, a hinge portion in the heavy chain constant region.

[0245] The immunoglobulin Fc region of the present invention may comprise a specific hinge sequence at the N-terminus.

[0246] As used herein, the term "hinge sequence" refers to a site located in a heavy chain that forms a dimer of an immunoglobulin Fc region through inter disulfide bonds.

[0247] The hinge sequence of the present invention may be a mutated hinge sequence having the following amino acid sequence with a partial deletion to have only one cysteine ​​residue, but is not limited thereto:

[0248] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (sequence number 119).

[0249] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 119 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention is one that contains only one cysteine ​​residue and is composed of 3 to 12 amino acids, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 120), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 121), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 122), Glu -Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 123), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 124), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 125), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 126), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 12 7), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 128), Pro-Ser-Cys-Pro (SEQ ID NO: 129), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 130), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 131), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 132) , Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (sequence number 133), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (sequence number 134), Glu-Ser-Lys-Pro-Ser-Cys-Pro (sequence number 135), Glu-Ser-Pro-Ser-Cys-Pro (sequence number 136), Glu-Pro-Ser-Cys (sequence number 137), Ser-Cys-Pro (sequence number 138).

[0250] More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 129 (Pro-Ser-Cys-Pro) or SEQ ID NO: 138 (Ser-Cys-Pro), but is not limited thereto.

[0251] The immunoglobulin Fc region of the present invention may be in a form in which two molecules of immunoglobulin Fc chains form a dimer due to the presence of a hinge sequence, and the conjugate of Chemical Formula 1 of the present invention may be in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0252] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide and may include the extreme amino terminus or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc region of the present invention may include, but is not limited to, a hinge sequence at the N-terminus.

[0253] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that excludes only the variable regions of the heavy and light chains of an immunoglobulin and includes a part or all of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 has been deleted.

[0254] For example, the immunoglobulin Fc region of the present invention may be, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination of at least one or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region) (e.g., a combination of a CH2 domain and a CH3 domain with a hinge region or a portion thereof, and a dimeric form of two polypeptides having the above combinations); or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0255] In the present invention, the immunoglobulin Fc region may be in a dimeric or multimeric form composed of single-chain immunoglobulins consisting of domains of the same origin, but is not limited thereto.

[0256] In one specific example, the immunoglobulin Fc region may be in a dimeric form, and one molecule of X is covalently linked to one Fc region in the dimeric form, and at this time, the immunoglobulin Fc and X are linked to each other via a non-peptide polymer. On the other hand, two molecules of X may be symmetrically linked to one Fc region in the dimeric form. At this time, the immunoglobulin Fc and X are linked to each other via a non-peptide linker. However, the present invention is not limited to the above example.

[0257] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means a sequence that differs from the naturally occurring amino acid sequence by deletion, insertion, non-conservative or conservative substitution of at least one amino acid residue, or a combination thereof.

[0258] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as suitable sites for modification.

[0259] Also, various kinds of derivatives are possible, such as removing the site that forms a disulfide bond, removing some amino acids at the N-terminus from the native Fc, or adding a methionine residue to the N-terminus of the native Fc. Also, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, for example.

[0260] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications may be made, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0261] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc region.

[0262] In addition, such an Fc region may be obtained from a natural source isolated from the living body of an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant product obtained from a transformed animal cell or a microorganism, or a derivative thereof. Here, the method of obtaining it from a natural source may be a method of obtaining it by separating the whole immunoglobulin from the living body of a human or animal and then treating it with a proteolytic enzyme. When treating with papain, it is cleaved into Fab and Fc, and when treating with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated from it using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0263] Furthermore, the immunoglobulin Fc region may be in a form having a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. For increasing, decreasing, or removing such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used. Here, an immunoglobulin Fc region from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (c1q) and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, it can be said that the form more suited to the original purpose as a drug carrier is an immunoglobulin Fc region from which the sugar chains have been removed or which has been non-glycosylated.

[0264] In the present invention, "deglycosylation" refers to an Fc region from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0265] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as bovine, goat, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment is of human origin.

[0266] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are the most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0267] Furthermore, in one specific embodiment, the immunoglobulin Fc region may be a human IgG4 Fc region in the form of a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between cysteines, which are the third amino acids of each monomer, and in this case, each monomer of the homodimer independently has / may have an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, i.e., two internal disulfide bonds (intra-chain form). Each monomer is composed of 221 amino acids, and the total number of amino acids forming the homodimer is 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc region is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 139 (consisting of 221 amino acids) through a disulfide bond between the third amino acid cysteine ​​of each monomer, and the monomers of the homodimer independently form an internal disulfide bond between the 35th and 95th cysteines and an internal disulfide bond between the 141st and 199th cysteines, but is not limited thereto.

[0268] F in the above formula 1 may include a monomer having the amino acid sequence of SEQ ID NO: 139, and F may be a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 139, but is not limited thereto.

[0269] As an example, the immunoglobulin Fc region may be, but is not limited to, a homodimer comprising the amino acid sequence of SEQ ID NO: 140 (composed of 442 amino acids).

[0270] Meanwhile, in the present invention, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin when forming a dimer or multimer. That is, a dimer or multimer can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and Fc fragments of IgE.

[0271] Furthermore, the above-mentioned conjugate may have an increased duration of efficacy compared to native glucagon, native GLP-1, or native GIP, or compared to X in which F is not modified, and such conjugates include not only the above-mentioned forms but also all forms such as those encapsulated in biodegradable nanoparticles.

[0272] A composition comprising the liver-targeted drug, for example, a composition comprising the peptide (e.g., the peptide itself or a form in which an immunoglobulin Fc region is bound to the peptide) may be for the prevention or treatment of a disease requiring drug action in the liver.

[0273] In the present invention, the term "prevention" means any action of suppressing or delaying the onset of a disease that requires drug action in the liver by administering a composition containing the liver-targeted drug, and "treatment" means any action of improving or benefiting the symptoms of a disease that requires drug action in the liver by administering a composition containing the liver-targeted drug.

[0274] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the administration route of the composition is not particularly limited, but may be any common route by which the composition can reach an in vivo target, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, or rectal administration.

[0275] In the present application, the term "diseases requiring drug action in the liver" refers to diseases for which the liver-targeting drug according to the present invention exhibits therapeutic activity, and which show preventive or therapeutic effects due to the increased distribution of the drug administered to the liver tissue. For example, diseases that can be targeted and treated by the liver-targeting drug of the present invention are included without limitation. An example of such a disease may be liver disease. In the present invention, the term "liver disease" refers to a disease that occurs in the liver, and may include, but is not limited to, metabolic liver disease or liver inflammation. Representative examples of the liver disease include simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, and liver cancer, and as long as abnormalities occur in the liver tissue and function, they may be liver diseases according to the present invention. In many cases, inflammation in the liver may occur due to causes such as viruses, alcohol, drugs, immune abnormalities, and metabolic diseases, and it is known that the progression and chronicization of liver inflammation can lead to diseases such as cirrhosis and liver cancer. The composition according to the present invention may be effective against liver diseases accompanied by or caused by liver inflammation, such as, but not limited to, liver inflammation, non-alcoholic steatohepatitis, or liver fibrosis. Meanwhile, the composition according to the present invention may also be effective in preventing or treating liver diseases not accompanied by inflammation, such as, but not limited to, simple steatosis, non-alcoholic fatty liver, and cirrhosis.

[0276] The liver disease for which the peptide or its conjugate of the present invention has a therapeutic effect may be, but is not limited to, a metabolic liver disease, which is caused by abnormal chemical reactions in the body that interfere with the body's metabolism, and includes simple steatosis, fatty liver, steatohepatitis, etc.

[0277] The composition according to the present invention may have a preventive or therapeutic effect on metabolic liver disease by reducing the amount of triglyceride and / or cholesterol in liver tissue upon administration, but is not limited thereto. The metabolic liver disease may or may not be accompanied by inflammation, and examples of liver diseases that can be treated with the composition according to the present invention include, but are not limited to, simple steatosis, non-alcoholic fatty liver, non-alcoholic steatohepatitis, etc.

[0278] "Nonalcoholic fatty liver disease (NAFLD)", a representative example of metabolic liver disease, refers to cases where fatty liver is present even though there is no history of alcohol intake and no relationship to alcohol intake. Fatty liver refers to the phenomenon in which neutral fat is abnormally deposited in liver cells, unlike normal cases. Approximately 5% of a normal liver is composed of adipose tissue, and neutral fat, fatty acid, phospholipid, cholesterol, and cholesterol ester are the main components of fat. Once fatty liver occurs, most of the components are replaced by neutral fat, and if the amount of neutral fat is 5% or more of the liver weight, it is diagnosed as fatty liver. Fatty liver is caused by a fat metabolism disorder in liver cells or a defect in the process of transporting excess fat, and is mainly caused by a fat metabolism disorder in the liver. Most of the fat accumulated in the fatty liver may be neutral fat (triglyceride).

[0279] Nonalcoholic fatty liver disease refers to a series of diseases including simple steatosis, which is characterized by excessive accumulation of fat in liver cells, nonalcoholic fatty liver, and nonalcoholic steatohepatitis (NASH), which is characterized by hepatocyte necrosis, inflammation, and fibrosis, but is not limited thereto, as long as it is treated with the composition of the present invention. Nonalcoholic fatty liver disease according to the present invention may be, but is not limited to, nonalcoholic steatohepatitis.

[0280] In addition, the liver disease for which the peptide or its conjugate of the present invention has a therapeutic effect may be, but is not limited to, liver inflammation. In the present invention, "liver inflammation" refers to a disease that causes inflammation in the liver, which is the main cause of liver disease, and is classified into acute hepatitis and chronic hepatitis depending on the cause and symptoms. The main causes are viruses, alcohol, drugs, immune disorders, metabolic diseases, etc.

[0281] In the present invention, "nonalcoholic steatohepatitis" is one of nonalcoholic fatty liver diseases, and is a representative example of liver disease accompanied by hepatocyte necrosis, inflammation, and fibrosis. The composition according to the present invention can suppress liver inflammation and fibrosis and show effects on nonalcoholic steatohepatitis, specifically, nonalcoholic steatohepatitis accompanied by fatty liver, liver fibrosis, or liver cirrhosis; or liver cancer caused by nonalcoholic steatohepatitis, but is not limited thereto.

[0282] In the present invention, "liver fibrosis" refers to the result of a wound healing process against repeated liver injury, and excessive fibrous connective tissue is formed in organs and tissues during reparative or reactive processes. Chronicity and deepening of liver inflammation are known to be one of the causes of the disease. Unlike cirrhosis, liver fibrosis is reversible, composed of thin fibrils, and is known to have no nodule formation. If the cause of liver injury disappears, normal recovery is possible. However, if such a process of liver fibrosis is repeated, crosslinking between ECM (extra cellular matrix) increases, progressing to irreversible liver cirrhosis with nodules. The composition according to the present invention can exhibit a preventive or therapeutic effect on liver fibrosis, specifically liver fibrosis associated with nonalcoholic steatohepatitis, but is not limited thereto.

[0283] In the present invention, "cholestasis" refers to a condition in which the flow of bile from the liver to the duodenum is slowed or blocked, and "cholestasis liver disease" refers to a condition in which bile formation in the liver is impeded by conditions such as various diseases, extended jugular feeding, or side effects of certain drugs (e.g., some antibiotics). Common symptoms of cholestasis include fatigue, pruritus (itch), jaundice, and xanthomas (deposition of cholesterol-rich material under the skin). The effects of cholestasis are extreme and widespread, which can lead to the deterioration of liver disease into systemic disease, liver failure, and the need for liver transplantation. Causes of cholestatic liver disease include acute hepatitis, inflammation of the bile duct, etc.

[0284] The cholestatic liver disease may include, but is not limited to, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), and Alagille syndrome (AS).

[0285] Primary biliary cirrhosis, also known as primary biliary cholangitis (PBC), is a chronic cholestatic liver disease of unknown etiology. Progressive bile duct damage due to portal and periportal inflammation can lead to progressive fibrosis and ultimately cirrhosis. To date, immunological, genetic and environmental factors are known to be potential causes of the disease. Primary biliary cirrhosis is primarily seen in middle-aged women, and symptoms may include fatigue, itching or unclear hyperlipidemia at the initial onset of primary biliary cirrhosis.

[0286] Primary sclerosing cholangitis (PSC) is a chronic progressive cholestatic liver disease caused by inflammation and fibrosis of the intrahepatic / extrahepatic biliary tract of unknown cause. Specifically, as the disease progresses as an inflammatory disease of the bile duct and biliary tract, fibrosis occurs, causing the bile duct wall to thicken and become narrow or stricture. Although the cause is still unknown, it is suspected that a complex combination of various factors, including genetic factors, environmental factors, and associated immune responses, may be the cause.

[0287] The "liver cirrhosis" of the present invention is a chronic disease that develops with repeated regeneration of liver cells and increase in fibrous tissue, and is pathologically accompanied by necrosis, inflammation, and fibrosis, and ultimately progresses to diseases such as liver cirrhosis complications such as liver failure and liver cancer, leading to death. In particular, since there are no subjective symptoms in the early stages and it is discovered at a fairly advanced stage, it is necessary to quickly treat liver fibrosis, which is a state before it progresses to liver cirrhosis, etc. The composition according to the present invention can exhibit a preventive or therapeutic effect against liver cirrhosis, specifically, liver cirrhosis accompanied by non-alcoholic steatohepatitis, but is not limited thereto.

[0288] The term "liver decompensation" as used herein refers to a state in which the liver is unable to perform normal physiological functions such as protein synthesis and metabolic functions due to weakened liver function caused by liver damage or liver disease such as viral hepatitis, cirrhosis, drugs, or alcohol. It is known that liver decompensation can be classified into acute liver failure and chronic liver failure depending on the rate of progression, and can cause various complications. The composition according to the present invention exhibits effects such as inhibition of inflammation and fibrosis, and therefore can exhibit preventive or therapeutic effects against liver failure.

[0289] The term "hepatocellular carcinoma" as used herein means a malignant tumor derived from hepatocytes, and can be divided into primary hepatic cancer (hepatocellular carcinoma) that arises from hepatic cells themselves and metastatic hepatic cancer that is the metastasis of cancer from other tissues to the liver, with approximately 90% or more of liver cancer being primary hepatic cancer. As main causes, alcohol, smoking, obesity, etc. are known to be involved in addition to hepatitis and chronic liver disease. The composition according to the present invention can exhibit preventive or therapeutic effects against liver cancer, specifically, liver cancer caused by non-alcoholic steatohepatitis, but is not limited thereto.

[0290] As disclosed in International Publication WO 2020 / 263063, the triple activity substance or its sustained-acting conjugate of the present invention, which is highly effective against liver diseases such as metabolic liver disease, simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure and liver cancer, is also excellent in targeting liver tissue and has excellent preventive or therapeutic effects against the liver diseases, and can be effectively provided as a therapeutic agent for liver diseases.

[0291] The pharmaceutical compositions of the present invention may further comprise a pharma- ceutically acceptable carrier, excipient, or diluent. Such pharma- ceutically acceptable carrier, excipient, or diluent may be non-natural.

[0292] In the present invention, the term "pharmaceutical acceptable" means a sufficient amount capable of exhibiting a therapeutic effect and causing no side effects, and can be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, number of administrations, treatment period, and drugs used in combination or concomitantly.

[0293] The pharmaceutical composition comprising the peptide of the present invention may further comprise a pharma- ceutically acceptable carrier, which is not particularly limited, and may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc. for oral administration, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. for injections, and may include bases, excipients, lubricants, preservatives, etc. for topical administration.

[0294] The composition of the present invention may be prepared in various dosage forms by mixing with the above-mentioned pharma- ceutically acceptable carriers. For example, when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and when administered by injection, it may be prepared in the form of unit-dose ampoules or multiple doses. In addition, it may be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0295] On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, preservatives, etc. may be further included.

[0296] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0297] Furthermore, the composition may be formulated into a unit dosage form suitable for administration into the body of a patient by a method commonly used in the pharmaceutical field, specifically, into a formulation form useful for administration of protein pharmaceuticals, and administered orally or via a parenteral administration route, including dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method commonly used in the art, but is not limited to these.

[0298] The conjugate may be mixed with various pharma- ceutical acceptable carriers, such as physiological saline or organic solvents, and drugs such as carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used to increase stability or water absorption.

[0299] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined depending on the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease.

[0300] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have different active ingredient contents depending on the severity of the disease. Specifically, the preferred total dose of the conjugate of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day. However, the dose of the conjugate is determined in consideration of various factors such as the age, body weight, health condition, sex, severity of disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these points into consideration, a person having ordinary skill in the art can determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0301] The pharmaceutical composition of the present invention has excellent in vivo durability and potency, and can significantly reduce the number and frequency of administration of the pharmaceutical preparation of the present invention.

[0302] Another embodiment of the present invention provides a physiologically active substance targeted to liver tissue, specifically a substance having binding ability to the glucagon receptor, examples of which include liver-targeted drugs.

[0303] All of the above-mentioned applies to the physiologically active substance targeted to the liver tissue, the substance having a binding ability to the glucagon receptor, the liver-targeted drug, etc.

[0304] Another aspect embodying the present invention provides a method for preventing or treating a disease requiring drug action in the liver, comprising administering the pharmaceutical composition or a bioactive substance targeted to liver tissue to an individual in need thereof.

[0305] All of the above applies to the pharmaceutical composition, the physiologically active substance targeted to the liver tissue, and the diseases, prevention and treatment requiring drug action in the liver.

[0306] In the present invention, the individual is an individual who requires a drug action in the liver, for example, an individual suspected of having a liver disease, and the individual suspected of having a liver disease means a mammal, including a rat, livestock, etc., including a human, who is suffering from or may be suffering from the disease, but includes, without limitation, an individual who can be treated with the composition containing the liver-targeting drug of the present invention.

[0307] The method of the present invention may include administering a pharmaceutical composition comprising the liver targeting drug in a pharmacologic effective amount. The appropriate total daily dose is determined by the treating physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, it is preferred that the specific therapeutically effective amount for a particular patient varies depending on a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether other formulations are used, the age, weight, general health, sex, and diet of the patient, the administration time, administration route, and excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or simultaneously with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0308] Another aspect of the present invention is to provide a use of the liver-targeted drug, specifically a biologically active substance targeted to liver tissue, for use in the manufacture of a medicament for the prevention or treatment of a disease requiring drug action in the liver.

[0309] All of the above applies to the liver disease, liver-targeted drugs, and bioactive substances targeted to liver tissue.

[0310] Another aspect of the present invention is to provide a method for inducing liver targeting of the liver-targeted drug by administering the liver-targeted drug, particularly a biologically active substance targeted to liver tissue, to an individual in need thereof.

[0311] All of the above applies to the liver-targeted drug, the bioactive substance targeted to liver tissue, and the individual.

[0312] Another aspect of the present invention is to provide a method for administering the liver-targeted drug, particularly a bioactive substance targeted to liver tissue, to an individual in need thereof to induce increased distribution of the bioactive substance within liver tissue.

[0313] All of the above applies to the liver-targeted drug, the bioactive substance targeted to liver tissue, and the individual.

[0314] The method of inducing liver targeting involves administering the substance through an appropriate administration route so that the substance is targeted to the liver, which may be, for example, subcutaneous (sc) administration, but is not limited thereto.

[0315] The present invention will be described in more detail with reference to the following examples, however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0316] Example 1: Preparation of triple actives A triple active compound was prepared that exhibited activity at all of the glucagon, GLP-1, and GIP receptors, and its sequence is shown in Table 1 below.

[0317] [Table 1] JPEG2025502749000006.jpg234160 JPEG2025502749000007.jpg233160 JPEG2025502749000008.jpg235160 JPEG2025502749000009.jpg234160 JPEG2025502749000010.jpg233160 JPEG2025502749000011.jpg148160

[0318] In the sequences shown in Table 1, the amino acid marked with X is the unnatural amino acid Aib (aminoisobutyric acid), and the underlined amino acids form a ring with each other. In Table 1, CA stands for 4-imidazoacetyl, and Y stands for tyrosine. The triple activation peptide is used as a triple activation peptide with an amidated C-terminus as necessary. Meanwhile, the peptide is produced by a solid-phase peptide synthesis method using a synthesizer, and in the case of a triple activation peptide with an amidated C-terminus, an amide resin is used for amidation of the C-terminus during production by the solid-phase peptide synthesis method using a synthesizer.

[0319] Example 2: Preparation of a long-acting triple-active conjugate In order to PEGylate the cysteine ​​residues of the triple-active substance of Example 1 (SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96) with 10 kDa PEG having a maleimide group and an aldehyde group at both ends, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the triple-active substance and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1 to 3, the protein concentration was 1 to 5 mg / ml, and the mixture was reacted at a low temperature for 0.5 to 3 hours. At this time, the reaction was carried out in an environment where 20 to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple-active substance mono-PEGylated to cysteine.

[0320] Next, the purified mono-PEGylated triplex active agent and immunoglobulin Fc (homodimer of SEQ ID NO: 139) were reacted at a molar ratio of 1:1-5 and a protein concentration of 10-50 mg / ml at 4-8°C for 12-18 hours. The reaction was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was supplemented with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-30% isopropanol. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing the triplex active agent and immunoglobulin Fc. This purified long-acting conjugate is a 1:1:1 molar intramolecular structure of a triple-active peptide, a polyethylene glycol (PEG) linker and an Fc dimer covalently linked together, with the PEG linker being linked to only one of the polypeptide chains of the Fc dimer.

[0321] Meanwhile, the immunoglobulin Fc is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 139 (consisting of 221 amino acids) through a disulfide bond between the cysteine ​​at the third amino acid of each monomer, and the monomers of the homodimer independently have an internal disulfide bond between the cysteines at the 35th and 95th positions and an internal disulfide bond between the cysteines at the 141st and 199th positions.

[0322] After preparation, the purity was analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography to be 95% or more.

[0323] Here, the triple activity complex of SEQ ID NO: 21 with its C-terminus amidated and the conjugate in which immunoglobulin Fc is linked via PEG are named "a conjugate comprising SEQ ID NO: 21 and immunoglobulin Fc" or "a long-acting conjugate of SEQ ID NO: 21", and these terms may be used interchangeably in the present application.

[0324] Here, the triple activity complex of SEQ ID NO: 22 in which the C-terminus is amidated and the conjugate in which immunoglobulin Fc is linked via PEG are named "a conjugate comprising SEQ ID NO: 22 and immunoglobulin Fc" or "a long-acting conjugate of SEQ ID NO: 22", and these terms may be used interchangeably in the present application.

[0325] Here, the conjugate in which the C-terminus of the triple activity form of SEQ ID NO: 42 is amidated and the immunoglobulin Fc is linked via PEG is named a "conjugate comprising SEQ ID NO: 42 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 42", and these terms may be used interchangeably in the present application.

[0326] Here, the triple activity complex of SEQ ID NO: 43, whose C-terminus is amidated, and the conjugate in which immunoglobulin Fc is linked via PEG are named "a conjugate comprising SEQ ID NO: 43 and immunoglobulin Fc" or "a long-acting conjugate of SEQ ID NO: 43", and these terms may be used interchangeably in the present application.

[0327] Here, the conjugate in which the C-terminus of the triple activity molecule of SEQ ID NO: 50 is amidated and the immunoglobulin Fc is linked via PEG is named a "conjugate comprising SEQ ID NO: 50 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 50", and these terms may be used interchangeably in the present application.

[0328] Here, the triple activity complex of SEQ ID NO: 77 with its C-terminus amidated and the conjugate in which immunoglobulin Fc is linked via PEG are named "a conjugate comprising SEQ ID NO: 77 and immunoglobulin Fc" or "a long-acting conjugate of SEQ ID NO: 77", and these terms may be used interchangeably in the present application.

[0329] Here, the conjugate in which the C-terminus of the triple activity form of SEQ ID NO: 96 is amidated and the immunoglobulin Fc is linked via PEG is named a "conjugate comprising SEQ ID NO: 96 and immunoglobulin Fc" or a "long-acting conjugate of SEQ ID NO: 96", and these terms may be used interchangeably in the present application.

[0330] Experimental Example 1: Measurement of in vitro activity of triple-active complex and its long-acting conjugate In order to measure the activity of the triple-active complex and its long-acting conjugate prepared in Examples 1 and 2, a method for measuring cellular activity in vitro was used using cell lines transformed with GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor, respectively.

[0331] The above cell lines were transformed into CHO (chinese hamster ovary) cells to express human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, and are suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity of each moiety was measured using the transformed cell lines.

[0332] To measure the GLP-1 activity of the triple active substance and its long-acting conjugate prepared in Examples 1 and 2, human GLP-1 was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple active substance and its long-acting conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing human GLP-1 receptor, and 5 μl of each of the serially diluted substances was added to the cells, and then 5 μl of a buffer containing a cAMP antibody was added and incubated at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to measure EC through the accumulated cAMP. 50 The values ​​were calculated and compared with each other, and the relative potencies compared to human GLP-1 are shown in Tables 2 and 3 below.

[0333] To measure the GCG activity of the triple activator and its long-acting conjugate prepared in Examples 1 and 2, human GCG was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its long-acting conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing human GCG receptor, and 5 μl of each of the serially diluted substances was added to the cells, and then 5 μl of a buffer containing a cAMP antibody was added and incubated at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to measure EC through the accumulated cAMP. 50 The values ​​were calculated and then compared to each other, and the relative potencies compared to human GCG are shown in Tables 2 and 3 below.

[0334] To measure the GIP activity of the triple active substance and its long-acting conjugate prepared in Examples 1 and 2, human GIP was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple active substance and its long-acting conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing human GIP receptor, and 5 μl of each of the serially diluted substances was added to the cells, and then 5 μl of a buffer containing a cAMP antibody was added and incubated at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to measure EC through accumulated cAMP. 50The values ​​were calculated and compared with each other. The relative potency compared to human GIP is shown in Tables 2 and 3 below. The triple activity peptides of SEQ ID NOs: 1 to 102, whose activities were confirmed in Table 2 below, and the triple activity peptides in the triple activity long-acting conjugates, whose activities were confirmed in Table 3 below, are triple activity peptides with amidated C-terminus.

[0335] [Table 2] JPEG2025502749000013.jpg233143 JPEG2025502749000014.jpg230142 JPEG2025502749000015.jpg230142

[0336] [Table 3]

[0337] The novel triple-activity long-acting conjugate prepared above has high activity on the glucagon receptor, which increases the targeting of hepatocytes. In addition, it activates all of the glucagon receptor, GLP-1 receptor, and GIP receptor, and can be used as a therapeutic agent for target diseases in the liver.

[0338] Experimental Example 2: Confirmation of tissue distribution of triple-active long-acting conjugate From three SD rats, the long-acting conjugate of SEQ ID NO: 42 was selected as a representative liver-targeting drug, and the tissue and organ distribution of the liver-targeting drug was compared.

[0339] Specifically, the triple activity long-acting conjugates were subcutaneously injected at 544 μg / kg each, and the organs were removed 4, 48, and 168 hours later, and the concentrations of each substance in the tissues (serum, brain, pancreas, heart, kidney, stomach, small intestine, large intestine, lung, liver, spleen, adipose tissue, and muscle) were measured and compared using ELISA.

[0340] As a result, the triple activation long-acting conjugate showed the strongest tissue distribution 48 hours after administration, and showed a particularly high distribution ratio in the liver. The tissue distribution was highest in the liver, followed by the heart, lung, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, and muscle. It was still detected at the highest rate in the liver 168 hours after administration, and was found to be present at a high rate in the liver even up to 7 days after administration. The results of the tissue distribution ratio of the SEQ ID NO: 42 long-acting conjugate compared to serum confirmed in the above example are summarized in Table 4 below.

[0341] [Table 4]

[0342] The above results suggest that the triple activation substance long-acting conjugate of the present invention has excellent tissue distribution in the liver compared to other tissues and can be used as a therapeutic substance for the target disease. Therefore, the triple activation substance long-acting conjugate can be used as a new application that can induce targeting to liver tissue to efficiently deliver the required amount of drug and optimize drug treatment. In addition, as disclosed in International Publication WO 2020 / 263063, the triple activation substance or its long-acting conjugate of the present invention, which is effective for liver diseases such as metabolic liver disease, simple steatosis, non-alcoholic fatty liver, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer, has excellent targeting to liver tissue and excellent preventive or therapeutic effects on the liver disease.

[0343] Furthermore, the above results suggest that distribution in the liver is high even 7 days after administration, supporting the convenience of the formulation being administered approximately once a week.

[0344] From the above description, it will be understood by those skilled in the art to which the present invention pertains that the present invention can be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A pharmaceutical composition comprising a liver-targeted drug, wherein the drug is highly distributed in the liver among the organs in the body of an individual to which the composition is administered, The pharmaceutical composition, wherein the liver-targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 1: Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-X aa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103) In the general formula 1, Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y); Xaa2 is glycine (Gly, G), alpha-methyl-glutamic acid, or Aib (aminoisobutyric acid); Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q), Xaa7 is threonine (Thr, T) or isoleucine (Ile, I), Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V); Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I); Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C); Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y); Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L); Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S); Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K); Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H); Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V); Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R); Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D); Xaa23 is isoleucine (Ile, I) or valine (Val, V), Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E); Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K) or methionine (Met, M); Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D); Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H); Xaa30 is cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), histidine (His, H), or is absent; R1 is cysteine ​​(Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; where: m is Cys, Pro or Gly-Pro; n is Cys, Gly, Ser, His-Gly, or absent.

2. The pharmaceutical composition according to claim 1, wherein the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is represented by the following chemical formula 1: [Chemical formula 1] X-L-F where: X is a peptide comprising the amino acid sequence of general formula 1; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; - indicates a covalent bond between X and L, and between L and F.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the internal organs are the liver, heart, lung, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney and brain, and among the organs, the liver has the highest distribution.

4. The pharmaceutical composition according to claim 1 or 2, for use in the prevention or treatment of a disease requiring drug action in the liver.

5. The pharmaceutical composition according to claim 1 or 2, wherein the liver-targeting drug has a T / S (tissue-to-serum) ratio in the liver after administration selected from one or more of the following: (a) T / S ratio of 25% to 50% between 2 and 5 hours after administration; (b) a T / S ratio of 40% to 60% at 40 to 50 hours after administration; and (c) T / S ratio of 45% to 75% at 160 to 180 hours after administration.

6. The pharmaceutical composition according to claim 5, wherein the liver-targeted drug has a T / S ratio in the liver after administration selected from one or more of the following: (a) T / S ratio of 35% to 45% at 4 hours after administration; (b) a T / S ratio of 45% to 55% at 2 days after administration; and (c) T / S ratio of 55% to 70% 7 days after administration.

7. The pharmaceutical composition according to claim 1 or 2, wherein the liver-targeted drug has a distribution ratio in the liver relative to the lung tissue after administration of 1:2-4.

8. The pharmaceutical composition of claim 7, wherein the liver-targeted drug has a distribution ratio in the liver relative to the lung tissue after administration of 1:3-4.

9. 9. The pharmaceutical composition of claim 8, wherein the liver-targeted drug has a distribution ratio in the liver relative to the lung tissue after administration of 1:3.5-4.

10. The pharmaceutical composition according to claim 7, wherein the distribution ratio is the distribution ratio at 40 to 180 hours after administration.

11. The pharmaceutical composition according to claim 9, wherein the distribution ratio is the distribution ratio from 2 to 7 days after administration.

12. The liver-targeted drug is (a) a distribution ratio in the liver relative to the heart of 1:1.5 to 3.0 at 2 to 5 hours after administration; (b) a distribution ratio in the liver relative to the heart of 1:2.0-3.0 at 40-50 hours after administration; and (c) the distribution ratio in the liver relative to the heart is 1:5.5-7.0 at 160 to 180 hours after administration;

13. The pharmaceutical composition of claim 1 or 2, wherein the liver-targeted drug has therapeutic activity against liver disease.

14. Xaa14 is leucine or methionine; 3. The pharmaceutical composition of claim 1, wherein Xaa15 is cysteine, aspartic acid, or leucine.

15. In the general formula 1, Xaa2 is glycine, alpha-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, tyrosine, 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; 3. The pharmaceutical composition of claim 1, wherein Xaa27 is leucine or lysine.

16. The pharmaceutical composition according to claim 1 or 2, wherein the peptide is a peptide comprising an amino acid sequence represented by the following general formula 2: Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa2 1-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 2, SEQ ID NO: 104) In the general formula 2, Xaa1 is 4-imidazoacetyl, histidine, or tyrosine; Xaa2 is glycine, alpha-methyl-glutamic acid, or Aib; Xaa10 is tyrosine or cysteine; Xaa13 is alanine, glutamine, tyrosine, or cysteine; Xaa14 is leucine, methionine, or tyrosine; Xaa15 is aspartic acid, glutamic acid, or leucine; Xaa16 is glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine, isoleucine, glutamic acid, cysteine, or lysine; Xaa18 is alanine, glutamine, arginine, or histidine; Xaa19 is alanine, 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; Xaa31 is proline or cysteine; Xaa40 is cysteine ​​or absent.

17. In the general formula 1, Xaa2 is glycine, alpha-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; Xaa23 is valine, Xaa24 is alanine, glutamine, cysteine, asparagine, or aspartic acid; Xaa27 is leucine or lysine; 3. The pharmaceutical composition of claim 1 or 2.

18. In the 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; Xaa30 is cysteine, lysine, or histidine; 17. The pharmaceutical composition of claim 16.

19. In the general formula 1, Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa7 is threonine, Xaa10 is tyrosine or cysteine; Xaa12 is lysine or isoleucine; Xaa13 is tyrosine, alanine, or cysteine; Xaa14 is leucine or methionine; Xaa15 is cysteine ​​or aspartic acid; Xaa16 is glutamic acid, 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, Xaa28 is cysteine, alanine, asparagine, or aspartic acid; 3. The pharmaceutical composition of claim 1 or 2.

20. In the general formula 1, Xaa1 is histidine or 4-imidazoacetyl; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa3 is glutamine, Xaa7 is threonine, Xaa10 is tyrosine, Xaa12 is isoleucine, Xaa13 is alanine or cysteine; Xaa14 is methionine, Xaa15 is aspartic acid, Xaa16 is glutamic acid, Xaa17 is isoleucine or lysine; Xaa18 is alanine or histidine; Xaa19 is glutamine or cysteine; Xaa20 is lysine, Xaa21 is aspartic acid, Xaa23 is valine, Xaa24 is asparagine, Xaa27 is leucine, Xaa28 is alanine or asparagine; Xaa29 is glutamine or threonine; Xaa30 is cysteine ​​or lysine or is absent; 3. The pharmaceutical composition of claim 1 or 2.

21. The pharmaceutical composition according to claim 1 or 2, wherein the peptide is a peptide comprising an amino acid sequence of the following general formula 3: Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe -Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40 (general formula 3, SEQ ID NO: 105), In the general formula 3, Xaa1 is histidine or tyrosine; Xaa2 is alpha-methyl-glutamic acid or Aib; Xaa13 is alanine, tyrosine or cysteine; Xaa17 is arginine, cysteine, or lysine; Xaa18 is alanine or arginine; Xaa19 is alanine or cysteine; Xaa21 is glutamic acid or aspartic acid, Xaa24 is glutamine or asparagine; Xaa28 is cysteine ​​or aspartic acid; Xaa29 is cysteine, histidine, or glutamine; Xaa30 is cysteine ​​or histidine; Xaa31 is proline or cysteine; Xaa40 is cysteine ​​or absent.

22. 3. The pharmaceutical composition of claim 1 or 2, wherein 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 is absent.

23. The pharmaceutical composition of claim 1 or 2, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102.

24. The pharmaceutical composition according to claim 1 or 2, wherein the Xaa16 amino acid and the Xaa20 amino acid of the general formula 1 together form a ring.

25. The pharmaceutical composition according to claim 1 or 2, wherein the peptide is amidated or has a free carboxyl group (-COOH) at its C-terminus.

26. 23. The pharmaceutical composition of claim 22, wherein the peptide is amidated at its C-terminus.

27. 24. The pharmaceutical composition of claim 23, wherein the peptide is amidated at its C-terminus.

28. The pharmaceutical composition of claim 2 , wherein F is an IgG Fc region.

29. 3. The pharmaceutical composition of claim 2, wherein L is polyethylene glycol.

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

31. The pharmaceutical composition according to claim 4, wherein the disease requiring drug action in the liver is a liver disease.

32. 31. The pharmaceutical composition of claim 30, wherein the liver disease is a metabolic liver disease.

33. 32. The pharmaceutical composition of claim 31, wherein the liver disease is at least one disease selected from the group consisting of simple steatosis, non-alcoholic fatty liver disease, liver inflammation, non-alcoholic fatty liver disease, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure, and liver cancer.

34. The pharmaceutical composition of claim 33, wherein the cholestatic liver disease is any one selected from the group consisting of primary biliary cirrhosis, primary sclerosing cholangitis, and combinations thereof.

35. Use of a pharmaceutical composition in the preparation of a medicament for inducing liver targeting of a liver-targeted drug, wherein the pharmaceutical composition contains a liver-targeted drug and has a high distribution rate of the drug in the liver among the organs in the body of an individual to which the pharmaceutical composition is administered; The liver-targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 1: Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-X aa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103) In the general formula 1, Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y); Xaa2 is glycine (Gly, G), alpha-methyl-glutamic acid, or Aib (aminoisobutyric acid); Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q), Xaa7 is threonine (Thr, T) or isoleucine (Ile, I), Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V); Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I); Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C); Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y); Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L); Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S); Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K); Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H); Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V); Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R); Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D); Xaa23 is isoleucine (Ile, I) or valine (Val, V), Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E); Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K) or methionine (Met, M); Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D); Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H); Xaa30 is cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), histidine (His, H), or is absent; R1 is cysteine ​​(Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; where: m is Cys, Pro or Gly-Pro; n is Cys, Gly, Ser, His-Gly, or absent.

36. Use of a pharmaceutical composition in the preparation of a medicament for inducing increased distribution of a liver-targeted drug in liver tissue, wherein the pharmaceutical composition contains a liver-targeted drug and has a high distribution rate of the drug in the liver among the organs in the body of an individual to which the pharmaceutical composition is administered; The liver-targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 1: Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-X aa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 103) In the general formula 1, Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or tyrosine (Tyr, Y); Xaa2 is glycine (Gly, G), alpha-methyl-glutamic acid, or Aib (aminoisobutyric acid); Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q), Xaa7 is threonine (Thr, T) or isoleucine (Ile, I), Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine ​​(Cys, C), or valine (Val, V); Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine (Ile, I); Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine (Ala, A), or cysteine ​​(Cys, C); Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine (Tyr, Y); Xaa15 is cysteine ​​(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or leucine (Leu, L); Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine (Ser, S); Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine ​​(Cys, C), or lysine (Lys, K); Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine (Arg, R), or histidine (His, H); Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), or valine (Val, V); Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R); Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu, L), cysteine ​​(Cys, C), or aspartic acid (Asp, D); Xaa23 is isoleucine (Ile, I) or valine (Val, V), Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine ​​(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic acid (Glu, E); Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K) or methionine (Met, M); Xaa28 is cysteine ​​(Cys, C), lysine (Lys, K), alanine (Ala, A), asparagine (Asn, N), or aspartic acid (Asp, D); Xaa29 is cysteine ​​(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine (His, H); Xaa30 is cysteine ​​(Cys, C), glycine (Gly, G), lysine (Lys, K), histidine (His, H), or is absent; R1 is cysteine ​​(Cys, C), GKKNDWKHNIT (SEQ ID NO: 106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or is absent; where: m is Cys, Pro or Gly-Pro; n is Cys, Gly, Ser, His-Gly, or absent.