Liver targeting substances 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. There are various diseases caused by various causes that require drug action in the liver, such as liver disease, hypoglycemia, and congenital hyperinsulinism.

[0003] 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).

[0004] 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]

[0005] [Patent Document 1] Korean Patent Publication Number KR10-2013-0131227 [Patent Document 2] International Publication WO 2016 / 108586 [Patent Document 3] International Publication WO 2017 / 003191 [Patent Document 4] International Patent Publication No. WO97 / 34631 [Patent Document 5] International Patent Publication No. 96 / 32478 [Patent Document 6] International Publication WO 2020 / 263063 [Patent Document 7] International Patent Publication WO2007 / 021129 [Non-patent literature]

[0006] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-Patent Document 2] Rice et al., 2000,Trends Genet. 16:276-277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-Patent Document 4] Devereux, J., et al, Nucleic Acids Research 12:387(1984) [Non-Patent Document 5] Atschul, [S.] [F.,] [ET AL,J MOLEC BIOL 215]:403(1990) [Non-Patent Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-Patent Document 7] [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073 [Non-Patent Document 8] Smith and Waterman,Adv. Appl. Math(1981)2:482 [Non-Patent Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 10] Gribskov et al (1986) Nucl. Acids Res. 14:6745 [Non-Patent Document 11] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0007] 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]

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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

[0014] 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 a drug action in the liver, including liver diseases, hypoglycemia, and congenital hyperinsulinism, while reducing the inconvenience of administering medication to patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] One embodiment of the present invention is a pharmaceutical composition comprising a liver-targeting drug, specifically 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. In one specific example, the liver-targeting drug is in the form of a peptide comprising the amino acid sequence of the following general formula 1 or a long-acting conjugate comprising the same. In another specific example, the liver-targeting drug is in the form of a peptide comprising the amino acid sequence of the following general formula 2 or a long-acting conjugate comprising the same.

[0016] In one embodiment, the liver targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 1:

[0017] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (general formula 1, sequence number: 46),

[0018] In the above formula, X1 is histidine (H), desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan (W), or tyrosine (Y), or is absent; X2 is alpha-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine; X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X13 is tyrosine (Y) or cysteine ​​(C); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D), glutamic acid (E) or cysteine ​​(C); X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methyl-glutamic acid, or cysteine ​​(C) or absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C) or absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C) or absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methyl-glutamic acid, or cysteine ​​(C) or absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C) or absent; X23 is isoleucine (I), valine (V), or arginine (R) or absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methyl-glutamic acid, or leucine (L) or absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R) or absent; X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent; X29 is lysine (K), alanine (A), glycine (G), or threonine (T) or absent; X30 is cysteine ​​(C) or absent.

[0019] One embodiment of the present invention is a pharmaceutical composition comprising a liver-targeting drug, which has a high distribution in the liver among the organs in the body of an individual to which it is administered. In one specific example, the liver-targeting drug is a peptide comprising an amino acid sequence represented by the following general formula 2:

[0020] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula 2, sequence number: 47)

[0021] In the above general formula 2 X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X15 is aspartic acid (D) or cysteine ​​(C); X16 is glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 2 is identical to SEQ ID NO: 12).

[0022] 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:

[0023] [Chemical formula 1] XLF

[0024] Where: X is a peptide comprising the amino acid sequence of general formula 2; 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.

[0025] 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.

[0026] 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.

[0027] 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 20% to about 40% at about 40 hours to about 50 hours after administration; and (b) a T / S ratio of about 25% to about 40% at about 160 hours to about 180 hours after administration.

[0028] 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 25% to about 35% about 2 days after administration; and (c) a T / S ratio of about 27% to about 35% about 7 days after administration.

[0029] 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.

[0030] 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 2.2 to about 3.2.

[0031] 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.

[0032] 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.

[0033] 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 1:1.6-3.0 40-50 hours after administration; and (b) the distribution ratio in the liver compared to the heart is 1:2.8-7 160-180 hours after administration.

[0034] 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.

[0035] 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.

[0036] 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, hypoglycemia, or congenital hyperinsulinism.

[0037] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the hypoglycemia is characterized in that it is acute hypoglycemia or chronic hypoglycemia.

[0038] 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.

[0039] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that a ring is formed between the amino acids X16 and X20 of the general formula 2.

[0040] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the peptide comprising the amino acid sequence of general formula 2 is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 19, 33, and 36 to 45.

[0041] As a pharmaceutical composition according to any one of the above-mentioned specific examples, the peptide comprising the amino acid sequence of general formula 2 is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, and 36 to 44.

[0042] In the pharmaceutical composition according to any one of the above-mentioned specific examples, the peptide comprising the amino acid sequence of general formula 1 is characterized in that it comprises any one of the amino acid sequences of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44.

[0043] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the peptide comprising the amino acid sequence of general formula 1 is characterized in that it comprises any one of the amino acid sequences of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44.

[0044] The composition according to any one of the above-mentioned specific examples is characterized in that, among the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in the general formula 1, each of the amino acids in at least one amino acid pair is substituted with glutamic acid or lysine capable of forming a ring.

[0045] The composition according to any one of the above-mentioned embodiments is characterized in that each of the amino acids of the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 is substituted with glutamic acid or lysine capable of forming a ring.

[0046] The composition according to any one of the above-mentioned specific examples is characterized in that, in the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in the general formula 1, at least one of the amino acid pairs forms a ring between each amino acid.

[0047] The 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: 1-44.

[0048] In a composition according to any one of the above embodiments, the peptide is characterized in that its C-terminus is amidated or has a free carboxyl group (-COOH).

[0049] The composition according to any one of the above embodiments is characterized in that the C-terminus of the peptide is amidated.

[0050] The composition according to any one of the above embodiments is characterized in that the C-terminus of the peptide is not modified.

[0051] 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.

[0052] 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:

[0053] [Chemical formula 3] JPEG2025500508000001.jpg3398

[0054] Here, X and F are as defined in Chemical Formula 1.

[0055] In the composition according to any one of the above embodiments, the ethylene glycol repeat unit is [OCH 2 CH 2 ]n, n being a natural number, and [OCH 2 CH 2 The average molecular weight of the n moieties, for example, the number average molecular weight, is set to be 1 to 100 kDa.

[0056] In the composition according to any one of the above embodiments, the value of n is the number of [OCH 2 CH 2 The average molecular weight of the n moieties, for example, the number average molecular weight, is set to 10 kDa.

[0057] In the pharmaceutical composition according to any one of the above embodiments, L is polyethylene glycol.

[0058] In the composition according to any one of the above embodiments, X is linked through a sulfur atom of a cysteine ​​in the peptide.

[0059] The pharmaceutical composition according to any one of the above embodiments is characterized in that F is an IgG Fc region.

[0060] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the immunoglobulin Fc region is derived from IgG4.

[0061] 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.

[0062] The composition according to any one of the above embodiments, wherein F comprises a monomer having the amino acid sequence of SEQ ID NO:70.

[0063] 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:70.

[0064] The composition according to any one of the above embodiments, wherein F is a homodimer comprising the amino acid sequence of SEQ ID NO:71.

[0065] 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.

[0066] The composition according to any one of the above embodiments is characterized in that the immunoglobulin Fc domain, F and X, is not glycosylated.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Specific details for carrying out the present invention are as follows. 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. Also, the following specific description is not considered to limit the scope of the present invention.

[0073] 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.

[0074] 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.

[0075] 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

[0076] 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.

[0077] 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.

[0078] 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.

[0079] The pharmaceutical composition may be for use in the prevention or treatment of a disease requiring a drug action in the liver.

[0080] 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.

[0081] The liver-targeting drug may have a T / S ratio (tissue-to-serum ratio) in the liver after administration selected from the following, but is not limited thereto: (a) a T / S ratio of about 20% to about 40%, about 24% to about 40%, about 25% to about 40%, about 25% to about 38%, about 25% to about 35%, about 25% to about 34%, about 25% to about 33%, about 26% to about 33%, about 26% to about 32%, about 27% to about 32%, about 28% to about 33% and about 35% to about 35%. and (b) a T / S ratio of about 25% to about 40%, about 26% to about 38%, about 27% to about 35%, about 27% to about 34%, about 27% to about 33%, about 28% to about 33%, about 29% to about 33%, about 29% to about 32%, about 30% to about 32%, or about 30.5% to about 31.5% at about 160 hours to about 180 hours after administration. The above characteristics may be, but are not limited to, one or more, or both selected from (a) and (b).

[0082] The liver targeting 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 25% to about 35%, about 26% to about 34%, about 27% to about 33%, about 28% to about 32%, about 29% to about 31%, about 29.5% to about 30.5%, or about 29.8% about 2 days after administration; and (c) a T / S ratio of about 27% to about 35%, about 27% to about 34%, about 28% to about 33%, about 29% to about 32%, about 30% to about 31.5%, or about 31% about 7 days after administration. The characteristic may be one or more selected from (a) or (b), or both, but is not limited thereto.

[0083] 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.

[0084] 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.

[0085] 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 4.0, about 1:2 to about 3.5, about 1:2.1 to about 3.4, about 1:2.2 to about 3.2, about 1:2.2 to about 3.3, about 1:2.3 to about 3.2, about 1:2.4 to about 3.2, about 1:2.4 to about 3.1, about 1:2.4 to about 3.0, about 1:2.5 to about 2.95, about 1:2.5 to about 2.9, about 1:2.55 to about 2.87, about 1:2.6 to 2.9, or about 1:2.5 to 3.9.

[0086] 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.

[0087] 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.

[0088] The distribution ratio in the liver compared to the lung tissue after the administration may be, but is not limited to, 1:about 2.4 to about 2.7, 1:about 2.4 to about 2.7, 1:about 2.5 to about 2.6, or 1:about 2.5 to 3.7 at about 2 days after administration, and 1:about 2.5 to 3, 1:about 2.7 to about 3, 1:about 2.8 to about 3.0, 1:about 2.8 to about 2.9, or 1:about 2.8 to about 3.9 at about 7 days after administration.

[0089] The distribution ratio in the liver relative to the heart after the administration may be, but is not limited to, (a) a distribution ratio in the liver relative to the heart of 1:about 1.6 to about 3.0 at about 40 to about 50 hours after administration; and (b) a distribution ratio in the liver relative to the heart of 1:about 2.8 to about 7.0 at about 160 to about 180 hours after administration.

[0090] the distribution ratio in the liver relative to the heart after said administration is ((a) about 40 hours to about 50 hours, about 45 hours to about 50 hours, or about 2 days after administration, the distribution ratio in the liver relative to the heart is 1: about 1.6 to 3.0, 1: about 1.6 to 2.5, 1: about 1.6 to about 2.2, 1: about 1.7 to about 2.1, 1: about 1.7 to about 2.0, 1: about 1.8 to 2.0, 1: about 1.8 to 2.0, 1: about 1.89 to 2.0, 1: about 1.8 to 2.0, or 1: about 1.8 to 2.9; and (b) 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 2.8 to about 4, 1: about 2.9 to about 3.8, 1: about 3.0 to about 3.8, 1: about 3.0 to about 3.6, 1: about 3.0 to about 3.5, 1: about 3.0 to about 3.4, 1: about 3.1 to about 3.4, 1: about 3.2 to about 3.4, 1: about 3.2 to about 3.3, 1: about 2.8 to about 7.0, or 1: about 3.3 to about 6.6.

[0091] 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.

[0092] 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.

[0093] The liver targeting drug may include a substance that can be targeted to liver tissue. Specifically, the liver targeting drug may be a peptide or protein containing glucagon or a derivative thereof that can induce liver targeting, and may include a peptide sequence that has activity against glucagon receptor. For example, the peptide may be in the form of a peptide containing the amino acid sequence of the general formula 1 or a long-acting conjugate containing the same, or in the form of a peptide containing the amino acid sequence of the general formula 2 or a long-acting conjugate containing the same, but is not limited thereto. The peptide is a peptide that has activity against glucagon receptor, and specifically, may be a glucagon derivative, but is not limited thereto.

[0094] Meanwhile, in the present invention, glucagon or its derivatives, which are liver-targeting drugs, means that they have binding power to glucagon receptors to such an extent that they can be targeted from blood to the liver when delivered into the blood of an animal. More specifically, when the organ distribution ratio of the drug is confirmed at least 6 hours, 30 hours, at least 48 hours, or at least 168 hours after delivery of the drug 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 the heart, lung, large intestine, spleen, pancreas, adipose tissue, small intestine, stomach, muscle, kidney, or brain, but is not particularly limited thereto as long as the distribution ratio is higher than that of the comparative organ.

[0095] The substances having activity against the glucagon receptor include a variety of substances having a significant level of activity against the glucagon receptor, for example, substances in the form of compounds or peptides.

[0096] Although not limited thereto, the substance having a significant level of activity against glucagon may be not only natural glucagon, but also a substance that exhibits in vitro activity against the glucagon receptor of 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the natural ligand of the receptor (natural glucagon).

[0097] The method for measuring the in vitro activity of glucagon or a glucagon derivative can be found in Experimental Example 1 of the present specification, but is not particularly limited thereto.

[0098] Examples of the substance having activity against the glucagon receptor include, but are not limited to, natural glucagon, its agonist, or a derivative thereof.

[0099] The glucagon derivatives according to the present invention include peptides having one or more differences in amino acid sequence compared to native glucagon, peptides in which the native glucagon sequence has been altered through modification, and mimetics of native glucagon that activate the glucagon receptor like native glucagon, and if the peptides are highly distributed in the liver among the organs in the body of an individual to which they are administered, they may fall within the scope of the present invention.

[0100] The glucagon derivatives include those described in International Publication Nos. WO 2016 / 108586 and WO 2017 / 003191, the entire specifications of which are incorporated herein by reference. In addition, the method for preparing the long-acting conjugate of the glucagon derivative peptide is described in WO 2017 / 003191, the entire specifications of which are incorporated herein by reference.

[0101] Such glucagon derivatives may have an altered pI relative to native glucagon and thus exhibit improved physical properties. In addition, the glucagon derivatives may have improved solubility while retaining the activity of activating the glucagon receptor, but are not limited thereto.

[0102] The glucagon derivative may also be non-naturally occurring.

[0103] On the other hand, native glucagon can have the following amino acid sequence:

[0104] 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: 1)

[0105] As used herein, the term "isoelectric point" or "pI" refers to the pH value at which a molecule such as a polypeptide or peptide has no (0) overall net charge. For a polypeptide having multiple charged functional groups, the sum of these charges is zero at the pI. At a pH higher than the pI, the overall net charge of the polypeptide should be negative, and at a pH value lower than the pI, the overall net charge of the polypeptide should be positive.

[0106] The pI can be determined by isoelectric focusing on immobilized pH gradient gels composed of polyacrylamide, starch or agarose, or by estimating the pI from the amino acid sequence using, for example, the pI / MW tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003).

[0107] In the present invention, the term "altered pI" means that a part of the amino acid sequence of native glucagon is replaced with negatively and positively charged amino acid residues to have a pI different from that of native glucagon, i.e., a pI that is decreased or increased. A peptide having such an altered pI can exhibit improved solubility and / or high stability at neutral pH as a glucagon derivative, but is not particularly limited thereto.

[0108] For example, the glucagon derivative may have a pI value different from that of natural glucagon (6.8), and may be less than 6.8, 6.7 or less, 6.5 or less, or more than 6.8, 7 or more, or 7.5 or more, but is not limited thereto, and is included in the scope of the present invention as long as it has a pI value different from that of natural glucagon. In particular, as long as it has a pI value different from that of natural glucagon and thus exhibits improved solubility at neutral pH compared to natural glucagon and thus has a low degree of aggregation, it may be included in the scope of the present invention, but is not limited thereto.

[0109] Examples of the pI value include, but are not limited to, those having a pI value of 4 to 6.5 and / or 7 to 9.5, 7.5 to 9.5, or 8.0 to 9.3. In this case, since the pI value is higher or lower than that of native glucagon, it can exhibit improved solubility and high stability at neutral pH compared to native glucagon. However, the pI value is not limited to these.

[0110] Specifically, a derivative of native glucagon can be obtained by modifying some amino acids in native glucagon through any one of substitution, addition, deletion and modification, or a combination of such methods.

[0111] Examples of glucagon derivatives produced by combining the above-mentioned methods include, but are not limited to, peptides that have at least one amino acid sequence different from that of native glucagon, have deamination at the N-terminal amino acid residue, and have the function of activating the glucagon receptor. Native glucagon derivatives applicable to the present invention can be produced by combining multiple methods for producing derivatives.

[0112] Such modifications to produce derivatives of native glucagon also include modifications with L- or D-amino acids, and / or non-natural amino acids; and / or modifications of the native sequence, such as modifications of side chain functional groups, intramolecular covalent bonds, e.g., side chain intercyclization, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. Also included are substitutions with non-natural compounds.

[0113] It also includes all of the additions of one or more amino acids to the amino and / or carboxy termini of native glucagon.

[0114] 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.

[0115] Amino acid derivatives can also be obtained in a similar manner, with 4-imidazoacetic acid being just a few examples.

[0116] Glucagon has a pI of about 7, is insoluble in solutions at physiological pH (pH 4-8), and tends to precipitate at neutral pH. In aqueous solutions below pH 3, glucagon initially dissolves but precipitates by gel formation within an hour. Gelled glucagon consists primarily of β-sheet fibrils, and such precipitated glucagon is unsuitable for use as an injectable drug because it would clog blood vessels if the gel were administered through a needle or intravenously. To slow the precipitation process, acidic (pH 2-4) dosage forms are usually used, which maintain glucagon in a relatively aggregate-free state for a short period of time. However, because glucagon fibril formation occurs very rapidly at low pH, such acidic dosage forms must be injected immediately after preparation.

[0117] The glucagon derivatives of the present invention include those developed by altering the pI of native glucagon through the substitution of negatively and positively charged amino acid residues to have an extended profile of action, and such derivatives are characterized by having an altered pI compared to native glucagon, thereby exhibiting improved solubility and / or increased stability at neutral pH.

[0118] In one specific embodiment, the glucagon or glucagon derivative may be a peptide comprising an amino acid sequence of the following general formula 1:

[0119] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (general formula 1, sequence number: 46)

[0120] In the above formula, X1 is histidine, desamino-histidyl, N-dimethyl-histidyl, beta-hydroxy imidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl, tryptophan, or tyrosine, or is absent; X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine; X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X13 is tyrosine or cysteine; X14 is leucine or cysteine; X15 is aspartic acid, glutamic acid or cysteine; X16 is glutamic acid, aspartic acid, serine, alpha-methyl-glutamic acid, cysteine, or absent; X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or is absent; X18 is alanine, aspartic acid, glutamine, glutamic acid, arginine, valine, cysteine, or absent; X19 is alanine, arginine, serine, valine, cysteine, or absent; X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, alpha-methyl-glutamic acid, cysteine, or is absent; X21 is aspartic acid, glutamic acid, leucine, valine, cysteine, or absent; X23 is isoleucine, valine, or arginine or is absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid, or leucine, or is absent; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine or is absent; X28 is glutamine, lysine, asparagine, arginine, or absent; X29 is lysine, alanine, glycine, or threonine or is absent; X30 may be cysteine ​​or may be absent.

[0121] More specifically, In the general formula 1, X1 is histidine, tryptophan, or tyrosine, or is absent; X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine or cysteine; X13 is tyrosine or cysteine; X14 is leucine or cysteine; X15 is aspartic acid or cysteine; X16 is glutamic acid, serine or cysteine; X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine; X18 is aspartic acid, glutamic acid, arginine, or cysteine; X19 is alanine or cysteine; X20 is glutamine, aspartic acid, lysine, or cysteine; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine; X23 is isoleucine, valine, or arginine; X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine; X27 is isoleucine, valine, alanine, methionine, glutamine, or arginine; X28 is glutamine, lysine, asparagine, or arginine; X29 is threonine; X30 may be cysteine ​​or may be absent.

[0122] As an example, the peptide may be, but is not limited to, one that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 44, specifically, one that is (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 44. As another specific example, the peptide of the present invention may be, but is not limited to, one that includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, or one that is (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] The above may be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0129] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11 to 17, 19 to 27, 29, 31, 33, and 35 to 44, and specifically may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11 to 17, 19 to 27, 29, 31, 33, and 35 to 44, but is not limited thereto.

[0130] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, 40 to 42, and 44, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, 17, 19 to 25, 27, 29, 33, 35 to 38, and 40 to 42, and 44, but is not limited thereto.

[0131] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, specifically, the peptide may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13, 15, 17, 20 to 24, 26 to 30, and 32 to 44, but is not limited thereto.

[0132] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, but is not limited thereto.

[0133] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44, and more specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 37, 38, and 44, but is not limited thereto.

[0134] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, and more specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, but is not limited thereto.

[0135] As a specific example, a pharmaceutical composition containing a liver-targeted drug and having a high distribution rate of the drug in the liver among the organs in the body of an individual to which the composition is administered is The liver targeting drug may be a peptide comprising an amino acid sequence of the following general formula 2:

[0136] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (general formula 2, sequence number: 47)

[0137] In the above general formula 2 X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X15 is aspartic acid (D) or cysteine ​​(C); X16 is glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 2 is identical to SEQ ID NO: 12).

[0138] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 19, 33, and 36 to 45, specifically, the peptide may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 19, 33, and 36 to 45, but is not limited thereto.

[0139] Specifically, in the general formula 2 X7 is threonine, valine, or cysteine; X10 is tyrosine or cysteine; X12 is lysine; X15 is aspartic acid; X16 is glutamic acid or serine; X17 is lysine or arginine; X20 is glutamine or lysine; X21 is aspartic acid or glutamic acid; X24 is glutamine; X30 may be cysteine ​​or may be absent, but is not particularly limited thereto.

[0140] As an example, the peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, and 36 to 44, specifically, the peptide may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, and 36 to 44, but is not limited thereto.

[0141] The glucagon derivative may include an intramolecular bridge (e.g., a covalent or non-covalent bridge), and may specifically include a ring, for example, a ring formed between the 16th and 20th amino acids of the glucagon derivative, but is not limited thereto.

[0142] Non-limiting examples of such rings can include lactam bridges (or lactam rings).

[0143] The glucagon derivatives also include all those modified to include a ring, such as an amino acid capable of forming a ring at a desired position.

[0144] Such a ring may be formed between the side chains of amino acids in the glucagon derivative, for example, a lactam ring may be formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0145] For example, the peptide comprising the amino acid sequence of general formula 1 or 2 may be, but is not limited to, one in which the amino acids in each amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 or 2 are substituted with glutamic acid or lysine, respectively. In the Xn (n is a natural number), n indicates the position of the amino acid from the N-terminus of the presented amino acid sequence.

[0146] In addition, the peptide comprising the amino acid sequence of general formula 1 or general formula 2 may be one in which each of the amino acids of the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 is substituted with glutamic acid or lysine capable of forming a ring, but is not limited thereto.

[0147] In addition, in the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in the general formula 1 or 2, 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.

[0148] In addition, in the general formula 1 or 2, X16 may be glutamic acid, X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but is not limited thereto.

[0149] Furthermore, the peptide may have a longer half-life in the body than native glucagon, but is not particularly limited thereto.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] In one specific example, the peptide may be amidated at the C-terminus, but is not limited thereto.

[0159] In one embodiment, but not limited to, the peptide may be non-glycosylated.

[0160] 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.

[0161] 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.

[0162] 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:

[0163] (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.

[0164] In addition, the peptide or glucagon derivative comprising the amino acid sequence of formula 1 or formula 2 may be in the form of a long-acting conjugate bound 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.

[0165] The peptide comprising the amino acid sequence of general formula 1 or general formula 2 in the form of a long-acting conjugate (conjugate form), or the peptide comprising the amino acid sequence of general formula 1 or general formula 2 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 organs in the body of an individual to which it is administered.

[0166] In the present application, the term "long-acting conjugate" refers to a form in which a biocompatible substance moiety or a carrier is bound to a physiologically active substance (e.g., a glucagon derivative), and 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 sequence as or include the amino acid sequence of general formula 1 or general formula 2 or SEQ ID NOs: 2 to 11, and 13 to 45. In the long-acting conjugate, the biocompatible substance moiety or carrier may be bound to the physiologically active substance by a covalent bond, but is not particularly limited thereto.

[0167] 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.

[0168] Alternatively, such conjugates may be non-naturally occurring.

[0169] In a specific embodiment of the present invention, the long-acting conjugate refers to a form in which a glucagon derivative and an immunoglobulin Fc region are linked to each other. Specifically, the conjugate may be a glucagon derivative and an immunoglobulin Fc region covalently linked to each other via a linker, but is not particularly limited thereto.

[0170] In one embodiment, but not limited to, the immunoglobulin Fc region and X may be non-glycosylated.

[0171] In one embodiment of the present invention, the persistent conjugate is a conjugate represented by the following formula 1:

[0172] [Chemical formula 1] XLF

[0173] 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.

[0174] X of the long-acting conjugate of Formula 1 may be, but is not limited to, the glucagon derivatives described above.

[0175] In one specific example, X of the persistent conjugate of formula 1 may be a peptide comprising the amino acid sequence of formula 2.

[0176] Specifically, X may be a peptide comprising an amino acid sequence of any one selected from the group consisting of SEQ ID NOs: 13, 15, 19, 33, and 36 to 45, or may be a peptide comprising an amino acid sequence of any one selected from the group consisting of SEQ ID NOs: 33, and 36 to 44, but is not limited thereto.

[0177] In addition, as an example, the X may be a peptide comprising the amino acid sequence of general formula 1, or the X may be a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 45, or any one of the amino acid sequences of SEQ ID NOs: 2 to 11 and 13 to 45, or the X may be a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42, and 44, but is not limited thereto.

[0178] In the conjugate, F is X, i.e., a liver targeting drug, specifically, a peptide having activity against the glucagon 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.

[0179] 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.

[0180] In the present invention, the term "long-acting conjugate of Chemical Formula 1" refers to a form in which a glucagon derivative and an immunoglobulin Fc region are linked to each other via a linker, and the conjugate can exhibit increased sustained efficacy compared to a glucagon derivative to which an immunoglobulin Fc region is not linked.

[0181] In the long-acting conjugate, F is X, ie, a substance that increases the half-life of the glucagon derivative, and corresponds to one of the moieties that constitute the conjugate of the present invention.

[0182] In the long-acting conjugate of Chemical Formula 1, the link between X, which is a peptide that is a glucagon derivative, and the immunoglobulin Fc region may be a physical or chemical bond, or a non-covalent or covalent bond, specifically, a covalent bond, but is not limited thereto.

[0183] In addition, the method of linking X, which is the glucagon derivative of the long-acting conjugate of Chemical Formula 1, to the immunoglobulin Fc region is not particularly limited, and the glucagon derivative and the immunoglobulin Fc region may be linked to each other via a linker.

[0184] In Formula 1, X and F are bonded to each other via L by a covalent bond.

[0185] 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.

[0186] Moreover, the X may be linked to F via a linker (L).

[0187] Alternatively, L may be a non-peptidic linker, such as a linker containing repeating ethylene glycol units.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following formula 2:

[0192] [Chemical formula 2] JPEG2025500508000002.jpg2035

[0193] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

[0194] In the persistent conjugate, the PEG moiety is -(CH 2 CH 2 O) n -Not only the structure, but also the linking elements and this -(CH 2 CH 2 O) n It may also include, but is not limited to, an oxygen atom between - and -.

[0195] As a specific example, the ethylene glycol repeat unit is, for example, [OCH 2 CH 2 ]n, where n is a natural number and the [OCH 2 CH 2The average molecular weight of the ]n moiety, for example, the number average molecular weight, is set to be 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 number of [OCH 2 CH 2 The average molecular weight of the n portion, 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 15 kDa, about 3 to about 13 kDa, about 3 to about The molecular weight may be, but is not limited 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, 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.

[0196] In addition, 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 General Formula 2 and an immunoglobulin domain (F) are covalently linked through a linker containing an ethylene glycol repeat unit, but is not limited thereto.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0203] 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.

[0204] The linker is linked to F, which is an immunoglobulin Fc region, and X, which is a peptide (glucagon derivative), through the reactive groups described above, and is converted into a linker linker.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of the peptide via a thioether bond, and the aldehyde group is linked to the -NH 2 The linkage can be achieved, but is not limited to, via a reductive alkylation reaction, which is just one example.

[0211] Through this reductive alkylation, the oxygen atom at one end of PEG is converted to the N-terminal amino group of the immunoglobulin Fc region with the -CH 2 CH 2 CH 2 -PEG-O-CH 2 CH 2 CH 2 It is possible to form a structure similar to that of NH-immunoglobulin Fc, in which one end of PEG is linked to a sulfur atom located at a cysteine ​​residue of a peptide through a thioether bond. The above-mentioned thioether bond is It can contain the structure JPEG2025500508000003.jpg4033.

[0212] However, the present invention is not particularly limited to the above example, which is merely an example.

[0213] In addition, in the conjugate, the reactive group of the linker is -NH 2However, this is just one example.

[0214] 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.

[0215] 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 at 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.

[0216] As a specific example, the conjugate of Formula 1 may have a structure of Formula 3 below.

[0217] [Chemical formula 3] JPEG2025500508000004.jpg3398

[0218] In the above formula 3, X is the peptide (glucagon derivative) 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.

[0219] 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.

[0220] In the above Chemical Formula 3, the value of n is the number of [OCH 2 CH 2The average molecular weight of the ]n moieties, for example, the number average molecular weight may be determined to be 1 to 100 kDa, or 1 to 20 kDa, or 10 kDa, but is not limited thereto.

[0221] 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.

[0222] 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.

[0223] In the conjugate, the reactive group of the non-peptidic polymer is -NH 2 However, this is just one example.

[0224] 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.

[0225] 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".

[0226] 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.

[0227] 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.

[0228] In one specific example, the F may be linked through the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0229] Such an immunoglobulin Fc region may include, but is not limited to, a hinge portion in the heavy chain constant region.

[0230] The immunoglobulin Fc region of the present invention may comprise a specific hinge sequence at the N-terminus.

[0231] 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.

[0232] 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:

[0233] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (sequence number 50).

[0234] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 50 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention is composed of 3 to 12 amino acids containing only one cysteine ​​residue, 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: 51), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 52), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 53), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 54), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 55), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 56), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 57), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 58), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 59), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 60), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser ( u-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 54), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 55), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 56), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 57), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 58), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 59), Pro-Ser-Cys-Pro (SEQ ID NO: 60), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 61), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 62), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 63) ), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (sequence number 64), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (sequence number 65), Glu-Ser-Lys-Pro-Ser-Cys-Pro (sequence number 66), Glu-Ser-Pro-Ser-Cys-Pro (sequence number 67), Glu-Pro-Ser-Cys (sequence number 68), Ser-Cys-Pro (sequence number 69).

[0235] More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 60 (Pro-Ser-Cys-Pro) or SEQ ID NO: 69 (Ser-Cys-Pro), but is not limited thereto.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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, may be removed, 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. WO97 / 34631 and WO96 / 32478, for example.

[0245] 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.

[0246] 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.

[0247] Furthermore, 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 or derivative thereof obtained from a transformed animal cell or a microorganism. Here, the method for obtaining it from a natural source may be a method in which the whole immunoglobulin is separated from the living body of a human or animal and then treated with a proteolytic enzyme. When treated with papain, it is cleaved at Fab and Fc, and when treated with pepsin, it is cleaved at pF'c and F(ab). 2 The resulting fragment is cleaved into Fc and pF'c. Fc or pF'c can then be separated 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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: 70 (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.

[0253] F in the above formula 1 may include a monomer having the amino acid sequence of SEQ ID NO: 70, and F may be a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 70, but is not limited thereto.

[0254] 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: 71 (composed of 442 amino acids).

[0255] 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.

[0256] Furthermore, the above-mentioned conjugates may have an increased duration of efficacy compared to native glucagon 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.

[0257] A composition comprising the liver-targeted drug, for example, a composition comprising the peptide (e.g., the peptide itself or a long-acting conjugate form in which an immunoglobulin Fc region is bound to the peptide) may be used for the prevention or treatment of a disease requiring drug action in the liver.

[0258] 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.

[0259] 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.

[0260] In the present application, the term "disease requiring drug action in the liver" refers to a disease for which the liver-targeting drug of the present invention exhibits therapeutic activity and which shows preventive or therapeutic effects due to the increased distribution of the drug administered to the liver tissue, such as, but not limited to, liver disease, hypoglycemia, or congenital hyperinsulinism, and includes, without limitation, diseases that can be targeted and treated by the liver-targeting drug of the present invention.

[0261] As disclosed in International Publication WO 2020 / 263063, a peptide having activity on the glucagon receptor or a long-acting conjugate thereof is effective for liver diseases such as metabolic liver disease, simple steatosis, nonalcoholic fatty liver, liver inflammation, nonalcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, liver cirrhosis, liver failure and liver cancer. In contrast, the peptide having activity on the glucagon receptor or a long-acting conjugate thereof, which is a liver-targeting drug having excellent targeting to liver tissue according to the present invention, also has a preventive or therapeutic effect on the liver disease and can be effectively provided as a therapeutic agent for liver disease.

[0262] In the present invention, the term "liver disease" refers to a disease that occurs in the liver, and may be any one selected from the group consisting of nonalcoholic fatty liver disease (NAFLD), liver fibrosis, liver inflammation, liver cirrhosis, liver decompensation, hepatocellular carcinoma, and cholestatic liver disease. For example, the non-alcoholic fatty liver disease is a group of diseases including simple steatosis, which is characterized by only excessive accumulation of fat in hepatocytes, non-alcoholic fatty liver, and non-alcoholic steatohepatitis (NASH), which is characterized by hepatocyte necrosis, inflammation, and fibrosis. The cholestatic liver disease may be primary biliary cirrhosis, primary sclerosing cholangitis, or a combination thereof, but is not limited to the above diseases as long as abnormalities occur in liver tissue and function.

[0263] In the present invention, the term "hypoglycemia" refers to a state in which blood sugar levels are lower than those of normal people. Usually, it refers to a state in which blood sugar levels are 50 mg / dl or less, but is not limited thereto. A common cause of hypoglycemia is when a person who uses oral hypoglycemic agents or insulin eats less food than usual or is overly active or exercises. Hypoglycemia can also occur due to drinking alcohol or the use of some drugs that lower blood sugar levels, serious physical illnesses, hormone deficiencies such as adrenal cortical hormones and glucagon, insulin-producing pancreatic tumors, autoimmune diseases against insulin, gastrectomy patients, and hereditary carbohydrate metabolic enzyme disorders.

[0264] In the present invention, the hypoglycemia includes both acute hypoglycemia and chronic hypoglycemia.

[0265] Symptoms of hypoglycemia include lethargy, trembling, paleness, cold sweat, dizziness, agitation, anxiety, palpitations, hunger, headache, fatigue, etc. If hypoglycemia continues for a long time, it can cause convulsions and seizures, and can even lead to shock and loss of consciousness.

[0266] More specifically, the hypoglycemia may be caused by persistent hyperinsulinism due to genetic defects. Known causes of hyperinsulinism due to genetic defects include mutations in the SUR or Kir6.2 genes on chromosome 11p15.1, mutations in the GK (glucokinase) gene on chromosome 7p15-p13 that increase GK activity, and mutations in the GDH (Glutamate dehydrogenase) gene that activate GDH, which increases ATP in beta islet cells.

[0267] Congenital hyperinsulnism, on the other hand, is one of the causes of severe and persistent hypoglycemia in newborns and children. It can be caused by a temporary increase in insulin secretion in low birth weight babies or babies born to diabetic pregnant women, or by abnormal function of pancreatic cells due to gene mutations.

[0268] Meanwhile, it is known that the liver can produce glucose through the decomposition of stored glycogen and gluconeogenesis. Therefore, if a pharmaceutical composition capable of inducing glycogenolysis and gluconeogenesis in the liver during hypoglycemia, such as glucagon and conjugates containing glucagon, can be targeted to the liver, it is expected that hypoglycemia can be improved more effectively and blood glucose normalized.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] Another embodiment of the present invention provides a bioactive substance targeted to liver tissue, in particular a bioactive substance that includes a substance that has binding ability to the glucagon receptor and is targeted to liver tissue.

[0281] 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, etc.

[0282] 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.

[0283] 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.

[0284] In the present invention, the individual is an individual suspected of having a disease requiring a drug action in the liver, and the individual suspected of having a disease requiring a drug action in the liver refers to mammals including rats, livestock, etc., including humans, who are suffering from or may suffer from the disease, but includes, without limitation, individuals who can be treated with the composition containing the liver-targeting drug of the present invention.

[0285] 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.

[0286] 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.

[0287] All of the above applies to diseases requiring drug action in the liver, liver-targeted drugs, and bioactive substances targeted to liver tissue.

[0288] 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.

[0289] All of the above applies to the liver-targeted drug, the bioactive substance targeted to liver tissue, and the individual.

[0290] 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.

[0291] All of the above applies to the liver-targeted drug, the bioactive substance targeted to liver tissue, and the individual.

[0292] 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.

[0293] 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.

[0294] Example 1: Production of glucagon and its derivative peptides Glucagon and its derivative peptides exhibiting activity at the glucagon receptor were prepared, and their sequences are shown in the following Table 1. Specifically, the amino acid sequence of natural glucagon of SEQ ID NO: 1 was replaced with negatively and positively charged amino acid residues to synthesize the glucagon derivative peptides shown in the following Table 1. The relative in vitro activities shown therein were measured by the method described in the following Experimental Example 1.

[0295] [Table 1] JPEG2025500508000006.jpg207160

[0296] In the sequences listed in Table 1, the amino acid X indicates the unnatural amino acid aminoisobutyric acid (Aib), the underline in the amino acid symbol indicates the formation of a lactam ring between the side chains of the underlined amino acid pair, and "-" indicates the absence of an amino acid residue at that position. In addition, in the column regarding the presence or absence of cyclicity, "-" indicates that no ring is formed in the sequence.

[0297] Example 2: Preparation of long-acting conjugates of glucagon derivative peptides As a representative glucagon derivative peptide, the glucagon derivative of SEQ ID NO: 37 was selected from the derivative peptides shown in Table 1 prepared in Example 1, and a conjugate was prepared by the following method.

[0298] Maleimide-PEG-aldehyde (NOF Japan), a linear modified polyethylene glycol having a molecular weight of 10 kDa in which the hydrogen atoms at both ends are substituted with a 3-(3-maleimidopropionamido)propyl group and a 3-oxopropyl group (propionaldehyde group), respectively, was reacted with a glucagon derivative peptide derivative of SEQ ID NO: 37 having a cysteine, and the cysteine ​​residue of this glucagon derivative peptide was PEGylated at the maleimide end of the maleimide-PEG-aldehyde.

[0299] Specifically, the glucagon derivative peptide of SEQ ID NO:37 and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1-5 and a protein concentration of 3-10 mg / ml at low temperature for 1-3 hours. The reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative mono-PEGylated to cysteine.

[0300] The immunoglobulin Fc fragment was produced by the method described in International Publication WO2007 / 021129 using an immunoglobulin Fc fragment (49.8 kDa, a homodimer in which two monomer chains of SEQ ID NO: 70 are linked by a disulfide bond) having a hinge region with the sequence Pro-Ser-Cys-Pro (SEQ ID NO: 60) at the N-terminus.

[0301] Next, the purified mono-PEGylated glucagon derivative peptide and immunoglobulin Fc fragment were reacted at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg / mL at 4-8°C for 12-18 hours. The reaction solution was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was added with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-20% 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 a long-acting conjugate of glucagon derivative peptide in which the polyethylene glycol terminal on the aldehyde side of the mono-PEGylated glucagon derivative peptide was linked to the N-terminal proline nitrogen of one chain in the two chains of the immunoglobulin Fc homodimer.

[0302] After preparation, the purity was analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography and was found to be 95% or more.

[0303] Here, the conjugate in which a glucagon derivative peptide and an immunoglobulin Fc fragment are linked via PEG is named a "long-acting conjugate of glucagon derivative peptide," a "conjugate comprising a glucagon derivative and immunoglobulin Fc," a "long-acting conjugate of a glucagon derivative," or a "long-acting glucagon derivative," and these terms may be used interchangeably in this application.

[0304] Experimental Example 1: Measurement of in vitro activity of long-acting conjugates of glucagon derivatives In order to measure the activity of the glucagon derivative prepared in Example 1, a method for measuring cell activity in vitro was used using cell lines transformed with glucagon (GCG) receptors.

[0305] Each of the above cell lines was transformed into CHO (chinese hamster ovary) to express the human GCG receptor gene, and is suitable for measuring the activity of GCG. Therefore, the activity against the GCG receptor was measured using each transformed cell line.

[0306] Specifically, a portion corresponding to the ORF in the cDNA of the human glucagon receptor gene (OriGene Technologies, Inc., USA) was used as a template, and PCR was performed using forward and reverse primers of SEQ ID NOs: 48 and 49, which contain an EcoRI cleavage site and an XhoI cleavage site, respectively.

[0307] In this case, the PCR reaction was repeated 30 times, with denaturation at 95° C. for 60 seconds, annealing at 55° C. for 60 seconds, and extension at 68° C. for 30 seconds. The amplified PCR product was electrophoresed on a 1.0% agarose gel, and a 450 bp band was eluted.

[0308] Forward primer (SEQ ID NO:48): 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' Reverse primer (SEQ ID NO:49): 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3'

[0309] The PCR product was cloned into a known animal cell expression vector, x0GC / dhfr, to prepare a recombinant vector, x0GC / GCGR.

[0310] The recombinant vector x0GC / GCGR was transformed into CHO DG44 cells cultured in 10% FBS-containing DMEM / F12 medium using lipofectamine, and selectively cultured in a selection medium containing 1 mg / mL G418 and 10 nM methotrexate. From this, monoclonal cell lines were selected by limiting dilution, and finally, cell lines showing excellent concentration-dependent cAMP response to glucagon were selected.

[0311] The activity of the glucagon derivative synthesized in Example 1 was measured in the cell line. Specifically, the transformed cell line was subcultured three or four times a week, and then cultured in a 384-well plate at 6 × 10 cells / well. 3 The subcultured cell lines were dispensed and cultured for 24 hours. The native glucagon was suspended at 200 nM and the glucagon derivatives were suspended at 1600 nM in HBSS (Hank's Balanced Salt Solution) buffer containing 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 0.1% BSA (bovine serum albumin), and 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and then serially diluted 10 times by 4 times. This was applied to a cAMP assay kit (LANCE cAMP 384 kit, PerkinElmer) and added to the cells, and the fluorescence value was measured. After the measurement, the highest fluorescence value was selected as 100%, and the EC 50 The values ​​were calculated and compared with those of native glucagon, and the results are shown in Table 1.

[0312] The glucagon derivatives prepared as above have high activity on glucagon receptors, and therefore can be targeted to increased hepatocytes, and can activate glucagon receptors, making them useful as therapeutic agents for target diseases in the liver.

[0313] Experimental Example 2: Measurement of pI of glucagon derivatives To confirm the improved physical properties of the glucagon derivative synthesized in Example 1, pI was estimated from the amino acid sequence using the pI / Mw tool (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003) on the ExPASy server.

[0314] As shown in Table 1, native glucagon of SEQ ID NO:1 has a pI of 6.8, while some glucagon derivatives according to the present invention have a pI in the range of about 4 to 6. Such glucagon derivatives have a lower or higher pI than native glucagon, and therefore can exhibit improved solubility and higher stability at neutral pH and the like, compared to native glucagon.

[0315] Such glucagon derivatives according to the present invention can increase patient compliance when used as therapeutic agents for diseases targeting the liver.

[0316] Experimental Example 3: Confirmation of tissue distribution of long-acting glucagon derivative conjugates The distribution of the long-acting conjugate of SEQ ID NO:37, selected as a representative example of the long-acting conjugate of glucagon derivative, in tissues and organs was compared in three SD rats.

[0317] Specifically, 1558μg / kg of each long acting glucagon derivative conjugate was subcutaneously injected, and the organs were removed at 4, 48, and 168 hours, 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 by ELISA. The T / S ratio (%) was calculated as tissue concentration / serum concentration*100. For example, the 48-hour data in Table 3 below is serum: 15500.8±2686.9, liver: 4625.4±1216.9, and the T / S ratio (%) was calculated as 4625.4 / 15500.8*100, which is about 29.8%. The concentrations were measured by ELISA after removing the organs.

[0318] As a result, the glucagon derivative long-acting conjugate was most strongly distributed in tissues 48 hours after administration, and showed a particularly high distribution ratio in the liver. The tissue distribution was highest in the liver, heart, lung, large intestine, spleen, small intestine, muscle, stomach, pancreas, adipose tissue, and kidney, in that order. 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. In addition, it was present at the highest rate in the liver 168 hours after administration, and the distribution ratio was higher in the lung than in the heart compared to 48 hours. The results of the tissue distribution ratio of the long-acting conjugate of SEQ ID NO: 37 compared to serum confirmed in the above example are summarized in Table 2 below.

[0319] [Table 2] JPEG2025500508000008.jpg103150

[0320] The above results suggest that the long-acting conjugate of the glucagon derivative of the present invention has superior tissue distribution in the liver compared to other tissues and can be used as a therapeutic agent for a target disease. Therefore, the long-acting conjugate of the glucagon derivative can be used in a new application that induces targeting to the liver tissue, efficiently delivers a required amount of the drug, and optimizes drug treatment.

[0321] Furthermore, the above results suggest that distribution in the liver is high even 7 days after administration, supporting the convenience of the formulation, which can be administered approximately once a week.

[0322] 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.

[0323] [Explanation regarding government-sponsored research and development] This research was supported by the National New Drug Development Project of the National Drug Development Corporation with funding from the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare (HN21C0601).

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 of the following general formula 2: Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X30 (General formula 2, Sequence number: 47) In the above general formula 2 X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X15 is aspartic acid (D) or cysteine ​​(C); X16 is glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 2 is identical to SEQ ID NO: 12).

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 containing the amino acid sequence of general formula 2; 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) a T / S ratio of 20% to 40% at 40 to 50 hours after administration; and (b) T / S ratio of 25% to 40% 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) a T / S ratio of 25% to 35% at 2 days after administration; and (b) T / S ratio of 27% to 37% 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. 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 1:2.2-3.

2.

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

10. The pharmaceutical composition according to claim 8, wherein the distribution ratio is the distribution ratio from 2 to 7 days after administration.

11. The liver-targeted drug is (a) a distribution ratio in the liver compared to the heart of 1:1.6-3.0 at 40-50 hours after administration; and (b) the distribution ratio in the liver relative to the heart is 1:2.8-7.0 at 160 hours to 180 hours after administration;

12. The pharmaceutical composition according to claim 1 or 2, wherein the liver-targeted drug has therapeutic activity for liver disease, hypoglycemia, or congenital hyperinsulinism.

13. The pharmaceutical composition according to claim 4, wherein the disease requiring drug action in the liver is liver disease, hypoglycemia, or congenital hyperinsulinism.

14. 14. The pharmaceutical composition of claim 13, wherein the hypoglycemia is acute or chronic hypoglycemia.

15. The pharmaceutical composition according to claim 1 or 2, wherein a ring is formed between the amino acids X16 and X20 of general formula 2.

16. 3. The pharmaceutical composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 19, 33, and 36-45.

17. 3. The pharmaceutical composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, and 36-44.

18. 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.

19. The pharmaceutical composition of claim 1 or 2, wherein the peptide is amidated at its C-terminus.

20. The pharmaceutical composition of claim 1 or 2, wherein the C-terminus of the peptide is not modified.

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

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

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

24. 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 the amino acid sequence of the following general formula 2: Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X30 (General formula 2, Sequence number: 47) In the above general formula 2 X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X15 is aspartic acid (D) or cysteine ​​(C); X16 is glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 2 is identical to SEQ ID NO: 12).

25. Use of a pharmaceutical composition in the preparation of a medicament for inducing increased distribution of a liver-targeted drug in liver tissue, the pharmaceutical composition comprising a liver-targeted drug, the drug having a high distribution 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 the amino acid sequence of the following general formula 2: Y-Aib-QGTF-X7-SD-X10-S-X12-Y-L-X15-X16-X17-R-A-X20-X21-F-V-X24-W-L-M-N-T-X30 (General formula 2, Sequence number: 47) In the above general formula 2 X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X15 is aspartic acid (D) or cysteine ​​(C); X16 is glutamic acid (E) or serine (S); X17 is lysine (K) or arginine (R); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X24 is valine (V) or glutamine (Q); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 2 is identical to SEQ ID NO: 12).