Pharmaceutical compositions for the prevention or treatment of metabolic bone diseases, comprising GLP-2 or its conjugates.

Abstract

The present invention relates to the use of GLP-2 or a long-acting conjugate thereof for the prevention or treatment of metabolic bone diseases.

Description

Technical Field

[0001] The present invention relates to the therapeutic use of GLP-2 and its long-acting conjugates for metabolic bone diseases.

Background Art

[0002] Glucagon-like peptide-2 (GLP-2) is a peptide hormone composed of 33 amino acids produced in L cells of the small intestine in response to ingested nutrients. GLP-2 promotes mucosal growth in the small intestine and large intestine, promotes the growth of intestinal cells and crypt cells, and suppresses apoptosis. In addition, GLP-2 increases nutrient absorption in the small intestine and decreases intestinal permeability. Furthermore, it suppresses gastric emptying and gastric acid secretion, increases intestinal blood flow velocity, and relaxes intestinal smooth muscle.

[0003] Metabolic bone disease (MBD) is a comprehensive term that means bone deformities caused by various categories of disorders, and it develops due to imbalances in calcium, phosphorus, magnesium, vitamin D, etc., and calcium deficiency is particularly known as a typical cause. Examples of metabolic bone diseases include osteoporosis, osteomalacia, rickets, osteitis fibrosa cystica, Paget's disease of bone, etc.

[0004] On the other hand, osteoporosis, which is a typical example of metabolic bone disease, is an inevitable symptom, to some extent, in the elderly, especially women after menopause, and in developed countries, as the population ages, the interest in osteoporosis and its therapeutic agents has been increasing. Osteoporosis means a pathological condition in which the bone mass is significantly reduced compared to normal people, and the ability to withstand body weight and mechanical pressure becomes weak, and it is a common metabolic disease in which bones are easily fractured even by a slight impact such as falling lightly indoors.

[0005] The bones of the human body repeat the process of being absorbed and regenerated, and metabolic bone diseases are considered to occur when the balance of such bone formation and bone resorption processes is disrupted. Promoting bone formation and suppressing bone degradation are known as important treatment mechanisms in the treatment of metabolic bone diseases such as osteoporosis. Therefore, drugs have been developed to maintain the current bone mass by increasing bone formation or preventing bone loss. As drugs for suppressing bone resorption, bisphosphate, calcitonin, estrogen, etc. are used, and parathyroid hormone is used to increase bone formation (Patent Document 1).

[0006] To date, the development of therapeutic agents for metabolic bone diseases using glucagon-like peptide-2 has been insufficient, so the development of therapeutic agents is still necessary.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention aims to provide a pharmaceutical composition containing GLP-2 (Glucagon-like peptide-2) for the prevention or treatment of metabolic bone disease.

[0010] Furthermore, the present invention aims to provide a method for preventing or treating metabolic bone disease, comprising the step of administering GLP-2 or a composition containing the same to an individual in need of GLP-2 or a composition containing the same.

[0011] Furthermore, the present invention aims to provide uses for GLP-2 or compositions containing the same in the manufacture of agents for the prevention or treatment of metabolic bone diseases. [Means for solving the problem]

[0012] One embodiment of the present invention is a composition for the prevention, treatment, or improvement of metabolic bone disease, comprising glucagon-like peptide-2 (GLP-2).

[0013] As a specific example, the present invention relates to a pharmaceutical composition containing GLP-2 for the prevention or treatment of metabolic bone disease.

[0014] As another specific example, the present invention relates to a food composition containing GLP-2 for the prevention or improvement of metabolic bone disease.

[0015] The present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic bone disease, comprising a pharmaceutically acceptable excipient and GLP-2 in a pharmaceutically effective amount, according to the above-described specific example.

[0016] A composition according to any of the above-mentioned specific examples, characterized in that the GLP-2 is either natural GLP-2 or a GLP-2 derivative in which at least one amino acid in the natural GLP-2 sequence has been modified by a method selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0017] A composition according to any of the above-described specific examples, wherein the GLP-2 derivative is modified at at least one amino acid among the amino acids at positions 1, 2, 30, and 34 in SEQ ID NO: 1.

[0018] A composition according to any of the above-described specific examples, wherein the GLP-2 derivative is a peptide containing an amino acid sequence represented by General Formula 1.

[0019] [General Formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 9)

[0020] Here, X1 is histidine, imidazoacetyl des-histidine, des-amino-histidine, β-hydroxyimidazolyl propionyl des-histidine, N-dimethyl-histidine or β-carboxyimidazolyl propionyl des-histidine, X2 is alanine, glycine or Aib (2-aminoisobutyric acid), X 30 is lysine or arginine, and X 34 is absent or is lysine, arginine, glutamine, histidine, 6-azido lysine or cysteine.

[0021] A composition according to any of the above-described specific examples, wherein the GLP-2 derivative is (1) X1 is imidazoacetyl des-histidine, X2 is glycine, X 30 is lysine, and X 34 is cysteine, or (2) X1 is imidazoacetyl des-histidine, X2 is glycine, X 30 is lysine, and X 34 is lysine, or (3) X1 is imidazoacetyl des-histidine, X2 is glycine, X 30 is arginine, and X 34(4) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 It is ricin, X 34 (5) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 That is arginine, X 34 (6) X1 is imidazoacetyldeshistidine, X2 is Aib, and X 30 It is ricin, X 34 (7) X1 is histidine, X2 is Aib, and X 30 It is ricin, X 34 It is characterized by being cysteine.

[0022] A composition according to any of the above-mentioned specific examples, characterized in that the GLP-2 derivative comprises an amino acid sequence represented by general formula 2.

[0023] [General formula 2] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 10)

[0024] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine, and X2 is alanine, glycine, or Aib (2-aminoisobutyric acid), and X 30 It is lysine or arginine, X 34 is at least one arbitrary amino acid, or at least one modified arbitrary amino acid.

[0025] A composition according to any of the above-mentioned specific examples, characterized in that the GLP-2 derivative is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

[0026] A composition according to any of the above-mentioned specific examples, characterized in that the metabolic bone disease is osteoporosis, osteoopenia, arthritis, periodontal disease, osteoplasia, or osteomalacia.

[0027] A composition according to any of the above-mentioned specific examples, characterized in that the composition promotes bone formation, inhibits bone breakdown, or promotes bone formation and inhibits bone breakdown.

[0028] A composition according to any of the above-mentioned specific examples, characterized in that the metabolic bone disease is osteoporosis.

[0029] A composition according to any of the above-mentioned specific examples, characterized in that the composition has at least one of the following properties when administered. (i) Increased blood concentration of osteocalcin (Bone Gla Protein, OC / BGP) (ii) Decreased blood concentration of osteoprotegerin (OPG) (iii) Decreased blood concentration of type 1 collagen C-telopeptide (CTX-1)

[0030] A composition according to any of the above-mentioned specific examples, characterized in that the GLP-2 is either unmodified or amidated at its C-terminus.

[0031] A composition according to any of the above-mentioned specific examples, characterized in that the GLP-2 is in the form of a sustained-release conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

[0032] A composition according to any of the above-mentioned specific examples, characterized in that the compound is represented by chemical formula (1).

[0033] X-La-F···(1)

[0034] Here, X is GLP-2 (natural GLP-2 or a GLP-2 derivative), L is a linker containing repeating units of ethylene glycol, a is 0 or a natural number, but if a is 2 or greater, each L is independent of the others, F is an immunoglobulin Fc region, and the "-" indicates a covalent bond.

[0035] A composition according to any of the above-mentioned specific examples, characterized in that F is an immunoglobulin Fc region.

[0036] A composition according to any of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region is non-glycosylated.

[0037] A composition according to any of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region is an IgG4 Fc region.

[0038] A composition according to any of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4.

[0039] A composition according to any of the above-mentioned specific examples, wherein F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0040] A composition according to any of the above-mentioned specific examples, characterized in that L is polyethylene glycol.

[0041] A composition according to any of the above-mentioned specific examples, characterized in that the chemical formula weight of the repeating unit portion (moiety) of ethylene glycol in L is in the range of 1 to 100 kDa.

[0042] Another aspect of the present invention is a method for preventing or treating metabolic bone disease, comprising the step of administering a pharmaceutically effective amount of GLP-2 or a GLP-2 sustained-release conjugate or a composition containing the same to an individual who requires such an amount.

[0043] A further aspect of the present invention is the use of GLP-2 or compositions containing the same for the prevention or treatment of metabolic bone diseases.

[0044] A further aspect of the present invention is the use of GLP-2 or a composition containing the same in the manufacture of a drug for the prevention or treatment of metabolic bone disease. [Effects of the Invention]

[0045] The composition containing GLP-2 according to the present invention is applicable to the treatment of bone-related diseases, including osteoporosis. [Brief explanation of the drawing]

[0046] [Figure 1] This figure shows the therapeutic effect of GLP-2 sustained-release conjugates on metabolic bone diseases, based on changes in the blood concentrations of osteocalcin (OC / BGP), osteoprotegerin (OPG), and type I collagen C-telopeptide (CTX-1). [Modes for carrying out the invention]

[0047] The present invention will be described in more detail below.

[0048] Furthermore, each description and embodiment disclosed herein applies to each other. That is, any combination of the various elements disclosed herein is included in the present invention. Moreover, the present invention is not limited to the following specific descriptions.

[0049] Furthermore, any person with ordinary skill in the art will be able to recognize and confirm many equivalents of the particular aspects of the present invention described in this application using only ordinary experiments. Moreover, these equivalents are also intended to be included in the present invention.

[0050] Throughout this specification, in addition to the usual one- and three-letter codes for naturally occurring amino acids, generally accepted three-letter codes are used for other amino acids such as Aib (α-aminoisobutyl acid), Sar (N-methylglycine), and α-methylglutamic acid. Furthermore, amino acids referred to by abbreviations in this specification are written according to IUPAC-IUB nomenclature. 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 His,H Isoleucine (Ile,I) Leucine Leu,L Lysine, K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Serine Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin Val,V

[0051] One embodiment of the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, comprising glucagon-like peptide-2 (GLP-2). Specifically, the bone disease is a metabolic bone disease, and more specifically, an osteopenic disease, but is not limited to these.

[0052] One specific example of the present invention is a pharmaceutical composition for the prevention or treatment of metabolic bone disease, comprising a pharmaceutically acceptable excipient and glucagon-like peptide-2 in a pharmaceutically effective amount, but is not limited to this.

[0053] In the present invention, "glucagon-like peptide-2" or "GLP-2 (Glucagon-like peptide-2)" refers to an agonist of the human glucagon-like peptide-2 receptor, and means a substance in the form of a polypeptide or a conjugate in which a non-polypeptide portion is linked to a polypeptide. In the present invention, glucagon-like peptide-2 or GLP-2 includes not only natural human GLP-2 but also its derivatives and their conjugates. The GLP-2 is an active ingredient contained in the pharmaceutical composition of the present invention, and may be included in the pharmaceutical composition in a pharmacologically effective amount.

[0054] The amino acid sequence of natural GLP-2 is as follows: GLP-2(1-33) HADGSFSDEMNTILDNLAARDFINWLIQTKITD(Sequence ID 1)

[0055] In this invention, "GLP-2 receptor agonist" means a substance that binds to the in vivo or isolated human glucagon-like peptide-2 (GLP-2) receptor and induces physiological activity identical or equivalent to that of native GLP-2. For example, examples of GLP-2 agonists include native GLP-2 or GLP-2 derivatives.

[0056] The "GLP-2 derivatives" in this invention include peptides having at least one difference in amino acid sequence compared to natural GLP-2, peptides modified by modification of the natural GLP-2 sequence, and / or mimics of natural GLP-2 that have the same preventive, therapeutic, and / or ameliorative functions for metabolic bone diseases as natural GLP-2. Specifically, the GLP-2 derivatives are modified in the natural GLP-2 sequence by substitution, addition, deletion, modification, and combinations thereof, of at least one amino acid, but are not limited to these.

[0057] In one specific embodiment, the GLP-2 of the present invention may have its N-terminal amino group substituted, removed, or modified, but is not limited thereto. To prevent binding at the N-terminus, which is an important site for the in vivo activity of GLP-2, when producing a sustained-type conjugate, the GLP-2 of the present invention may be produced by methods such as removing the α-amino group of the N-terminal histidine, synthesizing by substituting the N-terminal amino group with a hydroxyl or carboxyl group, removing the α-carbon of the N-terminal histidine and the N-terminal amino group attached to the α-carbon to leave only the imidazo-acetyl functional group, or modifying the N-terminal amino group with two methyl groups.

[0058] Specifically, GLP-2 derivatives include imidazoacetyl-deshistidyl-GLP-2 (CA-GLP-2), in which the α-carbon of the histidine residue, the first amino acid at the N-terminus of GLP-2, and the N-terminal amino group attached to the α-carbon are removed; desaminohistidyl GLP-2 (DA-GLP-2), in which the N-terminal amino group of GLP-2 is removed; β-hydroxyimidazopropionyldeshistidyl GLP-2 (HY-GLP-2), in which the N-terminal amino group of GLP-2 is replaced with a hydroxyl group; and N-dimethylhistidyl GLP-2 (N-dimethylhistidyl GLP-2), in which the N-terminal amino group of GLP-2 is modified with two methyl groups. This includes, but is not limited to, DM-GLP-2, or β-carboxyimidazopropionyl-deshistidyl GLP-2 (CX-GLP-2), in which the N-terminal amino group of GLP-2 is substituted with a carboxyl group. The following are examples of the structures of substances used in the preparation of GLP-2 derivatives, but are not limited to these.

[0059] TIFF0007846622000001.tif147150

[0060] In one specific embodiment, the GLP-2 derivative is modified in which at least one amino acid among amino acids 1, 2, 30, and 34 in Sequence ID No. 1 has been altered, but is not limited to this embodiment.

[0061] Specifically, the modification is selected from the group consisting of substitution, addition, removal, modification, and combination thereof of at least one amino acid, where the added amino acid may be a non-natural amino acid (e.g., a D-type amino acid), or it may be a substitution of a non-natural amino acid in addition to a natural amino acid. The added amino acid sequence is derived from natural GLP-2, but is not limited thereto.

[0062] Specifically, the GLP-2 derivatives of the present invention exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology in amino acid sequence with native GLP-2, and / or are in which a portion of one amino acid residue of GLP-2 is chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination), or modified (e.g., N-methylation).

[0063] In one specific embodiment, the GLP-2 derivative includes, but is not limited to, the amino acid sequence of general formula 1.

[0064] [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 9)

[0065] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine, and X2 is alanine, glycine, or Aib (2-aminoisobutyric acid), and X 30 It is lysine or arginine, X 34 It is either absent, or is lysine, arginine, glutamine, histidine, 6-azidrisine, or cysteine.

[0066] In other specific embodiments, the GLP-2 derivative includes, but is not limited to, the amino acid sequence of general formula 2.

[0067] [General formula 2] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 10)

[0068] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine, and X2 is alanine, glycine, or Aib (2-aminoisobutyric acid), and X 30 It is lysine or arginine, X 34 is at least one arbitrary amino acid, or at least one modified arbitrary amino acid.

[0069] Specifically, amino acid sequences of general formula 1 or 2 that are the same as sequence number 1 are excluded from GLP-2 derivatives, but are not limited to these.

[0070] Specifically, the GLP-2 derivatives of the present invention have, but are not limited to, substitution of alanine, the second amino acid of natural GLP-2, with glycine or Aib (2-aminoisobutyric acid), substitution of lysine, the 30th amino acid, with arginine, or a combination thereof. Furthermore, the GLP-2 derivatives have, but are not limited to, a thiol group (e.g., cysteine), an amino group (e.g., lysine, arginine, glutamine, or histidine), or an azide group (e.g., 6-azidrisine) introduced at the C-terminus (e.g., the 33rd amino acid).

[0071] When producing a sustained-release conjugate of a GLP-2 derivative, as described above, binding occurs at the introduced group, and this can be used to produce a GLP-2 conjugate with selectively adjusted binding sites. Specifically, one end of a non-peptide linker may be bound to the hydroxyl group, thiol group, amino group, or azide group of the GLP-2 derivative, and a substance capable of extending the in vivo half-life (e.g., an immunoglobulin Fc region) may be bound to the other end of the non-peptide linker. The thiol group, amino group, or azide group can be introduced by adding an amino acid to GLP-2, but is not limited to this. The thiol group can be introduced by adding cysteine ​​(C) to GLP-2, the amino group can be introduced by adding lysine (K), arginine (R), glutamine (Q), or histidine (H), and the azide group can be introduced by adding 6-azidolysine, AZ It can be introduced by adding K), but is not limited to these.

[0072] Specifically, the GLP-2 derivative according to the present invention has at least one residue that is cysteine, lysine, arginine, glutamine, histidine, or 6-azidrisine, but is not limited to these.

[0073] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine, and the introduction of a thiol group (e.g., cysteine) to the C-terminus. More specifically, it comprises imidazoacetyldeshistidine, from which the α-carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and has, for example, the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0074] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine, and the introduction of an amino group (e.g., lysine) to the C-terminus. More specifically, it comprises imidazoacetyldeshistidine, from which the α-carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and has, for example, the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0075] Specifically, the GLP-2 derivatives of the present invention include substitution of alanine, the second amino acid of natural GLP-2, with glycine; substitution of lysine, the 30th amino acid of natural GLP-2, with arginine; and introduction of an amino group (e.g., lysine) to the C-terminus. More specifically, they include imidazoacetyldeshistidine, from which the α-carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and have the amino acid sequence of, for example, SEQ ID NO: 4, but are not limited thereto.

[0076] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine, and the introduction of an azide group (e.g., 6-azidrisine) to the C-terminus. More specifically, it comprises imidazoacetyldeshistidine, in which the α-carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and has, for example, the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0077] Specifically, the GLP-2 derivatives of the present invention include substitution of alanine, the second amino acid of natural GLP-2, with glycine; substitution of lysine, the 30th amino acid of natural GLP-2, with arginine; and introduction of a thiol group (e.g., cysteine) to the C-terminus. More specifically, they include imidazoacetyldeshistidine, in which the α-carbon of the histidine residue, the first amino acid of the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and have the amino acid sequence of, for example, SEQ ID NO: 6, but are not limited thereto.

[0078] Specifically, the GLP-2 derivatives of the present invention include the substitution of alanine, the second amino acid of natural GLP-2, with 2-aminoisobutyric acid, and the introduction of a thiol group (e.g., cysteine) to the C-terminus, and have the amino acid sequence of, for example, SEQ ID NO: 8. More specifically, they include imidazoacetyldeshistidine, in which the α-carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the α-carbon have been removed, and have the amino acid sequence of, for example, SEQ ID NO: 7, but are not limited to these.

[0079] Table 1 shows the GLP-2 derivatives of Sequence IDs 2-8.

[0080] [Table 1]

[0081] In Table 1, ca H indicates that histidine is substituted with imidazoacetyldeshistidine, and Aib indicates 2-aminoisobutyric acid. AZ K stands for 6-azido-L-lysyine.

[0082] The GLP-2 derivative according to the present invention is a peptide containing the above-mentioned specific sequence, or a peptide (essentially) composed of the above-mentioned specific sequence, but is not limited to these.

[0083] Furthermore, even if the present application describes a peptide or GLP-2 derivative "composed of a specific sequence number," it does not exclude meaningless sequence additions before or after the amino acid sequence of the said sequence number, spontaneously occurring mutations, or silent mutations thereof, as long as they have the same or equivalent activity as the peptide or GLP-2 derivative composed of the amino acid sequence of the said sequence number. It goes without saying that products having such sequence additions or mutations are also included in this application.

[0084] Specifically, GLP-2 derivatives are defined as having either (1) X2 being glycine or Aib, or (2) X 30 (3) X2 is glycine or Aib, and X 30 This is lysine or arginine, but is not limited to these two.

[0085] Specifically, in general formula 1 or 2, (1) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 It is ricin, X 34 (2) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 It is ricin, X 34 (3) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 That is arginine, X 34 (4) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 It is ricin, X 34 (5) X1 is imidazoacetyldeshistidine, X2 is glycine, and X 30 That is arginine, X 34 (6) X1 is imidazoacetyldeshistidine, X2 is Aib, and X 30 It is ricin, X 34 (7) X1 is histidine, X2 is Aib, and X 30 It is ricin, X 34 These are some examples of the system, but they are not limited to these.

[0086] Such modifications for the production of agonists, fragments, variants, and derivatives of the native GLP-2 in the present invention include all modifications using L-type or D-type amino acids and / or non-native amino acids, and / or modifications by modifying or post-translational modifications of the native sequence (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonding, etc.).

[0087] In the present invention, GLP-2 or a GLP-2 derivative may be in a form in which its N-terminus and / or C-terminus are chemically modified, protected by an organic group, or modified by the addition of amino acids to its terminus or other parts, in order to protect it from protein-cutting enzymes in living organisms and improve its stability.

[0088] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so acetylation of the N-terminus and / or amidation of the C-terminus are performed to remove the charge, but the process is not limited to these methods.

[0089] Specifically, the GLP-2 or GLP-2 derivatives in the present invention are those whose C-terminus is either unmodified or amidated, but are not limited to these.

[0090] The GLP-2 or GLP-2 derivatives of the present invention can be synthesized by solid-phase synthesis, produced by recombinant methods, or manufactured by commercial commission.

[0091] In the present invention, the GLP-2 is in the form of a sustained-release conjugate in which a biocompatible substance capable of extending the in vivo half-life of natural GLP-2 or a GLP-2 derivative is bound to natural GLP-2 or a GLP-2 derivative, but is not limited to these forms. The sustained-release conjugate exhibits improved sustained efficacy compared to GLP-2 or its derivatives without a biocompatible substance (e.g., immunoglobulin Fc region) bound to it. In the present invention, a conjugate containing GLP-2 or its derivatives with an increased half-life due to the binding of a biocompatible substance is referred to as a "sustained-release conjugate." In the present invention, the sustained-release conjugate is used in combination with other conjugates.

[0092] Furthermore, such compound forms may not exist naturally.

[0093] Furthermore, in the persistent conjugate of GLP-2, the linkage between GLP-2 and a biocompatible substance (e.g., immunoglobulin Fc region) may be physical or chemical, and may be non-covalent or covalent, specifically covalent, but is not limited to these.

[0094] Furthermore, while the method of linking GLP-2 and a biocompatible substance (e.g., immunoglobulin Fc region) is not particularly limited, the GLP-2 and the biocompatible substance (e.g., immunoglobulin Fc region) are linked to each other via a linker.

[0095] Furthermore, Patent Document 2 relating to a persistent conjugate of GLP-2 is included in this application by reference.

[0096] In one specific embodiment, the persistent conjugate of GLP-2 of the present invention has the structure of chemical formula (1).

[0097] X-La-F···(1)

[0098] Here, X is GLP-2 (natural GLP-2 or a GLP-2 derivative), L is a linker containing repeating units of ethylene glycol, a is 0 or a natural number, but if a is 2 or greater, each L is independent of the others, F is an immunoglobulin Fc region, and the "-" indicates a covalent bond.

[0099] More specifically, X and L, and L and F may be linked to each other by covalent bonds, where the compound may be a compound in which X, L, and F are linked by covalent bonds in the order of chemical formula (1).

[0100] Furthermore, F may be directly linked to X (i.e., a is 0 in chemical formula (1)), or it may be linked via a linker L.

[0101] In the compound according to the present invention, F is a substance that can extend the half-life of X, i.e., GLP-2 or its derivative according to the present invention, and is a component that forms part of the compound according to the present invention.

[0102] The aforementioned F may be bonded to X by covalent or non-covalent chemical bonds, or it may be bonded to X via L by covalent, non-covalent chemical bonds, or a combination thereof.

[0103] The substance that can extend the half-life of F, i.e., X, is a biocompatible substance selected from the group consisting of, for example, high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited to these.

[0104] In the case of elastin, it may be human tropoelastin, which is a water-soluble precursor, or a polymer of some of its sequences or some of its repeating units, or, for example, an elastin-like polypeptide, but is not particularly limited to these.

[0105] Examples of the aforementioned polymers include polymers selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof. Examples of the polysaccharide include dextran, but the polymers are not limited to these.

[0106] In this specification, polyethylene glycol includes, but is not limited to, ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEGs).

[0107] Furthermore, the biocompatible substances include, but are not limited to, polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid.

[0108] Furthermore, fatty acids have the ability to bind to albumin in the body, but are not limited to this.

[0109] For example, F is an FcRn-binding substance, and more specifically, the FcRn-binding substance is an immunoglobulin Fc region, more specifically an IgG Fc region, and even more specifically a non-glycosylated IgG4 Fc region, but is not limited to these.

[0110] As a specific example of the present invention, F (for example, the immunoglobulin Fc region) is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but the invention is not limited to this.

[0111] In one specific embodiment, the persistent conjugate of the present invention is a compound in which GLP-2 or a derivative thereof is linked to an immunoglobulin Fc region, but is not limited to this.

[0112] In this invention, the "immunoglobulin Fc region" refers to the region of immunoglobulin excluding the variable regions of the heavy and light chains, and including the heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3). The immunoglobulin Fc region is a component that forms part of the compound of this invention. Specifically, it corresponds to F in chemical formula (1).

[0113] In this specification, the Fc region includes not only the natural sequence obtained by papain digestion of immunoglobulin, but also its derivatives, such as sequences that differ from the natural sequence in which at least one amino acid residue of the natural sequence is substituted by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0114] The aforementioned F (for example, the immunoglobulin Fc region) is a structure in which two polypeptide chains are linked by a disulfide bond, and the linkage is via a nitrogen atom of only one of the two chains, but is not limited to this. The linkage via the nitrogen atom may be linked to the ε-amino atom or the N-terminal amino group of lysine by reductive amination.

[0115] Reductive amination is a reaction in which an amine group or amino group in one reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) in another reactant to produce an amine, which is then reduced to form an amine bond. This is a well-known organic synthesis reaction in the field.

[0116] As a specific example of the GLP-2 sustained-release conjugate of the present invention, the sustained-release conjugate may be one in which the immunoglobulin Fc region is linked to a linker via its N-terminal nitrogen atom.

[0117] Such immunoglobulin Fc regions include, but are not limited to, a hinge region within the heavy chain constant region.

[0118] The immunoglobulin Fc region in the present invention may include a specific hinge sequence at its N-terminus.

[0119] In this invention, "hinge arrangement" refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region via an interdisulfide bond.

[0120] The hinge sequence in the present invention is a mutated hinge sequence having the following amino acid sequence, in which a portion of the sequence is deleted and only one cysteine ​​residue remains, but is not limited to this. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 11)

[0121] The aforementioned hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of Sequence ID No. 11 being deleted. The hinge sequence of the present invention consists 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 sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence ID 12) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (Sequence ID 13) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 14) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 15) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (Sequence ID 16) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 17) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 18) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 19) Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 20) Pro-Ser-Cys-Pro (Sequence ID 21) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (Sequence ID 22) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (Sequence No. 23) Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 24) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 25) Lys-Tyr-Gly-Pro-Pro-Cys-Pro (Sequence No. 26) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 27) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 28) Glu-Pro-Ser-Cys (SEQ ID NO: 29) Ser-Cys-Pro (SEQ ID NO: 30)

[0122] More specifically, the hinge sequence includes, but is not limited to, the amino acid sequence of SEQ ID NO: 21 (Pro-Ser-Cys-Pro) or SEQ ID NO: 30 (Ser-Cys-Pro).

[0123] In a more specific embodiment of the GLP-2 sustained-release conjugate of the present invention, the N-terminus of the immunoglobulin Fc region in the conjugate is proline, and the conjugate is such that the Fc region is linked to a linker via the nitrogen atom of the proline.

[0124] In one embodiment of the GLP-2 sustained-release conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer in which the two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of chemical formula (1) of the present invention is 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.

[0125] In this invention, "N-terminus" means the amino terminus of a protein or polypeptide, and includes the very end of the amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc region of this invention includes a hinge sequence at the N-terminus, but is not limited to this.

[0126] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes some or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding the variable regions of the heavy and light chains of the immunoglobulin, provided that it has substantially equivalent or improved effects to those of the natural immunoglobulin. In addition, it may be a region in which a very long partial amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0127] For example, the immunoglobulin Fc region of the present invention is a combination of 1) the CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) the CH1 domain and CH2 domain, 3) the CH1 domain and CH3 domain, 4) the CH2 domain and CH3 domain, 5) at least one of the CH1 domain, CH2 domain, CH3 domain and CH4 domain and an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of the heavy chain constant region and the light chain constant region, but is not limited to these.

[0128] Furthermore, in one embodiment of the GLP-2 sustained-release conjugate of the present invention, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, where the dimer of the Fc region F and X are covalently linked via a single linker L containing repeating units of ethylene glycol. In a specific example of this embodiment, X is covalently linked via linker L to only one of the two polypeptide chains of the dimer of such Fc region F. In a more specific example of this embodiment, of the two polypeptide chains of such a dimer of Fc region F, only one molecule of X is covalently linked via L to the polypeptide chain to which X is linked. In the most specific example of this embodiment, F is a homodimer.

[0129] In other embodiments of the persistent conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in the dimer form, where the immunoglobulin Fc and X may be linked to each other by a non-peptide linker. However, the present invention is not limited to these examples.

[0130] Furthermore, the immunoglobulin Fc region of the present invention includes not only natural amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means one in which at least one amino acid residue of a natural amino acid sequence is deleted, inserted, non-conservative or conservatively substituted, or has a different sequence due to a combination thereof.

[0131] For example, in the case of IgG Fc, amino acid residues 214-238, 297-299, 318-322, or 327-331, which are known to be important for binding, may be used as suitable modification sites.

[0132] Furthermore, various derivatives are used, such as derivatives in which the disulfide bond-forming site has been removed, derivatives in which several amino acids at the N-terminus of the natural Fc are deleted, and derivatives in which a methionine residue has been added to the N-terminus of the natural Fc. In addition, to eliminate the effector function, the complement binding site, such as the C1q binding site, may be removed, and the ADCC (antibody-dependent cell-mediated cytotoxicity) site may also be removed. Techniques for producing such immunoglobulin Fc region sequence derivatives are disclosed in Patent Documents 3 and 4, among others.

[0133] Amino acid exchanges in proteins and peptides that do not alter the overall molecular activity are known in the field (Non-Patent Literature 1). 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. Modifications may also be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0134] The aforementioned Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention, and may also have improved structural stability against heat, pH, etc., of the Fc region.

[0135] Furthermore, such Fc regions may be obtained from natural sources isolated from living organisms such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, or they may be recombinants or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from natural sources may involve isolating total immunoglobulins from living human or animal organisms and then treating them with proteolytic enzymes. Treatment with papain cleaves them into Fab and Fc, and treatment with pepsin cleaves them into pF'c and F(ab)2. These can then be separated into Fc or pF'c using methods such as size exclusion chromatography. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0136] Furthermore, the immunoglobulin Fc region may be in the form of natural glycans, increased glycans compared to natural ones, decreased glycans compared to natural ones, or glycans removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering techniques using microorganisms may be used to increase, decrease or remove such immunoglobulin Fc glycans. Here, the immunoglobulin Fc region from which glycans have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cell-mediated cytotoxicity or complement-dependent cell-mediated cytotoxicity is reduced or eliminated, thus not inducing unwanted immune responses in the body. For these reasons, immunoglobulin Fc regions from which glycans have been removed or which have been deglycosylated are suitable for their original purpose as drug carriers.

[0137] In this invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that is produced in prokaryotes, and more specifically in Escherichia coli, and has not been glycosylated.

[0138] On the other hand, the immunoglobulin Fc region may be of human origin, or of animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, and in more specific embodiments, it is of human origin.

[0139] Furthermore, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or an Fc region resulting from a combination thereof or a hybrid thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it may be 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 the most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited to these.

[0140] As a specific example, the immunoglobulin Fc region may be a fragment of human IgG4 Fc, and may be a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteine ​​of each monomer. Here, the homodimer has / may have two disulfide bonds (intra-chain form) in each monomer, namely between the 35th and 95th cysteine ​​and between the 141st and 199th cysteine.

[0141] Each monomer consists of 221 amino acids, and the total number of amino acids forming the homodimer is 442, but is not limited to these. Specifically, an immunoglobulin Fc fragment is formed when two monomers having the amino acid sequence of SEQ ID NO: 31 (consisting of 221 amino acids) form a homodimer through a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of the homodimer independently form an internal disulfide bond between the 35th and 95th cysteine ​​positions, and an internal disulfide bond between the 141st and 199th cysteine ​​positions, but is not limited to these.

[0142] On the other hand, "combination" in the present invention means that when forming a dimer or polymer, polypeptides encoding single-chain immunoglobulin Fc regions of the same origin are bound to single-chain polypeptides of different origins. That is, a dimer or polymer can be produced from at least two fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0143] In this invention, "hybrid" means that within the constant region of a single-chain immunoglobulin, there are sequences corresponding to immunoglobulin Fc fragments of at least two different origins. Various forms of hybrids are possible in this invention. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0144] On the other hand, IgG is also divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations thereof or hybridization thereof are also possible. Specifically, these are the IgG2 and IgG4 subclasses, and more specifically, the Fc region of IgG4, which has almost no effector function such as complement-dependent cytotoxicity (CDC).

[0145] Furthermore, the aforementioned conjugates exhibit improved efficacy persistence compared to natural GLP-2 or to X without F modification. Such conjugates include, but are not limited to, the forms described above, as well as forms encapsulated in biodegradable nanoparticles.

[0146] In chemical formula (1), L may be a non-peptide linker, such as a linker containing repeating units of ethylene glycol.

[0147] The "non-peptide linker" in the present invention includes a biocompatible polymer in which at least two repeating units are bonded together. The repeating units are linked to each other by any covalent bond other than peptide bonds. The non-peptide linker is a component that forms part of the compound of the present invention and corresponds to L in chemical formula (1).

[0148] The non-peptide linker used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes. The non-peptide linker in the present invention is used in combination with the non-peptide polymer.

[0149] The non-peptide linker is a linker containing repeating units of ethylene glycol, such as polyethylene glycol, but is not limited to these. Furthermore, derivatives of these known in the art and derivatives that can be easily produced by the art are also included in the present invention.

[0150] The repeating units of the non-peptide linker may be ethylene glycol repeating units, and specifically, the non-peptide linker may contain ethylene glycol repeating units and also contain functional groups used in the production of the conjugate at its termini. The persistent conjugate according to the present invention is in a form in which X and F are linked via the functional groups, but is not limited thereto. The non-peptide linker in the present invention contains two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0151] Specifically, the linker is polyethylene glycol (PEG) represented by chemical formula (2), but is not limited to this.

[0152] TIFF0007846622000003.tif2239...(2)

[0153] Here, n is 10 to 2400, n is 10 to 480, or n is 50 to 250, but is not limited to these ranges.

[0154] The PEG portion of the aforementioned persistent conjugate is -(CH2CH2O) n -Not just the structure, but also the connecting elements and their (CH2CH2O) n -This includes, but is not limited to, oxygen atoms interposed between them.

[0155] Furthermore, while the aforementioned conjugate in one specific embodiment has a structure in which GLP-2 or a GLP-2 derivative and an immunoglobulin Fc region (F) are covalently linked via a linker containing ethylene glycol repeating units, it is not limited to these.

[0156] The aforementioned polyethylene glycol encompasses, but is not limited to, ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEGs).

[0157] The non-peptide linker used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes. The molecular weight of the non-peptide polymer is in the range of greater than 0 and 200 kDa, specifically in the range of about 1 to 100 kDa, more specifically in the range of about 1 to 50 kDa, even more specifically in the range of about 1 to 20 kDa, even more specifically in the range of about 3.4 kDa to 10 kDa, and even more specifically in the range of about 3.4 kDa, but is not limited to these ranges.

[0158] In this invention, "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, any numerical value or range equivalent to the term "approximately" that follows it.

[0159] Furthermore, the non-peptide linker of the present invention, which is bound to the immunoglobulin Fc region, may be a combination of different polymers, not just one type of polymer.

[0160] In one specific embodiment, both ends of the non-peptide linker are bonded to the thiol, amino, and hydroxyl groups of the immunoglobulin Fc region, and to the thiol, amino, azide, and hydroxyl groups of GLP-2, but are not limited to these.

[0161] Specifically, the non-peptide linker may be a reactive group whose two ends are each bound to immunoglobulin Fc and GLP-2 or a derivative thereof, more specifically, a reactive group which is bound to the thiol group of cysteine ​​in the immunoglobulin Fc region and at least one selected from the group consisting of an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine, and a hydroxyl group located at the C-terminus, and is also bound to the thiol group of cysteine ​​in GLP-2 and at least one selected from the group consisting of an amino group of lysine, arginine, glutamine and / or histidine, an azide group of azidrisine, and a hydroxyl group, but is not limited to these.

[0162] More specifically, the reactive group of the non-peptidic polymer is at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited to these.

[0163] In the above, examples of aldehyde groups include, but are not limited to, propionaldehyde groups and butyraldehyde groups.

[0164] In the above, succinimide derivatives include, but are not limited to, succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidylpropionate, N-hydroxysuccinimide, hydroxysuccinimidyl, or succinimidyl carbonate.

[0165] The non-peptidic linker may be linked to immunoglobulin Fc and GLP-2 derivatives via the above-mentioned reactive group and converted into a non-peptidic polymer linkage.

[0166] Furthermore, the final product generated by reductive alkylation via aldehyde bonds is far more stable than that linked by amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH, and can form covalent bonds with lysine residues at high pH, ​​such as pH 9.0.

[0167] The terminal reactive groups of the non-peptide linker of the present invention may be the same or different. The non-peptide linker may have an aldehyde reactive group at its terminal, or it may have an aldehyde group and a maleimide reactive group at its terminal, or it may have an aldehyde group and a succinimide reactive group at its terminal, but is not limited to these.

[0168] As an example, one end may have a maleimide group, and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. As another example, one end may have a succinimidyl group, and the other end may have a propionaldehyde group or a butyraldehyde group.

[0169] When polyethylene glycol having a hydroxyl reactive group at the propione end is used as a non-peptide linker, the compound of the present invention can be produced by activating the hydroxyl group as one of the various reactive groups described above through known chemical reactions, or by using commercially available polyethylene glycol having modified reactive groups.

[0170] In one specific embodiment, the reactive group of the non-peptide linker is linked to a cysteine ​​residue of GLP-2 or a GLP-2 derivative, more specifically to the -SH group of cysteine, but is not limited to these embodiments.

[0171] When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of GLP-2 or a GLP-2 derivative via a thioether bond, and the aldehyde group can be linked to the -NH2 group of immunoglobulin Fc via a reductive alkylation reaction, but these are not the only examples.

[0172] Through such reductive alkylation, the N-terminal amino group of the immunoglobulin Fc domain is linked to the oxygen atom at one end of PEG via a linker functional group having the structure -CH2CH2CH2-, forming a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc. A thioether bond can then be used to form a structure in which one end of PEG is linked to the sulfur atom located at the cysteine ​​of GLP-2 or a GLP-2 derivative. The aforementioned thioether bond may have the structure of the following chemical formula.

[0173] TIFF0007846622000004.tif4234

[0174] However, this is not limited to the above example; it is merely one example.

[0175] Furthermore, in the aforementioned conjugate, the reactive group of the non-peptide linker may be linked to the -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is merely one example.

[0176] Furthermore, in the aforementioned conjugate, GLP-2 or a GLP-2 derivative may be linked to a non-peptide linker having a reactive group via its C-terminus, but this is merely one example.

[0177] In this invention, "C-terminus" refers to the carboxyl end of a peptide, and for the purposes of this invention, it refers to the position where the peptide binds to a non-peptidic polymer. For example, although not limited to these, it includes not only the very last amino acid residue of the C-terminus, but also all amino acid residues surrounding the C-terminus, specifically the 1st to 20th amino acid residues from the very end.

[0178] On the other hand, the persistent conjugates containing GLP-2 or GLP-2 derivatives according to the present invention include all forms of the peptide itself, its salts (e.g., pharmaceutically acceptable salts of the peptide), or its solvates.

[0179] Furthermore, the persistent conjugate may be in any form, as long as it is pharmaceutically acceptable.

[0180] The GLP-2 or GLP-2 derivative of the present invention, a sustained-release conjugate containing the same, or a composition containing the same may have applications for the prevention or treatment of metabolic bone diseases.

[0181] Specifically, the compositions of the present invention are used for the prevention or treatment of metabolic bone diseases, and in particular for the prevention or treatment of metabolic bone diseases induced by an imbalance between osteoblasts and osteoclasts, but are not limited to these. Examples of such diseases include, but are not limited to, osteoporosis, osteoopenia, arthritis, periodontal disease, osteoplasia, and osteomalacia.

[0182] The GLP-2 or derivative thereof according to the present invention, and the sustained-release conjugate containing the same, have the effect of promoting bone formation and / or inhibiting osteodecomposition by osteoclasts, and can therefore be used as an effective therapeutic agent for metabolic bone diseases.

[0183] Specifically, the compositions of the present invention exhibit, but are not limited to, at least one of the following properties upon administration. (i) Increased blood concentration of osteocalcin (Bone Gla Protein, OC / BGP) (ii) Decreased blood concentration of osteoprotegerin (OPG) (iii) Decreased blood concentration of type 1 collagen C-telopeptide (CTX-1)

[0184] Since the OC / BGP values ​​mentioned above are indicators of bone formation, an increase in their blood concentration signifies the bone formation-promoting effect of the sustained-release conjugate of the present invention. Since OPG and CTX-1 are indicators of the suppression of osteoclast differentiation and the activity of osteoclasts, a decrease in their blood concentration signifies the bone decomposition-inhibiting effect of the sustained-release conjugate of the present invention. Therefore, based on these properties, the compositions or derivatives of the present invention exert preventive or therapeutic effects on metabolic bone diseases by promoting bone formation and / or suppressing bone decomposition.

[0185] In this invention, "osteoporosis" refers to a condition in which the calcium content of bone tissue decreases, the compact bone thins, and the bone marrow cavity widens. As the condition progresses, the bones weaken, making them more susceptible to fractures even from minor impacts. Bone mass is influenced by various factors, including genetic factors, nutritional intake, hormonal changes, exercise, and lifestyle differences. Known causes of osteoporosis include aging, lack of exercise, low body weight, smoking, low-calcium diet, menopause, and oophorectomy. On the other hand, although there are individual differences, Black people have lower bone resorption levels and therefore more bone mass than Caucasians. Bone mass is generally highest between the ages of 14 and 18, and decreases by about 1% per year in old age. In particular, in women, bone loss continues to progress after the age of 30, and bone loss accelerates rapidly during menopause due to hormonal changes. In other words, during menopause, estrogen levels decrease rapidly. At this point, similar to the case of IL-7 (interleukin-7), a large amount of B lymphocytes are produced, and pre-B cells accumulate in the bone marrow. This increases the amount of IL-6, which in turn increases the activity of osteoclasts, ultimately leading to a decrease in bone mass. Furthermore, in patients with short bowel syndrome, insufficient nutrient supply leads to complications such as osteoporosis, osteopenia, and osteomalacia due to decreased bone density.

[0186] More specific types of osteoporosis include primary type 1 osteoporosis, primary type 2 osteoporosis, secondary osteoporosis, osteomalacia-like osteoporosis, osteoporosis due to menopause or oophorectomy, and osteoporosis due to short bowel syndrome, but are not limited to these.

[0187] In this invention, "osteopenia" refers to a condition in which bones become weaker or bone mineral density (BDM) decreases compared to normal.

[0188] In this invention, "arthritis" refers to a disease in which inflammatory changes occur within the joint, and includes osteoarthritis, rheumatoid arthritis, and the like.

[0189] In this invention, "periodontal disease" refers to an inflammatory condition of the tooth-supporting tissues caused by bacteria, and is divided into gingivitis and periodontitis. The cause of onset is that oral bacteria form plaque due to poor oral hygiene. If left untreated, plaque causes inflammation, which can lead to bleeding gums and bad breath. These symptoms are called gingivitis, and as gingivitis progresses, it becomes periodontitis. As periodontitis progresses, even mild stimuli such as brushing teeth can cause bleeding and swelling of the gums, and can change into acute inflammation, inducing pain. Such inflammation reduces the function of bone formation and increases the action of bone resorption, so the alveolar bone gradually decreases and is destroyed, ultimately leading to tooth loss. Therefore, the composition according to this invention is useful for periodontal disease, and especially for alveolar bone loss due to periodontal disease.

[0190] In this invention, "osteogenesis imperfecta" refers to a disease also known as incomplete osteogenesis, characterized by weak bone strength and easy fractures without any particular reason.

[0191] In the present invention, "osteomalacia" is characterized by temporary edema and pain in the knee area. The causes are vitamin D deficiency and calcium and phosphate metabolism disorders, and it can occur due to malabsorption of nutrients in the gastrointestinal tract, long-term use of pain relievers, insufficient exposure to sunlight, pregnancy, etc. This means that excessive formation of uncalcified osteoid tissue occurs in the bone, causing the bone to soften and leading to impaired movement, and the bone mineral density (BMD) is lower than normal.

[0192] In the present invention, “prevention” means any action that suppresses or delays the onset of metabolic bone disease by administering GLP-2 or its derivatives or compositions containing the same, and “treatment” means any action that improves or favorably alters the symptoms of metabolic bone disease by administering GLP-2 or a sustained-release conjugate of GLP-2 or compositions containing the same. In the present invention, “prevention or treatment of metabolic bone disease” includes the prevention and complete or partial treatment of metabolic bone disease. It also includes any other change in the patient that reduces, improves, alleviates the pain of symptoms of metabolic bone disease, reduces the incidence of metabolic bone disease, or improves treatment outcomes.

[0193] The GLP-2 or its derivatives or sustained-release conjugates of the present invention can maintain bone mass and bone density by promoting bone formation and / or suppressing bone breakdown and inhibiting bone loss, thereby exhibiting preventive or therapeutic effects on metabolic bone diseases.

[0194] In the present invention, "administration" means introducing a predetermined substance to a patient by any appropriate method, and the administration route of the composition is not limited to these, but can be any common route that can deliver the composition to a target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0195] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent. Such a pharmaceutically acceptable carrier, excipient, or diluent may be non-naturally occurring.

[0196] In this invention, "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect and without causing side effects, which can be easily determined by a person skilled in the art based on known factors in the medical field, such as the type of disease, the patient's age, weight, health condition, sex, sensitivity to the drug, route of administration, method of administration, number of administrations, duration of treatment, formulation, and drugs used simultaneously.

[0197] The pharmaceutical composition comprising GLP-2 or its sustained-release conjugate according to the present invention may further comprise a pharmaceutically acceptable carrier. The carrier is not limited to these, but for oral administration, it may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. For injectable preparations, it may be a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. For topical administration, it may include bases, excipients, lubricants, preservatives, etc.

[0198] The composition of the present invention can be manufactured in various forms by mixing it with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be manufactured in single-use ampoules or multi-dose forms. In addition, it can be manufactured in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0199] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The formulation may also further contain fillers, anti-agglomerates, lubricants, wetting agents, fragrances, preservatives, and the like.

[0200] Furthermore, the pharmaceutical composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized agents, and suppositories.

[0201] Furthermore, the composition may be formulated into a single-dose formulation suitable for internal administration to a patient by conventional methods in the pharmaceutical field, specifically into a formulation useful for the administration of protein drugs, and may be administered orally, or by parenteral administration routes including, but not limited to, skin, intravenous, intramuscular, intraarterial, intramedullary, intrameningeal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, vaginal, or rectal routes using methods of administration commonly used in the art.

[0202] Furthermore, the conjugate can be used in combination with various drug-acceptable carriers such as physiological saline or organic solvents. To improve stability and water absorption, carbohydrates such as glucose, sucrose, and dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, and other stabilizers can be used as drugs.

[0203] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug that is the active ingredient, along with various related factors such as the disease being treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

[0204] 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 involving multiple doses over a long period. The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Specifically, the total dose of GLP-2 or its sustained-release conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day.

[0205] However, the effective dose of GLP-2 or its sustained-release conjugate is determined by considering various factors such as the patient's age, weight, health status, sex, disease severity, diet, and excretion rate, as well as the route of administration and number of treatments of the pharmaceutical composition. Considering these factors, a person with ordinary knowledge in the art should be able to determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in dosage form, route of administration, and method of administration, as long as it produces the effects of the present invention.

[0206] In the present invention, the inclusion of GLP-2 or a GLP-2 derivative or a sustained-release conjugate containing the same in a pharmaceutically effective amount in a pharmaceutical composition means that the GLP-2 or GLP-2 derivative or a sustained-release conjugate containing the same is included in an amount sufficient to achieve the desired pharmacological activity (e.g., prevention, improvement, or treatment of metabolic bone disease), and that it is included at a pharmaceutically acceptable level, either at a level that does not cause toxicity or side effects to the administered individual or at a very low level. However, this does not limit the meaning of the pharmaceutically effective amount. Such a pharmaceutically effective amount is determined by comprehensively considering the number of administrations, the patient, the dosage form, and other factors.

[0207] Furthermore, since the pharmaceutical composition of the present invention exhibits excellent in vivo persistence and potency, the number and frequency of administrations of the pharmaceutical preparation of the present invention can be significantly reduced.

[0208] Specifically, the pharmaceutical composition of the present invention is administered once a week, once every two weeks, once every four weeks, or once a month, but is not limited to these doses.

[0209] Another embodiment of the present invention provides a food composition for the prevention or improvement of metabolic bone disease, comprising GLP-2 or a sustained-release conjugate thereof.

[0210] The aforementioned GLP-2 or its sustained-release conjugates, metabolic bone diseases, etc., are as described above.

[0211] The aforementioned food composition is used in the form of a functional food. When the composition of the present invention is used as a food additive, GLP-2 or a GLP-2 derivative or a sustained-release conjugate containing the same may be added as is, or it may be used together with other foods or food components, and is used as appropriate in the usual way. The amount of active ingredient mixed is appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

[0212] In this invention, "functional food" means a food manufactured or processed using specific components as raw materials or by methods such as extraction, concentration, purification, and mixing of specific components contained in food ingredients, for the purpose of supporting health, and which is designed and processed so that the biological regulatory functions such as biological defense, regulation of biological rhythms, prevention and recovery from disease are fully exerted in the body by the said components. The composition for the said functional food has functions related to disease prevention and recovery.

[0213] A further embodiment of the present invention provides a method for preventing or treating metabolic bone disease, comprising the step of administering a composition containing GLP-2 or a sustained-release conjugate thereof to an individual requiring such a composition.

[0214] The aforementioned GLP-2 or its sustained-release conjugates, metabolic bone diseases, etc., are as described above.

[0215] In the present invention, the individual referred to is an individual suspected of having metabolic bone disease, and the individual suspected of having metabolic bone disease means a mammal including humans, mice, livestock, etc., that has developed or is at risk of developing metabolic bone disease, but any individual that can be treated with the conjugate of the present invention or the composition containing it may be used. Specifically, any individual that is preferable for promoting bone formation and / or inhibiting bone decomposition may be used.

[0216] The method of the present invention may include the step of administering a pharmaceutical composition containing GLP-2 or its sustained-release conjugate in a pharmaceutically effective amount. The preferred total daily dose is determined by the attending physician within the bounds of sound medical judgment and can be administered in one or several doses. However, for the purposes of the present invention, the specific therapeutically effective dose for a particular patient is preferably different depending on the type and degree of response to be achieved, whether other formulations are used in some cases, the specific composition, the patient's age, weight, general health condition, sex, diet, administration time, route of administration, secretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and other factors, as well as similar factors well known in the pharmaceutical field.

[0217] Specifically, the GLP-2 or its sustained-release conjugate of the present invention is preferably administered in a total dose of approximately 0.0001 mg to 500 mg per kg of body weight per day, once a week, once every two weeks, once every four weeks, or once a month, but is not limited to these doses.

[0218] A further embodiment of the present invention provides the use of GLP-2 or its sustained-release conjugate or a composition containing the same for the prevention or treatment of metabolic bone disease.

[0219] The aforementioned GLP-2 or its sustained-release conjugates, metabolic bone diseases, etc., are as described above.

[0220] A further embodiment of the present invention provides the use of GLP-2 or its sustained-release conjugate or a composition containing it in the manufacture of a drug for the prevention or treatment of metabolic bone disease.

[0221] The aforementioned GLP-2 or its sustained-release conjugates, metabolic bone diseases, etc., are as described above.

[0222] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention, and the present invention is not limited to them. [Examples]

[0223] Fabrication of GLP-2 derivative sustained-release conjugates To pegylate the GLP-2 derivative CA GLP-2 RK (SEQ ID NO. 4 in Table 1) into ALD(2) PEG, a modified polyethylene glycol (in which both terminal hydrogens are substituted with propionaldehyde groups (3-oxopropyl groups), and the molecular weight of the ethylene glycol repeating unit (moiety) is 3.4 kDa, manufactured by NOF Corporation of Japan), the molar ratio of the GLP-2 derivative to ALD(2) PEG was set to 1:5 to 1:20, and the concentration of the GLP-2 derivative was set to 5 to 10 mg / ml. The reaction was carried out at 2 to 8°C for 4 to 16 hours. The reaction was carried out in 20 mM HEPES (pH 7.5) and ethanol, with 20 mM sodium cyanoborohydride (NaCNBH3) added as a reducing agent. The reaction mixture was purified using a Source 15S (GE, USA) column with a concentration gradient of a buffer containing sodium citrate (pH 2.0) and ethanol, and potassium chloride, to obtain a monopegged GLP-2 derivative.

[0224] Next, as described above, the molar ratio of the purified monopegged GLP-2 derivative to the immunoglobulin Fc fragment of SEQ ID NO: 31 was set to 1:2 to 1:6, and the total protein concentration was set to 30 to 35 mg / mL. The reaction was carried out at 2 to 8°C for 12 to 20 hours. In this reaction, 100 mM potassium phosphate buffer (pH 6.0) and isopropanol were added to the reaction mixture, along with 20 mM sodium cyanoborohydride as a reducing agent.

[0225] After the reaction was completed, the reaction solution was purified by applying it to a Source 15Q (GE, USA) column using a concentration gradient of bis-Tris (pH 6.5) buffer and sodium chloride, and then by applying it to a Source 15 ISO (GE, USA) column using a concentration gradient of ammonium sulfate and sodium citrate (pH 5.0-5.2). This yielded a persistent conjugate of the GLP-2 derivative, in which the GLP-2 derivative was covalently linked to an immunoglobulin Fc fragment by a polyethylene glycol linker. [Examples]

[0226] Confirmation of changes in serum osteocalcin, osteoprotegerin, and type 1 collagen C-telopeptide levels in osteoporosis model mice after administration of a sustained-release conjugate of a GLP-2 derivative. To measure the in vivo bone-protective effect of administering the GLP-2 derivative sustained-release conjugate prepared in Example 1, ovariectomized mice (OVX mice, Dae Han Bio Link Co., Ltd.), a model of osteoporosis, were used.

[0227] Specifically, osteocalcin (OC / BGP) is carboxylated osteocalcin that affinityfully binds to hydroxyapatite, a basic mineral component of bone. As a bone formation marker, its high levels in the blood indicate increased bone synthesis. Osteoprotegerin (OPG) is an osteoclast differentiation inhibitory marker. It competes with RANKL (Receptor activator of nuclear factor kappa-B ligand), which plays a crucial role in osteoclast differentiation, by binding to the osteoclast precursor receptor and inhibiting osteoclast differentiation. Therefore, its high levels in the blood indicate suppression of osteolysis. Type I collagen C-telopeptide (CTX-1) is a specific peptide sequence produced when cleaved by osteoclasts during bone resorption, and is therefore proportional to osteoclast activity.

[0228] Therefore, by observing the changes in blood levels of osteocalcin (OC / BGP), osteoprotegerin (OPG), and type I collagen C-telopeptide (CTX-1) after administration of the GLP-2 derivative sustained-release conjugate, the effects of the GLP-2 derivative sustained-release conjugate of the present invention on bone formation and bone decomposition were confirmed.

[0229] First, the ovaries of 7-week-old female mice (C57BL / 6J mice) were removed, and after a one-week recovery period, they were separated into groups of 10 mice each: a normal control group (where only skin incision and suturing were performed without ovarian removal, G1, Sham Vehicle), an ovariectomized control group (G2, OVX vehicle), a group administered teduglutide (56 μg / kg / BID) after ovariectomy (G3, OVX Teduglutide), and a group administered GLP-2 derivative sustained-release conjugate (6237 μg / kg / QW) after ovariectomy (G4, OVX GLP-2). (In this specification, the value of 6237 μg / kg per week for the dose of GLP-2 derivative sustained-release conjugate refers to the total mass of the GLP-2 derivative sustained-release conjugate used excluding the polyethylene glycol linker portion, i.e., the sum of the masses of only the polypeptide portion.) Each group was subcutaneously administered a vehicle, teduglutide, or a sustained-release GLP-2 derivative conjugate for 10 weeks. Subsequently, serum osteocalcin (OC / BGP), osteoprotegerin (OPG), and type I collagen C-telopeptide (CTX-1) levels were measured for each group.

[0230] The results of the study showed that, compared to the oophorectomy control group (OVX vehicle) and the teduglutide (56 μg / kg / BID) group, the group administered GLP-2 derivative sustained-release conjugate (6237 μg / kg / QW) showed a significant increase in serum osteocalcin (OC / BGP) levels (Figure 1(A)). Furthermore, serum osteoprotegerin (OPG) levels in the group administered GLP-2 derivative sustained-release conjugate (6237 μg / kg / QW) tended to increase compared to the oophorectomy control group (OVX vehicle), and were significantly higher than those in the teduglutide (56 μg / kg / BID) group (Figure 1(B)). Furthermore, serum C-terminal telopeptide (CTX-1) levels in the group administered with the GLP-2 derivative sustained-release conjugate (6237 μg / kg / QW) tended to decrease compared to the oophorectomy control group (OVX vehicle), and were confirmed to decrease significantly more than in the teduglutide (56 μg / kg / BID) administration group (Figure 1(C)).

[0231] These results suggest that administration of the GLP-2 derivative sustained-release conjugate of the present invention may provide preventive or therapeutic effects on metabolic bone diseases through a mechanism of reduced osteolysis and increased bone formation.

[0232] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification. Another aspect of the present invention may be as follows: [1] A pharmaceutical composition for the prevention or treatment of metabolic bone disease, Pharmacologically acceptable excipients, A pharmaceutical composition containing a pharmaceutically effective amount of glucagon-like peptide-2 (GLP-2). [2] The composition according to [1], wherein the GLP-2 is natural GLP-2 or a GLP-2 derivative. [3] The composition according to [2], wherein the GLP-2 derivative has been modified in at least one of the amino acids 1, 2, 30, and 34 in Sequence ID No. 1. [4] The composition according to [2], wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1. [General formula 1] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 9) Here, X 1 These are histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine. X 2 These are alanine, glycine, or Aib (2-aminoisobutyric acid), X 30 It is lysine or arginine, X 34 It is either absent, or is lysine, arginine, glutamine, histidine, 6-azidrisine, or cysteine. [5] The GLP-2 derivative is (1)X 1 is imidazoacetyldeshistidine, and X 2 It is glycine, X 30 It is ricin, X 34 Is it cysteine? (2)X 1 is imidazoacetyldeshistidine, and X 2 It is glycine, X 30 It is ricin, X 34 Is it ricin? (3)X 1 is imidazoacetyldeshistidine, and X 2 It is glycine, X 30 That is arginine, X 34 Is it ricin? (4)X 1 is imidazoacetyldeshistidine, and X 2 It is glycine, X 30 It is ricin, X 34 Is it 6-azidricin? (5)X 1 is imidazoacetyldeshistidine, and X 2 It is glycine, X 30 That is arginine, X 34 Is it cysteine? (6)X 1 is imidazoacetyldeshistidine, and X 2 Aib is X 30 It is ricin, X 34 Is it cysteine, or (7)X 1 is histidine, and X 2 Aib is X 30 It is ricin, X 34 The composition according to [4] above, wherein is cysteine. [6] The composition according to [2], wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 2. [General formula 2] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 10) Here, X 1 These are histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine. X 2 These are alanine, glycine, or Aib (2-aminoisobutyric acid), X 30 It is lysine or arginine, X 34 is at least one arbitrary amino acid, or at least one modified arbitrary amino acid. [7] The composition according to [2], wherein the GLP-2 derivative is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8. [8] The composition according to [1] or [2] above, wherein the metabolic bone disease is osteoporosis, osteoopenia, arthritis, periodontal disease, osteoplasia, or osteomalacia. [9] The composition according to [1] or [2], wherein the composition promotes bone formation, inhibits bone breakdown, or promotes bone formation and inhibits bone breakdown.

[10] The composition according to [9], wherein the metabolic bone disease is osteoporosis.

[11] The composition according to

[10] , wherein the composition has at least one of the following properties when administered. (i) Increased blood concentration of osteocalcin (Bone Gla Protein, OC / BGP) (ii) Decreased blood concentration of osteoprotegerin (OPG) (iii) Decreased blood concentration of C-telopeptide of collagen type 1 (CTX-1)

[12] The composition according to [1] or [2], wherein the GLP-2 is either unmodified or amidated at its C-terminus.

[13] The composition according to [1] or [2], wherein the GLP-2 is in the form of a sustained-release conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

[14] The compound is the composition described in

[13] , represented by the following chemical formula (1). X-La-F···(1) Here, X is GLP-2 (natural GLP-2 or GLP-2 derivative), L is a linker containing ethylene glycol repeating units. a is 0 or a natural number, but if a is 2 or greater, then each L is independent of the others. F is the immunoglobulin Fc region, The aforementioned "-" indicates a covalent bond.

[15] The composition according to

[14] , wherein the immunoglobulin Fc region is a non-glycosylated IgG4 Fc region.

[16] The composition according to

[14] , wherein F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[17] The composition according to

[14] , wherein L is polyethylene glycol.

[18] The composition according to

[14] , wherein the chemical formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

Claims

1. A pharmaceutical composition for the prevention or treatment of metabolic bone disease, Pharmacologically acceptable excipients, A pharmaceutical composition comprising a glucagon-like peptide-2 (GLP-2) derivative in a pharmaceutically effective amount, The GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1, The composition wherein the metabolic bone disease is osteoporosis, osteoopenia, arthritis, periodontal disease, osteoplasia, or osteomalacia. [General formula 1] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence No. 9) Here, X 1 It is imidazoacetyldeshistidine, X 2 It is glycine, X 30 It is arginine, X 34 It is lysine or cysteine.

2. The composition according to claim 1, wherein the GLP-2 derivative is the amino acid sequence of SEQ ID NO: 4 or 6.

3. The composition according to claim 1, wherein the GLP-2 derivative is the amino acid sequence of SEQ ID NO:

4.

4. The composition according to claim 1, wherein the composition promotes bone formation, inhibits bone breakdown, or promotes bone formation and inhibits bone breakdown.

5. The composition according to claim 1, wherein the metabolic bone disease is osteoporosis.

6. The composition according to claim 5, wherein the composition has at least one of the following properties when administered. (i) Increased blood concentration of osteocalcin (Bone Gla Protein, OC / BGP) (ii) Decreased blood concentration of osteoprotegerin (OPG) (iii) Decreased blood concentration of C-telopeptide of collagen type 1 (CTX-1)

7. The composition according to claim 1, wherein the GLP-2 derivative is either unmodified or amidated at its C-terminus.

8. The composition according to claim 1, wherein the GLP-2 derivative is in the form of a sustained-release conjugate to which a biocompatible substance capable of extending its in vivo half-life is bound.

9. The aforementioned compound is the composition according to claim 8, represented by the following chemical formula (1). X-La-F...(1) Here, X is a GLP-2 derivative, L is a linker containing ethylene glycol repeating units, a is 0 or a natural number, but if a is 2 or greater, then each L is independent of the others. F is the immunoglobulin Fc region, The aforementioned "-" indicates a covalent bond.

10. The composition according to claim 9, wherein the immunoglobulin Fc region is a non-glycosylated IgG4 Fc region.

11. The composition according to claim 9, wherein F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

12. The composition according to claim 9, wherein L is polyethylene glycol.

13. The composition according to claim 9, wherein the chemical formula amount of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

14. The composition according to claim 9, wherein L is polyethylene glycol and the GLP-2 derivative is the amino acid sequence of SEQ ID NO: 4.

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