Combination therapy of GLP-2 with insulinotropic peptides, TNFα inhibitors, or both for the prevention or treatment of intestinal disorders

Abstract

The present invention relates to combination therapy of GLP-2 with an insulinotropic peptide, a TNFα inhibitor, or both, for the prevention, amelioration, or treatment of intestinal disorders.

Description

[Technical field]

[0001] The present invention relates to combination therapy of GLP-2 with insulinotropic peptides, TNFα inhibitors, or both, for the prevention or treatment of intestinal disorders. [Background technology]

[0002] Bowel diseases include irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, or intestinal damage, etc. Among them, inflammatory bowel disease (IBD) is an inflammatory disease in which inflammation or ulcers occur in the gastrointestinal tract, and symptoms include increased expression of inflammatory cytokines, weight loss, shortened colon length, abdominal pain, fever, diarrhea, and / or bloody stool, and these symptoms can worsen and improve repeatedly.

[0003] GLP-1, a type of insulin-secreting peptide, is an incretin hormone secreted from L-cells in the ileum and large intestine. The main function of glucagon-like peptide-1 is to increase insulin secretion, and since insulin secretion is dependent on the concentration of glucose (glucose dependent secretion), hypoglycemia does not occur. Due to these characteristics, it is used in the treatment of type 2 diabetes, but since its half-life in the blood is very short at around 2 minutes, it has a major drawback in developing it into a drug. As a result, one of the GLP-1 agonists that has been developed and is commercially available is exendin-4, a GLP-1 analog purified from the salivary glands of the Gila monster. It has resistance to DPP-IV (Dipeptidyl peptidase-4) and higher physiological activity than glucagon-like peptide-1, and therefore has a longer half-life in the body of 2-4 hours than GLP-1 (US 5,424,286 A). However, the method of increasing DPP-IV resistance alone cannot be expected to maintain physiological activity for a sufficient period of time. For example, the currently available exendin-4 (exenatide) must be administered to patients via injection twice a day, and the administration of the drug induces vomiting and nausea, which is a major burden on patients.

[0004] GLP-2 is a peptide hormone consisting of 33 amino acids that is produced in the L-cells of the small intestine in response to ingested nutrients. GLP-2 induces mucosal growth in the small and large intestines, promotes growth and inhibits apoptosis of enterocytes and crypt cells. GLP-2 also increases nutrient absorption in the small intestine and decreases intestinal permeability. It inhibits gastric emptying and gastric acid secretion, increases intestinal blood flow rate, and relaxes intestinal smooth muscles.

[0005] TNFα is a cytokine that plays a central role in immune responses and is also associated with inflammatory responses, and it is known that abnormal regulation of TNFα is manifested in various diseases. To suppress such TNFα-induced responses, various TNFα inhibitors have been developed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US 5,424,286 A [Patent Document 2] Korean Patent Publication No. 10-2019-0037181 [Patent Document 3] International Patent Publication No. 97 / 34631 [Patent Document 4] International Patent Publication No. 96 / 32478 [Non-patent literature]

[0007] [Non-Patent Document 1] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0008] To date, the development of drugs for treating intestinal diseases using insulinotropic peptides, GLP-2, and / or TNFα inhibitors has been inadequate, and there is a need for the development of sustainable drugs. [Means for solving the problem]

[0009] One object of the present invention is to provide a therapeutic method for preventing, ameliorating or treating intestinal diseases using GLP-2 in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating intestinal diseases, comprising GLP-2, which is characterized in that the pharmaceutical composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0011] Another object of the present invention is to provide a combination comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing, ameliorating or treating intestinal diseases, comprising said combination.

[0013] Another object of the present invention is to provide a pharmaceutical kit for preventing, ameliorating or treating intestinal disorders, comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both.

[0014] Another object of the present invention is to provide a method for preventing, ameliorating or treating an intestinal disease, comprising administering and / or using said combination, pharmaceutical composition or pharmaceutical kit to an individual in need thereof.

[0015] Another object of the present invention is to provide a method for preventing, ameliorating or treating an intestinal disease, comprising the step of co-administering and / or co-using a composition containing a pharmacologic amount of GLP-2, a composition containing a pharmacologic amount of an insulinotropic peptide, a composition containing a pharmacologic amount of a TNFα inhibitor, or both, to an individual in need thereof.

[0016] Another object of the present invention is to provide a use of said combination, pharmaceutical composition or pharmaceutical kit for the prevention, amelioration or treatment of intestinal diseases; and / or for the manufacture of a medicament for the prevention, amelioration or treatment of intestinal diseases. Effect of the Invention

[0017] The combined administration therapy of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both according to the present invention has improved effects compared to single-administration therapy, and can be useful for preventing, improving, or treating intestinal diseases. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the inhibitory effects of combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulin-secreting peptide (long-acting GLP-1 derivative conjugate) on M1 polarization (A), macrophage differentiation (B), and monocyte migration (C). [Diagram 2] This figure shows changes in small intestinal length in rats with indomethacin (INN)-induced inflammatory bowel disease after combined administration of GLP-2 (long-acting GLP-2 derivative conjugate), insulinotropic peptide (long-acting GLP-1 derivative conjugate), TNFα inhibitor (anti-TNFα antibody), or both. [Diagram 3] FIG. 1 shows changes in ulcer area in rats with indomethacin-induced inflammatory bowel disease after administration of GLP-2 (long-acting GLP-2 derivative conjugate), insulinotropic peptide (long-acting GLP-1 derivative conjugate), TNFα inhibitor (anti-TNFα antibody), or both. [Figure 4] FIG. 1 shows changes in colon length in mice with ulcerative colitis induced by combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulinotropic peptide (long-acting GLP-1 derivative conjugate). [Diagram 5] FIG. 1 shows the disease activity index (DAI) in mice with ulcerative colitis induced by dextran sulfate sodium after combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulinotropic peptide (long-acting GLP-1 derivative conjugate). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] One embodiment of the present invention is a method of treatment for the prevention, amelioration or treatment of intestinal disorders using GLP-2 in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0020] One embodiment of the present invention is a composition for preventing, ameliorating, or treating an intestinal disease, which contains GLP-2, and is characterized in that the GLP-2 is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0021] In one embodiment, the present invention relates to a pharmaceutical composition for preventing, ameliorating or treating an intestinal disease, comprising a pharmacologic effective amount of GLP-2, wherein the pharmaceutical composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0022] In another embodiment, the present invention relates to a food composition for preventing or improving intestinal diseases, which contains GLP-2, and is characterized in that the food composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0023] The composition according to any one of the above-mentioned embodiments is characterized in that the insulinotropic peptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof.

[0024] The composition according to any one of the above-mentioned specific examples is characterized in that the insulinotropic peptide is an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is substituted with imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine.

[0025] In the composition according to any one of the above-mentioned specific examples, the insulinotropic peptide is characterized by being naturally occurring exendin-4, an exendin-4 derivative in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the N-terminus of exendin-4, has been removed, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been substituted with a hydroxyl group, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been modified with two methyl groups, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been substituted with a carboxyl group, an exendin-4 derivative in which the 12th amino acid (lysine) of exendin-4 has been substituted with serine, or an exendin-4 derivative in which the 12th amino acid (lysine) of exendin-4 has been substituted with arginine.

[0026] The composition according to any one of the above-mentioned embodiments is characterized in that the GLP-2 is a natural GLP-2 or a GLP-2 derivative.

[0027] In a composition according to any one of the above-mentioned specific examples, the GLP-2 derivative is characterized in that it is a GLP-2 derivative in which at least one amino acid in the native GLP-2 sequence has been altered in a manner selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof.

[0028] In a composition according to any one of the above-mentioned embodiments, the GLP-2 derivative is characterized in that at least one of the amino acids at positions 1, 2, 30 and 33 in SEQ ID NO:1 has been modified.

[0029] In the composition according to any one of the above-mentioned embodiments, the GLP-2 derivative is characterized in that it comprises an amino acid sequence represented by the following general formula 1:

[0030] [General formula 1] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0031] Where: X 1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 is lysine or arginine; X 34 is absent, lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine; However, among the amino acid sequences of general formula 1, the sequence identical to SEQ ID NO: 1 is excluded.

[0032] In a composition according to any one of the above embodiments, the GLP-2 derivative comprises: (1)X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is cysteine ​​or (2)X 1is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is lysine, (3) X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is arginine, and X 34 is lysine, (4) X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is 6-azidolysine, (5)X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is arginine, and X 34 is cysteine ​​or (6)X 1 is imidazoacetyldeshistidine, and X 2 is Aib and X 30 is lysine and X 34 is cysteine, or (7)X 1 is histidine, and X 2 is Aib and X 30 is lysine and X 34 is cysteine. In the composition according to any one of the above-mentioned embodiments, the GLP-2 derivative is characterized in that it comprises an amino acid sequence represented by the following general formula 2:

[0033] [General formula 2] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:10)

[0034] Where: X 1is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 is lysine or arginine; X 34 is any one or more amino acids or any one or more amino acids modified; However, among the amino acid sequences of general formula 2, the sequence identical to SEQ ID NO: 1 is excluded.

[0035] The composition according to any one of the above-mentioned specific examples is characterized in that the GLP-2 derivative is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8.

[0036] The composition according to any one of the above-mentioned embodiments is characterized in that GLP-2 and an insulinotropic peptide are administered in combination.

[0037] The composition according to any one of the above embodiments is characterized in that GLP-2 and a TNFα inhibitor are administered in combination.

[0038] The composition according to any one of the above-mentioned embodiments is characterized in that GLP-2, an insulinotropic peptide, and a TNFα inhibitor are administered in combination.

[0039] The composition according to any one of the above embodiments is characterized in that the TNFα inhibitor is a soluble TNF receptor, an anti-TNFα antibody or fragment thereof, or a combination thereof.

[0040] In a composition according to any one of the above-mentioned embodiments, the intestinal disease is characterized in that it is at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal damage.

[0041] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease and Behcet's disease.

[0042] The composition according to any one of the above-mentioned embodiments is characterized in that, when administered to an individual, the composition exhibits one or more of the following: inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration.

[0043] The composition according to any one of the above-mentioned embodiments is characterized in that, when administered to an individual, the composition causes at least one of an increase in small intestine length, a decrease in small intestinal inflammation, an increase in large intestine length, and a decrease in large intestinal inflammation.

[0044] The composition according to any one of the above-mentioned embodiments is characterized in that the insulinotropic peptide and GLP-2 are unmodified or amidated at their C-terminus.

[0045] In a composition according to any one of the above embodiments, (i) the insulinotropic peptide is in the form of a long-acting conjugate to which a biocompatible substance that increases its in vivo half-life is attached, or (ii) the GLP-2 is in the form of a long-acting conjugate to which a biocompatible substance that increases its in vivo half-life is attached, or (iii) Each of the insulinotropic peptide and GLP-2 is in the form of a long-acting conjugate to which a biocompatible substance that increases their in vivo half-life is bound.

[0046] In the composition according to any one of the above-mentioned embodiments, the conjugate is characterized by being represented by the following chemical formula (1):

[0047] X-La-F···(1)

[0048] Where: X is an insulinotropic peptide or GLP-2; L is a linker containing ethylene glycol repeat units; a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others; F is an immunoglobulin Fc region; The "-" is a covalent bond.

[0049] The composition according to any one of the previous embodiments, wherein the immunoglobulin Fc region is an aglycosylated IgG4 Fc region.

[0050] The pharmaceutical composition according to any one of the above-mentioned specific examples is characterized in that F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0051] The composition according to any one of the above embodiments, wherein L is polyethylene glycol.

[0052] The pharmaceutical composition according to any one of the above-mentioned embodiments is characterized in that the chemical formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

[0053] The composition according to any one of the above embodiments may further comprise a pharma- ceutically acceptable carrier, excipient or diluent.

[0054] The composition according to any one of the above-mentioned embodiments is characterized in that (i) GLP-2 and an insulinotropic peptide; (ii) GLP-2 and a TNFα inhibitor; or (iii) GLP-2, an insulinotropic peptide, and a TNFα inhibitor are administered in combination, either simultaneously, sequentially or in the reverse order.

[0055] Another embodiment of the invention is a combination comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both.

[0056] Another embodiment of the present invention is a pharmaceutical composition for preventing, ameliorating or treating an intestinal disease, comprising the above combination.

[0057] Another embodiment of the present invention is a pharmaceutical kit for preventing, ameliorating or treating an intestinal disease, comprising the above combination.

[0058] Another embodiment of the present invention is a method for preventing, ameliorating or treating an intestinal disease comprising administering and / or using the combination, pharmaceutical composition or pharmaceutical kit to an individual in need thereof.

[0059] Another embodiment of the present invention is a method for preventing, ameliorating or treating an intestinal disease, comprising the step of co-administering and / or co-using a composition containing a pharmacologic amount of GLP-2, a composition containing a pharmacologic amount of an insulinotropic peptide, a composition containing a pharmacologic amount of a TNFα inhibitor, or both, to an individual in need thereof.

[0060] Another embodiment of the present invention is the use of the combination, pharmaceutical composition or pharmaceutical kit for the prevention, amelioration or treatment of an intestinal disease, and / or for the manufacture of a medicament for the prevention or treatment of an intestinal disease.

[0061] In the following the invention will be explained in more detail.

[0062] Meanwhile, each description and embodiment disclosed in the present application can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present invention. In addition, the following specific description is not considered to limit the scope of the present invention.

[0063] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed by the present invention.

[0064] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids, such as Aib (2-aminoisobutyric acid), AZK (6-azidolysine), etc. Also, the amino acids referred to as abbreviations herein are described according to the IUPAC-IUB nomenclature system.

[0065] 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

[0066] One embodiment of the present invention provides a pharmaceutical composition for preventing, ameliorating, or treating an intestinal disease, comprising GLP-2, wherein the GLP-2 is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0067] In one embodiment of the present invention, the pharmaceutical composition is a pharmaceutical composition for preventing, ameliorating or treating an intestinal disease, which contains a pharmacologic effective amount of GLP-2, and the pharmaceutical composition may be characterized in that it is administered in combination with an insulinotropic peptide (e.g., a pharmacologic effective amount of an insulinotropic peptide), a TNFα inhibitor (e.g., a pharmacologic effective amount of a TNFα inhibitor), or both, but is not limited thereto.

[0068] Another aspect of the present invention provides a use of GLP-2 in combination with an insulinotropic peptide, a TNFα inhibitor, or both, for the prevention, amelioration, or treatment of intestinal disorders.

[0069] Another embodiment provides a pharmaceutical composition for preventing, ameliorating or treating intestinal disorders, which uses GLP-2 in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0070] Specifically, one aspect of the present invention provides a combination, pharmaceutical composition, or pharmaceutical kit comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both. In one embodiment, the combination, pharmaceutical composition, or pharmaceutical kit is for preventing, improving, or treating intestinal disorders.

[0071] In the present invention, the term "combination" refers to the use of combined administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both, and is understood to be synonymous with "combined use." This includes, but is not limited to, pharmaceutical compositions and pharmaceutical kits in which GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both are combined.

[0072] Specifically, (i) GLP-2 and an insulinotropic peptide; (ii) GLP-2 and a TNFα inhibitor; or (iii) GLP-2, an insulinotropic peptide, and a TNFα inhibitor may be co-administered simultaneously, sequentially, or in the reverse order.

[0073] The co-administration may include i) co-administration of GLP-2 with an insulinotropic peptide, a TNFα inhibitor, or both, in a pharmaceutical composition, either in the form of a mixture or in separate, isolated forms; ii) administering a pharmaceutical composition containing GLP-2 in combination with a pharmaceutical composition containing an insulinotropic peptide, a pharmaceutical composition containing a TNFα inhibitor, or both simultaneously, sequentially, or in reverse order; iii) A pharmaceutical composition containing two substances, GLP-2, an insulinotropic peptide, and a TNFα inhibitor, in the form of a mixture or in a separate, separated form, and the remaining substance may be co-administered simultaneously, sequentially, or in the reverse order, but is not limited thereto.

[0074] The combination or composition comprises: a) (i) a mixture of GLP-2 and an insulinotropic peptide, (ii) a mixture of GLP-2 and a TNFα inhibitor, or (iii) a mixture of GLP-2, an insulinotropic peptide, and a TNFα inhibitor; or b) (i) GLP-2 and insulinotropic peptide are administered in separate forms, (ii) GLP-2 and TNFα inhibitor are administered in separate forms, or (iii) GLP-2, insulinotropic peptide and TNFα inhibitor are administered in separate forms; or c) (i) a mixture of GLP-2 and an insulinotropic peptide and a TNFα inhibitor administered separately; (ii) a mixture of GLP-2 and a TNFα inhibitor and an insulinotropic peptide administered separately; or (iii) a mixture of an insulinotropic peptide and a TNFα inhibitor and a GLP-2 administered separately, but is not limited thereto.

[0075] When in separate forms as in b) above, (i) GLP-2 and insulinotropic peptide; (ii) GLP-2 and TNFα inhibitor; or (iii) GLP-2, insulinotropic peptide, and TNFα inhibitor may each be formulated together in a separate, separate form; or each may be formulated in a separate formulation and the separate formulations may be administered simultaneously, separately, sequentially, or in reverse order.

[0076] In addition, when they are in separate forms as in c) above, the mixture and the insulinotropic peptide, GLP-2, or TNFα inhibitor may be formulated together in separate, separate forms; or each may be formulated in a separate formulation and the separate formulations may be administered simultaneously, separately, sequentially, or in the reverse order.

[0077] In the present invention, "co-administration", "co-administered" or "co-administered" does not simply mean simultaneous administration, but should be understood as an administration form in which GLP-2 and insulinotropic peptide, TNFα inhibitor, or both act together on an individual, and each substance (insulinotropic peptide, GLP-2, and / or TNFα inhibitor) can perform the same or higher level of its original function. Therefore, when the term "co-administration" is used in this application, it should be understood that this indicates simultaneous, separate, sequential, or reverse order administration, and the order is not limited. When the administration is sequential, reverse, or separate, the order of administration is not particularly limited, but the interval between the administration of the second or more components should be such that the beneficial effect of the combination is not lost.

[0078] In the present invention, the term "composition comprising a combination" refers to a combination itself comprising the GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both, and may have therapeutic applications, but is not limited thereto. For example, the composition may have applications in the prevention, amelioration, or treatment of intestinal diseases, but is not limited thereto. In the present application, the term "composition comprising a combination" can be used interchangeably with the term "composition."

[0079] The composition comprising the combination according to the present invention is for co-administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both, and may be formulated in a single formulation, or in two or more separate formulations. Specifically, the GLP-2 and the insulinotropic peptide, the TNFα inhibitor, or both may be administered simultaneously, separately, sequentially, or in the reverse order, but is not limited thereto.

[0080] In the present invention, the term "kit" may comprise a combination or composition according to the present invention for the co-administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both. Specifically, the kit according to the present invention may include, but is not limited to, a) (i) GLP-2 and an insulinotropic peptide, (ii) a GLP-2 and a TNFα inhibitor, or (iii) a GLP-2, an insulinotropic peptide, and a TNFα inhibitor, all formulated into a single formulation; b) (i) separate formulations of GLP-2 and an insulinotropic peptide, (ii) separate formulations of GLP-2 and a TNFα inhibitor, or (iii) separate formulations of GLP-2, an insulinotropic peptide, and a TNFα inhibitor; or c) (i) a formulation of GLP-2 and an insulinotropic peptide formulated together and a separate formulation of a TNFα inhibitor, (ii) a formulation of GLP-2 and a TNFα inhibitor formulated together and a separate formulation of an insulinotropic peptide, or (iii) a formulation of an insulinotropic peptide and a TNFα inhibitor formulated together and a separate formulation of GLP-2, and may further include substances necessary for the co-administration of two or more substances.

[0081] The present invention has confirmed that when GLP-2 is used in combination with an insulinotropic peptide, a TNFα inhibitor, or both, the preventive, ameliorative or therapeutic effects on intestinal diseases are dramatically increased compared to the use of an insulinotropic peptide, GLP-2, or a TNFα inhibitor alone, and provides a treatment method using said combination use.

[0082] The term "insulinotropic peptide" refers to a peptide having an insulinotropic function and can stimulate the synthesis or expression of insulin in pancreatic beta cells. Examples of the insulinotropic peptide include, but are not limited to, GLP-1 (glucagon like peptide-1), exendin-3, or exendin-4. The insulinotropic peptide includes not only natural insulinotropic peptides, but also precursors, agonists, derivatives, fragments, and variants thereof, and also includes long-acting conjugates to which biocompatible substances that increase their in vivo half-life are bound. The insulinotropic peptide may be included in the pharmaceutical composition in a pharmacologic effective amount.

[0083] GLP-1 is a hormone secreted from the small intestine, and generally promotes insulin synthesis and secretion, inhibits glucagon secretion, and promotes intracellular glucose absorption. In the small intestine, glucagon precursor is broken down into three peptides: glucagon, GLP-1, and GLP-2. Here, GLP-1 means GLP-1(1-37), which is a form that does not have insulin secretion function, and is processed in the form of GLP-1(7-37) to become active GLP-1(7-37). The amino acid sequence of GLP-1(7-37) is as follows:

[0084] GLP-1(7-37): HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G (SEQ ID NO:32)

[0085] Exendin-3 and exendin-4 are GLP-1 analogs or derivatives consisting of 39 amino acids that show 53% amino acid sequence similarity with GLP-1 and correspond to insulinotropic peptides. The amino acid sequences of exendin-3 and exendin-4 are as follows:

[0086] Exendin-3: HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO:33) Exendin-4: HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO:34)

[0087] An insulinotropic peptide derivative has insulinotropic function and may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity in amino acid sequence to a natural insulinotropic peptide, or may have a form in which a certain group of amino acid residues of the insulinotropic peptide have been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination) or modified (e.g., N-methylation), but is not limited thereto.

[0088] In one specific embodiment, the insulinotropic peptide derivative of the present invention can be prepared by a method of removing the alpha-amino group of N-terminal histidine, a method of synthesizing by replacing the N-terminal amino group with a hydroxyl group or a carboxyl group, a method of removing the alpha carbon and the N-terminal amino group bonded to the alpha carbon of N-terminal histidine to leave only an imidazo-acetyl functional group, a method of modifying the N-terminal amino group with two methyl groups, etc. The insulinotropic peptide derivative prepared by the above methods may be an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is replaced with imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine.

[0089] As a specific example, the insulinotropic peptide derivative may be a GLP-1 derivative, which includes, but is not limited to, exendin-3, exendin-4, or derivatives thereof.

[0090] Examples of insulinotropic peptide derivatives include derivatives in which the N-terminal amino group or amino acid residue of exendin-4 is chemically mutated, such as imidazoacetyl-deshistidyl-exendin-4 (CA-exendin-4) in which the alpha carbon and the N-terminal amino group bound to the alpha carbon of the histidine residue, which is the first amino acid at the N-terminus of exendin-4, have been removed; desaminohistidyl exendin-4 (DA-exendin-4) in which the N-terminal amino group of exendin-4 has been removed; and beta-hydroxyimidazopropionyldeshistidylexendin-4 in which the N-terminal amino group of exendin-4 has been replaced with a hydroxyl group. exendin-4 (HY-exendin-4), N-dimethylhistidyl exendin-4 (DM-exendin-4) in which the N-terminal amino group of exendin-4 is modified with two methyl groups, or beta-carboxyimidazopropionyl-deshistidyl-exendin-4 (CX-exendin-4) in which the N-terminal amino group of exendin-4 is substituted with a carboxyl group, an exendin-4 derivative in which the 12th amino acid (lysine) of exendin-4 is substituted with serine, or an exendin-4 derivative in which the 12th amino acid (lysine) of exendin-4 is substituted with arginine, but is not limited thereto.

[0091] An insulinotropic peptide fragment refers to a form in which one or more amino acids have been removed and / or added to the N-terminus or C-terminus of a natural insulinotropic peptide, and the added amino acids may be non-naturally occurring amino acids (e.g., D-amino acids).

[0092] An insulinotropic peptide variant refers to a peptide that differs from a naturally occurring insulinotropic peptide by one or more amino acids, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity, and has insulinotropic function.

[0093] An insulinotropic peptide agonist refers to a substance that exhibits the same biological activity as insulinotropic peptide by binding to an in vivo receptor of insulinotropic peptide, regardless of the structure of insulinotropic peptide.

[0094] The methods for producing the derivatives, fragments, variants and agents of the present invention may be used independently or in combination. For example, the present invention includes insulinotropic peptides having one or more different amino acid sequences and deamination of the N-terminal amino acid residue.

[0095] In the present invention, the term "glucagon-like peptide-2" or "GLP-2" refers to an agonist of the glucagon-like peptide-2 receptor, and may be in the form of a polypeptide or a long-acting conjugate to which a biocompatible substance that increases its in vivo half-life is bound, but is not limited thereto.

[0096] In the present invention, glucagon-like peptide-2 or GLP-2 includes not only the sequence identical to that of natural human GLP-2, but also GLP-2 derivatives, and also includes long-acting conjugates in which natural GLP-2 or GLP-2 derivatives are bound to a biocompatible substance. The GLP-2 may be contained in a pharmaceutical composition containing an insulinotropic peptide in a pharma- ceutical effective amount or in a separate composition so that it can be administered or used in combination with the composition.

[0097] The amino acid sequence of native GLP-2 is as follows:

[0098] GLP-2(1-33) HADGSFSDEMNTILDNLAARDFINWLIQTKITD (SEQ ID NO:1)

[0099] In the present invention, the term "GLP-2 receptor agonist" refers to a substance that binds to an in vivo or isolated human glucagon-like peptide-2 receptor and induces a physiological activity equivalent or similar to that of native GLP-2. For example, the GLP-2 agonist may include native GLP-2 or a GLP-2 derivative.

[0100] In the present invention, the term "GLP-2 derivative" includes a peptide having one or more differences in the amino acid sequence compared to native GLP-2; a peptide obtained by modifying the native GLP-2 sequence; and / or a mimetic of native GLP-2 having the function of preventing, treating and / or improving intestinal diseases like native GLP-2. Specifically, the GLP-2 derivative may be a peptide in which at least one amino acid in the native GLP-2 sequence has been modified by a method selected from the group consisting of substitution, addition, deletion, modification and combinations thereof, but is not limited thereto.

[0101] The added amino acids may be non-natural amino acids (e.g., D-amino acids), and non-natural amino acids may be substituted in addition to natural amino acids. The added amino acid sequence may be derived from natural GLP-2, but is not limited thereto. In the present invention, modification of amino acids may mean, together with or independently of the substitution, addition, removal, or combination thereof, that a part of the amino acid residues is chemically replaced (e.g., alpha-methylation, alpha-hydroxylation, replacement with azido group), removed (e.g., deamination), and / or modified (e.g., N-methylation).

[0102] In one specific embodiment, the GLP-2 derivative of the present invention may have an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity to native GLP-2, or may have a form in which a certain group of amino acid residues in GLP-2 have been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation, substitution with azido group), removed (e.g., deamination) and / or modified (e.g., N-methylation), but is not limited thereto.

[0103] In one specific embodiment, the GLP-2 of the present invention may have an N-terminal amino group substituted, removed, or modified, but is not limited thereto. The GLP-2 of the present invention may be prepared by a method of removing the alpha-amino group of the N-terminal histidine to prevent binding to the N-terminus, which is an important site for the in vivo activity of GLP-2, a method of synthesizing the N-terminal amino group by replacing it with a hydroxyl group or a carboxyl group, a method of removing the alpha carbon of the N-terminal histidine and the N-terminal amino group bound to the alpha carbon to leave only an imidazo-acetyl functional group, a method of modifying the N-terminal amino group with two methyl groups, etc.

[0104] Specifically, the GLP-2 derivatives include imidazoacetyl-deshistidyl-GLP-2 (CA-GLP-2), in which the N-terminal amino group bound to the alpha carbon and alpha carbon of the histidine residue, which is the first amino acid at the N-terminus of GLP-2, has been removed; desaminohistidyl GLP-2 (DA-GLP-2), in which the N-terminal amino group of GLP-2 has been removed; beta-hydroxyimidazopropionyldeshistidyl GLP-2 (HY-GLP-2), in which the N-terminal amino group of GLP-2 has been replaced with a hydroxyl group; and N-dimethylhistidyl GLP-2 (N-dimethylhistidyl GLP-2), in which the N-terminal amino group of GLP-2 has been modified with two methyl groups. The GLP-2 derivative may be, but is not limited to, beta-carboxyimidazopropionyl-deshistidyl-GLP-2 (DM-GLP-2) or beta-carboxyimidazopropionyl-deshistidyl-GLP-2 (CX-GLP-2) in which the N-terminal amino group of GLP-2 is replaced with a carboxyl group. As a non-limiting example, the structure of a substance used in the preparation of a GLP-2 derivative is as follows:

[0105] JPEG2025513345000002.jpg133147

[0106] As used herein, imidazoacetyldeshistidyl(dine), desaminohistidyl(dine), beta-hydroxyimidazopropionyldeshistidyl(dine), N-dimethylhistidyl(dine), and beta-carboxyimidazopropionyldeshistidyl(dine) are used interchangeably to mean imidazoacetyl, des-amino-histidyl, beta-hydroxy-imidazopropionyl, dimethyl-histidyl, and beta-carboxyl-imidazopropionyl, respectively.

[0107] In a specific embodiment, the GLP-2 derivative may have at least one amino acid modification among the first, second, 30th and 33rd amino acids in SEQ ID NO: 1, but is not limited thereto. Specifically, the modification may be a modification selected from the group consisting of substitution, addition, removal, modification and combinations thereof of at least one amino acid, and the added amino acid may be a non-natural amino acid (e.g., D-amino acid), and a non-natural amino acid may be substituted in addition to natural amino acids. The added amino acid sequence may be derived from natural GLP-2, but is not limited thereto. In addition, in the present invention, the modification of amino acids means that some groups of amino acid residues are chemically replaced (e.g., alpha-methylation, alpha-hydroxylation, replacement with azido group), removed (e.g., deamination) and / or modified (e.g., N-methylation) together with or independently of the replacement, addition, removal or combinations thereof of at least one amino acid, but is not limited thereto.

[0108] In one specific embodiment, the GLP-2 derivative may comprise, but is not limited to, the amino acid sequence of general formula 1 below:

[0109] [General formula 1] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0110] Where: X 1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X30 is lysine or arginine; X 34 is absent, lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine.

[0111] In another specific embodiment, the GLP-2 derivative may comprise, but is not limited to, the amino acid sequence of general formula 2:

[0112] [General formula 2] X 1 X 2 DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:10)

[0113] Where: X 1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 is lysine or arginine; X 34 is any one or more amino acids or any one or more amino acids in which a modification has occurred.

[0114] Specifically, the amino acid may be a natural amino acid or a non-natural amino acid, and the modification of the amino acid is as described above.

[0115] In addition, among the amino acid sequences of the general formula 1 or 2, the sequence identical to SEQ ID NO: 1 is excluded from the GLP-2 derivatives, but is not limited thereto.

[0116] Specifically, the GLP-2 derivative of the present invention may have, but is not limited to, a substitution of the second amino acid, alanine, of natural GLP-2 with glycine or Aib (2-aminoisobutyric acid), a substitution of the 30th amino acid, lysine, with arginine, or a combination thereof. In addition, the GLP-2 derivative may have, but is 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-azidolysine) introduced at the C-terminus (e.g., the 33rd amino acid).

[0117] Since the conjugation occurs at the introduced group during the preparation of a long-acting conjugate of a GLP-2 derivative, it is possible to prepare a GLP-2 conjugate with a selectively adjusted conjugation position by using the introduced group. Specifically, one end of a linker may be bound to a hydroxyl group, thiol group, amino group, or azide group of a GLP-2 derivative, and a substance that increases the in vivo half-life (e.g., immunoglobulin Fc region) may be bound to the other end of the linker. The thiol group, amino group, or azide group may be introduced by adding an amino acid to GLP-2, but is not limited thereto. The thiol group may be introduced by adding cysteine ​​(C) to GLP-2; the amino group may be introduced by adding lysine (K), arginine (R), glutamine (Q), or histidine (H); and the azide group may be introduced by adding 6-azidolysine (AZK), but is not limited thereto.

[0118] Specifically, the GLP-2 derivative according to the present invention may have at least one residue which is, but is not limited to, cysteine, lysine, arginine, glutamine, histidine or 6-azidolysine.

[0119] Specifically, the GLP-2 derivative of the present invention comprises a substitution of the second amino acid of naturally occurring GLP-2, alanine, with glycine and introduction of a thiol group (e.g., cysteine) at the C-terminus, and more specifically, may comprise imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and an example of such a derivative may have the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0120] Specifically, the GLP-2 derivative of the present invention comprises a substitution of the second amino acid of naturally occurring GLP-2, alanine, with glycine and introduction of an amino group (e.g., lysine) at the C-terminus, and more specifically, may comprise imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and an example of such a derivative may have the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0121] Specifically, the GLP-2 derivative of the present invention includes a substitution of the second amino acid, alanine, of native GLP-2 with glycine, a substitution of the 30th amino acid, lysine, of native GLP-2 with arginine, and introduction of an amino group (e.g., lysine) at the C-terminus, and more specifically, may include imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and an example of such a derivative may have the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0122] Specifically, the GLP-2 derivative of the present invention comprises a substitution of the second amino acid of naturally occurring GLP-2, alanine, with glycine and introduction of an azide group (e.g., 6-azidolysine) at the C-terminus, and more specifically, may comprise imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and an example of such a derivative may have the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0123] Specifically, the GLP-2 derivative of the present invention includes a substitution of the second amino acid, alanine, of native GLP-2 with glycine, a substitution of the 30th amino acid, lysine, of native GLP-2 with arginine, and introduction of a thiol group (e.g., cysteine) at the C-terminus, and more specifically, may include imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and an example of such a derivative may have the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0124] Specifically, the GLP-2 derivative of the present invention comprises a substitution of the second amino acid of naturally occurring GLP-2, alanine, with 2-aminoisobutyric acid and introduction of a thiol group (e.g., cysteine) at the C-terminus, and may have, for example, the amino acid sequence of SEQ ID NO: 8, and more specifically, it may comprise imidazoacetyldeshistidine in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bound to the alpha carbon have been removed, and may have, for example, the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0125] The GLP-2 derivatives of SEQ ID NOs: 2 to 8 are shown in Table 1 below.

[0126] [Table 1]

[0127] In Table 1 above ca H stands for imidazoacetyldeshistidine instead of histidine, Aib stands for 2-aminoisobutyric acid, AZ K stands for 6-azido-L-lysyine.

[0128] The GLP-2 according to the present invention may be a peptide containing the specific sequence described above, or a peptide composed of (or essentially composed of) the specific sequence described above, but is not limited thereto.

[0129] On the other hand, even if the present application describes a peptide or GLP-2 that is "composed of (consists of) a specific sequence number," if the peptide or GLP-2 has the same or corresponding activity as the peptide or GLP-2 that consists of the amino acid sequence of the sequence number, this 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, and it is self-evident that even if the peptide or GLP-2 has such additions or mutations, it falls within the scope of the present application.

[0130] Specifically, the GLP-2 derivative is represented by the general formula 1 or 2: (1) X 2 is glycine or Aib, or (2) X 30 is lysine or arginine, or (3) X 2 is glycine or Aib, and X 30 may be, but is not limited to, lysine or arginine.

[0131] Specifically, the GLP-2 derivative is represented by the general formula 1 or 2: (1)X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is cysteine ​​or (2)X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is lysine, (3) X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is arginine, and X 34 is lysine, (4) X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is lysine and X 34 is 6-azidolysine, (5)X 1 is imidazoacetyldeshistidine, and X 2 is glycine and X 30 is arginine, and X 34 is cysteine ​​or (6)X 1 is imidazoacetyldeshistidine, and X 2 is Aib and X 30 is lysine and X 34 is cysteine, or (7)X 1 is histidine, and X 2 is Aib and X 30 is lysine and X 34 may be, but is not limited to, cysteine.

[0132] In the present invention, such modifications for the production of agonists, fragments, variants and derivatives of native GLP-2 include all modifications using L- or D-amino acids, and / or non-natural amino acids; and / or modifications of the native sequence or by post-translational modifications (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonds, etc.).

[0133] In the present invention, the GLP-2 or GLP-2 derivative may be in a modified form in which its N-terminus and / or C-terminus are chemically modified or protected with an organic group in order to protect it from in vivo protein-cleaving enzymes and increase its stability, or the GLP-2 or GLP-2 derivative may be in a modified form in which amino acids are added to the termini, etc.

[0134] In particular, in the case of chemically synthesized peptides, since the N- and C-termini are charged, in order to remove such charges, the N-terminus may be acetylated and / or the C-terminus may be amidated, but is not limited thereto. Specifically, in the present invention, the C-terminus of GLP-2 or a GLP-2 derivative may be unmodified or amidated, but is not limited thereto.

[0135] In the present invention, the term "Tumor Necrosis Factor alpha (TNFα) inhibitor" collectively refers to a substance that reduces the activity of TNFα, and includes, without limitation, a substance that is used in combination therapy with GLP-2 or with GLP-2 and insulinotropic peptide, and can prevent, improve, or treat intestinal diseases compared to using either alone. Specifically, the TNFα inhibitor may be a substance that reduces the activity of TNFα by binding to TNFα or binding to a TNFα receptor, and ultimately interfering with the binding between TNFα and its receptor; or a substance that reduces the activity of TNFα by reducing cellular TNFα production, and such a substance is not limited to its form, such as a compound, a nucleic acid, or a peptide.

[0136] As a specific example, the TNFα inhibitor is a soluble TNF receptor (e.g., etanercept), an anti-TNFα antibody or a fragment thereof (e.g., infliximab, adalimumab, certolizumab pegol, golimumab), a compound (e.g., thalidomide and its derivatives (e.g., lenalidomide, pomalidomide); xanthine and its derivatives (e.g., pentoxifylline), bupropion; 5-HT 2AThe TNFα inhibitor may be, but is not limited to, an agonist (e.g., (R)-DOI ((R)-2,5-Dimethoxy-4-iodoamphetamine), TCB-2, LSD (Lysergic acid diethylamide), LA-SS-Az (Lysergic acid 2,4-dimethylazetidide)), or a combination thereof. In addition to such substances, substances known in the art to reduce the activity of TNFα may also be used as the TNFα inhibitor in the present application. Specifically, the TNFα inhibitor may be, but is not limited to, a soluble TNF receptor, an anti-TNFα antibody or a fragment thereof, or a combination thereof.

[0137] In the present invention, the insulinotropic peptide and / or GLP-2 may be in the form of a long-acting conjugate to which a biocompatible substance that increases the half-life in vivo is bound, but is not limited thereto. The long-acting conjugate may show an increased duration of efficacy compared to a derivative of insulinotropic peptide or GLP-2 to which a biocompatible substance (e.g., immunoglobulin Fc region) is not bound, and in the present invention, a conjugate containing insulinotropic peptide or GLP-2, whose half-life is increased by binding a biocompatible substance to insulinotropic peptide or GLP-2, is referred to as "insulinotropic peptide long-acting conjugate or insulinotropic peptide long-acting conjugate" or "GLP-2 long-acting conjugate or GLP-2 long-acting conjugate", respectively. In the present invention, the term "long-acting conjugate" is used interchangeably with "conjugate".

[0138] In one embodiment, (i) the insulinotropic peptide is in the form of a long-acting conjugate to which a biocompatible substance that increases its in vivo half-life is attached, (ii) the GLP-2 is in the form of a long-acting conjugate to which a biocompatible substance that increases its in vivo half-life is attached, or (iii) Each of the insulinotropic peptide and GLP-2 may be in the form of a long-acting conjugate to which a biocompatible substance that increases their in vivo half-life is bound.

[0139] Alternatively, such conjugates may be non-naturally occurring.

[0140] Furthermore, in the long-acting conjugate, the link between the insulinotropic peptide or GLP-2 and the biocompatible substance (e.g., the immunoglobulin Fc region) may be a physical or chemical bond, or a non-covalent or covalent bond, and specifically may be a covalent bond, but is not limited thereto.

[0141] Furthermore, the method for linking the insulinotropic peptide or GLP-2 to the biocompatible substance (e.g., the immunoglobulin Fc region) in the long-acting conjugate is not particularly limited, and the insulinotropic peptide or GLP-2 and the biocompatible substance (e.g., the immunoglobulin Fc region) may be linked to each other via a linker.

[0142] In addition, Korean Patent Publication No. 10-2019-0037181 relating to a long-acting conjugate of GLP-2 is incorporated herein by reference.

[0143] In one specific embodiment, the long-acting conjugate of insulinotropic peptide or GLP-2 of the present invention may have a structure represented by the following chemical formula (1), but is not limited thereto.

[0144] X-La-F···(1)

[0145] Where: X is an insulinotropic peptide or GLP-2; L is a linker (e.g., a linker containing ethylene glycol repeat units); a is 0 or a natural number, provided that when a is 2 or more, each L is independent of the others; F is a biocompatible substance that increases the in vivo half-life of X (e.g., an immunoglobulin Fc region); The "-" is a chemical bond (eg, a covalent bond).

[0146] 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 according to the procedure of chemical formula (1).

[0147] Furthermore, F may be directly linked to X (ie, a is 0 in the formula (1)) or may be linked to X via a linker (L).

[0148] In the conjugate according to the present invention, F is X, that is, a substance that increases the half-life of the insulinotropic peptide or GLP-2 according to the present invention, and corresponds to one constituent of the moiety that constitutes the conjugate according to the present invention.

[0149] The F may be bonded to X by a covalent or non-covalent chemical bond, and F and X may be bonded to each other through L by a covalent or non-covalent chemical bond or a combination thereof.

[0150] The substance F that increases the in vivo half-life of X is a biocompatible substance, and may be, for example, selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0151] In the case of elastin, it may be human tropoelastin, which is a water-soluble precursor, or it may be a polymer of a partial sequence or a partial repeating unit of these, including, for example, all cases of elastin-like polypeptides, but is not limited to these.

[0152] Examples of the polymer include a polymer selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof. The polysaccharide may include dextran, but is not particularly limited thereto.

[0153] As used herein, polyethylene glycol is a term that encompasses all forms of ethylene glycol homopolymer, PEG copolymer, and monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto.

[0154] The biocompatible material also includes, but is not limited to, polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid.

[0155] Furthermore, the fatty acid may be one that has a binding ability to albumin in vivo, but is not particularly limited thereto.

[0156] As an example of the F, the F may be an FcRn-binding substance, specifically, the FcRn-binding substance may be an immunoglobulin Fc region, more specifically, an IgG Fc region, more specifically, a non-glycosylated IgG4 Fc region, but is not particularly limited thereto.

[0157] As a specific example of the present invention, the F (e.g., an immunoglobulin Fc region) may be a dimer consisting of two polypeptide chains, and one end of L may be linked to only one of the two polypeptide chains, but is not limited thereto.

[0158] In one specific embodiment, the long-acting conjugate of the present invention may be, but is not limited to, an insulinotropic peptide or GLP-2 linked to an immunoglobulin Fc region.

[0159] In the present invention, the "immunoglobulin Fc region" refers to a heavy chain constant region excluding the heavy and light chain variable regions of an immunoglobulin. Specifically, the immunoglobulin Fc region may include a heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3) portion, and more specifically, may further include a hinge region (meaning the whole or a part of the hinge region). The immunoglobulin Fc region may be one of the components constituting the moiety of the conjugate of the present invention. Specifically, it corresponds to F in the above chemical formula (1).

[0160] As used herein, the term "Fc region" refers not only to the native sequence obtained by papain digestion of immunoglobulin, but also to derivatives, substitutions, and variants thereof, such as sequences that differ from the native sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native sequence, provided that the derivatives, substitutions, and variants retain the ability to bind to FcRn.

[0161] The F (e.g., immunoglobulin Fc region) may be a structure in which two polypeptide chains are linked by a disulfide bond, and may be linked only through a nitrogen atom of one of the two chains, but is not limited thereto. The linkage through the nitrogen atom may be linked to the epsilon-amino atom of lysine or the amino group at the N-terminus by reductive amination.

[0162] 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.

[0163] In one embodiment, the immunoglobulin Fc region may be linked via its N-terminal nitrogen atom.

[0164] Such an immunoglobulin Fc region may include a hinge portion in the heavy chain constant region, but is not limited thereto.

[0165] The immunoglobulin Fc region of the present invention may comprise a specific hinge sequence at the N-terminus.

[0166] 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.

[0167] The hinge sequence of the present invention may be a mutated hinge sequence having the following amino acid sequence, in which a portion of the hinge sequence is deleted to have only one cysteine ​​residue, but is not limited thereto:

[0168] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (sequence number 11).

[0169] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 11 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention is composed of 3 to 12 amino acids and contains 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: 12), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 13), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 14), G lu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 15), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 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: No. 19), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 20), Pro-Ser-Cys-Pro (SEQ ID NO: 21), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 22), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 23), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 24) 4), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (sequence number 25), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (sequence number 26), Glu-Ser-Lys-Pro-Ser-Cys-Pro (sequence number 27), Glu-Ser-Pro-Ser-Cys-Pro (sequence number 28), Glu-Pro-Ser-Cys (sequence number 29), Ser-Cys-Pro (sequence number 30).

[0170] More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 21 (Pro-Ser-Cys-Pro) or SEQ ID NO: 30 (Ser-Cys-Pro), but is not limited thereto.

[0171] In a more specific embodiment of the long-acting conjugate of the present invention, the N-terminus of the immunoglobulin Fc domain in the conjugate is proline, and the conjugate has the Fc domain linked to a linker via the nitrogen atom of the proline.

[0172] In one specific embodiment, the immunoglobulin Fc region may be in a dimeric form in which two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of the present invention represented by chemical formula (1) 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.

[0173] 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.

[0174] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes a part or the whole of 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 an immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one, or may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0175] 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 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), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0176] In addition, as one embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region may be in a dimeric form, and one molecule of X may be covalently linked to one Fc region in the dimeric form, and in this case, the immunoglobulin Fc and X may be linked to each other via one and the same linker. Meanwhile, two molecules of X may be symmetrically linked to one Fc region in the dimeric form. In this case, the immunoglobulin Fc and X may be linked to each other via a linker. However, the present invention is not limited to the above examples.

[0177] 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 refers to a sequence that differs from the naturally occurring amino acid sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof.

[0178] 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.

[0179] 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 of the native Fc, or adding a methionine residue to the N-terminus of the native Fc. Also, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, among others.

[0180] 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 by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0181] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have increased structural stability against heat, pH, and the like of the Fc region.

[0182] 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 Fc or pF'c can 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.

[0183] 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 with 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.

[0184] In the present invention, "deglycosylation" refers to an Fc region from which sugars have been removed from the enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, and in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0185] 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.

[0186] 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.

[0187] In addition, in one specific example, the immunoglobulin Fc region may be a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteines of each monomer as a fragment of human IgG4 Fc, and in this case, the homodimer has / may have disulfide bonds between cysteines at positions 35 and 95 and between cysteines at positions 141 and 199 in each monomer, i.e., two disulfide bonds (intra-chain form).

[0188] The number of amino acids in each monomer is 221 amino acids, and the amino acids forming the homodimer are 442 amino acids in total, but are not limited thereto. Specifically, the immunoglobulin Fc region may be a homodimer containing the amino acid sequence of SEQ ID NO: 35 (consisting of 442 amino acids) (wherein 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 cysteine ​​that is the third amino acid 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.

[0189] Meanwhile, in the present invention, the term "combination" related to an immunoglobulin Fc region means that, when a dimer or multimer is formed, 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. That is, a dimer or multimer can be produced from two or more Fc regions selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc regions.

[0190] In the present invention, the term "hybrid" means that a single-chain immunoglobulin constant region contains sequences corresponding to immunoglobulin Fc regions of two or more different origins. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of 1 to 4 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.

[0191] On the other hand, IgG can also be divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the present invention also allows for combinations or hybridization of these subclasses. Specifically, these are the IgG2 and IgG4 subclasses, and most specifically, the Fc region of IgG4, which has almost no effector functions such as complement dependent cytotoxicity (CDC).

[0192] Furthermore, the above-mentioned conjugate may have an increased duration of efficacy compared to native insulinotropic peptide or native GLP-2, or compared to X where F is not modified, and such conjugates include, but are not limited to, not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles, etc.

[0193] In the above chemical formula (1), the linker L may be a peptidic linker or a non-peptidic linker (for example, a linker containing an ethylene glycol repeating unit).

[0194] When L is a peptidic linker, it may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not limited thereto. In the present invention, various known peptide linkers can be used to link F and X, and examples thereof include [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, where x may be a natural number of 1 or more. However, it is not limited to the above examples.

[0195] In the present invention, the term "non-peptidic linker" includes a biocompatible polymer having two or more repeating units linked together. The repeating units are linked together through any covalent bond other than 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 formula (1).

[0196] The non-peptide linker that can be used in the present invention is not limited as long as it is a polymer that is resistant to in vivo protease degradation.

[0197] In the present invention, the non-peptide linker can be used in combination with a non-peptide polymer.

[0198] In addition, the non-peptide linker may be selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides (e.g., dextran, etc.), polyvinyl ethyl ether, PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof, but is not limited thereto.

[0199] Furthermore, the non-peptidyl linker that can be used in the present invention can be any polymer that is resistant to in vivo proteolytic enzymes, and specifically, the molecular weight of the non-peptidyl polymer is in the range of more than 0 to about 100 kDa, in the range of about 1 to about 100 kDa, specifically, in the range of about 1 to 50 kDa, in the range of about 1 to about 20 kDa, in the range of about 1 to about 10 kDa, or about 3.4 kDa to 10 kDa, but is not limited thereto.

[0200] 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.

[0201] 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 for preparing a conjugate at its end. 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.

[0202] Specifically, the linker may include a repeating unit represented by the following chemical formula (4), such as, but not limited to, polyethylene glycol (PEG):

[0203] JPEG2025513345000004.jpg3750...(4)

[0204] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

[0205] In the persistent conjugate, the PEG moiety is -(CH 2 CH 2 O) n - Not only the structure, but also the linking elements and their -(CH 2 CH 2 O) n It may also contain an oxygen atom between - and -, but is not limited thereto.

[0206] In a specific embodiment, the conjugate may have a structure in which the insulinotropic peptide or GLP-2 and the immunoglobulin Fc region (F) are covalently linked via a linker containing an ethylene glycol repeat unit, but is not limited thereto.

[0207] 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.

[0208] In one embodiment, the ethylene glycol repeat unit is, for example, [OCH 2 CH 2 ] n The value of n is a natural number, and the [OCH 2 CH 2 ] n The average molecular weight of the moiety, for example, the number average molecular weight, may be 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 ] n The average molecular weight of the site, 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 11 kDa. a, 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, about 3.4 kDa, or about 10 kDa, but is not limited thereto.

[0209] 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, etc., including all numerical values ​​in a range equal to or similar to the numerical value following the term about.

[0210] Furthermore, the non-peptide linker of the present invention to be bound to the immunoglobulin Fc region can be made of not only one type of polymer, but also a combination of different types of polymers.

[0211] In one specific embodiment, both ends of the non-peptidic linker can be bound to a thiol group, an amino group, or a hydroxyl group of an immunoglobulin Fc region and a thiol group, an amino group, an azide group, or a hydroxyl group of GLP-2, but are not limited thereto.

[0212] Specifically, the non-peptidic linker may contain reactive groups at both ends capable of binding to immunoglobulin Fc and insulinotropic peptide or GLP-2, 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 ​​of GLP-2; an amino group of lysine, arginine, glutamine and / or histidine; an azide group of azidolysine; and / or a hydroxyl group, but are not limited thereto.

[0213] More specifically, the reactive group of the non-peptidic polymer may be, but is not limited to, one or more selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative.

[0214] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0215] In the above, the succinimide derivative may be, but is not limited to, succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, or succinimidyl carbonate.

[0216] The non-peptidic linker can be linked to a biocompatible substance (eg, immunoglobulin Fc) and an insulinotropic peptide or GLP-2 through a reactive group as described above, and converted into a non-peptidic polymer linker.

[0217] Furthermore, the final products generated by reductive alkylation with aldehyde bonds are much more stable than those linked with amide bonds: the aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0218] The terminal reactive groups of the non-peptidic linker of the present invention may be the same or different from each other. The non-peptidic linker may have an aldehyde reactive group at its terminal, and the non-peptidic linker may have an aldehyde group and a maleimide reactive group at each terminal, or an aldehyde group and a succinimide reactive group at each terminal, but is not limited thereto.

[0219] 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.

[0220] When polyethylene glycol having a hydroxy reactive group at the propionyl end is used as the non-peptidic 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 produced using commercially available polyethylene glycol having a modified reactive group.

[0221] In one specific embodiment, the reactive group of the non-peptidic linker may be linked to a cysteine ​​residue of the insulinotropic peptide or GLP-2, more specifically, to the -SH group of cysteine, but is not limited thereto.

[0222] If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of insulinotropic peptide or GLP-2 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.

[0223] 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 A structure similar to that of NH-immunoglobulin Fc can be formed, and one end of PEG can be linked to GLP-2 or a sulfur atom located at a cysteine ​​of GLP-2 through a thioether bond. The above-mentioned thioether bond is It can contain the structure JPEG2025513345000005.jpg3936.

[0224] However, the present invention is not particularly limited to the above example, which is merely an example.

[0225] In the conjugate, the reactive group of the non-peptide linker is -NH 2 However, this is just one example.

[0226] In the conjugate, the insulinotropic peptide or GLP-2 may be linked to a non-peptide linker having a reactive group via the C-terminus, which is just one example.

[0227] 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 at which a non-peptidic polymer can be bound. 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, and specifically, the first to 20th amino acid residues from the most terminal.

[0228] As a specific example, the conjugate of the above chemical formula (1) may have a structure of the following chemical formula (2) or (3).

[0229] JPEG2025513345000006.jpg44130...(2)

[0230] JPEG2025513345000007.jpg1079...(3)

[0231] In the formula (2) or (3), X is a peptide of the formula (1) described above; F is the immunoglobulin Fc segment; n may be a natural number, and in this case, the explanation regarding n is as described above.

[0232] As a specific example, the long-acting conjugate of the chemical formula (2) may have a structure in which X of an insulinotropic peptide or GLP-2 and F of an immunoglobulin Fc domain are covalently linked via an ethylene glycol repeat moiety, where X is linked to a succinimide ring in the chemical formula (2) and F is linked to an oxypropylene group in the chemical formula (2). The long-acting conjugate of the chemical formula (3) may have a structure in which X of an insulinotropic peptide or GLP-2 and F of an immunoglobulin Fc domain are covalently linked via an ethylene glycol repeat moiety, where X is linked to an oxypropylene group in the chemical formula (3) and F is linked to another oxypropylene group in the chemical formula (3).

[0233] In the chemical formula (2) or (3), the value of n is the same as the value of [OCH 2 CH 2 The 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.

[0234] In one embodiment, the succinimide ring of formula (2) or the site at which X is attached to the succinimide ring of formula (2) may be the sulfur atom of the C-terminal cysteine ​​of X. Additionally, the oxypropylene group of formula (3) or the site at which X is attached to the oxypropylene group of formula (3) may be the sulfur atom of the C-terminal cysteine ​​of X.

[0235] The site in F that is linked to the oxypropylene group of formula (2) or formula (3) is not particularly limited. In one embodiment of the present invention, the site of F that is 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 one specific embodiment of the present invention, the site of F that is linked to the oxypropylene group of formula (2) or formula (3) may be, but is not limited to, the N-terminal proline of F.

[0236] On the other hand, the insulinotropic peptide, GLP-2, long-acting conjugates thereof, or TNFα inhibitors according to the present invention include all of the forms thereof, their salts (e.g., pharma- ceutically acceptable salts), or solvates thereof.

[0237] Furthermore, the insulinotropic peptide, GLP-2, long-acting conjugates thereof, or TNFα inhibitor may be in any pharma- ceutically acceptable form.

[0238] 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.

[0239] 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.

[0240] In addition, the term "solvate" as used in the present invention refers to a complex formed between the peptide, compound or salt thereof according to the present invention and a solvent molecule.

[0241] The natural insulinotropic peptides and modified insulinotropic peptides, GLP-2 and GLP-2 derivatives, and TNFα inhibitors of the present invention may be synthesized by solid phase synthesis or produced by recombinant methods, and may be commercially manufactured or may be available as commercially available products.

[0242] In another embodiment, the combination, pharmaceutical composition, or pharmaceutical kit of the present invention comprises: (i) containing GLP-2 and insulinotropic peptide, Contains GLP-2 and TNFα inhibitors Contains GLP-2, insulinotropic peptides, and TNFα inhibitors; (ii) GLP-2; and a long-acting insulinotropic peptide conjugate to which a biological substance that increases the in vivo half-life is attached; comprising GLP-2, the insulinotropic peptide long-acting conjugate, and a TNFα inhibitor; (iii) a long-acting conjugate of GLP-2 to which a biocompatible substance that increases the in vivo half-life is attached; and an insulinotropic peptide; The GLP-2 long-acting conjugate and the TNFα inhibitor are included, The composition comprises the long-acting GLP-2 conjugate, an insulinotropic peptide, and a TNFα inhibitor, (iv) a long-acting conjugate of GLP-2 conjugated to a biocompatible substance that increases its in vivo half-life; and a long-acting conjugate of an insulinotropic peptide conjugated to a biocompatible substance that increases its in vivo half-life, The composition may include the long-acting conjugate of GLP-2, the long-acting conjugate of insulinotropic peptide, and a TNFα inhibitor.

[0243] The combination, pharmaceutical composition or pharmaceutical kit of the present invention can be used for the prevention, amelioration or treatment of intestinal diseases.

[0244] In the present invention, the term "prevention" refers to any action that prevents or delays the onset of a target disease, such as intestinal disease, by administering a combination comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both; or a composition that combines GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both; and "treatment" refers to any action that improves or benefits the symptoms of a target disease, such as intestinal disease, by administering the combination or composition of the present invention. Also, "amelioration" refers to any action that at least reduces a parameter associated with the condition to be treated by administering the combination or composition of the present invention, such as the severity of the symptoms.

[0245] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition is not particularly limited, but may be any common route by which the GLP-2, insulinotropic peptide, and TNFα inhibitor can reach their target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0246] In the method of the present invention, the GLP-2, insulinotropic peptide, and TNFα inhibitor may be administered by the same administration route or by different administration routes, and the administration routes of the co-administered drugs may be independent of each other.

[0247] The GLP-2 and insulinotropic peptides, TNFα inhibitors, or both may be used in combination to prevent or treat intestinal disorders.

[0248] The intestinal disease may be at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal damage, but the intestinal diseases prevented, ameliorated, or treated by the composition of the present invention include, without limitation, any of the following.

[0249] As a specific example, the intestinal disease may be inflammatory bowel disease. The inflammatory bowel disease (IBD) is an inflammatory disease in which inflammation or ulcers occur in the gastrointestinal tract, and may be accompanied by symptoms such as increased expression of inflammatory cytokines, weight loss, shortened length of the colon, abdominal pain, fever, diarrhea, and / or bloody stool, and the symptoms may worsen and improve repeatedly, but is not limited thereto. Specifically, the inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, Crohn's disease, and Bechet's disease, but is not limited thereto.

[0250] The combination or composition according to the present invention, when administered to an individual, exhibits one or more of the following effects: inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration, and can prevent, ameliorate, or treat intestinal diseases, but is not limited thereto.

[0251] The combination or composition according to the present invention, when administered to an individual, can prevent, ameliorate or treat intestinal diseases by causing at least one of an increase in the length of the small intestine, a decrease in inflammation in the small intestine, an increase in the length of the large intestine and a decrease in inflammation in the large intestine, but is not limited thereto.

[0252] 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.

[0253] In the present invention, the term "pharmaceutical acceptable" means a sufficient amount to exhibit a therapeutic effect and to cause no side effects, and 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, sex, the patient's sensitivity to the drug, the administration route, the administration method, the number of administrations, the treatment period, and drugs used in combination or simultaneously. The pharmaceutical composition of the present invention may further contain a pharmaceutical acceptable excipient. The excipient is not particularly limited to the above, but may be a binder, a lubricant, a disintegrant, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., when administered orally, and may be used in the form of an injection, a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, etc., when administered topically.

[0254] The composition of the present invention may be prepared in various dosage forms by mixing with the above-mentioned pharma- ceutically acceptable excipients. 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.

[0255] 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. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, a preservative, etc.

[0256] Furthermore, 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.

[0257] Furthermore, the composition can be formulated into a single-dose formulation suitable for administration into the body of a patient, specifically, into a formulation form useful for administration of a protein pharmaceutical, by a conventional method in the pharmaceutical field, and administered orally or via a parenteral administration route, including, but not limited to, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method commonly used in the art.

[0258] Furthermore, the conjugate may be used by mixing with various medicament-acceptable carriers, such as physiological saline or organic solvents, and in order to improve stability and water absorption, 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 as drugs.

[0259] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease, etc. Specifically, the composition of the present invention may contain the insulinotropic peptide, GLP-2, and / or TNFα inhibitor in a pharmacologic effective amount, but is not limited thereto.

[0260] The inclusion of the insulinotropic peptide, GLP-2, and / or TNFα inhibitor in a pharmacologic effective amount means a level at which the insulinotropic peptide, GLP-2, and / or TNFα inhibitor can achieve the desired pharmacological activity, and also means a pharmacologic level at which no toxicity or side effects are caused in an individual to which the insulinotropic peptide, GLP-2, and / or TNFα inhibitor is administered, or a pharmacologic level at which the toxicity or side effects are negligible, but is not limited thereto. Such a pharmacologic effective amount can be determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0261] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01 to 99% by weight to volume.

[0262] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the peptide or conjugate of the present invention may be about 0.0001 mg to 500 mg per kg of patient body weight per day, but is not limited thereto. 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, food 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.

[0263] 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, but is not limited thereto.

[0264] One aspect of the present invention provides a food composition for preventing or ameliorating intestinal diseases, which contains GLP-2, and is characterized in that the food composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0265] The insulinotropic peptide, GLP-2, TNFα inhibitor, prevention, amelioration, and intestinal disease are as described above.

[0266] The food composition is used as a health functional food. When the composition of the present invention is used as a food supplement additive, the insulinotropic peptide, GLP-2, its long-acting conjugate, TNFα inhibitor, or a combination thereof can be added directly or together with other foods or food ingredients, and can be used in a conventional manner. The amount of the active ingredient mixed can be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

[0267] The term "health functional food" in the present invention refers to a food that is manufactured and processed for the purpose of health supplementation by using specific ingredients as raw materials or by extracting, concentrating, refining, mixing, etc. of specific ingredients contained in food raw materials, and refers to a food that is designed and processed so that the ingredients can fully exert their bioregulatory functions in the living body, such as biodefense, regulating biological rhythms, and preventing and recovering from diseases, and the health food composition can perform functions related to disease prevention and recovery, etc.

[0268] Another aspect embodying the present invention provides a method for preventing, ameliorating or treating intestinal disorders comprising administering GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both to an individual in need thereof.

[0269] Also, as one specific example, the present invention may be a method for preventing, ameliorating or treating an intestinal disease, comprising administering and / or using the combination, pharmaceutical composition or pharmaceutical kit of the present invention to an individual in need thereof.

[0270] As a specific example, there is provided a method for preventing, ameliorating or treating an intestinal disease, comprising the step of administering and / or using in combination a composition containing a pharma- ceutical amount of GLP-2, a composition containing a pharma- ceutical amount of an insulinotropic peptide, a composition containing a pharma- ceutical amount of a TNFα inhibitor, or both, to an individual in need thereof.

[0271] The insulinotropic peptide, GLP-2, TNFα inhibitor, combined use, prevention, treatment, amelioration, intestinal disease, combination, pharmaceutical composition and pharmaceutical kit are as described above.

[0272] The individual in the present invention is an individual suspected of having an intestinal disease, and the individual suspected of having an intestinal disease means a mammalian animal, including rats, including humans, and livestock, that has or is at risk of developing the disease, but includes, without limitation, an individual that can be treated with the combination of GLP-2, insulinotropic peptide, TNFα inhibitor, or both of the present invention. In addition, the individual can be effectively treated by administering the GLP-2, insulinotropic peptide, TNFα inhibitor, or both of the present invention to an individual suspected of having an intestinal disease. The intestinal disease is as described above.

[0273] The method of the present invention can include administering a pharmacologic effective amount of a combination or pharmaceutical composition comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both. The method of the present invention can include, but is not limited to, administering GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both in a single formulation, or administering separate formulations simultaneously, separately, sequentially, or in reverse order.

[0274] The appropriate total daily dosage will be 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, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors including the type and extent of the response to be achieved, the specific composition, including if other formulations are used, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and excretion rate of the composition, the duration of treatment, drugs used in conjunction with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0275] Another embodiment of the present invention is the use of the combination, pharmaceutical composition, or pharmaceutical kit for the prevention, amelioration, or treatment of an intestinal disease, and / or for the manufacture of a medicament for the prevention, amelioration, or treatment of an intestinal disease. The prevention, amelioration, treatment, intestinal disease, combination, pharmaceutical composition, and pharmaceutical kit are as described above.

[0276] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0277] Example 1: Preparation of long-acting conjugate of GLP-1 derivative 1-1. Preparation of CA-Exendin-4-PEG conjugate A PEGylation peptide conjugate (mono-PEGylated CA-Exendin-4) was prepared by covalently bonding the amine group of the 27th lysine residue in CA-Exendin-4 (N-[2-(1H-Imidazol-5-yl)acetyl]-Exendin-4, Hanmi Fine Chemicals, Korea) with polyethylene glycol (3.4 kDa, ALD(2) PEG, Hanmi Fine Chemicals, Korea) having aldehyde (ALD) groups at both ends. Specifically, the molar ratio of CA-Exendin-4:ALD(2) PEG was 1:5 to 1:15, and the concentration of CA-Exendin-4 was 6 to 12 mg / ml, and the reaction was carried out at 4 to 10°C or room temperature for about 4 to 12 hours. The reaction was carried out in 0.1M HEPES pH 7.0-8.5, approximately 45% isopropanol, and sodium cyanoborohydride (NaCNBH3) was added at a concentration of 2-5mM. The reaction solution was purified using a Source 15S (Cytiva, USA) column using a buffer containing sodium citrate (pH 2.0-3.5), approximately 45% ethanol, and a potassium chloride concentration gradient.

[0278] 1-2. Preparation of CA-Exendin-4-PEG-Fc conjugate To prepare the CA-Exendin-4-PEG-Fc conjugate, the immunoglobulin Fc fragment was added to the mono-PEGylated CA-Exendin-4 obtained in Example 1-1 to adjust the total protein concentration to 10-50 mg / ml, and the mixture was reacted at 4-10° C. or room temperature for about 12-17 hours. At this time, the reaction solution was 0.1 M potassium phosphate pH 5.5-8.5, and 2-50 mM sodium cyanoborohydride was added as a reducing agent. After the reaction was completed, the CA-Exendin-4-PEG-Fc conjugate in the reaction solution was purified using two types of columns: hydrophobic binding and anion exchange column. In the case of Source 15Phenyl (Cytiva, USA), a hydrophobic binding column, unreacted immunoglobulin Fc fragments were separated and purified using Tris-HCl (pH 7.5) buffer and a NaCl concentration gradient, and in the case of Source 15Q (Cytiva, USA), an anion exchange column, over-reacted impurities were removed using Tris-HCl (pH 7.5) buffer and a NaCl concentration gradient, and the CA-Exendin-4-PEG-Fc conjugate was separated and purified.

[0279] Example 2: Preparation of long-acting conjugate of GLP-2 derivative 2-1. Preparation of CA GLP-2 RK-PEG conjugate To pegylate the GLP-2 derivative CA GLP-2 RK of SEQ ID NO: 4 in Table 1 with modified polyethylene glycol ALD (2) PEG (modified polyethylene glycol in which hydrogen at both ends is replaced with a propionaldehyde group (3-oxopropyl group) and the chemical formula weight of the ethylene glycol repeating unit moiety is 3.4 kDa, NOF Japan), the molar ratio of the GLP-2 derivative to ALD (2) PEG was 1:5 to 1:20, the concentration of the GLP-2 derivative was 5 to 10 mg / ml, and the reaction was carried out at 2 to 8 ° C for 4 to 16 hours. At this time, the reaction was carried out in 20 mM HEPES (pH 7.5) and ethanol, and 20 mM sodium cyanoborohydride as a reducing agent was added to the reaction. The reaction solution was purified into a mono-PEGylated GLP-2 derivative using a Source 15S (GE, USA) column with a buffer solution containing sodium citrate (pH 2.0) and ethanol and a potassium chloride concentration gradient.

[0280] 2-2. Preparation of CA GLP-2 RK-PEG-Fc conjugate Next, the purified monopegylated GLP-2 derivative and the immunoglobulin Fc fragment were reacted at a molar ratio of 1:2 to 1:6 at a total protein concentration of 30 to 35 mg / mL for 12 to 20 hours at 2 to 8° C. At this time, the reaction solution was prepared by adding 20 mM sodium cyanoborohydride as a reducing agent to 100 mM potassium phosphate buffer (pH 6.0) and isopropanol.

[0281] After the reaction was terminated, the reaction solution was applied to a Source 15Q (GE, USA) column using a bis-Tris pH 6.5 buffer and a sodium chloride gradient, and then applied to a Source 15 ISO (GE, USA) using an ammonium sulfate and sodium citrate pH 5.0-5.2 gradient to purify the long-acting conjugate of the GLP-2 derivative, in which the GLP-2 derivative is covalently linked to the immunoglobulin Fc fragment via a polyethylene glycol linker.

[0282] Example 3: Confirmation of the effect of suppressing M1 polarization, macrophage differentiation, and monocyte migration in THP-1 cells (monocytes) through combined treatment with a long-acting conjugate of a GLP-1 derivative and a long-acting conjugate of a GLP-2 derivative In order to confirm the inhibitory effects of the long-acting conjugates of GLP-1 and GLP-2 derivatives, respectively, and in combination, on M1 polarization, macrophage differentiation, and monocyte migration in THP-1 cells (monocytes), in vitro experiments were performed as follows.

[0283] [Experiment to confirm M1 polarization inhibition] THP-1 cells were treated with long-acting conjugates of GLP-1 derivatives (0.5 or 1μM), long-acting conjugates of GLP-2 derivatives (10μM), and long-acting conjugates of GLP-1 derivatives (0.5μM) + long-acting conjugates of GLP-2 derivatives (10μM) for 4 hours. To induce inflammation, cells were treated with 1μg / mL of lipopolysaccharides (LPS) for 2 hours, and RNA was extracted to measure the mRNA expression levels of pro-inflammatory cytokines (M1 polarization), TNF-α, IL-1β, and IL-6, by qPCR.

[0284] [Macrophage differentiation inhibition confirmation experiment] THP-1 cells were treated with long-acting conjugates of GLP-1 derivatives (0.1 or 1 μM), long-acting conjugates of GLP-2 derivatives (10 μM), or long-acting conjugates of GLP-1 derivatives (0.1 μM) + long-acting conjugates of GLP-2 derivatives (10 μM) for 48 h with phorbol 12-myristate 13-acetate (PMA) to induce THP-1 differentiation, and the number of adherent cells, which were differentiated cells, was counted.

[0285] [Monocyte migration inhibition experiment] THP-1 cells were treated with a long-acting conjugate of GLP-1 derivatives (0.1 or 1 μM), a long-acting conjugate of GLP-2 derivatives (10 μM), or a long-acting conjugate of GLP-1 derivatives (0.1 μM) + a long-acting conjugate of GLP-2 derivatives (10 μM) for 48 hours. The drug-treated THP-1 cells were transferred to the upper chamber of a Boyden chamber, and then cultured for 4 hours with 50 ng / mL of CCL-2, which can induce migration to the bottom chamber. The degree of migration was measured using a migration assay kit (Abcam).

[0286] Through the above experiments, we confirmed that the long-acting GLP-1 derivative conjugate and the long-acting GLP-2 derivative conjugate each had inhibitory effects on M1 polarization (Fig. 1, A), macrophage differentiation (Fig. 1, B), and monocyte migration (Fig. 1, C), and that these effects were further amplified when administered in combination.

[0287] Example 4: Confirmation of increase in small intestinal length and reduction in small intestinal inflammation in rats with indomethacin-induced inflammatory bowel disease by combined administration of a long-acting conjugate of a GLP-1 derivative, a long-acting conjugate of a GLP-2 derivative, and anti-TNFα mAb To measure the small intestine length and inflammation improvement efficacy in vivo by the combined administration of a long-acting conjugate of a GLP-1 derivative, a long-acting conjugate of a GLP-2 derivative, and an anti-TNFα mAb (BioXcell, Catalog # BE0244), rats with inflammatory bowel disease induced by indomethacin (INN) were used. Specifically, 8-week-old male SD rats were divided into groups of 6 rats each as follows:

[0288] - G1: Normal control group (no drug administered, Vehicle) - G2: Indomethacin control group (Indomethacin 7.5mg / kg; Day 1 and Day 2 (D1,2)) - G3: Group administered with long-acting GLP-2 derivative conjugate (3.1 mg / kg / single) followed by indomethacin (GLP-2 derivative long-acting conjugate 3.1 mg / kg / single; day 0 (D0) + indomethacin 7.5 mg / kg; D1, 2) - G4: Group administered with long-acting GLP-1 derivative conjugate (0.178mg / kg / single) followed by indomethacin (GLP-1 derivative long-acting conjugate 0.178mg / kg / single; D0 + Indomethacin 7.5mg / kg; D1,2) - G5: Group administered GLP-2 derivative long-acting conjugate (3.1mg / kg / single) and GLP-1 derivative long-acting conjugate (0.178mg / kg / single), followed by indomethacin ([GLP-2 derivative long-acting conjugate 3.1mg / kg / single + GLP-1 derivative long-acting conjugate 0.178mg / kg / single; D0] + Indomethacin 7.5mg / kg, D1,2) - G6: Group administered with Anti-TNFαmAb (0.2mg / kg / single) followed by indomethacin (Anti-TNFαmAb 0.2mg / kg / single; D0 + Indomethacin 7.5mg / kg; D1,2) - G7: Group administered GLP-2 derivative long-acting conjugate (3.1mg / kg / single) and Anti-TNFαmAb (0.2mg / kg / single), followed by indomethacin ([GLP-2 derivative long-acting conjugate 3.1mg / kg / single + Anti-TNFαmAb 0.2mg / kg / single; D0] + Indomethacin 7.5mg / kg; D1,2) - G8: Group administered GLP-2 derivative long-acting conjugate (3.1mg / kg / single), GLP-1 derivative long-acting conjugate (0.178mg / kg / single) and Anti-TNFαmAb (0.2mg / kg / single), followed by indomethacin ([GLP-2 derivative long-acting conjugate 3.1mg / kg / single + GLP-1 derivative long-acting conjugate 0.178mg / kg / single + Anti-TNFαmAb 0.2mg / kg / single; D0] + Indomethacin 7.5mg / kg; D1,2)

[0289] The numerical values ​​shown in the present specification for the dosage of a long-acting GLP-1 derivative conjugate or a long-acting GLP-2 derivative conjugate are values ​​obtained by subtracting the mass of the polyethylene glycol linker portion from the total mass of the long-acting GLP-1 derivative conjugate or long-acting GLP-2 derivative conjugate used, i.e., values ​​expressed based on the sum of the masses of only the polypeptide portions.

[0290] On the third day, the length of the small intestine was measured through autopsy, and the ulcer area was measured through Evans blue staining.

[0291] Compared to the indomethacin control group (G2), the indomethacin group after co-administration of the long-acting GLP-2 derivative conjugate and the long-acting GLP-1 derivative conjugate (G5) showed an increase in small intestine length of +25cm (Fig. 2) and a decrease in inflammation area (ulcer area) of 6.7% (Fig. 3).Compared to the indomethacin control group (G2), the indomethacin group after co-administration of the long-acting GLP-2 derivative conjugate and the anti-TNFαmAb (G7) showed an increase in small intestine length of +12cm (Fig. 2) and a decrease in inflammation area of ​​3.5% (Fig. 3). Furthermore, compared with the indomethacin control group G2, the group G8 administered indomethacin after combined administration of a long-acting GLP-2 derivative conjugate or a long-acting GLP-1 derivative conjugate and anti-TNFα mAb showed an increase in small intestine length of +32 cm (similar to the normal control group, Figure 2) and a decrease in the area of ​​inflammation of 7.2% (similar to the normal control group, Figure 3).

[0292] Example 5: Confirmation of increase in colon length and decrease in ulcerative colitis activity index in mice with dextran sulfate sodium-induced ulcerative colitis by combined administration of a long-acting conjugate of a GLP-1 derivative and a long-acting conjugate of a GLP-2 derivative To measure the efficacy of coadministration of a long-acting GLP-1 derivative conjugate and a long-acting GLP-2 derivative conjugate on the improvement of in vivo large intestine length and disease activity index (DAI), mice with ulcerative colitis induced by dextran sulfate sodium (DSS) were used. Specifically, 7-week-old male mice (C57BL / 6) were divided into groups of 10 mice each as follows: DSS was mixed with drinking water at 2% and administered, and one cycle refers to drinking water containing DSS for 5 days and regular drinking water for 5 days. After a total of 3 cycles, the drug was administered, and the first day on which drinking water containing DSS was administered in the first cycle was designated as day 0.

[0293] - G1: Normal control group (no drug administered, Vehicle) - G2: DSS control group (2% DSS, 3 cycles) - G3: Group administered DSS followed by GLP-1 derivative long-acting conjugate (0.301 mg / kg / Q2D) (2% DSS, 3 cycles + GLP-1 derivative long-acting conjugate 0.301 mg / kg / Q2D, 2 weeks) - G4: Group administered DSS followed by GLP-2 derivative long-acting conjugate (1.757 mg / kg / Q2D) (2% DSS, 3 cycles + GLP-2 derivative long-acting conjugate 1.757 mg / kg / Q2D, 2 weeks) - G5: Group administered with DSS, followed by GLP-1 derivative long-acting conjugate (0.301 mg / kg / Q2D) and GLP-2 derivative long-acting conjugate (1.757 mg / kg / Q2D) (2% DSS, 3 cycles + [GLP-1 derivative long-acting conjugate 0.301 mg / kg / Q2D + GLP-2 derivative long-acting conjugate 1.757 mg / kg / Q2D], 2 weeks) - G6: Group administered with Cyclosporine A (20mg / kg / QD, PO) after DSS (2% DSS, 3 cycles + Cyclosporine A 20mg / kg / QD, 2 weeks)

[0294] Then, on the 44th day, the length of the colon was measured through autopsy to assess the improvement of the disease (Figure 4).

[0295] The ulcerative colitis activity index was measured by scoring stool consistency and bleeding status. Stool consistency was measured as follows: normal (0 points), visible to the naked eye but soft when pressed (1 point), not round and soft like normal stool (2 points), no consistency and very soft stool (3 points), wet around the anus due to diarrhea, watery stool (4 points). Bleeding status was measured as follows: no bleeding (0 points), blood visible when pressing stool (2 points), blood visible without pressing stool (3 points), severe bleeding from rectum (4 points). Measurements were taken on the 21st, 23rd, 25th, 28th, 30th, 32nd, 35th, 37th, 39th, 42nd, and 44th days, and the improvement of the disease was measured by adding up the scores (Figure 5).

[0296] Compared to the G2 DSS control group, the colon length increased by +0.8 cm (18%) in the G5 group administered with the long-acting GLP-1 and long-acting GLP-2 derivatives after DSS administration (similar to the normal control group, Fig. 4), and the ulcerative colitis activity index decreased by 10 (Fig. 5). In addition, compared to the G6 group administered with Cyclosporine A (20 mg / kg / QD), which is used in clinical practice for the severe stage immediately before surgery, the colon length increased by +1.4 cm (25%) in the G5 group administered with the long-acting GLP-1 and long-acting GLP-2 derivatives after DSS administration (similar to the normal control group, Fig. 4), and the ulcerative colitis activity index decreased by 6 (Fig. 5).

[0297] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A pharmaceutical composition for the prevention or treatment of intestinal disease containing a pharmaceutically effective amount of GLP-2, The pharmaceutical composition is characterized by being administered in combination with an insulin secretion peptide, a TNFα inhibitor, or both.

2. The pharmaceutical composition according to claim 1, wherein the insulin secretion peptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, their activators, derivatives, fragments, variants, and combinations thereof.

3. The pharmaceutical composition according to claim 1, wherein the insulin secretion peptide is an insulin secretion peptide derivative in which the N-terminal histidine residue of the insulin secretion peptide is substituted with imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine.

4. The pharmaceutical composition according to claim 1, wherein the insulin secretion peptide is natural exendin-4, an exendin-4 derivative from which the alpha carbon of the histidine residue which is the first amino acid at the N-terminus of exendin-4 and the N-terminal amino group attached to the alpha carbon have been removed, an exendin-4 derivative from which the N-terminal amino group of exendin-4 has been removed, an exendin-4 derivative from which the N-terminal amino group of exendin-4 has been replaced with a hydroxyl group, an exendin-4 derivative from which the N-terminal amino group of exendin-4 has been modified with two methyl groups, an exendin-4 derivative from which the N-terminal amino group of exendin-4 has been replaced with a carboxyl group, an exendin-4 derivative from which the twelfth amino acid (lysine) of exendin-4 has been replaced with serine, or an exendin-4 derivative from which the twelfth amino acid (lysine) of exendin-4 has been replaced with arginine.

5. The pharmaceutical composition according to claim 1, wherein the GLP-2 is natural GLP-2 or a GLP-2 derivative.

6. The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative is a GLP-2 derivative that has undergone a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof of at least one amino acid in the natural GLP-2 sequence.

7. The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative is modified in at least one amino acid among the amino acids at positions 1, 2, 30, and 33 in SEQ ID NO:

1.

8. The pharmaceutical composition according to claim 5, 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 No. 9) Here, X 1 These are histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is 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; However, the amino acid sequence of general formula 1 that is identical to sequence number 1 is excluded.

9. The GLP-2 derivative is (1) X 1 is imidazoacetyl deshistidine, X 2 is glycine, X 30 is lysine, X 34 is cysteine, or (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, X 2 Aib is X 30 It is ricin, X 34 The pharmaceutical composition according to claim 8, wherein is cysteine.

10. The pharmaceutical composition according to claim 5, 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 No. 10) Here, X 1 These are histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X 2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 It is lysine or arginine; X 34 is one or more arbitrary amino acids or one or more modified arbitrary amino acids; However, the amino acid sequence in general formula 2 that is identical to sequence number 1 is excluded.

11. The pharmaceutical composition according to claim 5, wherein the GLP-2 derivative is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

12. The pharmaceutical composition according to claim 1, wherein GLP-2 and insulin secretory peptide are administered in combination.

13. The pharmaceutical composition according to claim 1, wherein GLP-2 and a TNFα inhibitor are administered in combination.

14. The pharmaceutical composition according to claim 1, wherein GLP-2, insulin secretion peptide, and a TNFα inhibitor are administered in combination.

15. The pharmaceutical composition according to claim 1, wherein the TNFα inhibitor is a soluble TNF receptor, an anti-TNFα antibody or a fragment thereof, or a combination thereof.

16. The pharmaceutical composition according to claim 1, wherein the intestinal disease is at least one selected from the group consisting of irritable bowel disease, enteritis, inflammatory bowel disease, colitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal injury.

17. The pharmaceutical composition according to claim 16, wherein the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, Crohn's disease, and Behçet's disease.

18. The pharmaceutical composition according to claim 1, wherein, upon administration to an individual, the composition exhibits one or more of the following effects: inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration.

19. The pharmaceutically active composition according to claim 1, wherein, upon administration to an individual, it causes at least one of the following: an increase in the length of the small intestine, a decrease in inflammation of the small intestine, an increase in the length of the large intestine, and a decrease in inflammation of the large intestine.

20. The pharmaceutical composition according to claim 1, wherein the insulin secretion peptide and GLP-2 are either unmodified or amidated at their C-terminus.

21. (i) The insulin secretory peptide is in the form of a sustained-release conjugate to which a biocompatible substance that increases its in vivo half-life is attached, (ii) The GLP-2 is in the form of a sustained-release compound to which a biocompatible substance that increases its in vivo half-life is bound, (iii) The pharmaceutical composition according to claim 1, wherein each of the insulin secretion peptide and GLP-2 is in the form of a sustained-release conjugate to which a biocompatible substance that increases the in vivo half-life of the insulin secretion peptide is bound.

22. The compound is the pharmaceutical composition according to claim 21, represented by the following chemical formula (1): [Chemical 1] X-La-F...(1) Here, X is either an insulin secretory peptide or GLP-2; L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; F is the immunoglobulin Fc region; The aforementioned "-" indicates a covalent bond.

23. The pharmaceutical composition according to claim 22, wherein the immunoglobulin Fc region is a non-glycosylated IgG4 Fc region.

24. The pharmaceutical composition according to claim 22, wherein F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the polypeptide chains of the two polypeptide chains.

25. The pharmaceutical composition according to claim 22, wherein L is polyethylene glycol.

26. The pharmaceutical composition according to claim 22, wherein the chemical formula amount of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

27. The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

28. (i) GLP-2 and insulin secretory peptide; (ii) GLP-2 and TNFα inhibitor; or (iii) GLP-2, insulin secretory peptide, and TNFα inhibitor are administered in combination simultaneously, sequentially, or in reverse order, as described in any one of claims 1 to 27.

29. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for the prevention or treatment of intestinal disease.

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