Combination therapy of GLP-2 with insulinotropic peptide, TNF alpha inhibitor or both for preventing or treating bowel disease

Abstract

The present disclosure relates to combination therapies comprising GLP-2, and an insulinotropic peptide, a TNF [alpha] inhibitor, or both, for the prevention, amelioration or treatment of bowel disease.

Description

Technical Field

[0001] The present invention relates to a combination therapy for preventing or treating intestinal diseases by administering glucagon-like peptide 2 (GLP-2) in combination with an insulinotropic peptide, a TNFα inhibitor, or both. Background Art

[0002] Intestinal diseases include irritable bowel disease, enteritis, inflammatory bowel disease, enterocolitis, colitis, pancreatitis, ileitis, intestinal atrophy, intestinal damage, etc. Among them, inflammatory bowel disease (IBD) is an inflammatory disease characterized by inflammation or ulcers in the gastrointestinal tract, and can show symptoms such as increased expression of inflammatory cytokines, weight loss, shortened colon length, abdominal pain, fever, diarrhea and / or blood in the stool. These symptoms will repeatedly worsen and improve.

[0003] Glucagon-like peptide 1 (GLP-1), as a class of insulinotropic peptides, is an incretin hormone secreted by L cells in the ileum and colon. The main effect of GLP-1 is to increase insulin secretion. It causes glucose-dependent insulin secretion, thereby preventing hypoglycemia. Due to this feature, GLP-1 is used as a therapy for type 2 diabetes; however, due to its very short half-life in the blood (about 2 minutes), it has obvious limitations on drug development. Therefore, an exemplary GLP-1 agonist developed and marketed is exendin-4, which is a GLP-1 analog purified from the saliva of the glia monster lizard. It has higher physiological activity than GLP-1 and resistance to dipeptidyl peptidase-4 (DPP-IV), and a longer half-life of 2 to 4 hours compared to GLP-1 (US 5,424,286 A). However, sufficient duration of physiological activity cannot be expected only by increasing resistance to DPP-IV. For example, exendin-4 (exenatide) currently available on the market still needs to be administered to patients by injection twice a day, and side effects such as vomiting and nausea caused by its administration are still a significant burden on patients.

[0004] GLP-2 is a 33-amino acid peptide hormone produced by L cells in the small intestine in response to digested nutrients. GLP-2 triggers mucosal growth in the small and large intestines, promotes the growth of enterocytes and crypt cells, and inhibits apoptosis. In addition, GLP-2 increases the absorption of nutrients in the small intestine and reduces intestinal permeability. It inhibits gastric emptying and gastric acid secretion, increases intestinal blood flow, and relaxes intestinal smooth muscle.

[0005] TNFα is a cytokine that plays a central role in immune responses and is also involved in inflammatory responses. Abnormal regulation of TNFα is known to occur in various diseases. In order to inhibit such responses caused by TNFα, various TNFα inhibitors have been developed. Summary of the invention

[0006]

Technical issues

[0007] To date, the development of therapeutic agents for intestinal diseases using insulinotropic peptides, GLP-2 and / or TNFα inhibitors has been insufficient. Therefore, there is a need for the development of long-acting therapies.

[0008]

Technical solution

[0009] An object of the present invention is to provide a therapy for preventing, ameliorating or treating intestinal diseases by combining GLP-2 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, which comprises GLP-2, wherein the pharmaceutical composition is characterized by being administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0011] It is a further object of the present invention 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, improving or treating intestinal diseases, which comprises the combination.

[0013] Another object of the present invention is to provide a drug kit for preventing, improving or treating intestinal diseases, which comprises 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, improving or treating intestinal diseases, which comprises administering and / or using the 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, improving or treating intestinal diseases, which comprises administering to an individual in need thereof a composition containing a pharmaceutically effective amount of an insulinotropic peptide, a composition containing a pharmaceutically effective amount of a TNFα inhibitor, or a combination of both and / or using a composition containing a pharmaceutically effective amount of GLP-2.

[0016] Another object of the present invention is to provide the use of the combination, pharmaceutical composition or pharmaceutical kit for preventing, improving or treating intestinal diseases; and / or the use of the combination, pharmaceutical composition or pharmaceutical kit in the preparation of a medicament for preventing, improving or treating intestinal diseases.

[0017]

Beneficial effects

[0018] The combination therapy of the present invention comprising GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both provides enhanced effects compared to single therapies and can be effectively used for preventing, ameliorating, or treating intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are results demonstrating the inhibitory effects of combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulinotropic peptide (long-acting GLP-1 derivative conjugate) on M1 polarization (A), macrophage differentiation (B), and monocyte migration (C).

[0020] Figure 2 Shown are changes in small intestine length in an indomethacin (INN)-induced inflammatory bowel disease rat model after administration of GLP-2 (long-acting GLP-2 derivative conjugate) with an insulinotropic peptide (long-acting GLP-1 derivative conjugate), a TNFα inhibitor (anti-TNFα antibody), or both in combination.

[0021] Figure 3 Shown are changes in ulcer area in an indomethacin-induced inflammatory bowel disease rat model after administration of GLP-2 (long-acting GLP-2 derivative conjugate) with an insulinotropic peptide (long-acting GLP-1 derivative conjugate), a TNFα inhibitor (anti-TNFα antibody), or both in combination.

[0022] Figure 4 Shown are changes in colon length in a dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model following combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulinotropic peptide (long-acting GLP-1 derivative conjugate).

[0023] Figure 5 Shown is the disease activity index (DAI) in the DSS-induced ulcerative colitis mouse model following combined administration of GLP-2 (long-acting GLP-2 derivative conjugate) and insulinotropic peptide (long-acting GLP-1 derivative conjugate).

[0024] Description of the preferred embodiment

[0025] One aspect of the present invention provides a therapy for preventing, ameliorating or treating intestinal diseases by combining GLP-2 with an insulinotropic peptide, a TNFα inhibitor or both.

[0026] One aspect of the present invention provides a composition for preventing, improving or treating intestinal diseases, which comprises GLP-2, wherein the GLP-2 is characterized by being administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0027] In one embodiment, the present invention provides a pharmaceutical composition for preventing, improving or treating intestinal diseases, comprising a pharmaceutically effective amount of GLP-2, wherein the pharmaceutical composition is characterized by being administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0028] In another embodiment, the present invention provides a food composition for preventing or improving intestinal diseases, which comprises GLP-2, wherein the food composition is characterized by being administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0029] In the composition according to any of the preceding embodiments, the insulinotropic peptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, agents, derivatives, fragments and variants thereof, and combinations thereof.

[0030] In the composition according to any of the preceding embodiments, the insulinotropic peptide is an insulinotropic peptide derivative, wherein the N-terminal histidine residue of the insulinotropic peptide is substituted by imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine.

[0031] In the composition according to any of the foregoing embodiments, the insulinotropic peptide is natural exendin-4; an exendin-4 derivative in which the α carbon of the first amino acid at the N-terminus of exendin-4, i.e., the histidine residue, and the N-terminal amino group connected to the α carbon are removed; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is removed; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is substituted with a hydroxyl group; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is modified with two methyl groups; an exendin-4 derivative 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.

[0032] In the composition according to any of the preceding embodiments, the GLP-2 is native GLP-2 or a GLP-2 derivative.

[0033] In the composition according to any of the preceding embodiments, the GLP-2 derivative is a GLP-2 derivative in which at least one amino acid of the sequence of native GLP-2 has been modified by a modification selected from substitution, addition, deletion, chemical modification and a combination thereof.

[0034] In the composition according to any of the preceding embodiments, the GLP-2 derivative is a GLP-2 derivative in which at least one of the amino acids at positions 1, 2, 30 and 33 of SEQ ID NO: 1 is modified.

[0035] In the composition according to any one of the preceding embodiments, the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1:

[0036] [General formula 1]

[0037] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0038] X1 is histidine, imidazoacetyl deshitidine, deaminohistidine, β-hydroxyimidazopropionyl deshitidine, N-dimethylhistidine or β-carboxyimidazopropionyl deshitidine;

[0039] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0040] X 30 is lysine or arginine; and

[0041] X 34 absent or lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine;

[0042] The prerequisite is that among the amino acid sequences of general formula 1, sequences identical to SEQ ID NO: 1 are excluded.

[0043] In the composition according to any of the preceding embodiments, the GLP-2 derivative comprises an amino acid sequence represented by Formula 1, wherein:

[0044] (1) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is cysteine;

[0045] (2) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is lysine;

[0046] (3) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is lysine;

[0047] (4) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azidolysine;

[0048] (5) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is cysteine;

[0049] (6) X1 is imidazoacetyl desyl histidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine; or

[0050] (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It's cysteine.

[0051] In the composition according to any one of the aforementioned embodiments, the GLP-2 derivative comprises an amino acid sequence represented by the following Formula 2;

[0052] [General formula 2]

[0053] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 10)

[0054] in,

[0055] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0056] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0057] X 30 is lysine or arginine; and

[0058] X 34 is any one or more amino acids or any one or more amino acids modified therein;

[0059] The prerequisite is that among the amino acid sequences of general formula 2, the sequence identical to SEQ ID NO: 1 is excluded.

[0060] In the composition according to any one of the preceding embodiments, the GLP-2 derivative is a peptide selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2 to 8.

[0061] In the composition according to any of the preceding embodiments, GLP-2 is administered in combination with an insulinotropic peptide.

[0062] In the composition according to any of the preceding embodiments, GLP-2 is administered in combination with a TNFα inhibitor.

[0063] In the composition according to any of the preceding embodiments, GLP-2 is administered in combination with both an insulinotropic peptide and a TNFα inhibitor.

[0064] In the composition according to any of the preceding embodiments, the TNFα inhibitor is a soluble TNF receptor, an anti-TNFα inhibitor or a fragment thereof, or a combination thereof.

[0065] In the composition according to any one of the preceding embodiments, the intestinal disease is at least one selected from irritable bowel disease, enteritis, inflammatory bowel disease, enterocolitis, colitis, pancreatitis, ileitis, intestinal atrophy, and intestinal injury.

[0066] In the composition according to any one of the preceding embodiments, the inflammatory bowel disease is at least one selected from ulcerative colitis, Crohn's disease and Behcet's disease.

[0067] In the composition according to any one of the preceding embodiments, the composition causes at least one of inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration when administered to a subject.

[0068] In the composition according to any of the preceding embodiments, the composition, when administered to a subject, causes at least one of 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.

[0069] In the composition according to any of the preceding embodiments, in the insulinotropic peptide and GLP-2, the C-terminus is unmodified or is amidated.

[0070] In the composition according to any of the preceding embodiments,

[0071] (i) the insulinotropic peptide is in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of the insulinotropic peptide is combined with the long-acting conjugate;

[0072] (ii) GLP-2 is in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of GLP-2 is bound to the long-acting conjugate; or

[0073] (iii) The insulinotropic peptide and GLP-2 are each in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of the insulinotropic peptide and GLP-2, respectively, is bound to the long-acting conjugate.

[0074] In the composition according to any one of the preceding embodiments, the conjugate is represented by the following Formula 1:

[0075] [Formula 1]

[0076] X-La-F

[0077] in,

[0078] X is insulinotropic peptide or GLP-2;

[0079] L is a linker containing ethylene glycol repeating units;

[0080] a is 0 or a natural number, wherein when a is 2 or greater, each L is independent of each other;

[0081] F is an immunoglobulin Fc region; and

[0082] “-” represents a covalent bond.

[0083] In the composition according to any of the preceding embodiments, the immunoglobulin Fc region is an aglycosylated IgG4 Fc region.

[0084] In the composition according to any one of the preceding embodiments, F is a dimer composed of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0085] In the composition according to any of the preceding embodiments, L is polyethylene glycol.

[0086] In the composition according to any of the preceding embodiments, the formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 kDa to 100 kDa.

[0087] In the composition according to any of the preceding embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0088] In the composition according to any of the preceding embodiments, (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 simultaneously, sequentially or in reverse order.

[0089] Yet another aspect of the invention provides a combination comprising GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both.

[0090] Another aspect of the present invention provides a pharmaceutical composition for preventing, improving or treating intestinal diseases, which comprises a combination.

[0091] Another aspect of the present invention provides a drug kit for preventing, improving or treating intestinal diseases, which comprises GLP-2, and an insulinotropic peptide, a TNFα inhibitor or both.

[0092] Another aspect of the present invention provides a method for preventing, improving or treating intestinal diseases, which comprises administering and / or using a combination, a pharmaceutical composition or a pharmaceutical kit to an individual in need thereof.

[0093] Another aspect of the present invention is a method for preventing, improving or treating intestinal diseases, which comprises administering to an individual in need thereof a composition containing a pharmaceutically effective amount of an insulinotropic peptide, a composition containing a pharmaceutically effective amount of a TNFα inhibitor, or a combination of both and / or using a composition containing a pharmaceutically effective amount of GLP-2.

[0094] Another aspect of the present invention provides the use of a combination, a pharmaceutical composition or a drug kit for preventing, improving or treating intestinal diseases; and / or the use of a combination, a pharmaceutical composition or a drug kit in the preparation of a medicament for preventing, improving or treating intestinal diseases. DETAILED DESCRIPTION

[0095] Hereinafter, the present invention will be described in more detail.

[0096] At the same time, the description and implementation disclosed herein may also be applicable to other descriptions and other implementations, respectively. In other words, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it is not intended that the scope of the present invention be limited by the following specific description.

[0097] Furthermore, those skilled in the art will recognize or be able to ascertain using no more than routine experimentation numerous equivalents to the specific aspects of the invention described in this application. Additionally, such equivalents are intended to be encompassed by the present invention.

[0098] In the whole specification, the conventional single letter and three letter codes of naturally occurring amino acids are used, and the three letter codes commonly accepted for other amino acids such as α-aminoisobutyric acid (Aib) and 6-azidolysine (AZK) are also used. In addition, the amino acids referred to by the abbreviations herein are described according to the IUPAC-IUB nomenclature.

[0099] Alanine (Ala, A) Arginine (Arg, R)

[0100] Asparagine (Asn, N) Aspartic acid (Asp, D)

[0101] Cysteine ​​(Cys, C) Glutamic acid (Glu, E)

[0102] Glutamine (Gln, Q) Glycine (Gly, G)

[0103] Histidine (His, H) Isoleucine (Ile, I)

[0104] Leucine (Leu, L) Lysine (Lys, K)

[0105] Methionine (Met, M) Phenylalanine (Phe, F)

[0106] Proline (Pro, P) Serine (Ser, S)

[0107] Threonine (Thr, T) Tryptophan (Trp, W)

[0108] Tyrosine (Tyr, Y) Valine (Val, V)

[0109] One aspect of the present invention provides a pharmaceutical composition for preventing or treating intestinal diseases, which comprises GLP-2, wherein the pharmaceutical composition is characterized by being administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

[0110] In one embodiment of the present invention, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating intestinal diseases, which comprises a pharmaceutically effective amount of GLP-2, wherein the pharmaceutical composition is characterized in being administered in combination with an insulinotropic peptide (such as a pharmaceutically effective amount of an insulinotropic peptide), a TNFα inhibitor (such as a pharmaceutically effective amount of a TNFα inhibitor), or both, but is not limited thereto.

[0111] Another aspect of the present invention provides use of GLP-2 and insulinotropic peptide, TNFα inhibitor or a combination thereof in preventing, improving or treating intestinal diseases.

[0112] Another embodiment provides a pharmaceutical composition for preventing, improving or treating intestinal diseases, which comprises GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both.

[0113] Specifically, one aspect of the present invention provides a combination, a pharmaceutical composition or a drug kit, which includes GLP-2, and an insulinotropic peptide, a TNFα inhibitor or both. One embodiment provides a combination, a pharmaceutical composition or a drug kit for preventing, ameliorating or treating intestinal diseases.

[0114] As used herein, the term "combination" refers to the use of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both for combined administration, and can be understood to be synonymous with "combination use". This also includes the form of a pharmaceutical composition and a pharmaceutical kit in which GLP-2 is used in combination with an insulinotropic peptide, a TNFα inhibitor, or both, but is not limited thereto.

[0115] 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 administered in combination simultaneously, sequentially or in reverse order.

[0116] The combination can be:

[0117] i) a combination of GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both, administered as a mixture or in separate formulations;

[0118] ii) a combination of administering a pharmaceutical composition comprising GLP-2 simultaneously, sequentially or in reverse order with a pharmaceutical composition comprising an insulinotropic peptide, a pharmaceutical composition comprising a TNFα inhibitor, or a combination of both; or

[0119] iii) A combination in which a pharmaceutical composition comprising two substances selected from GLP-2, an insulinotropic peptide and a TNFα inhibitor is administered in combination with the remaining substances simultaneously, sequentially or in reverse order in the form of a mixture or separate preparations, but is not limited thereto.

[0120] The combination or composition may be:

[0121] a) (i) administering GLP-2 and an insulinotropic peptide as a mixture, (ii) administering GLP-2 and a TNFα inhibitor as a mixture, or (iii) administering GLP-2, an insulinotropic peptide, and a TNFα inhibitor as a mixture;

[0122] b) administering GLP-2 and an insulinotropic peptide in separate formulations, (ii) administering GLP-2 and a TNFα inhibitor in separate formulations, or (iii) administering GLP-2, an insulinotropic peptide, and a TNFα inhibitor in separate formulations; or

[0123] c) administering GLP-2 and an insulinotropic peptide as a mixture and a TNFα inhibitor in a single form, (ii) administering GLP-2 and a TNFα inhibitor as a mixture and an insulinotropic peptide in a single form, or (iii) administering an insulinotropic peptide and a TNFα inhibitor as a mixture and GLP-2 in a single form, but are not limited thereto.

[0124] In the case where they are in separate formulations as in b) above, (i) GLP-2 and the insulinotropic peptide; (ii) GLP-2 and a TNFα inhibitor; or (iii) GLP-2, an insulinotropic peptide, and a TNFα inhibitor may each be formulated together in a separate formulation or formulated into separate formulations, and these formulations may be administered simultaneously, separately, sequentially or in reverse order.

[0125] Furthermore, in the case where they are in separate preparations as in c) above, the mixture, and the insulinotropic peptide, GLP-2 or TNFα inhibitor may be formulated together individually in separate preparations or as separate preparations, and these preparations may be administered simultaneously, separately, sequentially or in reverse order.

[0126] As used herein, the terms "combination administration", "administered in combination" or "combination administration" not only mean simultaneous administration, but should be understood as an administration form in which GLP-2 and an insulinotropic peptide, a TNFα inhibitor or both act on a subject together so that each substance (insulinotropic peptide, GLP-2 and / or TNFα inhibitor) can function at a level equal to or higher than its original function. Therefore, when the term "combination use" is used herein, this should be understood that combined use refers to simultaneous, separate, sequential or reverse administration, and the order is not limited. When the administration is sequential administration, reverse administration or separate administration, the order of administration is not particularly limited, but the administration interval of the second component or subsequent components should be such that the beneficial effects of the combination are not lost.

[0127] As used herein, the term "composition comprising a combination" may be a combination containing GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both, and may also include the combination and have therapeutic uses, but is not limited thereto. For example, the composition can be used to prevent, improve, or treat intestinal diseases, but is not limited thereto. As used herein, the term "composition comprising a combination" can be used interchangeably with "composition".

[0128] The composition comprising the combination according to the present invention is used for the combined administration of GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both, and GLP-2 and the insulinotropic peptide, the TNFα inhibitor, or both can be prepared as a single preparation or as two or more separate preparations. Specifically, the composition can be a composition in which GLP-2 and an insulinotropic peptide, a TNFα inhibitor, or both are administered simultaneously, separately, sequentially, or in reverse order, but administration is not limited thereto.

[0129] As used herein, the term "kit" may include a combination or composition according to the present invention so as to administer GLP-2 in combination with an insulinotropic peptide, a TNFα inhibitor, or both. Specifically, the kit according to the present invention may include a) (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, formulated as 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 single formulation of GLP-2 and an insulinotropic peptide and a separate formulation of a TNFα inhibitor, (ii) a single formulation of GLP-2 and a TNFα inhibitor and a separate formulation of an insulinotropic peptide, or (iii) a single formulation of an insulinotropic peptide and a TNFα inhibitor and a separate formulation of GLP-2, and may further include substances required for the combined administration of two or more substances, but the components are not limited thereto.

[0130] The present invention demonstrates that the effect of preventing, improving or treating intestinal diseases is significantly enhanced when GLP and insulinotropic peptide, TNFα inhibitor or both are used in combination compared to when insulinotropic peptide, GLP-2 and TNFα inhibitor are used alone, and thus provides a method for using combination therapy.

[0131] "Insulinotropic peptide" refers to a peptide that has insulin secretion function and can stimulate the synthesis or expression of insulin in pancreatic beta cells. The insulinotropic peptide can be, for example, glucagon-like peptide-1 (GLP-1), exendin-3 or exendin-4, but is not limited to this. Insulinotropic peptides include not only natural insulinotropic peptides, but also their precursors, agonists, derivatives, fragments and variants, and also include long-acting conjugates bound to biocompatible substances that can increase their half-life in vivo. The insulinotropic peptide can be contained in a pharmaceutical composition in a pharmaceutically effective amount.

[0132] GLP-1 is a hormone secreted by the small intestine and generally promotes insulin biosynthesis and secretion, inhibits glucagon secretion, and promotes intracellular glucose uptake. In the small intestine, the 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 into the GLP-1 (7-37) form to become active GLP-1 (7-37). The amino acid sequence of GLP-1 (7-37) is as follows.

[0133] GLP-1(7-37):

[0134] HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G(SEQ ID NO:32)

[0135] Exendin-3 and exendin-4 are GLP-1 analogs or derivatives, consisting of 39 amino acids, have 53% amino acid sequence similarity to GLP-1, and correspond to insulinotropic peptides. The amino acid sequences of exendin-3 and exendin-4 are as follows.

[0136] Exendin-3:

[0137] HSDGT FTSDL SKQME EEAVR LFIEW LKNGGPSSGAPPPS(SEQ ID NO:33)

[0138] Exendin-4:

[0139] HGEGT FTSDL SKQME EEAVR LFIEW LKNGGPSSGA PPPS(SEQ ID NO:34)

[0140] The insulinotropic peptide derivative may have insulin secretion function and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with the natural insulinotropic peptide in the amino acid sequence, and / or may be in a form in which certain amino acid residue groups of the insulinotropic peptide can be chemically substituted (e.g., α-methylated and α-hydroxylated), removed (e.g., deaminated) or modified (e.g., N-methylated), but are not limited thereto.

[0141] In a specific embodiment, the insulinotropic peptide derivative of the present invention can be prepared by a method of removing the α-amino group of the N-terminal histidine, a method of replacing the N-terminal amino group with a hydroxyl group or a carboxyl group and synthesizing it, a method of removing the α-carbon of the N-terminal histidine and the N-terminal amino group bound to the α-carbon to leave only the imidazo-acetyl functional group, a method of modifying the N-terminal amino group with two methyl groups, etc. The insulinotropic peptide derivative prepared by these methods can be an insulinotropic peptide derivative in which the N-terminal histidine residue of the insulinotropic peptide is substituted with imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine.

[0142] In one embodiment, the insulinotropic peptide derivative may be a GLP-1 derivative, and the GLP-1 derivative includes exendin-3, exendin-4 or a derivative thereof, but is not limited thereto.

[0143] In addition, examples of the insulinotropic peptide derivative may be derivatives obtained by chemically modifying the N-terminal amino group or amino acid residue of exendin-4, and may be imidazoacetyl-deshistidyl-exendin-4 (CA-exendin-4) in which the α carbon of the first N-terminal amino acid histidine residue of exendin-4 and the N-terminal amino group bound to the α carbon are removed; desaminohistidyl exendin-4 (DA-exendin-4) in which the N-terminal amino group of exendin-4 is removed; β-hydroxyimidazopropionylhistidyl in which the N-terminal amino group of exendin-4 is substituted with a hydroxyl group -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 β-carboxyimidazole-propionyl-des-histidyl 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 twelfth amino acid (lysine) of exendin-4 is substituted with serine; or an exendin-4 derivative in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine, but are not limited thereto.

[0144] The insulinotropic peptide fragment refers to a form in which one or more amino acids are removed and / or added from the N-terminus or C-terminus of the natural insulinotropic peptide, and the added amino acids may also be amino acids that do not exist in nature (eg, D-amino acids).

[0145] An insulinotropic peptide variant refers to a peptide that differs from a natural insulinotropic peptide in one or more amino acids, exhibits at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity with a natural insulinotropic peptide, and has insulin secretion function.

[0146] An insulinotropic peptide agonist refers to a substance that exhibits the same biological activity as an insulinotropic peptide by binding to the in vivo receptor of the insulinotropic peptide regardless of the structure of the insulinotropic peptide.

[0147] The methods for preparing the derivatives, fragments, variants and agonists of the present invention can be used independently or in combination. For example, insulinotropic peptides in which one or more amino acids are different and the N-terminal amino acid residue is deaminated are also included in the present invention.

[0148] As used herein, the term "glucagon-like peptide-2" or "GLP-2" is an agonist of the glucagon-like peptide-2 receptor and may be in the form of a polypeptide or in the form of a long-acting conjugate conjugated to a biocompatible substance capable of increasing the in vivo half-life of the polypeptide, but is not limited thereto.

[0149] In the present invention, glucagon-like peptide-2 or GLP-2 is a concept that includes not only the same sequence as natural human GLP-2, but also GLP-2 derivatives and long-acting conjugates of biocompatible substances combined with natural GLP-2 or GLP-2 derivatives. GLP-2 can be contained in a composition or a separate composition in a pharmaceutically effective amount so as to be administered or used in combination with a pharmaceutical composition including an insulinotropic peptide.

[0150] The amino acid sequence of native GLP-2 is as follows.

[0151] GLP-2 (1-33):

[0152] HADGSFSDEMNTILDNLAARDFINWLIQTKITD(SEQ ID NO:1)

[0153] As used herein, the term "GLP-2 receptor agonist" refers to a substance that binds to the human glucagon-like peptide-2 receptor in vivo or isolated to exhibit physiological activities equivalent to or similar to natural GLP-2. For example, a GLP-2 agonist may include natural GLP-2 or a GLP-2 derivative.

[0154] As used herein, the term "GLP-2 derivative" includes peptides having one or more amino acid sequence differences compared to native GLP-2; peptides modified by modifying the sequence of native GLP-2; and / or mimetics of native GLP-2, which are similar to native GLP-2 and have the function of preventing, treating and / or improving intestinal diseases. Specifically, the GLP-2 derivative may be a derivative in which at least one or more amino acids in the sequence of native GLP-2 have undergone a modification selected from substitution, addition, deletion, modification and a combination thereof, but is not limited thereto.

[0155] The added amino acid may also be a non-natural amino acid (e.g., a D-amino acid), and substitution with a non-natural amino acid is possible in addition to the natural amino acid. 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 may also mean that certain groups of amino acid residues are chemically substituted (e.g., α-methylated, α-hydroxylated, and substituted with an azide group), removed (e.g., deaminized), and / or modified (e.g., N-methylated), together with the substitution, addition, or removal, or a combination thereof, of at least one amino acid, or independently of the substitution, addition, or removal, or a combination thereof of at least one amino acid; but is not limited thereto.

[0156] In a specific embodiment, the GLP-2 derivative of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with native GLP-2 in the amino acid sequence, and / or may be in a form in which certain groups of amino acid residues in GLP-2 are chemically substituted (e.g., α-methylated, α-hydroxylated and substituted with an azide group), removed (e.g., deaminated) and / or modified (e.g., N-methylated), but are not limited thereto.

[0157] In a specific embodiment, the N-terminal amino group of the GLP-2 of the present invention may be substituted, removed or modified, but is not limited thereto. In order to prevent binding to the N-terminus, which is an important site for the in vivo activity of GLP-2, during the preparation of the long-acting conjugate, the GLP-2 of the present invention may be prepared by a method of removing the α-amino group of the N-terminal histidine, a method of replacing the N-terminal amino group with a hydroxyl group or a carboxyl group and synthesizing, a method of removing the α-carbon of the N-terminal histidine and the N-terminal amino group bound to the α-carbon to leave only the imidazoacetyl functional group, a method of modifying the N-terminal amino group with two methyl groups, and the like.

[0158] Specifically, the GLP-2 derivative can be imidazoacetyl-deshistidyl-GLP-2 (CA-GLP-2) in which the α carbon of the first amino acid histidine residue at the N-terminus of GLP-2 and the N-terminal amino group bound to the α carbon are removed; deaminohistidyl GLP-2 (DA-GLP-2) in which the N-terminal amino group of GLP-2 is removed; β-hydroxyimidazopropionyl dehistidyl GLP-2 (HY-GLP-2) in which the N-terminal amino group of GLP-2 is substituted with a hydroxyl group; N-dimethylhistidyl GLP-2 (DM-GLP-2) in which the N-terminal amino group of GLP-2 is modified with two methyl groups; or β-carboxyimidazopropionyl-deshistidyl GLP-2 (CX-GLP-2) in which the N-terminal amino group of GLP-2 is substituted with a carboxyl group, but is not limited thereto. As a non-limiting example, the structure of the substance used to prepare the GLP-2 derivative is as follows.

[0159]

[0160] Herein, imidazoacetyl dehistidyl (dine), deaminohistidyl (dine), β-hydroxyimidazopropionyl dehistidyl (dine), N-dimethylhistidyl (dine) and β-carboxyimidazopropionyl dehistidyl (dine) may be used with the same meanings as imidazoacetyl, deamino-histidyl, β-hydroxy-imidazopropionyl, dimethyl-histidyl and β-carboxy-imidazopropionyl, respectively.

[0161] In a specific embodiment, the GLP-2 derivative may undergo modification of at least one amino acid among the amino acids at positions 1, 2, 30 and 33 in SEQ ID NO: 1, but is not limited thereto. Specifically, the modification may be a modification selected from substitution, addition, removal and modification of at least one amino acid and a combination thereof, in which case the added amino acid may also be a non-natural amino acid (e.g., D-type amino acid), and substitution with non-natural amino acids is also possible 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 may mean that certain groups of amino acid residues are chemically substituted (e.g., α-methylated, α-hydroxylated, substituted with an azide group), removed (e.g., deaminized) and / or modified (e.g., N-methylated), together with substitution, addition or removal or a combination thereof of at least one amino acid, or independently of substitution, addition or removal or a combination thereof of at least one amino acid, but is not limited thereto.

[0162] In a specific embodiment, the GLP-2 derivative may include an amino acid sequence represented by the following general formula 1, but is not limited thereto:

[0163] [General formula 1]

[0164] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0165] in,

[0166] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0167] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0168] X 30 is lysine or arginine; and

[0169] X 34 Absent, or lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine.

[0170] In another specific embodiment, the GLP-2 derivative may include an amino acid sequence represented by the following general formula 2, but is not limited thereto:

[0171] [General formula 2]

[0172] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX34 (SEQ ID NO: 10)

[0173] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0174] X2 is alanine, glycine or 2-aminoisobutyric acid (Aib);

[0175] X 30 is lysine or arginine; and

[0176] X 34 is one or more arbitrary amino acids or one or more arbitrary amino acids that undergo variation.

[0177] 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.

[0178] Furthermore, among the amino acid sequences represented by Formula 1 or Formula 2, the sequence identical to SEQ ID NO: 1 may be excluded from the GLP-2 derivative, but the GLP-2 derivative is not limited thereto.

[0179] Specifically, the GLP-2 derivative of the present invention may have a substitution of the second amino acid, i.e., alanine, of the natural GLP-2 with glycine or Aib (2-aminoisobutyric acid); a substitution of the 30th amino acid, i.e., lysine, with arginine; or a combination thereof, but not limited thereto. In addition, the GLP-2 derivative may also have a thiol group (e.g., cysteine), an amino group (e.g., lysine, arginine, glutamine, or histidine) or an azido group (e.g., 6-azidolysine) introduced to the C-terminus (e.g., the 33rd amino acid), but the GLP-2 derivative is not limited thereto.

[0180] When preparing a long-acting conjugate of a GLP-2 derivative, the binding occurs at the introduced group, thereby allowing the preparation of a GLP-2 conjugate with a selectively controlled binding position. Specifically, one end of the linker can be bound to a hydroxyl group, a thiol group, an amino group, or an azide group of the GLP-2 derivative, and the other end of the linker can be bound to a substance that can increase the half-life in vivo (e.g., an immunoglobulin Fc region). A thiol group, an amino group, or an azide group can be introduced by adding amino acids to GLP-2, but the introduction is not limited thereto. A thiol group can be introduced by adding cysteine ​​(C) to GLP-2; an amino group can be introduced by adding lysine (K), arginine (R), glutamine (Q), or histidine (H) to GLP-2; and an azide group can be introduced by adding 6-azidolysine (AZK) to GLP-2, but the introduction is not limited thereto.

[0181] Specifically, the GLP-2 derivative according to the present invention may have at least one residue, cysteine, lysine, arginine, glutamine, histidine or 6-azidolysine, but is not limited thereto.

[0182] Specifically, the GLP-2 derivative of the present invention includes the substitution of the second amino acid, i.e., alanine, of native GLP-2 with glycine and the introduction of a thiol group (e.g., cysteine) into the C-terminus, and more specifically, may include the α carbon of the first N-terminal amino acid histidine residue and the imidazoacetyl dehistidine in which the N-terminal amino group bound to the α carbon is removed, and may have, for example, an amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0183] Specifically, the GLP-2 derivative of the present invention may include the replacement of the second amino acid, i.e., alanine, of native GLP-2 with glycine and the introduction of an amino group (e.g., lysine) into the C-terminus, and more specifically, may include the α carbon of the first N-terminal amino acid histidine residue and the imidazoacetyl dehistidine in which the N-terminal amino group bound to the α carbon is removed, and may have, for example, an amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0184] Specifically, the GLP-2 derivative of the present invention may include substitution of the second amino acid, i.e., alanine, of native GLP-2 with glycine, substitution of the 30th amino acid, i.e., lysine, of native GLP-2 with arginine, and introduction of an amino group (e.g., lysine) into the C-terminus, and more specifically, may include an imidazoacetyl-dehistidine in which the α carbon of the first N-terminal amino acid histidine residue and the N-terminal amino group bound to the α carbon are removed, and may have, for example, an amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0185] Specifically, the GLP-2 derivative of the present invention may include substitution of the second amino acid, i.e., alanine, of native GLP-2 with glycine and introduction of an azido group (e.g., 6-azidolysine) into the C-terminus, and more specifically, may include an α carbon of the first N-terminal amino acid histidine residue and an imidazoacetyl-dehistidine in which the N-terminal amino group bound to the α carbon is removed, and may have, for example, an amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0186] Specifically, the GLP-2 derivative of the present invention may include substitution of the second amino acid, i.e., alanine, of native GLP-2 with glycine, substitution of the 30th amino acid, i.e., lysine, of native GLP-2 with arginine, and introduction of a thiol group (e.g., cysteine) into the C-terminus, and more specifically, may include an α carbon of the first N-terminal amino acid histidine residue and an imidazoacetyl dehistidine in which the N-terminal amino group bound to the α carbon is removed, and may have, for example, an amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0187] Specifically, the GLP-2 derivative of the present invention may include substitution of the second amino acid, i.e., alanine, of native GLP-2 with 2-aminoisobutyric acid and introduction of a thiol group (e.g., cysteine) into the C-terminus, and may have, for example, an amino acid sequence of SEQ ID NO: 8, and more specifically, the GLP-2 derivative may include an α carbon of the first N-terminal amino acid histidine residue and an imidazoacetyl-dehistidine in which the N-terminal amino group bound to the α carbon is removed, and may have, for example, an amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0188] The GLP-2 derivatives of SEQ ID NOs: 2 to 8 are shown in Table 1 below.

[0189] [Table 1]

[0190]

[0191] In Table 1, ca H indicates substitution of histidine by imidazoacetyl dehistidine; Aib indicates 2-aminoisobutyric acid; and AZK indicates 6-azido-L-lysine.

[0192] The GLP-2 derivative according to the present invention may be a peptide including the above-mentioned specific sequence, or may be a peptide (mainly) consisting of the above-mentioned specific sequence, but is not limited thereto.

[0193] At the same time, although the peptide or GLP-2 "composed of (consisting of) a specific sequence number" is described herein, this does not exclude the addition of meaningless sequences before and after the amino acid sequence of the corresponding sequence number, or naturally occurring mutations, or silent mutations thereof, as long as the peptide or GLP-2 exhibits the same or equivalent activity as the peptide or GLP-2 composed of the amino acid sequence of the corresponding sequence number, and it is obvious that the peptide or GLP-2 with such sequence additions or mutations also falls within the scope of the present application.

[0194] Specifically, the GLP-2 derivative can be a compound of Formula 1 or Formula 2, wherein (1) X2 can be glycine or Aib, (2) X 30 can be lysine or arginine, or (3) X2 can be glycine or Aib, and X 30 It may be lysine or arginine, but the GLP-2 derivative is not limited thereto.

[0195] Specifically, the GLP-2 derivative may be,

[0196] (1) X1 can be imidazoacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It can be cysteine;

[0197] (2) X1 can be imidazoacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It can be lysine;

[0198] (3) X1 can be imidazoacetyl desyl histidine, X2 can be glycine, X 30 can be arginine, and X 34 It can be lysine;

[0199] (4) X1 can be imidazoacetyl desyl histidine, X2 can be glycine, X 30 can be lysine, and X 34 It may be 6-azidolysine;

[0200] (5) X1 can be imidazoacetyl desyl histidine, X2 can be glycine, X 30 can be arginine, and X 34 It can be cysteine;

[0201] (6) X1 can be imidazoacetyl desyl histidine, X2 can be Aib, X 30 can be lysine, and X 34 may be cysteine; or

[0202] (7) X1 can be histidine, X2 can be Aib, X 30 can be lysine, and X 34 The GLP-2 derivative may be cysteine, but is not limited thereto.

[0203] In the present invention, these modifications for preparing agonists, fragments, variants and derivatives of natural GLP-2 include all modifications using L- or D-type amino acids and / or non-natural amino acids; and / or modifications due to modifications to the natural sequence or post-translational modifications (e.g., methylation, acetylation, ubiquitination and intramolecular covalent binding).

[0204] In the present invention, in order to resist in vivo proteolytic enzymes and improve its stability, the N-terminus and / or C-terminus of GLP-2 or a GLP-2 derivative may be chemically modified with an organic group, or additional amino acids may be added to the N-terminus of GLP-2 or a GLP-2 derivative.

[0205] Specifically, in the case of a chemically synthesized peptide, since the N-terminus and the C-terminus are charged, in order to remove these charges, the N-terminus may be acetylated and / or the C-terminus may be amidated, but the modification is not particularly limited thereto. Specifically, in the present invention, GLP-2 or a GLP-2 derivative may have its C-terminus unmodified or amidated, but is not limited thereto.

[0206] As used herein, the term "tumor necrosis factor alpha (TNFα) inhibitor" refers to any substance that reduces TNFα activity, and includes but is not limited to substances used in combination therapy with GLP-2 or with both GLP-2 and insulinotropic peptides, wherein the combination therapy is more effective in preventing, improving or treating intestinal diseases than when used alone. Specifically, a TNFα inhibitor can be a substance that reduces TNFα activity by binding to TNFα or TNFα receptors, thereby ultimately blocking the interaction between TNFα and its receptor; it can also be a substance that reduces TNFα activity by reducing TNFα production in cells, and the form of these substances is not limited, such as compounds, nucleic acids or peptides.

[0207] In a specific example, the TNFα inhibitor can be a soluble TNF receptor [e.g., etanercept], an anti-TNFα antibody or fragment thereof [e.g., infliximab, adalimumab, certolizumab pegol, and golimumab], a compound [e.g., thalidomide and its derivatives (e.g., lenalidomide and pomalidomide); xanthines and their derivatives (e.g., pentoxifylline); bupropion; 5-HT 2A Agonists (e.g., (R)-2,5-dimethoxy-4-iodoamphetamine ((R)-DOI), TCB-2, lysergic acid diethylamide (LSD), lysergic acid 2,4-dimethylazetidide (LA-SS-Az)) or combinations thereof, but not limited thereto. In addition to these substances, other substances known in the art that reduce TNFα activity can also be used as TNFα inhibitors in the present invention. Specifically, the TNFα inhibitor can be a soluble TNF receptor, an anti-TNFα antibody or a fragment thereof, or a combination thereof, but not limited thereto.

[0208] In the present invention, the insulinotropic peptide and / or GLP-2 may be in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of each of the insulinotropic peptide and / or GLP-2 is bound to the long-acting conjugate, but is not limited thereto. The long-acting conjugate may exhibit enhanced efficacy persistence compared to an insulinotropic peptide or GLP-2 derivative that is not bound to a biocompatible substance (e.g., an immunoglobulin Fc region). In the present invention, a conjugate containing an insulinotropic peptide or GLP-2 is referred to as a "long-acting conjugate of an insulinotropic peptide or a long-acting conjugate of an insulinotropic peptide" or "a long-acting conjugate of GLP-2 or a long-acting conjugate of GLP-2", and the long-acting conjugate has an increased half-life by binding a biocompatible substance to an insulinotropic peptide or GLP-2. In the present invention, a long-acting conjugate may be used interchangeably with a conjugate.

[0209] In one embodiment,

[0210] (i) the insulinotropic peptide may be in the form of a long-acting conjugate, to which a biocompatible substance capable of increasing the in vivo half-life of the insulinotropic peptide is bound,

[0211] (ii) GLP-2 may be in the form of a long-acting conjugate, to which a biocompatible substance capable of increasing the in vivo half-life of GLP-2 is bound; or

[0212] (iii) Each of the insulinotropic peptide and GLP-2 is in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of each of the insulinotropic peptide and GLP-2 is bound to the long-acting conjugate.

[0213] Also, these conjugates may be non-naturally occurring conjugates.

[0214] In addition, the linkage between the insulinotropic peptide or GLP-2 and the biocompatible substance (e.g., immunoglobulin Fc region) in the long-acting conjugate can be a physical bond or a chemical bond, or a non-covalent bond or a covalent bond, and specifically can be a covalent bond, but is not limited thereto.

[0215] In addition, there is no particular limitation on the method of connecting the insulinotropic peptide or GLP-2 and the biocompatible substance (e.g., immunoglobulin Fc region) in the long-acting conjugate, but the insulinotropic peptide or GLP-2 and the biocompatible substance (e.g., immunoglobulin Fc region) can be connected to each other through a linker.

[0216] In addition, Korean Patent Publication No. 10-2019-0037181 regarding a long-acting conjugate of GLP-2 is incorporated herein by reference.

[0217] In a specific embodiment, the long-acting conjugate of the insulinotropic peptide or GLP-2 of the present invention may have a structure represented by the following Formula 1, but is not limited thereto.

[0218] [Formula 1]

[0219] X-La-F

[0220] in,

[0221] X is insulinotropic peptide or GLP-2;

[0222] L is a linker (e.g., a linker comprising ethylene glycol repeating units);

[0223] a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others;

[0224] F is a biocompatible substance (e.g., an immunoglobulin Fc region) that can increase the in vivo half-life of X; and

[0225] "-" is a chemical bond (eg, a covalent bond).

[0226] More specifically, X and L and L and F may be linked to each other by a covalent bond, and specifically, the conjugate may be a conjugate in which X, L and F are linked to each other by a covalent bond in the order of Formula 1.

[0227] In addition, F can be directly connected to X (ie, a is 0 in Formula 1), or connected via a linker (L).

[0228] In the conjugate according to the invention, F is a substance capable of increasing the half-life of X, ie the insulinotropic peptide or GLP-2 according to the invention, and corresponds to a constituent part constituting the part of the conjugate of the invention.

[0229] F may be bonded to X via a covalent chemical bond or a non-covalent chemical bond, and F and X may also be bonded to each other via L via a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0230] F is a biocompatible substance capable of increasing the in vivo half-life of X, and may be selected from, for example, high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, FcRn binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, sugars, heparin and elastin, but is not particularly limited thereto.

[0231] The elastin may be human tropoelastin, which is a water-soluble precursor, may be a polymer of a certain sequence or a certain repeating unit thereof, and includes, for example, all elastin-like polypeptides, but is not particularly limited thereto.

[0232] Examples of high molecular weight polymers may include high molecular weight polymers selected from the following: polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyethoxylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, oligonucleotides and combinations thereof, and the polysaccharides may include dextran, but are not particularly limited thereto.

[0233] In the present specification, polyethylene glycol is a term encompassing all forms of ethylene glycol homopolymers, PEG copolymers or monomethyl-substituted PEG polymers (mPEG), but is not particularly limited thereto.

[0234] In addition, the biocompatible material also includes polyamino acids such as polylysine, polyaspartic acid, and polyglutamic acid, but is not limited thereto.

[0235] Furthermore, the fatty acid may be a fatty acid having a binding affinity to albumin in vivo, but is not particularly limited thereto.

[0236] As an example of F, F may be an FcRn-binding substance, and the FcRn-binding substance may specifically be an immunoglobulin Fc region, more specifically an IgG Fc region, and even more specifically an aglycosylated IgG4 Fc region, but F is not particularly limited thereto.

[0237] As a specific example of the present invention, F (eg, immunoglobulin Fc region) is a dimer composed of two polypeptide chains, and may have a structure in which one end of L is connected to only one of the two polypeptide chains, but is not limited thereto.

[0238] In a specific embodiment, the long-acting conjugate of the present invention may be a long-acting conjugate in which an insulinotropic peptide or GLP-2 is linked to an immunoglobulin Fc region, but is not limited thereto.

[0239] In the present invention, "immunoglobulin Fc region" means the heavy chain constant region excluding the heavy chain and light chain variable regions of the immunoglobulin. Specifically, the immunoglobulin Fc region may include a portion of the heavy chain constant region 2 (CH2) and / or a portion of the heavy chain constant region 3 (CH3), and more specifically may also include a hinge region (referring to all or part of the hinge region). The immunoglobulin Fc region may be a component of a portion of the conjugate of the present invention. Specifically, the immunoglobulin Fc region corresponds to F in Formula 1.

[0240] In this specification, the Fc region includes not only the native sequence obtained by papain digestion of immunoglobulin, but also its derivatives, substitutions and modifications, for example, one or more amino acid residues in the native sequence are changed by deletion, insertion, non-conservative or conservative substitution or a combination thereof to be different from the sequence of the native sequence. The premise of these derivatives, substitutions and modifications is to have the ability to bind to FcRn.

[0241] F (e.g., immunoglobulin Fc region) has a structure in which two polypeptide chains are linked by a disulfide bond, and may have a structure in which a bond is formed only through a nitrogen atom in one of the two chains, but is not limited thereto. The bond through the nitrogen atom may be linked by reductive amination of the ε-amino atom of lysine or the N-terminal amino group.

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

[0243] As a specific example, the immunoglobulin Fc region can be linked via its N-terminal nitrogen atom.

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

[0245] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

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

[0247] In the present invention, the hinge sequence may be mutated to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto:

[0248] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 11).

[0249] The hinge sequence may include only one cysteine ​​residue by deleting the cysteine ​​residue at position 8 or 11 in the hinge sequence of SEQ ID NO: 11. The hinge sequence of the present invention may consist of 3 to 12 amino acids and include only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (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), Glu-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). NO: 17), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 18), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 19), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 20), Pro-Ser-Cys-Pro (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), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO:25), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO:26), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO:27), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO:28), Glu-Pro-Ser-Cys (SEQ ID NO:29) and Ser-Cys-Pro (SEQ ID NO:29) NO:30).

[0250] More specifically, the hinge sequence may include 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.

[0251] In a more specific form of the long-acting conjugate of the present invention, the N-terminus of the immunoglobulin Fc region in the conjugate is proline, and the conjugate has the immunoglobulin Fc region connected to the linker via the nitrogen atom of the proline.

[0252] In one embodiment, the immunoglobulin Fc region may be in the form of a dimer, wherein the two chains of the immunoglobulin Fc region form a homodimer or a heterodimer due to the presence of the hinge sequence. The conjugate represented by Formula 1 of the present invention may be in the form of one end of the linker connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0253] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide, and may include the last residue at the amino terminus, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the last residue at the terminus. The immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.

[0254] In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region, which includes part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), and does not include the heavy chain and light chain variable regions of the immunoglobulin, and the precursor is that it has substantially the same or improved effects compared to the natural type. In addition, the immunoglobulin Fc region may also be a region in which a portion of a relatively long amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0255] For example, the immunoglobulin Fc region of the present invention can be 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 two or more of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and an immunoglobulin hinge region (or a portion of the hinge region), or 6) a dimer of each domain of the heavy chain constant region and the light chain constant region, but is not limited thereto.

[0256] In addition, as an embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region can be in the form of a dimer, and one molecule of X can be covalently linked to one Fc region in the form of a dimer. Specifically, the immunoglobulin Fc and X can be connected to each other through the same connector. At the same time, two X molecules can also be symmetrically connected to one Fc region in the form of a dimer. Specifically, the immunoglobulin Fc and X can be connected to each other through a connector. However, the immunoglobulin Fc region is not limited to the above examples.

[0257] In addition, the immunoglobulin Fc region of the present invention includes not only the natural amino acid sequence, but also its sequence derivatives. The amino acid sequence derivative refers to a sequence that differs from the natural amino acid sequence in one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution or a combination thereof.

[0258] For example, in 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, can be used as suitable sites for modification.

[0259] In addition, various types of derivatives can be obtained by removing sites capable of forming disulfide bonds, removing several amino acids at the N-terminus of the native Fc, or adding a methionine residue to the N-terminus of the native Fc. In addition, in order to eliminate effector functions, complement binding sites (e.g., C1q binding sites) can also be removed, and antibody-dependent cell-mediated cytotoxicity (ADCC) sites can also be removed. International Patent Application Publication No. WO 97 / 34631, International Patent Application Publication No. 96 / 32478, etc. disclose such techniques for preparing sequence derivatives of immunoglobulin Fc regions.

[0260] It is known in the art that amino acid exchanges in proteins and peptides do not substantially alter the activity of the molecule (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, amino acids may be modified by phosphorylation, sulfation, acrylate, glycosylation, methylation, farnesylation, acetylation and amidation.

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

[0262] In addition, such an Fc region can also be obtained from a natural type isolated in vivo from an animal such as a human, a cow, a goat, a pig, a mouse, a rabbit, a hamster, a rat, a guinea pig or the like, and Fc can also be a recombinant obtained from a transformed animal cell or a microorganism or a derivative thereof. Specifically, the method for obtaining the Fc region from the natural type can be a method for obtaining the Fc region by isolating the whole immunoglobulin from a living body of a human or an animal and then treating the immunoglobulin with a protease. When treated with papain, the immunoglobulin is cleaved into Fab and Fc, and when treated with pepsin, the immunoglobulin is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated therefrom by size exclusion chromatography or a similar method. In a more specific embodiment, the Fc region is a recombinant immunoglobulin Fc region, wherein the human-derived Fc region is obtained from a microorganism.

[0263] In addition, the immunoglobulin Fc region can be a form of native sugar chains, sugar chains increased compared to the native type, sugar chains reduced compared to the native type, or sugar chains removed. Conventional methods using microorganisms such as chemical methods, enzymatic methods, and genetic engineering methods can be used to increase, reduce, or remove immunoglobulin Fc sugar chains. Specifically, the immunoglobulin Fc region from which sugar chains are removed from Fc has a significantly reduced ability to bind complement (c1q), has reduced or eliminated antibody-dependent cellular toxicity or complement-dependent cellular toxicity, and therefore does not induce unnecessary immune responses in the body. In this regard, a form more suitable for its original purpose as a drug carrier is a deglycosylated or non-glycosylated immunoglobulin Fc region.

[0264] In the present invention, the term "deglycosylated" refers to an Fc region from which sugars are removed by an enzyme, and the term "non-glycosylated" refers to an Fc region produced by a prokaryotic animal, more specifically E. coli, and not glycosylated.

[0265] Meanwhile, the immunoglobulin Fc region may be of human origin or animal origin such as cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig or the like, and in a more specific embodiment is of human origin.

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

[0267] In addition, as one embodiment, the immunoglobulin Fc region is a fragment of human IgG4 Fc, and may be a homodimer in which two monomers are linked by a disulfide bond (interchain form) between the amino acid cysteine ​​at position 3 of each monomer, and specifically, the homodimer has / can have a disulfide bond between the cysteines at positions 35 and 95 and has / can have a disulfide bond between the cysteines at positions 141 and 199, i.e., two disulfide bonds (intrachain form) in each monomer.

[0268] The number of amino acids in each monomer may be 221, and the amino acids forming the homodimer may be 442 amino acids in total, but is not limited thereto. Specifically, the immunoglobulin Fc region may be a homodimer comprising an amino acid sequence of SEQ ID NO: 35 (consisting of 442 amino acids) (specifically, two monomers having an amino acid sequence of SEQ ID NO: 31 (consisting of 221 amino acids) may form a homodimer through a disulfide bond between the 3rd amino acid cysteine ​​as each monomer, and the monomers of the homodimer may each independently form an intramolecular disulfide bond between the cysteines at positions 35 and 95 and an intramolecular disulfide bond between the cysteines at positions 141 and 199), but is not limited thereto.

[0269] Meanwhile, in the present invention, the term "combination" related to the 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. In other words, the dimer or multimer can be prepared from two or more Fc regions selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc regions.

[0270] In the present invention, the term "hybrid" refers to the presence of sequences corresponding to immunoglobulin Fc regions from two or more different sources in a single-chain immunoglobulin constant region. In the present invention, a variety of hybrids are possible. In other words, the hybrid of the domains may consist of one to four domains selected from CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include a hinge.

[0271] Meanwhile, IgG can also be divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and in the present invention, combinations thereof or hybrids thereof can also be used. Specifically, the immunoglobulin Fc region is IgG2 and IgG4 subclasses, and most specifically, the Fc region of IgG4 - which has almost no effector functions such as complement dependent cytotoxicity (CDC).

[0272] Compared to native insulinotropic peptide, native GLP-2, or compared to X not modified with F, the above conjugates can exhibit enhanced efficacy durability, and such conjugates include not only the above forms but also all forms encapsulated in biodegradable nanoparticles, but are not limited thereto.

[0273] In Formula 1, the linker L may be a peptide linker or a non-peptide linker (eg, a linker comprising ethylene glycol repeating units).

[0274] When L is a peptide connector, the peptide connector may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not particularly limited thereto. In the present invention, various known peptide connectors can be used to connect F and X, and examples include [GS]x connectors, [GGGS]x connectors, and [GGGGS]x connectors, where x can be a natural number of 1 or greater. However, the peptide connector is not limited to these examples.

[0275] In the present invention, the term "non-peptide linker" includes a biocompatible polymer that combines two or more repeating units. These repeating units are connected to each other by any covalent bond that is not a peptide bond. The non-peptide linker can be a component of a part of the conjugate of the present invention and corresponds to L in Formula 1.

[0276] The non-peptide linker used in the present invention can be, without limitation, any polymer that is resistant to proteolytic enzymes in vivo.

[0277] In the present invention, non-peptide linker can be used interchangeably with non-peptide polymer.

[0278] In addition, although not particularly limited, the non-peptide linker can be selected from polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyethoxylated polyols, polyvinyl alcohol, polysaccharides (e.g., dextran), polyvinyl ethyl ether, biodegradable polymers such as polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipopolymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0279] In addition, the non-peptide linker that can be used in the present invention may be, but is not limited to, any polymer that is resistant to proteases in vivo, but specifically, the molecular weight of the non-peptide polymer is in the range of greater than 0 kDa to about 100 kDa, about 1 kDa to about 100 kDa, specifically in the range of about 1 kDa to about 50 kDa, about 1 kDa to about 20 kDa, about 1 kDa to about 10 kDa, or about 3.4 kDa to 10 kDa, but is not limited thereto.

[0280] Although not particularly limited, the non-peptide linker may be a linker comprising ethylene glycol repeating units, for example, polyethylene glycol and derivatives thereof known in the art and derivatives that can be easily prepared at the level of skill in the art are also included within the scope of the present invention.

[0281] The repeating unit of the non-peptide connector may be an ethylene glycol repeating unit. Specifically, the non-peptide connector may include an ethylene glycol repeating unit and include a functional group for preparing a conjugate at its end. The long-acting conjugate according to the present invention may be in the form of X and F connected by the functional group, but is not limited thereto. In the present invention, the non-peptide connector may include two or three or more functional groups, and the corresponding functional groups may be the same or different from each other, but are not limited thereto.

[0282] Specifically, the linker may include a repeating unit represented by the following Formula 4. Examples thereof include polyethylene glycol (PEG), but are not limited thereto:

[0283] [Formula 4]

[0284]

[0285] Wherein, n=10 to 2400, n=10 to 480, or n=50 to 250, but not limited thereto.

[0286] In the long-acting conjugate, the PEG moiety may include not only -(CH2CH2O) n - structure, including between the connecting element and -(CH2CH2O) n - oxygen atoms between, but not limited thereto.

[0287] Furthermore, in a specific embodiment, the conjugate may be a structure in which the insulinotropic peptide or GLP-2 is covalently linked to the immunoglobulin Fc region (F) via a linker containing ethylene glycol repeating units, but is not limited thereto.

[0288] The term "polyethylene glycol" encompasses all forms of ethylene glycol homopolymers, PEG copolymers, and monomethyl-substituted PEG polymers (mPEG), but is not particularly limited thereto.

[0289] In one embodiment, the ethylene glycol repeating unit may be composed of, for example, [OCH2CH2] n , where n is a natural number and can be determined so that [OCH2CH2] n The average molecular weight (e.g., number average molecular weight) of the moiety is greater than 0 kDa to about 100 kDa, but is not limited thereto. As another example, the value of n is a natural number, and the peptide conjugate [OCH2CH2] n The average molecular weight (e.g., number average molecular weight) of the moiety can be from about 1 kDa to about 100 kDa, from about 1 kDa to about 80 kDa, from about 1 kDa to about 50 kDa, from about 1 kDa to about 30 kDa, from about 1 kDa to about 25 kDa, from about 1 kDa to about 20 kDa, from about 1 kDa to about 15 kDa, from about 1 kDa to about 13 kDa, from about 1 kDa to about 11 kDa, from about 1 kDa to about 10 kDa, from about 1 kDa to about 8 kDa, from about 1 kDa to about 5 kDa, from about 1 kDa to about 3.4 kDa, from about 3 kDa to about 30 kDa, from about 3 kDa to about 27 kDa, from about 3 kDa to about 25 kDa, from about 3 kDa to about 22 kDa, from about 3 kDa to about 20 kDa, from about 3 kDa to about 18 kDa, from about 3 kDa to about 16 kDa, from about 3 kDa to about 15 kDa, from about 3 kDa to about 13 kDa, from about 3 kDa to about 11 kDa, about 3 kDa to about 10 kDa, about 3 kDa to about 8 kDa, about 3 kDa to about 5 kDa, about 3 kDa to about 3.4 kDa, about 8 kDa to about 30 kDa, about 8 kDa to about 27 kDa, about 8 kDa to about 25 kDa, about 8 kDa to about 22 kDa, about 8 kDa to about 20 kDa, about 8 kDa to about 18 kDa, about 8 kDa to about 16 kDa, about 8 kDa to about 15 kDa, about 8 kDa to about 13 kDa, about 8 kDa to about 11 kDa, about 8 kDa to about 10 kDa, about 9 kDa to about 15 kDa, about 9 kDa to about 14 kDa, about 9 kDa to about 13 kDa, about 9 kDa to about 12 kDa, about 9 kDa to about 11 kDa, about 9.5 kDa to about 10.5 kDa, about 3.4 kDa or about 10 kDa, but is not limited thereto.

[0290] As used herein, the term "about" includes all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​that are equal to or similar to the numerical value following the term "about", but is not limited thereto.

[0291] Furthermore, the non-peptide linker of the present invention that binds to the immunoglobulin Fc region may be not only one type of polymer but also a combination of different types of polymers.

[0292] In a specific embodiment, both ends of the non-peptide linker can be linked to the thiol group, amino group or hydroxyl group of the immunoglobulin Fc region, and to the thiol group, amino group, azide group or hydroxyl group of GLP-2, but are not limited thereto.

[0293] Specifically, the non-peptide linker may include reactive groups connected to the immunoglobulin Fc and the insulinotropic peptide or GLP-2 at both ends, specifically, a reactive group capable of binding to the thiol group of cysteine; an amino group located at the N-terminus of lysine, arginine, glutamine and / or histidine; and / or a hydroxyl group located at the C-terminus of the immunoglobulin Fc region and capable of binding to the thiol group of cysteine; an amino group of lysine, arginine, glutamine and / or histidine; an azide group of azidolysine; and / or a hydroxyl group in GLP-2, but is not limited thereto.

[0294] More specifically, the reactive group of the non-peptide polymer may be one or more selected from an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0295] In the above, examples of the aldehyde group include a propionaldehyde group or a butyraldehyde group, but are not limited thereto.

[0296] In the above, the succinimide derivative may be succinimidyl carboxymethyl, succinimidyl pentanoate, succinimidyl methylbutyrate, succinimidyl methylpropionate, succinimidyl butyrate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl or succinimidyl carbonate, but is not limited thereto.

[0297] The non-peptide linker can be converted into a non-peptide polymer linkage by linking a biocompatible substance (eg, immunoglobulin Fc) to the insulinotropic peptide or GLP-2 via a reactive group as described above.

[0298] In addition, the final product produced by reductive alkylation via an aldehyde bond is more stable than the final product produced by amide bond ligation. At low pH, the aldehyde reactive group selectively reacts with the N-terminus, while at high pH (e.g., at pH 9.0), it can form a covalent bond with a lysine residue.

[0299] The terminal reactive groups of the non-peptide linker of the present invention may be the same or different from each other. The non-peptide linker may have an aldehyde reactive group at the end, and in addition, the non-peptide linker may have an aldehyde group and a maleimide reactive group at each end, or may have an aldehyde group and a succinimide reactive group at each end, but is not limited thereto.

[0300] For example, the non-peptide linker may have a maleimide group at one end and an aldehyde group, propionaldehyde group or butyraldehyde group at the other end. In another example, the non-peptide linker may have a succinimide group at one end and a propionaldehyde group or butyraldehyde group at the other end.

[0301] In the case of using polyethylene glycol having a hydroxyl group at the propionic acid end as a non-peptide linker, the conjugate of the present invention can be prepared by activating the hydroxyl group into various reactive groups by known chemical reactions, or using commercially available polyethylene glycol having a modified reactive group.

[0302] In a specific embodiment, the reactive group of the non-peptide linker can be linked to a cysteine ​​residue in the insulinotropic peptide or GLP-2, more specifically, to a -SH group of cysteine, but is not limited thereto.

[0303] If maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group in the insulinotropic peptide or GLP-2 through a thioether bond, and the aldehyde group can be linked to the -NH2 group in the immunoglobulin Fc through a reductive alkylation reaction, but the non-peptide linker is not limited to this, and this corresponds to an example.

[0304] Through this reductive alkylation, the N-terminal amino group of the immunoglobulin Fc region can be connected to the oxygen atom at one end of the PEG through a linker functional group having a -CH2CH2CH2- structure to form a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and the structure in which the PEG end is connected to the sulfur atom located in GLP-2 or at the cysteine ​​of GLP-2 can be formed through a thioether bond. The above-mentioned thioether bond may include the following structure:

[0305]

[0306] However, the non-peptide linker is not particularly limited to the above-mentioned examples, but this corresponds to one example.

[0307] In addition, in the conjugate, the reactive group of the non-peptide linker can also be linked to -NH2 located at the N-terminus of the Fc region of an immunoglobulin, and this corresponds to an example.

[0308] Furthermore, in the conjugate, the insulinotropic peptide or GLP-2 can also be linked to a non-peptide linker having a reactive group via the C-terminus, and this corresponds to an example.

[0309] In the present invention, the term "C-terminus" refers to the carboxyl end of a peptide, and for the purpose of the present invention, refers to a position capable of binding to a non-peptide polymer. For example, although the C-terminus is not limited, it may include the terminal amino acid residue of the C-terminus and the amino acid residues around the C-terminus, and specifically may include the first to twentieth amino acid residues from the end.

[0310] In one embodiment, the conjugate represented by Formula 1 may have a structure represented by Formula 2 or Formula 3 below.

[0311] [Formula 2]

[0312]

[0313] [Formula 3]

[0314] X-(CH2)3[OCH2CH2] n O(CH2)3-F

[0315] In Formula 2 or Formula 3, X may be the above-mentioned peptide in Formula 1;

[0316] F may be an immunoglobulin Fc fragment; and

[0317] n can be a natural number. Specifically, the description of n is as described above.

[0318] In one embodiment, the long-acting conjugate represented by Formula 2 may have a structure in which X of the insulinotropic peptide or GLP-2 is covalently linked to F of the immunoglobulin Fc region through an ethylene glycol repeating unit, wherein X may be linked to the succinimide ring in Formula 2 and F may be linked to a propylene oxide group in Formula 2. In addition, the long-acting conjugate represented by Formula 3 may have a structure in which X of the insulinotropic peptide or GLP-2 is covalently linked to F of the immunoglobulin Fc region through an ethylene glycol repeating unit, and X may be linked to a propylene oxide group in Formula 3 and F may be linked to another propylene oxide group in Formula 3.

[0319] In Formula 2 or Formula 3, the value of n can be determined so that [OCH2CH2] n The average molecular weight (eg, number average molecular weight) of the moiety is from 1 kDa to 100 kDa, or from 1 kDa to 20 kDa, or 10 kDa, but is not limited thereto.

[0320] In one embodiment, the succinimide ring in Formula 2 or the site where X is connected to the succinimide ring in Formula 2 may be the sulfur atom of the C-terminal cysteine ​​of X. In addition, the oxypropylene group in Formula 3 or the site where X is connected to the oxypropylene group in Formula 3 may be the sulfur atom of the C-terminal cysteine ​​of X.

[0321] The site in F that is connected to the oxypropylene group in Formula 2 or Formula 3 is not particularly limited. In one embodiment of the present invention, the site where F is connected to the oxypropylene group may be the N-terminal nitrogen or a nitrogen atom of a residue in F (e.g., the epsilon nitrogen of lysine). In a specific embodiment of the present invention, the site where F is connected to the oxypropylene group in Formula 2 or Formula 3 may be the N-terminal proline of F, but is not limited thereto.

[0322] Meanwhile, the insulinotropic peptide, GLP-2, the long-acting conjugate thereof or the TNFα inhibitor according to the present invention includes all forms of itself, its salt (eg, pharmaceutically acceptable salt) or its solvate.

[0323] Furthermore, the insulinotropic peptide, GLP-2, a long-acting conjugate thereof or a TNFα inhibitor may be in any pharmaceutically acceptable form.

[0324] The type of the salt is not particularly limited. However, the salt is preferably in a form that is safe and effective for a subject (eg, a mammal), but is not particularly limited thereto.

[0325] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium and potassium, or alkaline earth metals such as magnesium and ammonium.

[0326] As used herein, the term "solvate" refers to a complex formed by a peptide, a compound or a salt thereof according to the present invention and solvent molecules.

[0327] The natural insulinotropic peptides and modified insulinotropic peptides, GLP-2 and GLP-2 derivatives, and TNFα inhibitors of the present invention can be synthesized by solid phase synthesis, can be produced by recombinant methods, can be prepared by commercial request, and commercially available products can be used.

[0328] In another embodiment, the combination, pharmaceutical composition or pharmaceutical kit of the present invention may include:

[0329] (i) GLP-2 and insulinotropic peptides,

[0330] GLP-2 and TNFα inhibitors, or

[0331] GLP-2, insulinotropic peptide, and TNFα inhibitors;

[0332] (ii) GLP-2, and a long-acting conjugate of an insulinotropic peptide bound to a biocompatible substance capable of increasing its half-life in vivo, or

[0333] GLP-2, insulinotropic peptide long-acting conjugates, and TNFα inhibitors;

[0334] (iii) a long-acting conjugate of GLP-2 combined with a biocompatible substance capable of increasing its half-life in vivo, and an insulinotropic peptide,

[0335] A long-acting conjugate of GLP-2 and a TNFα inhibitor, or

[0336] A long-acting conjugate of GLP-2, an insulinotropic peptide, and a TNFα inhibitor; or

[0337] (iv) a long-acting conjugate of GLP-2 combined with a biocompatible substance capable of increasing its half-life in vivo, and a long-acting conjugate of an insulinotropic peptide combined with a biocompatible substance capable of increasing its half-life in vivo, or

[0338] GLP-2 long-acting conjugate, insulinotropic peptide long-acting conjugate and TNFα inhibitor.

[0339] The combination, pharmaceutical composition or pharmaceutical kit of the present invention can be used to prevent, improve or treat intestinal diseases.

[0340] As used herein, the term "prevention" refers to any effect of inhibiting or delaying the onset of a target disease (e.g., intestinal disease) by administering a composition comprising GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or a combination of the two; or a composition comprising GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both. The term "treatment" refers to any effect of improving or benefiting the symptoms of a target disease (e.g., intestinal disease) by administering a composition comprising GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or a combination of the two; or a composition comprising GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both. The term "improvement" refers to any effect of at least reducing a parameter (e.g., the severity of the symptom) associated with the condition treated by administering the combination or composition of the present invention.

[0341] As used herein, the term "administer" refers to the introduction of a given substance into a patient's body by any appropriate method, and although the route of administration of the composition is not particularly limited, it may be any general route by which GLP-2, insulinotropic peptides and TNFα inhibitors can reach the target in vivo. Administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration or the like.

[0342] In the method of the present invention, 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 drugs administered in combination may be independent of each other.

[0343] GLP-2 can be used in combination with insulinotropic peptides, TNFα inhibitors, or both to prevent or treat enteropathy.

[0344] The intestinal disease may be at least one selected from irritable bowel disease, enteritis, inflammatory bowel disease, enterocolitis, colitis, pancreatitis, ileitis, intestinal atrophy and intestinal damage, but the intestinal diseases prevented, improved or treated by the composition of the present invention are not limitedly included.

[0345] In a specific example, the intestinal disease can be inflammatory bowel disease. Inflammatory bowel disease (IBD) refers to an inflammatory disease characterized by gastrointestinal inflammation or ulcers, and may show symptoms such as increased expression of inflammatory cytokines, weight loss, shortened colon length, abdominal pain, fever, diarrhea and / or blood in the stool. These symptoms may repeatedly worsen and improve, but are not limited to this. Specifically, inflammatory bowel disease can be at least one selected from ulcerative colitis, Crohn's disease and Behcet's disease, but is not limited to this.

[0346] The combination or composition according to the present invention can prevent, improve or treat intestinal diseases by exhibiting one or more of the following effects: inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation and inhibition of monocyte migration, but is not limited thereto.

[0347] When administered to a subject, the combination or composition according to the present invention prevents, improves or treats intestinal diseases by causing at least one effect among the following: weight gain, increased small intestine length, decreased gastrointestinal motility and increased nutrient absorption, but not limited thereto.

[0348] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. Such a pharmaceutically acceptable carrier, excipient or diluent may be a non-naturally occurring carrier, excipient or diluent.

[0349] As used herein, the term "pharmaceutically acceptable" refers to an amount sufficient to show a therapeutic effect without causing side effects, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health status and gender, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of administrations, the treatment period, and the drugs used in combination or simultaneously. The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable excipient. Although the excipient is not particularly limited, adhesives, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavoring agents, etc. can be used for oral administration; buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. can be used in the mixture for injection; and bases, excipients, lubricants, preservatives, etc. can be used for topical administration.

[0350] The formulation of the composition of the present invention can be prepared differently by combining the composition with a pharmaceutically acceptable excipient as described above. For example, the composition can be prepared in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and the composition can be prepared in unit dose ampoules or multiple dosage forms for injection. In addition, the composition can also be formulated into solutions, suspensions, tablets, pills, capsules, sustained release formulations, etc.

[0351] Meanwhile, examples of carriers, excipients and diluents suitable for preparations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil or the like. In addition, fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives, etc. may be further included.

[0352] Furthermore, the pharmaceutical composition of the present invention may have any formulation selected from tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations and suppositories.

[0353] In addition, the composition can also be formulated into a unit dosage form suitable for administration to a patient, specifically a preparation that can be used to administer a protein drug according to conventional methods in the pharmaceutical field, and administered by an oral administration route or a parenteral administration route using an administration method commonly used in the art, including skin, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal routes, but not limited thereto.

[0354] In addition, the conjugate can also be used in combination with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, other stabilizers or the like can be used as agents to improve stability or absorption.

[0355] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined according to the drug type of the active ingredient together with various relevant factors such as the target disease, administration route, age, sex and weight of the patient, and severity of the disease. Specifically, the composition of the present invention may include a pharmaceutically effective amount of an insulinotropic peptide, a GLP-2 or a TNFα inhibitor, but is not limited thereto.

[0356] Including insulinotropic peptide, GLP-2 or TNFα inhibitor in a pharmaceutically effective amount refers to an amount sufficient to obtain the desired pharmacological activity from insulinotropic peptide, GLP-2 and / or TNFα inhibitor, and may refer to a pharmaceutically acceptable amount that does not cause toxicity or side effects to the subject of administration or causes insignificant toxicity or side effects, but is not limited thereto. Such a pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, patients, formulation, etc.

[0357] Although not particularly limited, the pharmaceutical composition of the present invention may include the ingredient (active ingredient) in an amount of 0.01 w / v % to 99 w / v %.

[0358] The total effective amount of the composition of the present invention can be administered to the patient in a single dose, or it can be administered to the patient in a graded treatment regimen of multiple doses over a long period of time. The pharmaceutical composition of the present invention may include an active ingredient, the content of which varies depending on the severity of the disease. Specifically, the preferred total dose of the peptide or conjugate of the present invention can be about 0.0001 mg to 500 mg per 1 kg of patient body weight per day, but is not limited thereto. However, the effective dose of the conjugate to the patient is determined by taking into account various factors such as the patient's age, weight, health status, gender, diet and excretion rate and the severity of the disease, as well as the route of administration of the pharmaceutical composition and the number of treatments. Therefore, taking this into account, those skilled in the art will be able to determine an appropriate effective dose according to the specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration, as long as it exhibits the effects of the present invention.

[0359] The pharmaceutical composition of the present invention has excellent in vivo persistence and efficacy so that the number and frequency of administration of the pharmaceutical preparation of the present invention can be significantly reduced, but is not limited thereto.

[0360] Another aspect embodying the present invention provides a food composition for preventing, ameliorating or treating intestinal diseases, which comprises GLP-2, wherein the food composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor or both.

[0361] Insulinotropic peptides, GLP-2, TNFα inhibitors, prevention, treatment, improvement and intestinal diseases are as described above.

[0362] The food composition can be used as a health functional food. In the case where the composition of the present invention is used as a food supplement additive, the insulinotropic peptide, GLP-2, a long-acting conjugate containing it, or a combination thereof can be added as it is or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient used in combination can be appropriately determined according to the purpose of use (prevention, health care or therapeutic treatment).

[0363] As used herein, the term "health functional food" refers to food manufactured and processed for the purpose of health supplementation using specific ingredients as raw materials or by methods such as extraction, concentration, purification and mixing of specific ingredients contained in food materials, and refers to food designed and processed to fully exert biological regulatory functions such as biological defense, biological rhythm regulation, disease prevention and rehabilitation of diseases related to living bodies through the above ingredients. The composition for health food can perform functions related to disease prevention, disease rehabilitation, etc.

[0364] Another aspect of the present invention provides a method for preventing, ameliorating or treating intestinal diseases, comprising administering GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both to a subject in need thereof.

[0365] In another embodiment, the method may be a method for preventing, improving or treating intestinal diseases, comprising administering and / or using the combination, pharmaceutical composition or pharmaceutical kit of the present invention in a subject in need thereof.

[0366] In another embodiment, the method is a method for preventing, ameliorating or treating intestinal diseases, comprising administering and / or using a composition comprising a pharmaceutically effective amount of GLP-2, a composition comprising a pharmaceutically effective amount of an insulinotropic peptide, a composition comprising a pharmaceutically effective amount of a TNFα inhibitor, or both in a subject in need thereof.

[0367] Insulinotropic peptides, GLP-2, TNFα inhibitors, combined use, prevention, treatment, improvement, intestinal diseases, combinations, pharmaceutical compositions and pharmaceutical kits are as described above.

[0368] In the present invention, the subject is a subject suspected of having an intestinal disease, wherein the subject suspected of having an intestinal disease refers to a mammal that has developed or may develop the disease, including mice, livestock, and humans. However, subjects that can be treated by the GLP-2 combination of the present invention, insulinotropic peptide, TNFα inhibitor, or both are included without limitation. In addition, by administering the GLP-2 combination of the present invention, insulinotropic peptide, TNFα inhibitor, or both to a subject suspected of having an intestinal disease, the subject can be effectively treated. The intestinal disease is as described above.

[0369] The method of the present invention may include administering a combination or pharmaceutical composition in a pharmaceutically effective amount, which includes GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both. The method according to the present invention may be administering GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both as a single formulation, or administering GLP-2, and an insulinotropic peptide, a TNFα inhibitor, or both as separate formulations administered simultaneously, separately, sequentially, or in reverse order, but the method is not limited thereto.

[0370] The appropriate total daily amount can be determined by the treating physician within the scope of sound medical judgment, and can be administered at once or in divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is preferably applied differently, depending on the type and degree of the desired response, in some cases, whether other agents are used in combination and the specific composition, the patient's age, weight, general health, sex and diet, administration time, administration route and secretion rate of the composition, treatment period and drugs used or used simultaneously with the specific composition, and similar factors well known in the medical field.

[0371] Another aspect of the present invention is the use of a combination, a pharmaceutical composition or a pharmaceutical kit for preventing, improving or treating intestinal diseases and / or the use of a combination, a pharmaceutical composition or a pharmaceutical kit in the preparation of a medicament for preventing, improving or treating intestinal diseases. The prevention, improvement, treatment, intestinal diseases, combinations, pharmaceutical compositions and pharmaceutical kits are as described above.

[0372] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0373] Example

[0374] Example 1: Preparation of long-acting GLP-1 derivative conjugate

[0375] 1-1. Preparation of CA-exendin-4-PEG conjugate

[0376] The PEGylated peptide conjugate (mono-PEGylated CA-exendin-4) was prepared by covalent bonding between the amine group of the 27th lysine residue in the CA-exendin-4 (N-[2-(1H-imidazol-5-yl)acetyl]-exendin-4, HanmiFine Chemical, Korea) peptide and polyethylene glycol (3.4 kDa, ALD(2)PEG, Hanmi Fine Chemical, 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, the concentration of CA-exendin-4 was 6 mg / mL to 12 mg / mL, and the reaction was performed at a temperature of 4°C to 10°C to room temperature for about 4 to 12 hours. At this time, the reaction was carried out in 0.1M HEPES and about 45% isopropanol at a pH of 7.0 to 8.5, and sodium cyanoborohydride (NaCNBH3) was added at a concentration of 2mM to 50mM for reaction. The reaction mixture was purified using a Source 15S (Cytiva, USA) column with a buffer (pH 2.0 to 3.5) including sodium citrate and about 45% ethanol and a potassium chloride concentration gradient.

[0377] 1-2. Preparation of CA-exendin-4-PEG-Fc conjugate

[0378] In order to prepare 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 a total protein concentration of 10 mg / mL to 50 mg / mL, followed by reaction at 4°C to 10°C to room temperature for about 12 to 17 hours. At this time, the reaction solution was 0.1 M potassium phosphate at a pH of 5.5 to 8.5, and 2 mM to 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 mixture was purified using two columns, namely a hydrophobic binding column and an anion exchange column. The hydrophobic binding column Source 15Phenyl (Cytiva, USA) used Tris-HCl (pH 7.5) buffer and NaCl concentration gradient to separate and purify the unreacted immunoglobulin Fc fragment, while the anion exchange column Source 15Q (Cytiva, USA) used Tris-HCl (pH 7.5) buffer and NaCl concentration gradient to separate and purify the CA-exendin-4-PEG-Fc conjugate by removing over-reacted impurities.

[0379] Example 2: Preparation of long-acting GLP-2 derivative conjugates

[0380] 2-1. Preparation of CAGLP-2RK-PEG conjugate

[0381] In order to PEGylate the GLP-2 derivative CA GLP-2RK of SEQ ID NO: 4 in Table 1 with modified polyethylene glycol ALD(2)PEG (modified polyethylene glycol in which hydrogen atoms at both ends are substituted with propionaldehyde (3-oxopropyl) groups and the formula weight of the ethylene glycol repeating unit portion is 3.4 kDa, manufactured by NOF Corporation of Japan), the reaction was carried out at a molar ratio of the GLP-2 derivative to ALD(2)PEG of 1:5 to 1:20, a concentration of the GLP-2 derivative of 5 mg / mL to 10 mg / mL, and a temperature of 2°C to 8°C for 4 to 16 hours. At this time, the reaction was carried out in 20 mM HEPES buffer and ethanol at pH 7.5, and 20 mM sodium cyanoborohydride was added as a reducing agent for the reaction. The reaction mixture was purified using a Source 15S (GE, USA) column using a buffer solution including sodium citrate at pH 2.0 and a concentration gradient of ethanol and potassium chloride.

[0382] 2-2. Preparation of CA GLP-2RK-PEG-Fc conjugate

[0383] Next, the purified mono-PEGylated GLP-2 derivative was reacted with the immunoglobulin Fc fragment at a molar ratio of 1:2 to 1:6. The total protein concentration was adjusted to 30 mg / mL to 35 mg / mL, and the reaction was carried out at a temperature of 2°C to 8°C for 12 to 20 hours. During the reaction, 20 mM sodium cyanoborohydride was added as a reducing agent to the reaction solution containing 100 mM potassium phosphate buffer (pH 6.0) and isopropanol.

[0384] After the reaction is completed, the reaction mixture is applied to a Source15Q (GE, USA) column with a bis-Tris buffer (pH 6.5) and a sodium chloride concentration gradient, and applied to a Source15ISO (GE, USA) column with an ammonium sulfate and sodium citrate concentration gradient from pH 5.0 to 5.2 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 through a polyethylene glycol linker.

[0385] Example 3: Confirmation of M1 polarization inhibition, macrophage differentiation inhibition, and monocyte migration inhibition in THP-1 cells (monocytes) by combined treatment with a long-acting GLP-1 derivative conjugate and a long-acting GLP-2 derivative conjugate

[0386] In vitro experiments were performed as follows to demonstrate the inhibitory effects on M1 polarization, macrophage differentiation and monocyte migration in THP-1 cells (monocytes) after treatment with a long-acting GLP-1 derivative conjugate, a long-acting GLP-2 derivative conjugate and a combination of both conjugates.

[0387]

Experiment to confirm M1 polarization inhibition

[0388] THP-1 cells were treated with long-acting GLP-1 derivative conjugates (0.5 μM or 1 μM), long-acting GLP-2 derivative conjugates (10 μM), and a combination of long-acting GLP-1 derivative conjugates (0.5 μM) and long-acting GLP-2 derivative conjugates (10 μM) for 4 hours. To induce inflammation, 1 μg / mL of lipopolysaccharide (LPS) was used for 2 hours, and RNA was extracted to measure the mRNA expression levels of proinflammatory cytokines TNFα, IL-1β, and IL-6 (M1 polarization markers) by qPCR.

[0389]

Macrophage differentiation inhibition confirmation experiment

[0390] THP-1 cells were treated for 48 hours with a long-acting GLP-1 derivative conjugate (0.1 μM or 1 μM), a long-acting GLP-2 derivative conjugate (10 μM), and a combination of a long-acting GLP-1 derivative conjugate (0.1 μM) and a long-acting GLP-2 derivative conjugate (10 μM) to induce THP-1 differentiation using phorbol 12-myristate 13-acetate (PMA). The number of differentiated cells (i.e., adherent cells) was then counted.

[0391]

Monocyte migration inhibition assay

[0392] THP-1 cells were treated for 48 hours with a long-acting GLP-1 derivative conjugate (0.1 μM or 1 μM), a long-acting GLP-2 derivative conjugate (10 μM), and a combination of a long-acting GLP-1 derivative conjugate (0.1 μM) and a long-acting GLP-2 derivative conjugate (10 μM). The treated THP-1 cells were transferred to the upper chamber of the Boyden chamber, and 50 ng / mL CCL-2 was added to the lower chamber to induce migration, followed by incubation for 4 hours. The degree of migration was measured using a migration assay kit (Abcam).

[0393] Through the above experiments, it was confirmed that the long-acting GLP-1 derivative conjugate and the long-acting GLP-2 derivative conjugate each had an effect on M1 polarization ( Figure 1 , (A)), macrophage differentiation ( Figure 1 , (B)) and monocyte migration ( Figure 1, (C)), and it was confirmed that these effects were further enhanced when the two were administered in combination.

[0394] Example 4: Examination of increased small intestinal length and reduced inflammation in an indomethacin-induced inflammatory bowel disease rat model following combined administration of a long-acting GLP-1 derivative conjugate, a long-acting GLP-2 derivative conjugate, and an anti-TNFα mAb

[0395] In order to measure the in vivo efficacy of the combined administration of long-acting GLP-1 derivative conjugate, long-acting GLP-2 derivative conjugate and anti-TNFα mAb (BioXcell, catalog number BE0244) in increasing the length of the small intestine and reducing inflammation, an indomethacin (INN)-induced inflammatory bowel disease rat model was used. Specifically, 8-week-old male SD rats (6 rats per group) were divided as follows:

[0396] -G1: Normal control group (no drug group, vehicle)

[0397] -G2: Indomethacin control group (Indomethacin 7.5 mg / kg, administered on days 1 and 2 (D1 and D2))

[0398] -G3: a group administered with a long-acting GLP-2 derivative conjugate (3.1 mg / kg / single dose) followed by indomethacin (long-acting GLP-2 derivative conjugate 3.1 mg / kg / single dose, D0; indomethacin 7.5 mg / kg, D1 and D2)

[0399] -G4: administration of long-acting GLP-1 derivative conjugate (0.178 mg / kg / single dose) followed by administration of indomethacin (long-acting GLP-1 derivative conjugate 0.178 mg / kg / single dose, D0; indomethacin 7.5 mg / kg, D1 and D2)

[0400] -G5: A group that was administered with a combination of a long-acting GLP-2 derivative conjugate (3.1 mg / kg / single dose) and a long-acting GLP-1 derivative conjugate (0.178 mg / kg / single dose) followed by indomethacin (long-acting GLP-2 derivative conjugate 3.1 mg / kg / single dose, long-acting GLP-1 derivative conjugate 0.178 mg / kg / single dose, D0; indomethacin 7.5 mg / kg, D1 and D2)

[0401] -G6: group administered with anti-TNFα mAb (0.2 mg / kg / single) followed by indomethacin (anti-TNFα mAb 0.2 mg / kg / single, D0; indomethacin 7.5 mg / kg, D1 and D2)

[0402] -G7: A group that was administered with a combination of a long-acting GLP-2 derivative conjugate (3.1 mg / kg / single dose) and anti-TNFα mAb (0.2 mg / kg / single dose) followed by indomethacin (long-acting GLP-2 derivative conjugate 3.1 mg / kg / single dose, anti-TNFα mAb 0.2 mg / kg / single dose, D0; indomethacin 7.5 mg / kg, D1 and D2)

[0403] -G8: The group administered with a combination of a long-acting GLP-2 derivative conjugate (3.1 mg / kg / single dose), a long-acting GLP-1 derivative conjugate (0.178 mg / kg / single dose) and an anti-TNFα mAb (0.2 mg / kg / single dose), followed by administration of indomethacin (long-acting GLP-2 derivative conjugate 3.1 mg / kg / single dose, long-acting GLP-1 derivative conjugate 0.178 mg / kg / single dose, anti-TNFα mAb 0.2 mg / kg / single dose, D0; indomethacin 7.5 mg / kg, D1 and D2).

[0404] In the present specification, the dose of the long-acting GLP-1 derivative conjugate or the long-acting GLP-2 derivative conjugate refers to the value obtained by subtracting the mass of the polyethylene glycol linker part from the total mass of the long-acting GLP-1 derivative conjugate or the long-acting GLP-2 derivative conjugate used, that is, the value based on the total mass of the polypeptide part.

[0405] On day 3, small intestinal length was measured by necropsy and ulcer area was determined by Evans blue staining.

[0406] The group administered with a combination of a long-acting GLP-2 derivative conjugate and a long-acting GLP-1 derivative conjugate followed by indomethacin (G5) showed an increase in small intestine length of 25 cm ( Figure 2 ) and the ulcer area decreased by 6.7% ( Figure 3 The group that received a combination of a long-acting GLP-2 derivative conjugate and anti-TNFα mAb followed by indomethacin (G7) showed an increase in small intestine length of 12 cm ( ) compared to the indomethacin control group (G2). Figure 2 ) and the ulcer area decreased by 3.5% ( Figure 3 In addition, the group administered with a combination of a long-acting GLP-2 derivative conjugate, a long-acting GLP-1 derivative conjugate, and anti-TNFα mAb followed by indomethacin (G8) showed an increase in small intestine length of 32 cm compared to the indomethacin control group (G2), which was similar to the normal control group ( Figure 2 ), and the ulcer area decreased by 7.2%, which was also similar to the normal control group ( Figure 3 ).

[0407] Example 5: Confirmation of increased colon length and decreased disease activity index (DAI) in the dextran sodium sulfate-induced ulcerative colitis mouse model after combined administration of a long-acting GLP-1 derivative conjugate and a long-acting GLP-2 derivative conjugate

[0408] In order to measure the in vivo efficacy of the increase in colon length and improvement in disease activity index (DAI) after the combined administration of a long-acting GLP-1 derivative conjugate and a long-acting GLP-2 derivative conjugate, a dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model was used. Specifically, 7-week-old male mice (C57BL / 6) were divided into groups of 10 mice per group as follows. DSS was administered by mixing 2% DSS into drinking water, wherein 1 cycle consisted of 5 days of DSS-containing water followed by 5 days of regular water. After a total of 3 cycles, the drug was administered, and the first day of administration of DSS-containing water in the first cycle was designated as day 0.

[0409] -G1: Normal control group (no drug group, vehicle)

[0410] -G2: DSS control group (2% DSS, 3 cycles)

[0411] -G3: group administered with a long-acting GLP-1 derivative conjugate (0.301 mg / kg / Q2D) after DSS administration (2% DSS, 3 cycles; long-acting GLP-1 derivative conjugate 0.301 mg / kg / Q2D, 2 weeks)

[0412] -G4: group administered with long-acting GLP-2 derivative conjugate (1.757 mg / kg / Q2D) after DSS administration (2% DSS, 3 cycles; long-acting GLP-2 derivative conjugate 1.757 mg / kg / Q2D, 2 weeks)

[0413] -G5: group administered with long-acting GLP-1 derivative conjugate (0.301 mg / kg / Q2D) and long-acting GLP-2 derivative conjugate (1.757 mg / kg / Q2D) after DSS administration (2% DSS, 3 cycles; long-acting GLP-1 derivative conjugate 0.301 mg / kg / Q2D, long-acting GLP-2 derivative conjugate 1.757 mg / kg / Q2D, 2 weeks)

[0414] -G6: group administered with cyclosporine A (20 mg / kg / QD, PO) after DSS administration (2% DSS, 3 cycles; cyclosporine A 20 mg / kg / QD, 2 weeks)

[0415] On day 44, necropsy was performed and colon length was measured to assess disease improvement ( Figure 4 ).

[0416] The Disease Activity Index (DAI) for ulcerative colitis was measured by assessing stool consistency and bleeding status using scale scores. Stool consistency scale scores were given as follows: normal (0 points); stool shape was visible but softened differently from the normal group after compression (1 point); stool was not round and softened easily unlike the normal group (2 points); stool was unformed and very soft (3 points); anal wetness due to diarrhea with watery stools instead of solid stools (4 points). Bleeding status scores were as follows: no bleeding (0 points); blood was visible after compression of the stool (2 points); blood was visible without compression of the stool (3 points); and severe bleeding was confirmed by the rectum (4 points). Measurements were made on days 21, 23, 25, 28, 30, 32, 35, 37, 39, 42, and 44, and the sum of these values ​​was used to assess disease improvement ( Figure 5 ).

[0417] Compared with the DSS control group (G2), the group (G5) administered with the long-acting GLP-1 derivative conjugate and the long-acting GLP-2 derivative conjugate after DSS administration showed an increase in colon length of approximately 0.8 cm (18%), which was similar to the normal control group ( Figure 4 ), and the DAI of ulcerative colitis decreased by 10 points ( Figure 5 In addition, compared with the cyclosporine A (20 mg / kg / QD) administration group (G6), which is commonly used in severe clinical stages before surgery, the long-acting GLP-1 derivative conjugate and long-acting GLP-2 derivative conjugate administration groups (G5) after DSS administration showed an increase in colon length of approximately 1.4 cm (25%), which was similar to the normal control group ( Figure 4 ), and the disease activity index decreased by 6 points ( Figure 5 ).

[0418] Based on the above description, it will be understood by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic features. Therefore, it should be understood that the above-mentioned embodiments are not restrictive, but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by their previous description, and therefore all changes and modifications falling within the limits of the claims or the equivalents of such limits are intended to be covered by the claims.

Claims

1. A pharmaceutical composition for preventing or treating intestinal diseases, comprising a pharmaceutically effective amount of GLP-2, The pharmaceutical composition is administered in combination with an insulinotropic peptide, a TNFα inhibitor, or both.

2. The pharmaceutical composition according to claim 1, wherein the insulinotropic peptide is selected from glucagon-like peptide-1 (GLP-1), exendin-3, exendin-4, agents, derivatives, fragments and variants thereof, and combinations thereof.

3. The pharmaceutical composition according to claim 1, wherein the insulinotropic peptide is an insulinotropic peptide derivative, wherein the N-terminal histidine residue of the insulinotropic peptide is substituted by imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine.

4. The pharmaceutical composition according to claim 1, wherein the insulinotropic peptide is natural exendin-4; an exendin-4 derivative in which the α carbon of the first amino acid at the N-terminus of exendin-4, i.e., the histidine residue, and the N-terminal amino group connected to the α carbon are removed; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is removed; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is substituted with a hydroxyl group; an exendin-4 derivative in which the N-terminal amino group of exendin-4 is modified with two methyl groups; an exendin-4 derivative 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. 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 in which at least one amino acid in the sequence of the native GLP-2 has been modified by a modification selected from substitution, addition, deletion, modification and a combination thereof.

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

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] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9) in, X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine; X2 is alanine, glycine or 2-aminoisobutyric acid (Aib); X 30 is lysine or arginine; and X 34 absent or lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine; The prerequisite is that among the amino acid sequences of general formula 1, sequences identical to SEQ ID NO: 1 are excluded.

9. The pharmaceutical composition according to claim 8, wherein the GLP-2 derivative comprises an amino acid sequence represented by Formula 1, wherein: (1) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is cysteine; (2) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It is lysine; (3) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is lysine; (4) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azidolysine; (5) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It is cysteine; (6) X1 is imidazoacetyl desyl histidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine; or (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It's 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] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:10) in, X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine; X2 is alanine, glycine or 2-aminoisobutyric acid (Aib); X 30 is lysine or arginine; and X 34 is any one or more amino acids or any one or more amino acids modified therein; The prerequisite is that among the amino acid sequences of general formula 2, the sequence identical to SEQ ID NO: 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 SEQ ID NOs: 2 to 8.

12. The pharmaceutical composition according to claim 1, wherein GLP-2 is administered in combination with an insulinotropic peptide.

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

14. The pharmaceutical composition according to claim 1, wherein GLP-2 is administered in combination with an insulinotropic peptide and a TNFα inhibitor.

15. The pharmaceutical composition of claim 1, wherein the TNFα inhibitor is a soluble TNF receptor, an anti-TNFα inhibitor 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 irritable bowel disease, enteritis, inflammatory bowel disease, enterocolitis, 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 ulcerative colitis, Crohn's disease and Behcet's disease.

18. The pharmaceutical composition according to claim 1, wherein the composition causes at least one of inhibition of M1 polarization in monocytes, inhibition of macrophage differentiation, and inhibition of monocyte migration when administered to a subject.

19. The pharmaceutical composition of claim 1, wherein the composition, when administered to a subject, causes at least one of 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 in the insulinotropic peptide and GLP-2, the C-terminus is unmodified or is amidated.

21. The pharmaceutical composition according to claim 1, wherein: (i) the insulinotropic peptide is in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of the insulinotropic peptide is combined with the long-acting conjugate; (ii) the GLP-2 is in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of the GLP-2 is combined with the long-acting conjugate; or (iii) The insulinotropic peptide and GLP-2 are each in the form of a long-acting conjugate, and a biocompatible substance capable of increasing the in vivo half-life of the insulinotropic peptide and GLP-2, respectively, is bound to the long-acting conjugate.

22. The pharmaceutical composition according to claim 21, wherein the conjugate is represented by the following formula 1: [Formula 1] X-La-F in, X is insulinotropic peptide or GLP-2; L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, wherein when a is 2 or greater, each L is independent of each other; F is an immunoglobulin Fc region; and "-" represents 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 the F is a dimer consisting of two polypeptide chains, and one end of L is connected to only one of the two polypeptide chains.

25. The pharmaceutical composition of claim 22, wherein said L is polyethylene glycol.

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

27. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

28. A pharmaceutical composition according to any one of claims 1 to 27, wherein (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 simultaneously, sequentially or in reverse order.

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