Pharmaceutical composition for preventing or treating graft versus host disease containing GLP-2 derivative or long-acting conjugate thereof

Abstract

The present invention relates to the prophylactic or therapeutic use of GLP-2 derivatives and / or long-acting conjugates thereof for graft versus host disease. In addition, the present invention relates to the prophylactic or therapeutic use of a GLP-2 derivative and / or a long-acting conjugate thereof in combination with a steroid for steroid refractory graft versus host disease.

Description

Technical Field

[0001] The present invention relates to the use of GLP-2 derivatives and / or long-acting conjugates thereof for the prevention or treatment of graft-versus-host disease. The present invention also relates to the use of GLP-2 derivatives and / or long-acting conjugates thereof for the prevention or treatment of steroid-refractory graft-versus-host disease, wherein the GLP-2 derivatives and / or long-acting conjugates thereof are used in combination with steroids. Background Art

[0002] Graft-versus-host disease (GVHD) is an immune response that occurs when T lymphocytes in the peripheral blood or bone marrow of the donor, injected during an allogeneic transplant, recognize the host's body (i.e., the patient receiving the allogeneic transplant) as foreign and attack it. In other words, GVHD is caused by the injected live lymphocytes and, due to the immune response, can lead to symptoms such as liver dysfunction, skin lesions, jaundice, diarrhea, fever, and pancytopenia. In severe cases, the patient may die.

[0003] Several approaches have been proposed for treating graft-versus-host disease. These include removing T cells from transplanted bone marrow cells, administering antibodies against CD80, CD86, and similar targets to inhibit the reaction between T cells and antigen-presenting cells, and administering antibodies against cytokines such as IL-2 and IFN-γ. Furthermore, the administration of immunosuppressant compounds such as cyclosporine A, rapamycin, FK-506, and steroid preparations has also been utilized. Among these approaches, the most widely used is the administration of immunosuppressant compounds that inhibit T cell activation.

[0004] In particular, steroid-refractory graft-versus-host disease (GVHD) is fatal in transplant recipients due to direct organ damage caused by GVHD itself and opportunistic infections caused by the potent immunosuppressants used to treat the disease. Various approaches have been tried as conventional treatments for acute GVHD that does not respond to standard-dose steroids, including high-dose steroids, monoclonal or polyclonal antibodies, and immunotoxins, but response rates and survival rates remain low. According to previous reports, the percentage of patients with steroid-refractory GVHD who show improvement beyond a partial response is approximately 60%, but when high-dose steroids are used as second-line therapy, the survival rate is 35%. This indirectly suggests that survival is difficult in patients who do not respond to high-dose steroids used as second-line treatment for steroid-refractory GVHD.

[0005] Recently, the JAK1 / 2 inhibitor ruxolitinib has been approved by the FDA as a treatment for steroid-refractory graft-versus-host disease (SR-GVHD) that has an inadequate response to steroids. Although a large number of immunosuppressant compounds have been developed to date, cyclosporine A has demonstrated the most favorable clinical efficacy and is widely used for organ transplant rejection, including acute graft-versus-host disease.

[0006] However, although cyclosporine A can completely suppress T cell activation and treat the disease when used in high doses, it has the problem of severe side effects, including nephrotoxicity. Summary of the Invention

[0007] [Technical Issues]

[0008] There is a need to develop new drugs that can prevent or treat graft-versus-host disease or steroid-refractory graft-versus-host disease.

[0009] [Technical solution]

[0010] The object of the present invention is to provide a composition for preventing, improving or treating graft-versus-host disease, which contains a glucagon-like peptide-2 (GLP-2) derivative and / or a long-acting conjugate thereof.

[0011] Another object of the present invention is to provide a method for preventing, improving or treating graft-versus-host disease, which comprises administering a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same to a subject in need thereof.

[0012] Yet another object of the present invention is to provide use of a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same in the preparation of a medicament for preventing or treating graft-versus-host disease.

[0013] Yet another object of the present invention is to provide a composition for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same.

[0014] Yet another object of the present invention is to provide a method for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises administering a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same to a subject in need thereof.

[0015] Yet another object of the present invention is to provide use of a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same in the preparation of a medicament for preventing or treating steroid-refractory graft-versus-host disease.

[0016] Yet another object of the present invention is to provide a therapy for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, comprising using a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same in combination with a steroid.

[0017] Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating steroid-refractory graft-versus-host disease, comprising a GLP-2 derivative and / or a long-acting conjugate thereof, wherein the pharmaceutical composition administers the GLP-2 derivative or the long-acting conjugate thereof in combination with a steroid.

[0018] Yet another object of the present invention is to provide a combination comprising a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid.

[0019] Yet another object of the present invention is to provide a pharmaceutical composition containing the combination for preventing, improving or treating steroid-refractory graft-versus-host disease.

[0020] Yet another object of the present invention is to provide a pharmaceutical kit for preventing, improving or treating steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid.

[0021] Yet another object of the present invention is to provide a method for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises administering and / or using the combination, pharmaceutical composition or pharmaceutical kit to a subject in need thereof.

[0022] Yet another object of the present invention is to provide a method for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises administering and / or using in combination a pharmaceutically effective amount of a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing them and a pharmaceutically effective amount of a composition containing a steroid or both to a subject in need thereof.

[0023] Yet another object of the present invention is to provide the use of the combination, pharmaceutical composition or pharmaceutical kit for preventing, improving or treating steroid-refractory graft-versus-host disease; and / or the use of the combination, pharmaceutical composition or pharmaceutical kit in the preparation of a medicament for preventing, improving or treating steroid-refractory graft-versus-host disease.

[0024] [Beneficial effects]

[0025] The GLP-2 derivatives, long-acting conjugates thereof, or compositions containing GLP-2 thereof of the present invention can be used to prevent, treat, and ameliorate graft-versus-host disease. Combination therapy with the GLP-2 derivatives and / or long-acting conjugates of the present invention and steroids demonstrates improved efficacy compared to single-dose therapy with these drugs, and thus is effectively used to prevent, ameliorate, or treat steroid-refractory graft-versus-host disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a graph showing changes in survival rate when a long-acting conjugate of a GLP-2 derivative is administered in an allogeneic transplant-induced GVHD model;

[0027] Figure 2 is a graph showing changes in GVHD scores when a long-acting conjugate of a GLP-2 derivative is administered in an allogeneic transplant-induced GVHD model;

[0028] Figure 3 is a graph showing changes in the length of the small intestine (top) and colon (bottom) when a long-acting conjugate of a GLP-2 derivative is administered in a GVHD model induced by allogeneic transplantation;

[0029] Figure 4 is a graph showing changes in survival rate when a long-acting conjugate of a GLP-2 derivative is administered in an allogeneic transplant-induced SR-GVHD model;

[0030] Figure 5 is a graph showing changes in GVHD scores when a long-acting conjugate of a GLP-2 derivative is administered in an SR-GVHD model induced by allogeneic transplantation;

[0031] Figure 6 is a graph showing changes in survival rate by administering a long-acting conjugate of a GLP-2 derivative and steroids in an allogeneic transplant-induced SR-GVHD model;

[0032] Figure 7 is a graph showing changes in GVHD scores by administering a long-acting conjugate of a GLP-2 derivative and steroids in an allogeneic transplant-induced SR-GVHD model;

[0033] Figure 8 is a graph comparing the survival rates of a group administered with a long-acting conjugate of a GLP-2 derivative and steroids and a group administered with ruxolitinib and steroids in an allogeneic transplant-induced SR-GVHD model; and

[0034] Figure 9This graph compares the GVHD scores of the group administered with a long-acting conjugate of a GLP-2 derivative and steroids and the group administered with ruxolitinib and steroids in an allogeneic transplant-induced SR-GVHD model. DETAILED DESCRIPTION

[0035] One aspect of the present invention is a composition for preventing, treating or ameliorating graft-versus-host disease, comprising a GLP-2 derivative and / or a long-acting conjugate thereof.

[0036] As a specific example, the present invention is a pharmaceutical composition for preventing or treating graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0037] As another specific example, the present invention is a food composition for preventing or improving graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0038] Another aspect of the present invention is a composition for preventing, improving or treating steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0039] As a specific example, the present invention is a pharmaceutical composition for preventing or treating steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0040] As another specific example, the present invention is a food composition for preventing or improving steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0041] Yet another aspect of the present invention is a therapy for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, which comprises using a GLP-2 derivative and / or a long-acting conjugate thereof in combination with a steroid.

[0042] Yet another aspect of the present invention is a composition for preventing, treating or ameliorating steroid-refractory graft-versus-host disease, which comprises administering a combination of a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid.

[0043] As a specific example, the present invention is a pharmaceutical composition for preventing or treating steroid-refractory graft-versus-host disease, which administers a combination of a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid.

[0044] As another specific example, the present invention is a food composition for preventing or improving steroid-refractory graft-versus-host disease, which administers a combination of a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid.

[0045] The composition according to any one of the aforementioned specific embodiments, wherein the present invention contains a pharmaceutically effective amount of a GLP-2 derivative, and the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1:

[0046] [General formula 1]

[0047] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 10)

[0048] In general formula 1,

[0049] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0050] X2 is alanine, glycine, or Aib (2-aminoisobutyric acid);

[0051] X 30 is lysine or arginine; and

[0052] X 34 is one or more arbitrary amino acids or one or more modified arbitrary amino acids,

[0053] However, among the amino acid sequences represented by the general formula 1, sequences identical to SEQ ID NO: 1 are excluded.

[0054] The composition according to any one of the preceding embodiments, wherein the GLP-2 derivative comprises an amino acid sequence represented by the following Formula 2:

[0055] [Formula 2]

[0056] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0057] In general formula 2,

[0058] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0059] X2 is alanine, glycine, or Aib (2-aminoisobutyric acid);

[0060] X 30 is lysine or arginine; and

[0061] X 34 Lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine is absent or

[0062] However, among the amino acid sequences represented by the general formula 2, sequences identical to SEQ ID NO: 1 are excluded.

[0063] The composition according to any one of the preceding embodiments, wherein in the GLP-2 derivative,

[0064] (1) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's cysteine,

[0065] (2) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's lysine,

[0066] (3) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It's lysine,

[0067] (4) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azidolysine,

[0068] (5) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It's cysteine,

[0069] (6) X1 is imidazoacetyl desahistidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine, or

[0070] (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It's cysteine.

[0071] The composition according to any one of the preceding embodiments, wherein the GLP-2 derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

[0072] The composition according to any one of the preceding embodiments, wherein the C-terminus of the GLP-2 derivative is unmodified or amidated.

[0073] The composition according to any of the preceding embodiments, wherein the graft-versus-host disease is acute graft-versus-host disease or chronic graft-versus-host disease.

[0074] The composition according to any of the preceding embodiments, wherein the graft-versus-host disease is caused by an allogeneic transplant, and the allogeneic transplant comprises at least one of a hematopoietic stem cell transplant (HSCT), a bone marrow transplant (BMT), a peripheral hematopoietic stem cell transplant (PBSCT), an umbilical cord blood transplant (UCBT), or a blood transfusion.

[0075] The composition according to any one of the preceding embodiments, wherein the composition is administered to the subject together with, before or after an allogeneic transplant.

[0076] The composition according to any one of the preceding embodiments, wherein the composition causes one or more of the following in a subject to which the composition is administered: an increase in survival rate, an increase in small intestine length, and an increase in colon length.

[0077] The composition according to any one of the preceding embodiments, wherein the GLP-2 derivative is in the form of a long-acting conjugate, which is combined with a biocompatible substance capable of increasing the in vivo half-life of the GLP-2 derivative.

[0078] The composition according to any one of the preceding embodiments, wherein the conjugate is represented by the following Chemical Formula 1:

[0079] [Chemical Formula 1]

[0080] XL a -F

[0081] In Chemical Formula 1,

[0082] X is a GLP-2 derivative;

[0083] L is a linker containing ethylene glycol repeating units;

[0084] a is 0 or a natural number, but when a is 2 or greater, each L is independent of each other;

[0085] F is an immunoglobulin Fc region; and

[0086] “-” is a covalent bond.

[0087] The composition according to any one of the preceding embodiments, wherein the GLP-2 derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

[0088] The composition according to any one of the preceding embodiments, wherein the C-terminus of the GLP-2 derivative is unmodified or amidated.

[0089] The composition according to any of the preceding embodiments, wherein the GLP-2 derivative has an amino acid sequence comprising SEQ ID NO: 4, L is polyethylene glycol, and the immunoglobulin Fc region is an aglycosylated IgG4 Fc region.

[0090] The composition according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is an aglycosylated IgG4 Fc region.

[0091] The composition according to any of the preceding embodiments, wherein F is a dimer composed of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0092] The composition according to any of the preceding embodiments, wherein L is polyethylene glycol.

[0093] The composition according to any of the preceding embodiments, wherein the formula weight of the ethylene glycol repeat unit portion in L is in the range of 1 to 100 kDa.

[0094] The composition according to any one of the preceding embodiments, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

[0095] The composition according to any of the preceding embodiments, wherein the composition is administered to the subject together with, before or after an allogeneic transplant.

[0096] The composition according to any one of the preceding embodiments, wherein the composition causes one or more of the following in a subject to which the composition is administered: an increase in survival rate, an increase in small intestine length, and an increase in colon length.

[0097] The composition according to any of the preceding embodiments, wherein the steroid-refractory graft-versus-host disease is steroid-refractory acute graft-versus-host disease or steroid-refractory chronic graft-versus-host disease.

[0098] The composition according to any of the preceding embodiments, wherein the steroid-refractory graft-versus-host disease is caused by an allogeneic transplant, and the allogeneic transplant comprises at least one of a hematopoietic stem cell transplant (HSCT), a bone marrow transplant (BMT), a peripheral blood stem cell transplant (PBSCT), an umbilical cord blood transplant (UCBT), or a blood transfusion.

[0099] The composition according to any of the preceding embodiments, wherein the graft-versus-host disease is steroid-refractory graft-versus-host disease, and the pharmaceutical composition is administered in combination with a steroid.

[0100] The composition according to any of the preceding embodiments, wherein the steroid-refractory graft-versus-host disease is steroid-refractory acute graft-versus-host disease or steroid-refractory chronic graft-versus-host disease.

[0101] The composition according to any one of the preceding embodiments, wherein the combined administration is simultaneous, sequential or reverse order combined administration of the GLP-2 derivative or its long-acting conjugate; and the steroid.

[0102] Yet another aspect of the present invention is a method for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, which comprises administering a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same to a subject in need thereof, wherein the GLP-2 derivative, the long-acting conjugate thereof and the composition are as described in any one of the aforementioned specific examples.

[0103] Yet another aspect of the present invention is the use of a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same for preventing, improving or treating steroid-refractory graft-versus-host disease, wherein the GLP-2 derivative, the long-acting conjugate thereof and the composition are as described in any one of the aforementioned specific examples.

[0104] Yet another aspect of the present invention is the use of a GLP-2 derivative, a long-acting conjugate thereof and / or a pharmaceutical composition containing the same in the preparation of a medicament for preventing or treating steroid-refractory graft-versus-host disease, wherein the GLP-2 derivative, the long-acting conjugate thereof and the composition are as described in any one of the aforementioned specific examples.

[0105] Yet another aspect of the present invention is a pharmaceutical composition containing the combination for preventing, improving or treating steroid-refractory graft-versus-host disease.

[0106] Yet another aspect of the present invention is a pharmaceutical kit containing the combination for preventing, improving or treating steroid-refractory graft-versus-host disease.

[0107] Yet another aspect of the present invention is a method for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, which comprises administering and / or using the combination, pharmaceutical composition or pharmaceutical kit to a subject in need thereof.

[0108] Yet another aspect of the present invention is a method for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, which comprises administering and / or using in combination to a subject in need thereof a pharmaceutically effective amount of a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing them, and a pharmaceutically effective amount of a composition containing a steroid or both, wherein the GLP-2 derivative, the long-acting conjugate thereof, the composition and the combination are as described in any of the foregoing specific examples.

[0109] Yet another aspect of the present invention is the use of the combination, pharmaceutical composition or pharmaceutical kit for preventing, improving or treating steroid-refractory graft-versus-host disease; and / or the use of the combination, pharmaceutical composition or pharmaceutical kit in the preparation of a medicament for preventing or treating steroid-refractory graft-versus-host disease. DETAILED DESCRIPTION

[0111] Hereinafter, the present invention will be described in more detail.

[0112] At the same time, each description and each embodiment disclosed in this application can also be applied to other descriptions and other embodiments respectively. In other words, all combinations of the various elements disclosed in this application fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific description described below.

[0113] Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described herein. Additionally, such equivalents are intended to be encompassed by the present invention.

[0114] Throughout this specification, the conventional one-letter and three-letter codes for the naturally occurring amino acids are used, as well as the recognized three-letter codes for other amino acids, such as Aib (2-aminoisobutyric acid), AZ K (6-azidolysine), etc. In addition, the amino acids abbreviated in the present specification are described according to the IUPAC-IUB nomenclature.

[0115] Alanine Ala, A Arginine Arg, R

[0116] Asparagine Asn, N Aspartic acid Asp, D

[0117] Cysteine Cys, C Glutamic acid Glu, E

[0118] Glutamine Gln, Q Glycine Gly, G

[0119] Histidine His, H Isoleucine Ile, I

[0120] Leucine Leu, L Lysine Lys, K

[0121] Methionine Met, M Phenylalanine Phe, F

[0122] Proline (Pro, P) Serine (Ser, S)

[0123] Threonine Thr, T Tryptophan Trp, W

[0124] Tyrosine Tyr, Y Valine Val, V

[0125] One aspect of the present invention is a composition for preventing, improving or treating graft-versus-host disease, comprising a glucagon-like peptide-2 (GLP-2) derivative. Specifically, the composition can be a pharmaceutical composition for preventing or treating graft-versus-host disease.

[0126] As a specific example of the present invention, the composition may include, but is not limited to, a pharmaceutical composition for preventing or treating steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative.

[0127] In the present invention, the term "graft-versus-host disease (GVHD)" refers to a disease in which an immune response occurs when T lymphocytes in the peripheral blood or bone marrow of a donor injected during an allogeneic transplant recognize the body of the host (i.e., the patient receiving the allogeneic transplant) as a foreign body and attack the body. In other words, graft-versus-host disease (GVHD) is a disease caused by injected live lymphocytes, and due to the immune response, symptoms such as liver dysfunction, skin lesions, jaundice, diarrhea, fever, and pancytopenia may occur, and in severe cases, the patient may die. Specifically, graft-versus-host disease is caused by an allogeneic transplant, and here, the allogeneic transplant may include, but is not limited to, at least one of hematopoietic stem cell transplantation (e.g., bone marrow transplantation, peripheral blood stem cell transplantation, and umbilical cord blood transplantation) and blood transfusion.

[0128] As a specific example of the present invention, the graft-versus-host disease may be, but is not limited to, acute graft-versus-host disease (aGVHD) or chronic graft-versus-host disease (cGVHD).

[0129] Chronic graft-versus-host disease (GVHD) typically develops more than 100 days after allogeneic transplantation and rarely occurs within 80 days or more than a year after transplantation. Chronic GVHD has a long incubation period and slowly manifests its effects on target organs. It is the leading cause of late mortality after allogeneic hematopoietic stem cell transplantation.

[0130] Acute graft-versus-host disease (GVHD) usually occurs within 100 days after allogeneic transplantation and is a major complication of allogeneic hematopoietic stem cell transplantation, occurring within 30 to 40 days after allogeneic hematopoietic stem cell transplantation and affecting the skin, liver, and gastrointestinal tract. This acute GVHD occurs in three stages: the first stage occurs before bone marrow transplantation and is a stage in which the patient's tissues are damaged. In some cases, bacterial infection, etc., stimulates the secretion of inflammatory cytokines, and antigen-presenting cells are activated by these inflammatory cytokines; the second stage is a stage in which T cells in the transplanted bone marrow cells are activated, and the already activated patient's antigen-presenting cells differentiate T cells into Th1 (T helper 1) cells, ultimately increasing the secretion of cytokines such as IL-2 and IFN-γ; the third stage is a stage in which the patient's organs are destroyed, and cytotoxic T cells and natural killer cells are activated by cytokines secreted by activated Th1 cells. These cells attack the patient's organs, leading to GVHD.

[0131] As a specific example of the present invention, graft-versus-host disease may be steroid-refractory graft-versus-host disease (SR-GVHD) that does not respond adequately to steroids, and may specifically be steroid-refractory acute graft-versus-host disease (SR-aGVHD) or steroid-refractory chronic graft-versus-host disease (SR-cGVHD), but is not limited thereto.

[0132] As a specific example of the present invention, steroid-refractory acute graft-versus-host disease (SR-aGVHD) refers to acute graft-versus-host disease that does not respond to standard doses of steroids in first-line therapy after allogeneic hematopoietic stem cell transplantation. Steroid-refractory acute graft-versus-host disease (SR-aGVHD) occurs in 10% to 30% of patients after allogeneic hematopoietic stem cell transplantation and is a disease with a low survival rate.

[0133] As a specific example of the present invention, the chronic graft-versus-host disease may be steroid-refractory chronic graft-versus-host disease (SR-cGVHD).

[0134] In the present invention, the term "glucagon-like peptide-2" or "GLP-2" refers to an agonist of the glucagon-like peptide-2 receptor, which may be in the form of a polypeptide or a long-acting conjugate, which is combined with a biocompatible substance capable of increasing the half-life of the polypeptide in vivo, but is not limited thereto. In the present invention, GLP-2 includes not only natural (e.g., human) GLP-2, but also its derivatives and conjugates. GLP-2 is the active ingredient contained in the pharmaceutical composition of the present invention and may be contained in the pharmaceutical composition in a pharmaceutically effective amount.

[0135] In the present invention, a "GLP-2 receptor agonist" refers to a substance that binds to the human glucagon-like peptide-2 receptor in vivo or isolated and induces physiological activity comparable to or similar to that of native GLP-2. For example, a GLP-2 agonist may include native GLP-2 or a GLP-2 derivative.

[0136] The amino acid sequence of natural GLP-2 is as follows:

[0137] GLP-2 (1-33)

[0138] HADGSFSDEMNTILDNLAARDFINWLIQTKITD(SEQ ID NO:1)

[0139] In the present invention, "GLP-2 derivatives" include peptides having one or more differences in amino acid sequence compared to native GLP-2, peptides in which the native GLP-2 sequence is modified; and / or mimetics of native GLP-2 that have the function of preventing, treating and / or improving graft-versus-host disease.

[0140] Specifically, the GLP-2 derivatives of the present invention may have a modification selected from substitution, addition, deletion, modification and a combination thereof in at least one amino acid in the natural GLP-2 sequence, but are not limited thereto. The added amino acid may also be a non-natural amino acid (e.g., a D-type amino acid), and in addition to natural amino acids, substitution of non-natural amino acids is also possible. The added amino acid sequence may be derived from natural GLP-2, but is not limited thereto. In the present invention, the modification of amino acids may refer to, but is not limited to, substitution, addition, deletion or a combination thereof of at least one amino acid; or independently, chemical substitution (e.g., α-methylation, α-hydroxylation, azide group substitution), deletion (e.g., deamination) and / or modification (e.g., N-methylation) of some groups of amino acid residues.

[0141] In a specific embodiment, the GLP-2 derivative of the present invention may exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology (or identity) with native GLP-2 in the amino acid sequence, and / or may be in a form in which some groups of the amino acid residues of the GLP-2 derivative are chemically substituted (e.g., α-methylation, α-hydroxylation, azide group substitution), deleted (e.g., deamination) and / or modified (e.g., N-methylation), but is not limited thereto.

[0142] In specific embodiments, the GLP-2 derivative may have, but is not limited to, a substituted, deleted, or modified N-terminal amino group. The GLP-2 derivative of the present invention can be prepared by deleting the α-amino group of the N-terminal histidine, synthesizing the N-terminal amino group by replacing the hydroxyl group or carboxyl group, deleting 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, or modifying the N-terminal amino group with two methyl groups.

[0143] Specifically, the GLP-2 derivative may be imidazoacetyl-dehistidyl-GLP-2 (CA-GLP-2), in which the α carbon of the histidine residue as the first amino acid at the N-terminus and the N-terminal amino group bound to the α carbon are missing; deaminohistidyl GLP-2 (DA-GLP-2), in which the N-terminal amino group of GLP-2 is missing; 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-dehistidyl 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 non-limiting examples, the structures of substances used to prepare GLP-2 derivatives are as follows.

[0144]

[0145] In the present invention, imidazoacetyl des histidyl (histidine), desaminohistidyl (histidine), β-hydroxyimidazopropionyl des histidyl (histidine), N-dimethylhistidyl (histidine) and β-carboxyimidazopropionyl des histidyl (histidine) are used with the same meanings as imidazoacetyl, des-amino-histidyl, β-hydroxy-imidazopropionyl, dimethyl-histidyl and β-carboxy-imidazopropionyl, respectively.

[0146] In a specific embodiment, the GLP-2 derivative may include, but is not limited to, modifications of at least one of amino acids 1, 2, 30, and 33 in SEQ ID NO: 1. Specifically, in the present invention, the modification of the amino acid may be a modification selected from substitution, addition, deletion, modification, and combinations thereof of at least one amino acid, and the added amino acid may be a non-natural amino acid (e.g., a D-amino acid), and substitution of 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 the present invention, the modification of the amino acid may refer to, but is not limited to, substitution, addition, deletion, or combinations thereof of at least one amino acid; or independently, chemical substitution (e.g., α-methylation, α-hydroxylation, azide group substitution), deletion (e.g., deamination), and / or modification (e.g., N-methylation) of some groups of the amino acid residue.

[0147] In a specific embodiment, the GLP-2 derivative may include, but is not limited to, an amino acid sequence represented by the following general formula 1:

[0148] [Formula 1]

[0149] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO: 10)

[0150] In general formula 1,

[0151] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0152] X2 is alanine, glycine, or Aib (2-aminoisobutyric acid);

[0153] X 30 is lysine or arginine; and

[0154] X 34 is one or more arbitrary amino acids or one or more modified arbitrary amino acids,

[0155] However, among the amino acid sequences represented by the general formula 1, sequences identical to SEQ ID NO: 1 are excluded.

[0156] In particular, the amino acid can be a natural amino acid or an unnatural amino acid, and the modifications are as described above.

[0157] In a specific embodiment, the GLP-2 derivative may include, but is not limited to, an amino acid sequence represented by the following formula 2:

[0158] [Formula 2]

[0159] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9)

[0160] In general formula 2,

[0161] X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine;

[0162] X2 is alanine, glycine, or Aib (2-aminoisobutyric acid);

[0163] X 30 is lysine or arginine; and

[0164] X 34 Lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine is absent or

[0165] However, among the amino acid sequences represented by the general formula 2, sequences identical to SEQ ID NO: 1 are excluded.

[0166] Specifically, the GLP-2 derivatives of the present invention may have, but are not limited to, a substitution of the second amino acid alanine of native GLP-2 with glycine or Aib (2-aminoisobutyric acid), a substitution of the 30th amino acid lysine with arginine, or a combination thereof. The GLP-2 derivatives may have a sulfhydryl group (e.g., cysteine), an amino group (e.g., lysine, arginine, glutamine, or histidine), or an azide group (e.g., 6-azidolysine) introduced into the C-terminus (e.g., the 33rd amino acid), but are not limited thereto.

[0167] Since binding occurs in the introduced groups when preparing long-acting conjugates of GLP-2 derivatives, this can be utilized to prepare GLP-2 conjugates with selectively controlled binding sites. Specifically, one end of the non-peptide linker can be bound to the hydroxyl group, sulfhydryl group, amino group or azide group of the GLP-2 derivative, and a substance that can increase the half-life in vivo (e.g., immunoglobulin Fc region) can be bound to the other end of the non-peptide linker. The sulfhydryl group, amino group or azide group can be introduced by adding amino acids to GLP-2, but is not limited thereto. The sulfhydryl group can be introduced by adding cysteine (C) to GLP-2; the amino group can be introduced by adding lysine (K), arginine (R), glutamine (Q) or histidine (H) to GLP-2; and the azide group can be introduced by adding 6-azidolysine ( AZ K) to introduce, but not limited to this.

[0168] Specifically, the GLP-2 derivative may have, but is not limited to, at least one residue that is cysteine, lysine, arginine, glutamine, histidine or 6-azidolysine.

[0169] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with glycine and introducing a sulfhydryl group (e.g., cysteine) at the C-terminus, more specifically, may include imidazoacetyl dehistidine, wherein the α carbon of the histidine residue as the first amino acid at the N-terminus and the N-terminal amino group bound to the α carbon are deleted, and may have, for example, the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0170] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with glycine and introducing an amino group (e.g., lysine) at the C-terminus, more specifically, may include imidazoacetyl dehistidine, wherein the α carbon of the histidine residue as the first amino acid at the N-terminus and the N-terminal amino group bound to the α atom carbon are deleted, and may have, for example, the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0171] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with glycine, replacing the 30th amino acid lysine of natural GLP-2 with arginine, and introducing an amino group (e.g., lysine) at the C-terminus. More specifically, it may include imidazoacetyl dehistidine, in which the α carbon of the histidine residue as the first amino acid of the N-terminus and the N-terminal amino group bound to the α carbon are deleted, and may have, for example, the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0172] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with glycine and introducing an azide group (e.g., 6-azidolysine) at the C-terminus, more specifically, may include imidazoacetyl dehistidine, wherein the α carbon of the histidine residue as the first amino acid at the N-terminus and the N-terminal amino group bound to the α carbon are deleted, and may have, for example, the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0173] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with glycine, replacing the 30th amino acid lysine of natural GLP-2 with arginine, and introducing a sulfhydryl group (e.g., cysteine) at the C-terminus. More specifically, it may include imidazoacetyl dehistidine, in which the α carbon of the histidine residue as the first amino acid of the N-terminus and the N-terminal amino group bound to the α carbon are deleted, and may have, for example, the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0174] Specifically, the GLP-2 derivative of the present invention may include replacing the second amino acid alanine of natural GLP-2 with 2-aminoisobutyric acid and introducing a sulfhydryl group (e.g., cysteine) at the C-terminus, and may have, for example, an amino acid sequence of SEQ ID NO: 8. More specifically, it may include imidazoacetyl dehistidine, in which the α carbon of the histidine residue as the first amino acid at the N-terminus and the N-terminal amino group bound to the α carbon are deleted, and may have, for example, an amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0175] The GLP-2 derivatives of SEQ ID NOs: 2 to 8 are listed in Table 1 below.

[0176] [Table 1]

[0177] name sequence SEQ ID NO: CA GLP-2KC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 2 CA GLP-2KK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDK]]> 3 CA GLP-2RK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDK]]> 4 <![CDATA[CA GLP-2K AZ K]]> <![CDATA[ ca HGDGFSDEMNTILDNLAARDFINWLIQTKITD AZ K]]> 5 CA GLP-2RC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDC]]> 6 CA GLP-2Aib <![CDATA[ ca HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 7 GLP-2Aib HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC 8

[0178] In Table 1, caH represents histidine substituted with imidazoacetyl desohistidine, Aib represents 2-aminoisobutyric acid, and AZ K represents 6-azido-L-lysine.

[0179] The GLP-2 derivative according to the present invention may be a peptide including the above-mentioned specific sequence, or a peptide consisting (substantially) of the above-mentioned specific sequence, but is not limited thereto.

[0180] At the same time, even if a peptide or GLP-2 derivative is described in the present application as 'composed of (consisting of) a specific sequence number', meaningless sequence additions, possible naturally occurring mutations or silent mutations before and after the amino acid sequence of the corresponding sequence number are not excluded, as long as the peptide or GLP-2 derivative has the same or equivalent activity as the peptide or GLP-2 derivative composed of the amino acid sequence of the corresponding sequence number, and obviously, in the case of having such sequence additions or mutations, the peptide or GLP-2 derivative also falls within the scope of the present invention.

[0181] Specifically, the GLP-2 derivative may be a derivative represented by the general formula 1 or 2, wherein (1) X2 is glycine or Aib, (2) X 30 is lysine or arginine, or (3) X2 is glycine or Aib and X 30 It is lysine or arginine, but is not limited thereto.

[0182] Specifically, the GLP-2 derivative can be a derivative represented by Formula 1 or 2, wherein

[0183] (1) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's cysteine,

[0184] (2) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's lysine,

[0185] (3) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It's lysine,

[0186] (4) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 is 6-azidolysine,

[0187] (5) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34It's cysteine,

[0188] (6) X1 is imidazoacetyl desahistidine, X2 is Aib, X 30 is lysine, and X 34 is cysteine, or

[0189] (7) X1 is histidine, X2 is Aib, X 30 is lysine, and X 34 It is, but not limited to, cysteine.

[0190] In the present invention, modifications used to prepare agonists, fragments, variants and derivatives of natural GLP-2 include modifications using L- or D-amino acids and / or non-natural amino acids; and / or modifications of the natural sequence or post-translational modifications (e.g., methylation, acylation, ubiquitination and intramolecular covalent bonding).

[0191] In the present invention, the GLP-2 derivative may be in a modified form, wherein the N-terminus and / or C-terminus are chemically modified or protected with an organic group, or amino acids are added to the termini of the GLP-2 derivative to protect the GLP-2 derivative from the effects of protein-cleaving enzymes in the body and increase its stability.

[0192] In particular, in the case of chemically synthesized peptides, since the N- and C-termini are charged, the N-terminus may be acetylated and / or the C-terminus may be amidated to remove the charge, but the present invention is not particularly limited thereto. Specifically, in the present invention, the GLP-2 derivative may have an unmodified or amidated C-terminus, but is not limited thereto.

[0193] The GLP-2 derivatives of the present invention can be synthesized by solid phase synthesis, can be produced by recombinant methods, and can be commercially manufactured as required.

[0194] In the present invention, the GLP-2 derivative may be in the form of a long-acting conjugate that is bound to a biocompatible substance capable of increasing the half-life of the GLP-2 derivative in vivo, but is not limited thereto. Compared to a GLP-2 derivative that is not bound to a biocompatible substance (e.g., an immunoglobulin Fc region), the long-acting conjugate may exhibit an increased duration of therapeutic effect. In the present invention, a conjugate containing a GLP-2 derivative has an increased half-life due to the binding of a biocompatible substance to the GLP-2 derivative, and is referred to as a "long-acting conjugate of a GLP-2 derivative" or a "long-acting conjugate of a GLP-2 derivative." In the present invention, the terms "long-acting conjugate" and "conjugate" are used interchangeably.

[0195] Also, such conjugates may not occur naturally.

[0196] In the GLP-2 derivative long-acting conjugate, the connection between the GLP-2 derivative and the biocompatible substance (e.g., immunoglobulin Fc region) can be a physical or chemical bond, or a non-covalent or covalent bond, and specifically can be a covalent bond, but is not limited thereto.

[0197] In the GLP-2 derivative long-acting conjugate, the method of linking the GLP-2 derivative and the biocompatible substance (eg, immunoglobulin Fc region) is not particularly limited, but the GLP-2 derivative and the biocompatible substance may be linked to each other via a linker.

[0198] Korean Patent Publication No. 10-2019-0037181 regarding long-acting conjugates of GLP-2 derivatives is incorporated herein by reference.

[0199] In a specific embodiment, the long-acting conjugate of the GLP-2 derivative of the present invention may have a structure represented by the following Chemical Formula 1, but is not limited thereto:

[0200] [Chemical Formula 1]

[0201] XL a -F

[0202] In Chemical Formula 1,

[0203] X may be a GLP-2 derivative described herein;

[0204] L can be a linker (e.g., a linker comprising ethylene glycol repeating units);

[0205] a can be 0 or a natural number, but when a is 2 or greater, each L is independent of each other;

[0206] F can be a biocompatible substance (e.g., an immunoglobulin Fc region) that can increase the in vivo half-life of X; and

[0207] "-" may be a chemical bond (eg, a covalent bond).

[0208] More specifically, X and L, and L and F may be linked to each other via a covalent bond. In this case, the conjugate may be a conjugate in which X, L, and F are each linked via a covalent bond in the order of Chemical Formula 1.

[0209] F may be directly linked to X (eg, a is 0 in Chemical Formula 1), or may be linked through a linker (L).

[0210] In the conjugate according to the present invention, X, ie the GLP-2 derivative, refers to the GLP-2 derivative described and / or defined herein.

[0211] In the conjugate according to the present invention, F is a substance capable of increasing the half-life of X (ie, the GLP-2 derivative according to the present invention), and corresponds to one component constituting a part of the conjugate of the present invention.

[0212] F and X may be bonded to each other 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.

[0213] F is a biocompatible substance that can increase the half-life of X, and can be selected from, for example, high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, sugars, heparin, and elastin, but is not particularly limited thereto.

[0214] The elastin may be human tropoelastin, which is a water-soluble precursor, and may be a polymer of some sequences or some repeating units in these sequences, and for example, includes all elastin-like polypeptides, but is not particularly limited thereto.

[0215] Examples of high molecular weight polymers include high molecular weight polymers selected from polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides (e.g., dextran), polyethylene ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, oligonucleotides, and any combination thereof, and the polysaccharide may include dextran, but is not particularly limited thereto.

[0216] Polyethylene glycol is a term that includes, but is not specifically limited to, all forms of ethylene glycol homopolymers, PEG copolymers, or monomethyl substituted PEG polymers (mPEG).

[0217] Biocompatible materials include, but are not limited to, polyamino acids, such as polylysine, polyaspartic acid, and polyglutamic acid.

[0218] The fatty acids may be, but are not particularly limited to, those that bind to albumin in vivo.

[0219] As an example of F, F may be an FcRn-binding substance, and specifically, the FcRn-binding substance may be an immunoglobulin Fc region, more specifically, an IgG Fc region, and even more specifically, an aglycosylated IgG4 Fc region, but is not particularly limited thereto.

[0220] 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.

[0221] In a specific embodiment, the long-acting conjugate of the present invention may be a GLP-2 derivative linked to the Fc region of an immunoglobulin, but is not limited thereto.

[0222] In the present invention, "immunoglobulin Fc region" refers to 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 heavy chain constant region 2 (CH2) and / or a heavy chain constant region 3 (CH3) portion, and more specifically, may further include a hinge region (the entire hinge region or a portion of the hinge region). The immunoglobulin Fc region may be a component that forms part of the conjugate of the present invention. The immunoglobulin Fc region specifically corresponds to F in Chemical Formula 1.

[0223] In this specification, the Fc region includes not only the native sequence obtained by papain digestion of immunoglobulins, but also derivatives, substitution products, and variants, for example, sequences in which one or more amino acid residues in the native sequence are altered by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, and thus the sequence differs from the native form. It is assumed that the derivatives, substitution products, and variants have the ability to bind to FcRn.

[0224] F (e.g., immunoglobulin Fc region) is a structure in which two polypeptide chains are linked by a disulfide bond, and may be a structure in which the two polypeptide chains are linked only by a nitrogen atom of one of the two chains, but is not limited thereto. Linkage via a nitrogen atom can be achieved by reductive amination to the epsilon amino atom of lysine or the N-terminal amino group.

[0225] Reductive amination refers to a reaction in which an amino group or an amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to form an amine, which is then reduced to form an amine bond. It is an organic synthesis reaction well known in the art.

[0226] As a specific example, the immunoglobulin Fc region can be linked via its N-terminal nitrogen atom.

[0227] Such an immunoglobulin Fc region may include, but is not limited to, a hinge region in a heavy chain constant region.

[0228] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

[0229] The term "hinge sequence" of the present invention refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region through a disulfide bond.

[0230] 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 not limited thereto:

[0231] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro(SEQ ID NO:11)。

[0232] The hinge sequence may be a sequence in which the 8th or 11th cysteine residue of the hinge sequence of SEQ ID NO: 11 is deleted, thereby comprising only one cysteine residue. The hinge sequence of the present invention may consist of 3 to 12 amino acids, which 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), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 18), 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-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:30).

[0233] Still more specifically, the hinge sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 21 (Pro-Ser-Cys-Pro) or SEQ ID NO: 30 (Ser-Cys-Pro).

[0234] 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 is a conjugate in which the Fc region is linked to the linker via the nitrogen atom of proline.

[0235] In a specific 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 heterodimer due to the presence of a hinge sequence. The conjugate represented by Chemical Formula 1 of the present invention may be in the form of a linker connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0236] The term "N-terminus" of the present invention refers to the amino terminus of a protein or polypeptide and may include the very end of the amino terminus, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acids from the very end. The immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.

[0237] The immunoglobulin Fc region of the present invention may be an extended Fc region comprising part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 2 (CL1), excluding only the heavy and light chain variable regions of an immunoglobulin, as long as it has substantially the same or improved effects compared to a native form. The immunoglobulin Fc region may be a region in which a relatively long portion of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0238] 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 more of a CH1 domain, a CH2 domain, a CH3 domain, or a CH4 domain and an immunoglobulin hinge region (or a portion of a hinge region); or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region, but is not limited thereto.

[0239] As an embodiment of the long-acting conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer, and one X molecule may be covalently linked to one Fc region in the dimer form. In this case, the immunoglobulin Fc and X may be linked to each other via the same linker. Alternatively, two X molecules may be symmetrically bound to one Fc region in the form of a dimer. In this case, the immunoglobulin Fc and X may be linked to each other via a linker. However, the immunoglobulin Fc region is not limited to the above examples.

[0240] The immunoglobulin Fc region of the present invention includes not only the native amino acid sequence but also its sequence derivatives. An amino acid sequence derivative refers to a sequence that differs from the native amino acid sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native amino acid sequence.

[0241] For example, for IgG Fc, amino acid residues 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 modification sites.

[0242] Various types of derivatives are possible, such as derivatives in which sites capable of forming disulfide bonds are deleted, derivatives in which several amino acids at the N-terminus of the native Fc are deleted, or derivatives in which a methionine residue is added to the N-terminus of the native Fc. To eliminate effector functions, complement binding sites, such as the C1q binding site, may be deleted, or ADCC (antibody-dependent cell-mediated cytotoxicity) sites may be deleted. Techniques for preparing such sequence derivatives of immunoglobulin Fc regions are disclosed in WO 97 / 34631, WO 96 / 32478, and the like.

[0243] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are known in the art (H. Neurath, RL Hill, The proteins, Academic Press, New York, 1979). The most common exchanges occur between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications can be achieved by phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0244] The above-mentioned Fc derivatives exhibit biological activities comparable to those of the Fc region of the present invention, and may have enhanced structural stability of the Fc region against heat, pH, and the like.

[0245] This Fc region can be obtained from the natural form isolated from living animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or can be a recombinant form obtained from transformed animal cells or microorganisms, or a derivative thereof. Here, the method for obtaining the Fc region from the natural form can be a method of isolating the whole immunoglobulin from a living human or animal body and then treating it with a protease. When treated with papain, the immunoglobulin is cut into Fab and Fc, and when treated with pepsin, the immunoglobulin is cut into pF'c and F(ab)2. Fc or pF'c can be separated by size exclusion chromatography, etc. In a more specific embodiment, the Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism, which is a humanized Fc region.

[0246] The immunoglobulin Fc region can be in the form of native sugar chains, sugar chains increased compared to native sugar chains, sugar chains reduced compared to native sugar chains, or a form in which sugar chains have been deleted. Conventional methods, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms, can be used to increase or decrease such immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region in which sugar chains have been deleted from the Fc significantly reduces the binding ability to complement (C1q), reduces or eliminates antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, and therefore does not induce unnecessary immune responses in the body. In this regard, the form that is more suitable for its original purpose as a drug carrier is the immunoglobulin Fc region in which sugar chains have been deleted or the aglycosylated immunoglobulin Fc-region.

[0247] In the present invention, "sugar chain deleted or deglycosylated" refers to an Fc region in which sugars have been enzymatically deleted, and aglycosylation refers to an Fc region produced in a prokaryotic organism, in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0248] Meanwhile, the immunoglobulin Fc region may be of animal origin, such as human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, and in a more specific embodiment, may be of human origin.

[0249] The immunoglobulin Fc region can be derived from IgG, IgA, IgD, IgE or IgM, or a combination thereof or a hybrid thereof. In a more specific embodiment, the immunoglobulin Fc region is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In a still more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region. In the most specific embodiment, the immunoglobulin Fc region is, but is not limited to, an aglycosylated Fc region derived from human IgG4.

[0250] As a specific example, the immunoglobulin Fc region can be a fragment of human IgG4 Fc, which can be a homodimer in which two monomers are linked by a disulfide bond (interchain form) between the third amino acid cysteine of each monomer. In this case, the homodimer has / can have two disulfide bonds (intrachain form), namely, a disulfide bond between the cysteines at positions 35 and 95 and between the cysteines at positions 141 and 199 in each monomer.

[0251] The number of amino acids in each monomer can be composed of 221 amino acids, and the number of amino acids forming a homodimer can be composed of a total of 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc region can be a homodimer (here, two monomers having an amino acid sequence of SEQ ID NO: 31 (consisting of 221 amino acids) form a homodimer through a disulfide bond between the third amino acid cysteine of each monomer, and the monomers of the homodimer independently form an internal disulfide bond between the cysteines at positions 35 and 95, and independently form an internal disulfide bond between the cysteines at positions 141 and 199), which includes the amino acid sequence of SEQ ID NO: 32 (consisting of 442 amino acids), but is not limited thereto.

[0252] Meanwhile, in the present invention, the term "combination" in relation to immunoglobulin Fc regions refers to the formation of a dimer or multimer by a polypeptide encoding a single-chain immunoglobulin Fc region from the same source and a single-chain polypeptide from a different source. In other words, a dimer or multimer can be prepared from two or more Fc regions selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc regions.

[0253] In the present invention, the term "hybrid" refers to the presence of sequences corresponding to immunoglobulin Fc regions from two or more different sources within a single-chain immunoglobulin constant region. In the present invention, various types of hybridization are possible. In other words, hybridization of domains consisting of one to four of the CH1, CH2, CH3, and CH4 domains of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible, and may include a hinge.

[0254] Meanwhile, IgG can also be divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and in the present invention, a combination or hybrid of these subclasses is also possible. Specifically, this includes IgG2 and IgG4 subclasses, and most specifically, the Fc region of IgG4, which has almost no effector functions such as complement-dependent cytotoxicity (CDC).

[0255] The conjugates may have increased duration of efficacy compared to native GLP-2 or compared to X in which F is unmodified, and such conjugates include, but are not limited to, not only the forms described above, but also forms encapsulated in biodegradable nanoparticles.

[0256] In Chemical Formula 1, the linker L may be a peptide linker or a non-peptide linker (eg, a linker containing ethylene glycol repeating units).

[0257] When L is a peptide linker, the peptide linker 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 linkers can be used to link F and X, examples of which include [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, where x can be a natural number of 1 or greater. However, the peptide linker is not limited to these examples.

[0258] In the present invention, a "non-peptidyl linker" includes a biocompatible polymer having two or more repeating units bonded thereto. The repeating units are linked to each other by arbitrary covalent bonds rather than peptide bonds. In the present invention, the non-peptide linker contains a reactive group at the end and can form a conjugate by reacting with another component constituting the conjugate. The non-peptide linker can be a component that forms part of the conjugate of the present invention and corresponds to L in Chemical Formula 1.

[0259] As the non-peptide linker that can be used in the present invention, any non-peptide polymer that is resistant to in vivo proteases can be used without limitation.

[0260] In the present invention, non-peptide linker can be used interchangeably with non-peptide polymer.

[0261] Although not particularly limited thereto, the non-peptide linker can be selected from fatty acids, sugars, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. The high molecular weight polymer can be selected from biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides (e.g., dextran), polyethylene ethyl ether, polylactic acid (PLA) and polylactic glycolic acid (PLGA), lipopolymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof.

[0262] As the non-peptide linker that can be used in the present invention, any polymer that is resistant to in vivo proteases can be used without limitation. Specifically, the molecular weight of the non-peptide polymer can be, but is not limited to, in the range of greater than 0 to about 100 kDa, in the range of about 1 to about 100 kDa, particularly in the range of about 1 to about 50 kDa, in the range of about 1 to about 20 kDa, in the range of about 1 to about 10 kDa, or in the range of about 3.4 kDa to 10 kDa.

[0263] Although not particularly limited thereto, the non-peptide linker may be a linker containing ethylene glycol repeating units, for example, polyethylene glycol, and these derivatives known in the art and derivatives that can be easily prepared within the technical level of the art are also included in the scope of the present invention.

[0264] The repeating unit of the non-peptide linker can be an ethylene glycol repeating unit. Specifically, the non-peptide linker can contain an ethylene glycol repeating unit and a functional group used when preparing the conjugate at the end. The long-acting conjugate according to the present invention can be in a form in which X and F are connected by a functional group, but is not limited thereto. In the present invention, the non-peptide linker can contain two or three or more functional groups, and each functional group can be the same or different from each other, but is not limited thereto.

[0265] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following Chemical Formula 4:

[0266] [Chemical Formula 4]

[0267]

[0268] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

[0269] In long-acting conjugates, the PEG moiety may contain, but is not limited to, -(CH2CH2O) n - structure and between the connecting element and -(CH2H2O) n - between the oxygen atoms.

[0270] In a specific embodiment, the conjugate may be a structure in which the GLP-2 derivative and the immunoglobulin Fc region (F) are covalently linked via a linker containing ethylene glycol repeating units, but is not limited thereto.

[0271] Polyethylene glycol is a term that includes, but is not specifically limited to, all forms of ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0272] As a specific example, the ethylene glycol repeating unit can be composed of, for example, [OCH2CH2] n and the value of n is a natural number and can be determined so that [OCH2CH2] n The average molecular weight of the moiety, for example, the number average molecular weight is greater than 0 to about 100 kDa, but is not limited thereto. As another example, the value of n is a natural number, and [OCH2CH2] nThe average molecular weight of the moiety, for example, a number average molecular weight of about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to 25 kDa, about 1 to 20 kDa, about 1 to 15 kDa, about 1 to 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 15 kDa, about 3 to about 13 kDa, about 3 ... kDa, about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa, about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, about 3.4 or about 10 kDa, but are not limited thereto.

[0273] In the present invention, the term "about" includes ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values within the range equal to or similar to the value after the term "about", but is not limited thereto.

[0274] As the non-peptide linker of the present invention that binds to the immunoglobulin Fc region, not only one polymer but also a combination of different kinds of polymers can be used.

[0275] In a specific embodiment, both ends of the non-peptide linker can be bound to the sulfhydryl group, amino group or hydroxyl group of the immunoglobulin Fc region and the sulfhydryl group, amino group, azide group or hydroxyl group of GLP-2, but are not limited thereto.

[0276] Specifically, the non-peptide linker may contain, but is not limited to, a reactive group at each of the two ends that can bind to immunoglobulin Fc and GLP-2 or a derivative thereof, specifically, a reactive group that can bind to the sulfhydryl group of cysteine in the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine; and / or a hydroxyl group located at the C-terminus and bound to the sulfhydryl group of cysteine of GLP-2; an amino group of lysine, arginine, glutamine and / or histidine; an azide group of azidolysine; and / or a hydroxyl group.

[0277] More specifically, the reactive group of the non-peptide polymer may be, but is not limited to, one or more selected from an aldehyde group, a maleimide group, and a succinimide derivative.

[0278] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0279] Among the above, as the succinimide derivatives, succinimidylcarboxymethyl, succinimidylvalerate, succinimidylmethylbutyrate, succinimidylmethylpropionate, succinimidylbutyrate, succinimidylpropionate, N-hydroxysuccinimide, hydroxysuccinimidyl or succinimidyl carbonate can be used, but the succinimide derivatives are not limited thereto.

[0280] The non-peptide linker is linked to a biocompatible substance (eg, immunoglobulin Fc) and a GLP-2 derivative via the reactive group as described above, and can be converted into a non-peptide polymer linking portion.

[0281] The final products produced by reductive alkylation of aldehyde bonds are much more stable than those linked via amide bonds. The aldehyde-reactive group reacts selectively at the N-terminus at low pH values and can form covalent bonds with lysine residues at high pH values, for example, pH 9.0.

[0282] The terminal reactive groups of the non-peptide linker of the present invention may be the same as or different from each other. The non-peptide linker may have an aldehyde reactive group at the terminal, and the non-peptide linker may have an aldehyde group and a maleimide reactive group at the terminal, or may have an aldehyde group or a succinimide reactive group at the terminal, but is not limited thereto.

[0283] For example, the non-peptide linker can have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. As another example, the non-peptide linker can have a succinimide group at one end and a propionaldehyde group or a butyraldehyde group at the other end.

[0284] In the case of using polyethylene glycol having a hydroxyl-reactive group at the propionic acid terminus as a non-peptide linker, the conjugate of the present invention can be prepared by activating the hydroxyl group with various reactive groups through known chemical reactions, or by using commercially available polyethylene glycol having modified reactive groups.

[0285] In a specific embodiment, the reactive group of the non-peptide linker can be linked to a cysteine residue of GLP-2, more specifically, to a -SH group of cysteine, but is not limited thereto.

[0286] If maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of the GLP-2 derivative via a thioether bond, and the aldehyde group can be linked to the -NH2 group of the immunoglobulin Fc via a reductive alkylation reaction, but the non-peptide linker is not limited thereto and this is just an example.

[0287] By this reductive alkylation, the N-terminal amino group of the immunoglobulin Fc region can be linked 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 a structure in which one end of the PEG is linked to the sulfur atom on the cysteine of GLP-2 through a thioether bond. The above-mentioned thioether bond may include structure.

[0288] However, the non-peptide linker is not particularly limited to the above examples, and this is just one example.

[0289] In the conjugate, the reactive group of the non-peptide linker may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is an example.

[0290] In the conjugate, the GLP-2 derivative may be linked via the C-terminus to a non-peptide linker having a reactive group, but this is an example.

[0291] In the present invention, "C-terminus" refers to the carboxyl terminus of a peptide, and for the purposes of the present invention, refers to a position capable of binding to a non-peptide polymer. For example, the C-terminus may include, but is not limited to, not only the terminal amino acid residue at the C-terminus, but may also include all amino acid residues surrounding the C-terminus, specifically, the first to twentieth amino acid residues from the terminal end.

[0292] As a specific example, the conjugate represented by Chemical Formula 1 may have a structure represented by the following Chemical Formula 2 or 3.

[0293] [Chemical Formula 2]

[0294]

[0295] [Chemical Formula 3]

[0296] X-(CH2)3[OCH2CH2] n O(CH2)3-F

[0297] In Chemical Formula 2 or 3, X may be the above-mentioned GLP-2 derivative;

[0298] F may be an immunoglobulin Fc fragment; and

[0299] n can be a natural number. In this case, the description of n is as described above.

[0300] As a specific example, the long-acting conjugate represented by Chemical Formula 2 is a structure in which X of the GLP-2 derivative and F of the immunoglobulin Fc region are covalently linked via an ethylene glycol repeating moiety, and X can be respectively linked to the succinimide ring of Chemical Formula 2, and F can be linked to an oxypropylene group of Chemical Formula 2. The long-acting conjugate represented by Chemical Formula 3 is a structure in which X of the GLP-2 derivative and F of the immunoglobulin Fc region are covalently linked via an ethylene glycol repeating moiety, and X can be respectively linked to an oxypropylene group of Chemical Formula 3, and F can be linked to another oxypropylene group of Chemical Formula 3.

[0301] As a specific example, in Chemical Formula 2 or 3, the value of n can be determined so that [OCH2CH2] n The average molecular weight of the moiety, for example, the number average molecular weight is 1 to 100 kDa, or 1 to 20 kDa, or 10 kDa, but is not limited thereto.

[0302] In an embodiment, the succinimide ring of Chemical Formula 2 or the site where X is linked to the succinimide ring of Chemical Formula 2 may be the sulfur atom of the C-terminal cysteine of X. The oxypropylene group of Chemical Formula 3 or the site where X is linked to the oxypropylene group of Chemical Formula 3 may be the sulfur atom of the C-terminal cysteine of X.

[0303] The site in F that is linked to the oxypropylene group of Chemical Formula 2 or Chemical Formula 3 is not particularly limited. In an embodiment of the present invention, the site at which F is linked to the oxypropylene group may be the N-terminal nitrogen of F or a nitrogen atom of an internal residue (e.g., the epsilon nitrogen of lysine). In a specific embodiment of the present invention, the site at which F is linked to the oxypropylene group of Chemical Formula 2 or Chemical Formula 3 may be, but is not limited to, the N-terminal proline of F.

[0304] Meanwhile, the GLP-2 derivative or the long-acting conjugate thereof according to the present invention includes all forms, such as itself, their salts (eg, pharmaceutically acceptable salts), and their solvates.

[0305] The GLP-2 derivative or its long-acting conjugate may be in any pharmaceutically acceptable form.

[0306] The type of salt is not particularly limited. However, a salt that is safe and effective for a subject (eg, a mammal) may be preferred, but is not particularly limited thereto.

[0307] In the present invention, 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, toluene p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Salts derived from suitable bases can include salts of alkali metals (such as sodium and potassium), alkaline earth metals (such as magnesium), ammonium etc.

[0308] The term "solvate" used in the present invention refers to a complex formed by the peptide, compound or salt thereof according to the present invention and solvent molecules.

[0309] The GLP-2 derivatives of the present invention can be synthesized by solid phase synthesis, can be produced by recombinant methods, or can be commercially manufactured as required, or commercially available products can be used.

[0310] GLP-2 derivatives, their long-acting conjugates and / or compositions containing them can be used to prevent, improve or treat graft-versus-host disease in a subject. GLP-2 derivatives, their long-acting conjugates and / or compositions containing them can be used to prevent, improve or treat steroid-refractory graft-versus-host disease in a subject.

[0311] GLP-2 derivatives, long-acting conjugates thereof and / or compositions containing them can be used to prevent, improve or treat steroid-refractory graft-versus-host disease in a subject, and can be administered in combination with steroids.

[0312] An aspect of implementing the present invention is a pharmaceutical composition for preventing, ameliorating or treating steroid-refractory graft-versus-host disease, comprising a GLP-2 derivative and / or a long-acting conjugate thereof, wherein the GLP-2 derivative and / or a long-acting conjugate thereof and a steroid are administered in combination.

[0313] Specifically, aspects of the present invention provide a combination, pharmaceutical composition, or pharmaceutical kit comprising a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid. As a specific example, a combination, pharmaceutical composition, or pharmaceutical kit for preventing, ameliorating, or treating steroid-refractory graft-versus-host disease is provided.

[0314] In the present invention, the term "combination" refers to the combined administration of a GLP-2 derivative and / or its long-acting conjugate with a steroid and can be understood to have the same meaning as "used in combination." This also includes, but is not limited to, pharmaceutical compositions and pharmaceutical kits in which a GLP-2 derivative and / or its long-acting conjugate is used in combination with a steroid.

[0315] Specifically, the GLP-2 derivative and / or its long-acting conjugate and the steroid can be administered in combination simultaneously, sequentially or in reverse order.

[0316] The combined administration may be

[0317] i) combined administration using a pharmaceutical composition containing a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid in a mixture or in different isolated forms, or

[0318] ii) administering a pharmaceutical composition containing a GLP-2 derivative and / or a long-acting conjugate thereof and a pharmaceutical composition containing a steroid simultaneously, sequentially or in reverse order in combination, but not limited thereto.

[0319] The combination or composition can be

[0320] a) administered as a mixture containing a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid, or

[0321] b) Administration in the form of separation of the GLP-2 derivative and / or its long-acting conjugate from the steroid, but not limited thereto.

[0322] In the case of the separated forms in b), the GLP-2 derivative and / or its long-acting conjugate and the steroid can be formulated together in different separated forms; or can be formulated into different preparations so that the different preparations are administered simultaneously, separately, sequentially or in reverse order.

[0323] In the present invention, "administered in combination", "used in combination" or "using in combination" should be understood not only to mean simultaneous administration, but also to mean a form of administration in which the GLP-2 derivative and / or its long-acting conjugate and the steroid act together in a subject so that each substance performs at a level equal to or higher than its original function. Therefore, when the term "used in combination" is used herein, it should be understood that combined use refers to administration simultaneously, separately, sequentially or in reverse order, and the order is not limited. In the case of sequential, reverse order or separate administration, the order of administration is not particularly limited, but the interval between the second and subsequent administrations of the components should be such that the beneficial effects of the combined use are not lost.

[0324] The preferred dosage of the pharmaceutical composition according to the present invention and the composition used in combination varies according to the patient's condition and body weight, disease extent, drug form, route of administration and cycle, but those of ordinary skill in the art can appropriately select. However, in order to achieve the desired effect, the pharmaceutical composition and the composition used in combination can be used at 0.0001 to 500 mg / kg per day, particularly 0.001 to 500 mg / kg. The dosage can be applied once a day or divided into several dosage administrations. However, the scope of the present invention is not limited by dosage.

[0325] In the present invention, the term "composition comprising a combination" may include, but is not limited to, a combination comprising a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid itself, or a composition comprising such a combination and having therapeutic use. For example, such a composition may be used to prevent, improve, or treat steroid-refractory graft-versus-host disease, but is not limited thereto. In this application, the term "composition comprising a combination" may be used interchangeably with "composition."

[0326] The composition containing the combination according to the present invention is used for administering the GLP-2 derivative and / or its long-acting conjugate in combination with a steroid, and the GLP-2 derivative and / or its long-acting conjugate and the steroid can be formulated as a single preparation.

[0327] The term "kit" in the present invention may include a combination or composition according to the present invention for administering a GLP-2 derivative and / or a long-acting conjugate thereof in combination with a steroid. Specifically, the kit according to the present invention may include a) a single formulation of a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid; or b) separate formulations of a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid, and may further contain two or more substances required for combined administration, but is not limited thereto.

[0328] The present invention provides a combination therapy to verify that the effect of preventing, improving or treating steroid-refractory graft-versus-host disease is significantly improved when a GLP-2 derivative and / or a long-acting conjugate thereof is used in combination with a steroid, compared with the case where a GLP-2 derivative and / or a long-acting conjugate thereof is used alone.

[0329] In the present invention, term " steroid " is the general name of the fat-soluble compound with steroid core, as sterol, bile acid and sex hormone.Steroid is a kind of lipid and organic compound that does not contain fatty acid, and has common basic structure, wherein three rings of each free six carbon atoms are combined with a ring (i.e. four rings) of five carbon atoms.Steroid is divided into sugar regulating steroid and mineral steroid according to its function, i.e. the steroid that participates in fluid and blood pressure regulation.Steroid also can be divided into synthetic hormone steroid and artificial synthetic steroid that are produced naturally in vivo.The example of steroid hormone comprises corticosteroid, and it comprises glucocorticoid and mineralocorticoid.

[0330] In the present invention, the corticosteroid is a drug closely related to Cortisol, which is a hormone naturally produced in the adrenal cortex, and examples thereof include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, deltasone, triamcinolone acetonide, mometasone, and fluticasone, but are not limited thereto.

[0331] Corticosteroids work by blocking the production of substances (such as prostaglandins) that cause allergic and inflammatory responses, but they can interfere with the function of white blood cells, which destroy foreign substances and enable the immune system to function properly, leading to side effects such as increased susceptibility to infection.

[0332] Corticosteroids can be used with other medications and are available for both short-term and long-term use. However, when used long-term and absorbed into the system, they can cause side effects such as increased appetite and weight gain, fat deposits in areas such as the chest and face, a buildup of water and salts – leading to swelling and edema, high blood pressure, diabetes, osteoporosis, and cataracts.

[0333] In the present invention, steroids include corticosteroids, and steroids are active ingredients contained in the pharmaceutical composition of the present invention and can be contained in the pharmaceutical composition in a pharmaceutically effective amount.

[0334] The combination, pharmaceutical composition or pharmaceutical kit of the present invention can be used to prevent, improve or treat steroid-refractory graft-versus-host disease.

[0335] In the present invention, the term "prevention" refers to any action that completely or partially inhibits or delays the onset of graft-versus-host disease and / or steroid-refractory graft-versus-host disease by administering a GLP-2 derivative, a long-acting conjugate of a GLP-2 derivative, and / or a composition containing them, or a combination or composition containing a GLP-2 derivative and / or its long-acting conjugate and a steroid. The term "treatment" refers to any action that completely or partially improves or benefits the symptoms of graft-versus-host disease and / or steroid-refractory graft-versus-host disease, and includes alleviating / improving symptoms, alleviating symptom distress, reducing the incidence of graft-versus-host disease and / or steroid-refractory graft-versus-host disease, or other changes that increase the patient's response to treatment. The term "improvement" refers to any action that at least reduces a parameter associated with the condition being treated (e.g., the severity of symptoms) by administering a GLP-2 derivative, a long-acting conjugate thereof, or a composition containing them.

[0336] In the present invention, term " administration " refers to that predetermined substance is introduced into patient by any appropriate method, and the route of administration of composition is not specifically limited, but composition can be applied by any general route, and by these routes, composition can reach the target in living body. For example, the composition can be applied by oral route or parenteral route, including intradermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary artery, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal route, but route of administration is not limited thereto. Meanwhile, because peptide can be digested when orally administered, it is desirable that oral composition be formulated to be coated with active agent or prevent degradation in the stomach. Specifically, composition of the present invention can be applied in the form of injection. In the present invention, composition can be applied by any device that can transport active ingredient to target cell.

[0337] In the present invention, a "subject" refers to a subject requiring an allogeneic transplant, specifically a subject suspected of developing graft-versus-host disease and / or steroid-refractory graft-versus-host disease due to an allogeneic transplant, and is a subject who has or is at risk of developing such disease. Specifically, the subject may be a mammal, including humans, mice, livestock, and the like, but includes any subject that can be treated with the GLP-2 derivatives, long-acting conjugates thereof, and / or compositions containing the same, of the present invention, without limitation. Humans may be excluded from the subject list of the present invention, but the subject list is not limited thereto.

[0338] In specific embodiments, the composition of the present invention can be administered to a subject together with, before, or after an allogeneic transplant. For example, the composition of the present invention can be administered within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, or within 1 week, 2 weeks, or 1 month before an allogeneic transplant, or within 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, or within 1 week, 2 weeks, or 1 month after an allogeneic transplant, but is not limited thereto.

[0339] In a specific embodiment, the composition of the present invention can prevent, treat or improve graft-versus-host disease and / or steroid-refractory graft-versus-host disease by causing one or more of increased survival rate, increased small intestine length and increased colon length in a subject administered the composition, but is not limited thereto.

[0340] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent. Such a pharmaceutically acceptable carrier, excipient or diluent may not be naturally occurring.

[0341] In the present invention, the term "pharmaceutically acceptable" refers to an amount sufficient to show a therapeutic effect and not causing side effects, and those of ordinary skill in the art can easily determine based on factors known in the medical field, such as disease type, patient's age, weight, health and sex, patient's sensitivity to drugs, route of administration, method of administration, number of administrations, treatment period, and the drugs mixed or used simultaneously. The pharmaceutical composition of the present invention can additionally contain a pharmaceutically acceptable excipient. Excipient is not specifically limited to this, but when administered orally, adhesives, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, etc. can be used. In the case of injection, buffer, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers can be used in the mixture. In the case of topical administration, alkali, excipients, lubricants, and preservatives can be used.

[0342] The preparation of the composition of the present invention can be prepared in various ways by mixing the composition with the above-mentioned pharmaceutically acceptable excipients. For example, when administered orally, the composition can be prepared in the form of tablets, lozenges, capsules, tinctures (elixirs), suspensions, syrups, wafers, etc. In the case of injection, the composition can be prepared into unit dose ampoules or multiple dosage forms. The composition can be formulated as a solution, suspension, tablet, pill, capsule, sustained-release formulation, etc.

[0343] Meanwhile, as examples of carriers, excipients and diluents suitable for preparation, glucose, fructose, galactose, lactose, maltose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, amino acid (e.g., methionine), gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, sodium acetate, cellulose, methylcellulose, microcrystalline cellulose, polysorbate, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used. Said composition can additionally contain fillers, anticoagulants, lubricants, wetting agents, flavorings, preservatives etc.

[0344] The pharmaceutical composition of the present invention may have any one formulation selected from tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, and suppositories.

[0345] The composition can be formulated into a unit dosage form suitable for administration into a patient, in particular, formulated into a preparation for administering a protein drug according to conventional methods in the pharmaceutical field, and administered orally or parenterally using administration methods commonly used in the art, including intradermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary artery, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal or rectal routes, but not limited thereto.

[0346] GLP-2 derivatives or conjugates thereof can be used by mixing with various pharmaceutically acceptable carriers, such as saline solutions or organic solvents. Carbohydrates such as glucose, sucrose, or dextrose; antioxidants such as ascorbic acid or glutathione; chelating agents, low molecular weight proteins, or other stabilizers can be used as agents to increase stability or absorption.

[0347] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of active ingredient, as well as various related factors, such as the disease to be treated, the route of administration, the patient's age, sex, and weight, and the severity of the disease. Specifically, the composition of the present invention may contain a pharmaceutically effective amount of a GLP-2 derivative, but is not limited thereto.

[0348] "Containing a pharmaceutically effective amount of a GLP-2 derivative, its long-acting conjugate, and / or steroid" means that the desired pharmacological activity of the GLP-2 derivative, its long-acting conjugate, and / or steroid can be achieved. This may mean, but is not limited to, no or minimal toxicity or side effects in the administered subject at a pharmaceutically acceptable level. This pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, the patient, the formulation, and other factors.

[0349] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain 0.01% to 99% weight / volume of the GLP-2 derivative and / or its long-acting conjugate based on the total volume of the composition, and may contain 1% to 99.99% weight / volume of a pharmaceutically acceptable excipient.

[0350] The total effective amount of the composition of the present invention can be administered to the patient in a single dose or in multiple doses over a long period of time through a fractionated treatment regimen. Depending on the severity of the disease, the pharmaceutical composition of the present invention may have different contents of active ingredients. Specifically, the preferred total dose of the GLP-2 derivative and / or conjugate of the present invention may be, but is not limited to, about 0.0001 mg to 500 mg per kg of patient body weight per day. However, since the dose of the conjugate is determined as an effective dose for the patient by considering not only the route of administration and the number of treatments of the pharmaceutical composition, but also various factors such as the patient's age, weight, health status and gender, severity of the disease, diet and excretion rate. Taking these points into account, a person of ordinary skill in the art will be able to determine the appropriate effective dose for the specific use of the composition of the present invention. There are no particular restrictions on the formulation, route of administration and method of administration of the pharmaceutical composition of the present invention, as long as the effects of the present invention can be demonstrated.

[0351] The pharmaceutical composition of the present invention has excellent in vivo duration and efficacy, and thus can significantly reduce the number and frequency of administration of the pharmaceutical preparation of the present invention, but is not limited thereto.

[0352] Another aspect of the present invention provides a food composition for preventing or improving graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0353] Yet another aspect of the present invention provides a food composition for preventing or improving steroid-refractory graft-versus-host disease, which contains a GLP-2 derivative and / or a long-acting conjugate thereof.

[0354] The food composition for preventing or improving steroid-refractory graft-versus-host disease is administered in combination with a steroid.

[0355] The GLP-2 derivatives, long-acting conjugates thereof, steroids, prevention, improvement, graft-versus-host disease or steroid-refractory graft-versus-host disease, etc. are as described above.

[0356] 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 peptide (peptide itself or its long-acting conjugate) can be added as is or used together with other foods or food ingredients, and the composition can be appropriately used according to a conventional method. The amount of the active ingredient mixed can be appropriately determined according to the intended purpose (prevention, health or treatment).

[0357] In the present invention, the term "health functional food" refers to a food manufactured or processed to supplement health by using specific ingredients as raw materials or by extracting, concentrating, purifying, or mixing specific ingredients contained in food raw materials. The food is designed and processed so that the ingredients can fully exert bioregulatory functions such as biodefense, biorhythm regulation, disease prevention, and recovery from disease. This health food composition can exert functions related to disease prevention and recovery.

[0358] Yet another aspect of the present invention provides a method for preventing, improving or treating graft-versus-host disease, which comprises administering a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing the same to a subject in need thereof.

[0359] Yet another aspect of the present invention provides a method for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises administering a GLP-2 derivative, a long-acting conjugate thereof and / or a composition containing them, or a composition containing a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid to a subject in need thereof.

[0360] As another specific example, the method can be a method for preventing, improving or treating graft-versus-host disease and / or steroid-refractory graft-versus-host disease, which comprises administering and / or using the combination, pharmaceutical composition or pharmaceutical kit of the present invention to a subject in need thereof.

[0361] As yet another specific example, the method is a method for preventing, improving or treating graft-versus-host disease, which comprises administering and / or using a pharmaceutically effective amount of a composition containing a GLP-2 derivative and / or a long-acting conjugate thereof to a subject in need thereof, and a method for preventing, improving or treating steroid-refractory graft-versus-host disease, which comprises administering and / or using a pharmaceutically effective amount of a composition containing a steroid to a subject in need thereof.

[0362] The GLP-2 derivatives, long-acting conjugates thereof, steroids, combined use, compositions, subjects, prevention, treatment, improvement, graft-versus-host disease and / or steroid-refractory graft-versus-host disease, etc. are as described above.

[0363] The methods of the present invention may include administering a GLP-2 derivative, a long-acting conjugate thereof, and / or a composition containing them, or a composition containing a GLP-1 derivative and / or a long-acting conjugate thereof and a steroid in a pharmaceutically effective amount. The treating physician may determine the appropriate total amount to be used per day within the scope of sound medical judgment, and may administer the drug once or more in a single dose. However, for the purposes of the present invention, it is expected that a specific therapeutically effective amount will be applied differently to a specific patient based on various factors, such as the type and extent of the response to be achieved, the specific composition, including in some cases whether other formulations are used, the patient's age, weight, general health, sex and diet, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, and the drugs used together with or concurrently with the specific composition, as well as similar factors well known in the medical field.

[0364] Yet another aspect of the present invention provides use of GLP-2 derivatives, long-acting conjugates thereof and / or compositions containing the same in preventing, improving or treating graft-versus-host disease.

[0365] Yet another aspect of the present invention provides use of a GLP-2 derivative, a long-acting conjugate thereof, and / or a composition containing the same in preventing, improving, or treating steroid-refractory graft-versus-host disease.

[0366] Yet another aspect of the present invention provides use of a composition comprising a GLP-2 derivative and / or a long-acting conjugate thereof and a steroid in preventing, improving or treating steroid-refractory graft-versus-host disease.

[0367] The GLP-2 derivative, its long-acting conjugate, composition, steroid, graft-versus-host disease or steroid-refractory graft-versus-host disease, etc. are as described above.

[0368] Yet another aspect of the present invention provides use of a GLP-2 derivative, a long-acting conjugate thereof, and / or a composition containing the same in the preparation of a medicament for preventing, improving, or treating steroid-refractory graft-versus-host disease.

[0369] Yet another aspect of the present invention provides the use of the combination, pharmaceutical composition or pharmaceutical kit in preventing, improving or treating steroid-refractory graft-versus-host disease and / or in preparing a medicament for preventing, improving or treating steroid-refractory graft-versus-host disease.

[0370] The GLP-2 derivatives, long-acting conjugates thereof, prevention, improvement, treatment, steroids, steroid-refractory graft-versus-host disease, combinations, pharmaceutical compositions or pharmaceutical kits, etc. are as described above.

[0371] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0372] Example 1: Preparation of GLP-2 derivatives

[0373] In the present invention, seven GLP-2 derivatives having the amino acid sequences of SEQ ID NOs: 2 to 8 were prepared, as shown in Table 2 below.

[0374] [Table 2]

[0375] name sequence SEQ ID NO: CA GLP-2KC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 2 CA GLP-2KK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTKITDK]]> 3 CA GLP-2RK <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDK]]> 4 <![CDATA[CA GLP-2K AZ K]]> <![CDATA[ ca HGDGFSDEMNTILDNLAARDFINWLIQTKITD AZ K]]> 5 CA GLP-2RC <![CDATA[ ca HGDGSFSDEMNTILDNLAARDFINWLIQTRITDC]]> 6 CA GLP-2Aib <![CDATA[ ca HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC]]> 7 GLP-2Aib HAibDGSFSDEMNTILDNLAARDFINWLIQTKITDC 8

[0376] Example 2: Preparation of long-acting conjugates of GLP-2 derivatives

[0377] As a representative GLP-2 derivative, the GLP-2 derivative of SEQ ID NO: 4 was selected, and the GLP-2 derivative CA GLP-2RK was pegylated with a modified polyethylene glycol, ALD(2)PEG (a modified polyethylene glycol in which hydrogen atoms at both ends are replaced by propionaldehyde groups (3-oxopropyl groups), and the formula weight of the ethylene glycol repeating unit portion is 3.4 kDa, NOF, Japan). The molar ratio of the GLP-2 derivative to ALD(2)PEG was set to 1:5 to 1:20, the concentration of the GLP-2 derivative was adjusted to 5 to 10 mg / ml, and the reaction was carried out at 2° C. to 8° C. for 4 to 16 hours. At this time, the reaction was carried out in 20 mM HEPES (pH 7.5) and ethanol, and 20 mM sodium cyanoborohydride was added as a reducing agent for the reaction. The reaction solution was applied using a Source 15S (GE, USA) column using a buffer solution containing sodium citrate (pH 2.0) and ethanol and a potassium chloride concentration gradient to purify the mono-PEGylated GLP-2 derivative.

[0378] Next, the molar ratio of the purified mono-PEGylated GLP-2 derivative to the immunoglobulin Fc fragment was set to 1:2 to 1:6, the total protein concentration was adjusted to 30 to 35 mg / mL, and the reaction was carried out at 2°C to 8°C for 12 to 20 hours. At this time, the reaction solution consisted of 100 mM potassium phosphate buffer (pH 6.0) and isopropanol, with 20 mM sodium cyanoborohydride added as a reducing agent.

[0379] After the reaction was completed, the reaction solution was applied to a Source 15Q (GE, USA) column using a bis-Tris pH 6.5 buffer and a sodium chloride concentration gradient, and applied to a Source 15ISO (GE, USA) using 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, which is a conjugate of the GLP-2 derivative covalently linked to the immunoglobulin Fc fragment through a polyethylene glycol linker.

[0380] In addition to the GLP-2 derivative of SEQ ID NO: 4, conjugates of other GLP-2 derivatives of SEQ ID NOs: 2, 3, and 5 to 8 were prepared by the same method as described above.

[0381] Example 3: Examination of the effect of treating and preventing acute graft-versus-host disease by administering a long-acting conjugate of a GLP-2 derivative

[0382] To establish a mouse allogeneic transplant model for graft-versus-host disease (GVHD), female C57BL / 6j donor mice were purchased from OrientBio (Korea), and female BALB / C donor and recipient mice were purchased from DBL (Korea). Mice were housed in a sterile environment with a 12-h light / dark cycle. Food and water were provided ad libitum, and all procedures were performed with approval from the Animal Experimentation Committee.

[0383] To mobilize hematopoietic stem cells (HSCs) into the peripheral blood of C57BL / 6j or BALB / C mice (donors), 10 μg of G-CSF (granulocyte colony-stimulating factor) (PeproTech, USA) was subcutaneously injected for 5 days, and then 1.5×10 7 1.5×10 7 Splenocytes (200 μL volume) were injected into BALB / C mice (recipients) via the tail vein.

[0384] For transplantation, BALB / C mice (recipients) were administered busulfan (18.8 mg / kg) (Sigma-Aldrich, USA) by intraperitoneal injection once a day for 4 days 7 days before spleen cell injection, and subsequently cyclophosphamide (100 mg / kg) (Sigma Aldrich, USA) was administered for 2 days.

[0385] In order to examine the efficacy of treating and preventing GVHD, the long-acting conjugate of the GLP-2 derivative prepared in Example 1 was administered to BALB / C mice (recipients) 14 days before spleen cell injection. Specifically, the groups were divided into

[0386] - Group 1: BALB / C→BALB / C isogenic transplant model - vehicle administered subcutaneously (n=10 for survival, n=4 for histology),

[0387] - Group 2: C57BL / 6j→BALB / C allograft model - vehicle administered subcutaneously (n=10 for survival, n=5 for histology), and

[0388] - Group 3: C57BL / 6j→BALB / C allograft model - GLP-2 derivative long-acting conjugate (4613 μg / kg administered subcutaneously once a week) (n=11 for survival, n=5 for histology).

[0389] The numerical values expressed herein as the dose of the long-acting GLP-2 derivative conjugate are based on the value obtained by excluding the mass of the polyethylene glycol linker portion from the total mass of the long-acting GLP-2 derivative conjugate used, i.e., the sum of the masses of only the polypeptide portion.

[0390] The composition (e.g., liquid preparation) containing or not containing the long-acting conjugate of the GLP-2 derivative herein may contain 2 to 200 mM sodium acetate (pH 5.5 to 6.5), 0.002% to 0.2% (w / v) polysorbate, 1% to 20% (w / w) sucrose and / or 0.01 to 1 mg / mL methionine as excipients, and the content of each ingredient may be appropriately adjusted within the above range.

[0391] In the GVHD animal model established above, GVHD induction was evaluated by scoring five medical symptoms: weight loss, activity, posture, hair texture, and skin integrity. Specifically, each symptom was scored on a scale of 0 to 2, with 10 being the highest score, and this was used to evaluate the signs of disease (Table 3). In addition, survival was determined, and small intestinal and colon tissues were collected and observed for pathological changes.

[0392] [Table 3]

[0393] Macroscopic evaluation criteria for the pathogenesis of GVHD in animal models

[0394]

[0395]

[0396] Statistical analysis was performed using one-way ANOVA (***p<0.001, **p<0.01, *p<0.05) to compare the control group and the experimental group.

[0397] like Figure 1As shown in Figure 2, the survival rate of mice with allogeneic transplants induced with GVHD (Group 2) steadily decreased after allogeneic transplantation, and all mice died within 7 days. However, the survival rate of the group administered with the long-acting conjugate of the GLP-2 derivative (Group 3) increased. It was observed that in addition to the changes in survival rate, mice receiving the allogeneic transplant showed clinical symptoms of acute GVHD, such as weight loss and decreased activity, starting from day 3. The group administered with the long-acting conjugate of the GLP-2 derivative showed improvement or recovery in the evaluation items of the signs of the disease ( Figure 2 ).like Figure 3 As shown, histological findings revealed that the small intestine and colon lengths of the allogeneic transplanted mice (Group 2) as the control group were shortened. However, the GLP-2 derivative long-acting conjugate-administered group effectively maintained the small intestine and colon lengths comparable to those of the syngeneic transplanted mice (Group 1).

[0398] The results showed that long-acting conjugates of GLP-2 derivatives can exhibit efficacy in preventing and treating graft-versus-host disease.

[0399] Example 4: Examination of the effect of switching from steroid administration to administration of a long-acting conjugate of a GLP-2 derivative for the treatment of steroid-refractory graft-versus-host disease

[0400] Steroids are recommended as a first-line treatment for graft-versus-host disease (GVHD), but 40% to 50% of GVHD patients develop steroid-refractory graft-versus-host disease (SR-GVHD), which has a high mortality rate. The present invention aimed to evaluate the therapeutic efficacy and responsiveness of a long-acting conjugate of a GLP-2 derivative against SR-GVHD.

[0401] To establish a mouse allogeneic transplant model for graft-versus-host disease (GVHD), female C57BL / 6j donor mice were purchased from OrientBio (Korea), and female BALB / C recipient mice were purchased from DBL (Korea). Mice were housed in a sterile environment with a 12-h light / dark cycle. Food and water were provided ad libitum, and all procedures were performed with approval from the Animal Experimentation Committee.

[0402] To mobilize hematopoietic stem cells (HSCs) into the peripheral blood of 13-week-old C57BL / 6j mice (donors), 10 μg of G-CSF (granulocyte colony-stimulating factor) (PeproTech, USA) was subcutaneously injected for 5 days, and then 1.5×10 7 1.5×10 7 Splenocytes (200 μL volume) were injected into BALB / C mice (recipients) via the tail vein.

[0403] For transplantation, 13-week-old BALB / C mice (recipients) were prepared and administered busulfan (18.8 mg / kg) (Sigma-Aldrich, USA) by intraperitoneal injection once a day for 4 days 7 days before spleen cell injection, and then cyclophosphamide (100 mg / kg) (Sigma Aldrich, USA) was administered for 2 days.

[0404] To examine the responsiveness and therapeutic effect of switching from steroid administration to administration of a long-acting conjugate of a GLP-2 derivative against SR-GVHD, spleen cells were injected into BALB / C mice (recipients), and 3 days later, a long-acting conjugate of a GLP-2 derivative or a steroid was administered.

[0405] - Group 1: C57BL / 6j→BALB / C allograft model - subcutaneous administration of GLP-2 derivatives with long-acting conjugated vehicle (n=15)

[0406] - Group 2: C57BL / 6j→BALB / C allograft model - Apraglutide 469 μg / kg (administered subcutaneously every other day) (n=15)

[0407] - Group 3: C57BL / 6j → BALB / C allograft model - Prednisolone 2 mg / kg (once a day, intraperitoneally administered for 3 to 16 days) → GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneously administered starting on day 17, replacing steroids) (n=15)

[0408] - Group 4: C57BL / 6j→BALB / C allograft model - GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneously administered) (n=15)

[0409] The numerical values expressed herein as the dose of the long-acting GLP-2 derivative conjugate are based on the value obtained by excluding the mass of the polyethylene glycol linker portion from the total mass of the long-acting GLP-2 derivative conjugate used, i.e., the sum of the masses of only the polypeptide portion.

[0410] The same criteria as in Table 3 were used to evaluate the signs of disease, determine the survival rate, and collect small intestinal and colon tissues and observe pathological changes.

[0411] like Figure 4As shown in Figure 2, the survival rate of allogeneic transplanted mice induced with GVHD (Group 1) steadily decreased after allogeneic transplantation, and the survival rate on day 25 was 21.4%. The survival rate of the apalutamide-administered group (Group 2) was 35.7%, and the survival rate of the GLP-2 derivative long-acting conjugate-administered group was improved to 85.7%. When the survival rate dropped to 75.0% after 16 days of steroid administration, the GLP-2 derivative long-acting conjugate was administered instead of steroid from day 17 (Group 3), and the survival rate on day 25 was maintained at 75% ( Figure 1 ).

[0412] In addition to changes in survival rate, mice that received allogeneic transplants at 13 weeks of age also developed clinical symptoms of GVHD, such as weight loss and decreased activity starting on day 7. The group administered with the long-acting conjugate of the GLP-2 derivative maintained clinical scores when evaluating signs of disease, and the evaluation items did not deteriorate. The group administered with the steroid showed a worsening macroscopic condition by day 16 and was judged to be steroid-refractory. When the long-acting conjugate of the GLP-2 derivative was administered instead of the steroid starting on day 17, the evaluation scores showed improvement and recovery ( Figure 5 ).

[0413] The results indicate that GLP-2 derivatives or their long-acting conjugates can show efficacy in treating steroid-refractory graft-versus-host disease.

[0414] Example 5: Examination of the effect of treating steroid-refractory graft-versus-host disease (SR-GVHD) by combined administration of a steroid and a long-acting conjugate of a GLP-2 derivative

[0415] Steroids have the advantage of being standard treatments for graft-versus-host disease (GVHD) with both injectable and oral agents, and are used as primary and combination therapies. Therefore, the present invention aimed to evaluate the therapeutic effects and responsiveness of combined administration of a steroid and a long-acting conjugate of a GLP-2 derivative for SR-GVHD.

[0416] To establish a mouse allogeneic transplant model for GVHD, female C57BL / 6j donor mice were purchased from OrientBio (Korea), and female BALB / C recipient mice were purchased from DBL (Korea). Mice were housed in a sterile environment with a 12-h light / dark cycle. Food and water were provided ad libitum, and all procedures were performed with approval from the Animal Experimentation Committee.

[0417] To mobilize hematopoietic stem cells (HSCs) into the peripheral blood of 12-week-old C57BL / 6j mice (donors), 10 μg of G-CSF (granulocyte colony-stimulating factor) (PeproTech, USA) was subcutaneously injected for 5 days, and then 1.5×107 1.5×10 7 Splenocytes (200 μL volume) were injected into BALB / C mice (recipients) via the tail vein.

[0418] For the severe GVHD model, 12-week-old BALB / C mice (recipients) were prepared and busulfan (18.8 mg / kg) (Sigma-Aldrich, USA) was administered by intraperitoneal injection once a day for 4 days 7 days before spleen cell injection, and cyclophosphamide (100 mg / kg) (Sigma Aldrich, USA) was subsequently administered for 2 days in preparation for transplantation.

[0419] In order to examine the responsiveness and therapeutic effect on SR-GVHD obtained by combined administration of steroids and a long-acting conjugate of a GLP-2 derivative, spleen cells were injected into BALB / C mice (recipients), and 3 days later, a long-acting conjugate of a GLP-2 derivative or a steroid was administered. Specifically

[0420] - Group 1: C57BL / 6j→BALB / C allograft model - subcutaneous administration of GLP-2 derivatives with long-acting conjugated vehicle (n=15)

[0421] - Group 2: C57BL / 6j→BALB / C allograft model - Prednisolone 2-mg / kg (once a day, intraperitoneally) (n=15)

[0422] - Group 3: C57BL / 6j→BALB / C allograft model - GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneously administered) (n=15)

[0423] - Group 4: C57BL / 6j → BALB / C allograft model - Prednisolone 2 mg / kg (once a day, intraperitoneally administered for 3 to 9 days) → GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneously administered starting on day 10 instead of steroids) (n=15)

[0424] - Group 5: C57BL / 6j→BALB / C allograft model - Prednisolone 2 mg / kg (once a day, intraperitoneal administration) + GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneous administration) (n=15)

[0425] like Figure 6As shown, the survival rate of allogeneic transplanted mice (Group 1) induced with severe GVHD steadily decreased after allogeneic transplantation, and the survival rate on day 24 was 7.7%. It was found that the survival rate of the steroid administration group (Group 2) was 30.7%, and the survival rate of the GLP-2 derivative long-acting conjugate administration group (Group 3) was 23.1%. When the survival rate dropped to 53.8% after 9 days of steroid administration, the GLP-2 derivative long-acting conjugate was administered instead of the steroid from day 10 (Group 4), and the survival rate on day 24 remained at 46.2%. The survival rate of Group 5, which received a combination of steroid and GLP-2 derivative long-acting conjugate, was 46.2%, which was better than that of the steroid administration group or the GLP-2 derivative long-acting conjugate administration group alone.

[0426] The severity of GVHD was analyzed using clinical scores of mice that received allogeneic transplants at 12 weeks of age to induce severe GVHD. Control allogeneic transplant mice (Group 1) administered with vehicle developed clinical symptoms of GVHD, such as weight loss and decreased activity, after 3 days, and their macroscopic condition continued to deteriorate. The GLP-2 derivative long-acting conjugate administration group maintained the clinical score in the evaluation of signs of onset, with mild deterioration in the evaluation items. The steroid administration group was judged to be steroid-refractory because no improvement in macroscopic condition was observed within 9 days, and when the GLP-2 derivative long-acting conjugate was administered instead of steroids starting from the 10th day, the evaluation score improved, and the macroscopic condition showed signs of recovery. In addition, the group administered with a combination of steroids and GLP-2 derivative long-acting conjugates had the most favorable GVHD treatment effect, with an improvement in the evaluation score ( ) compared to the steroid administration group alone or the GLP-2 derivative long-acting conjugate administration group alone. Figure 7 ).

[0427] Example 6: Comparison of the effects of combined administration of steroids, long-acting conjugates of GLP-2 derivatives, and / or ruxolitinib in the treatment of steroid-refractory graft-versus-host disease (SR-GVHD)

[0428] Ruxolitinib is a selective JAK1 / 2 inhibitor that was first approved by the FDA for the treatment of acute and chronic steroid-refractory graft-versus-host disease (SR-GVHD). Therefore, the aim was to compare and evaluate the therapeutic effects of combining a long-acting conjugate of a GLP-2 derivative with steroids with combining ruxolitinib with steroids for the treatment of SR-GVHD.

[0429] For the severe GVHD model, 12-week-old BALB / C mice (recipients) were prepared and busulfan (18.8 mg / kg) (Sigma-Aldrich, USA) was administered by intraperitoneal injection once a day for 4 days 7 days before spleen cell injection, and cyclophosphamide (100 mg / kg) (Sigma Aldrich, USA) was subsequently administered for 2 days in preparation for transplantation.

[0430] To mobilize hematopoietic stem cells (HSCs) into the peripheral blood of 12-week-old C57BL / 6j mice (donors), 10 μg of G-CSF (granulocyte colony-stimulating factor) (PeproTech, USA) was subcutaneously injected for 5 days, and then 1.5×10 7 1.5×10 7 Splenocytes (200 μL volume) were injected into BALB / C mice (recipients) via the tail vein.

[0431] In order to compare and evaluate the responsiveness and therapeutic effect on SR-GVHD obtained by combined administration of a GLP-2 derivative long-acting conjugate and steroids with that obtained by combined administration of ruxolitinib and steroids, spleen cells were injected into BALB / C mice (recipients), and steroids, ruxolitinib, or a GLP-2 derivative long-acting conjugate were administered one day later. Specifically

[0432] - Group 1: C57BL / 6j→BALB / C allograft model - subcutaneous administration of GLP-2 derivatives with long-acting conjugated vehicle (n=15)

[0433] - Group 2: C57BL / 6j→BALB / C allograft model - prednisolone 2 mg / kg (once a day, intraperitoneal administration) (n=15)

[0434] - Group 3: C57BL / 6j→BALB / C allograft model - Ruxolitinib 30 mg / kg (once a day, oral administration) (n=15)

[0435] - Group 4: C57BL / 6j→BALB / C allograft model - GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneously administered) (n=15)

[0436] - Group 5: C57BL / 6j→BALB / C allograft model - prednisolone 2 mg / kg (once a day, intraperitoneal administration) + ruxolitinib 30 mg / kg (once a day, oral administration) (n=15)

[0437] - Group 6: C57BL / 6j→BALB / C allograft model - Prednisolone 2 mg / kg (once a day, intraperitoneal administration) + GLP-2 derivative long-acting conjugate 4613 μg / kg (once a week, subcutaneous administration) (n=15)

[0438] like Figure 8 As shown, the survival rate of allogeneic transplanted mice (Group 1) induced with severe GVHD steadily decreased after allogeneic transplantation, with a survival rate of 14.3% on day 14. The survival rate of the group administered with steroids alone (Group 2) was 7.1%, the group administered with ruxolitinib alone was 21.4% (Group 3), and the group administered with the long-acting conjugate of a GLP-2 derivative alone (Group 4) was 21.4%. The survival rate of the group administered with a combination of steroids and a long-acting conjugate of a GLP-2 derivative (Group 6) was 71.4%, which was superior to the 50.0% survival rate of the group administered with a combination of steroids and ruxolitinib (Group 5).

[0439] In order to compare the efficacy of combined administration of steroids in a severe GVHD model, the severity of GVHD was analyzed using the clinical scores of mice receiving allogeneic transplants. The allogeneic transplanted mice administered with excipients (Group 1), the control group, developed clinical symptoms of GVHD after 2 days, such as weight loss and decreased activity, and their macroscopic condition continued to deteriorate. In the steroid administration group, the evaluation items did not deteriorate until the 5th day, but then deteriorated rapidly, and the group was judged to be steroid-refractory (Group 2). The evaluation items of the group administered only with ruxolitinib (Group 3) continued to deteriorate, similar to those of the control group, but only the evaluation scores and clinical symptoms of the group administered with the long-acting conjugate of the GLP-2 derivative remained good. Compared with the group administered with the combination of steroids and ruxolitinib (Group 5), the group administered with the long-acting conjugate of steroids and GLP-2 derivatives (Group 6) had a better GVHD evaluation score ( Figure 9 ).

[0440] The results indicate that GLP-2 derivatives or long-acting conjugates thereof can be used in combination with steroids (standard therapeutic agents) to treat steroid-refractory graft-versus-host disease and can show therapeutic efficacy.

[0441] From the above description, it will be understood by those skilled in the art that the present invention can be implemented in other specific forms without changing its technical ideas or basic features. In this regard, it should be understood that the above embodiments are illustrative in all aspects and not restrictive. The scope of the present invention is defined by the appended claims rather than by the preceding description thereof, and therefore, all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating graft-versus-host disease, comprising a pharmaceutically effective amount of a glucagon-like peptide-2 (GLP-2) derivative, wherein The GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1: [Formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:10) In general formula 1, X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 is lysine or arginine; and X 34 is one or more arbitrary amino acids or one or more modified arbitrary amino acids, However, among the amino acid sequences represented by the general formula 1, sequences identical to SEQ ID NO: 1 are excluded.

2. The pharmaceutical composition according to claim 1, wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 2: [Formula 2] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (SEQ ID NO:9) In general formula 2, X1 is histidine, imidazoacetyl dehistidine, deaminohistidine, β-hydroxyimidazopropionyl dehistidine, N-dimethylhistidine or β-carboxyimidazopropionyl dehistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 is lysine or arginine; and X 34 Lysine, arginine, glutamine, histidine, 6-azidolysine, or cysteine is absent or However, among the amino acid sequences represented by the general formula 2, sequences identical to SEQ ID NO: 1 are excluded.

3. The pharmaceutical composition according to claim 2, wherein in the GLP-2 derivative, (1) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's cysteine, (2) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is lysine, and X 34 It's lysine, (3) X1 is imidazoacetyl desyl histidine, X2 is glycine, X 30 is arginine, and X 34 It's 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's cysteine, (6) X1 is imidazoacetyl desahistidine, 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. 4 . The pharmaceutical composition according to claim 1 , wherein the GLP-2 derivative comprises an amino acid sequence selected from SEQ ID NOs: 2 to 8. The pharmaceutical composition according to claim 1 , wherein the C-terminus of the GLP-2 derivative is unmodified or amidated.

6. The pharmaceutical composition of claim 1, wherein the composition causes one or more of an increase in survival rate, an increase in small intestine length, and an increase in colon length in a subject administered the composition.

7. The pharmaceutical composition according to claim 1, wherein the GLP-2 derivative is in the form of a long-acting conjugate combined with a biocompatible substance capable of increasing the in vivo half-life of the GLP-2 derivative.

8. The pharmaceutical composition according to claim 7, wherein the conjugate is represented by the following Chemical Formula 1: [Chemical Formula 1] X-L a -F In Chemical Formula 1, X is a GLP-2 derivative; L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, but when a is 2 or greater, each L is independent of each other; F is an immunoglobulin Fc region; and "-" is a covalent bond.

9. The pharmaceutical composition according to claim 8, wherein the GLP-2 derivative comprises an amino acid sequence selected from SEQ ID NOs: 2 to 8.

10. The pharmaceutical composition according to claim 8, wherein the C-terminus of the GLP-2 derivative is unmodified or amidated. The pharmaceutical composition according to claim 8 , wherein the immunoglobulin Fc region is an aglycosylated IgG4 Fc region. 12 . The pharmaceutical composition according to claim 8 , 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.

13. The pharmaceutical composition of claim 8, wherein L is polyethylene glycol.

14. The pharmaceutical composition according to claim 8, wherein the formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

15. The pharmaceutical composition according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the composition is administered to a subject together with, before or after an allogeneic transplant. 17 . The pharmaceutical composition according to any one of claims 1 to 15 , wherein the graft-versus-host disease is acute graft-versus-host disease or chronic graft-versus-host disease.

18. The pharmaceutical composition according to any one of claims 1 to 15, wherein the graft-versus-host disease is caused by allogeneic transplantation, and the allogeneic transplantation comprises at least one of hematopoietic stem cell transplantation, bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood transplantation, or blood transfusion.

19. The pharmaceutical composition according to any one of claims 1 to 15, wherein the graft-versus-host disease is steroid-refractory graft-versus-host disease.

20. The pharmaceutical composition according to claim 19, wherein the steroid-refractory graft-versus-host disease is steroid-refractory acute graft-versus-host disease or steroid-refractory chronic graft-versus-host disease.

21. The pharmaceutical composition according to any one of claims 1 to 15, wherein the graft-versus-host disease is steroid-refractory graft-versus-host disease, and the pharmaceutical composition is administered in combination with a steroid.

22. The pharmaceutical composition according to claim 21, wherein the steroid-refractory graft-versus-host disease is steroid-refractory acute graft-versus-host disease or steroid-refractory chronic graft-versus-host disease.

23. The pharmaceutical composition according to claim 21, wherein the combined administration is simultaneous, sequential or reverse order combined administration of the GLP-2 derivative or its long-acting conjugate; and the steroid.

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