Pharmaceutical compositions for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or combinations thereof, comprising GLP-2 derivatives or their sustained-release conjugates.

Abstract

To provide a composition for the prevention, improvement, or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a glucagon-like peptide-2 (GLP-2) derivative and / or a sustained-release conjugate thereof. [Solution] The present invention relates to the prophylactic or therapeutic use of GLP-2 and its sustained-release conjugates against mucositis induced by radiotherapy, chemotherapy, or a combination thereof. The GLP-2, its sustained-release conjugates, or compositions comprising the same of the present invention may be applied to the prevention, treatment, and improvement of mucositis induced by radiotherapy and / or chemotherapy.

Description

[Technical Field]

[0001] The present invention relates to the prophylactic or therapeutic use of GLP-2 derivatives and their sustained-release conjugates for mucositis induced by radiotherapy, chemotherapy, or combinations thereof. [Background technology]

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

[0003] GLP-2 has shown promising potential as a therapeutic agent in experimental models of various intestinal diseases and intestinal injuries due to its characteristics such as energy absorption and protection, and activation of intestinal cell function. As a hormone that regulates nutrient absorption, GLP-2 has potential as a therapeutic agent for short bowel syndrome (SBS). SBS occurs due to congenital or acquired causes such as bowel resection surgery, and leads to nutritional deficiencies due to a reduction in the absorption area of ​​the small intestine. GLP-2 has been confirmed to enhance nutrient absorption and gastrointestinal absorption in experimental models of rodents with SBS (Ljungmann K et al., Am. J. Physiol. Gastrointest Liver Physiol., 2001, 281(3):G779-85).

[0004] On the other hand, during radiotherapy and / or chemotherapy, organs with rapid cell growth, such as mucosa, are damaged in addition to the target cells (e.g., cancer cells), leading to side effects such as mucositis. Therefore, there is a need for the development of drugs that can prevent or treat mucositis induced by radiotherapy and / or chemotherapy.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] One object of the present invention is to provide a composition for preventing, ameliorating or treating mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a glucagon-like peptide-2 (GLP-2) derivative and / or a sustained conjugate thereof.

[0008] Another object of the present invention is to provide a method for preventing, ameliorating or treating mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising administering to an individual in need thereof a GLP-2 derivative, a sustained conjugate thereof, and / or a composition containing the same.

[0009] Another object of the present invention is to provide uses for GLP-2 derivatives, their sustained-release conjugates, and / or compositions containing them in the manufacture of agents for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof. [Means for solving the problem]

[0010] One embodiment of the present invention is a composition for the prevention, treatment, or improvement of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a glucagon-like peptide-2 (GLP-2) derivative and / or a sustained-release conjugate thereof.

[0011] As one specific example, the present invention is characterized by a pharmaceutical composition comprising a GLP-2 derivative and / or a sustained-release conjugate thereof, for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof.

[0012] As another specific example, the present invention is characterized by a food composition for the prevention or improvement of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a GLP-2 derivative and / or a sustained-release conjugate thereof.

[0013] As a specific example of the composition described above, the present invention is a pharmaceutical composition for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a pharmaceutically acceptable excipient and a pharmaceutically effective amount of a GLP-2 derivative, wherein the GLP-2 derivative comprises an amino acid sequence represented by the following general formula 1:

[0014] [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 10)

[0015] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 It is lysine or arginine; X 34 is one or more arbitrary amino acids or one or more arbitrary amino acids that have undergone a transformation; However, sequences identical to sequence number 1 are excluded from the amino acid sequence of general formula 1.

[0016] A composition comprising any one of the aforementioned specific examples, characterized in that the above deformation involves substitution, addition, deletion, modification, or a combination thereof in at least one amino acid.

[0017] A composition according to one of the above-mentioned specific examples, characterized in that the GLP-2 derivative contains an amino acid sequence represented by the following general formula 2:

[0018] [General formula 2] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 9)

[0019] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 It is lysine or arginine; X34 is absent or is lysine, arginine, glutamine, histidine, 6-azido lysine, or cysteine, provided that the sequence identical to SEQ ID NO: 1 is excluded from the amino acid sequence of General Formula 2.

[0020] A composition according to any one of the foregoing specific examples, wherein the GLP-2 derivative (1) X1 is imidazoacetyl deshistidine, X2 is glycine, X 30 is lysine, X 34 is cysteine, or (2) X1 is imidazoacetyl deshistidine, X2 is glycine, X 30 is lysine, X 34 is lysine, or (3) X1 is imidazoacetyl deshistidine, X2 is glycine, X 30 is arginine, X 34 is lysine, or (4) X1 is imidazoacetyl deshistidine, X2 is glycine, X 30 is lysine, X 34 is 6-azido lysine, or (5) X1 is imidazoacetyl deshistidine, X2 is glycine, X 30 is arginine, X 34 is cysteine, or (6) X1 is imidazoacetyl deshistidine, X2 is Aib, X 30 is lysine, X 34 is cysteine, or (7) X1 is histidine, X2 is Aib, X 30 is lysine, X 34 is cysteine.

[0021] A composition according to any one of the foregoing specific examples, wherein the GLP-2 derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8.

[0022] A composition comprising one of the aforementioned specific examples, characterized in that the GLP-2 derivative is either unmodified or amidated at its C-terminus.

[0023] A composition comprising one of the above-mentioned specific examples, characterized in that the mucositis is oral mucositis, gastrointestinal mucositis, or a combination thereof.

[0024] A composition comprising one of the above-mentioned specific examples, characterized in that the chemotherapy is chemotherapy using an anticancer agent.

[0025] A composition according to one of the above-mentioned specific examples, characterized in that the anticancer agent is a cytotoxic anticancer agent, a targeted anticancer agent, an immunosuppressant, or a combination thereof.

[0026] A composition comprising any one of the above-mentioned specific examples, characterized in that the cytotoxic anticancer agent is a nucleoside analog, a folate antagonist, an antimetabolite, a topoisomerase I inhibitor, anthracycline, podophyllotoxin, taxane, vinca alkaloid, alkylating agent, platinum compound, or a combination thereof.

[0027] A composition according to any one of the above-mentioned specific examples, characterized in that the anticancer agent is 5-fluorouracil (5-FU), cyclophosphamide (CPA), docetaxel, doxorubicin, vincristine, prednisone, etoposide, ifosfamide, methotrexate, paclitaxel, gemcitabine, vinorelbine, leucovorin, irinotecan, oxaliplatin, or a combination thereof.

[0028] A composition comprising one of the above-mentioned specific examples, characterized in that the composition is administered within one day before radiotherapy or chemotherapy; or within one day after radiotherapy or chemotherapy.

[0029] A composition comprising any one of the above-mentioned specific examples, characterized in that the composition causes one or more of the following in an administered individual: an increase in small intestinal weight, a decrease in the level of small intestinal weight loss, suppression of inflammation, suppression of monocyte differentiation into macrophages, and suppression of monocyte migration.

[0030] A composition comprising one of the aforementioned specific examples, characterized in that the GLP-2 derivative is in the form of a sustained-release conjugate to which a biocompatible substance that increases the in vivo half-life is bound.

[0031] A composition comprising any one of the aforementioned specific examples, characterized in that the compound is represented by the following chemical formula (1):

[0032] X-La-F···(1)

[0033] Here, X is a GLP-2 derivative; L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; F is the immunoglobulin Fc region; The above "-" indicates a covalent bond.

[0034] A composition comprising one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region is non-glycosylated.

[0035] A composition comprising one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region includes a hinge region.

[0036] A composition comprising one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc region is an IgG4Fc region.

[0037] A composition comprising one of the above-mentioned specific examples, wherein F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0038] A composition comprising one of the above-mentioned specific examples, characterized in that L is polyethylene glycol.

[0039] A composition comprising one of the above-mentioned specific examples, characterized in that the chemical formula weight of the ethylene glycol repeating unit (moiety) in L is in the range of 1 to 100 kDa.

[0040] Another aspect of the present invention is a method for preventing, improving or treating mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising the step of administering a GLP-2 derivative, a sustained-release conjugate thereof, and / or a composition containing the same to an individual in need thereof, wherein the GLP-2 derivative comprises an amino acid sequence represented by the above general formula 1 (excluding the sequence identical to SEQ ID NO: 1 from the amino acid sequence of general formula 1).

[0041] Another aspect of the present invention is the use of a GLP-2 derivative, a sustained-release conjugate thereof, or a composition containing the same for the prevention, improvement, or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, wherein the GLP-2 derivative comprises an amino acid sequence represented by the general formula 1 (excluding the sequence identical to SEQ ID NO: 1 from the amino acid sequence of general formula 1).

[0042] Another aspect of the present invention is the use of a GLP-2 derivative, a sustained-release conjugate thereof, or a pharmaceutical composition containing the same in the manufacture of a drug for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, wherein the GLP-2 derivative comprises an amino acid sequence represented by the above general formula 1 (excluding the amino acid sequence of general formula 1 that is identical to sequence number 1). [Effects of the Invention]

[0043] The GLP-2 derivative of the present invention, its sustained-release conjugate, or a composition containing the same may be applied to the prevention, treatment, and improvement of mucositis induced by radiotherapy and / or chemotherapy. [Brief explanation of the drawing]

[0044] [Figure 1] This study compares and analyzes changes in small intestinal weight after administration of a sustained-release GLP-2 derivative conjugate in a rat model in which mucositis was induced by chemotherapy (Docetaxel and Cyclophosphamide, TC). [Figure 2]This report presents the results of a comparative analysis of small intestinal weight changes in model rats in which mucositis was induced by chemotherapy (TC), comparing administration of a GLP-2 derivative (teduglutide) and a GLP-2 derivative sustained-release conjugate concurrently with chemotherapy induction (A) and administration of the GLP-2 derivative sustained-release conjugate one day before chemotherapy induction (B). [Figure 3] This study confirmed the effects of a sustained-release GLP-2 derivative conjugate on M1 polarization (A), macrophage differentiation (B), and monocyte migration (C) in THP-1 cells (monocytes). [Modes for carrying out the invention]

[0045] The present invention will be described in more detail below.

[0046] On the other hand, each description and embodiment disclosed herein may also apply to each other different descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the following specific descriptions.

[0047] Furthermore, a person with ordinary skill in the art can recognize and confirm numerous equivalents to the specific modes of the present invention described in this application using only ordinary experiments. Such equivalents are intended to be included in the present invention.

[0048] Throughout this specification, in addition to the usual one- and three-letter codes for naturally occurring amino acids, generally accepted three-letter codes are used for other amino acids such as Aib (2-aminoisobutyric acid) and AZK (6-azidolysine). Furthermore, amino acids referred to by abbreviations in this specification are described according to IUPAC-IUB nomenclature.

[0049] Alanine (Ala,A) and Arginine (Arg,R) Asparagine Asn,N; Aspartic acid Asp,D Cysteine ​​(Cys,C), Glutamic acid (Glu,E) Glutamine Gln,Q Glycine Gly,G Histidine His,H Isoleucine Ile,I Leucine (Lu,L) and Lysine (Lys,K) Methionine Met,M Phenylanine Phe,F Proline (Pro,P) and Serine (Se,S) Threonine Thr,T Tryptophan Trp,W Tyrosine (Tyr,Y) and Valine (V,V)

[0050] One embodiment of the present invention is a composition comprising a glucagon-like peptide-2 (GLP-2) derivative for the prevention, treatment, or improvement of mucositis induced by radiotherapy, chemotherapy, or a combination thereof. Specifically, the above composition may be a pharmaceutical composition for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof.

[0051] One specific example of the present invention is, but is not limited to, a pharmaceutical composition for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a pharmaceutically acceptable excipient and a GLP-2 derivative in a pharmaceutically effective amount.

[0052] In this invention, the term "mucositis" refers to inflammation and / or ulceration of the mucous membrane of the oral cavity and / or the digestive tract, and "mucositis induced by radiotherapy, chemotherapy, or a combination thereof" refers to mucositis that occurs as a side effect of radiotherapy or chemotherapy alone, or a combination of radiotherapy and chemotherapy regardless of the order or number of treatments. Specifically, it may be, but is not limited to, oral mucositis, gastrointestinal mucositis, or a combination thereof. The above gastrointestinal mucositis may be, but is not limited to, intestinal inflammatory disease and / or intestinal ulcer disease. Gastrointestinal mucositis may present with symptoms such as anal ulcer, rectal ulcer, rectal bleeding, abdominal pain, dysphagia, vomiting, bloating, or nutrient deficiency or weight loss due to decreased nutrient absorption caused by a decrease in the length of the villi and the depth of the crypts in the small intestine.

[0053] The above-mentioned radiotherapy refers to the prevention or treatment of disease using radiation, and includes all radiotherapy that induces mucositis, either alone or in conjunction with chemotherapy. Furthermore, the above-mentioned chemotherapy refers to the prevention or treatment of disease using chemical drugs, and includes all chemotherapy that induces mucositis, either alone or in conjunction with radiotherapy.

[0054] In one specific embodiment, the radiotherapy and chemotherapy described above may be for the prevention or treatment of cancer, which include lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic sac cancer (e.g., ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small intestine cancer, colorectal cancer (e.g., colon cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor), breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian carcinoma, extratesticular germ cell tumor, ovarian germ cell tumor, low-grade ovarian tumor), testicular tumor, and prostate cancer (e.g., hormone-dependent prostate cancer). This includes, but is not limited to, other cancers such as hormone-independent prostate cancer, liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic cholangiocarcinoma), thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, endometrial cancer, uterine sarcoma), brain tumors (e.g., myelocyte tumor, glioma, pineal astrocytoma, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (e.g., basal cell carcinoma, malignant melanoma), sarcoma (e.g., rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma), malignant bone tumors, bladder cancer, hematological cancers (e.g., multiple myeloma, leukemia, malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorders), and cancers of unknown primary origin.

[0055] The chemotherapy for the prevention or treatment of the above-mentioned cancers may be chemotherapy using anticancer drugs, and mucositis may be induced as a side effect. The above-mentioned "use of anticancer drugs" includes the use of anticancer drugs alone or in combination, and also includes inducing mucositis by administering other substances (e.g., drugs other than anticancer drugs, drugs that suppress the side effects of anticancer drugs, etc.) together with anticancer drugs, sequentially or in reverse order.

[0056] In one specific embodiment, the anticancer agent that induces mucositis may be, but is not limited to, a cytotoxic anticancer agent, a targeted anticancer agent, an immunosuppressant, or a combination thereof.

[0057] The above-mentioned cytotoxic anticancer drugs are drugs that attack rapidly differentiating cells, and specifically include nucleoside analogs [azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, fluorouracil, gemcitabine]. ), mercaptopurine, nelarabine, pentostatin, tegafur, thioguanine, etc., antifolates [methotrexate, pemetrexed, lartitrexed, etc.], antimetabolites [hydroxyurea, etc.], topoisomerase II) Inhibitors [such as irinotecan and topotecan], anthracyclines [such as daunorubicin, doxorubicin, epirubicin, and idarubicin], podophyllotoxins [such as etoposide and teniposide], taxanes [such as cabazitaxel, docetaxel, and paclitaxel] [e.g., Paclitaxel], Vinca alkaloids [e.g., Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine], Alkylating agents [e.g., Bendamustine, Busulfan, Carmustine, Chlorambucil, Chlormeth [e.g., cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, streptozotocin, temozolomide, etc.], platinum compounds [e.g., carboplatin, cisplatin, nedaplatin (N)] Other drugs such as edaplatin, oxaliplatin, etc., or altretamine, bleomycin, bortezomib, dactinomycin, estramustine, ixabepilone, mitomycin, procarbazine, etc., may be used, but are not limited to these.

[0058] Furthermore, targeted anticancer drugs are drugs that selectively attack cancer cells that have specific parts that differ from other cells (e.g., normal cells). Specifically, they are monoclonal antibodies [e.g., alemtuzumab, bevacizumab, cetuximab, denosumab, gemtuzumab ozogamicin, ibritumomab tiuxetan]. [e.g., tumomabtiuxetan), ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, tositumomab, trastuzumab, etc.], tyrosine kinase inhibitors [e.g., afatiuxetan], tyrosine kinase inhibitors [e.g., afatiuxetan] Nib (Afatinib), Aflibercept, Axitinib, Bosutinib, Crizotinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Nilotinib, Pazopanib, Ponatinib, Regorafenib [Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, Vandetanib, etc.], mTOR inhibitors [e.g., Everolimus, Temsirolimus, etc.], Retinoids [Alitretinoin, Bexarotene, Isotretinoin]This may include, but is not limited to, tamibarotene, tretinoin, immunomodulators, lenalidomide, pomalidomide, thalidomide, histone deacetylase inhibitors, panobinostat, romidepsin, valproate, vorinostat, or other drugs such as anagrelide, arsenic trioxide, asparaginase, BCG vaccine, denileukin diftitox, vemurafenib, etc.

[0059] Furthermore, immunosuppressants are drugs that utilize the body's immune system to attack cancer cells. Specifically, they may be drugs that prevent cancer cells from evading the body's immune system or that allow immune cells to better recognize and attack cancer cells. More specifically, they may be, but are not limited to, immune checkpoint inhibitors [such as atezolizumab, ipilimumab, avelumab, nivolumab, pembrolizumab, and durvalumab], immune cell enhancers [such as blinatumomab], or other drugs [such as alemtuzumab, ofatumumab, and elotuzumab].

[0060] Furthermore, a single drug may be both a targeted anticancer agent and an immunosuppressant.

[0061] In one specific embodiment, the anticancer agent that induces mucositis may be, but is not limited to, 5-fluorouracil (5-FU), cyclophosphamide (CPA), docetaxel, doxorubicin, vineristine, prednisone, etoposide, ifosfamide, methotrexate, paclitaxel, gemcitabine, vinorelbine, leucovorin, irinotecan, oxaliplatin, or a combination thereof.

[0062] In one specific embodiment, the composition of the present invention can prevent or treat mucositis induced by radiotherapy, chemotherapy, or a combination thereof by causing one or more of the following in administered individuals: an increase in small intestinal weight, suppression of small intestinal weight loss (i.e., a decrease in the level of small intestinal weight loss), suppression of inflammation, suppression of monocyte differentiation into macrophages, and suppression of monocyte migration.

[0063] In the present invention, the term "glucagon-like peptide-2" or "GLP-2 (Glucagon-like peptide-2)" refers to an agonist of the glucagon-like peptide-2 receptor, and may, but is not limited to, a polypeptide form or a sustained-release conjugate to which a biocompatible substance that increases its in vivo half-life is bound. In the present invention, GLP-2 includes not only natural (e.g., human) GLP-2 but also its derivatives and their conjugates. The above GLP-2 is an active ingredient contained in the pharmaceutical composition of the present invention and may be included in the pharmaceutical composition in a pharmaceutically effective amount.

[0064] In the present invention, "GLP-2 receptor agonist" refers to a substance that binds to a human glucagon-like peptide-2 receptor in vivo or isolated from it, and induces physiological activity equivalent 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.

[0065] The amino acid sequence of natural GLP-2 is as follows:

[0066] GLP-2(1-33)

[0067] HADGSFSDEMNTILDNLAARDFINWLIQTKITD (Sequence ID 1)

[0068] In the present invention, "GLP-2 derivative" includes peptides having one or more differences in amino acid sequence compared to natural GLP-2, while having functions to prevent, treat, or improve mucositis induced by radiotherapy, chemotherapy, or a combination thereof; peptides obtained by modifying the natural GLP-2 sequence; and / or mimics of natural GLP-2 that have functions to prevent, treat, and / or improve mucositis induced by radiotherapy, chemotherapy, or a combination thereof, similar to natural GLP-2.

[0069] Specifically, the GLP-2 derivative of the present invention may, but is not limited to, a modified form of natural GLP-2 in which at least one amino acid has been substituted, added, deleted, modified, or modified in combination thereof. The added amino acid may be a non-natural amino acid (e.g., a D-type amino acid), and substitution with non-natural amino acids is also possible in addition to natural amino acids. The added amino acid sequence may, but is not limited to, be derived from natural GLP-2. Furthermore, in the present invention, amino acid modification may, but is not limited to, mean that a group of amino acid residues has been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation, substitution with an azido group), deleted (e.g., deamination), and / or modified (e.g., N-methylation), together with, or independently of, the substitution, addition, deletion, or combination thereof of at least one amino acid.

[0070] In one specific embodiment, the GLP-2 derivative of the present invention may / may exhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology (or identity) with native GLP-2 in terms of amino acid sequence, or may be in a form in which some groups of amino acid residues of the GLP-2 derivative are chemically substituted (e.g., alpha-methylation, alpha-hydroxylation, substitution with an azido group), removed (e.g., deamination), and / or modified (e.g., N-methylation).

[0071] In one specific embodiment, the GLP-2 derivative may, but is not limited to, have its N-terminal amino group substituted, removed, or modified. The GLP-2 derivative of the present invention may be produced by methods such as removing the alpha-amino group of the N-terminal histidine, synthesizing by substituting the N-terminal amino group with a hydroxyl or carboxyl group, removing the alpha carbon and the N-terminal amino group attached to the alpha carbon of the N-terminal histidine to leave only the imidazo-acetyl active group, or modifying the N-terminal amino group with two methyl groups, in order to prevent binding at the N-terminus, which is an important site for the bioactivity of the GLP-2 derivative, during the production of the sustained-release conjugate.

[0072] Specifically, GLP-2 derivatives include imidazoacetyl-deshistidyl-GLP-2 (CA-GLP-2), in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bonded to the alpha carbon are removed; desaminohistidyl GLP-2 (DA-GLP-2), in which the N-terminal amino group of GLP-2 is removed; beta-hydroxyimidazopropionyldeshistidyl GLP-2 (HY-GLP-2), in which the N-terminal amino group of GLP-2 is replaced with a hydroxyl group; and N-dimethylhistidyl GLP-2 (N-dimethylhistidyl GLP-2), in which the N-terminal amino group of GLP-2 is modified with two methyl groups. The derivative may be DM-GLP-2, or a derivative of β-carboxyimidazopropionyl-deshistidyl GLP-2 (CX-GLP-2) in which the N-terminal amino group of GLP-2 is substituted with a carboxyl group. The following is one non-restrictive example of a material structure used in the production of a GLP-2 derivative.

[0073] JPEG2026103884000002.jpg151167Des-amino-histidyl: Des-amino-histidyl Beta-hydroxy-imidazopropionyl Beta-carboxyl-imidazopropionyl: β-carboxyl-imidazopropionyl Imidazoacetyl Dimethyl-histidyl: Dimethyl-histidyl

[0074] In one specific embodiment, the GLP-2 derivative may include, but is not limited to, a modification of at least one amino acid of amino acids 1, 2, 30, and 33 in SEQ ID NO: 1. Specifically, in the present invention, the amino acid modification may be a modification selected from the group consisting of substitution, addition, removal, modification, and combinations thereof of at least one amino acid, in which case the added amino acid may be a non-natural amino acid (e.g., a D-type amino acid), and substitution of non-natural amino acids in addition to natural amino acids is also possible. The added amino acid sequence may, but is not limited to, originate from natural GLP-2. Furthermore, in the present invention, the amino acid modification may mean, together with, at least one amino acid substitution, addition, removal, or combination thereof, or independently thereof, that a group of amino acid residues are chemically substituted (e.g., alpha-methylation, alpha-hydroxylation, substitution with an azido group), removed (e.g., deamination), and / or modified (e.g., N-methylation).

[0075] In one specific embodiment, the GLP-2 derivative may, but is not limited to, the amino acid sequence of the following general formula 1:

[0076] [General formula 1] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30ITDX 34 (Sequence ID 10)

[0077] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 It is lysine or arginine; X 34 is one or more arbitrary amino acids or one or more arbitrary amino acids that have undergone a transformation; However, sequences identical to sequence number 1 are excluded from the amino acid sequence of general formula 1.

[0078] Specifically, the above amino acids may be natural amino acids or non-natural amino acids, and the above modifications are as described above.

[0079] In one specific embodiment, the GLP-2 derivative may, but is not limited to, the amino acid sequence of the following general formula 2:

[0080] [General formula 2] X1X2DGSFSDEMNTILDNLAARDFINWLIQTX 30 ITDX 34 (Sequence ID 9)

[0081] Here, X1 is histidine, imidazoacetyldeshistidine, desaminohistidine, β-hydroxyimidazopropionyldeshistidine, N-dimethylhistidine, or β-carboxyimidazopropionyldeshistidine; X2 is alanine, glycine, or Aib (2-aminoisobutyric acid); X 30 It is lysine or arginine; X34 It is either absent or is lysine, arginine, glutamine, histidine, 6-azidrisine, or cysteine; However, sequences identical to sequence number 1 are excluded from the amino acid sequence of general formula 2.

[0082] Specifically, the GLP-2 derivatives of the present invention may, but are not limited to, substitution of alanine, the second amino acid of natural GLP-2, with glycine or Aib (2-aminoisobutyric acid), substitution of lysine, the 30th amino acid, with arginine, or a combination thereof. Furthermore, the above GLP-2 derivatives may, but are not limited to, have a thiol group (e.g., cysteine), an amino group (e.g., lysine, arginine, glutamine, or histidine), or an azide group (e.g., 6-azidrisine) introduced at the C-terminus (e.g., the 33rd amino acid).

[0083] Since binding occurs in the introduced group during the production of sustained-release conjugates of GLP-2 derivatives, GLP-2 conjugates with selectively modified binding sites can be produced using this method. Specifically, one end of a non-peptide linker can be bound to the hydroxyl group, thiol group, amino group, or azide group of the GLP-2 derivative, and a substance that increases the in vivo half-life (e.g., an immunoglobulin Fc region) can be bound to the other end of the non-peptide linker. The thiol group, amino group, or azide group can be introduced by adding an amino acid to GLP-2, but is not limited to this. The thiol group can be introduced by adding cysteine ​​(C) to GLP-2; the amino group can be introduced by adding lysine (K), arginine (R), glutamine (Q), or histidine (H); and the azide group can be introduced by adding 6-azidolysine, AZ It may be introduced with the addition of K), but is not limited to this.

[0084] Specifically, the GLP-2 derivative may, but is not limited to, contain at least one residue of cysteine, lysine, arginine, glutamine, histidine, or 6-azidrisine.

[0085] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine and the introduction of a thiol group (e.g., cysteine) at the C-terminus. More specifically, it may also include imidazoacetyldeshistidine, which has the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the alpha carbon removed. For example, it may have the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0086] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine and the introduction of an amino group (e.g., lysine) at the C-terminus. More specifically, it may also include imidazoacetyldeshistidine, from which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the alpha carbon have been removed. For example, it may have the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0087] Specifically, the GLP-2 derivative of the present invention includes the substitution of alanine, the second amino acid of natural GLP-2, with glycine, the substitution of lysine, the 30th amino acid of natural GLP-2, with arginine, and the introduction of an amino group (e.g., lysine) at the C-terminus. More specifically, it may also include imidazoacetyldeshistidine, in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the alpha carbon have been removed. For example, it may have the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0088] Specifically, the GLP-2 derivative of the present invention comprises the substitution of alanine, the second amino acid of natural GLP-2, with glycine and the introduction of an azide group (e.g., 6-azidrisine) at the C-terminus. More specifically, it may also include imidazoacetyldeshistidine, which has the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the alpha carbon removed. For example, it may have the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0089] Specifically, the GLP-2 derivative of the present invention includes the substitution of alanine, the second amino acid of natural GLP-2, with glycine, the substitution of lysine, the 30th amino acid of natural GLP-2, with arginine, and the introduction of a thiol group (e.g., cysteine) at the C-terminus. More specifically, it may also include imidazoacetyldeshistidine, in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group attached to the alpha carbon have been removed. For example, it may have the amino acid sequence of SEQ ID NO: 6, but is not limited thereto.

[0090] Specifically, the GLP-2 derivative of the present invention involves the substitution of alanine, the second amino acid of natural GLP-2, with 2-aminoisobutyric acid and the introduction of a thiol group (e.g., cysteine) at the C-terminus, and may, for example, have the amino acid sequence of SEQ ID NO: 8. More specifically, it may also include imidazoacetyldeshistidine, in which the alpha carbon of the histidine residue, the first amino acid at the N-terminus, and the N-terminal amino group bonded to the alpha carbon have been removed, and may, for example, have the amino acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0091] The GLP-2 derivatives of Sequence IDs 2-8 are shown in Table 1 below.

[0092] [Table 1]

[0093] In Table 1 above, ca H is substituted with imidazoacetyldeshistidine instead of histidine, and Aib is 2-aminoisobutyric acid. AZ K stands for 6-azido-L-lysyine.

[0094] The GLP-2 derivative according to the present invention may be a peptide containing the above-mentioned specific sequence, or a peptide composed (essentially) of the above-mentioned specific sequence, but is not limited thereto.

[0095] On the other hand, even if the present application describes a peptide or GLP-2 derivative "composed of a specific sequence number," if it has the same or corresponding activity as the peptide or GLP-2 derivative consisting of the amino acid sequence of the said sequence number, it does not exclude meaningless additions of sequences before or after the amino acid sequence of the said sequence number, or spontaneously occurring mutations, or silent mutations. It is obvious that even if such additions or mutations of sequences are present, they still fall within the scope of the present application.

[0096] Specifically, GLP-2 derivatives are those in general formula 1 or 2 where (1) X2 is glycine or Aib, or (2) X 30 (3) X2 is glycine or Aib, and X 30 This may be lysine or arginine, but is not limited to these.

[0097] Specifically, GLP-2 derivatives are in general formula 1 or general formula 2. (1) X1 is imidazoacetyldeshistidine, X2 is glycine, X 30 It is ricin, X 34 Is it cysteine? (2) X1 is imidazoacetyldeshistidine, X2 is glycine, X 30 It is ricin, X 34 Is it ricin? (3) X1 is imidazoacetyldeshistidine, X2 is glycine, X 30 That is arginine, X 34 Is it ricin? (4) X1 is imidazoacetyldeshistidine, X2 is glycine, X 30 It is ricin, X 34 Is it 6-azidricin? (5) X1 is imidazoacetyldeshistidine, X2 is glycine, X 30 That is arginine, X 34 Is it cysteine? (6) X1 is imidazoacetyldeshistidine, X2 is Aib, X 30 It is ricin, X 34 Is it cysteine, or (7) X1 is histidine, X2 is Aib, X 30 It is ricin, X 34 It may be cysteine, but is not limited to it.

[0098] In the present invention, such modifications for the production of activators, fragments, variants, and derivatives of natural GLP-2 include modifications using L-type or D-type amino acids and / or non-natural amino acids; and / or modifications of the natural sequence by modification or post-translational modification (e.g., methylation, acylation, ubiquitination, intramolecular covalent bonding, etc.).

[0099] In the present invention, the GLP-2 derivative may be chemically modified or protected with an organic group at its N-terminus and / or C-terminus, or modified by adding amino acids to the terminals of the GLP-2 derivative, in order to protect it from protein-cutting enzymes in living organisms and to increase its stability.

[0100] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so the N-terminus can be acetylated and / or the C-terminus can be amidated to remove such charges, but this is not limited to this. Specifically, in the present invention, the GLP-2 derivative may have its C-terminus unmodified or amidated, but this is not limited to this.

[0101] The GLP-2 derivative of the present invention can be synthesized through a solid-phase synthesis method, produced by a recombinant method, and manufactured on a commercial basis.

[0102] In the present invention, the GLP-2 derivative may, but is not limited to, be in the form of a sustained-release conjugate to which a biocompatible substance that increases its in vivo half-life is bound. The sustained-release conjugate can exhibit increased efficacy and persistence compared to a GLP-2 derivative without a biocompatible substance (e.g., immunoglobulin Fc region) bound to it. In the present invention, a conjugate containing a GLP-2 derivative in which a biocompatible substance is bound to the GLP-2 derivative to increase its half-life is referred to as a "GLP-2 derivative sustained-release conjugate" or "GLP-2 derivative sustained-release conjugate." In the present invention, the sustained-release conjugate is used interchangeably with the conjugate.

[0103] On the other hand, such combinations may be non-naturally occurring.

[0104] Furthermore, in a GLP-2 derivative sustained-release conjugate, the linkage between the GLP-2 derivative and a biocompatible substance (e.g., an immunoglobulin Fc region) may be physical or chemical, non-covalent or covalent, and may specifically be covalent, but is not limited to these.

[0105] Furthermore, the method of linking the GLP-2 derivative and the biocompatible substance (e.g., immunoglobulin Fc region) in the GLP-2 derivative sustained-release conjugate is not particularly limited, but the GLP-2 derivative and the biocompatible substance (e.g., immunoglobulin Fc region) may be linked to each other via a linker.

[0106] Furthermore, Korean Published Patent No. 10-2019-0037181 relating to a GLP-2 derivative sustained-type conjugate is included in this application by reference.

[0107] In one specific embodiment, the persistent conjugate of the GLP-2 derivative of the present invention may have the structure of the following chemical formula (1), but is not limited thereto.

[0108] X-La-F···(1)

[0109] Here, X is a GLP-2 derivative as described in this application; L is a linker (for example, a linker containing ethylene glycol repeating units); a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; F is a biocompatible substance (e.g., an immunoglobulin Fc region) that increases the in vivo half-life of X; The above "-" may represent a chemical bond (for example, a covalent bond).

[0110] More specifically, X and L, and L and F may be linked to each other by covalent bonds, in which case the above compound may be a compound in which X, L, and F are linked to each other by covalent bonds in the order of chemical formula (1).

[0111] Furthermore, F may be directly linked to X (i.e., a is 0 in the above chemical formula (1)) or linked through a linker (L).

[0112] In the compound according to the present invention, F is X, that is, a substance that increases the half-life of the GLP-2 derivative according to the present invention, and corresponds to one of the components of the compound according to the present invention.

[0113] The above F may be bonded to X by a covalent or non-covalent chemical bond, or F and X may be bonded to each other through L by a covalent, non-covalent chemical bond, or a combination thereof.

[0114] The substance F that increases the half-life of X is a biocompatible substance, and may be selected from the group consisting of, for example, high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0115] In the case of elastin as described above, it may be human tropoelastin, which is a water-soluble precursor, or a polymer of some of its sequences or repeating units, and includes, but is not particularly limited to, elastin-like polypeptides.

[0116] Examples of the above-mentioned polymeric polymers include polymeric polymers selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides (e.g., dextran), polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof. The above-mentioned polysaccharides may include dextran, but are not particularly limited thereto.

[0117] The above term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologous polymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0118] Furthermore, the above-mentioned biocompatible substances include, but are not limited to, polylysine, polyaspartic acid, and polyglutamic acid, which are polyamino acids. Furthermore, the fatty acids may be those that have the ability to bind to albumin in the body, but are not particularly limited to this.

[0119] As an example of F mentioned above, F may be an FcRn-binding substance, and more specifically, the FeRn-binding substance may be an immunoglobulin Fc region, more specifically, an IgGFc region, and even more specifically, a non-glycosylated IgG4Fc region, but is not particularly limited thereto.

[0120] As a specific example of the present invention, the above-mentioned F (for example, the immunoglobulin Fc region) may be a dimer consisting of two polypeptide chains, and one end of L may be linked to only one of the two polypeptide chains, but is not limited thereto.

[0121] Furthermore, L may be a peptidic linker or a non-peptidic linker (for example, a linker containing ethylene glycol repeating units).

[0122] When L is a peptide linker, it may contain one or more amino acids, for example, 1 to 1000 amino acids, but is not particularly limited thereto. Various known peptide linkers may be used to link F and X in the present invention, including, for example, [GS]x linkers, [GGGS]x linkers and [GGGGS]x linkers, where x may be one or more natural numbers. However, the present invention is not limited to the above examples.

[0123] In the present invention, the "non-peptide linker" includes a biocompatible polymer in which two or more repeating units are bonded together. The repeating units are linked to each other not by peptide bonds, but by any covalent bonds. In the present invention, the non-peptide linker contains a reactive group at its terminus and can form a conjugate through reaction with other components constituting the conjugate. The non-peptide linker may be one component that forms a part of the conjugate of the present invention.

[0124] The non-peptide linker used in this invention is not limited to any non-peptide polymer that is resistant to in vivo proteolytic enzymes.

[0125] In the present invention, the non-peptide linker may be mixed with the non-peptide polymer.

[0126] Specifically, the non-peptide linker may be selected from the group consisting of fatty acids, saccharides, high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0127] The above polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, and the above polysaccharide may be dextran, but is not limited thereto.

[0128] Furthermore, although not particularly limited thereto, the polymer in the present invention may be in the range of greater than 0 and about 100 kDa, more specifically in the range of about 1 to about 100 kDa, and more specifically in the range of about 1 to about 20 kDa, but is not limited thereto.

[0129] The non-peptide linker may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides (e.g., dextran), polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, oligonucleotides, and combinations thereof, although this is not particularly limited.

[0130] The non-peptide linker may be a linker containing ethylene glycol repeating units, such as polyethylene glycol, and derivatives of these already known in the art and derivatives that can be easily produced in the art are also included in the scope of the present invention.

[0131] The repeating units of the non-peptide linker described above may be ethylene glycol repeating units. Specifically, the non-peptide linker may contain ethylene glycol repeating units while also containing active groups at its termini that are used in the production of the conjugate. The persistent conjugate according to the present invention may be in a form in which X and F are linked through the active groups, but is not limited thereto. In the present invention, the non-peptide linker may contain two or more active groups, and each active group may be the same or different from each other, but is not limited thereto.

[0132] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula (2):

[0133] JPEG2026103884000004.jpg1726...(2)

[0134] Here, n can be between 10 and 2400, between 10 and 480, or between 50 and 250, but is not limited to these ranges.

[0135] In the above persistent conjugate, the PEG site is -(CH2CH2O) n -Not just the structure, but also the connecting elements and their (CH2CH2O) n This may include, but is not limited to, oxygen atoms interposed between the two.

[0136] Furthermore, in one specific embodiment, the above-mentioned conjugate may have a structure in which GLP-2 and the immunoglobulin Fc region (F) are covalently linked through a linker containing ethylene glycol repeating units, but is not limited thereto.

[0137] The above term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologous polymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0138] The non-peptide linker used in the present invention is not limited to any polymer that is resistant to in vivo proteolytic enzymes, but is not limited to a molecular weight of more than 0 and 200 kDa, more specifically, in the range of about 1 to 100 kDa, more specifically, in the range of about 1 to 50 kDa, more specifically, in the range of about 1 to 20 kDa, more specifically, in the range of about 3.4 kDa to 10 kDa, or even more specifically, about 3.4 kDa.

[0139] In this invention, the term "approximately" includes, but is not limited to, a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and any numerical value within a range equivalent to or similar to the numerical value that follows the term "approximately".

[0140] Furthermore, the non-peptide linker of the present invention that binds to the immunoglobulin Fc region may be not only one type of polymer, but also a combination of different types of polymers.

[0141] In one specific embodiment, both ends of the non-peptide linker can, but are not limited to, a thiol group, amino group, or hydroxyl group of the immunoglobulin Fc region and a thiol group, amino group, azide group, or hydroxyl group of GLP-2.

[0142] Specifically, the non-peptide linker may include, but is not limited to, a reactive group at each end that can be bound to immunoglobulin Fc and GLP-2 or its derivatives, specifically, a thiol group of cysteine ​​in the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine, and / or histidine; and / or a hydroxyl group located at the C-terminus, and a reactive group that can be bound to the thiol group of cysteine ​​of GLP-2; an amino group of lysine, arginine, glutamine, and / or histidine; an azide group of azidrisine; and / or a hydroxyl group.

[0143] More specifically, the reactive group of the non-peptidic polymer may be one or more selected from the group consisting of the aldehyde group, the maleimide group, and succinimide derivatives, but is not limited thereto.

[0144] In the above, the propionaldehyde group or the butyraldehyde group can be given as examples of the aldehyde group, but are not limited to these.

[0145] In the above, the succinimide derivative may be, but is not limited to, succinimidyl carboxymethyl, succinimidyl valverate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidylpropionate, N-hydroxysuccinimide, hydroxysuccinimidyl, or succinimidyl carbonate.

[0146] The above-mentioned non-peptidic linker may be linked to immunoglobulin Fc and GLP-2 derivatives through the above-mentioned reactive groups and converted into a linkage portion of the non-peptidic polymer.

[0147] Furthermore, the final product generated by reductive alkylation via aldehyde bonds is far more stable than that linked by amide bonds. The aldehyde reactive group reacts selectively with the N-terminus at low pH, and can form covalent bonds with lysine residues under high pH conditions, such as pH 9.0.

[0148] The terminal reactive groups of the non-peptidic linker of the present invention may be the same or different from each other. The non-peptidic linker may have an aldehyde group reactive group at its terminal, or it may have an aldehyde group and a maleimide reactive group at its terminal, or an aldehyde group and a succinimide reactive group at its terminal, but is not limited thereto.

[0149] For example, one end may have a maleimide group, and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. Another example is having a succinimidyl group at one end and a propionaldehyde group or a butyraldehyde group at the other end.

[0150] When polyethylene glycol having a hydroxyl reactive group at the propione end is used as a non-peptide linker, the hydroxyl group can be activated with the various reactive groups by known chemical reactions, or the compound of the present invention can be produced using commercially available polyethylene glycol having modified reactive groups.

[0151] In one specific embodiment, the reactive group of the non-peptide linker may be linked to a cysteine ​​residue of GLP-2, more specifically, to the -SH group of cysteine, but is not limited thereto.

[0152] 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 immunoglobulin Fc via a reductive alkylation reaction, but this is not the only example.

[0153] Through such reductive alkylation, the N-terminal amino group of the immunoglobulin Fc domain is linked to the oxygen atom at one end of PEG via a linker group having the structure -CH2CH2CH2-, forming a structure similar to -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and a structure can be formed in which one end of PEG is linked to the sulfur atom located at the cysteine ​​of GLP-2 via a thioether bond. The thioether bond described above is It may contain the structure of JPEG2026103884000005.jpg3840.

[0154] However, this is not limited to the examples mentioned above, and is merely one example.

[0155] Furthermore, in the above-mentioned conjugate, the reactive group of the non-peptide linker may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region; this is just one example.

[0156] Furthermore, in the above-mentioned conjugate, the GLP-2 derivative may be linked to a non-peptide linker having a reactive group via its C-terminus; this is just one example.

[0157] In this invention, "C-terminus" refers to the carboxyl end of a peptide, and for the purposes of this invention, it refers to a position where a non-peptide polymer can be bound. For example, although not limited to this, it may include not only the terminal amino acid residue of the C-terminus, but also any of the amino acid residues surrounding the C-terminus, and specifically, it may include the 1st to 20th amino acid residues from the end.

[0158] In one specific embodiment, the persistent conjugate of the present invention may be a conjugation of a GLP-2 derivative and an immunoglobulin Fc region, but is not limited thereto.

[0159] In the present invention, the "immunoglobulin Fc region" refers to the region of the immunoglobulin that includes the heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), excluding the heavy chain and light chain variable regions. The above immunoglobulin Fc region may be one component of the conjugate of the present invention. Specifically, it corresponds to F in the above chemical formula (1).

[0160] In this specification, the term "Fc region" includes not only the native sequence obtained from the digestion of immunoglobulins into papain, but also its derivatives, such as sequences that differ from the native sequence due to deletions, insertions, non-conservative or conservative substitutions, or combinations thereof, of one or more amino acid residues in the native sequence.

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

[0162] Reductive amination is a reaction in which an amine group or amino group in one reactant reacts with an aldehyde (i.e., an active group capable of reductive amination) in another reactant to produce an amine, followed by the formation of an amine bond through a reduction reaction. This is a widely known organic synthesis reaction in the field.

[0163] As one specific example of the GLP-2 sustained-release conjugate of the present invention, the above-mentioned sustained-release conjugate may have the immunoglobulin Fc region linked to a linker through its N-terminal nitrogen atom.

[0164] Such immunoglobulin Fc regions may, but are not limited to, include a hinge region within the heavy chain constant region.

[0165] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at its N-terminus.

[0166] In this invention, the term "hinge sequence" refers to a site located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an interdisulfide bond.

[0167] In the present invention, the above hinge sequence may be a mutated version in which a portion of the following amino acid sequence is deleted, resulting in a single cysteine ​​residue, but is not limited thereto:

[0168] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 11).

[0169] The above hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 11 being deleted. The hinge sequence of the present invention may consist of 3 to 12 amino acids and contain only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 12), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 13), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 14), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-C ys-Pro-Pro (SEQ ID NO: 15), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 16), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 17), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 18), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 19), Glu-Pro-Ser-Cy s-Pro (SEQ ID NO: 20), Pro-Ser-Cys-Pro (SEQ ID NO: 21), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 22), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 23), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 24), Glu-Ser -Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 25), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 26), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 27), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 28), Glu-Pro-Ser-Cys (SEQ ID NO: 29), Ser-Cys-Pro (SEQ ID NO: 30).

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

[0171] In one more specific form of the GLP-2 derivative sustained-release conjugate of the present invention, the N-terminus of the immunoglobulin Fc region within the conjugate is proline, and the conjugate is such that the Fc region is linked to a linker via the nitrogen atom of the proline.

[0172] In one embodiment of the GLP-2 derivative sustained-release conjugate of the present invention, the immunoglobulin Fc region may be in the form of a dimer in which two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of chemical formula (1) of the present invention may be in the form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0173] In this invention, the term "N-terminus" means the amino terminus of a protein or polypeptide, and may include the very end of the amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc region of this invention may, but is not limited to, include a hinge sequence at its N-terminus.

[0174] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), excluding only the heavy chain and light chain variable regions of the immunoglobulin, as long as it produces an effect substantially equivalent to or improved upon that of the natural type. Alternatively, it may be a region from which a considerably long portion of the amino acid sequence corresponding to CH2 and / or CH3 has been removed.

[0175] For example, the immunoglobulin Fc region of the present invention may be, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains from the CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of the heavy chain constant region and a light chain constant region.

[0176] Furthermore, as one specific example, the immunoglobulin Fc region may be in a dimeric form, and one molecule of X may be covalently linked to one of the Fc regions in the dimeric form, in which case the immunoglobulin Fc and X may be linked to each other by a non-peptide polymer. On the other hand, it is also possible for two molecules of X to be symmetrically linked to one of the Fc regions in the dimeric form. In that case, the immunoglobulin Fc and X may be linked to each other by a non-peptide linker. However, the invention is not limited to the above examples.

[0177] Furthermore, 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 means having 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.

[0178] For example, in the case of IgGFc, amino acid residues 214-238, 297-299, 318-322, or 327-331, which are known to be important for binding, may be used as appropriate sites for deformation.

[0179] Furthermore, a variety of derivatives are possible, such as by removing sites capable of forming disulfide bonds, removing several amino acids from the N-terminus of the native Fc, or adding a methionine residue to the N-terminus of the native Fc. In addition, complement binding sites, such as Clq binding sites, may be removed to eliminate effector function, and ADCC (antibody-dependent cell-mediated cytotoxicity) sites may also be removed. Techniques for producing such immunoglobulin Fc region sequence derivatives are disclosed in International Patent Publication WO97 / 34631, International Patent Publication 96 / 32478, and others.

[0180] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in this field (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0181] The above-mentioned Fc derivative may exhibit biological activity equivalent to that of the Fc region of the present invention, and may also have increased structural stability of the Fc region with respect to heat, pH, etc.

[0182] Furthermore, such Fc regions may be obtained from native types isolated from living animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or they may be recombinant types or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from native types may involve isolating whole immunoglobulin from living human or animal organisms and then treating it with proteolytic enzymes. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. These can then be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the Fc region is derived from humans, and the recombinant immunoglobulin Fc region is obtained from microorganisms.

[0183] Furthermore, the immunoglobulin Fc region may be in the form of natural glycans, increased glycans compared to the natural form, decreased glycans compared to the natural form, or glycans removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used to increase, decrease, or remove such immunoglobulin Fc glycans. Here, the immunoglobulin Fc region from which glycans have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cell-mediated cytotoxicity or complement-dependent cell-mediated cytotoxicity is reduced or eliminated, thus not inducing unwanted immune responses in vivo. In this respect, the form that is more in line with the original purpose as a drug carrier is the immunoglobulin Fc region from which glycans have been removed or which has been deglycosinized.

[0184] In this invention, "deglycosylation" refers to the Fc region from which sugar has been removed by an enzyme, and "aglycosylation" refers to the Fc region that has not been glycosylated, produced in prokaryotes, and more specifically in Escherichia coli.

[0185] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as from a cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, and in a more specific embodiment, it may be of human origin.

[0186] Furthermore, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are most abundant in human blood, and in a more specific embodiment, it may be derived from IgG, which is known to improve the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region may be an IgG4Fc region, and in the most specific embodiment, the immunoglobulin Fc region may be a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0187] As a specific example, the immunoglobulin Fc region may be a fragment of human IgG4Fc, and may be a homodimer in which two monomers are linked by disulfide bonds (inter-chain form) between cysteine, which is the third amino acid of each monomer. In this case, the homodimer may have / have disulfide bonds between cysteine ​​positions 35 and 95 and between cysteine ​​positions 141 and 199, i.e., two disulfide bonds (intra-chain form), and the number of amino acids in each monomer may be 221, but is not limited to this.

[0188] Each monomer may consist of 221 amino acids, and the homodimer may consist of a total of 442 amino acids, but is not limited to this. Specifically, the immunoglobulin Fc region is formed when two monomers having the amino acid sequence of SEQ ID NO: 31 (composed of 221 amino acids) form a homodimer through a disulfide bond between the cysteine, which is the third amino acid of each monomer. The monomers of the homodimer may each independently form an internal disulfide bond between cysteine ​​positions 35 and 95, and an internal disulfide bond between cysteine ​​positions 141 and 199, but is not limited to this.

[0189] On the other hand, in the present invention, "combination" means that when forming a dimer or polymer, polypeptides encoding single-chain immunoglobulin Fc regions of the same origin form a bond with single-chain polypeptides of different origins. That is, dimers or polymers can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0190] In the present invention, "hybrid" is a term that means that a single-chain immunoglobulin constant region contains sequences corresponding to two or more immunoglobulin Fc fragments of different origins. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of one to four domains from the CH1, CH2, CH3, and CH4 groups of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include hinges.

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

[0192] Furthermore, the aforementioned conjugates may have increased efficacy compared to natural GLP-2 or to X without F modification. Such conjugates include, but are not limited to, the above-described forms, as well as forms encapsulated in biodegradable nanoparticles.

[0193] On the other hand, GLP-2 derivatives and their sustained-release conjugates include the peptide and the compound itself, a salt thereof (for example, a pharmaceutically acceptable salt of the peptide), or a solvate thereof.

[0194] Furthermore, peptides, compounds, GLP-2 derivatives, or their sustained conjugates may be in any pharmaceutically acceptable form.

[0195] The type of salt used is not particularly limited. However, it is preferable, though not particularly limited, that it be in a form that is safe and effective for individuals, such as mammals.

[0196] The term "pharmaceutically acceptable" above means a substance that can be effectively used for its desired purpose without causing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0197] In this invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable inorganic acids, organic acids, or salts derived from bases. Examples of suitable acids include hydrochloric acid, bromate, 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 may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0198] Furthermore, the term "solvate" as used in this invention refers to a complex formed by the peptide, compound, or salt thereof according to the present invention with a solvent molecule.

[0199] The above-mentioned GLP-2 derivatives, sustained-release conjugates, and compositions containing the same may be used for the prevention, improvement, or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof.

[0200] In the present invention, the term “prevention” means all actions that completely or partially suppress or delay the onset of mucositis induced by radiotherapy, chemotherapy, or a combination thereof by administration of a GLP-2 derivative, a sustained-release conjugate of a GLP-2 derivative, and / or a composition containing the same; and “treatment” means all actions that completely or partially improve or benefit the symptoms of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, including reduction, improvement, alleviation of the distress of the symptoms, reduction in the incidence of mucositis, or other changes in the patient that increase the treatment outcome.

[0201] In the present invention, the term "administration" means introducing a given substance to a patient by any appropriate method, and the route of administration of the composition is not particularly limited, but may be administered through any common route that allows the composition to reach a target in the body, for example, orally, or by parenteral administration routes including, but not limited to, skin, intravenous, intramuscular, intraarterial, intrabone marrow, intrathecal cavity, intraventricular, pulmonary, percutaneous, subcutaneous, intraabdominal, intranasal, gastrointestinal, topical, sublingual, vaginal, or rectal routes. On the other hand, since peptides are digested when administered orally, it is preferable that oral compositions be coated with the active agent or formulated in a dosage form that protects them from degradation in the gastrointestinal tract. Specifically, in the present invention, the composition may be administered in the form of an injection. Furthermore, in the present invention, the composition may be administered by any device that allows the active ingredient to move to target cells.

[0202] In one specific embodiment, the composition of the present invention may be administered sequentially or in reverse order with radiotherapy and / or chemotherapy. Specifically, the composition of the present invention may be administered before (for example, within 1, 2, 3, 4, 5, or 6 days before radiotherapy and / or chemotherapy; or within 1 week or 1 month) the administration of radiotherapy and / or chemotherapy. Also, specifically, the composition of the present invention may be administered after (for example, within 1, 2, 3, 4, 5, or 6 days after radiotherapy and / or chemotherapy; within 1 week or 1 month; or on the 1st day (24 hours)) the administration of radiotherapy and / or chemotherapy.

[0203] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent. Such a pharmaceutically acceptable carrier, excipient, or diluent may be unnaturally occurring.

[0204] In this invention, the term "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect and without causing side effects, which can be readily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of doses, the duration of treatment, and drugs used in combination or concurrently.

[0205] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier. The carrier is not particularly limited, but for oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. may be used; for injection, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used; and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

[0206] The composition of the present invention may be manufactured in various forms by mixing it with pharmaceutically acceptable carriers as described above. For example, when administered orally, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and when administered by injection, it can be manufactured in the form of unit dose ampoules or multi-dose formulations. In addition, it can be manufactured in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0207] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. Furthermore, fillers, anti-flocculants, lubricants, wetting agents, fragrances, preservatives, and the like may be included.

[0208] Furthermore, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid preparations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0209] Furthermore, the above composition may be formulated into a unit-dose type preparation suitable for intracellular administration to a patient by conventional methods in the pharmaceutical field, specifically in a dosage form useful for the administration of protein pharmaceuticals, and administered using a method of administration commonly used in this industry, but is not limited thereto.

[0210] Furthermore, the above-mentioned GLP-2 derivatives or their sustained-release conjugates are used in mixture with various carriers that are acceptable as pharmaceuticals, such as physiological saline or organic solvents. Carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers are used as pharmaceuticals to increase stability and water absorption.

[0211] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined according to the type of drug that is the active ingredient, along with various relevant factors such as the disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the composition of the present invention may, but is not limited to, contain a pharmaceutically effective amount of a GLP-2 derivative or its sustained-release conjugate.

[0212] In the present invention, the inclusion of a GLP-2 derivative or its sustained-release conjugate in a pharmaceutical composition as a pharmaceutically effective amount means the extent to which the desired pharmacological activity is obtained by the GLP-2 derivative or its sustained-release conjugate, and also means a level that is pharmaceutically acceptable, either to the extent that no toxicity or side effects occur in the administered individual, or to a level that is only minimal. Such a pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, the patient, the dosage form, and other factors.

[0213] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol involving multiple doses over a long period. The pharmaceutical composition of the present invention may have different active ingredient content depending on the severity of the disease. Specifically, the preferred total dose of the GLP-2 derivative or its sustained-release conjugate of the present invention may be about 0.0001 mg to 500 mg, about 0.001 mg to 100 mg, and more preferably about 0.01 mg to 10 mg per kg of patient body weight per day, but is not limited thereto.

[0214] However, the effective dose for a patient is determined by considering a variety of factors, including not only the route of administration and number of treatments of the pharmaceutical composition, but also the patient's age, weight, health status, sex, disease severity, diet, and excretion rate. Considering these points, a person with ordinary skill in the art can determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in terms of dosage form, route of administration, and method of administration, as long as it achieves the effects of the present invention.

[0215] The pharmaceutical composition of the present invention exhibits excellent in vivo persistence and potency, and can significantly reduce the number and frequency of administrations of the pharmaceutical formulation of the present invention.

[0216] Another embodiment of the present invention is to provide a food composition for the prevention or improvement of mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising a GLP-2 derivative and / or a sustained-release conjugate thereof.

[0217] The above-mentioned GLP-2 derivatives, their sustained-release conjugates, or mucositis induced by radiotherapy, chemotherapy, or combinations thereof have been explained previously.

[0218] The above food composition is used as a functional food for health. When the composition of the present invention is used as a food additive, the above peptide (the peptide itself or its sustained-release conjugate) can be added as is or used together with other foods or food components, and can be suitably used by conventional methods. The amount of active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

[0219] In this invention, the term "health functional food" refers to a food manufactured or processed by methods such as extracting, concentrating, purifying, or mixing specific components as raw materials or food ingredients for the purpose of health support. It refers to a food that is designed and processed so that the above components can fully exert biological regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery from disease. The above-mentioned health food composition can perform functions related to disease prevention and recovery from disease.

[0220] Another embodiment of the present invention provides a method for preventing, improving, or treating mucositis induced by radiotherapy, chemotherapy, or a combination thereof, comprising the step of administering the above-mentioned GLP-2 derivative, its sustained-release conjugate, and / or a composition containing the same to an individual in need.

[0221] The above-mentioned GLP-2 derivatives, their sustained-release conjugates, compositions, or mucositis induced by radiotherapy, chemotherapy, or combinations thereof have been explained previously.

[0222] Specifically, the above GLP-2 derivative may, but is not limited to, include the amino acid sequence represented by the above general formula 1 (excluding the sequence identical to sequence number 1 from the amino acid sequence of general formula 1).

[0223] In the present invention, an individual is an individual suspected of having mucositis induced by radiotherapy, chemotherapy, or a combination thereof, and may be a mammal including rats, livestock, etc., including humans who have the disease or are likely to develop it, but is not limited to individuals that can be treated with the GLP-2 derivative of the present invention, its sustained-release conjugate, and / or pharmaceutical compositions containing the same. Furthermore, humans may be excluded from the individuals of the present invention, but are not limited thereto.

[0224] The method of the present invention may include administering a pharmaceutically effective dose of a GLP-2 derivative, its sustained-release conjugate, and / or a composition containing the same. The appropriate total daily dose is determined by the treating physician within the bounds of sound medical judgment and can be administered in one or several divided doses. However, for the purposes of the present invention, it is preferable that the specific therapeutic effective dose for a particular patient be applied differently depending on various factors, including the type and degree of the reaction to be achieved, the specific composition (including whether different formulations are used), the patient's age, weight, general health status, sex and diet, administration time, administration route and secretion rate of the composition, duration of treatment, drugs used with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field.

[0225] Another embodiment of the present invention provides the use of GLP-2 derivatives, their sustained-release conjugates, and / or compositions containing them for the prevention, improvement, or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof.

[0226] As previously explained, GLP-2, its sustained-release conjugates, compositions, or mucositis induced by radiotherapy, chemotherapy, or combinations thereof are all relevant.

[0227] Specifically, the above GLP-2 derivative may, but is not limited to, include the amino acid sequence represented by the above general formula 1 (excluding the sequence identical to sequence number 1 from the amino acid sequence of general formula 1).

[0228] Another embodiment of the present invention provides the use of GLP-2 derivatives, their sustained-release conjugates, and / or pharmaceutical compositions containing them in the manufacture of agents for the prevention or treatment of mucositis induced by radiotherapy, chemotherapy, or a combination thereof.

[0229] The above-mentioned GLP-2 derivatives, their sustained-release conjugates, or mucositis induced by radiotherapy, chemotherapy, or combinations thereof have been explained previously.

[0230] Specifically, the above GLP-2 derivative may, but is not limited to, include the amino acid sequence represented by the above general formula 1 (excluding the sequence identical to sequence number 1 from the amino acid sequence of general formula 1).

[0231] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto. [Examples]

[0232] Example 1. Production of a GLP-2 derivative sustained-release conjugate To pegylate the GLP-2 derivative CAGLP-2RK of Sequence ID No. 4 in Table 1 with modified polyethylene glycol ALD(2)PEG (a modified polyethylene glycol in which both terminal hydrogens are substituted with propylaldehyde groups (3-oxopropyl groups) and the molecular weight of the ethylene glycol repeating unit (moiety) is 3.4 kDa, manufactured by NOF Corporation of Japan), the molar ratio of the GLP-2 derivative to ALD(2)PEG was set to 1:5 to 1:20, and the concentration of the GLP-2 derivative was set to 5 to 10 mg / ml. The reaction was carried out at 2 to 8°C for 4 to 16 hours. The reaction was carried out with 20 mM HEPES pH 7.5 and ethanol, and the reaction was continued with the addition of 20 mM sodium cyanoborohydride as a reducing agent. The reaction mixture was purified to obtain the monopegylated GLP-2 derivative using a Source 15S (GE, USA) column with a buffer solution containing sodium citrate pH 2.0 and ethanol, and a potassium chloride concentration gradient.

[0233] Next, the molar ratio of the purified monopegylated GLP-2 derivative to the immunoglobulin Fc fragment of SEQ ID NO: 32 was adjusted to 1:2 to 1:6, and the total protein concentration was set to 30 to 35 mg / mL. The reaction was then carried out at 2 to 8°C for 12 to 20 hours. 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.

[0234] After the reaction was completed, the reaction mixture was subjected to a concentration gradient of bis-Tris pH 6.5 buffer and sodium chloride on a Source 15Q (GE, USA) column, and then subjected to a concentration gradient of ammonium sulfate and sodium citrate pH 5.0-5.2 on a Source 15ISO (GE, USA) column to purify the persistent conjugate of the GLP-2 derivative, which is a conjugate in which the GLP-2 derivative is covalently linked to an immunoglobulin Fc fragment by a polyethylene glycol linker.

[0235] Example 2: Confirmation of small intestinal weight increase in a rat model of gastrointestinal mucositis induced by chemotherapy with administration of a sustained-release conjugate of a GLP-2 derivative. To measure the in vivo small intestine-improving efficacy of the GLP-2 derivative sustained-release conjugate produced in Example 1 above, rats in which gastrointestinal mucositis was induced by chemotherapy (Docetaxel + Cyclophosphamide, TC) were used.

[0236] Specifically, 7-week-old male rats (SD rats) were divided into three groups: a normal control group (a group that did not receive chemotherapy, G1, (SHarm) Vehicle), a control group with chemotherapy-induced gastrointestinal mucositis (G2, (TC) Vehicle), and a group in which gastrointestinal mucositis was induced by chemotherapy and then administered the GLP-2 derivative sustained-release conjugate (3.1 mg / kg / QW (0.5 mg / kg / QM HED)) prepared in Example 1 (G3, (TC) GLP-2 derivative sustained-release conjugate). (In this application, the numerical values ​​indicated for the dose of the GLP-2 derivative sustained-release conjugate are based on the total mass of the GLP-2 derivative sustained-release conjugate used minus the mass of the polyethylene glycol linker portion, i.e., the sum of the masses of only the polypeptide portion.) Then, G2 and G3 were administered Docetaxel 36 mg / kg and Cyclophosphamide 300 mg / kg to induce gastrointestinal mucositis (D0). The following day (D1), each group was administered either a vehicle or a GLP-2 derivative sustained-release conjugate subcutaneously, and the small intestine weight was measured on D2, D3, and D4.

[0237] As a result, we confirmed that the group administered a sustained-release conjugate of a GLP-2 derivative showed a significant recovery in small intestinal weight within a short period of time compared to the control group with chemotherapy-induced gastrointestinal mucositis (Figure 1).

[0238] These results suggest that the GLP-2 derivative sustained-release conjugate of the present invention is effective in preventing and treating chemotherapy-induced gastrointestinal mucositis through rapid recovery of the small intestine.

[0239] Example 3: Confirmation of changes in small intestinal weight in a rat model of gastrointestinal mucositis induced by chemotherapy with administration of GLP-2 derivatives and GLP-2 derivative sustained-release conjugates.

[0240] To measure the in vivo small intestinal improvement efficacy of GLP-2 derivatives and the sustained-release GLP-2 derivative conjugate produced in Example 1 above, rats in which gastrointestinal mucositis was induced by chemotherapy (Docetaxel + Cyclophosphamide, TC) were used. Teduglutide was selected as a representative example of the GLP-2 derivative and used in the experiment.

[0241] Specifically, five 7-week-old male rats (SD rats) were used in each group: a normal control group (no chemotherapy, G1, Vehicle), a control group with chemotherapy-induced gastrointestinal mucositis (G2, (TC) vehicle), and a control group with chemotherapy-induced gastrointestinal mucositis (D0) simultaneously with GLP-2 derivative (Teduglutide 0.1 mg / kg / BID, E). max The patients were divided into three groups: one group administered (G3, (TC)Teduglutide (D0)), one group administered the GLP-2 derivative sustained-release conjugate (3.1 mg / kg / QW (0.5 mg / kg / QM HED)) manufactured in Example 1 simultaneously with the induction of gastrointestinal mucositis by chemotherapy (D0) (G4, (TC)GLP-2 derivative sustained-release conjugate (D0)), and one group administered the GLP-2 derivative sustained-release conjugate (3.1 mg / kg / QW (0.5 mg / kg / QM HED)) manufactured in Example 1 one day before the induction of gastrointestinal mucositis by chemotherapy (D-1) (G5, (TC)GLP-2 derivative sustained-release conjugate (D-1)). The group in which gastrointestinal mucositis was induced by chemotherapy was administered Docetaxel 36 mg / kg and Cyclophosphamide 300 mg / kg to induce gastrointestinal mucositis.

[0242] As a result, the group administered a sustained-release GLP-2 derivative conjugate (G4) showed significantly less small intestinal weight loss due to chemotherapy-induced gastrointestinal mucositis and significantly greater small intestinal weight increase upon recovery compared to the group administered a GLP-2 derivative (G3) (Figure 2, A). Furthermore, when comparing the group administered a sustained-release GLP-2 derivative conjugate one day before inducing gastrointestinal mucositis by chemotherapy (D-1) (G5, (TC)GLP-2 derivative sustained-release conjugate (D-1)) with the group administered a sustained-release GLP-2 derivative conjugate simultaneously with the induction of gastrointestinal mucositis by chemotherapy (D0) (G4, (TC)GLP-2 derivative sustained-release conjugate (D0)), it was confirmed that administering a sustained-release GLP-2 derivative conjugate one day before inducing gastrointestinal mucositis by chemotherapy resulted in significantly less small intestinal weight loss due to chemotherapy (Figure 2, B).

[0243] These results suggest that administration of the GLP-2 derivative sustained-release conjugate of the present invention exhibits superior prophylactic or therapeutic efficacy against chemotherapy-induced gastrointestinal mucositis compared to administration of GLP-2 derivatives, through prevention of small intestinal damage and faster recovery, and that administering the GLP-2 derivative sustained-release conjugate one day before chemotherapy yields an even higher prophylactic effect.

[0244] Example 4: Confirmation of the effects of a sustained-release GLP-2 derivative conjugate on M1 polarization, macrophage differentiation, and monocyte migration in THP-1 cells (monocytes). To confirm the inhibitory effects of the GLP-2 derivative sustained-release conjugate produced in Example 1 above on M1 polarization, macrophage differentiation, and monocyte migration in THP-1 cells (monocytes), the following in vitro experiments were conducted.

[0245] [Experiment to confirm the suppression of M1 polarization] THP-1 cells were treated with the GLP-2 derivative sustained conjugate (10 μM) prepared in Example 1 for 4 hours. Then, after treating with 1 μg / mL of lipopolysaccharide (LPS) for 2 hours to induce inflammation, RNA was extracted and the mRNA expression levels of pro-inflammatory cytokines (M1 polarization), TNF-α, IL-1β, and IL-6 were measured through qPCR.

[0246] [Macrophage differentiation inhibition confirmation experiment] THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) (10 μM), which is used to induce the differentiation of THP-1, together with the GLP-2 derivative sustained conjugate prepared in Example 1 for 48 hours. Then, adherent cells, which are cells differentiated into macrophages, were counted.

[0247] [Monocyte migration inhibition experiment] THP-1 cells were treated with the GLP-2 derivative sustained conjugate (10 μM) prepared in Example 1 for 48 hours. Then, the THP-1 cells treated with the drug were transferred to the upper chamber of a Boyden chamber, and then 50 ng / mL of CCL-2, which can induce migration to the bottom chamber, was added and cultured for 4 hours. Then, the degree of migration was measured using a migration assay kit (abcam).

[0248] Through the above experiments, the effects of inhibiting M1 polarization (Figure 3, A), macrophage differentiation (Figure 3, B), and monocyte migration (Figure 3, C) of each GLP-2 derivative sustained conjugate were confirmed.

[0249] Such results suggest that the GLP-2 derivative sustained conjugate of the present invention can suppress inflammation, inhibit the differentiation of monocytes into macrophages that induce inflammation, inhibit the migration of monocytes to the inflammation site, and is effective in the prevention and treatment of gastrointestinal mucositis.

[0250] From the above description, a person skilled in the art in which the present invention pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

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