Pharmaceutical composition for preventing or treating chronic kidney disease containing a glucagon derivative

Abstract

The present invention relates to a glucagon derivative and its use.

Description

[Technical field]

[0001] The present invention relates to a composition containing a glucagon derivative and uses thereof. [Background technology]

[0002] Glucagon is produced and secreted by the pancreas when blood glucose levels drop due to various causes such as drug treatment, disease, hormone or enzyme deficiency. It is known that the secreted glucagon acts to break down glycogen in the liver to release glucose, thereby raising blood glucose levels to normal levels. It is also known that glucagon not only suppresses fat synthesis in the liver, but also promotes the burning of fatty acids, thereby lowering blood lipid levels. Furthermore, glucagon is known to act directly and indirectly on white fat to promote fat burning and fat browning, thereby exerting an effective weight loss effect (Non-Patent Document 1). Such glucagon is active by acting on the glucagon receptor.

[0003] In recent years, there has been an increasing need for glucagon derivatives that act on the glucagon receptor and have improved physical properties in order to improve efficacy and reduce side effects. Therefore, the present inventors have developed a glucagon derivative that acts on the glucagon receptor and a conjugate thereof (Patent Document 1).

[0004] The kidney is an organ located below the diaphragm and behind the lungs, which removes waste products from the body and regulates the amount of water and salt, electrolytes, and acid-base balance. Chronic kidney disease is one of the diseases caused by kidney damage or dysfunction.

[0005] Chronic kidney disease, also known as chronic kidney disease, is a condition in which protein is continuously excreted in the urine (proteinuria) and kidney function is reduced, and it is known that patients with diabetes and high blood pressure are at high risk of developing chronic kidney disease. In patients with chronic kidney disease, reduced kidney function causes waste products to accumulate in the body, leading to increased blood pressure, anemia, weak bones, nerve damage, and an increased risk of developing cardiovascular disease.

[0006] Such chronic kidney diseases are progressive and difficult to detect in the early stages, so early diagnosis through testing is important. If treatment is delayed, the disease will progress to chronic renal failure, requiring dialysis or a kidney transplant.

[0007] The treatment of chronic kidney disease is performed by reducing the risk and maintaining or restoring kidney function. In particular, it is known that 60% of patients with chronic kidney disease are accompanied by hypertension, and since hypertension is closely related to chronic kidney disease, the treatment of chronic kidney disease using antihypertensive drugs has been studied, and treatment methods using angiotensin converting enzyme inhibitors, angiotensin receptor blockers, etc. have been studied. However, it has also been reported that the administration of conventional antihypertensive drugs causes side effects such as renal dysfunction, hyperkalemia, and ischemic kidney damage.

[0008] Therefore, efforts are ongoing to develop new therapeutic agents and methods for chronic kidney disease that lower blood pressure and restore kidney function. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2018 / 004283 [Patent Document 2] International Publication No. 2016 / 108586 [Patent Document 3] International Publication No. 2017 / 003191

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Non-licensed literature

[0010] [Non-licensed document 1] Nat Rev Endocrinol. 11, 329-38 (2015); Physiol Rev. 97, 721-66 (2017) [Non-licensed document 2] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed document 3] Rice et al., 2000, Trends Genet. 16: 276-277

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0011] There is a need for the development of new therapeutic agents and methods for chronic kidney disease that lower blood pressure and restore kidney function. [Means for solving the problem]

[0012] The present invention aims to provide a pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative or a conjugate thereof. The present invention also aims to provide a method for preventing or treating chronic kidney disease, comprising the step of administering a glucagon derivative or a conjugate thereof, or a composition comprising the same.

[0013] A further object of the present invention is to provide use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for preventing or treating chronic kidney disease.

[0014] A further object of the present invention is to provide a use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for the manufacture of a medicament having an application in the prevention or treatment of chronic kidney disease.

[0015] A further object of the present invention is to provide a pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof.

[0016] A further object of the present invention is to provide a therapeutic use of a glucagon derivative or a conjugate thereof in combination with a GLP-1 receptor agonist or a conjugate thereof. Effect of the Invention

[0017] Since the glucagon derivative of the present invention has the effects of lowering blood pressure and restoring renal function, it can be used as a therapeutic drug for patients with chronic kidney disease, and further effects can be expected by using it in combination with a GLP-1 receptor agonist. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the effect of a long-acting conjugate of the glucagon derivative of the present invention represented by SEQ ID NO: 37 on reducing urinary albumin excretion in an essential hypertension (SHR) model. [Diagram 2] FIG. 1 shows the systolic (left) and diastolic (right) blood pressure lowering effects of a long-acting conjugate of the glucagon derivative of SEQ ID NO: 37 of the present invention in an essential hypertension (SHR) model. [Diagram 3] FIG. 1 shows changes in urinary ACR (albumin to creatinine ratio) following coadministration of a long-acting conjugate of the glucagon derivative of the present invention represented by SEQ ID NO: 37 and a long-acting conjugate of CA exendin-4 in an essential hypertension (SHR) model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] One embodiment of the present invention is a composition comprising a glucagon derivative or a conjugate thereof, the conjugate being a substance in which a peptide having activity against a glucagon derivative is covalently bound to an immunoglobulin Fc region via a linker.

[0020] The composition according to one embodiment is a pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative or a conjugate thereof.

[0021] In another embodiment of the composition, the glucagon derivative is a peptide comprising the amino acid sequence of general formula 1:

[0022] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (General formula 1, Sequence number 46)

[0023] In the above formula, X1 is tyrosine (Y), X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine, X7 is threonine (T), valine (V) or cysteine ​​(C), X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), and X15 is aspartic acid (D), glutamic acid (E) or cysteine ​​(C). X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, cysteine ​​(C), or absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or absent; X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C); is absent, X19 is alanine (A), arginine (R), serine (S), valine (V) or cysteine ​​(C) or is absent, X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid or cysteine ​​(C) or is absent, X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V) or cysteine ​​(C) or is absent, and X23 is isoleucine (I). , valine (V) or arginine (R), or absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid, leucine (L), or absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or absent; X28 is glutamine (Q), lysine (K),X29 is threonine (T), and X30 is cysteine ​​(C) or absent (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 or SEQ ID NO: 12).

[0024] In another specific example of the composition, the glucagon derivative comprises any one of amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 11 and 13 to 45.

[0025] In the composition according to any of the above-mentioned embodiments, the peptide is in the form of a long-acting conjugate, and the long-acting conjugate is characterized in that it is represented by chemical formula (1).

[0026] XLF···(1)

[0027] Here, X is a peptide comprising the amino acid sequence of general formula 1, L is a linker comprising an ethylene glycol repeat unit, F is an immunoglobulin Fc region, and - indicates a covalent bond linkage between X and L and between L and F.

[0028] In the composition according to any of the above-mentioned embodiments, in the general formula 1, X2 is Aib (aminoisobutyric acid), X7 is threonine (T), valine (V) or cysteine ​​(C), X10 is tyrosine (Y), X12 is lysine (K), X13 is tyrosine (Y), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), X16 is glutamic acid (E) or serine (S), X17 is lysine (K), arginine (R) or cysteine ​​(C), and X18 is arginine ( X19 is alanine (A) or cysteine ​​(C), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X23 is valine (V), X24 is glutamine (Q), X27 is methionine (M), X28 is asparagine (N), X29 is threonine (T), and X30 is cysteine ​​(C) or absent.

[0029] In the composition according to any of the above-mentioned specific examples, the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11, 13-25, 27, 29, 31, 33 and 35-45.

[0030] In a composition according to any of the previous embodiments, the peptide is characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42 and 44.

[0031] In a composition according to any of the above-mentioned embodiments, the composition is characterized by having at least one of the following properties: (a) Lowering blood pressure compared to vehicle-treated controls (b) Albumin excretion is reduced compared to the vehicle-treated control group (c) Reduced proteinuria compared to vehicle-treated controls (d) Kidney function is restored compared to vehicle-treated controls

[0032] The composition according to any of the above-mentioned embodiments is characterized in that, when administered to an individual, the composition exerts an effect of lowering blood pressure and / or restoring kidney function in the individual.

[0033] A composition according to any of the above embodiments, wherein the glucagon derivative is characterized in that the C-terminus is unmodified or amidated.

[0034] In a composition according to any of the above embodiments, the glucagon derivative is characterized in that a ring is formed between the amino acid residues.

[0035] In a composition according to any of the previous embodiments, the immunoglobulin Fc region is characterized in that it is aglycosylated.

[0036] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it is selected from the group consisting of: (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of at least one of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region; and (f) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0037] In any of the above-mentioned specific examples of the composition, the immunoglobulin Fc region is characterized in that it has a disulfide bond-forming site removed, some amino acids at the N-terminus of a native Fc are deleted, a methionine residue is added to the N-terminus of a native Fc, a complement binding site removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site removed.

[0038] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE or IgM.

[0039] In a composition according to any of the above-mentioned embodiments, the immunoglobulin Fc region is characterized in that it is a hybrid of domains of different origins derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

[0040] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region is in a dimeric form.

[0041] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

[0042] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region is an IgG4 Fc region.

[0043] In a composition according to any of the previous embodiments, the immunoglobulin Fc region is characterized in that it is an aglycosylated Fc region derived from human IgG4.

[0044] In a composition according to any of the above-mentioned specific examples, the immunoglobulin Fc region is characterized in that it has a structure in which two polypeptide chains are linked by disulfide bonds, and is linked only via a nitrogen atom of one of the two chains.

[0045] The composition according to any of the previous embodiments, wherein the immunoglobulin Fc region comprises a monomer having the amino acid sequence of SEQ ID NO:67.

[0046] In a composition according to any of the above embodiments, the immunoglobulin Fc region is characterized in that it is a homodimer consisting of monomers of the amino acid sequence of SEQ ID NO:67.

[0047] In a composition according to any of the previous embodiments, the immunoglobulin Fc region is characterized in that it is linked by the nitrogen atom of its N-terminal proline.

[0048] In a composition according to any of the above-mentioned embodiments, the conjugate is characterized in that one end of L is linked to F and X by covalent bonds formed by reaction of one end of L with an amino group or a thiol group of F, and the other end of L with an amino group or a thiol group of X, respectively.

[0049] The composition according to any of the previous embodiments, wherein L is polyethylene glycol.

[0050] In any of the above-mentioned specific examples of the composition, the chemical formula weight of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

[0051] In the composition according to any of the above specific examples, the ethylene glycol repeating unit is [OCH2CH2]n, where n is a natural number, and is determined so that the average molecular weight, for example the number average molecular weight, of the [OCH2CH2]n portion in the conjugate is 1 to 100 kDa.

[0052] The composition according to any one of the above-mentioned specific examples is characterized in that an X1 molecule is covalently linked to any one of the Fc region chains of the dimeric immunoglobulin Fc region via a linker containing an ethylene glycol repeat unit.

[0053] The composition according to any one of the above-mentioned specific examples is characterized in that one end of the linker is linked only to one of the two Fc region chains of the dimeric immunoglobulin Fc region.

[0054] A composition according to any of the above embodiments, further comprising a glucagon like peptide 1 receptor agonist.

[0055] In a composition according to any of the above-mentioned embodiments, the GLP-1 receptor agonist is characterized in that it is selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof.

[0056] In a composition according to any of the above-mentioned embodiments, the GLP-1 receptor agonist is characterized in that the N-terminal histidine residue is a GLP-1 receptor agonist derivative in which the histidine residue is replaced with a substance selected from the group consisting of des-amino-histidyl, dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl and β-carboxyimidazopropionyl.

[0057] In the composition according to any of the above-mentioned specific examples, the GLP-1 receptor agonist is characterized in that it is selected from the group consisting of natural exendin-4, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been removed, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been replaced with a hydroxyl group, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been modified with a dimethyl group, and exendin-4 derivatives in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been deleted.

[0058] The composition according to any of the previous embodiments, wherein the GLP-1 receptor agonist is imidazo-acetyl exendin-4 (CA exendin-4).

[0059] In the composition according to any of the above-mentioned embodiments, the GLP-1 receptor agonist is characterized in that it is in the form of a long-acting conjugate in which the GLP-1 receptor agonist is linked to an immunoglobulin Fc region.

[0060] In a composition according to any of the above-mentioned embodiments, the GLP-1 receptor agonist is characterized in that imidazo-acetyl exendin-4 (CA exendin-4) is linked to an immunoglobulin Fc region via a linker comprising an ethylene glycol repeat unit.

[0061] Another embodiment of the invention is a method for preventing or treating chronic kidney disease comprising the step of administering to an individual a glucagon derivative, a conjugate thereof, or a composition comprising same.

[0062] Yet another embodiment of the present invention is a method for preventing or treating chronic kidney disease, comprising the step of administering to an individual a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof, or a composition containing them.

[0063] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for the prevention or treatment of chronic kidney disease.

[0064] Yet another embodiment of the present invention relates to the use of a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof, or a composition containing them, for the prevention or treatment of chronic kidney disease.

[0065] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition comprising same, for the manufacture of a medicament having application in the prevention or treatment of chronic kidney disease.

[0066] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof, or a composition containing the same, for the manufacture of a medicament for the prevention or treatment of chronic kidney disease.

[0067] The present invention will now be described in more detail.

[0068] It should be noted that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, any combination of various elements disclosed in the present invention is included in the present invention. In addition, the present invention is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which the present invention belongs and the contents of the present invention.

[0069] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system. Alanine Ala,A Arginine Arg,R Asparagine Asn,N Aspartic acid Asp,D Cysteine ​​Cys,C Glutamic acid Glu,E Glutamine Gln,Q Glycine Gly,G Histidine Isoleucine Ile,I Leucine Leu,L Lysine Lys,K Methionine Met,M Phenylalanine Phe,F Proline Pro,P Ser,S Threonine Thr,T Tryptophan Trp,W Tyrosine Tyr,Y Valin,V

[0070] In this specification, "Aib" is used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid are used interchangeably.

[0071] One embodiment of the present invention is a composition comprising a glucagon derivative or a conjugate thereof. Specifically, the composition is a pharmaceutical composition for preventing or treating chronic kidney disease comprising a glucagon derivative or a conjugate thereof.

[0072] In one embodiment, the pharmaceutical composition for preventing or treating chronic kidney disease may be a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a pharma- ceutical effective amount of a glucagon derivative or a conjugate thereof.

[0073] The composition containing the glucagon derivative or its conjugate according to the present invention as an active ingredient lowers blood pressure and restores renal function by acting on the glucagon receptor, and exerts the effect of preventing or treating chronic kidney disease.

[0074] The composition of the present invention exhibits an effect of preventing or treating chronic kidney disease.

[0075] In the present invention, "chronic kidney disease (CKD)" is an independent disease name that is used interchangeably with chronic kidney disease and is defined as a state in which structural or functional abnormalities of the kidney exist for more than 3 months, or the glomerular filtration rate has decreased to 60 mL / min / 1.73 m2 or less for more than 3 months, regardless of the presence or absence of kidney damage. Chronic kidney disease is divided into 5 stages according to its progression and applied clinically. When the kidney is damaged by high blood sugar or when high blood pressure continues for a long time, the risk of developing chronic kidney disease increases. In addition, glomerulonephritis, polycystic kidney disease, autoimmune diseases (e.g., systemic lupus erythematosus), etc. are also known to be causes of chronic kidney disease.

[0076] Patients with chronic kidney disease suffer from a condition in which protein is excreted in urine due to a decrease in glomerular filtration rate (proteinuria) and a condition in which renal function is reduced, and hypertension in particular is known to be a cause of chronic kidney disease, but is also known to be a complication of chronic kidney disease. Patients with chronic kidney disease often suffer from glomerular hypertension and proteinuria, and therefore it is known that lowering blood pressure and reducing proteinuria are necessary and important for the treatment of chronic kidney disease.

[0077] The chronic kidney disease includes at least one disease selected from the group consisting of nephrotic syndrome, renal tubular dysfunction, renal hypertension, uremia, renal failure, and chronic renal failure, but is not limited thereto.

[0078] The glucagon derivative or its conjugate according to the present invention, or a composition containing same, has the effect of lowering blood pressure and / or restoring kidney function.

[0079] The lowering of blood pressure means, but is not limited to, a decrease in systolic blood pressure (SBP) and diastolic blood pressure (DBP).

[0080] The recovery of kidney function means recovery of the kidney's inherent function of filtering waste products from the blood, and can be measured by various methods, including, but not limited to, measuring albumin excretion, glomerular filtration rate, proteinuria, creatine concentration, etc.

[0081] In the present invention, proteinuria means a condition in which the urine of a patient with chronic kidney disease who has a low glomerular filtration rate contains a higher than normal amount of protein (e.g., albumin, globulin, etc.). Albumin is a protein that accounts for about 40% of urinary protein, and the proteinuria level can be confirmed by measuring the albumin level in urine. The glucagon derivative or its conjugate according to the present invention, or a composition containing the same, may restore renal function and reduce the amount of protein (e.g., albumin) excreted outside the body. Therefore, in the present invention, reduction of proteinuria is used in the same sense as recovery of renal function.

[0082] The glucagon derivative or its conjugate according to the present invention, or a composition containing the same, may exert a blood pressure lowering effect on an individual when administered to the individual. The glucagon derivative or its conjugate, or a composition containing the same, may restore the kidney function of an individual when administered to the individual. The blood pressure lowering and the kidney function restoration may be performed independently or together by the glucagon derivative or its conjugate according to the present invention, or a composition containing the same.

[0083] In the present invention, the term "individual" refers to a mammal, including humans, mice, livestock, etc., having kidney damage, abnormal kidney function, and / or chronic kidney disease, and is not particularly limited as long as beneficial effects can be obtained by lowering blood pressure and / or restoring kidney function.

[0084] The spontaneously hypertensive rat (SHR) model used in the examples of the present invention is known as a chronic kidney disease-related model in which kidney damage due to congenital hypertension is induced, and is used to evaluate the efficacy of antihypertensive therapeutic agents. In the examples of the present invention, the antihypertensive and renal function recovery effects of the peptide glucagon derivative or its long-acting conjugate according to the present invention were confirmed in the model. This suggests that it is useful for the prevention or treatment of chronic kidney disease.

[0085] The glucagon derivative or conjugate thereof, or a composition containing same according to the present invention has at least one of the following properties in an individual to which it is administered, but is not limited thereto. (a) Lowering blood pressure compared to vehicle-treated controls (b) Albumin excretion is reduced compared to the vehicle-treated control group (c) Reduced proteinuria compared to vehicle-treated controls (d) Kidney function is restored compared to vehicle-treated controls

[0086] The glucagon derivative or its conjugate according to the present invention, or a composition containing the same, has the above-mentioned properties and thus exerts a preventive or therapeutic effect against chronic kidney diseases.

[0087] In the present invention, "glucagon" refers to a type of hormone secreted from α cells of the pancreas, which plays a role in increasing the glucose concentration and raising blood sugar levels in the body by promoting the decomposition of glycogen. Native glucagon has the following amino acid sequence: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1)

[0088] The glucagon derivatives of the present invention include peptides having at least one difference in amino acid sequence compared to native glucagon, peptides altered by modifying the native glucagon sequence, and mimetics of native glucagon that can activate glucagon receptors like native glucagon. Such derivatives of native glucagon may have the effect of lowering blood pressure and / or restoring kidney function in vivo.

[0089] The glucagon derivative may also be a non-naturally occurring one.

[0090] The glucagon derivative in the present invention means a peptide that acts on the glucagon receptor at a significant level compared to natural glucagon. In the present invention, the glucagon derivative is also referred to as a "peptide having activity on the glucagon receptor", a "glucagon analogue" or a "peptide".

[0091] The glucagon derivative of the present invention is a peptide having activity against the glucagon receptor, and examples of the activity against the receptor include, but are not limited to, in vitro activity against the receptor of 0.1% or more, 0.2% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more, compared to the natural product.

[0092] Specifically, the glucagon derivative of the present invention has an in vitro activity against the glucagon receptor of about 0.001% or more, about 0.01% or more, about 0.1% or more, 0.2% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or about 200% or more, or any range that is significantly increased, as long as it is within the range. Specifically, the peptide having activity against the glucagon receptor of the present invention has a relative activity against the glucagon receptor of 5% or more, 10% or more, 20% or more, or 30% or more, as compared to the natural ligand of the receptor (natural glucagon), but is not limited thereto.

[0093] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about."

[0094] As a method for measuring the in vitro activity of such peptides, various methods known in the art (eg, Patent Documents 1, 2, and 3) can be used, but the methods are not particularly limited thereto.

[0095] For purposes of the present invention, the glucagon derivatives include agonists of the glucagon receptor, or analogs or fragments in which at least one amino acid of native glucagon has been mutated in a manner selected from the group consisting of substitutions, additions, deletions, modifications, and combinations thereof.

[0096] In the present invention, the term "glucagon receptor agonist" has the same meaning as "agonist" or "agonist drug", and refers to a substance that binds to the glucagon receptor and performs the same function as glucagon in the actual living body.

[0097] In the present invention, the term "glucagon analog" refers to a non-natural glucagon that is different from natural glucagon. Such glucagon analogs include analogs in which some amino acids of natural glucagon have been modified in the form of addition, deletion, or substitution.

[0098] In the present invention, the term "fragment" refers to a form in which at least one amino acid is deleted at the N-terminus or C-terminus of native glucagon or a glucagon derivative, which exerts a preventive or therapeutic effect on chronic kidney disease in the body.

[0099] The glucagon derivative of the present invention may be one in which some amino acids of natural glucagon have been modified by any of substitution, addition, deletion and modification or a combination thereof. The amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Conservative substitutions with amino acids having such similar properties are expected to exhibit the same or similar activity.

[0100] Examples of glucagon derivatives prepared by combining the above-mentioned methods include peptides that have at least one amino acid sequence different from that of native glucagon, have deamidated N-terminal amino acid residues, and have activation function for the glucagon receptor, but are not limited to these. Derivatives of native glucagon used in the present invention can be prepared by combining various methods for preparing derivatives.

[0101] Also included in the present invention are all modifications using L- or D-amino acids and / or non-natural amino acids for the preparation of peptides, and / or modifications of natural sequences, such as modifications of side chain functional groups, covalent bonds within the molecule, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. Furthermore, said modifications include all substitutions with non-natural compounds.

[0102] Furthermore, all of the above include those having at least one amino acid added to the N- and / or C-terminus.

[0103] As mentioned above, the amino acids to be substituted or added can be any of the 20 amino acids commonly found in human proteins, as well as unusual or unnatural amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides and typical peptide sequences containing these amino acids can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0104] Amino acid derivatives are similarly available, such as 4-imidazoacetic acid.

[0105] The glucagon derivative of the present invention may be prepared by using the above-mentioned preparation methods alone or in combination.

[0106] In a specific embodiment, the glucagon derivative may be a peptide comprising the amino acid sequence of general formula 1.

[0107] X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-X29-X30 (General formula 1, Sequence number 46)

[0108] In the above formula, X1 is tyrosine, X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine or D-serine, X7 is threonine, valine or cysteine, X10 is tyrosine or cysteine, X12 is lysine or cysteine, X13 is tyrosine or cysteine, X14 is leucine or cysteine, X15 is aspartic acid, glutamic acid or cysteine, and X16 is glutamic acid, asparagine X17 is aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine ​​or valine or is absent, X18 is alanine, aspartic acid, glutamic acid, arginine, valine or cysteine ​​or is absent, X19 is alanine, arginine, serine, valine or cysteine ​​or is absent; X20 is lysine, histidine, glutamine, aspartic acid, arginine, α-methylglutamic acid, cysteine, or is not present; X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine, or is not present; X23 is isoleucine, valine, or arginine, or is not present; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, α-methylglutamic acid, or leucine, or is not present; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or is not present; X28 is glutamine, lysine, asparagine, or arginine, or is not present; X29 is threonine (T); and X30 is cysteine ​​or is not present (except when the amino acid sequence of general formula 1 is the same as SEQ ID NO: 1 or SEQ ID NO: 12).

[0109] More specifically, in general formula 1, X2 is Aib (aminoisobutyric acid), X7 is threonine (T), valine (V) or cysteine ​​(C), X10 is tyrosine (Y), X12 is lysine (K), X13 is tyrosine (Y), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), X16 is glutamic acid (E) or serine (S), X17 is lysine (K), arginine (R) or cysteine ​​(C), and X18 is arginine (R). X19 is alanine (A) or cysteine ​​(C), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X23 is valine (V), X24 is glutamine (Q), X27 is methionine (M), X28 is asparagine (N), X29 is threonine (T), and X30 is cysteine ​​(C) or absent.

[0110] In another embodiment of the present invention, the glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, specifically, but not limited to, an amino acid sequence (essentially) selected from the group consisting of SEQ ID NOs: 2 to 45. Specifically, the glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 11, 13 to 45, more specifically, but not limited to, an amino acid sequence (essentially) selected from the group consisting of SEQ ID NOs: 2 to 11, 13 to 45.

[0111] As another specific example, the glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 11, 13 to 25, 27, 29, 31, 33, and 35 to 45, or is (essentially) composed of the amino acid sequence, but is not limited thereto.

[0112] Alternatively, the glucagon derivative comprises or consists (essentially) of an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42 and 44, but is not limited to these.

[0113] Even if the present invention describes "consisting of a specific sequence number," it does not exclude meaningless sequence additions before or after the amino acid sequence of the sequence number, naturally occurring mutations, or silent mutations thereof, as long as the peptide has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number, and it goes without saying that those having such sequence additions or mutations are also included in the present invention. In other words, even if there is a difference in a part of the sequence, as long as it shows a certain level or more of homology and shows activity against the glucagon receptor, it is included in the present invention.

[0114] For example, the glucagon derivative of the present invention has at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more sequence identity when compared with natural glucagon, but is not limited to a specific sequence as long as it has a chronic kidney disease preventive or therapeutic effect. The glucagon derivative of the present invention is disclosed in Patent Documents 1, 2, and 3.

[0115] Furthermore, the glucagon derivative of the present invention comprises any one of the amino acid sequences of SEQ ID NOs: 2 to 45, is (essentially) composed of any one of the amino acid sequences of SEQ ID NOs: 2 to 45, or comprises a peptide having at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more sequence identity to any one of the amino acid sequences of SEQ ID NOs: 2 to 45, but is not limited to a specific sequence as long as it has an effect of preventing or treating chronic kidney disease.

[0116] In the present invention, "homology" or "identity" means the degree to which two given amino acid sequences or nucleotide sequences are related to each other, and is expressed as a percentage.

[0117] Homology and identity are often used interchangeably.

[0118] Whether any two peptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0119] Homology, similarity or identity of peptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 9, for example. In summary, the GAP program defines the number of similar sequence symbols (i.e., amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix of Non-Patent Document 11 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 10, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in the present invention indicates the relevance between sequences.

[0120] The glucagon derivative of the present invention may include an intramolecular bridge (e.g., a covalent or non-covalent bridge), specifically, a ring between amino acid residues, for example, a ring between the 12th and 16th amino acids or between the 16th and 20th amino acids of the peptide, but is not limited thereto.

[0121] Examples of such rings include, but are not limited to, lactam bridges (or lactam rings).

[0122] The glucagon derivatives also include all those modified to contain a ring by including amino acids that form a ring at a desired position.

[0123] Such a ring may be formed between the amino acid side chains in the glucagon derivative, for example, a lactam ring formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0124] For example, a peptide containing the amino acid sequence of general formula 1 may be, but is not limited to, one in which the amino acids in each amino acid pair of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 are substituted with glutamic acid or lysine, respectively. In the above, n in Xn (n is a natural number) indicates the amino acid position from the N-terminus of the presented amino acid sequence.

[0125] Furthermore, a peptide comprising the amino acid sequence of general formula 1 may have each amino acid pair of X12 and X16, X16 and X20, or X17 and X21 substituted with glutamic acid or lysine to form a ring, but is not limited thereto.

[0126] Furthermore, at least one of the amino acid pairs of X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 in general formula 1 may form a ring (e.g., a lactam ring) between each amino acid in each amino acid pair, but is not limited thereto.

[0127] Furthermore, in general formula 1, X16 may be glutamic acid, X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but are not limited thereto.

[0128] The glucagon derivative of the present invention may have an unmodified N-terminus and / or C-terminus, but also includes those in which the N-terminus and / or C-terminus are chemically modified, protected with an organic group, or modified by adding an amino acid to the terminus of the glucagon derivative in order to protect it from a protease in the body and improve its stability. When the C-terminus is unmodified, the terminus of the glucagon derivative of the present invention has a carboxyl group, but is not particularly limited thereto.

[0129] In particular, in the case of chemically synthesized glucagon derivatives, the N- and C-termini are charged, and in order to remove the charges, acetylation of the N-terminus and / or amidation of the C-terminus are performed, but this is not particularly limited to these.

[0130] Specifically, the N-terminus or C-terminus of the glucagon derivative of the present invention has an amino group (-NH2) or a carboxy group (-COOH), but is not limited thereto.

[0131] In the present invention, the glucagon derivative is in the form of a conjugate to which a biocompatible substance capable of extending the in vivo half-life is bound, but is not limited thereto.

[0132] In the present invention, the term "long-acting conjugate" or "conjugate" refers to a structure in which the glucagon derivative and a biocompatible substance are bound, and the duration of efficacy is improved compared to that of a glucagon derivative not bound to the biocompatible substance. The biocompatible substance in the long-acting conjugate is covalently linked to the glucagon derivative, but is not particularly limited thereto. In the present invention, the glucagon derivative, which is one component of the conjugate, is a peptide or fragment in which at least one amino acid in the natural glucagon sequence is substituted, added, deleted, modified, or mutated by a combination thereof, and does not lose its activity by binding to a biocompatible substance, but any substance having a chronic kidney disease preventive or therapeutic effect can be used as one component of the conjugate of the present invention.

[0133] For example, the glucagon derivative contained in the conjugate is a peptide containing the amino acid sequence of the above-mentioned general formula 1. Alternatively, the glucagon derivative contained in the conjugate is a peptide containing the amino acid sequence of any one of SEQ ID NOs: 2 to 45. Alternatively, the glucagon derivative contained in the conjugate is a peptide containing the amino acid sequence of any one of SEQ ID NOs: 2 to 11 and 13 to 45, but is not limited thereto.

[0134] The term "biocompatible substance" as used herein means a substance that can be bound to the glucagon derivative of the present invention, which is a physiologically active substance, and can improve the duration of efficacy of the physiologically active substance compared to a physiologically active substance that is not bound to a biocompatible substance moiety or carrier. The biocompatible substance is linked to the physiologically active substance by a covalent bond, but is not limited thereto.

[0135] The biocompatible substance is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin, but is not particularly limited thereto.

[0136] For example, the FcRn-binding substance is an immunoglobulin Fc region, more specifically, an IgG Fc region, but is not particularly limited thereto.

[0137] At least one amino acid side chain in the peptides of the invention is conjugated to such biocompatible substances to improve solubility and / or half-life in vivo and / or increase bioavailability. Such modifications also reduce the clearance of therapeutic proteins and peptides.

[0138] The biocompatible materials mentioned above may be water soluble (amphiphilic or hydrophilic), non-toxic, and / or pharma- ceutical acceptable.

[0139] The long-acting conjugate of a glucagon derivative, which is an active ingredient contained in the composition of the present invention, will be described in more detail below.

[0140] The long-acting conjugate of the glucagon derivative of the present invention is a form in which the glucagon derivative and the immunoglobulin Fc region are linked to each other. Specifically, the conjugate is a form in which the glucagon derivative and the immunoglobulin Fc region are covalently linked to each other via a linker, but is not particularly limited thereto.

[0141] The long-acting conjugate of a glucagon derivative means a glucagon derivative having a longer-lasting effect than a glucagon derivative not bound to the immunoglobulin Fc region. In the present invention, the conjugate of the peptide of general formula 1 represented by chemical formula (1) is referred to as a "long-acting conjugate" and is used interchangeably with "long-acting conjugate of a glucagon derivative."

[0142] Specifically, the glucagon derivative or peptide is in the form of a long-acting conjugate, and the long-acting conjugate is a conjugate represented by chemical formula (1).

[0143] XLF···(1)

[0144] Here, X is a peptide (glucagon derivative) comprising the amino acid sequence of general formula 1, L is a linker comprising an ethylene glycol repeat unit, F is an immunoglobulin Fc region, and - indicates a covalent bond linkage between X and L and between L and F.

[0145] The long-acting conjugate of the present invention is characterized in that it contains a peptide containing the amino acid sequence of general formula 1 (glucagon derivative) as a part of the conjugate.

[0146] The conjugate of the present invention, even in the form of a conjugate, exhibits significant activity against the glucagon receptor, similar to glucagon derivatives, and therefore exerts efficacy in preventing or treating chronic kidney diseases by lowering blood pressure and recovering renal function.

[0147] Specifically, the conjugates of the invention have in vitro activity towards the glucagon receptor of about 0.01% or more, 0.1% or more, 0.2% or more, 0.5% or more, 0.7% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to native glucagon, but are not limited to these.

[0148] For purposes of the present invention, the peptides (glucagon derivatives) or conjugates thereof have, but are not limited to, about 0.01% or more, about 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, or 200% or more activity at the glucagon receptor compared to native glucagon.

[0149] The composition of the present invention may be (i) a glucagon derivative having activity against the glucagon receptor, or (ii) a long-acting conjugate of the glucagon derivative, which exerts an excellent effect in preventing or treating chronic kidney disease based on its improved persistence in the body.

[0150] In said conjugate, F is a biocompatible substance that increases the half-life, specifically an agent that increases the half-life of X, i.e., the glucagon derivative of the present invention, which is a component of said conjugate of the present invention.

[0151] The F's may be bonded to X by a covalent or non-covalent chemical bond, or may be bonded to X via L by a covalent or non-covalent chemical bond, or a combination thereof.

[0152] More specifically, X and L, and L and F may be linked to each other by a covalent bond, and the conjugate is a conjugate in which X, L, and F are linked to each other by a covalent bond in the order of chemical formula (1).

[0153] In the present invention, F may be an immunoglobulin Fc region.

[0154] The "immunoglobulin Fc region" refers to a region excluding the variable regions of the heavy and light chains of an immunoglobulin, including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3). The immunoglobulin Fc region may be a component constituting a part of the conjugate of the present invention. Specifically, it corresponds to F in chemical formula (1).

[0155] In the present specification, the Fc region includes not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as variants of sequences different from the native sequence, for example, sequences in which at least one amino acid residue of the native sequence is deleted, inserted, non-conservative or conservatively substituted, or a combination thereof. The derivatives, substitutions, and variants are premised on having the ability to bind to FcRn. In the present invention, F is a human immunoglobulin region, but is not limited thereto. The F has a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only via the nitrogen atom of one of the two chains, but is not limited thereto. The linkage via the nitrogen atom may be linked to the ε-amino atom of lysine or the amino group at the N-terminus by reductive amination.

[0156] Reductive amination refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to produce an amine, which is then reduced to form an amine bond, and is an organic synthesis reaction well known in the art.

[0157] In one specific example, but not limited to, the F is linked via the nitrogen atom of the N-terminal proline.

[0158] The immunoglobulin Fc region is a component that constitutes a part of the conjugate of the present invention represented by chemical formula (1), and specifically corresponds to F in chemical formula (1).

[0159] Such an immunoglobulin Fc region includes, but is not limited to, a hinge portion in a heavy chain constant region.

[0160] The term "hinge sequence" as used herein means a site located in a heavy chain that forms a dimer of an immunoglobulin Fc region through inter disulfide bonds.

[0161] The hinge sequence in the present invention is a hinge sequence having the following amino acid sequence, which has been mutated by deleting a portion thereof to have only one cysteine ​​residue, but is not limited thereto. Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 47)

[0162] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 47 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention is one that contains only one cysteine ​​residue and is composed of 3 to 12 amino acids, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 48) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 49) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 50) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 51) Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 52) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 53) Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 54) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 55) Glu-Pro-Ser-Cys-Pro (SEQ ID NO:56) Pro-Ser-Cys-Pro (SEQ ID NO: 57) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 58) Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 59) Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 60) Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 61) Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 62) Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 63) Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 64) Glu-Pro-Ser-Cys (SEQ ID NO: 65) Ser-Cys-Pro (SEQ ID NO:66)

[0163] More specifically, the hinge sequence includes, but is not limited to, the amino acid sequence of SEQ ID NO: 57 (Pro-Ser-Cys-Pro) or SEQ ID NO: 66 (Ser-Cys-Pro).

[0164] The immunoglobulin Fc region of the present invention is in a form in which two immunoglobulin Fc chain molecules form a dimer due to the presence of a hinge sequence, and the conjugate of the present invention represented by chemical formula (1) is in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0165] In the present invention, the "N-terminus" refers to the amino terminus of a protein or polypeptide, and includes the extreme amino terminus, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc region of the present invention includes a hinge sequence at the N-terminus, but is not limited thereto.

[0166] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes a part or the whole of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding the variable regions of the heavy and light chains of an immunoglobulin, so long as it has an effect substantially equivalent to or improved from that of a natural one. Furthermore, it may be a region in which a very long part of the amino acid sequence corresponding to CH2 and / or CH3 is deleted.

[0167] For example, the immunoglobulin Fc region of the present invention is 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 at least one of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a part of the hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region, but is not limited thereto.

[0168] The immunoglobulin Fc region of the present invention is in the form of a dimer or multimer composed of single-chain immunoglobulins consisting of domains of the same origin, but is not limited thereto.

[0169] Moreover, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, in which the Fc region dimer F and X are covalently linked via one and the same linker L containing an ethylene glycol repeat unit. In a specific example of this embodiment, X is covalently linked via the linker L only to one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently linked via L to one of the polypeptide chains to which X is linked, among the two polypeptide chains of the Fc region dimer F. In the most specific example of this embodiment, the F is a homodimer.

[0170] In another specific embodiment, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains, but is not limited to this.

[0171] In another embodiment of the long-acting conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in the form of a dimer. Here, the immunoglobulin Fc region and X may be linked to each other via a non-peptide linker. However, the present invention is not limited to the above example.

[0172] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof. An amino acid sequence derivative means a sequence in which at least one amino acid residue of the naturally occurring amino acid sequence is different due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0173] For example, in the case of IgG Fc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, are used as sites suitable for modification.

[0174] Various derivatives are also used, such as derivatives in which the site forming disulfide bonds has been removed, derivatives in which several amino acids have been deleted from the N-terminus of native Fc, and derivatives in which a methionine residue has been added to the N-terminus of native Fc. Furthermore, in order to eliminate effector functions, complement binding sites, such as C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in Patent Documents 4 and 5, etc.

[0175] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (Non-Patent Document 12). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications may be made by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.

[0176] The above-mentioned Fc derivatives may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have improved structural stability against heat, pH, and the like of the Fc region.

[0177] In addition, such an Fc region may be obtained from a natural source isolated from the living body of an animal such as a human or a cow, a goat, a pig, a mouse, a rabbit, a hamster, a rat, or a guinea pig, or may be a recombinant product obtained from a transformed animal cell or a microorganism, or a derivative thereof. Here, the method for obtaining it from a natural source may be a method for obtaining it by isolating the whole immunoglobulin from the living body of a human or an animal and then treating it with a protease. 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 be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0178] Furthermore, the immunoglobulin Fc region may have a natural sugar chain, an increased sugar chain compared to the natural one, a decreased sugar chain compared to the natural one, or a form in which the sugar chain has been removed. For increasing, decreasing, or removing such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms are used. Here, an immunoglobulin Fc region from which the sugar chains have been removed from Fc has a significantly reduced binding ability to complement (c1q), and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. For these reasons, an immunoglobulin Fc region from which the sugar chains have been removed or which has been deglycosylated is in a form suitable for its original purpose as a drug carrier.

[0179] In the present invention, "deglycosylation" refers to an Fc region from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0180] On the other hand, the immunoglobulin Fc region is of human origin or animal origin, such as bovine, goat, porcine, mouse, rabbit, hamster, rat, guinea pig, and in a more specific embodiment, of human origin.

[0181] The immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or may be an Fc region derived from a combination of these or a hybrid of these. In a more specific embodiment, it is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to extend the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0182] Furthermore, in a specific embodiment, the immunoglobulin Fc region is a human IgG4 Fc region, and may be in the form of a homodimer in which two monomers are linked by a disulfide bond between the third amino acid cysteines of each monomer (inter-chain form), and here each monomer of the homodimer independently has / may have an internal disulfide bond between the 35th and 95th cysteines, and an internal disulfide bond between the 141st and 199th cysteines, i.e., two internal disulfide bonds (intra-chain form).

[0183] Each monomer is composed of 221 amino acids, and the amino acids forming the homodimer are composed of a total of 442 amino acids, but are not limited thereto. Specifically, the immunoglobulin Fc region is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 67 (composed of 221 amino acids) via a disulfide bond between the cysteine ​​that is the third amino acid of each monomer, and the monomers of the homodimer independently form an internal disulfide bond between the 35th and 95th cysteines, and an internal disulfide bond between the 141st and 199th cysteines, but are not limited thereto.

[0184] F in formula (1) includes a monomer having the amino acid sequence of SEQ ID NO:67, and F is a homodimer of the monomer having the amino acid sequence of SEQ ID NO:67, but is not limited thereto.

[0185] As an example, but not limited to, the immunoglobulin Fc region is a homodimer comprising the amino acid sequence of SEQ ID NO:68 (consisting of 442 amino acids).

[0186] In one non-limiting example, the immunoglobulin Fc region and X are aglycosylated.

[0187] On the other hand, the term "combination" in the present invention means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin binds to a single-chain polypeptide of a different origin. That is, a dimer or multimer can be produced from at least two regions selected from the group consisting of the Fc regions of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE.

[0188] In the present invention, the term "hybrid" means that sequences corresponding to immunoglobulin Fc regions of at least two different origins are present in a single-chain immunoglobulin constant region. In the present invention, various forms of hybrids are possible. That is, hybrids of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0189] On the other hand, IgG is also divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations or hybridizations of these are also possible. Specifically, these are the IgG2 and IgG4 subclasses, and more specifically, the Fc region of IgG4, which has almost no effector functions such as complement dependent cytotoxicity (CDC).

[0190] In addition, the above-mentioned conjugates have improved durability of efficacy compared to native glucagon or compared to X where F is not modified, and such conjugates include not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles.

[0191] On the other hand, in the chemical formula (1), L may be a non-peptide linker, for example, a linker containing an ethylene glycol repeating unit.

[0192] The "non-peptidic linker" of the present invention includes a biocompatible polymer having at least two repeating units bonded together. The repeating units are linked to each other by any covalent bond except a peptide bond. The non-peptidic linker is a component that forms part of the conjugate of the present invention and corresponds to L in chemical formula (1).

[0193] The non-peptide linker used in the present invention may be any polymer that is resistant to in vivo protease. The non-peptide linker in the present invention is used in combination with a non-peptide polymer.

[0194] The non-peptidic linker in the present invention contains a reactive group at its terminal and can form a conjugate by reacting with other components constituting the conjugate. When a non-peptidic linker having reactive functional groups at both terminals forms a conjugate by binding to X and F in chemical formula (1) via each reactive group, the non-peptidic linker or non-peptidic polymer is also called a non-peptidic polymer linker moiety or non-peptidic linker linker moiety.

[0195] The non-peptide linker is a linker containing an ethylene glycol repeat unit, such as polyethylene glycol, but is not limited thereto. In addition, the present invention also includes derivatives thereof known in the art and derivatives that can be easily prepared at the technical level in the art.

[0196] The repeating unit of the non-peptidic linker may be an ethylene glycol repeating unit, and specifically, the non-peptidic linker may include an ethylene glycol repeating unit and a functional group at its end that is used for preparing a conjugate until it is configured as a conjugate. The long-lasting conjugate according to the present invention is in a form in which X and F are linked via the functional group, but is not limited thereto. The non-peptidic linker according to the present invention includes two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0197] Specifically, the linker is polyethylene glycol (PEG) represented by chemical formula (2), but is not limited thereto.

[0198] TIFF2024512895000002.tif2027...(2)

[0199] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited to these.

[0200] The PEG moiety in the persistent conjugate includes, but is not limited to, the -(CH2CH2O)n- structure as well as the oxygen atom(s) intervening between the linking element and the -(CH2CH2O)n-.

[0201] As a specific example, the ethylene glycol repeating unit is represented by [OCH2CH2]n, where the value of n is a natural number and is determined so that the average molecular weight of the [OCH2CH2]n portion in the persistent conjugate, for example the number average molecular weight, is greater than 0 to approximately 100 kDa, but is not limited thereto. In another specific example, the n value is a natural number, and the average molecular weight, for example, the number average molecular weight, of the [OCH2CH2]n portion in the long-acting conjugate is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 1 5 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa , about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, or about 10 kDa, but is not limited to these.

[0202] In a specific embodiment, the conjugate has a structure in which the glucagon derivative (X) and the immunoglobulin Fc region (F) are covalently linked via a linker containing an ethylene glycol repeating unit, but is not limited thereto.

[0203] In another specific embodiment of the long-acting conjugate, L may be a linker containing an ethylene glycol repeat unit, and F may be a dimeric immunoglobulin Fc region. More specifically, but not limited to, an X1 molecule is covalently linked to any one of the Fc regions of the dimeric immunoglobulin Fc region via the linker containing an ethylene glycol repeat unit. In yet another specific embodiment, one end of the linker containing an ethylene glycol repeat unit is linked only to any one of the two Fc region chains of the dimeric immunoglobulin Fc region, but not limited to this.

[0204] The polyethylene glycol includes, but is not limited to, ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG).

[0205] The formula weight of the ethylene glycol repeating unit moiety in L is in the range of, but not limited to, 1 to 100 kDa. In a specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker having reactive groups at both ends to be bound to F, specifically an immunoglobulin Fc region, and X, specifically a glucagon derivative.

[0206] The non-peptidyl linker used in the present invention may be any polymer that is resistant to endogenous proteases and contains an ethylene glycol repeating unit. The molecular weight of the non-peptidyl polymer is in the range of more than 0 to about 100 kDa, about 1 to about 100 kDa, specifically about 1 to about 20 kDa, or about 1 to about 10 kDa, but is not limited thereto. In addition, the non-peptidyl linker of the present invention that is bound to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0207] In the present invention, "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0208] Specifically, the non-peptide linker may have reactive groups at both ends when not bound to F and X, and be bound to F and X via the reactive groups.

[0209] In one specific example, both ends of the linker are bound to a thiol group, an amino group, or a hydroxyl group of an immunoglobulin Fc region, and a thiol group, an amino group, an azide group, or a hydroxyl group of a glucagon derivative (X), but this is not limited thereto.

[0210] Specifically, the linker has reactive groups at both ends which are bonded to the immunoglobulin Fc region and the glucagon derivative (X), more specifically, the linker is bonded to at least one selected from the group consisting of 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 a hydroxyl group located at the C-terminus, and is bonded to at least one selected from the group consisting of a thiol group of cysteine, an amino group of lysine, arginine, glutamine and / or histidine, an azide group of azidolysine, and a hydroxyl group in the glucagon derivative (X), but is not limited thereto.

[0211] More specifically, the reactive group of the linker is at least one selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative, but is not limited thereto.

[0212] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0213] In the above, examples of succinimide derivatives include, but are not limited to, succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, and succinimidyl carbonate.

[0214] The linker may be linked to an immunoglobulin Fc region, F, and a glucagon derivative, X, via the reactive groups described above, to form a linker linker.

[0215] Furthermore, the final products generated by reductive amination with aldehyde bonds are much more stable than those linked with amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0216] The reactive groups at both ends of the non-peptide linker may be the same or different. For example, the non-peptide linker may have aldehyde groups at both ends, or may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. However, the non-peptide linker is not particularly limited thereto, as long as F, specifically, the immunoglobulin Fc region and X are bound to each end of the non-peptide linker.

[0217] For example, the non-peptide linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end.

[0218] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups can be activated into the various reactive groups described above by known chemical reactions, or a commercially available polyethylene glycol having modified reactive groups can be used to prepare the long-acting protein conjugate of the present invention.

[0219] In a specific embodiment, the non-peptidyl polymer is linked to a cysteine ​​residue of X, more specifically, to a -SH group of cysteine, but is not limited thereto.

[0220] Examples of the non-peptide polymer include, but are not limited to, those in which the non-peptide polymer is linked to the 7th cysteine ​​residue, the 10th cysteine ​​residue, the 12th cysteine ​​residue, the 13th cysteine ​​residue, the 14th cysteine ​​residue, the 15th cysteine ​​residue, the 16th cysteine ​​residue, the 17th cysteine ​​residue, the 18th cysteine ​​residue, the 19th cysteine ​​residue, the 20th cysteine ​​residue, the 21st cysteine ​​residue, or the 30th cysteine ​​residue in the peptide corresponding to X.

[0221] Specifically, the reactive group of the non-peptidic polymer may be linked to the -SH group of the cysteine ​​residue. The reactive group is as described above. When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X by a thioether bond, and the aldehyde group can be linked to the -NH2 group of F, specifically, immunoglobulin Fc, by reductive amination, but these are merely examples and are not limited thereto.

[0222] By such reductive alkylation, the N-terminal amino group of the immunoglobulin Fc fragment can be linked to the oxygen atom located at one end of PEG via a linker functional group having the structure -CH2CH2CH2- to form a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and by a thioether bond, a structure can be formed in which one end of PEG is linked to the sulfur atom located at the cysteine ​​of the peptide of general formula 1. The above-mentioned thioether bond is It may have a structure of TIFF2024512895000003.tif3125.

[0223] However, the present invention is not limited to the above example, which is merely one example.

[0224] In another specific embodiment, the non-peptidyl polymer is linked to a lysine residue of X, more specifically, but not limited to, an amino group of a lysine.

[0225] Furthermore, in the above-mentioned conjugate, the reactive group of the non-peptidyl polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is merely one example.

[0226] Furthermore, in said conjugates, the glucagon derivative according to the invention may be linked via its C-terminus to a linker having a reactive group, but this is just one example.

[0227] The "C-terminus" in the present invention means the carboxy terminus of a peptide, and for the purposes of the present invention, means the position to be bound to a linker. For example, but not limited to, it includes not only the most terminal amino acid residue of the C-terminus, but also all amino acid residues around the C-terminus, specifically the 1st to 20th amino acid residues from the most terminal.

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

[0229] TIFF2024512895000004.tif41116...(3)

[0230] In chemical formula (3), X is the peptide of general formula 1 described above, F is a human immunoglobulin Fc region, and n is a natural number, where n is as described above.

[0231] As a specific example, the long-acting conjugate of chemical formula (3) may have a structure in which X of the peptide of general formula 1 of SEQ ID NO:46 and F of the human immunoglobulin Fc region are covalently linked via an ethylene glycol repeating unit, and X may be linked to the succinimide ring of chemical formula (3), and F may be linked to an oxypropylene group of chemical formula (3).

[0232] In chemical formula (3), the value of n is determined so that the average molecular weight of the [OCH2CH2]n portion in the peptide conjugate, for example, the number average molecular weight, is 1 to 100 kDa, 1 to 20 kDa, or 10 kDa, but is not limited thereto.

[0233] X of the conjugate may be a peptide having activity at the glucagon receptor.

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

[0235] F in formula (3) is a human immunoglobulin Fc region, and the site of F linked to the oxypropylene group is not particularly limited. In one embodiment of the present invention, the site of F linked to the oxypropylene group may be the N-terminal nitrogen or a nitrogen atom of an internal residue of F (e.g., the ε nitrogen of lysine). In a specific embodiment of the present invention, the site of F linked to the oxypropylene group in formula (3) is the N-terminal proline of F, but is not limited thereto.

[0236] In one specific example, but not limited to, the F is linked via the nitrogen atom of the N-terminal proline.

[0237] The glucagon derivatives include those described in Patent Documents 2 and 3, the entire text of which is incorporated herein by reference. A method for preparing a long-acting conjugate of the glucagon derivative is described in Patent Document 3, the entire text of which is incorporated herein by reference.

[0238] In addition, the above-mentioned conjugates have improved efficacy over X in which F is not modified, and such conjugates include not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles.

[0239] Unless otherwise specified in the present specification, the detailed description of the specification and the claims regarding the "peptide" according to the present invention or the "conjugate" in which the peptide is covalently linked to a biocompatible substance are applicable to the category including not only the peptide or conjugate, but also the salt of the peptide or conjugate (e.g., a pharma- ceutically acceptable salt of the peptide) or a solvate thereof. Thus, even if the specification only describes a "peptide" or a "conjugate", the description also applies to a specific salt thereof, a specific solvate thereof, and a specific solvate of a specific salt thereof. These salt forms may be, for example, any pharma-ceutically acceptable salt. The type of the salt is not particularly limited. However, it is preferable that the salt is in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0240] The term "pharmacologically acceptable" refers to a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reactions, and the like, within the scope of medical judgment.

[0241] The "pharmaceutically acceptable salt" in the present invention includes salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Suitable salts derived from bases include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0242] Further, the term "solvate" in the present invention means a complex formed between the peptide, conjugate or salt thereof according to the present invention and a solvent molecule.

[0243] Furthermore, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0244] In particular, the peptides of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other method known in the art. Thus, the glucagon derivatives of the present invention can be synthesized in many ways, including, for example, (a) synthesizing the peptide stepwise in solid or solution phase methods or by fragment assembly, and isolating and purifying the final peptide product, (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture, (c) expressing a nucleic acid construct encoding the peptide in a cell-free test tube and recovering the expression product, or any combination of (a), (b) and (c) to obtain fragments of the peptide, and then recovering the peptide by ligating the fragments to obtain the peptide.

[0245] The composition according to the present invention may contain a glucagon derivative or a conjugate thereof, specifically, a pharmacologically effective amount of a glucagon derivative or a conjugate thereof. It may further contain a pharma- ceutically acceptable carrier. The composition according to the present invention is used for the prophylaxis or treatment of chronic kidney disease, and has the effect of lowering blood pressure and / or restoring kidney function, but is not limited thereto.

[0246] In the present invention, "pharmacologically acceptable" means a sufficient amount to exert a therapeutic effect and to cause no side effects, and can be easily determined by a person skilled in the art based on factors known in the medical field, such as the type of disease, the patient's age, weight, health condition, sex, sensitivity to the drug, administration route, administration method, number of doses, treatment period, combination, and drugs used concomitantly.

[0247] The pharma- ceutically acceptable carrier may be a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye, flavoring, etc., for oral administration, a buffer, preservative, soothing agent, solubilizer, isotonic agent, stabilizer, etc., for injection, a base, excipient, lubricant, preservative, etc., for topical administration. The pharmaceutical composition of the present invention may be prepared in various forms by mixing with the pharma- ceutically acceptable carrier as described above. For example, for oral administration, it may be prepared in the form of a tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc., and for injection, it may be prepared in the form of a disposable ampoule or a multiple dose form. It may also be prepared in the form of a solution, suspension, tablet, pill, capsule, sustained release preparation, etc.

[0248] Examples of carriers, excipients, and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, etc.

[0249] In addition, the composition of the present invention may have any dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0250] Furthermore, the conjugate can be used by mixing it with various medicament-acceptable carriers such as physiological saline or organic solvents, and in order to improve stability and absorbability, carbohydrates such as glucose, sucrose, dextran, antioxidants such as ascorbic acid and glutathione, chelating agents, low molecular weight proteins, other stabilizers, etc. can be used as drugs.

[0251] The dosage and frequency of administration of the composition of the present invention are determined by the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, the severity of the disease, etc. Specifically, the composition of the present invention contains the glucagon derivative or its conjugate in a pharmacologic effective amount, but is not limited thereto.

[0252] The term "containing a pharmacologic effective amount of the glucagon derivative or its conjugate" means that the glucagon derivative or its conjugate is contained to such an extent that the desired pharmacological activity (e.g., prevention or treatment of chronic kidney disease) can be obtained, and also means that the glucagon derivative or its conjugate is contained at a pharmacologic acceptable level that is free of or has only slight toxicity or side effects on the individual to which it is administered, but is not limited thereto. Such a pharmacologic effective amount is determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0253] The pharmaceutical composition of the present invention contains the above-mentioned ingredient (active ingredient) in an amount of 0.01 to 99% by weight by volume, but is not particularly limited thereto.

[0254] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The composition of the present invention may have a different content of active ingredients depending on the severity of the disease. Specifically, the total dose of the conjugate of the present invention is preferably about 0.0001 mg to 500 mg per kg of body weight per day. However, the dose of the conjugate is determined by taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, diet, and excretion rate of the patient as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these into consideration, a person having ordinary knowledge in the art would be able to determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route, and administration method as long as it exhibits the effects of the present invention.

[0255] For example, but not limited to, the compositions of the present invention may be administered once a week, once every two weeks, once every four weeks, or once a month.

[0256] Another specific embodiment of the composition according to the present invention further comprises, but is not limited to, a GLP-1 receptor agonist (glucagon like peptide 1 receptor agonist).

[0257] Specifically, the composition of the present invention is a pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative and a GLP-1 receptor agonist, more specifically, a pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative, a GLP-1 receptor agonist and a pharma- ceutical acceptable excipient, but is not limited thereto.

[0258] The pharmaceutical composition of the present invention comprising (i) a glucagon derivative and (ii) a GLP-1 receptor agonist is administered in a mixture of (i) the glucagon derivative and (ii) the GLP-1 receptor agonist, or (b) the glucagon derivative and (ii) the GLP-1 receptor agonist are administered in separate forms, but is not limited thereto.

[0259] For example, the glucagon derivative and the GLP-1 receptor agonist may be formulated in one preparation or may be formulated separately. When the glucagon derivative and the GLP-1 receptor agonist are in a separated form, the glucagon derivative and the GLP-1 receptor agonist may be formulated in separate preparations and administered simultaneously, separately, sequentially, or in the reverse order.

[0260] The term "combined administration" in the present invention should be understood to mean not only simultaneous administration, but also a form of administration in which the glucagon derivative and the GLP-1 receptor agonist act together on an individual, and each substance performs a function at a level equal to or higher than its original function. Therefore, the term "combined administration" in the present invention should be understood to mean simultaneous, separate, sequential, or reversed administration of the glucagon derivative and the GLP-1 receptor agonist. When the administration is sequential, reverse, or separate, the order of administration is not particularly limited, but the interval between administrations of the secondary components must not lose the beneficial effect of the combination.

[0261] The glucagon derivative and GLP-1 receptor agonist of the present invention, or a composition containing the same, are provided in the form of a kit, but are not limited thereto. The "kit" of the present invention may contain the composition according to the present invention for co-administration of the glucagon derivative and the GLP-1 receptor agonist. Specifically, the kit according to the present invention may contain the glucagon derivative and the GLP-1 receptor agonist formulated in one formulation, may contain separate formulations of the glucagon derivative and the GLP-1 receptor agonist, or may further contain substances necessary for co-administration of the two substances, but are not limited thereto.

[0262] The glucagon derivative and the GLP-1 receptor agonist are in the form of a long-acting conjugate linked to an immunoglobulin Fc region, but are not limited thereto.

[0263] GLP-1 (glucagon-like peptide-1) is a hormone secreted from the small intestine and is known to promote glucose uptake into cells. In the small intestine, glucagon precursors are broken down into three peptides: glucagon, GLP-1, and GLP-2. Here, GLP-1 means GLP-1(1-37), which is a form without insulin secretion function, but is processed into the form of GLP-1(7-37) to become the active form GLP-1(7-37). The amino acid sequence of GLP-1(7-37) is as follows: GLP-1(7-37): HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G (SEQ ID NO: 69)

[0264] The term "GLP-1 receptor agonist" refers to an agonist that acts on the GLP-1 receptor, and includes various substances, for example, substances in the form of compounds or peptides, but is not limited to these.

[0265] The GLP-1 receptor agonist may be not only native GLP-1, but also a substance (e.g., a peptide) having a significant level of activity against the GLP-1 receptor, such as an in vitro activity against the GLP-1 receptor that is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the natural ligand of the receptor (native GLP-1), but is not limited thereto.

[0266] Methods for measuring the in vitro activity of such GLP-1 receptor agonists can be carried out by various in vitro activity assays known in the art.

[0267] GLP-1 receptor agonists according to the present invention include, but are not limited to, peptides that have a significant level of activity at the GLP-1 receptor and have at least one difference in amino acid sequence compared to native GLP-1, peptides in which the native GLP-1 sequence has been altered by modification, mimetics of native GLP-1, and the like.

[0268] Specifically, in the present invention, the GLP-1 receptor agonist includes, but is not limited to, those selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof.

[0269] The term "agonist" in the present invention has the same meaning as "acting drug" or "agonist", and refers to a substance that binds to a receptor and performs the same function as a natural ligand in an actual living body.

[0270] In the present invention, the term "derivative" refers to a form having a significant level of activity against the GLP-1 receptor and in which a portion of the sequence of native GLP-1 has been altered and some groups of amino acid residues have been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination) or modified (e.g., N-methylation), but is not limited to these.

[0271] The GLP-1 receptor agonists of the present invention include derivatives in which the N-terminal amino group has been removed (desamino-histidyl derivatives), derivatives in which the amino group has been replaced with a hydroxyl group (beta-hydroxy imidazopropionyl derivatives), derivatives in which the amino group has been modified with two methyl residues (dimethyl-histidyl derivatives), derivatives in which the N-terminal amino group has been replaced with a carboxyl group (beta-carboxyimidazopropionyl derivatives), and derivatives in which the alpha carbon of the N-terminal histidine residue has been removed to leave only the imidazoacetyl group and remove the positive charge of the amino group (imidazoacetyl derivatives). Derivatives in which the N-terminal amino group has been mutated to other forms are also included in the present invention.

[0272] As a specific example, in the present invention, the GLP-1 receptor agonist is a derivative of exendin-4 in which the amino group or amino acid residue at the N-terminus is chemically mutated, more specifically, an exendin-4 derivative in which the α-amino group or α-carbon present at the α-carbon of the histidine residue, which is the first amino acid at the N-terminus of exendin-4, is substituted or removed, and further specifically, desamino-histidyl-exendin-4 (DA-exendin-4) in which the amino group at the N-terminus is removed, beta-hydroxyimidazopropionyl-exendin-4 (HY-exendin-4) or beta-carboxyimidazopropionyl-exendin-4 (HY-exendin-4) in which the amino group at the N-terminus is substituted with a hydroxyl group or a carboxyl group, Exendin-4 modified with two methyl residues (Dimethyl-histidyl-exendin-4, DM-exendin-4), or imidazoacetyl-exendin-4 (CA-exendin-4) in which the alpha carbon of the N-terminal histidine residue has been removed, but are not limited to these.

[0273] Examples of GLP-1 receptor agonists of the present invention include, but are not limited to, GLP-1 receptor agonist derivatives in which the N-terminal histidine residue is replaced with a substance selected from the group consisting of des-amino-histidyl, dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl and β-carboxyimidazopropionyl.

[0274] In the present invention, the term "GLP-1 fragment" refers to a form of native GLP-1 that has a significant level of activity against the GLP-1 receptor and in which at least one amino acid has been added or deleted to the N-terminus or C-terminus of native GLP-1, and the added amino acid may be a non-naturally occurring amino acid (e.g., a D-amino acid).

[0275] In the present invention, the term "GLP-1 variant" refers to a peptide that has a significant level of activity at the GLP-1 receptor and differs from native GLP-1 in at least one amino acid sequence, and is used interchangeably with "analog".

[0276] Specifically, the GLP-1 variant may be one in which some amino acids have been altered from natural GLP-1 by any of substitution, addition, deletion and modification, or a combination thereof.

[0277] Examples of GLP-1 variants produced by combining the above methods include, but are not limited to, peptides that have at least one amino acid sequence different from that of native GLP-1, have deamidated amino acid residues at the N-terminus, and have activation function against the GLP-1 receptor.

[0278] Also included are all those in which at least one amino acid has been added to the N- and / or C-terminus of natural GLP-1. As mentioned above, the amino acids to be substituted or added can be any of the 20 amino acids commonly observed in human proteins, as well as unusual or unnatural amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea. Amino acid derivatives can also be obtained, such as 4-imidazoacetic acid.

[0279] Furthermore, such modifications for producing derivatives or variants include all modifications using L- or D-amino acids and / or non-natural amino acids, and / or modifications of the natural sequence, such as modifications of side chain functional groups, covalent bonds within the molecule, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. Furthermore, the modifications include all substitutions with non-natural compounds.

[0280] Exendin-3 and exendin-4 of the present invention are GLP-1 receptor agonists consisting of 39 amino acids that show 53% amino acid sequence similarity with GLP-1, and the amino acid sequences of exendin-3 and exendin-4 are as follows:

[0281] Exendin-3: HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 70) Exendin-4: HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS (SEQ ID NO: 71)

[0282] An exendin agonist means a substance that binds to an exendin receptor in the body and exhibits the same biological activity as exendin, regardless of the structure of exendin. An exendin derivative has at least 80% amino acid sequence homology with natural exendin, and is a form in which some groups of amino acid residues have been chemically substituted (e.g., alpha-methylation, alpha-hydroxylation), removed (e.g., deamination) or modified (e.g., N-methylation), but is not limited to these.

[0283] Exendin derivatives include derivatives in which the N-terminal amino group has been removed (desamino-histidyl derivatives), derivatives in which the amino group has been replaced with a hydroxyl group (beta-hydroxy imidazopropionyl derivatives), derivatives in which the amino group has been modified with two methyl residues (dimethyl-histidyl derivatives), derivatives in which the N-terminal amino group has been replaced with a carboxyl group (beta-carboxyimidazopropionyl derivatives), and derivatives in which the alpha carbon of the N-terminal histidine residue has been removed to leave only the imidazoacetyl group and remove the positive charge of the amino group (imidazoacetyl derivatives). Derivatives in which the N-terminal amino group has been mutated to other forms are also included in the present invention.

[0284] As a specific example, in the present invention, the exendin derivative is a derivative in which the amino group or amino acid residue at the N-terminus of exendin-4 is chemically mutated, more specifically, an exendin-4 derivative in which the α-amino group or α-carbon present at the α-carbon of the histidine residue, which is the first amino acid at the N-terminus of exendin-4, is substituted or removed, and further specifically, desamino-histidyl-exendin-4 (DA-exendin-4) in which the amino group at the N-terminus is removed, beta-hydroxyimidazopropionyl-exendin-4 (HY-exendin-4) in which the amino group is substituted with a hydroxy group or a carboxy group, beta-carboxyimidazopropionyl-exendin-4 (HY-exendin-4), beta-carboxyimidazopropionyl-exendin-4 (HY-exendin-4), Exendin-4 modified with two methyl residues (Dimethyl-histidyl-exendin-4, DM-exendin-4), or imidazoacetyl-exendin-4 (CA-exendin-4) in which the alpha carbon of the N-terminal histidine residue has been removed, but are not limited to these.

[0285] Examples of the exendin derivatives of the present invention include, but are not limited to, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been removed, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been replaced with a hydroxyl group, exendin-4 derivatives in which the amino group at the N-terminus of exendin-4 has been modified with a dimethyl group, and exendin-4 derivatives in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been deleted.

[0286] An exendin fragment refers to a form in which at least one amino acid has been added or deleted to the N-terminus or C-terminus of a native exendin, and the added amino acid may be a non-naturally occurring amino acid (e.g., a D-amino acid).

[0287] An exendin variant refers to a peptide that has at least one amino acid sequence that differs from that of a native exendin, and more specifically, refers to an exendin in which some amino acids of the native exendin have been altered by any of the following methods, or a combination of these: substitution, addition, deletion, and modification.

[0288] The methods for producing the agonists, derivatives, fragments and variants described above may be used alone or in combination, for example, insulinotropic peptides having at least one different amino acid sequence and deamination of the N-terminal amino acid residue.

[0289] Specific examples of the GLP-1 receptor agonist of the present invention include, but are not limited to, [(1H-Imidazol-4-yl)-acetyl1]GEGTFTSDL SKQMEEEAVR LFIEWLKNGGPSSGAPPPS (SEQ ID NO: 72) (Bachem), or HGEGTFTSDV SSYLEEQAAK EFIAWLVKG (SEQ ID NO: 73) (Bachem). The above amino acid sequences are written in the direction from the N-terminus to the C-terminus. The GLP-1 receptor agonist of the present invention is disclosed in the specification of Patent Document 6.

[0290] In the present invention, the GLP-1 receptor agonist is in the form of a long-acting conjugate in which the GLP-1 receptor agonist is linked to an immunoglobulin Fc region, but is not limited thereto. Specifically, the GLP-1 receptor agonist is imidazo-acetyl exendin-4 (CA exendin-4) linked to an immunoglobulin Fc region via a linker containing an ethylene glycol repeating unit, more specifically, a long-acting conjugate of the GLP-1 receptor agonist in which one end of a polyethylene glycol linker is linked to lysine (Lys) of the GLP-1 receptor agonist of SEQ ID NO: 72, and the other end of the polyethylene glycol linker is linked to an immunoglobulin Fc region, but is not limited thereto.

[0291] In the present invention, the GLP-1 receptor agonist is the GLP-1 receptor agonist "efpeglenatide", but is not limited thereto.

[0292] Here, the "long-acting conjugate" and the related "immunoglobulin Fc region", "linker containing ethylene glycol repeating units" and the like are as described above.

[0293] The composition of the present invention may contain any of the following (i) to (iv). (i) Glucagon derivatives and GLP-1 receptor agonists (ii) Long-acting conjugates of glucagon derivatives and GLP-1 receptor agonists (iii) Long-acting conjugate of glucagon derivative and GLP-1 receptor agonist (iv) Long-acting conjugates of glucagon derivatives and long-acting conjugates of GLP-1 receptor agonists

[0294] The glucagon derivative or its conjugate according to the present invention, the GLP-1 receptor agonist or its conjugate, or a composition containing them exerts a blood pressure lowering effect and / or restores renal function when administered to an individual, but is not limited thereto. Specifically, the combined administration of the glucagon derivative or its conjugate according to the present invention and the GLP-1 receptor agonist or its conjugate exerts the effects of lowering blood pressure, reducing albumin excretion, reducing proteinuria, and / or restoring renal function, as compared to a vehicle control group.

[0295] The pharmaceutical composition of the present invention contains the glucagon derivative or a conjugate thereof and the GLP-1 receptor agonist or a conjugate thereof in an amount of 0.01 to 99% by weight by volume, but is not particularly limited thereto.

[0296] Specifically, the composition of the present invention contains 0.01-5 nmol / kg, 0.05-4 nmol / kg, 0.05-3.5 nmol / kg, or 0.09-2.99 nmol / kg of glucagon derivative and 0.01-5 nmol / kg, 0.05-4 nmol / kg, 0.05-3.5 nmol / kg, or 0.09-2.99 nmol / kg of GLP-1 receptor agonist, but is not limited thereto. Also, the composition of the present invention contains the glucagon derivative and the GLP-1 receptor agonist in a molar ratio of 0.01-50, 0.02-40, or 0.03-33.22, but is not limited thereto.

[0297] The pharmaceutical composition of the present invention is administered to an individual in an amount of 0.1 to 20 mg, or 0.5 to 16 mg, of a glucagon derivative or a conjugate thereof, and a GLP-1 receptor agonist or a conjugate thereof, but is not limited thereto.

[0298] The above also applies to other embodiments or aspects of the present invention, but is not limited thereto.

[0299] Another embodiment of the invention is a method for preventing or treating chronic kidney disease comprising the step of administering to an individual a glucagon derivative, a conjugate thereof, or a composition comprising same.

[0300] The method for preventing or treating chronic kidney disease according to the present invention may further comprise the step of administering to an individual a GLP-1 receptor agonist, a conjugate thereof, or a composition containing same.

[0301] Yet another embodiment of the present invention is a method for preventing or treating chronic kidney disease, comprising the step of co-administering to an individual a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof.

[0302] The methods include, but are not limited to, administering to an individual a composition comprising a glucagon derivative or conjugate thereof and a GLP-1 receptor agonist or conjugate thereof, or co-administering a glucagon derivative or conjugate thereof and a GLP-1 receptor agonist or conjugate thereof in separate formulations.

[0303] The methods of the present invention include, but are not limited to, administering (i) a glucagon derivative and (ii) a GLP-1 receptor agonist in a single formulation, or administering separate formulations simultaneously, separately, sequentially or in reverse order.

[0304] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, compositions, blood pressure reduction, renal function recovery, chronic kidney disease, prevention and treatment are as described above.

[0305] The individual in the present invention refers to mammals including humans, mice, livestock, etc., and is an individual that has developed or is at risk of developing chronic kidney disease, but is not limited to these.

[0306] In the present invention, "administration" means introducing a predetermined substance into a patient (individual) by any appropriate method, and the administration route of the glucagon derivative, GLP-1 receptor agonist, conjugate thereof, or composition is not particularly limited to these, and can be any general route as long as it can deliver the glucagon derivative, GLP-1 receptor agonist, conjugate thereof, or composition to a target in the body, and examples of such route include intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0307] Furthermore, the glucagon derivative, GLP-1 receptor agonist, conjugate or composition thereof can be formulated into a unit-dose preparation suitable for administration to the body of a patient by a conventional method in the pharmaceutical field, specifically into a preparation form useful for administration of a protein pharmaceutical, and administered orally or via a parenteral administration route including dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, but is not limited to these, using an administration method conventionally used in the art.

[0308] The method of the present invention may include administering a pharmacologic effective amount of a composition comprising said glucagon derivative, GLP-1 receptor agonist or conjugate thereof. The appropriate total daily dose will be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and extent of the response to be achieved, whether or not other formulations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0309] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, or compositions containing the same of the present invention are administered once a week, once every two weeks, once every four weeks, or once a month, but are not limited thereto.

[0310] Specifically, when a glucagon derivative and a GLP-1 receptor agonist are administered, they are administered at about 1-200 μg, 3-180 μg, or 5-160 μg per kg of patient body weight, and when two substances are administered in combination, they are administered at a total amount of about 2-400 μg, about 6-360 μg, or 10-320 μg per kg of patient body weight, but are not limited thereto. Also, when a glucagon derivative and a GLP-1 receptor agonist are administered in combination, they are administered at a molar ratio of 0.01-50, 0.02-40, or 0.03-33.22, but are not limited thereto.

[0311] Specifically, the glucagon derivative is administered at 0.09 to 2.99 nmol / kg and the GLP-1 receptor agonist is administered at 0.09 to 2.99 nmol / kg, or the glucagon derivative and the GLP-1 receptor agonist are administered at a molar ratio of 0.01 to 50, 0.02 to 40, or 0.03 to 33.22, but the present invention is not limited to these.

[0312] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for the prevention or treatment of chronic kidney disease.

[0313] Yet another embodiment of the present invention relates to the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the prevention or treatment of chronic kidney disease.

[0314] Yet another embodiment of the present invention is the combined use of a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the prevention or treatment of chronic kidney disease.

[0315] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, compositions, blood pressure reduction, renal function recovery, chronic kidney disease, prevention and treatment are as described above.

[0316] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for the manufacture of a medicament having an application in the prevention or treatment of chronic kidney disease.

[0317] Yet another embodiment of the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the manufacture of a medicament for the prevention or treatment of chronic kidney disease.

[0318] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, compositions, blood pressure reduction, renal function recovery, chronic kidney disease, prevention and treatment are as described above.

[0319] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition containing the same, for lowering blood pressure and / or restoring kidney function.

[0320] Yet another embodiment of the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for lowering blood pressure and / or restoring kidney function.

[0321] Yet another embodiment of the present invention is the combined use of a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for lowering blood pressure and / or restoring kidney function.

[0322] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, compositions, blood pressure reduction, renal function recovery, chronic kidney disease, prevention and treatment are as described above.

[0323] Yet another embodiment of the present invention is the use of a glucagon derivative or a conjugate thereof, or a composition containing same, for the manufacture of a medicament having an application in lowering blood pressure and / or restoring kidney function.

[0324] Yet another embodiment of the present invention is the use of a composition comprising a glucagon derivative or a conjugate thereof and a GLP-1 receptor agonist or a conjugate thereof for the manufacture of a medicament for lowering blood pressure and / or restoring kidney function.

[0325] The glucagon derivatives, GLP-1 receptor agonists, conjugates thereof, compositions, blood pressure reduction, renal function recovery, chronic kidney disease, prevention and treatment are as described above.

[0326] The present invention will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present invention and are not intended to limit the present invention. EXAMPLES

[0327] Preparation of glucagon derivatives and activity measurement The present inventors confirmed whether or not the activity against the glucagon receptor can provide a preventive or therapeutic effect against chronic kidney disease by lowering blood pressure and restoring renal function.

[0328] First, natural glucagon and glucagon derivatives having activity equivalent thereto were prepared as shown in Table 1 below. Glucagon derivatives were synthesized with reference to Patent Documents 1, 2 and 3, and their pI and in vitro activity were measured.

[0329] [Table 1] JPEG2024512895000006.jpg104150

[0330] In the sequences of Table 1, the amino acid marked with X represents the unnatural amino acid aminoisobutyric acid (Aib), the underline in the amino acid symbol represents the formation of a lactam ring between the side chains of the amino acid pair shown in bold and underlined type, and "-" represents the absence of an amino acid residue at that position. In the column indicating the presence or absence of ring formation, "-" represents the absence of a ring in the sequence. EXAMPLES

[0331] Preparation of long-acting conjugates of glucagon derivatives The glucagon derivative of SEQ ID NO: 37 prepared in the above Example was linked to an immunoglobulin Fc region using polyethylene glycol as a linker to prepare a long-acting glucagon derivative conjugate.

[0332] Maleimide-PEG-aldehyde (NOF, Japan), which is a linear modified polyethylene glycol with a molecular weight of 10 kDa in which hydrogen at both ends is replaced with a 3-(3-maleimidopropionamido)propyl group and a 3-oxopropyl group (propionaldehyde group), was reacted with the glucagon derivative of SEQ ID NO: 37, and the cysteine ​​residue of the glucagon derivative peptide was PEGylated at the maleimide end of the maleimide-PEG-aldehyde. Specifically, the molar ratio of the glucagon derivative peptide of SEQ ID NO: 37 to maleimide-PEG-aldehyde was set to 1:1-5, and the protein concentration was set to 3-10 mg / ml, and the reaction was carried out at a low temperature for 1-3 hours. Here, the reaction was carried out in an environment in which 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction mixture was applied to SP Sepharose HP (GE Healthcare, USA) to purify the glucagon derivative monopegylated to cysteine.

[0333] On the other hand, the immunoglobulin Fc region was prepared by the method described in Patent Document 7 using an immunoglobulin Fc region (49.8 kDa, homodimer of two chains of SEQ ID NO: 67 linked by a disulfide bond) having a hinge region with the Pro-Ser-Cys-Pro sequence (SEQ ID NO: 57) at the N-terminus.

[0334] Next, the mono-PEGylated glucagon derivative purified as described above was reacted with the immunoglobulin Fc region at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg / ml at 4-8°C for 12-18 hours. The reaction was carried out in an environment where 100 mM potassium phosphate buffer (pH 6.0) was supplemented with 10-50 mM sodium cyanoborohydride and 10-20% isopropanol as reducing agents. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify a long-acting conjugate of glucagon derivative peptide in which the polyethylene glycol terminal on the aldehyde side of the mono-PEGylated glucagon derivative peptide was linked to the N-terminal proline nitrogen of one of the two chains of the immunoglobulin Fc homodimer. The purified conjugate has a structure in which a glucagon derivative, a polyethylene glycol (PEG) linker, and a dimeric immunoglobulin Fc domain are covalently linked in a 1:1:1 molar ratio within a molecule, with the PEG linker linked to the N-terminal proline nitrogen of one of the two polypeptide chains of the immunoglobulin Fc domain.

[0335] After preparation, the purity was analyzed by reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography and was found to be 95% or more.

[0336] The immunoglobulin Fc region is a homodimer in which two monomers having the amino acid sequence of SEQ ID NO:67 (consisting of 221 amino acids) form a disulfide bond between the third amino acid cysteine ​​of each monomer, and each monomer of the homodimer independently has an internal disulfide bond between the 35th and 95th cysteines, and an internal disulfide bond between the 141st and 199th cysteines.

[0337] Here, the conjugate in which the glucagon derivative peptide is linked to the immunoglobulin Fc region via PEG was named "long-acting conjugate of glucagon derivative." EXAMPLES

[0338] Improved renal function in a rat model of chronic kidney disease In order to confirm the effect of the long-acting conjugate of glucagon derivative prepared in the above example on improving chronic kidney disease (CKD), a spontaneous hypertensive rat (SHR) model, which is known as a model of hypertension and CKD, was used. SHR is a model in which renal dysfunction occurs due to hypertension, and it is known that the etiology of CKD is very similar to that of humans.

[0339] First, 16-week-old SHRs, which are known to develop renal dysfunction due to elevated blood pressure, were divided into a vehicle control group and a group administered a long-acting conjugate of the glucagon derivative of SEQ ID NO: 37 (3.9 nmol / kg, Q3D, subcutaneously), and were repeatedly administered for 6 weeks. Normal rats and a vehicle control group were used as negative controls. To evaluate renal function during the 6-week repeated administration, urinary albumin excretion was measured on days 0, 13, 28, and 46. One-way ANOVA was used for statistical analysis (*~***p<0.05~0.001 vs. SHR vehicle control group).

[0340] As a result, it was confirmed that urinary albumin excretion began to decrease 13 days after administration of the long-acting glucagon derivative conjugate compared to the SHR vehicle control group, and a significant difference was observed from the 28th day of administration onwards (Figure 1). EXAMPLES

[0341] Antihypertensive effect in a rat model of chronic kidney disease A spontaneous hypertensive rat (SHR) model, known as a CKD disease model, was used to confirm the antihypertensive effect of the long-acting conjugate of the glucagon derivative of SEQ ID NO: 37 prepared in Example 2 in chronic kidney disease. SHR is a model known to have a gradual increase in blood pressure depending on age in weeks, and is used to evaluate the efficacy of various antihypertensive therapeutic agents.

[0342] First, a telemetry device was surgically inserted into the aorta of 15-week-old SHR with hypertensive blood pressure, and then the animals were divided into a vehicle control group and a group administered with a long-acting conjugate of a glucagon derivative (2 nmol / kg, Q3D, subcutaneously), and the animals were repeatedly administered for one week. The changes in systolic (SBP) and diastolic (DBP) blood pressure during the one-week repeated administration were monitored at two-hour intervals using the telemetry device inserted into the aorta. To evaluate the blood pressure lowering effect of the administered long-acting conjugate of a glucagon derivative of SEQ ID NO: 37, the change in the average blood pressure during the one-week administration period relative to the average blood pressure for two days before administration was calculated. Statistical analysis was performed using t-test (*p<0.05 vs. SHR vehicle control group).

[0343] As a result, it was confirmed that in the vehicle control group, blood pressure increased compared to before administration, whereas in the group administered with the long-acting conjugate of the glucagon derivative of SEQ ID NO: 37, both systolic and diastolic blood pressures decreased. In particular, it was confirmed that there was a significant difference in systolic blood pressure compared to the vehicle control group (Figure 2).

[0344] Thus, it was confirmed that the long-acting conjugate of the glucagon derivative of the present invention also has a therapeutic effect on lowering blood pressure in chronic kidney disease (CKD). EXAMPLES

[0345] Confirmation of the effect of improving renal function by combined administration with GLP-1 receptor agonist Based on the therapeutic effect of the glucagon derivative of the present invention on chronic kidney disease, the therapeutic effect of chronic kidney disease when a GLP-1 receptor agonist is used in combination with the glucagon derivative of the present invention was further confirmed.

[0346] Example 5-1: Preparation of a long-acting conjugate of GLP-1 receptor agonist A 3.4 kDa propion-ALD2 PEG (IDB, Korea) having propionaldehyde groups at both ends was site-specifically reacted with lysine of the GLP-1 receptor agonist (imidazo-acetyl exendin-4 (CA exendin-4)) of SEQ ID NO: 72, and then mono-PEGylated imidazo-acetyl exendin-4 (CA exendin-4) was purified from the reaction mixture using a cation exchange column to obtain a conjugate in which PEG and imidazo-acetyl exendin-4 (CA exendin-4) are bound in a 1:1 ratio. To prepare an imidazo-acetyl exendin-4 (CA exendin-4)-immunoglobulin Fc region conjugate in which the N-terminus of the immunoglobulin Fc region was specifically linked to mono-pegylated imidazo-acetyl exendin-4 (CA exendin-4), the reaction was carried out under conditions of pH 5.0 to 8.2. After the coupling reaction, a two-step purification method using a hydrophobic column and an anion exchange column was performed to finally purify the site-specifically linked GLP-1 receptor agonist-immunoglobulin Fc region conjugate.

[0347] Here, the conjugate in which the GLP-1 receptor agonist of sequence number 72 (imidazo-acetylexendin-4 (CA exendin-4)) is linked to the immunoglobulin Fc region via PEG was named a "long-acting conjugate of GLP-1 receptor agonist" or a "long-acting conjugate of CA-Exd4 derivative."

[0348] The immunoglobulin Fc region used in the preparation of the long-acting conjugate of the GLP-1 receptor agonist was the same as the Fc region used in Example 2.

[0349] Example 5-2: Confirmation of the effect of combined administration with GLP-1 receptor agonist In order to confirm the effect of improving chronic kidney disease (CKD) by combined administration of the long-acting conjugate of the glucagon derivative of sequence number 37 prepared in Example 2 and the long-acting conjugate of the GLP-1 receptor agonist (long-acting conjugate of the CA-Exd4 derivative) prepared in Example 5-1, a spontaneous hypertensive rat (SHR) model, which is a CKD disease model, was used.

[0350] First, 16-week-old SHRs, known to develop renal dysfunction due to elevated blood pressure, were divided into a vehicle control group, a group administered a long-acting conjugate of a glucagon derivative (3.0 nmol / kg, Q3D, subcutaneously), and a group administered a combination of a long-acting conjugate of a glucagon derivative (3.0 nmol / kg, Q3D, subcutaneously) and a long-acting conjugate of a GLP-1 receptor agonist (6.9 nmol / kg, Q3D, subcutaneously), and were administered repeatedly for 10 weeks. Normal rats and a vehicle control group were used as negative controls. To evaluate renal function and the progression of CKD during the 10-week repeated administration, urinary ACR (albumin to creatinine ratio) was measured on days 0, 14, 28, 42, 56, and 70. One-way ANOVA was used for statistical analysis (*~***p<0.05~0.001 vs. SHR vehicle control group).

[0351] As a result, in the vehicle control group, ACR continued to increase, whereas in the group administered with the long-acting glucagon derivative conjugate, the increase in ACR was delayed.In addition, in the group administered with the long-acting glucagon derivative conjugate and the long-acting GLP-1 receptor agonist conjugate in combination, ACR was significantly decreased compared to the vehicle control group (Figure 3).

[0352] Thus, it was confirmed that the therapeutic effect of a long-acting conjugate of a glucagon derivative on chronic kidney disease (CKD) was further improved by coadministration with a long-acting conjugate of a GLP-1 receptor agonist.

[0353] All of these experimental results suggest that the glucagon derivative of the present invention has the effects of lowering blood pressure and restoring renal function, and is effective in treating chronic kidney disease, and that further therapeutic effects can be expected when administered in combination with a GLP-1 receptor agonist.

[0354] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications and alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative and a pharma- ceutical acceptable excipient, The glucagon derivative is a peptide comprising an amino acid sequence of the following general formula 1: X1-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-X29-X30 (general formula 1, sequence number 46) In the above formula, X1 is tyrosine (Y); X2 is α-methyl-glutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S) or D-serine; X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y) or cysteine ​​(C); X12 is lysine (K) or cysteine ​​(C); X13 is tyrosine (Y) or cysteine ​​(C); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D), glutamic acid (E) or cysteine ​​(C); X16 is glutamic acid (E), aspartic acid (D), serine (S), α-methylglutamic acid, cysteine ​​(C), or is absent; X17 is aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamic acid (E), arginine (R), valine (V), cysteine ​​(C), or absent; X19 is alanine (A), arginine (R), serine (S), valine (V), cysteine ​​(C), or absent; X20 is lysine (K), histidine (H), glutamine (Q), aspartic acid (D), arginine (R), α-methylglutamic acid, cysteine ​​(C), or is absent; X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), cysteine ​​(C), or absent; X23 is isoleucine (I), valine (V), arginine (R), or is absent; X24 is valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), α-methylglutamic acid, leucine (L), or absent; X27 is isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), arginine (R), or absent; X29 is threonine (T); X30 is cysteine ​​(C) or absent (However, this does not include the case where the amino acid sequence of the general formula 1 is the same as SEQ ID NO: 1 or SEQ ID NO: 12).

2. The composition of claim 1 , wherein the peptide is in the form of a persistent conjugate, and the persistent conjugate is represented by the following chemical formula (1): 【Chemistry 1】 wherein X is a peptide comprising the amino acid sequence of general formula 1; L is a linker comprising an ethylene glycol repeat unit; F is an immunoglobulin Fc region, - indicates a covalent bond link between X and L, and between L and F.

3. In the general formula 1, X2 is Aib (aminoisobutyric acid), X7 is threonine (T), valine (V) or cysteine ​​(C); X10 is tyrosine (Y); X12 is lysine (K); X13 is tyrosine (Y); X14 is leucine (L) or cysteine ​​(C); X15 is aspartic acid (D); X16 is glutamic acid (E) or serine (S); X17 is lysine (K), arginine (R) or cysteine ​​(C); X18 is arginine (R); X19 is alanine (A) or cysteine ​​(C); X20 is glutamine (Q) or lysine (K); X21 is aspartic acid (D) or glutamic acid (E); X23 is valine (V); X24 is glutamine (Q), X27 is methionine (M), X28 is asparagine (N); X29 is threonine (T); 3. The composition of claim 1 or 2, wherein X30 is cysteine ​​(C) or is absent.

4. The composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11, 13-25, 27, 29, 31, 33 and 35-45.

5. 4. The composition of claim 3, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 23, 27, 33, 35, 37, 38, 40, 41, 42 and 44.

6. The composition of claim 1 or 2, wherein the composition has at least one of the following characteristics: (a) Lowering blood pressure (b) Decreased albumin excretion (c) Decreased proteinuria (d) Kidney function recovery

7. The composition according to claim 1 or 2, which exerts a blood pressure lowering effect and a kidney function restoring effect in an individual when administered to the individual.

8. The composition according to claim 1 or 2, wherein the peptide is amidated at the C-terminus.

9. The composition according to claim 1 or 2, wherein the peptide has a ring formed between amino acid residues.

10. The composition of claim 2 , wherein the immunoglobulin Fc region is non-glycosylated.

11. The composition of claim 2 , wherein the immunoglobulin Fc region is an IgG4 Fc region.

12. The composition according to claim 2, wherein the immunoglobulin Fc region is a dimer consisting of two polypeptide chains, and one end of L is linked to only one of the two polypeptide chains.

13. 3. The composition of claim 2, wherein the persistent conjugate is linked to F and X by covalent bonds formed by reacting one end of L with an amino group or a thiol group of F and the other end of L with an amino group or a thiol group of X, respectively.

14. The composition of claim 2 , wherein L is polyethylene glycol.

15. The composition according to claim 2, wherein the chemical formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

16. The composition of claim 1 or 2, further comprising a glucagon like peptide 1 receptor agonist.

17. 17. The composition of claim 16, wherein the GLP-1 receptor agonist is selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists, derivatives, fragments, variants, and combinations thereof.

18. The composition of claim 16, wherein the GLP-1 receptor agonist is a GLP-1 receptor agonist derivative in which the N-terminal histidine residue is substituted with a substance selected from the group consisting of des-amino-histidyl, dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl and β-carboxyimidazopropionyl.

19. The composition of claim 16, wherein the GLP-1 receptor agonist is selected from the group consisting of native exendin-4, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been removed, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been replaced with a hydroxy group, an exendin-4 derivative in which the N-terminal amino group of exendin-4 has been modified with a dimethyl group, and an exendin-4 derivative in which the alpha carbon of the first amino acid (histidine) of exendin-4 has been deleted.

20. The composition according to claim 16, wherein the GLP-1 receptor agonist is in the form of a long-acting conjugate in which the GLP-1 receptor agonist is linked to an immunoglobulin Fc region.

21. The composition of claim 20, wherein the GLP-1 receptor agonist is imidazo-acetyl exendin-4 (CA exendin-4) linked to an immunoglobulin Fc region via a linker comprising an ethylene glycol repeat unit.

22. A pharmaceutical composition for preventing or treating chronic kidney disease, comprising a glucagon derivative and a pharma- ceutical acceptable excipient, The glucagon derivative comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-11 and 13-45.

23. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered simultaneously, separately, sequentially or in reverse order with a GLP-1 receptor agonist.

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