Novel GLP-1 receptor antagonist and pharmaceutical composition containing same for preventing or treating congenital hyperinsulinism or hypoglycemia

Abstract

The present invention relates to novel GLP-1 receptor antagonist analogs and acylated GLP-1 receptor analogs, and their use in the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of congenital hyperinsulinism or hypoglycemia, comprising a GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog conjugate. [Background technology]

[0002] Hypoglycemia refers to a state in which blood sugar is lower than normal (60-120 mg / dl when fasting, 140 mg / dl or less two hours after a meal), but hypoglycemia is usually considered to be present when blood sugar is 50 mg / dl or less.

[0003] Hypoglycemia can occur when oral hypoglycemic agents or insulin are administered in excessive doses, when fasting is maintained for a long period of time, or when the amount of activity or exercise is excessive. Hypoglycemia can also occur due to factors such as severe physical illness, deficiency of hormones such as adrenal cortical hormones or glucagon, insulin-producing pancreatic tumors, autoimmune diseases against insulin, patients who have undergone gastrectomy, and those with hereditary carbohydrate metabolic enzyme disorders.

[0004] Congenital hyperinsulinism (CHI) is one of the most common causes of severe and persistent hypoglycemia in newborns and children. Insulin is a hormone that regulates blood sugar in the human body. When blood sugar rises due to food intake, it lowers blood sugar. However, patients with CHI are unable to perform this regulatory role, and instead secrete insulin from the pancreas regardless of blood sugar levels. As a result, patients fall into hypoglycemia.

[0005] Hypoglycemia, or hypoglycemia due to congenital hyperinsulinism, prevents brain cells from maintaining their primary energy sources of glucose, ketones, and lactose, cutting off the body's energy supply from proteins and fats, damaging brain cells and resulting in seizures, learning disabilities, cerebral palsy, blindness, and even death.

[0006] Hypoglycemia can occur due to transient excessive insulin secretion and can also occur in newborns experiencing fetal distress. The cause of abnormal insulin secretion is unclear, but such cases usually improve within a few days to a few months. Transient hypoglycemia can also occur in pregnant women with diabetes who have poor glucose regulation. However, once lactation progresses successfully and the hypoglycemia resolves, it does not recur. Another cause is persistent hyperinsulinism due to several genetic defects. Studies have reported that genetic causes of hyperinsulinism include mutations in the SUR or Kir6.2 genes on chromosome 11p15.1, mutations in the GK (glucokinase) gene on chromosome 7p15-p13 that increase GK activity, and mutations in the GDH (glutamate dehydrogenase) gene that activate GDH, resulting in increased ATP in beta islet cells.

[0007] In addition, obesity metabolic surgery, which is performed to treat severe obesity and complications caused by obesity, is divided into laparoscopic adjustable gastric banding (LAGB), Roux-en-Y gastric bypass (RYGB), sleeve gastrectomy (SG), and biliary pancreatic diversion (BPD). However, hypoglycemia due to dumping syndrome has been reported as a complication of obesity metabolic surgery.

[0008] GLP-1 receptor antagonists are drugs administered to stimulate appetite (KR 10-2001-0089563 A). A representative example is exendin-3(9-39) (Indraneel Banerjee, Mark J. Dunne, in Encyclopedia of Endocrine Diseases (Second Edition), 2019). Exendin-3(9-39) suppresses insulin secretion and has been reported to be effective in treating acute hypoglycemia (Calabria AC, Li C, Gallagher PR, Stanley CA, De Leon DD. GLP-1 receptor antagonist exendin-(9-39) elevates fasting blood glucose levels in congenital hyperinsulinism owing to inactivating mutations in the ATP-sensitive K+ channel. Diabetes. 2012;61(10):2585-2591). doi:10.2337 / db12-0166). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Indraneel Banerjee, Mark J. Dunne, in Encyclopedia of Endocrine Diseases (Second Edition), 2019 [Non-patent document 2] Calabria AC, Li C, Gallagher PR, Stanley CA, De Leon DD. GLP-1 receptor antagonist exendin-(9-39) elevates fasting blood glucose levels in congenital hyperinsulinism owing to inactivating mutations in the ATP-sensitive K+ channel. Diabetes. 2012;61(10):2585-2591. doi:10.2337 / db12-0166 [Non-licensed document 3] Montrose-Rafizaden et al., 1997, J. Biol. Chem. 272(34):21201-21206

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[0010] The development of therapeutic agents for congenital hyperinsulinism and hypoglycemia using GLP-1 receptor antagonists is still incomplete, and the development of effective therapeutic agents is required. [Means for solving the problem]

[0011] One object of the present invention is to provide a GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog conjugate.

[0012] Another object of the present invention is to provide use of the GLP-1 receptor antagonist analog, the acylated GLP-1 receptor antagonist analog conjugate, or a composition containing the same for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, comprising the GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog conjugate.

[0014] Another object of the present invention is to provide a method for preventing or treating congenital hyperinsulinism or hypoglycemia, which comprises administering the pharmaceutical composition to an individual in need thereof.

[0015] Another object of the present invention is to provide a use of the GLP-1 receptor antagonist analog, the acylated GLP-1 receptor antagonist analog conjugate, or a composition containing the same for providing a medicament for the prevention or treatment of congenital hyperinsulinism or hypoglycemia. [Effects of the Invention]

[0016] The GLP-1 receptor antagonist analog conjugate and acylated GLP-1 receptor antagonist analog conjugate of the present invention can act on the GLP-1 receptor to function as an antagonist, thereby having a preventive or therapeutic effect against congenital hyperinsulinism or hypoglycemia. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows the results of measuring blood glucose levels over time for an acylated GLP-1 receptor antagonist analogue conjugate according to the present invention (SEQ ID NO: 17). [Figure 2] FIG. 1 shows the results of measuring blood glucose levels in sleeve gastrectomized rats following administration of an acylated GLP-1 receptor antagonist analogue conjugate according to the present invention (SEQ ID NO: 17). [Figure 3] FIG. 1 shows the results of measuring plasma insulin levels in sleeve gastrectomized rats after administration of an acylated GLP-1 receptor antagonist analog conjugate according to the present invention (SEQ ID NO: 17). DETAILED DESCRIPTION OF THE INVENTION

[0018] One aspect of the present invention is a novel GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue.

[0019] In one embodiment, the GLP-1 receptor antagonist analogue is characterized by being represented by the following general formula 1:

[0020] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-EE-X13-A-X15-R-X17-FI-X20-WL-X23-X24-GGPSSGAPPPS-X36 (general formula 1, sequence number 25)

[0021] In the general formula 1, X1 is threonine or absent; X2 is phenylalanine or absent; X3 is threonine or absent; X4 is serine or absent; X5 is aspartic acid or absent; X6 is leucine, valine, or absent; X7 is serine or absent; X8 is alanine, lysine, serine, or absent; X9 is glutamine or tyrosine; X10 is methionine or leucine; X13 is glutamic acid or glutamine; X15 is alanine or valine; X17 is leucine or glutamic acid; X20 is glutamic acid or alanine; X23 is lysine, valine, or an acylated amino acid; X24 is asparagine, lysine, or an acylated amino acid; X36 is cysteine, lysine, an acylated amino acid, or absent; The - indicates a peptide bond.

[0022] In another embodiment, the GLP-1 receptor antagonist analogue is In the general formula 1, X5 is aspartic acid; X6 is leucine, valine; X7 is serine; X8 is lysine or serine; X15 is valine; X23 is lysine or valine; X24 is characterized by being asparagine or lysine.

[0023] In another embodiment, the GLP-1 receptor antagonist analogue is In the general formula 1, X1-X4 do not exist; X5 is aspartic acid; X6 is leucine; X7 is serine; X8 is serine; X9 is tyrosine; X10 is leucine; X13 is glutamic acid; X15 is valine; X17 is leucine; X20 is glutamic acid; X23 is lysine; X24 is asparagine; X36 is characterized as being lysine or an acylated amino acid.

[0024] As an analog according to any one of the above-mentioned specific examples, the acylated amino acid is one of the amino acids represented by K(1) to K(4) below:

[0025] [K(1)] C20diacid-γGlu-(AEEA)2-Lys JPEG2025534686000002.jpg56137[K(2)] C18diacid-γGlu-(AEEA)2-Lys JPEG2025534686000003.jpg66140[K(3)] C16acid-γGlu-(AEEA)2-Lys JPEG2025534686000004.jpg65141[K(4)] C16diacid-γGlu-(AEEA)2-Lys JPEG2025534686000005.jpg59141.

[0026] The analog according to any one of the above embodiments is characterized in that an acyl group is attached to one or more amino acids directly or via a linker.

[0027] In an analog according to any one of the above embodiments, the linker comprises AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid).

[0028] An analog according to any one of the above embodiments, wherein the linker is γGlu-(AEEA) 2 .

[0029] In the analog according to any one of the above specific examples, the linker comprises 0 to 3 AEEA, and 0 to 3 gamma-glutamates are linked to the AEEA.

[0030] As an analog according to any one of the above embodiments, the GLP-1 receptor antagonist analog is characterized by an amidated C-terminus.

[0031] As an analog according to any one of the above embodiments, the GLP-1 receptor antagonist analog is characterized in that it is acylated with a C1-C30 straight or branched chain acyl group containing one or two carboxylic acids.

[0032] An analog according to any one of the above-mentioned embodiments is characterized in that the acyl group is a C4 to C30 fatty acid or dicarboxylic acid.

[0033] As an analog according to any one of the above-mentioned embodiments, the GLP-1 receptor antagonist analog is characterized in that it is acylated at an amino acid or lysine residue located at the C-terminus.

[0034] As an analog according to any one of the above embodiments, the GLP-1 receptor antagonist analog is characterized by an amidated C-terminus.

[0035] As an analog according to any one of the above-mentioned specific examples, the GLP-1 receptor antagonist analog is characterized by comprising any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 22.

[0036] As an analog according to any one of the above-mentioned specific examples, the GLP-1 receptor antagonist analog is characterized by comprising any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3, 7 to 12, 21, and 22.

[0037] As an analog according to any one of the above-mentioned embodiments, the GLP-1 receptor antagonist analog is characterized by comprising any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 17, 19, 21, and 22.

[0038] As an analog according to any one of the above-mentioned embodiments, the GLP-1 receptor antagonist analog is characterized by having any one of the following structures (i) to (iv): (i) JPEG2025534686000006.jpg51137(ii) JPEG2025534686000007.jpg49130(iii) JPEG2025534686000008.jpg56137(iv) JPEG2025534686000009.jpg53138.

[0039] Another aspect of the present invention is a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, comprising a pharmaceutically effective amount of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog.

[0040] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0041] In another embodiment, the pharmaceutical composition is characterized in that it increases blood glucose levels in an individual upon administration.

[0042] The pharmaceutical composition according to any one of the above embodiments is characterized in that the pharmaceutical composition reduces insulin secretion in an individual upon administration.

[0043] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the hypoglycemia is postbariatric hypoglycemia (PBH).

[0044] In the pharmaceutical composition according to any one of the above-mentioned embodiments, the post-obesity hypoglycemia is characterized by being caused by bariatric surgery.

[0045] Another aspect of the present invention is the use of the GLP-1 receptor antagonist analogue, the acylated GLP-1 receptor antagonist analogue, or a composition containing the same for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0046] Another aspect of the present invention is the use of the GLP-1 receptor antagonist analogue, the acylated GLP-1 receptor antagonist analogue, or a composition containing the same to provide a medicament for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0047] Specific details for implementing the present invention are as follows. Meanwhile, each description and embodiment disclosed in this application also applies to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of the present invention. Furthermore, the following specific description is not intended to limit the scope of the present invention.

[0048] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, amino acids referred to as abbreviations herein are described according to the IUPAC-IUB nomenclature system.

[0049] Alanine A Arginine R Asparagine N Aspartic acid D Cysteine ​​C Glutamic Acid E Glutamine Q Glycine G Histidine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V

[0050] One aspect of the present invention is a GLP-1 receptor antagonist analogue or a conjugate of said GLP-1 receptor antagonist analogue that is acylated.

[0051] GLP-1 is a hormone secreted from the small intestine in response to food intake, which promotes insulin secretion in the pancreas and suppresses glucagon secretion, helping to lower blood sugar levels. It also acts as a satiety factor, slowing gastrointestinal digestion and slowing the gastrointestinal transit time of food digestion, thereby reducing food intake.

[0052] The development of diabetes and obesity treatments is underway, taking advantage of the blood sugar regulation and weight loss effects of GLP-1. A representative example is exendin-4, a GLP-1 receptor agonist.

[0053] Native GLP-1 has the following sequence (SEQ ID NO: 23):

[0054] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0055] In the present invention, the term "GLP-1 receptor antagonist" refers to a substance that acts on the GLP-1 receptor to suppress or attenuate the activity of GLP-1. Known GLP-1 receptor antagonists include exendin-3(9-39) (Montrose-Rafizaden et al., 1997, J. Biol. Chem. 272(34):21201-21206).

[0056] Exendin-3(9-39) has the following sequence (SEQ ID NO: 24):

[0057] DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

[0058] In the present invention, the term "GLP-1 receptor antagonist analog" or "analog" refers to a substance that exhibits antagonistic activity against the GLP-1 receptor (GLP-1R). It may be, but is not limited to, a peptide having one or more differences in its amino acid sequence compared to native GLP-1, which may be modified from the native GLP-1 sequence by any one of partial sequence substitution, addition, deletion, and modification, or a combination of these methods. The GLP-1 receptor antagonist analog may be non-naturally occurring.

[0059] Furthermore, such modifications for producing the GLP-1 receptor antagonist analogues include 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, intramolecular covalent bonds, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, etc. Furthermore, the modifications include substitution with non-natural compounds.

[0060] Furthermore, the GLP-1 receptor antagonist analogue may be, but is not limited to, one in which one or more amino acids are added to the amino and / or carboxy terminus of natural GLP-1.

[0061] The substituted or added amino acids can be atypical or non-naturally occurring amino acids as well as the 20 amino acids commonly found in human proteins. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing such 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.

[0062] In one specific embodiment, the GLP-1 receptor antagonist analog of the present invention may be, but is not limited to, a GLP-1 receptor antagonist analog represented by the following general formula 1:

[0063] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-EE-X13-A-X15-R-X17-FI-X20-WL-X23-X24-GGPSSGAPPPS-X36 (general formula 1, sequence number 25)

[0064] In the general formula 1, X1 is threonine or absent; X2 is phenylalanine or absent; X3 is threonine or absent; X4 is serine or absent; X5 is aspartic acid or absent; X6 is leucine, valine, or absent; X7 is serine or absent; X8 is alanine, lysine, serine, or absent; X9 is glutamine or tyrosine; X10 is methionine or leucine; X13 is glutamic acid or glutamine; X15 is alanine or valine; X17 is leucine or glutamic acid; X20 is glutamic acid or alanine; X23 is lysine, valine, or an acylated amino acid; X24 is asparagine, lysine, or an acylated amino acid; X36 is cysteine, lysine, an acylated amino acid, or absent; The - indicates a peptide bond.

[0065] In one embodiment, the GLP-1 receptor antagonist analogue is In the general formula 1, X5 is aspartic acid; X6 is leucine, valine; X7 is serine; X8 is lysine or serine; X15 is valine; X23 is lysine or valine; X24 may be, but is not limited to, asparagine or lysine.

[0066] The GLP-1 receptor antagonist analog may comprise any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 3, 5, 7 to 12, and 17 to 22, more specifically, any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3, 7 to 12, 21, and 22, but is not limited thereto.

[0067] In another embodiment, the GLP-1 receptor antagonist analogue is In the general formula 1, X1-X4 do not exist; X5 is aspartic acid; X6 is leucine; X7 is serine; X8 is serine; X9 is tyrosine; X10 is leucine; X13 is glutamic acid; X15 is valine; X17 is leucine; X20 is glutamic acid; X23 is lysine; X24 is asparagine; X36 may be, but is not limited to, lysine or an acylated amino acid.

[0068] In another embodiment, the GLP-1 receptor antagonist analog may have any one of the following structures (i) to (iv), but is not limited thereto: (i) JPEG2025534686000010.jpg51137(ii) JPEG2025534686000011.jpg52138(iii) JPEG2025534686000012.jpg55136(iv) JPEG2025534686000013.jpg53139.

[0069] Alternatively, the GLP-1 receptor antagonist analogue may comprise any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 17, 19, 21, and 22, but is not limited thereto.

[0070] In one embodiment, the GLP-1 receptor antagonist analogue comprising the amino acid sequence of SEQ ID NO: 17 may have the following structure, but is not limited thereto: JPEG2025534686000014.jpg55147.

[0071] In one embodiment, the GLP-1 receptor antagonist analogue comprising the amino acid sequence of SEQ ID NO: 19 may have the following structure, but is not limited thereto: JPEG2025534686000015.jpg55147.

[0072] In one embodiment, the GLP-1 receptor antagonist analogue comprising the amino acid sequence of SEQ ID NO: 21 may have the following structure, but is not limited thereto: JPEG2025534686000016.jpg58142.

[0073] In one embodiment, the GLP-1 receptor antagonist analog comprising the amino acid sequence of SEQ ID NO: 22 may have the following structure, but is not limited to: JPEG2025534686000017.jpg55143.

[0074] In the present invention, the GLP-1 receptor antagonist analog may be, but is not limited to, an acylated GLP-1 receptor antagonist analog conjugate having an acyl group attached thereto, and may refer to both acylated and non-acylated GLP-1 receptor antagonist analogs.

[0075] Acylation is known as a method for improving the pharmacokinetic and pharmacodynamic properties of peptide drugs. Peptide drugs have the problem of being difficult to exert their efficacy due to enzymatic degradation in the body. In response to this problem, peptide acylation, which attaches a fatty acid to the peptide, blocks the enzyme's action site, thereby increasing the stability and half-life of the peptide drug. For purposes of the present invention, the GLP-1 receptor antagonist analog of the present invention may be in an acylated form to increase its half-life.

[0076] In the present invention, the term "acylated GLP-1 receptor antagonist analog conjugate", "acylated GLP-1 receptor antagonist analog", "acylated analog", or "conjugate" may be used interchangeably.

[0077] An acyl group may be attached to an amino acid of the acylated GLP-1 receptor antagonist analog conjugate of the present invention via a linker, and the linker may include AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), more specifically, γGlutamate may be further linked, but is not limited thereto. Specific examples of linkers include (γGlu) m-(AEEA) n The m and n may each independently be an integer of 0, 1, 2, 3 or more, but are not limited thereto.

[0078] An acyl group may be bound to an amine group, hydroxy group, thiol group, carboxyl group, or the like of an amino acid in a GLP-1 receptor antagonist analog through an amine group, hydroxy group, thiol group, or the like of the linker, but the type and length of the linker are not limited as long as the acyl group can be bound to the GLP-1 receptor antagonist analog to contribute to the structural stability and increased half-life of the analog. Furthermore, the linker may be covalently bound to the acyl group or may be repeatedly linked to the acyl group one, two, three, or more times, but is not limited thereto.

[0079] The acyl group utilized in the acylation may be a carbon chain of any length, linear or branched, including linear aliphatic chains, branched aliphatic chains, chains containing cyclic alkyl moieties, hydrophobic natural products such as steroids, alkyl chains, or alkyl chains containing acyl moieties.

[0080] In one specific embodiment, the acylated GLP-1 receptor antagonist analog may be acylated with a C1-C30 linear or branched acyl group containing one or more, two or more, specifically one or two, carboxylic acids. For example, the acyl group may be, but is not limited to, a fatty acid or dicarboxylic acid, specifically, a C4-C30 fatty acid or dicarboxylic acid. More specific examples include, but are not limited to, C16, C18, C20, C22, C24, C26, C28, or C30 fatty acid or dicarboxylic acid. Other examples of acyl groups include, but are not limited to, bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, and cholesterol acid; succinic acid or a succinic acid derivative; maleic acid or a maleic acid derivative; and the like.

[0081] The acylated GLP-1 receptor antagonist analog of the present invention may be one in which an acyl group is attached to a GLP-1 receptor antagonist analog by a method known in the art, or may be one produced by synthesizing a peptide using an acylated amino acid, but is not limited thereto.

[0082] In the present invention, the acylated GLP-1 receptor antagonist analog may be in a form in which an acyl group is directly attached to an amino acid residue of the GLP-1 receptor antagonist analog. For example, the acyl group may be attached via an ester, thioester, or amide bond, but is not limited thereto. Specifically, the acylated GLP-1 receptor antagonist analog may be acylated to an amino acid residue having an amine, hydroxyl, or thiol group. For example, the acylated GLP-1 receptor antagonist analog may be acylated to an amino acid or lysine residue located at the C-terminus, but is not limited thereto.

[0083] In the present invention, the acylated amino acid of the acylated GLP-1 receptor antagonist analogue may include, but is not limited to, any one of the amino acids represented by K(1) to K(4) below:

[0084] [K(1)] C20diacid-γGlu-(AEEA)2-Lys JPEG2025534686000018.jpg57140[K(2)] C18diacid-γGlu-(AEEA)2-Lys JPEG2025534686000019.jpg65139[K(3)] C16acid-γGlu-(AEEA)2-Lys JPEG2025534686000020.jpg61133[K(4)] C16diacid-γGlu-(AEEA)2-Lys JPEG2025534686000021.jpg56135.

[0085] The acylated GLP-1 receptor antagonist analog conjugates of the present invention may include, but are not limited to, an acylated amino acid at position 8, 23, 24, or 36.

[0086] In a specific embodiment, the GLP-1 receptor antagonist analog according to the present invention may comprise any one of the amino acid sequences of SEQ ID NOs: 1 to 22, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 22, but is not limited thereto.

[0087] Specifically, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences of SEQ ID NOs: 1 to 12, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, but is not limited thereto.

[0088] More specifically, the GLP-1 receptor antagonist analog may comprise any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3, 7 to 12, 21, and 22, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7 to 12, 21, and 22, but is not limited thereto.

[0089] More specifically, the GLP-1 receptor antagonist analog may comprise any one of the amino acid sequences of SEQ ID NOs: 17, 19, 21, and 22, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, and 22, but is not limited thereto.

[0090] In another specific embodiment, the acylated GLP-1 receptor antagonist analog conjugate according to the present invention may comprise any one of the sequences of SEQ ID NOs: 13 to 22, specifically, may be (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 22, but is not limited thereto.

[0091] Even if the present application describes a "peptide consisting of a specific sequence number," if the peptide has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, this does not exclude meaningless additions of sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof, and it is self-evident that even if the peptide has such additions or mutations of sequences, it falls within the scope of the present application.

[0092] Furthermore, the GLP-1 receptor antagonist analogs of the present invention may comprise an amino acid sequence that has 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequences of SEQ ID NOs: 1 to 22, but are not limited thereto as long as they can act on the GLP-1 receptor and exhibit antagonistic activity.

[0093] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, and can be expressed as a percentage.

[0094] Homology or identity of conserved polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or a portion of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0095] The terms homology and identity can often be used interchangeably.

[0096] Whether any two peptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program using default parameters, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444, or using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later). (The GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073) can be used to determine homology, similarity, or identity. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used.

[0097] Peptide homology, similarity, or identity can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48:443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a sequence as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used in this application, the terms "homology" or "identity" indicate the relatedness between sequences.

[0098] The GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention may exhibit activity that is approximately 1% or more, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, 200% or more, 250% or more, 300% or more, or 350% or more compared to the activity (100%) of the known GLP-1 receptor antagonist exendin-3(9-39).

[0099] In this application, the term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., including all numerical values ​​in a range that is equal to or similar to the numerical value following the term about.

[0100] The activity of such a GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate can be measured by methods known in the art and is not limited to a specific method. For example, the antagonistic activity of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog of the present invention can be analyzed by measuring the concentration of cAMP, the production of which is inhibited by the GLP-1 receptor antagonist analog.

[0101] Furthermore, the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate according to the present invention may have an unmodified N-terminus and / or C-terminus of the peptide, but the scope of the present invention also includes modified forms in which the N-terminus and / or C-terminus are chemically modified or protected with an organic group, or amino acids are added to the peptide termini, etc., to protect them from in vivo protease cleavage and increase their stability.

[0102] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, and therefore, to remove such charges, the N-terminus may be acetylated and / or the C-terminus may be amidated, but is not limited thereto.

[0103] The GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate according to the present invention may be amidated at the C-terminus, or may be amidated and acylated at the C-terminus at the same time, but is not limited thereto.

[0104] Unless otherwise specified herein, the description and claims of the invention relating to the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate according to the present invention apply not only to the analog or conjugate, but also to a category encompassing all salts of the analog or conjugate (e.g., pharmaceutically acceptable salts of the peptide), or solvates thereof. Therefore, the description in the specification also applies to specific salts, specific solvates, and specific solvates of specific salts. Such salt forms may be, for example, any pharmaceutically acceptable salt. The type of salt is not particularly limited. However, a form that is safe and effective for individuals, e.g., mammals, is preferred, but is not particularly limited thereto.

[0105] The term "pharmaceutically acceptable" means, within the scope of medical judgment, a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, or allergic reaction.

[0106] As used herein, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0107] The term "solvate" used in the present invention refers to a complex formed between the peptide, conjugate, or salt thereof according to the present invention and a solvent molecule.

[0108] Depending on the length, the analogs of the present invention can be synthesized by methods well known in the art, for example, by an automated peptide synthesizer, or can be produced by genetic engineering techniques.

[0109] Specifically, the analogs of the present invention can be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, GLP-1 receptor antagonist analogs or acylated GLP-1 receptor antagonist analog conjugates according to the present invention can be synthesized in a number of ways, including, for example, the following:

[0110] (a) synthesis of peptides stepwise or by fragment assembly by means of solid-phase or liquid-phase techniques, followed by isolation and purification of the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method in which peptide fragments are obtained by any combination of (a), (b) and (c), the fragments are then ligated to obtain a peptide, and the peptide is recovered.

[0111] The above may also be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0112] Another embodiment of the present invention provides a composition comprising the GLP-1 receptor antagonist analogue or acylated GLP-1 receptor antagonist analogue conjugate.

[0113] The GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue conjugate is as described above.

[0114] In particular, the composition may be a pharmaceutical composition, and more particularly, the composition may be used for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0115] A specific embodiment of the present invention is a pharmaceutical composition for treating or preventing congenital hyperinsulinism or hypoglycemia, comprising a pharmaceutically effective amount of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate.

[0116] The term "a pharmaceutically effective amount" of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate refers to an amount sufficient to achieve a desired pharmacological activity (e.g., prevention, amelioration, or treatment of congenital hyperinsulinism or hypoglycemia), and also refers to a pharmaceutically acceptable amount that does not cause or produces negligible toxicity or side effects in the individual to whom it is administered, but is not limited thereto. Such a pharmaceutically effective amount can be determined by comprehensively considering factors such as the frequency of administration, the patient, and the dosage form.

[0117] In the present invention, the term "prevention" means any action that suppresses or delays the onset of a target disease, such as congenital hyperinsulinism or hypoglycemia, by administering the GLP-1 receptor antagonist analogue or acylated GLP-1 receptor antagonist analogue conjugate, or a composition containing them, and "treatment" means any action that improves or provides benefit to the symptoms of a target disease, such as congenital hyperinsulinism or hypoglycemia, by administering the GLP-1 receptor antagonist analogue or acylated GLP-1 receptor antagonist analogue conjugate, or a composition containing them.

[0118] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition is not particularly limited, but the composition may be administered via any common route that can deliver the composition to an in vivo target, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0119] Congenital hyperinsulinism is a disease that causes severe and persistent hypoglycemia in newborns and children. It can be caused by a temporary increase in insulin secretion in low birth weight babies or babies born to diabetic pregnant women, or abnormal function of pancreatic cells due to gene mutations.

[0120] Hypoglycemia in the present invention refers to a state in which blood glucose levels are lower than those of normal individuals. It usually refers to a state in which blood glucose levels are 50 mg / dL or less, but is not limited thereto. In the present invention, hypoglycemia includes both acute hypoglycemia and chronic hypoglycemia.

[0121] Symptoms of hypoglycemia include lethargy, trembling, paleness, cold sweats, dizziness, agitation, anxiety, palpitations, hunger, headache, fatigue, etc. If hypoglycemia persists for a long time, convulsions and seizures may occur, leading to shock and fainting.

[0122] Specifically, in the present invention, the hypoglycemia may be postbariatric hypoglycemia (PBH), more specifically, hypoglycemia caused by bariatric surgery, but is not limited thereto.

[0123] In the present invention, "post-obesity hypoglycemia" or "hypoglycemia due to obesity metabolic surgery" refers to hypoglycemia experienced by patients after obesity metabolic surgery such as sleeve gastrectomy, duodenal bypass, or Roux-en-Y gastric bypass, and may also develop due to dumping syndrome, one of the complications.

[0124] To date, there is no known cure for hypoglycemia caused by obesity or obesity metabolic surgery, and all researchers agree that dietary and lifestyle modifications are the most important treatments. The principle of dietary therapy is to reduce the amount of food consumed at one time and eat high-protein, high-fat, low-carbohydrate, low-water meals in small, well-chewed portions (5-6 times a day). If dumping syndrome causes hypoglycemia, abdominal pain, or cramps, symptoms can be controlled with medications such as sedatives, anticonvulsants, glucose injections, sedatives, and autonomic blockers.

[0125] The GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention can exert an antagonizing effect on the GLP-1 receptor, thereby increasing / increasing blood glucose levels in the body or reducing insulin secretion, thereby exerting a preventive or therapeutic effect on congenital hyperinsulinism or hypoglycemia, but is not limited thereto.

[0126] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to exhibit a therapeutic effect and not cause side effects, and can be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, treatment period, and drugs used in combination or concomitantly.

[0127] The pharmaceutical composition comprising the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention may further comprise a pharmaceutically acceptable carrier. The carrier is not particularly limited, but may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavorings, etc. for oral administration, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. for injections, and bases, excipients, lubricants, preservatives, etc. for topical administration.

[0128] The composition of the present invention may be prepared in various dosage forms by mixing with the aforementioned pharmaceutically acceptable carriers. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it may be prepared in the form of unit-dose ampoules or multiple-dose forms. It may also be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0129] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, the formulation may further contain fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, preservatives, etc.

[0130] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0131] Furthermore, the composition may be formulated into a unit dosage form suitable for administration into the body of a patient by a method conventional in the pharmaceutical field, specifically, into a dosage form useful for administering peptide pharmaceuticals, and administered orally or parenterally using an administration method conventionally used in the art, including, but not limited to, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes.

[0132] In addition, the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate may be mixed with various pharmaceutically acceptable carriers, such as physiological saline or organic solvents, and to increase stability and water absorption, drugs such as carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used.

[0133] The dosage and frequency of the pharmaceutical composition of the present invention are determined depending on the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex, and weight of the patient, and the severity of the disease.

[0134] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01 to 99% weight to volume.

[0135] The total effective amount of the composition of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary in the content of the active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention may be about 0.3 to 0.9 mg per kg of patient body weight per day. The dosage can be determined based on the mass of the GLP-1 receptor antagonist analog; or the mass of the acylated GLP-1 receptor antagonist analog conjugate excluding the mass of the fatty acid moiety, i.e., the sum of the masses of only the polypeptide moieties. However, the effective dose of the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate is determined by taking into consideration various factors, such as the administration route and frequency of treatment of the pharmaceutical composition, as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate, and taking these factors into consideration, a person skilled in the art would be able to determine an appropriate effective dose depending on the 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, or administration method, as long as it exhibits the effects of the present invention.

[0136] The pharmaceutical composition of the present invention has excellent in vivo durability and potency, and can be administered less frequently and less frequently than other drugs, but is not particularly limited thereto.

[0137] Another embodiment of the present invention provides a method for preventing or treating congenital hyperinsulinism or hypoglycemia, comprising administering to an individual the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate, or a composition containing the same.

[0138] The GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate, the composition containing the same, and the prevention and treatment of congenital hyperinsulinism and hypoglycemia are as described above.

[0139] In the present invention, the individual is one suspected of having congenital hyperinsulinism or hypoglycemia. The individual suspected of having congenital hyperinsulinism or hypoglycemia refers to mammals, including rats, livestock, and humans, that are suffering from or may suffer from the disease. However, the individual includes, without limitation, individuals that can be treated with the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention, or the composition containing the same. Furthermore, administering a pharmaceutical composition containing the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention to an individual suspected of having congenital hyperinsulinism or hypoglycemia can effectively treat the individual. Congenital hyperinsulinism or hypoglycemia is as described above.

[0140] The methods of the present invention can include administering a pharmaceutically effective amount of a pharmaceutical composition containing a peptide. A suitable total daily dose will be determined by the treating physician within the scope of sound medical judgment and can be administered in single or divided doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will vary depending on a variety of factors, including the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the time and route of administration, the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0141] In the method of the present invention, the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate, or a composition containing the same, can be administered via a common route that can deliver it to an in vivo target, and the administration route includes, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, pulmonary administration, and rectal administration.

[0142] Yet another embodiment of the present invention provides the use of a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate, or said composition, for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0143] The GLP-1 receptor antagonist analog, the acylated GLP-1 receptor antagonist analog conjugate, the composition, congenital hyperinsulinism, and hypoglycemia are as described above.

[0144] Another embodiment of the present invention provides the use of a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate, or said composition, in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0145] The GLP-1 receptor antagonist analog, the acylated GLP-1 receptor antagonist analog conjugate, the composition, congenital hyperinsulinism, and hypoglycemia are as described above.

[0146] On the other hand, in this specification, unless the context otherwise requires, the terms "comprises", "contains", and the like shall be understood to mean the inclusion of a specified integer or group of integers, but not the exclusion of other integers or sets of integers. Example

[0147] The present invention will be described in more detail with reference to the following examples. These examples are merely for the purpose of explaining the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0148] Example 1: Preparation of GLP-1 receptor (GLP-1R) antagonist analogs and acylated GLP-1 receptor (GLP-1R) antagonist analog conjugates The GLP-1R antagonist analogs were synthesized using an automated peptide synthesizer (Symphony X, Gyros Protein Tech.) using solid-phase synthesis. Rink amide resin was used for C-terminal amidation. Each amino acid was synthesized in order from the C-terminus to the N-terminus.

[0149] The amino acids were sequentially coupled using Fmoc (9H-fluoren-9-ylmethoxycarbonyl) protected amino acids (4 equivalents in peptide-resin ratio), HOBt (1-hydroxybenzotriazole, 4 equivalents in peptide-resin ratio), and DIC (diisopropylcarbodiimide, 8 equivalents in peptide-resin ratio).

[0150] For the acylated GLP-1R antagonist analogue conjugates, peptides were synthesized using acylated amino acids K(1) to K(4) in the same manner as above.

[0151] [K(1)] C20diacid-γGlu-(AEEA)2-Lys JPEG2025534686000022.jpg58144[K(2)] C18diacid-γGlu-(AEEA)2-Lys JPEG2025534686000023.jpg64136[K(3)] C16acid-γGlu-(AEEA)2-Lys JPEG2025534686000024.jpg62136[K(4)] C16diacid-γGlu-(AEEA)2-Lys JPEG2025534686000025.jpg58138.

[0152] The Fmoc protecting group was removed in the automated synthesizer by adding 8 mL of 20% piperidine / DMF (2 x 5 min) to the reaction vessel containing the resin. Each step was followed by a 12 mL DMF wash (6 x 10 s) to remove any remaining impurities. After synthesis was complete, the N-terminus was acetylated with 6 mL of 34% acetic anhydride / DMF and 3.5 mL of 14% N,N-diisopropyl / DMF. Any remaining protecting groups were deprotected during cleavage of the peptide from the resin.

[0153] The sequences of the GLP-1R antagonist analogues and acylated GLP-1R antagonist analogue conjugates prepared through this process are shown in Table 1 below.

[0154] [Table 1] JPEG2025534686000027.jpg41153

[0155] In Table 1 above, K(1) to (4) mean that the amino acid corresponding to the position is an acylated amino acid having the above structure.

[0156] The synthesized GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates were purified using reversed-phase chromatography. The purity of the synthesized peptides was confirmed using analytical liquid chromatography (RP-HPLC). If the purity was 90% or higher, it was deemed suitable for use in experiments. In addition, the molecular weight and information of the peptides were confirmed using liquid chromatography / mass spectrometry (LC / MS).

[0157] The synthesized peptides were stored at −20° C. until use in experiments.

[0158] Example 2: Confirmation of in vitro activity of GLP-1 receptor (GLP-1R) antagonist analogs and acylated GLP-1 receptor (GLP-1R) antagonist analog conjugates To measure the activity of GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates, we used an in vitro method to measure cellular activity using cell lines transformed with human GLP-1R (hGLP-1R). These cell lines were CHO (Chinese hamster ovary) transformed to express human GLP-1R, and are suitable for measuring the activity of GLP-1R antagonists.

[0159] To measure the activity of GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates, human GLP-1 was diluted 3-fold from 9000 pM to 0.15 pM. To confirm antagonistic activity, a comparison substance [Exendin-3(9-39)amide (Tocris Bioscience, UK, Cat No. 2081), SEQ ID NO: 24], GLP-1R antagonist analogs (SEQ ID NOs: 1-12), and acylated GLP-1R antagonist analog conjugates (SEQ ID NOs: 13-22) were serially diluted 3-fold from 9000 nM to 0.15 nM in a plate containing 0.1 nM GLP-1. The human GLP-1R-expressing CHO cells were cultured in a 384-well plate for 24 hours, after which the culture medium was removed and 10 μL of each serially diluted substance was added to the plate and incubated at room temperature for 30 minutes. Then, 5 μL of Eu-cAMP tracer was added to each well, followed by 5 μL of buffer containing cAMP antibody, and the mixture was incubated at room temperature for 60 minutes. After the incubation, the cell lysate was applied to a LANCE Ultra cAMP kit (PerkinElmer, USA), and the half-maximal inhibitory concentration (IC) was determined based on the degree to which cAMP production was inhibited by the GLP-1R antagonist analog and acylated GLP-1R antagonist analog conjugate. 50 The relative activity values ​​were calculated and compared with each other. The relative activity values ​​of the comparative substances are shown in Table 2 below.

[0160] [Table 2] JPEG2025534686000029.jpg64165

[0161] From the above experimental results, it was confirmed that the GLP-1R antagonist analog and acylated GLP-1R antagonist analog conjugate of the present invention prepared in Example 1 act on the human GLP-1 receptor (GLP-1R) and are capable of antagonizing the GLP-1 receptor.

[0162] Example 3: Confirmation of in vitro activity of acylated GLP-1 receptor (GLP-1R) antagonist analog conjugates To measure the activity of acylated GLP-1R antagonist analog conjugates, we used an in vitro method to measure cellular activity using cell lines transformed with human (hGLP-1R) or mouse (mGLP-1R) GLP-1R. These cell lines were CHO (Chinese hamster ovary) transformed to express human and mouse GLP-1R, respectively, and are suitable for measuring the activity of GLP-1R antagonists.

[0163] To measure the activity of the acylated GLP-1R antagonist analog conjugates, human GLP-1 was diluted 3-fold from 9000 pM to 0.15 pM. To confirm antagonistic activity, a comparison substance [Exendin-3(9-39)amide (Tocris Bioscience, UK, Cat No. 2081), SEQ ID NO: 24] and acylated GLP-1R antagonist analog conjugates (SEQ ID NOs: 17, 19, 21, and 22) were serially diluted 3-fold from 9000 nM to 0.15 nM on a plate containing 0.1 nM GLP-1.

[0164] The acylated GLP-1R antagonist analogue conjugates of SEQ ID NOs: 17, 19, 21, and 22 used in this example have the following structures (i) to (iv), respectively:

[0165] (i) JPEG2025534686000030.jpg50136(ii) JPEG2025534686000031.jpg50133(iii) JPEG2025534686000032.jpg55137(iv) JPEG2025534686000033.jpg51132.

[0166] Furthermore, to confirm the effect of fatty acids that bind to albumin, the analog conjugates were diluted in buffer containing 1% human serum albumin and mouse serum albumin. CHO cells expressing human GLP-1R and mouse GLP-1R were cultured in 384-well plates for 24 hours, respectively, and then the culture medium was removed. 10 μL of each serially diluted substance was added to the plate and incubated at room temperature for 30 minutes. 5 μL of Eu-cAMP tracer was then added, followed by 5 μL of a buffer containing a cAMP antibody, and the mixture was incubated at room temperature for 60 minutes. The cell lysates after the reaction were applied to a LANCE Ultra cAMP kit (PerkinElmer, USA), and the half-maximal inhibitory concentration (IC) was determined based on the degree to which cAMP production was inhibited by the GLP-1R antagonist analog and the acylated GLP-1R antagonist analog conjugate. 50 The relative activity values ​​were calculated and compared with each other. The relative activity values ​​of the comparative substances are shown in Table 3 below.

[0167] [Table 3]

[0168] The GLP-1R antagonist analog and acylated GLP-1R antagonist analog conjugates prepared in Example 1 can act on both human and mouse GLP-1R receptors to exert antagonistic effects. In particular, as shown in Table 3, among the acylated GLP-1R antagonist analog conjugates, SEQ ID NOs: 21 and 22 having C16 fatty acids were found to have superior activity compared to the comparative substances, followed by SEQ ID NO: 17 having C18 fatty acids and SEQ ID NO: 19 having C20 fatty acids in that order.

[0169] Example 4: Blood glucose-regulating effect of GLP-1 receptor (GLP-1R) antagonist analogue conjugate (SEQ ID NO: 17) To confirm the effect of the acylated GLP-1 receptor antagonist analog conjugate (SEQ ID NO: 17) on glucose tolerance, C57BL / 6N mice were used as an animal model. Experiments were conducted on 8-week-old male rats. Mice were housed in groups of five and had free access to drinking water during the study period. Lights were turned off from 6 PM to 6 AM.

[0170] The test groups were: Group 1: vehicle (subcutaneous administration) - control group (vehicle); Group 2: Exendin-3(9-39) 3690 μg / kg (1095.0 nMol / kg, subcutaneous administration); Group 3: GLP-1R antagonist analog conjugate (SEQ ID NO: 17) 3690 μg / kg (1061.9 nMol / kg, subcutaneous administration); Group 4: GLP-1R antagonist analog conjugate (SEQ ID NO: 17) 7380 μg / kg (2123.8 nMol / kg, subcutaneous administration). Drug administration was completed 24 hours before the oral glucose tolerance test (OGTT) for Groups 1, 3, and 4, and 30 minutes before for Group 2. Here, the dosage is expressed based on the total mass of the acylated GLP-1 receptor antagonist analogue conjugate minus the mass of the fatty acid moiety, i.e., the sum of the masses of only the polypeptide moiety.

[0171] For the OGTT, all groups were fasted for 4 hours, and then 2 g / kg of glucose was orally administered at a time. Blood samples were collected from the tail vein at designated time intervals (0, 15, 30, 60, and 120 minutes) and blood glucose levels were measured using a OneTouch blood glucose meter (OneTouch Select®, LifeScan, USA). Statistical analysis was performed using an unpaired t-test ( # ~ ### p < 0.05 ~ 0.001) was used to compare between the control and test groups.

[0172] As a result, it was confirmed that the GLP-1R antagonist analog conjugate of SEQ ID NO: 17 increased blood glucose levels in vivo compared to the control group at all doses (Figure 1), suggesting that the GLP-1R antagonist analog or acylated GLP-1R antagonist analog conjugate according to the present invention may have a therapeutic effect on congenital hyperinsulinism due to its blood glucose regulating ability.

[0173] Example 5: Confirmation of the blood glucose-regulating effect of a GLP-1 receptor (GLP-1R) antagonist analog conjugate (SEQ ID NO: 17) in a vertical sleeve gastrectomy (VSG)-induced postbariatric hypoglycemia (PBH) rat model SD rats were subjected to sham or VSG surgery to establish the PBH rat model. The rats were fed a high-fat diet (HFD) for 19 weeks, and 10 rats were randomly divided into two groups: a vehicle control group and a GLP-1R antagonist analog conjugate treatment group. Vehicle and GLP-1R antagonist analog conjugate (SEQ ID NO: 17) were subcutaneously administered at 3720 μg / kg (1070.5 nMol / kg), respectively, followed by a mixed meal tolerance test (MMTT) 4 hours later. For the MMTT, all groups were fasted for 5 hours, and then 2 mL of mixed meal (Ensure Plus®, Abbott, USA) was orally administered. Blood samples were collected at designated intervals (0, 15, 30, 45, 60, and 120 minutes) and blood glucose levels were measured (GDoctor, Allmedicus Co., Korea). Statistical analysis was performed using one-way ANOVA (*~***p < 0.05~0.001) to compare the VSG rat vehicle (control) and test groups. The doses were expressed based on the total mass of the acylated GLP-1 receptor antagonist analog conjugate, excluding the mass of the fatty acid moiety, i.e., the sum of the mass of only the polypeptide moiety.

[0174] As a result, it was confirmed that the GLP-1R antagonist analog conjugate of SEQ ID NO: 17 significantly increased the blood glucose level in the body compared to the PBH rats administered with the vehicle control group (FIG. 2).

[0175] In addition, plasma was separated from the blood sample 15 minutes after the MMTT, and insulin secretion was measured using a rat insulin ELISA kit (Multi species GLP-1 total ELISA, Merck Millipore, USA). The results confirmed that insulin secretion was reduced by 47% in the group administered with the GLP-1R antagonist analog conjugate of SEQ ID NO: 17 compared to the vehicle control group (Figure 3). This suggests that the blood glucose-regulating ability of the GLP-1R antagonist analog or acylated GLP-1R antagonist analog conjugate of the present invention may be effective in treating hypoglycemia, particularly post-obesity hypoglycemia.

[0176] From the above examples, it was confirmed that the GLP-1 receptor analogs and acylated GLP-1 receptor analogs prepared in the present invention function as antagonists of the GLP-1 receptor, and based on this activity, they can be used as useful therapeutic agents for congenital hyperinsulinism and hypoglycemia, particularly post-obesity hypoglycemia.

[0177] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. GLP-1 receptor antagonist analogs represented by the following general formula 1: X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - In the general formula 1, X1 is threonine or absent; X2 is phenylalanine or absent; X3 is threonine or absent; X4 is serine or absent; X5 is aspartic acid or absent; X6 is leucine, valine, or absent; X7 is serine or absent; X8 is alanine, lysine, serine, or absent; X9 is glutamine or tyrosine; X10 is methionine or leucine; X13 is glutamic acid or glutamine; X15 is alanine or valine; X17 is leucine or glutamic acid; X20 is glutamic acid or alanine; X23 is lysine, valine, or an acylated amino acid; X24 is asparagine, lysine, or an acylated amino acid; X36 is cysteine, lysine, an acylated amino acid, or absent; The - indicates a peptide bond.

2. In the general formula 1, X5 is aspartic acid; X6 is leucine, valine; X7 is serine; X8 is lysine or serine; X15 is valine; X23 is lysine or valine; 2. The GLP-1 receptor antagonist analogue of claim 1, wherein X24 is asparagine or lysine.

3. In the general formula 1, X1 to X4 are absent; X5 is aspartic acid; X6 is leucine; X7 is serine; X8 is serine; X9 is tyrosine; X10 is leucine; X13 is glutamic acid; X15 is valine; X17 is leucine; X20 is glutamic acid; X23 is lysine; X24 is asparagine; 2. The GLP-1 receptor antagonist analogue of claim 1, wherein X36 is lysine or an acylated amino acid.

4. The GLP-1 receptor antagonist analogue according to claim 1, wherein the acylated amino acid is any one of the amino acids represented by the following K(1) to K(4): [K(1)) C20diacidd-γGlu-(AEE｡) 2 -ys [K(2)) C14diacid-γGlu--AEE｡) 2 -ys KK(3)) C16acid-γGlu-(AEEA) 2 -ys [K(4)) C16diacid-γGlu-(AEE｡) 2 -ys 。

5. The GLP-1 receptor antagonist analogue according to claim 1, wherein the GLP-1 receptor antagonist analogue has an acyl group attached to one or more amino acids via a linker containing AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid).

6. 2. The GLP-1 receptor antagonist analogue according to claim 1, wherein the C-terminus of the GLP-1 receptor antagonist analogue is amidated.

7. 2. The GLP-1 receptor antagonist analogue of claim 1, wherein the GLP-1 receptor antagonist analogue is acylated with a C1-C30 linear or branched chain acyl group containing one or two carboxylic acids.

8. 8. The GLP-1 receptor antagonist analogue of claim 7, wherein the acyl group is a C4 to C30 fatty acid or dicarboxylic acid.

9. 8. The GLP-1 receptor antagonist analogue according to claim 7, wherein the GLP-1 receptor antagonist analogue is acylated at an amino acid or lysine residue located at the C-terminus.

10. 8. The GLP-1 receptor antagonist analogue according to claim 7, wherein the C-terminus of the GLP-1 receptor antagonist analogue is amidated.

11. The GLP-1 receptor antagonist analogue according to claim 1, which comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 22.

12. The GLP-1 receptor antagonist analogue according to claim 1, comprising any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3, 7 to 12, 21, and 22.

13. The GLP-1 receptor antagonist analogue according to claim 1, comprising any one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 17, 19, 21, and 22.

14. The GLP-1 receptor antagonist analogue according to claim 1, which has any one of the following structures (i) to (iv): (i) (ii) (iii) (iv) 。

15. A pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, comprising the GLP-1 receptor antagonist analogue according to any one of claims 1 to 14.

16. 16. The pharmaceutical composition of claim 15, wherein the hypoglycemia is Postbariatric Hypoglycemia (PBH).

17. 17. The pharmaceutical composition of claim 16, wherein the post-obesity hypoglycemia is due to bariatric surgery.

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