NOVEL GLP-1 RECEPTOR ANTAGONIST AND PHARMACEUTICAL COMPOSITION COMPRISING SAME FOR PREVENTING OR TREATING CONGENERATIVE HIGH INSULINEmia OR

Abstract

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

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, which contains a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate. Background Art

[0002] Hypoglycemia refers to a condition where the blood glucose level is lower than that of normal individuals (60 - 120 mg / dL during fasting and 140 mg / dL or lower two hours after eating). A blood glucose level of 50 mg / dL or lower is generally defined as hypoglycemia.

[0003] Hypoglycemia may be caused by excessive oral hypoglycemic agents or insulin, prolonged fasting, or excessive physical activity or exercise. Hypoglycemia may also occur due to factors such as severe physical illnesses, hormone deficiencies such as adrenal cortical hormones or glucagon, insulin-producing pancreatic tumors, and insulin autoimmune diseases, and may also occur in patients who have undergone gastrectomy and individuals with genetic disorders of carbohydrate-metabolizing enzymes.

[0004] Meanwhile, congenital hyperinsulinism (CHI) is one of the most common causes of severe and persistent hypoglycemia in neonates and children. Insulin is a hormone that regulates blood glucose in the human body. When the blood glucose level rises due to food intake, it can lower the blood glucose level. However, patients with congenital hyperinsulinism cannot perform this regulatory function and secrete insulin from the pancreas regardless of the blood glucose level. Therefore, these patients experience hypoglycemia.

[0005] The occurrence of hypoglycemia or hypoglycemia caused by congenital hyperinsulinism leads to the inability to maintain glucose, ketones, and lactate (substances mainly used by brain cells), and blocks the energy supply of proteins or fats in the body. This can cause brain cell damage, which may lead to seizures, learning disabilities, cerebral palsy, blindness, and even death in severe cases.

[0006] Hypoglycemia may be caused by temporary excess insulin secretion and may also occur in infants who have experienced fetal distress. The cause of abnormal insulin secretion is unknown, but in this case, the condition improves within a few days to months. Hypoglycemia may also occur temporarily when a mother with diabetes has poor blood sugar regulation, but once hypoglycemia is relieved by proper breastfeeding, hypoglycemia will not recur. Another cause is persistent hyperinsulinemia caused by various genetic defects. Studies have reported that causes of hyperinsulinemia caused by genetic defects include mutations in the SUR gene or the Kir6.2 gene on chromosome 11p15.1, increased glucokinase (GK) activity caused by mutations in the GK gene on chromosomes 7p15-p13, and increased ATP levels in pancreatic beta cells caused by activation of glutamate dehydrogenase (GDH) caused by mutations in the GDH gene.

[0007] Bariatric surgery for the treatment of severe obesity complications is divided into laparoscopic adjustable gastric banding (LAGB), Roux-en-Y gastric bypass (RYGB), sleeve gastrectomy (SG), and biliopancreatic diversion (BPD). Dumping syndrome-induced hypoglycemia has been reported as a complication of bariatric surgery.

[0008] GLP-1 receptor antagonists are drugs used to increase appetite (KR 10-2001-0089563 A). Exendin-3 (9-39) is a representative example thereof (Indraneel Banerjee, Mark J. Dunne, Encyclopedia of Endocrine Diseases (Second edition), 2019), and administration of exendin-3(9-39) has been reported to inhibit insulin secretion and exhibit therapeutic effects in acute hypoglycemia (Calabria AC, Li C, Gallagher PR, Stanley CA, De Leon DD. GLP-1 receptor antagonist exendin-(9-39) elevatesfasting blood glucose levels in congenital hyperinsulinism owing toinactivating mutations in the ATP-sensitive K+ channel). Diabetes. 2012;61(10):2585-2591. doi:10.2337 / db12-0166).

[0009] Disclosure Technical Problem Therapeutic agents for congenital hyperinsulinism and hypoglycemia using GLP-1 receptor antagonists are still underdeveloped, and thus there is a need to develop effective therapeutic agents.

[0010] Technical Solution One aspect of the present invention is to provide a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate.

[0011] Another aspect of the present invention is to provide the use of a GLP-1 receptor antagonist analogue, an acylated GLP-1 receptor antagonist analogue conjugate, or a composition containing the analogue or conjugate in the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0012] Still another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, which contains a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate.

[0013] Still another aspect of the present invention is to provide a method for preventing or treating congenital hyperinsulinism or hypoglycemia, which comprises administering the pharmaceutical composition to a subject in need thereof.

[0014] Still another aspect of the present invention is to provide the use of a drug for preventing or treating congenital hyperinsulinism or hypoglycemia, which drug contains a GLP-1 receptor antagonist analogue, an acylated GLP-1 receptor antagonist analogue conjugate, or a composition containing the analogue or conjugate.

[0015] Advantageous Effects The GLP-1 receptor antagonist analogue and the acylated GLP-1 receptor antagonist analogue conjugate of the present invention can act as antagonists by acting on the GLP-1 receptor, thereby producing an effect in the prevention or treatment of congenital hyperinsulinism or hypoglycemia. Description of the Drawings

[0016] Figure 1 Shows the results of measuring blood glucose levels over time in an acylated GLP-1 receptor antagonist analogue conjugate (SEQ ID NO: 17) according to the present invention.

[0017] Figure 2 Shows the results of measuring blood glucose levels in vertical sleeve gastrectomy rats by administering an acylated GLP-1 receptor antagonist analogue conjugate (SEQ ID NO: 17) according to the present invention.

[0018] Figure 3 Shows the results of measuring plasma insulin levels in rats with vertical sleeve gastrectomy by administering the acylated GLP-1 receptor antagonist analogue conjugate (SEQ ID NO: 17) according to the present invention. SUMMARY OF THE INVENTION

[0019] One aspect of the present invention relates to a novel GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue.

[0020] In one embodiment, the GLP-1 receptor antagonist analogue can be represented by the following general formula 1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-E-E-X13-A-X15-R-X17-F-I-X20-W-L-X23-X24-G-G-P-S-S-G-A-P-P-P-S-X36 (General formula 1, SEQ ID NO: 25) Wherein, in 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; and The symbol "-" represents a peptide bond.

[0021] In another embodiment, regarding the GLP-1 receptor antagonist analogue, In general formula 1, X5 is aspartic acid; X6 is leucine or valine; X7 is serine; X8 is lysine or serine; X15 is valine; X23 is lysine or valine; X24 is asparagine or lysine.

[0022] In yet another embodiment, with respect to the GLP-1 receptor antagonist analogs, In 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; and X36 is lysine or an acylated amino acid.

[0023] In the GLP-1 receptor antagonist analogs according to any one of the foregoing embodiments, the acylated amino acid can be any one of the amino acids represented by K(1) to K(4): [K(1)]C20 diacid-γGlu-(AEEA)2-Lys; [K(2)]C18 diacid-γGlu-(AEEA)2-Lys; [K(3)]C16 acid-γGlu-(AEEA)2-Lys; [K(4)]C16 diacid-γGlu-(AEEA)2-Lys.

[0024] .

[0025] In the GLP-1 receptor antagonist analogs according to any one of the foregoing embodiments, the acyl group can be directly or through a linker connected to at least one amino acid of the GLP-1 receptor antagonist analog.

[0026] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the linker may contain (2-(2-aminoethoxy)ethoxy)acetic acid (AEEA).

[0027] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the linker may be γGlu-(AEEA)2.

[0028] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the linker may contain 0 to 3 AEEA molecules, and 0 to 3 γ-glutamic acid molecules may be linked to AEEA.

[0029] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the C-terminus of the GLP-1 receptor antagonist analog may be amidated.

[0030] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the GLP-1 receptor antagonist analog may be acylated with a C1 to C30 straight-chain or branched-chain acyl group containing one or two carboxylic acids.

[0031] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the acyl group may be a C4 to C30 fatty acid or a dicarboxylic acid.

[0032] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the amino acid at the C-terminus or the lysine residue of the GLP-1 receptor antagonist analog may be acylated.

[0033] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the C-terminus of the GLP-1 receptor antagonist analog may be amidated.

[0034] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences selected from SEQ ID NO: 1 to 22.

[0035] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences selected from SEQ ID NO: 3, 7 to 12, 21 and 22.

[0036] In the GLP-1 receptor antagonist analog according to any one of the foregoing embodiments, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences selected from SEQ ID NO: 17, 19, 21 and 22.

[0037] In the GLP-1 receptor antagonist analogs according to any one of the foregoing embodiments, the GLP-1 receptor antagonist analogs may have any one of the following structures (i) to (iv): (i) (ii) (iii) (iv) .

[0038] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, which contains a pharmaceutically effective amount of a GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog.

[0039] In one embodiment, the pharmaceutical composition may further contain a pharmaceutically acceptable carrier.

[0040] In another embodiment, when administered, the pharmaceutical composition can increase the blood glucose level of a subject.

[0041] In the pharmaceutical composition according to any one of the foregoing embodiments, the pharmaceutical composition can reduce the secretion of insulin in a subject when administered.

[0042] In the pharmaceutical composition according to any one of the foregoing embodiments, the hypoglycemia can be post-bariatric hypoglycemia (PBH).

[0043] In the GLP-1 receptor antagonist analogs according to any one of the foregoing embodiments, post-bariatric hypoglycemia may be caused by bariatric surgery.

[0044] Still another aspect of the present invention relates to the use of a GLP-1 receptor antagonist analog, an acylated GLP-1 receptor antagonist analog, or a composition containing the same for preventing or treating congenital hyperinsulinism or hypoglycemia.

[0045] Still another aspect of the present invention relates to the use of a GLP-1 receptor antagonist analog, an acylated GLP-1 receptor antagonist analog, or a composition containing the same in providing a medicament for preventing or treating congenital hyperinsulinism or hypoglycemia. Detailed Embodiments

[0046] In the following text, the details for implementing the present invention will be described. Each description and embodiment disclosed in this application can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this disclosure. In addition, the scope of the present invention is not limited by the following specific descriptions.

[0047] Throughout the description herein, not only the typical one-letter and three-letter codes of natural amino acids are used, but also the three-letter codes generally permitted for other amino acids, such as 2-aminoisobutyric acid (Aib), N-methylglycine (Sar), and α-methylglutamic acid. The amino acids mentioned herein are abbreviated as follows according to the nomenclature rules of IUPAC-IUB.

[0048] 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 One aspect of the present invention relates to a GLP-1 receptor antagonist analogue or a conjugate in which the GLP-1 receptor antagonist analogue is acylated.

[0049] GLP-1 is a hormone secreted in the small intestine stimulated by food intake, which promotes insulin secretion in the pancreas and inhibits glucagon secretion, thereby helping to lower blood glucose concentration. In addition, GLP-1 reduces the digestive action of the gastrointestinal tract by acting as a satiety factor and reduces food intake by delaying the time for digested food to pass through the gastrointestinal tract.

[0050] The blood glucose-regulating and weight-loss effects of GLP-1 are being used in the development of therapeutic agents for diabetes and obesity, and the known GLP-1 receptor agonist exendin-4 is a typical example.

[0051] Native GLP-1 has the following sequence (SEQ ID NO:23): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG As used herein, the term "GLP-1 receptor antagonist" refers to a substance that acts on the GLP-1 receptor to inhibit or weaken GLP-1 activity. Exendin-3(9-39) is a known GLP-1 receptor antagonist (Montrose-Rafizaden et al., 1997,J. Biol. Chem. 272(34):21201-21206).

[0052] The Gila monster exendin-3(9-39) has the following sequence (SEQ ID NO:24): DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS As used herein, the term "GLP-1 receptor antagonist analog" or "analog" refers to a substance that antagonizes the GLP-1 receptor (GLP-1R). A GLP-1 receptor antagonist analog is, but not limited to, a peptide that has at least one difference in amino acid sequence compared to native GLP-1 and can be obtained by altering the native GLP-1 sequence by any one change or a combination of substitutions, additions, deletions, and modifications in a portion of the sequence. A GLP-1 receptor antagonist analog can be non-naturally occurring.

[0053] In addition, such changes for preparing GLP-1 receptor antagonist analogs include all of the following changes: changes using L- or D-amino acids and / or non-natural amino acids; and / or changes resulting from modifying the sequence in its native form, such as changes in side-chain functional groups, intramolecular covalent bonding (e.g., loop formation between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanation, biotinylation, etc. Such changes also include substitution with non-natural compounds.

[0054] A GLP-1 receptor antagonist analog can be obtained by adding one or more amino acids to the amino and / or carboxyl terminus of native GLP-1, but is not limited thereto.

[0055] The amino acids that are substituted or added can be not only the 20 amino acids common in human proteins, but also non-typical or non-naturally occurring amino acids. Commercial sources of non-typical amino acids can include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides including these amino acids and typical peptide sequences can be synthesized and purchased from companies specializing in commercial peptide synthesis, such as the American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0056] According to a specific aspect, the GLP-1 receptor antagonist analog of the present invention can be a GLP-1 receptor antagonist analog represented by, but not limited to, the following general formula 1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-E-E-X13-A-X15-R-X17-F-I-X20-W-L-X23-X24-G-G-P-S-S-G-A-P-P-P-S-X36 (General formula 1, SEQ ID NO: 25) Wherein in 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 acylated amino acid; X24 is asparagine, lysine or acylated amino acid; X36 is cysteine, lysine, acylated amino acid or absent; and The symbol "-" represents a peptide bond.

[0057] In a specific embodiment, regarding the GLP-1 receptor antagonist analog, In General formula 1, X5 is aspartic acid; X6 can be leucine or valine; X7 can be serine; X8 can be lysine or serine; X15 can be valine; X23 can be lysine or valine; X24 can be asparagine or lysine, but not limited thereto.

[0058] The GLP-1 receptor antagonist analogs can include any one of the amino acid sequences selected from SEQ ID NO: 1 to 3, 5, 7 to 12, and 17 to 22. More specifically, it can include any one of the amino acid sequences selected from SEQ ID NO: 3, 7 to 12, 21, and 22, but is not limited thereto.

[0059] In another specific embodiment, regarding the GLP-1 receptor antagonist analogs, In General Formula 1, X1 to X4 may be absent; X5 may be aspartic acid; X6 may be leucine; X7 may be serine; X8 may be serine; X9 may be tyrosine; X10 may be leucine; X13 may be glutamic acid; X15 may be valine; X17 may be leucine; X20 may be glutamic acid; X23 may be lysine; X24 may be asparagine; X36 may be lysine or an acylated amino acid, but is not limited thereto.

[0060] In another embodiment, the GLP-1 receptor antagonist analogs can have any one of the following structures (i) to (iv), but are not limited thereto: (i) (ii) (iii) (iv) .

[0061] Optionally, the GLP-1 receptor antagonist analogs can include any one of the amino acid sequences selected from SEQ ID NO: 17, 19, 21, and 22, but are not limited thereto.

[0062] In one embodiment, the GLP-1 receptor antagonist analog including the amino acid sequence of SEQ ID NO: 17 can have the following structure, but is not limited thereto.

[0063] .

[0064] In one embodiment, the GLP-1 receptor antagonist analog comprising the amino acid sequence of SEQ ID NO:19 may have the following structure, but is not limited thereto.

[0065] 。

[0066] In one embodiment, the GLP-1 receptor antagonist analog comprising the amino acid sequence of SEQ ID NO:21 may have the following structure, but is not limited thereto.

[0067] 。

[0068] 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 thereto.

[0069] 。

[0070] In the present invention, the GLP-1 receptor antagonist analog may be an acylated GLP-1 receptor antagonist analog conjugate having a linked acyl group, but is not limited thereto. In the present invention, the GLP-1 receptor antagonist analog may refer to an acylated GLP-1 receptor antagonist analog and a non-acylated GLP-1 receptor antagonist analog.

[0071] Acylation is known as a method for improving the pharmacokinetic and pharmacodynamic properties of peptide drugs. Peptide drugs have problems in showing efficacy due to enzymatic degradation in vivo. Therefore, the enzyme action site is blocked by peptide acylation through the linkage of a fatty acid to the peptide, thereby enhancing the stability of the peptide drug and prolonging the half-life of the peptide drug. For the purposes of the present invention, the GLP-1 receptor antagonist analog of the present invention may be in an acylated form to prolong the half-life.

[0072] As used herein, the term "acylated GLP-1 receptor antagonist analog conjugate" may be used interchangeably with "acylated GL-1 receptor antagonist analog", "acylated analog", or "conjugate".

[0073] In the acylated GLP-1 receptor antagonist analog conjugate, the acyl group may be linked to the amino acid of the GLP-1 receptor antagonist analog through a linker, where the linker may include [2-(2-aminoethoxy)ethoxy]acetic acid (AEEA), and more specifically, may include an additional linkage of γ-glutamate, but is not limited thereto. Specific examples of the linker may be (γGlu) m -(AEEA) n where m and n may each independently be 0, 1, 2, 3 or larger integers, but are not limited thereto.

[0074] The acyl group can be linked to the amino group, hydroxyl group, mercapto group, carboxyl group, etc. of the amino acid of the GLP-1 receptor antagonist analogue through the amino group, hydroxyl group, mercapto group, etc. of the linker. However, the linker is not limited to a specific type or length, as long as the acyl group can be linked to the GLP-1 receptor antagonist analogue to contribute to structural stability and the extension of the half-life. In addition, the linker can be covalently linked to the acyl group and can be linked to the acyl group once, twice, three times or more than three times, but is not limited thereto.

[0075] The acyl group used in acylation can have a carbon chain of any length or can have a straight chain or a branched chain. Specifically, examples of the chain can include a straight-chain aliphatic chain, a branched-chain aliphatic chain, a chain containing a cycloalkyl moiety, a hydrophobic natural product such as a steroid, an aralkyl chain or an alkyl chain containing an acyl moiety.

[0076] In a specific embodiment, the acylated GLP-1 receptor antagonist analogue can be acylated with a C1-C30 straight-chain or branched-chain acyl group containing one or more, particularly one or two carboxylic acids. As an example, the acyl group can be a fatty acid or a dicarboxylic acid, specifically, a C4-C30 fatty acid or dicarboxylic acid, but is not limited thereto. As a more specific example, the acyl group can be a C16, C18, C20, C22, C24, C26, C28 or C30 fatty acid or dicarboxylic acid. Other examples of the acyl group can include bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid and cholestenic acid, succinic acid or succinic acid derivatives, maleic acid or maleic acid derivatives, etc.

[0077] The acylated GLP-1 receptor antagonist analogue of the present invention can be obtained by linking an acyl group to the GLP-1 receptor antagonist analogue by a method known in the art, or can be prepared by synthesizing a peptide using an acylated amino acid, but is not limited thereto.

[0078] In the present invention, the acylated GLP-1 receptor antagonist analogue can be in a form in which the acyl group is directly linked to the amino acid residue of the GLP-1 receptor antagonist analogue. For example, the linking of the acyl group can be carried out through an ester, thioester or amide bond, but is not limited thereto. Specifically, in the acylated GLP-1 receptor antagonist analogue, the amino acid residue having an amine, hydroxyl or mercapto group can be acylated. For example, in the acylated GLP-1 receptor antagonist analogue, the amino acid at the C-terminus or the lysine residue can be acylated.

[0079] In the present invention, the acylated amino acid of the acylated GLP-1 receptor antagonist analogue can include, but is not limited to, any one of the following amino acids represented by K(1) to K(4): [K(1)]C20 diacid-γGlu-(AEEA)2-Lys [K(2)]C18 diacid-γGlu-(AEEA)2-Lys [K(3)]C16 acid-γGlu-(AEEA)2-Lys [K(4)]C16 diacid-γGlu-(AEEA)2-Lys 。

[0080] The acylated GLP-1 receptor antagonist analog conjugate of the present invention may contain an acylated amino acid at position 8, 23, 24 or 36, but is not limited thereto.

[0081] According to a specific aspect, the GLP-1 receptor antagonist analog according to the present invention may include any one of the amino acid sequences of SEQ ID NO: 1 to 22, and specifically, may (substantially) consist of an amino acid sequence selected from SEQ ID NO: 1 to 22, but is not limited thereto.

[0082] Specifically, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences of SEQ ID NO: 1 to 12, and specifically, may (substantially) consist of an amino acid sequence selected from SEQ ID NO: 1 to 12, but is not limited thereto.

[0083] More specifically, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences selected from SEQ ID NO: 3, 7 to 12, 21 and 22, and specifically, may (substantially) consist of a sequence selected from SEQ ID NO: 3, 7 to 12, 21 and 22, but is not limited thereto.

[0084] More specifically, the GLP-1 receptor antagonist analog may include any one of the amino acid sequences selected from SEQ ID NO: 17, 19, 21 and 22, and specifically, may (substantially) consist of the amino acid sequences selected from SEQ ID NO: 17, 19, 21 and 22, but is not limited thereto.

[0085] According to another specific aspect, the acylated GLP-1 receptor antagonist analog conjugate according to the present invention may include any one of the sequences of SEQ ID NO: 13 to 22, specifically, may (substantially) consist of an amino acid sequence selected from SEQ ID NO: 13 to 22, but is not limited thereto.

[0086] Although this text describes it as "a peptide consisting of a specific serial number", this wording does not exclude the addition of nonsense sequences, naturally occurring mutations or their silent mutations upstream or downstream of the amino acid sequence of the corresponding serial number, as long as the peptide has an equivalent or corresponding activity to the peptide consisting of the amino acid sequence of the corresponding serial number, and it is obvious that the peptide with such sequence addition or mutation belongs to the scope of the present invention.

[0087] In addition, the GLP-1 receptor antagonist analogs of the present invention may include amino acid sequences having at least 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% homology or identity with the amino acid sequences of SEQ ID NOs: 1 to 22, but are not limited thereto, as long as the analog can act on the GLP-1 receptor to antagonize the GLP-1 receptor.

[0088] As used herein, the terms "homology" or "identity" refer to the degree of correlation between two given amino acid sequences or nucleotide sequences, and this term can be expressed as a percentage.

[0089] The sequence homology or identity of conserved polynucleotides can be determined by standard alignment algorithms and the default gap penalties established by the programs to be used can be used together. Basically, homologous or identical sequences can hybridize along their entire sequence or a part thereof under medium or highly stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons taking into account the codon degeneracy in polynucleotides.

[0090] The two terms homology and identity can often be used interchangeably.

[0091] Homology, similarity or identity between any two peptide sequences can be determined by, for example, known computer algorithms such as the "FASTA" program, using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Optionally, these can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is available in the European Molecular Biology Open Software Suite (EMBOSS) package (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (including 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 [CARILLOETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity or identity can be determined using BLAST of the National Center for Biotechnology Information database or ClustalW.

[0092] Homology, similarity or identity between peptides can be determined by comparing sequence information using the GAP computer program, such as Needleman et al., (1970), J Mol Biol. 48:443, as known in Smith and Waterman, Adv.Appl. Math (1981) 2:482. Briefly, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of symbols (i.e., nucleotides or amino acids) in a similar alignment by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include: (1) the unitary comparison matrix of Gribskov et al. (1986) Nucl. Acids Res.14:6745 (containing an identity value of 1 and a non-identity value of 0) and the weighted comparison matrix (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), as disclosed in Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation, pp.353-358 (1979); (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 opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, the terms "homology" or "identity" as used herein denote the relatedness between sequences.

[0093] Compared with the activity (100%) of exendin-3(9-39), a known GLP-1 receptor antagonist, the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention can exhibit an activity of about 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.

[0094] The term "about" as used herein refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., including all values equal to or similar to the range described after this term, but not limited thereto.

[0095] The activity of a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate can be measured by methods known in the art, and the measurement is not limited to a specific method. For example, the antagonistic effect of the GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue of the present invention can be analyzed by measuring the concentration of cAMP, wherein the production of cAMP is inhibited by the GLP-1 receptor antagonist analogue.

[0096] In addition, the GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue conjugate according to the present invention may be in a form in which the N-terminus and / or C-terminus of the peptide is not modified. However, those variants in which the N-terminus and / or C-terminus of the peptide is chemically modified or protected by an organic group, or an amino acid is added to the end of the peptide to protect it from proteases in vivo while increasing its stability may also fall within the scope of the present invention.

[0097] In particular, chemically synthesized peptides have charged N- and C-termini. Therefore, in order to eliminate these charges, the N-terminus can be acetylated and / or the C-terminus can be amidated, but it is not particularly limited thereto.

[0098] In the GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue conjugate according to the present invention, the C-terminus can be amidated, or the C-terminus can be both amidated and acylated, but it is not limited thereto.

[0099] Unless otherwise specified herein, the detailed description or claims regarding the GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue conjugate according to the present invention can also be applied to a range that includes not only the corresponding analogue or conjugate, but also salts (e.g., pharmaceutically acceptable salts of peptides) or solvates thereof of the corresponding analogue or conjugate. Therefore, the corresponding descriptions herein also apply to its specific salts, its specific solvates, and specific solvates of its specific salts. For example, these salts can be in the form of any pharmaceutically acceptable salt. The type of salt is not particularly limited. However, the salt is preferably in a form that is safe and effective for a subject (e.g., a mammal), but it is not particularly limited thereto.

[0100] The term "pharmaceutically acceptable" refers to a substance that can be effectively used for the desired purpose without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of medical and pharmaceutical decisions.

[0101] As used herein, the term "pharmaceutically acceptable salt" refers to salts derived from pharmaceutically acceptable inorganic acids, organic acids or bases. Suitable examples of acids can include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Suitable examples of salts derived from bases can include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, ammonium, etc.

[0102] As used herein, the term "solvate" refers to a complex formed by a peptide, conjugate or salt thereof of the present invention with solvent molecules.

[0103] Analogues of the present invention can be synthesized by methods well known in the art according to their length, such as an automated peptide synthesizer, or can be produced by genetic engineering techniques.

[0104] Specifically, analogues of the present invention can be prepared by standard synthetic methods, recombinant expression systems or any other methods known in the art. Thus, GLP-1 receptor antagonist analogues or acylated GLP-1 receptor antagonist analogue conjugates according to the present invention can be synthesized by a variety of methods including the following methods.

[0105] Method (a): Synthesize the peptide stepwise or by fragment assembly through a solid-phase or liquid-phase process, and isolate and purify the final peptide product; Method (b): Express a nucleic acid construct encoding the peptide in a host cell and collect the expression product from the host cell culture; Method (c): Perform cell-free in vitro expression of a nucleic acid construct encoding the peptide and collect the expression product therefrom; or Method for obtaining peptide fragments by any combination of (a), (b) and (c): ligate the fragments to obtain the peptide, and then collect the corresponding peptide.

[0106] The above description can be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0107] Another aspect of the present invention provides a composition containing a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate according to the present invention.

[0108] The GLP-1 receptor antagonist analogue or the acylated GLP-1 receptor antagonist analogue conjugate is as described above.

[0109] Specifically, the composition can be a pharmaceutical composition, and more specifically, the composition can be used for preventing or treating congenital hyperinsulinism or hypoglycemia.

[0110] A specific aspect of the present invention relates to a pharmaceutical composition for preventing or treating congenital hyperinsulinism or hypoglycemia, which contains a pharmaceutically effective amount of a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate.

[0111] Containing a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate in a pharmaceutically effective amount may mean the degree at which targeted drug activity (e.g., preventing, alleviating, or treating congenital hyperinsulinism or hypoglycemia) can be obtained in the subject to be administered, or the level at which toxicity or side effects are absent or slightly present, up to a pharmaceutically acceptable level, but not limited thereto. The effective amount of the drug can be determined by comprehensively considering the number of administrations, the patient, the formulation, etc.

[0112] As used herein, the term "prevention" refers to any action of inhibiting or delaying the development of a target disease (e.g., congenital hyperinsulinism or hypoglycemia) by administering a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate or a composition containing the analogue or conjugate, while the term "treatment" refers to any action of alleviating or favorably altering the symptoms of a target disease (e.g., congenital hyperinsulinism or hypoglycemia) by administering a GLP-1 receptor antagonist analogue or an acylated GLP-1 receptor antagonist analogue conjugate or a combination containing the analogue or conjugate.

[0113] As used herein, the term "administration" refers to introducing a predetermined substance into a patient by any appropriate method, and the administration route of the composition can include but is not limited to any typical route through which the composition can reach the target in the body. For example, the administration route can be intraperitoneal administration, intravenous administration, intramuscular administration, or subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, and intralung administration, rectal administration, etc.

[0114] The congenital hyperinsulinism of the present invention is one of the etiologies leading to severe and persistent hypoglycemia in newborns and children. Congenital hyperinsulinism may be caused by a temporary increase in insulin secretion in low birth weight infants or infants of diabetic mothers, pancreatic cell dysfunction caused by gene mutations, etc.

[0115] The hypoglycemia of the present invention refers to a situation where the blood glucose level is lower than that of normal people. This term generally refers to a situation where the blood glucose level is 50 mg / dL or lower, but is not particularly limited thereto. In the present invention, hypoglycemia includes acute hypoglycemia and chronic hypoglycemia.

[0116] Symptoms of hypoglycemia include lack of energy, body tremors, pale complexion, cold sweats, dizziness, restlessness, anxiety, palpitations, hunger, headache, fatigue, etc. If hypoglycemia persists for a long time, it may cause convulsions or seizures and lead to shock, which may result in fainting.

[0117] Specifically, the hypoglycemia in the present invention can be post-bariatric hypoglycemia (PBH), and more specifically, hypoglycemia caused by bariatric surgery, but is not limited thereto.

[0118] "Post-bariatric hypoglycemia" or "hypoglycemia caused by bariatric surgery" refers to hypoglycemia that occurs in a patient after undergoing bariatric surgery, such as sleeve gastrectomy, duodenal switch, and Roux-en-Y gastric bypass, and may also be caused by dumping syndrome, one of the complications.

[0119] Currently, there is no clear treatment method for post-bariatric hypoglycemia or hypoglycemia caused by bariatric surgery. All researchers believe that changing diet and lifestyle is the most important treatment method. The principles of diet therapy include reducing the amount of food ingested at one time and chewing small amounts of high-protein, high-fat, low-carbohydrate, and low-fluid foods 5-6 times. If hypoglycemia, abdominal pain, or convulsions occur due to dumping syndrome, the symptoms can be controlled by administering drugs such as sedatives, anticonvulsants, glucose injection solutions, sedatives, or autonomic nervous system blockers.

[0120] The GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention antagonizes the GLP-1 receptor, and thus can increase the blood glucose level in the body and / or reduce the secretion of insulin, thereby showing a preventive or therapeutic effect on congenital hyperinsulinism or hypoglycemia, but is not limited thereto.

[0121] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent. As used herein, the term "pharmaceutically acceptable" refers to having characteristics with a sufficient amount to show a therapeutic effect without causing adverse reactions, and those skilled in the art can easily determine it based on factors well-known in the medical field, such as the type of disease, the age, weight, health status, and gender of the patient, the drug sensitivity of the patient, the administration route, the administration method, the administration frequency, the treatment duration, the combination or co-use of drugs.

[0122] The pharmaceutical composition containing the GLP-1 receptor antagonist analog or the acylated GLP-1 receptor antagonist analog conjugate of the present invention may further comprise a pharmaceutically acceptable carrier. Regarding the carrier, although there is no particular limitation, binders, lubricants, disintegrants, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. can be used for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed for injection. In addition, alkalis, excipients, lubricants, preservatives, etc. can be used for topical administration.

[0123] The formulations of the compositions of the present invention can be prepared in various ways by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the compositions of the present invention can be formulated into forms such as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, the compositions can be formulated into single-dose ampoules or multi-dose forms. The compositions can be formulated into forms such as solutions, suspensions, tablets, pills, capsules or sustained-release formulations.

[0124] Meanwhile, examples of carriers, excipients and diluents suitable for preparation can include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, mineral oil, etc. The composition can also contain fillers, anticoagulants, lubricants, humectants, flavoring agents, preservatives, etc.

[0125] In addition, the pharmaceutical composition of the present invention can have any one of the formulations selected from tablets, pills, powders, granules, capsules, suspensions, oral liquid preparations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, freeze-dried preparations and suppositories.

[0126] The composition can be formulated into a unit dosage form preparation suitable for administration into a patient's body according to conventional methods in the pharmaceutical field, and can be specifically formulated into a preparation for peptide drugs, and can be administered by an oral administration route or a parenteral administration route, including percutaneous, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transpulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, intravaginal or rectal routes, by the administration methods commonly used in the art, but the formulation method and the administration method are not limited thereto.

[0127] The GLP-1 receptor antagonist analogs or acylated GLP-1 receptor antagonist analog conjugate of the present invention can be used by mixing with various pharmaceutically acceptable carriers such as normal saline or organic solvents, and for improving stability or absorption, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as drugs.

[0128] The dosage and the number of administrations of the pharmaceutical composition of the present invention are determined according to the type of the drug as the active ingredient and several related factors, such as the disease to be treated, the administration route, the age, sex and body weight of the patient, and the severity of the disease.

[0129] Although not particularly limited, the pharmaceutical composition of the present invention may contain the ingredient (active ingredient) in an amount of 0.01 to 99% (w / v).

[0130] The total effective amount of the composition of the present invention can be administered to a patient in a single dose, or can be administered in multiple doses over a long term by a divided treatment regimen. In the pharmaceutical composition of the present invention, the content of the active ingredient can vary according to the severity of the disease. Specifically, the preferred total dose of the GLP-1 receptor antagonist analog or its acylated GLP-1 receptor antagonist analog conjugate of the present invention can be about 0.3 to 0.9 mg per 1 kg of the patient's body weight per day. The dose can be determined based on the mass value of the GLP-1 receptor antagonist analog or the mass value of the GLP-1 receptor antagonist analog (excluding the fatty acid part, i.e., the total mass value of only the polypeptide part) in the acylated GLP-1 receptor antagonist analog conjugate. However, regarding the dose of the GLP-1 receptor antagonist analog or the acylated GLP-1 receptor antagonist analog conjugate, the effective dose for the patient is determined by considering various factors, including the patient's age, body weight, health condition and sex, the severity of the disease, diet and excretion rate, and the administration route and the number of treatments of the pharmaceutical composition. Therefore, considering these factors, those skilled in the art will be able to determine an appropriate effective dose according to the specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its formulation, administration route and administration method, as long as the pharmaceutical composition can exhibit the beneficial effects of the present invention.

[0131] Compared with other drugs, the pharmaceutical composition of the present invention can exhibit an excellent duration of in vivo efficacy and potency, and allows for a reduction in the number and frequency of administrations, but is not particularly limited thereto.

[0132] Another aspect of the present invention provides a method for preventing or treating congenital hyperinsulinism or hypoglycemia, the method comprising administering to a subject a GLP-1 receptor antagonist analog, an acylated GLP-1 receptor antagonist analog conjugate or a composition containing the analog or conjugate.

[0133] GLP-1 receptor antagonist analogs or acylated GLP-1 receptor antagonist analog conjugates, compositions containing the same, congenital hyperinsulinism, hypoglycemia, prevention and treatment are as described above.

[0134] The subjects described herein refer to subjects suspected of having congenital hyperinsulinism or hypoglycemia, wherein subjects suspected of having congenital hyperinsulinism or hypoglycemia refer to mammals, including humans, rats, livestock, etc., that have or are at risk of developing the corresponding diseases, but including but not limited to any subject as long as the subject can be treated with a GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog conjugate or a composition containing the analog. A pharmaceutical composition containing the GLP-1 receptor antagonist analog or acylated GLP-1 receptor antagonist analog conjugate of the present invention can be administered to a subject suspected of having congenital hyperinsulinism or hypoglycemia, and thus can effectively treat the subject. Congenital hyperinsulinism or hypoglycemia is as described above.

[0135] The method of the present invention may include administering a pharmaceutically effective amount of a peptide-containing pharmaceutical composition. The appropriate total daily dose can be determined by a doctor within the scope of accurate medical judgment and can be administered in one or more divided doses. For the purposes of the present invention, the specific therapeutically effective dose for a particular patient can preferably be applied differently according to various factors well known in the medical field, including the type and degree of response to be achieved, the particular composition, including whether other preparations are used occasionally in combination, the patient's age, weight, general health, sex and diet, the time of administration, the route of administration, the secretion rate of the composition, the duration of treatment, other drugs used in combination with or simultaneously with the composition of the present invention, and similar factors well known in the medical field.

[0136] In the method of the present invention, a GLP-1 receptor antagonist analog or an acylated GLP-1 receptor antagonist analog conjugate or a composition containing the same can be administered by typical routes to reach the target in vivo, and examples of the route of administration can include intraperitoneal administration, intravenous administration, intramuscular administration or subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0137] Yet another aspect of the present invention provides the use of a GLP-1 receptor antagonist analog, an acylated GLP-1 receptor antagonist analog conjugate or a composition for the prevention or treatment of congenital hyperinsulinism or hypoglycemia.

[0138] GLP-1 receptor antagonist analogs, acylated GLP-1 receptor antagonist analog conjugates and compositions, congenital hyperinsulinism and hypoglycemia are as described above.

[0139] Yet another aspect of the present invention provides the use of a GLP-1 receptor antagonist analogue, an acylated GLP-1 receptor antagonist analogue conjugate or a composition in the preparation of a medicament (or pharmaceutical composition) for preventing or treating congenital hyperinsulinism or hypoglycemia.

[0140] The GLP-1 receptor antagonist analogue, the acylated GLP-1 receptor antagonist analogue conjugate and the composition, congenital hyperinsulinism and hypoglycemia are as described above.

[0141] Unless the context clearly requires otherwise, expressions such as "comprising", "including", "containing", etc. shall be understood to include the stated integer or group of integers, but not to exclude other integers or sets of integers.

[0142] Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are given to specifically illustrate the present invention, and the scope of the present invention is not limited to these exemplary embodiments.

[0143] Example 1: Preparation of GLP-1 Receptor (GLP-1R) Antagonist Analogs and Acylated GLP-1 Receptor (GLP-1R) Antagonist Analog Conjugates SEQ ID NO Regarding the synthesis of the GLP-1R antagonist analogue, an automatic peptide synthesizer (Symphony X, Gyros Protein Tech) using solid-phase synthesis was employed. Rink amide resin was used for C-terminal amidation. The corresponding amino acids were synthesized sequentially from the C-terminus to the N-terminus.

[0144] Fmoc (9H-fluoren-9-ylmethoxycarbonyl)-protected amino acids (4 equivalents relative to the peptide resin), HOBt (1-hydroxybenzotriazole, 4 equivalents relative to the peptide resin) and DIC (diisopropylcarbodiimide, 8 equivalents relative to the peptide resin) were used for the sequential ligation of the amino acids.

[0145] Regarding the acylated GLP-1R antagonist analogue conjugate, peptides were synthesized by the above method using acylated amino acids K(1) to K(4).

[0146] [K(1)]C20 diacid-γGlu-(AEEA)2-Lys [K(2)]C18 diacid-γGlu-(AEEA)2-Lys [K(3)]C16 acid-γGlu-(AEEA)2-Lys [K(4)]C16 diacid-γGlu-(AEEA)2-Lys 。

[0147] The Fmoc protecting group was removed by adding 8 mL of 20% piperidine / DMF (2 x 5 minutes) to the reaction vessel containing the resin in an automatic synthesizer. To remove residual impurities, it was washed with 12 mL of DMF after each step (6 x 10 seconds). After synthesis was completed, the N-terminus was acetylated with 6 mL of 34% acetic anhydride / DMF and 3.5 mL of 14% N,N-diisopropyl / DMF. When the peptide was cleaved from the resin, the unremoved protecting groups were deprotected together.

[0148] The sequences of the GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugate prepared by this method are shown in Table 1 below.

[0149] Table 1 Amino Acid Sequence TFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 1 TSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 2 DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS 3 DLSAQMEEEAVRLFIEWLKNGGPSSGAPPPS 4 DLSSQMEEEAVRLFIEWLKNGGPSSGAPPPS 5 QMEEEAVRLFIEWLKNGGPSSGAPPPS 6 TSDVSSYLEEEAVRLFIAWLVKGGPSSGAPPPS 7 DVSSYLEEEAVRLFIAWLVKGGPSSGAPPPS 8 DVSSYLEEQAVREFIAWLVKGGPSSGAPPPS 9 DLSSYLEEEAVRLFIEWLKNGGPSSGAPPPS 10 DVSSYLEEEAVRLFIEWLKNGGPSSGAPPPS 11 DVSSYLEEEAVRLFIAWLVKGGPSSGAPPPSC 12 Example 2: In Vitro Activity of GLP-1 Receptor (GLP-1R) Antagonist Analogs and Acylated GLP-1R Receptor (GLP-1R) Antagonist Analog Conjugates 13 <![CDATA[DLSSYLEEEAVRLFIEWL K(1) NGGPSSGAPPPS]]> 14 <![CDATA[DLSSYLEEEAARLFIEWL K(2) NGGPSSGAPPPS]]> 15 <![CDATA[DVSSYLEEEAVRLFIAWLV K(2) GGPSSGAPPPS]]> 16 <![CDATA[DVSSYLEEEAARLFIAWLV K(2) GGPSSGAPPPS]]> 17 <![CDATA[DLSSYLEEEAVRLFIEWLKNGGPSSGAPPPS K(2) > 18 <![CDATA[DVSSYLEEEAVRLFIAWLVKGGPSSGAPPPS K(2) > 19 <![CDATA[DLSSYLEEEAVRLFIEWLKNGGPSSGAPPPS K(1) > 20 <![CDATA[DVSSYLEEEAVRLFIAWLVKGGPSSGAPPPS K(1) > 21 <![CDATA[DLSSYLEEEAVRLFIEWLKNGGPSSGAPPPS K(3) > 22 <![CDATA[DLSSYLEEEAVRLFIEWLKNGGPSSGAPPPS K(4) > In Table 1, K(1) to K(4) respectively indicate that the amino acids at the corresponding sites are acylated amino acids with the above structure.

[0150] The synthesized GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates were purified by reverse-phase chromatography. The purity of the synthesized peptides was checked by analytical liquid chromatography (RP-HPLC), and a purity of 90% or higher was determined to be a suitable level for testing. In addition, the molecular weight and information of the peptides were identified using liquid chromatography / mass spectrometry (LC / MS).

[0151] The synthesized peptides were stored at -20 °C until used for testing.

[0152] Ultra SEQ ID NO To determine the activity of the GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates, the in vitro cell activity was measured using a cell line transfected with human GLP-1R (hGLP-1R). Each cell line was obtained by transfecting Chinese hamster ovary (CHO) cells to express human GLP-1R and was suitable for determining the activity of GLP-1R antagonists.

[0153] To determine the activity of GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugate, human GLP-1 was diluted from 9000 pM to 0.15 pM in 3-fold dilutions. To identify antagonism, reference substance [Exendin-3(9-39) amide (Tocris Bioscience, UK, catalog number 2081), SEQ ID No:24], GLP-1R antagonist analogs (SEQ ID No:1 to 12) and acylated GLP-1R antagonist analog conjugates (SEQ ID NO:13 to 22) were serially diluted from 9000 pM to 0.15 pM in 3-fold dilutions on a plate, with 0.1 nM GLP-1 added. CHO cells expressing human GLP-1R were cultured in a 384-well plate for 24 hours. Thereafter, the culture was removed, 10 μL of each serial dilution was added to the plate, and then incubated at room temperature for 30 minutes. Then, 5 μL of Eu-cAMP tracer was added, followed by 5 μL of buffer containing cAMP antibody, and then incubated at room temperature for 60 minutes. After the reaction was completed, the cell lysate was applied to the LANCE In Vitro Activity Compared with Control Substances (%) cAMP kit (PerkinElmer, USA), and the half-maximal inhibitory concentration (IC 50 ) was calculated by the degree of inhibition of cAMP by GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates, and then compared with each other. As a result, the relative activities compared with the reference substance are shown in Table 2 below.

[0154] Table 2 Relative activity percentage of GLP-1R analogs and acylated analog conjugates Control Substance (SEQ ID NO: 24) SEQ ID NO: 1 SEQ ID NO: 2 100 SEQ ID NO: 3 69.1 SEQ ID NO: 4 69.8 SEQ ID NO: 5 134.7 SEQ ID NO: 6 74.3 SEQ ID NO: 7 85.0 SEQ ID NO: 8 13.1 SEQ ID NO: 9 195.5 SEQ ID NO: 10 238.5 SEQ ID NO: 11 188.6 SEQ ID NO: 12 249.8 SEQ ID NO: 13 166.1 SEQ ID NO: 14 189.3 SEQ ID NO: 15 4.8 SEQ ID NO: 16 1.7 SEQ ID NO: 17 14.3 SEQ ID NO: 18 13.5 SEQ ID NO: 19 91.8 SEQ ID NO: 20 19.8 SEQ ID NO: 21 33.6 SEQ ID NO: 22 15.1 Example 3: In Vitro Activity of Acylated GLP-1 Receptor (GLP-1R) Antagonist Analog Conjugates 380.1 Example 4: Blood Glucose Regulation of GLP-1 Receptor (GLP-1R) Antagonist Analog Conjugate (SEQ ID NO:17) 251.9 The test results confirmed that the GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugates prepared in Example 1 were able to act on the human GLP-1 receptor (GLP-1R) to antagonize the GLP-1 receptor.

[0155] Figure 1 To determine the activity of the acylated GLP-1R antagonist analog conjugate, in vitro cell activity was measured using cell lines transfected with human (hGLP-1R) or mouse (mGLP-1R) GLP-1R. Each cell line was obtained by transfecting Chinese hamster ovary (CHO) cells to express human and mouse GLP-1R respectively, and was suitable for determining the activity of GLP-1R antagonists.

[0156] To determine the activity of the acylated GLP-1R antagonist analog conjugates, human GLP-1 was diluted from 9000 pM to 0.15 pM in 3-fold dilutions. To identify the antagonism, the reference substance [Exendin-3(9-39) amide (Tocris Bioscience, UK, catalog number 2081), SEQ ID NO:24] and the acylated GLP-1R antagonist analog conjugates (SEQ ID No:17, 19, 21, and 22) were serially diluted from 9000 pM to 0.15 pM in 3-fold dilutions on a plate, with 0.1 M GLP-1 added.

[0157] The acylated GLP-1R antagonist analog conjugates of SEQ ID NO:17, 19, 21, and 22 used in this example have the following structures (i) to (iv), respectively.

[0158] (i) (ii) (iii) (iv) .

[0159] In addition, to identify the effect of the fatty acid with binding affinity for albumin, the analog conjugates were diluted and prepared in a buffer containing 1% human serum albumin and mouse serum albumin. CHO cells expressing human GLP-1R or mouse GLP-1R were cultured in a 384-well plate for 24 hours. Thereafter, the culture was removed, 10 μL of each serially diluted substance was added to the plate, and then incubated at room temperature for 30 minutes. Then, 5 μL of Eu-cAMP tracer was added, followed by 5 μL of buffer containing cAMP antibody, and then incubated at room temperature for 60 minutes. After the reaction was completed, the cell lysate was applied to the LANCE Ultra cAMP kit (PerkinElmer, USA), and the half-maximal inhibitory concentration (IC 50 ) was calculated by the degree of inhibition of cAMP by the GLP-1R antagonist analogs and the acylated GLP-1R antagonist analog conjugates, and then compared with each other. As a result, the relative activities compared with the reference substance are shown in Table 3 below.

[0160] Table 3 Relative activity ratio of the acylated GLP-1R antagonist analog conjugates The GLP-1R antagonist analogs and acylated GLP-1R antagonist analog conjugate prepared in Example 1 can act on human and mouse GLP-1R receptors to antagonize the receptors. In particular, as clearly stated in Table 3, in terms of potency, among the acylated GLP-1R antagonist analog conjugates, SEQ ID NO: 21 and 22 with C16 fatty acids showed excellent activity as antagonists compared to the reference substances, followed by SEQ ID NO: 17 with C18 fatty acid, and then SEQ ID NO: 19 with C20 fatty acid.

[0161] ​ ​ To study 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. The experiment was conducted on male mice at 8 weeks of age. During the study, five mice were housed in each group and had free access to water. The lights were turned off from 6:00 p.m. to 6:00 a.m.

[0162] The experimental groups were designated as Group 1: excipient (subcutaneous administration) - control slurry (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), and Group 4: GLP-1R antagonist analog conjugate (SEQ ID NO: 17) 7380 μg / kg (2123.8 nmol / kg, subcutaneous administration). Groups 1, 3, and 4 completed drug administration 24 hours before the oral glucose tolerance test (OGTT), and Group 2 completed administration 30 minutes before the oral glucose tolerance test. In particular, the dose is expressed as a value based on the value obtained by subtracting the mass of the fatty acid moiety from the total mass of the acylated GLP-1 receptor antagonist analog conjugate, that is, the value obtained by simply adding the masses of each polypeptide moiety.

[0163] For the OGTT, all groups were orally administered 2 g / kg glucose once after a 4-hour fast, and then blood samples were continuously collected from the tail vein at specific time intervals (0, 15, 30, 60, and 120 minutes), and the blood glucose levels were measured using a OneTouch blood glucose meter (OneTouch Select®, LifeScan, USA). Unpaired t-tests were used for statistical analysis to compare the control group and the experimental groups ( # ~ ### p < 0.05 to 0.001).

[0164] The results confirmed that, compared with the control group, the GLP-1R antagonist analogue conjugate of SEQ ID NO:17 increased the blood glucose level in vivo at all doses ( ​ ). This indicates that the GLP-1R antagonist analogue or acylated GLP-1R antagonist analogue conjugate according to the present invention can show an effect in the treatment of congenital hyperinsulinism through its blood glucose-regulating ability.

[0165] Example 5: Glycemic regulation of GLP-1 receptor (GLP-1R) antagonist analog conjugate (SEQ ID NO: 17) in a rat model of postprandial hypoglycemia (PBH) induced by vertical sleeve gastrectomy (VSG) Figure 2 A PBH rat model was established by performing sham surgery or VSG surgery on SD rats. The rats were fed a high-fat diet (HFD) for 19 weeks and randomly divided into two groups of 10 animals each (a vehicle control group and a GLP-1R antagonist analogue conjugate administration group). Four hours after subcutaneous administration of the vehicle and the GLP-1R antagonist analogue conjugate (SEQ ID NO:17), a mixed meal tolerance test (MMTT) was performed at a dose of 3720 μg / kg (1070.5 nmol / kg). For the MMTT, all groups orally received 2 mL of a mixed meal (Ensure Plus®, Abbott, USA) once after a 5-hour fast, and then blood samples were continuously collected at specific time intervals (0, 15, 30, 45, 60, and 120 minutes) to measure the blood glucose level (GDoctor, Allmedicus Co., Korea). Statistical analysis was performed using one-way ANOVA to compare the vehicle group (control group) and the test group in VSG rats (*~***p<0.05~0.001). In particular, the dose is expressed as a value based on the value obtained by subtracting the mass of the fatty acid moiety from the total mass of the acylated GLP-1 receptor antagonist analogue conjugate, that is, the value obtained by simply adding the masses of each polypeptide moiety.

[0166] The results confirmed that, compared with PBH rats administered the vehicle, the GLP-1R antagonist analogue conjugate of SEQ ID NO:17 significantly increased the blood glucose level in vivo ( Figure 3 ).

[0167] Furthermore, 15 minutes after the MMTT, plasma was isolated from the blood samples, and the insulin secretion level was measured using a rat insulin ELISA kit (Multi-Species GLP-1 Total ELISA, Merck Millipore, USA). The results confirmed that, compared with the vehicle control group, the GLP-1R antagonist analogue conjugate of SEQ ID NO:17 showed a 47% reduction in insulin secretion ( ​) This indicates that the GLP-1R antagonist analog or acylated GLP-1R antagonist analog conjugate according to the present invention can play a role in treating hypoglycemia, especially post-weight loss hypoglycemia, through its blood glucose regulating ability.

[0168] It is confirmed from the above examples that the GLP-1 receptor antagonist analog and acylated GLP-1 receptor antagonist analog conjugate prepared in the present invention can not only act as an antagonist of the GLP-1 receptor, but also, based on these activities, be used as a useful therapeutic agent for congenital hyperinsulinism, hypoglycemia, especially post-weight loss hypoglycemia.

[0169] Although the present invention has been described with reference to specific exemplary embodiments, those skilled in the art to which the present invention pertains can understand that the present invention can be embodied in other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the above exemplary embodiments should be construed as being exemplary and not limiting the present disclosure. The scope of the present invention should be construed such that the appended claims, rather than the meaning and scope of the detailed description, and all variations or modifications derived from equivalent concepts fall within the scope of protection of the present invention.

Claims

1. A GLP-1 receptor antagonist analogue represented by the following general formula 1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-E-E-X13-A-X15-R-X17-F-I-X20-W-L-X23-X24-G-G-P-S-S-G-A-P-P-P-S-X36 (General formula 1, SEQ ID NO: 25) Among them, In 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; and The symbol "-" represents a peptide bond.

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

3. The GLP-1 receptor antagonist analogue according to claim 1, wherein, In 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; and 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 K(1) to K(4): [K(1)] C20 diacid-γGlu-(AEEA)2-Lys; [K(2)] C18 diacid-γGlu-(AEEA)2-Lys; [K(3)] C16 acid-γGlu-(AEEA)2-Lys; [K(4)] C16 diacid-γGlu-(AEEA)2-Lys 。 5. The GLP-1 receptor antagonist analogue according to claim 1, wherein the acyl group is linked to at least one amino acid of the GLP-1 receptor antagonist analogue through a linker containing (2-(2-aminoethoxy)ethoxy)acetic acid (AEEA).

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

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

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

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

10. 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, wherein the GLP-1 receptor antagonist analogue comprises any one of the amino acid sequences selected from SEQ ID NO: 1 to 22.

12. The GLP-1 receptor antagonist analogue according to claim 1, wherein the GLP-1 receptor antagonist analogue comprises any one of the amino acid sequences selected from SEQ ID NO: 3, 7 to 12, 21 and 22.

13. The GLP-1 receptor antagonist analogue according to claim 1, wherein the GLP-1 receptor antagonist analogue comprises any one of the amino acid sequences selected from SEQ ID NO: 17, 19, 21 and 22.

14. The GLP-1 receptor antagonist analogue according to claim 1, wherein the GLP-1 receptor antagonist analogue 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, which contains the GLP-1 receptor antagonist analogue according to any one of claims 1 to 14.

16. The pharmaceutical composition according to claim 15, wherein the hypoglycemia is post-bariatric hypoglycemia (PBH).

17. The pharmaceutical composition according to claim 16, wherein the post-bariatric hypoglycemia is caused by bariatric surgery.

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