Glucagon-like peptide-1 receptor antagonist

Abstract

Provided herein are GLP-1 receptor antagonist peptides and pharmaceutical compositions for treating hypoglycemia. Also provided herein are methods of treating atypical hypoglycemia in patients with hyperinsulinemia, including patients who have become hyperinsulinemic after bariatric surgery.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 395,783, filed on August 5, 2022, the disclosure of which is hereby expressly incorporated herein by reference. Incorporation by Reference of Electronically Submitted Materials This application includes a Sequence Listing submitted electronically in XML file format, which is hereby incorporated by reference in its entirety. A copy of the XML created on August 1, 2023, is named 32993 - 392613_SL.xml and is 281 kilobytes in size.

Background Art

[0002] Glucagon - like peptide - 1 (GLP - 1) plays an important role in regulating human blood glucose levels. Its actions include stimulating insulin synthesis and secretion, inhibiting glucagon secretion, and inhibiting food intake. Normally, the body maintains the concentration of glucose in the blood within the range of about 70 - 110 milligrams per deciliter (mg / dL), or 3.9 - 6.1 millimoles per liter (mmol / L) per liter. However, there is a possibility that a state may occur where the glucose level becomes too low and leads to hypoglycemia. Hypoglycemia is most often caused by drugs taken to control diabetes. A much less common cause of hypoglycemia, referred to herein as "atypical hypoglycemia," can occur independently of exogenous insulin administration.

[0003] Since alcohol can block the formation of glucose in the liver, atypical hypoglycemia can occur in people who drink large amounts without eating. Furthermore, people with advanced liver diseases such as viral hepatitis, cirrhosis, or liver cancer may not be able to store and produce sufficient glucose. Atypical hypoglycemia can also occur in infants and children with congenital mutations that result in hyperinsulinemia.

[0004] More recently, over the past decade, there has been a growing recognition that atypical hypoglycemia can occur as a complication following surgery performed for the purpose of resolving excessive forms of obesity. A small percentage (less than 10%) of patients who undergo Roux-en-Y gastric bypass (RYGB) surgery subsequently develop hyperinsulinemic hypoglycemia, and blood glucose levels can drop low enough (20 - 40 mg / dL) to cause seizures, changes in mental status, loss of consciousness, cognitive dysfunction, physical impairment, and death.

[0005] One approach for treating hyperinsulin-induced hypoglycemia is to administer a GLP-1 receptor antagonist. Such peptide antagonists block the ability of inappropriately elevated concentrations of plasma GLP-1 to stimulate insulin secretion, normalize plasma glucose, and function to reduce the risk of cognitive impairment, vascular disease, and potential sudden death in patients experiencing atypical hypoglycemia.

[0006] Exendin-4 (SEQ ID NO: 1) is a 39-amino acid agonist of the glucagon-like peptide 1 (GLP-1) receptor. Exendin-4 is present in the saliva of the Gila monster (Heloderma suspectum). Ex-4(9-39)a (SEQ ID NO: 2) is an N-terminal truncated derivative of exendin-4 known to function as a GLP-1 receptor antagonist. However, Ex-4(9-39)a has two significant limitations with respect to its potential use for treating chronic atypical hypoglycemia, its non-human amino acid sequence and its relatively short duration of action in vivo.

[0007] According to one embodiment of the present disclosure, there is provided a novel and optimized series of GLP-1 antagonists for use as drug candidates in the treatment of atypical hypoglycemia, including hyperinsulin-induced hypoglycemia resulting from bariatric surgery or as a result of a congenital mutation. SUMMARY OF THE INVENTION

[0008] As disclosed herein, compositions and methods are provided for treating patients experiencing atypical hypoglycemia, and more specifically, in one embodiment, for treating patients experiencing hyperinsulinemia-induced hypoglycemia. According to one embodiment, compositions and methods are provided for treating hyperinsulinemia-induced hypoglycemia occurring after bariatric surgery. In one embodiment, the method comprises administering an effective amount of a glucagon-like peptide-1 receptor antagonist (GLP1RA) to increase blood glucose levels and alleviate associated acute symptoms and chronic outcomes related to hypoglycemia.

[0009] According to one embodiment, the GLP-1 receptor antagonist peptide is provided as comprising the amino acid sequence R 10 -DX 10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 5), wherein X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 15 is Glu or dGlu, X 16 is Trp, dTrp, dGlu or Glu, X 23 is Ile or dIle, X 40 is absent or is an acylated amino acid, R 10 is NH2, -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH, R 20 is COOH or CONH2, and optionally, the peptide comprises one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28 relative to the native exendin-4 sequence (SEQ ID NO: 1), or optionally, comprises a substitution of Lys at position 12 relative to the native exendin-4 sequence (SEQ ID NO: 1), and optionally, X10 or X 11 When one of them is Trp or dTrp, the other is not Trp or dTrp, and optionally, X 40 If X does not exist, R 10 is not NH2. In one embodiment, a GLP-1 receptor antagonist of SEQ ID NO: 5 is provided, where X 40 is acylated Lys, and the acyl group of the acylated Lys is a C16-C18 acid or diacid optionally linked to the Lys side chain via a spacer.

[0010] In one embodiment, a GLP-1 receptor antagonist is provided, and this antagonist is R 10 -DX 10 X 11 X 12 YLX 15 X 16 QAVREFX 23 X 24 contains the amino acid sequence of WLVRGGPSSGAPPPS (SEQ ID NO: 98), where X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 12 is Arg, Lys or Ser, X 15 is Glu or dGlu, X 16 is Trp, dTrp, dGlu or Glu, X 23 is Ile or dIle, X 24 is Ala or Glu, R 10 is -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH. According to one embodiment, DVWRYLX 15 EQAVREFIEWLVRGGPSSGAPPPSX 40 R 20A GLP-1 receptor antagonist peptide having the amino acid sequence of (SEQ ID NO: 96) is provided, wherein, X 15 is dGlu, X 40 is acylated Lys, and R 20 is COOH or CONH2, and the acyl group of the acylated Lys is a C16-C18 acid or diacid optionally bonded to the Lys side chain via a spacer. In one embodiment, the spacer comprises a minipeg or a γ-glutamic acid subunit, or any plurality or combination of such minipeg or γGlu molecules. In one embodiment, the peptide of SEQ ID NO: 96 is modified by one, two or three amino acid substitutions including, for example, substitution of amino isobutyric acid (Aib) at one or more of positions 16, 18, 19, 24, 26 or 28 relative to the native exendin-4 sequence of SEQ ID NO: 1, or substitution of Lys at position 12.

[0011] According to one embodiment, the amino acid sequence DX 10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 5) is provided, where X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 15 is Glu or dGlu, X 16 is Trp, dTrp, dGlu or Glu, X 23 is Ile or dIle, X 40 is an acylated amino acid, and R 20is COOH or CONH2, and optionally, the peptide contains one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28 relative to the native exendin-4 sequence (SEQ ID NO: 1), or optionally, contains an N-terminal extension of the peptide of SEQ ID NO: 5 by X7X8 (where X7 is an acylated amino acid (e.g., Lys) and X8 is Gly or C1-C3 N-alkyl Gly), and the numbering of the positions is relative to the native exendin-4 sequence (SEQ ID NO: 1), and optionally, X 10 or X 11 is Trp or dTrp, provided that the other is not Trp or dTrp.

[0012] According to one embodiment, DVWRYLX 15 EQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 a GLP-1 receptor antagonist peptide having the amino acid sequence of (SEQ ID NO: 9), wherein X 15 is dGlu, X 40 is acylated Lys, and R 20 is COOH or CONH2, and the acyl group of the acylated Lys is optionally a C16-C18 acid or diacid bonded to the Lys side chain via a spacer. In one embodiment, the spacer comprises a minipeg or a γ-glutamic acid subunit, or any plurality or combination of such minipeg or γGlu molecules. In one embodiment, the peptide of SEQ ID NO: 9 is modified by, for example, one, two or three amino acid substitutions including substitution by amino isobutyric acid (Aib) at one or more of positions 16, 18, 19, 24, 26 or 28 relative to the native exendin-4 sequence of SEQ ID NO: 1, or by addition of the dipeptide X7X8 (where X7 is an acylated amino acid (e.g., Lys) and X8 is Gly or C1-C3 N-alkyl Gly) to the N-terminus of SEQ ID NO: 9, and the numbering of the positions is relative to the native exendin-4 sequence (SEQ ID NO: 1).

[0013] In one embodiment, 1 to 3 amino acids are added to the N-terminus of the peptide of SEQ ID NO: 5 or an analog thereof. In one embodiment, one of the amino acids comprising the N-terminal extension is an acylated amino acid. In one embodiment, the N-terminal extension is a dipeptide X7X8, where X7 is an acylated lysine, optionally the lysine is in the D-configuration, and X8 is any amino acid. In one embodiment, the N-terminal extension is a self-cleaving dipeptide attached to the N-terminal α-amine of a 9-29 exendin-4 analog (e.g., the peptide of SEQ ID NO: 5), forming a prodrug of any of the GLP-1 antagonists of the present disclosure. One advantage of using a prodrug derivative of a GLP-1 antagonist is that such an approach extends the biological half-life of the peptide based on a strategy that inhibits the recognition of the prodrug by the GLP-1 receptor. In one embodiment, the prodrug derivative comprises a self-cleaving dipeptide (A-B) covalently attached to the GLP-1 antagonist, which is cleaved under physiological conditions and in the absence of enzymatic activity to restore full activity to the GLP-1 antagonist. In one embodiment, the GLP-1 antagonist is modified by covalently attaching one or more dipeptides (A-B) to the amine of the GLP-1 antagonist, where A is an amino acid or hydroxy acid and B is an N-alkylated amino acid attached to the GLP-1 antagonist via an amide bond between the carboxyl portion of B and the amine of the GLP-1 antagonist. In one embodiment, A-B is attached to the GLP-1 antagonist via an amide bond between the carboxyl of A-B and the amine of the GLP-1 antagonist, having the structure:

[0014]

Chemical Structure

[0015] comprising, wherein R1, R2, R4 and R8 are H, C1-C 18 alkyl, C2-C 18 alkenyl, (C1-C 18 alkyl)OH, (C1-C 18(Alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2 + )NH2, (C0-C4 alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C 12 alkyl(W1)C1-C 12 alkyl, independently selected from the group consisting of, W1 is a heteroatom selected from the group consisting of N, S and O, R3 is C1-C 18 alkyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)NH2, (C1-C 18 alkyl)SH, (C0-C4 alkyl)(C3-C6) cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl), or R4 and R3, together with the atom to which they are attached, form a pyrrolidine ring, R5 is NHR6 or OH, R6 is H, C1-C8 alkyl, R7 is selected from the group consisting of H and OH, and the chemical cleavage half-life (t 1 / 2 ) of A-B from the GLP-1 antagonist is at least about 1 hour to about 1 week in PBS under physiological conditions. In one embodiment, the dipeptide A-B covalently binds to the N-terminal α amine of the GLP-1 antagonist amino acid sequence. In one embodiment, R1 is H, C1-C4 alkyl, (C1-C4 alkyl)OH or (C1-C4 alkyl)NH2, R2 is H, R3 is C1-C6 alkyl, R4 is H, C1-C4 alkyl or (CH2)(C6 aryl)R7, R5 is NH2, R7 is H or OH, and R8 is hydrogen.

[0016] The present disclosure further provides a pharmaceutical composition comprising any one of the GLP-1 antagonist peptides and variant peptides described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, a method for treating a patient suffering from atypical hypoglycemia is provided, the method comprising administering to a patient in need of treatment a pharmaceutical composition comprising a GLP-1 antagonist peptide or variant peptide described herein in an amount effective to increase blood glucose levels.

[0017] According to one embodiment, a method for treating atypical hypoglycemia is provided, the method comprising administering any one of the GLP-1 receptor antagonist peptides disclosed herein in a therapeutically effective amount to increase blood glucose levels. In one embodiment, the GLP-1 receptor antagonist is acylated with a fatty acid or diacid group of a size sufficient to bind with high affinity to serum albumin. In one embodiment, the method comprises administering an acylated GLP-1 receptor antagonist, wherein the GLP-1 antagonist is acylated with a fatty acid or diacid group of a size sufficient to bind with high affinity to serum albumin. In one embodiment, the N-terminal α-amine of the GLP-1 receptor antagonist is acylated, and optionally a second acylation is performed at the C-terminus. In one embodiment, the GLP-1 receptor antagonist is acylated at the C-terminus, optionally the acylated amino acid is the C-terminal amino acid, optionally the GLP-1 antagonist is further modified by the attachment of a self-cleaving dipeptide via an amide bond, and optionally the amino acids of the dipeptide are acylated with a fatty acyl group of a size sufficient to bind with high affinity to serum albumin. In one embodiment, the acylated amino acid is added at position 40 of the C-terminus of SEQ ID NO: 11, and optionally the added acylated amino acid is Lys acylated with a C14-C20 fatty acid or fatty diacid optionally attached to the amino acid side chain via any one of the spacers disclosed herein.

[0018] In one embodiment, a pharmaceutical composition for treating atypical hypoglycemia comprises any of the GLP-1 receptor antagonists disclosed herein in combination with any existing therapeutic agent useful for treating hypoglycemia. For example, the pharmaceutical composition may comprise a GLP-1 receptor antagonist of the present invention and one or more of the following glucose supplements (e.g., dextrose), hyperglycemic agents such as glucagon and glucagon analogs, and inhibitors of insulin secretion (e.g., diazoxide, octreotide).

Brief Description of the Drawings

[0019]

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[0020] **Definitions** In describing and claiming the present invention, the following terms are used in accordance with the definitions set forth below.

[0021] As used herein, the term "about" means up to 10 percent greater or less than the recited value or range of values, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values prefaced by the term "about" is also intended to encompass embodiments of the recited absolute value or range of values.

[0022] As used herein, the term "amino acid" encompasses any molecule containing both an amino functional group and a carboxyl functional group, where the amino group and carboxylate group are attached to the same carbon (alpha carbon). The alpha carbon may optionally have one or two additional organic substituents. Amino acids can be designated by their three-letter code, one-letter code, or in some cases by the name of their side chain. For example, a non-standard amino acid containing a cyclohexane group attached to the alpha carbon is called "cyclohexane" or "cyclohexyl". For the purposes of the present disclosure, a designation of an amino acid without specifying its stereochemistry is intended to encompass either the L- or D-form of the amino acid, or a racemic mixture thereof. However, when an amino acid is designated by its three-letter code (i.e., Lys), such a designation is intended to specify the natural L-form of the amino acid, while the D-form is specified by including a lower-case d before the three-letter or one-letter code (i.e., dLys or dK).

[0023] As used herein, the term "hydroxy acid" refers to an amino acid modified such that the alpha carbon amino group is replaced with a hydroxyl group. As used herein, the term "non-coded amino acid" encompasses any amino acid that is not an L-isomer of any of the following twenty amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr.

[0024] "Bioactive polypeptide" refers to a polypeptide that can exert biological effects in vitro and / or in vivo. As used herein, a general reference to a peptide is intended to include peptides having modified amino and carboxy termini. For example, an amino acid sequence that specifies a standard amino acid is intended to include the standard amino acids at the N- and C-termini, as well as the corresponding C-terminal amino acids modified to include an amide group in place of the corresponding hydroxy acid at the N-terminus and / or terminal carboxylic acid.

[0025] As used herein, an "acylated" amino acid is an amino acid that contains an acyl group that is non-natural relative to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods for producing acylated amino acids and acylated peptides are known in the art and include acylating the amino acid prior to incorporation into the peptide or chemically acylating the peptide after peptide synthesis. In some embodiments, the acyl group confers to the peptide one or more of (i) an extended half-life in circulation, (ii) a delayed onset of action, (iii) an extended duration of action, (iv) improved resistance to proteases, and (v) increased potency at the GLP-1 receptor.

[0026] As used herein, an "alkylated" amino acid is an amino acid that contains an alkyl group that is non-natural relative to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods for producing alkylated amino acids and alkylated peptides are known in the art and include alkylating the amino acid prior to incorporation into the peptide or chemically alkylating the peptide after peptide synthesis. Without being bound by any particular theory, alkylation of a peptide is thought to achieve similar effects as acylation of a peptide, such as an extended half-life in circulation, a delayed onset of action, an extended duration of action, and improved resistance to proteases.

[0027] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various kinds of wetting agents. This term also encompasses any agent approved by the regulatory authorities of the federal government of the United States or listed in the United States Pharmacopeia for use in animals including humans.

[0028] As used herein, the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound and are not biologically or otherwise undesirable. Many of the compounds disclosed herein are capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0029] As used herein, the term "hydrophilic moiety" refers to any compound that is readily water-soluble or readily absorbs water and is acceptable in vivo (i.e., biocompatible) by mammalian species without toxic effects. Examples of hydrophilic moieties include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymers, polyvinyl alcohol, polyvinyl pyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxyethyl methacrylate, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses such as hydroxymethyl cellulose or hydroxyethyl cellulose and their copolymers, and natural polymers such as albumin, heparin, and dextran.

[0030] As used herein, the term "treating" includes alleviating symptoms associated with a particular disorder or condition, and / or preventing or removing such symptoms. For example, as used herein, the term "treating hypoglycemia" generally refers to maintaining or increasing blood glucose levels to near normal levels.

[0031] As used herein, an "effective" amount or "therapeutically effective amount" of a GLP-1 receptor antagonist refers to an amount of the GLP-1 antagonist that is non-toxic but sufficient to provide the desired effect. For example, one desired effect is the prevention or treatment of hypoglycemia. The amount that is "effective" will vary for each subject depending on, for example, the age and general condition of the individual, the mode of administration, etc. Thus, it is not always possible to specify the exact "effective amount." However, the appropriate "effective" amount in any given case can be determined by one of ordinary skill in the art using routine experimentation.

[0032] The term "parenteral" means by a route other than through the gastrointestinal tract, such as intranasal, inhalation, subcutaneous, intramuscular, intraspinal, or intravenous. As used herein, the term "derivative" is intended to encompass chemical modifications to a compound (e.g., an amino acid), including, for example, chemical modifications in vitro by introducing a nitro group to a side chain at one or more positions of a polypeptide, such as to a tyrosine residue, or iodine to a tyrosine residue, or by converting a free carboxyl group to an ester or amide group, or by converting an amino group to an amide by acylation, or by acylating a hydroxy group to yield an ester, or by alkylating a primary amine to yield a secondary amine or by attachment of a hydrophilic moiety to an amino acid side chain. Other derivatives are obtained by oxidation or reduction of the side chain of an amino acid residue in a polypeptide.

[0033] As used herein, the term "identity" relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to obtain a percentage. Thus, two copies of exactly the same sequence have 100% identity, while two sequences with amino acid deletions, additions, or substitutions relative to each other have lower identity. One of ordinary skill in the art will recognize that several computer programs, such as those using algorithms like BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403 - 410), are available for determining sequence identity.

[0034] As used herein, the term "selectivity" of a molecule for a first receptor as compared to a second receptor refers to the following ratio, the EC of the molecule in the second receptor 50 divided by the EC of the molecule in the first receptor 50 For example, a molecule having an EC of 1 nM in a first receptor 50 and an EC of 100 nM in a second receptor 50 has 100 - fold selectivity for the first receptor as compared to the second receptor.

[0035] As used herein, an amino acid "modification" refers to the substitution of an amino acid, or the derivation of an amino acid by the addition and / or removal of chemical groups to / from an amino acid, including substitution by any of the 20 amino acids commonly found in human proteins, as well as non - canonical or non - natural amino acids. Commercial sources of non - canonical amino acids include Sigma - Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Non - canonical amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids.

[0036] As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue with a different amino acid residue. As used herein, the term "conservative amino acid substitution" refers to the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gln, cysteic acid, and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; ornithine (Orn) IV. Large aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, as defined herein as an exchange at one of acetylphenylalanine.

[0037] Throughout this application, whenever a specific amino acid position (e.g., position 28) is referred to by number, it refers to the amino acid at that position in native exendin-4 (SEQ ID NO: 1) or the corresponding amino acid position of any analog thereof. For example, a reference to "position 28" herein means the corresponding position 27 for an analog of exendin-4 in which the first amino acid of SEQ ID NO: 1 is deleted. Thus, 9-39 exendin-4 represents an N-terminally truncated exendin-4 peptide in which the first 8 amino acids are deleted. Further, references to positions greater than 39 (native exendin-4 has only 39 amino acids) are intended to refer to the amino acid position in an analog having a C-terminal amino acid extension after the corresponding position 39 of SEQ ID NO: 1.

[0038] As used herein, the general term "polyethylene glycol chain" or "PEG chain" has the general formula H(OCH2CH2) nOH (wherein n is at least 2), which refers to a mixture of branched or linear condensation polymers of ethylene oxide and water. The terms "polyethylene glycol chain" or "PEG chain" are used in combination with a numerical suffix to indicate an approximate average molecular weight. For example, PEG-5,000 refers to a polyethylene glycol chain having an average total molecular weight of about 5,000 daltons.

[0039] As used herein, the term "pegylation" and similar terms refer to a compound modified from its natural state by linking a polyethylene glycol chain to the compound. A "pegylated polypeptide" is a polypeptide having a PEG chain covalently attached to the polypeptide.

[0040] As used herein, the term "miniPEG" or "OEG" refers to the structure:

[0041]

Chemical formula

[0042] defines a functionalized polyethylene compound containing. As used herein, a "linker" or "spacer" is a bond, molecule, or group of molecules that binds two separate entities to each other. A linker can provide an optimal spacing between the two entities or can further provide a labile linkage that allows the two entities to be separated from each other. Labile bonds include photocleavable groups, acid-labile moieties, base-labile moieties, and enzymatically cleavable groups.

[0043] As used herein, a "dimer" is a complex containing two subunits covalently bonded to each other via a linker. The term dimer, when used without any limiting words, encompasses both homodimers and heterodimers. A homodimer contains two identical subunits, and a heterodimer contains two different subunits, but the two subunits are substantially similar to each other.

[0044] As used herein, the term "C16-C20 fatty acid" refers to the structure CO(CH2) 14~20 CH3, and the term "C16-C20 diacid" refers to the structure -CO(CH2) 14~20 COOH, wherein the prefix "C16-C20" indicates a variable value for the total number of carbons included in the specified compound. For example, C18 diacid has the structure -CO(CH2) 16 COOH. As used herein, a general reference to an acylated amino acid includes both an amino acid having a side chain acylated with a fatty acid and an amino acid having a side chain acylated with a diacid.

[0045] The physiological conditions disclosed herein are intended to include a temperature of about 35-40 °C and a pH of about 7.0 to about 7.4, and more typically include a pH of 7.2-7.4 and a temperature of 36-38 °C. Since physiological pH and temperature are tightly regulated within a highly defined range in humans, the rate of conversion of the dipeptide / drug conjugate (prodrug) to the drug exhibits high reproducibility within and between patients.

[0046] As used herein, the term "C1-C n alkyl" (where n may be from 1 to 6) refers to a branched or straight-chain alkyl group having from 1 to the specified number of carbon atoms. Typical C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

[0047] As used herein, the term "C2-C nThe term "alkenyl" (where n may be from 2 to 6) represents an olefinically unsaturated branched or straight-chain group having from 2 to the specified number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl (-CH2-CH=CH2), 1,3-butadienyl (-CH=CHCH=CH2), 1-butenyl (-CH=CHCH2CH3), hexenyl, pentenyl, and the like.

[0048] 「C2~C n The term "alkynyl" (where n may be from 2 to 6) refers to an unsaturated branched or straight-chain group having from 2 to n carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.

[0049] As used herein, the term "aryl" refers to a monocyclic or bicyclic carbocyclic system having one or two aromatic rings, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. The size of the aryl ring and the presence of substituents or linking groups are indicated by specifying the number of carbons present. For example, the term "(C1-C3 alkyl)(C6-C 10 aryl)" refers to a 5- to 10-membered aryl bonded to the parent moiety via a 1- to 3-membered alkyl chain.

[0050] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in the aromatic ring. The size of the heteroaryl ring and the presence of substituents or linking groups are indicated by specifying the number of carbons present. For example, the term "(C1-C n alkyl)(C5-C6 heteroaryl)" refers to a 5- or 6-membered heteroaryl bonded to the parent moiety via an alkyl chain from 1 to "n" members.

[0051] As used herein, the term "halo" refers to one or more members of the group consisting of fluorine, chlorine, bromine, and iodine. As used herein, the term "patient", unless otherwise specified, is intended to include any warm-blooded vertebrate domesticated animal (e.g., but not limited to, livestock, horses, cats, dogs and other pets) and humans, and is not limited to an individual under the direct care of a physician.

[0052] As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" refers to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in its natural or native environment, and means that the purity has increased as a result of separation from other components of the original composition. The term "purified peptide" is used herein to describe a peptide separated from other compounds including, but not limited to, nucleic acids, lipids and carbohydrates.

[0053] As used herein, the term "peptide" includes sequences of two or more amino acids, typically less than 50 amino acids, where the amino acids are either naturally occurring or encoded, or are non-naturally occurring or non-encoded amino acids. Non-naturally occurring amino acids refer to amino acids that do not occur naturally in vivo but can nevertheless be incorporated into the peptide structures described herein. As used herein, "non-coded" refers to an amino acid that is not any of the L-isomers of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr.

[0054] As used herein, "partially non-peptidic" refers to a molecule in which a part of the molecule is a compound or substituent that has biological activity and does not contain an amino acid sequence. "Peptidomimetic" refers to a chemical compound that has a structure different from the general structure of an existing peptide but functions in a manner similar to the existing peptide, for example, by mimicking the biological activity of the peptide. Peptidomimetics typically contain naturally occurring amino acids and / or non-natural amino acids, but can also include modifications to the peptide backbone. For example, peptidomimetics can include sequences of naturally occurring amino acids with insertions or substitutions of non-peptidic moieties, such as retro-inverso fragments, or non-peptidic bonds such as azapeptide bonds (where CO is replaced by NH) or pseudopeptide bonds (e.g., where NH is replaced by CH2), or the incorporation of ester bonds (e.g., depsipeptides in which one or more of the amide (-CONHR-) bonds are replaced by ester (COOR) bonds). Alternatively, peptidomimetics may contain no naturally occurring amino acids at all.

[0055] As used herein, the terms "charged amino acid" or "charged residue" refer to an amino acid that contains a side chain that is negatively charged (i.e., deprotonated) or positively charged (i.e., protonated) in an aqueous solution at physiological pH. For example, negatively charged amino acids include aspartic acid, glutamic acid, cysteic acid, homocysteic acid, and homoglutamic acid, while positively charged amino acids include arginine, lysine, and histidine. Charged amino acids include charged amino acids among the 20 amino acids commonly found in human proteins, as well as non-canonical or non-naturally occurring amino acids.

[0056] As used herein, the term "acidic amino acid" refers to an amino acid that contains a second acidic moiety (other than the α-carboxylic acid of the amino acid), for example, containing a side chain carboxylic acid or sulfonic acid group.

[0057] As used herein, the term "prodrug" is defined as any compound that undergoes chemical modification before exhibiting its full pharmacological effect. As used herein, "dipeptide" results from the linkage of an α-amino acid or α-hydroxy acid to another amino acid via a peptide bond.

[0058] As used herein, the term "chemical cleavage" encompasses non-enzymatic reactions that result in the cleavage of a covalent chemical bond, unless otherwise specified. As used herein, the term "atypical hypoglycemia" defines a state of hypoglycemia that occurs in a patient independent of exogenous insulin administration. Abbreviations: Lowercase k = D-isomer of lysine Uppercase K = L-isomer of lysine γE = L-isomer of γ-glutamic acid (miniPEG)2 = COCH2OCH2CH2OCH2CH2NH COC 16 H 32 CO2H = (C18 diacid) (N-Me)G = sarcosine Embodiments According to one embodiment of the present disclosure, compositions and methods are provided for treating patients suffering from a state of hypoglycemia (i.e., atypical hypoglycemia) that occurs independent of exogenous insulin administration. According to the present disclosure, a composition comprising a GLP-1 antagonist is administered in an amount sufficient to increase blood glucose levels and / or alleviate associated acute symptoms and chronic outcomes associated with hypoglycemia in a patient suffering from atypical hypoglycemia. In one embodiment, a patient experiencing atypical hypoglycemia also has a state of hyperinsulinemia. In one embodiment, the hyperinsulin state occurs after the patient has undergone bariatric surgery.

[0059] According to one embodiment, a method of treating a patient suffering from atypical hypoglycemia includes administering an acylated GLP-1 receptor antagonist peptide, wherein the GLP-1 receptor antagonist peptide is acylated at the N-terminus and / or C-terminus with a fatty acid or fatty diacid group of a size sufficient to bind with high affinity to serum albumin. In one embodiment, a C14-C20 acyl group is covalently bonded to the N-terminal α amine of the GLP-1 receptor antagonist peptide.

[0060] In one embodiment, the GLP-1 receptor antagonist is acylated at the N-terminus of the GLP-1 receptor antagonist, and optionally, an acetyl group is covalently bonded to the N-terminal α amine of the glucagon peptide. The acyl group of the acylated glucagon peptide may be of any size, e.g., a carbon chain of any length, and may be straight-chain or branched-chain. In some particular embodiments of the present invention, the acyl group is a C12-C30 fatty acid or fatty diacid. For example, the acyl group may be any one of a C12 fatty acid / diacid, a C14 fatty acid / diacid, a C16 fatty acid / diacid, a C18 fatty acid / diacid, a C20 fatty acid / diacid, a C22 fatty acid / diacid, a C24 fatty acid / diacid, a C26 fatty acid / diacid, a C28 fatty acid / diacid, or a C30 fatty acid / diacid. In some embodiments, the acyl group is a C14-C20 fatty acid, e.g., a C14 fatty acid or fatty diacid, a C16 fatty acid or fatty diacid, or a C16 fatty acid or fatty diacid.

[0061] According to one embodiment, the acylated GLP-1 receptor antagonist peptide includes DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK (SEQ ID NO: 3), or an analog thereof having at least 95% sequence identity with SEQ ID NO: 3 while retaining GLP-1 receptor antagonist activity.

[0062] According to one embodiment, the acylated GLP-1 receptor antagonist peptide of the present disclosure includes an analog of the peptide DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK (SEQ ID NO: 3), and the GLP-1 receptor antagonist differs from SEQ ID NO: 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, the modifications being selected from amino acid substitutions, additions, or modifications of the amino acid structure, including but not limited to acylation and / or amidation of the C-terminal amino acid. In one embodiment, the amino acid modification is an amino acid substitution at one or more of positions 12, 16, 18, 19, 24, 26, 27, and 28 of the peptide, and / or a substitution with a d-isomer at one or more of positions 15, 16, 17, 19, 20, 21, and 23 (numbering relative to native exendin-4 (SEQ ID NO: 1)). In one embodiment, the analog of SEQ ID NO: 3 is further modified by acylation of the C-terminal amino acid side chain with a C16-C18 fatty acid or diacid, optionally via a spacer.

[0063] According to one embodiment, there is provided a GLP-1 receptor antagonist comprising the amino acid sequence of DVSRYLEEQAVREFIEWLVRGGPSSGAPPPSK (SEQ ID NO: 4), or an amino acid sequence that differs from SEQ ID NO: 4 by 1, 2, 3, 4, or 5 amino acid substitutions while retaining GLP-1 receptor antagonist activity, provided that the GLP-1 antagonist peptide does not include the sequence of SEQ ID NO: 3. In one embodiment, the following, i. Substitution of 1, 2, or 3 amino acids with Trp or dTrp at any of positions 7, 10, 11, 13, or 16, i. Substitution of 1, 2, or 3 amino acids with the corresponding amino acid in the D conformation at any of positions 15, 16, or 23, ii. Substitution of 1, 2, or 3 amino acids with Aib at any of positions 16, 18, 19, 24, 26, or 28, iii. An acylated amino acid at position 12, optionally substituted with an acylated lysine, iv. Substitution with dGlu, Asp, homoglutamic acid or homocysteic acid at the 16 - position, v. Substitution with cyclopropane, cyclopentane, cyclohexane or phenylglycine at the 19 - position, vi. Substitution with dArg, homolysine or citrulline at the 20 - position, vii. Substitution with an amide of the native C - terminal carboxyl group viii. Addition of an N - terminal extension of 1 - 3 amino acids, where optionally one of the N - terminal extension amino acids is acylated, ix. Substitution of the C - terminal amino acid with an acylated amino acid, x. Any combination of i) - x) A peptide different from the peptide of SEQ ID NO: 4 is provided by one or more of the above.

[0064] According to one embodiment, R 10 -DVX 11 X 12 YLX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 (SEQ ID NO: 23) A GLP - 1 receptor antagonist comprising the amino acid sequence is provided, where R 10 is NH2, -CO(CH2) 14~20 CH3, -CO(CH2) 14~20 COOH or an N - terminal extension of 1, 2 or 3 amino acids, where one of the N - terminal extension amino acids is optionally acylated with a C16 - C18 fatty acid or diacid via a spacer, and optionally, R 10 is a dipeptide of the structure: X7X8 (where X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1 - C4 N - alkylated Gly), X 11 is Trp, dTrp or Ser, X 12 is Arg or an acylated amino acid, optionally acylated Lys, X 15 is Glu or dGlu, X 16 is Glu, dGlu, Asp, homoglutamic acid or homocysteic acid, X 19 is Val, cyclopropane, cyclopentane, cyclohexane or phenylglycine, X 20 is Arg, homolysine or citrulline, X 23 is Ile, or dIle, X 40 is an acylated amino acid, optionally acylated Lys, R 20 is COOH or CONH2, optionally, 1, 2 or 3 amino acids selected from positions 7, 10, 13, or 16 are replaced by Trp or dTrp, and as an optional condition, neither position 10 nor position 11 is Trp or dTrp, and as a further optional condition, R 10 and X 12 cannot both contain an acylated amino acid, and optionally, 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26, or 28 are replaced by Aib.

[0065] In one embodiment, only one of positions 7, 12 and 40 of the peptide of SEQ ID NO: 23 contains an acylated amino acid. In one embodiment, two of positions 7, 12 and 40 of the peptide of SEQ ID NO: 23 contain an acylated amino acid. In one embodiment, the acylated amino acids at positions 7, 12 and 40 are, independently, amino acids having a structure of formula I (optionally, Lys) or formula II (optionally, Ser), and each of formula I and II is

[0066]

Chemical formula

[0067] as follows. In one embodiment, a peptide of SEQ ID NO: 23 is provided, wherein R10 is X7X8, where X7 is acylated Lys or acylated dLys, and X 12 is Arg, and X 40 is acylated Lys. In one embodiment, a peptide of SEQ ID NO: 23 is provided, where R 10 is NH2, and X 12 is acylated Lys, and X 40 is acylated Lys. In one embodiment, a peptide of SEQ ID NO: 23 is provided, where R 10 is NH2, and X 12 is Arg, and X 40 is acylated Lys. In one embodiment, the acylated amino acid of the peptide of SEQ ID NO: 23 is an acylated Lys residue, and optionally, the acylated Lys residue is independently acylated with a C14 - C24 fatty acid or fatty diacid, or a C16 - C18 fatty acid or fatty diacid, and optionally, the fatty acid or fatty diacid is attached to the side chain of the Lys residue via any of the spacer molecules disclosed herein.

[0068] According to one embodiment, R 10 -DVX 11 X 12 YLEX 16 QAVREFIEWLVRGGPSSGAPPPSX 40 -R 20 (SEQ ID NO: 24) A GLP - 1 receptor antagonist comprising the amino acid sequence is provided, wherein, R 10 is NH2, -CO(CH2) 14~20 CH3, -CO(CH2) 14~20 COOH, or a dipeptide of the structure: X7X8 (where X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1 - C4 N - alkylated Gly), X 11 is Trp, dTrp or Ser, X 12 is Arg or an acylated amino acid, optionally acylated Lys, X 16is Glu or Asp, X 40 is an acylated amino acid, optionally acylated Lys, R 20 is COOH or CONH2, and optionally, 1, 2, or 3 amino acids selected from positions 16, 17, 18, 20, or 21 are substituted with the corresponding amino acids in the D - conformation, and / or 1, 2, or 3 amino acids at any of positions 16, 18, 19, 24, 26, or 28 are substituted with Aib.

[0069] According to one embodiment, R 10 -DVX 11 X 12 YLEX 16 QAVREFIEWLVRGGPSSGAPPPSX 40 -R 20 A GLP - 1 receptor antagonist is provided that comprises the amino acid sequence of (SEQ ID NO: 24), wherein R 10 is a dipeptide of the structure: X7X8 (where X7 is acylated Lys or acylated dLys, and X8 is Gly or sarcosine), X 11 is Trp or dTrp, X 12 is Arg, X 16 is Glu, X 40 is acylated Lys, R 20 is COOH or CONH2, and the acylated Lys residue of the peptide is independently optionally acylated with a C16 - C18 fatty acid or a fatty diacid via a spacer disclosed herein, and optionally, 1, 2, or 3 amino acids at any of positions 16, 18, 19, 24, 26, or 28 are substituted with Aib.

[0070] In one embodiment, a GLP - 1 receptor antagonist is provided, which antagonist is DX 10 X 11 X 12YLX 15 X 16 QAVREFX 23 X 24 comprises the amino acid sequence of WLVRGGPSSGAPPPS (SEQ ID NO: 98), wherein, X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 12 is Arg, Lys or Ser, X 15 is Glu or dGlu, X 16 is Trp, dTrp, dGlu or Glu, X 23 is Ile or dIle, X 24 is Ala or Glu, the GLP-1 receptor antagonist is acylated with a fatty acid or a diacid group of a size sufficient to bind to serum albumin with high affinity, and optionally, the amino acids of the GLP-1 receptor antagonist are acylated with a C16-C18 fatty acid or a C16-C18 fatty diacid.

[0071] In one embodiment, a GLP-1 receptor antagonist is provided, the antagonist comprising R 10 -DX 10 X 11 X 12 YLX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS-R 20 (SEQ ID NO: 98), or a sequence different from SEQ ID NO: 98 by one or two amino acid substitutions, wherein, X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 12is Arg, Lys or Ser, and X 15 is Glu or dGlu, and X 16 is Trp, dTrp, dGlu or Glu, and X 23 is Ile or dIle, and X 24 is Ala or Glu, and R 10 is NH2, -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH, and R 20 is COOH or CONH2. In one embodiment, the sequence of SEQ ID NO: 98 is provided, where X 10 is Val, and X 11 is Trp, dTrp or Ser, and X 12 is Arg, Lys or Ser, and X 15 is Glu or dGlu, and X 16 is dGlu or Glu, and X 23 is Ile, and X 24 is Ala or Glu, and R 10 is -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH, and R 20 is COOH, and optionally, the peptide comprises one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28 relative to the native exendin-4 sequence (SEQ ID NO: 1).

[0072] In one embodiment, a GLP-1 receptor antagonist is provided, which antagonist is R 10 -DX 10 X 11 X 12 YLX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS-R20 comprising the amino acid sequence of (SEQ ID NO: 98), wherein, X 10 is Val, X 11 is Ser, X 12 is Arg, X 15 is Glu or dGlu, X 16 is dGlu or Glu, X 23 is Ile, X 24 is Glu, R 10 is -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH, R 20 is COOH or CONH2.

[0073] In one embodiment, the GLP-1 antagonist peptide is R 10 -DVSSYLEEQAVREFIAWLVKGGPSSGAPPPS (SEQ ID NO: 3) or comprises an amino acid sequence different from SEQ ID NO: 3 by 1, 2 or 3 amino acid substitutions while retaining GLP-1 agonist activity, wherein R 10 is -CO(CH2) 14~20 CH3 or -CO(CH2) 14~20 COOH bonded to the N-terminal α amine of the amino acid sequence of SEQ ID NO: 3, R 20 is COOH or CONH2, and optionally, R 20 is COOH.

[0074] In one embodiment, the GLP-1 antagonist peptide is DVX 11 RYLQX 15 X 16 AVREFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 comprising the amino acid sequence of (SEQ ID NO: 25), wherein, X11 is Trp, dTrp or Ser, X 15 is Glu or dGlu, X 16 is Glu or dGlu, X 23 is Ile or dIle, X 40 is an acylated amino acid and may be Lys acylated with a C16-C18 fatty acid or a diacid, optionally via a spacer, R 20 is COOH or CONH2. Optionally, the GLP-1 antagonist of SEQ ID NO: 25 is further modified by one or more Aib substitutions at any of positions 16, 18, 19, 24, 26, or 28 relative to the numbering of the native exendin-4 sequence of SEQ ID NO: 1, or optionally by substitution of an acylated Lys at position 12. In one embodiment, a peptide of SEQ ID NO: 25 is provided that further comprises substitution of an acylated Lys at position 12 and optional substitution of Aib at position 27.

[0075] In one embodiment, the GLP-1 antagonist peptide is DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R2 (SEQ ID NO: 6) or X7X8DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 comprises the amino acid sequence of (SEQ ID NO: 1), wherein X7 is an acylated Lys or an acylated dLys, X8 is Gly or sarcosine, X 11 is Trp or dTrp, X 40 is an acylated amino acid, optionally an acylated Lys, and the acylated Lys residue comprises a C16-C18 fatty acid or a diacid covalently attached to the Lys side chain, optionally via a spacer, R 20is COOH or CONH2. Optionally, the peptides of SEQ ID NO: 6 and SEQ ID NO: 26 can be further modified by Aib substitution at any one of positions 16, 18, 19, 24, 26 or 28 based on the numbering of native exendin-4 (SEQ ID NO: 1), or the peptide of SEQ ID NO: 6 is optionally substituted with acylated Lys at position 12.

[0076] In one embodiment, the GLP-1 antagonist peptide is DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 6) or DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 7) or SEQ ID NO: 6 or SEQ ID NO: 7 and contains amino acids with one or two amino acid substitutions different therefrom, wherein X 11 is Trp or dTrp, X 40 has an acyl group of a size sufficient to bind with high affinity to serum albumin bound to the amino acid side chain, optionally the acyl group is bound via a spacer, and R 20 is COOH or CONH2 which may have an Aib substitution at any one of positions 16, 18, 19, 24, 26 or 28 relative to the numbering of native exendin-4 (SEQ ID NO: 1), and optionally the carboxy group of the C-terminal amino acid is substituted with an amide (i.e., R 20 is CONH2) amino acid. In one embodiment, X 40 is an amino acid containing a structure of formula I (optionally Lys), formula II (optionally Ser), or formula III (optionally Cys), and each of formulas I, II, and III is

[0077]

Chemical formula

[0078] is as follows. In one embodiment, the GLP-1 antagonist peptide is DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 -COOH (SEQ ID NO: 9) or DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 -NH2 (SEQ ID NO: 10) amino acid sequence, where X 11 is Trp or dTrp, and X 40 is an amino acid having an acyl group optionally attached to the side chain of the amino acid via a spacer. In one embodiment, X 40 is acylated Lys.

[0079] According to one embodiment, DVWX 12 YLEEQAVREFIEWLVRGGPSSGAPPPSX 40 a GLP-1 receptor antagonist peptide having the amino acid sequence of -NH2 (SEQ ID NO: 8) is provided, wherein, X 12 is acylated Lys, X 40 is acylated Lys having an amide that replaces the C-terminal carboxylic acid, and the acyl group of the acylated Lys is a C16-C18 acid or diacid optionally attached to the Lys side chain via a spacer.

[0080] In yet another aspect, the amino acid at position 1 of any of the GLP-1 receptor antagonists disclosed herein is modified to inhibit proteolytic degradation of the peptide. In one embodiment, the inhibition of protease is i. acylating the side chain of the N-terminal amino acid, ii. replacing the N-terminal amino acid with its D-stereoisomer, iii. modifying the N-terminal α-amino group, including, for example, covalently bonding an acetyl group to the α-amino group or removing the α-amino group, or iv. achieved by any combination of i) - iii). In one embodiment, DVX having up to three amino acid substitutions relative to SEQ ID NO: 6 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 A peptide comprising the sequence of R2 (SEQ ID NO: 6) is provided, where X 11 is Trp or dTrp, X 40 is an acylated amino acid, R 20 is COOH or CONH2, and this peptide exhibits antagonist activity against human GLP-1.

[0081] In one embodiment, the GLP-1 receptor antagonist is DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 27) DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 28) DV(dW)RYLE(Aib)QAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 29) DVWRYLE(Aib)QAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 30 DV(dW)RYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 31) DVWRYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 32) DV(dW)RYLEEQA(Aib)REFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 33) DVWRYLEEQA(Aib)REFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 34) DV(dW)RYLEEQAVREFI(Aib)WLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 35) DVWRYLEEQAVREFI(Aib)WLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 36) DV(dW)RYLEEQAVREFIEW(Aib)VRGGPSSGAPPPSX40 R20, (SEQ ID NO: 37) DVWRYLEEQAVREFIEW(Aib)VRGGPSSGAPPPSX40 R20, (SEQ ID NO: 38) DV(dW)RYLEEQAVREFIEWLV(Aib)GGPSSGAPPPSX40 R20, (SEQ ID NO: 39) DVWRYLEEQAVREFIEWLV(Aib)GGPSSGAPPPSX40 R20, (SEQ ID NO: 40) DV(dW)RYLEEQAV(dR)EFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO: 41) DVWRYLEEQAV(dR)EFIEWLVRGGPSSGAPPPSX 40 R 20 、(SEQ ID NO: 42) and comprises a peptide selected from the group consisting of wherein X 40 is an acylated amino acid, optionally Lys acylated with a C16 - C18 fatty acid or a diacid, and R 20 is COOH or CONH2, and the peptide exhibits antagonist activity against human GLP - 1.

[0082] In one embodiment, the GLP - 1 receptor antagonist is R 10 -DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPS - R 20 、(SEQ ID NO: 100) R 10 -DVWRYLEEQAVREFIEWLVRGGPSSGAPPPS - R 20 、(SEQ ID NO: 101) R 10 -DV(dW)RYLE(Aib)QAVREFIEWLVRGGPSSGAPPPS - R 20 、(SEQ ID NO: 102) R 10 -DVWRYLE(Aib)QAVREFIEWLVRGGPSSGAPPPS - R 20 、(SEQ ID NO: 103) R10 -DV(dW)RYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 104) R 10 -DVWRYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 105) R 10 -DV(dW)RYLEEQA(Aib)REFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 106) R 10 -DVWRYLEEQA(Aib)REFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 107) R 10 -DV(dW)RYLEEQAVREFI(Aib)WLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 108) R 10 -DVWRYLEEQAVREFI(Aib)WLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 109) R 10 -DV(dW)RYLEEQAVREFIEW(Aib)VRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 110) R 10 -DVWRYLEEQAVREFIEW(Aib)VRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 111) R 10 -DV(dW)RYLEEQAVREFIEWLV(Aib)GGPSSGAPPPS-R 20 ,(SEQ ID NO: 112) R 10 DVWRYLEEQAVREFIEWLV(Aib)GGPSSGAPPPS-R 20 ,(SEQ ID NO: 113) R 10 -DV(dW)RYLEEQAV(dR)EFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 114) R 10-DVWRYLEEQAV(dR)EFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO: 115) comprising a peptide selected from the group consisting of wherein R 10 is -CO(CH2)14~20CH3 or -CO(CH2)14~20COOH attached to the N-terminal α-amine of the polypeptide, and R 20 is COOH or CONH2, and the peptide exhibits antagonist activity against human GLP-1.

[0083] Further exemplified species of the present disclosure include those listed in Table 1.

[0084]

Table 1-1

[0085]

Table 1-2

[0086]

Table 1-3

[0087]

Table 1-4

[0088]

Table 1-5

[0089]

Table 1-6

[0090]

Table 1-7

[0091] In one embodiment, dimers and multimers are prepared that include two or more GLP-1 receptor antagonist peptides of the present disclosure, including homo- or hetero-multimers or homo- or hetero-dimers. The two or more GLP-1 receptor antagonist peptides can be joined together using standard linking agents and procedures known to those of skill in the art. For example, dimers can be formed between two peptides via the use of bifunctional thiol cross-linking agents and bifunctional amine cross-linking agents, particularly for GLP-1 receptor antagonist peptides that include or are substituted with cysteine, lysine, ornithine, homocysteine, or acetylphenylalanine residues. The dimer can be a homodimer or a heterodimer. In an exemplary embodiment, the linker that joins two (or more) analogs is PEG, such as 5 kDa PEG, 20 kDa PEG. In some embodiments, the linker is a disulfide bond. For example, each monomer of the dimer can include a Cys residue (e.g., located at the terminus or internally), and the sulfur atoms of each Cys residue are involved in the formation of a disulfide bond. In an exemplary aspect, each monomer of the dimer is joined via a thioether bond. In an exemplary aspect, the ε-amine of a Lys residue of one monomer is bonded to a Cys residue, which is then bonded via a chemical moiety to the epsilon amine of a Lys residue of the other monomer.

[0092] In some embodiments, the monomers are connected via terminal amino acids (e.g., N-terminus or C-terminus, optionally, the amino acid is added to the terminus of the peptide to be dimerized), via internal amino acids, or via the terminal amino acids of at least one monomer and the internal amino acids of at least one other monomer. In some embodiments, the monomers of the multimer are joined together in a "tail-to-tail" orientation in which the C-terminal amino acids of each monomer are joined together. Alternatively, in one embodiment, the multimer is joined together in a "head-to-head" orientation in which the N-terminal amino acids of each monomer are joined together.

[0093] In one embodiment, the C-terminal amino acid of any of the GLP-1 antagonist peptides disclosed herein can be modified to replace the native carboxyl group with an amide. In one embodiment, the C-terminal amino acid of any of the GLP-1 antagonist peptides disclosed herein contains a native amino acid carboxyl group. Pharmaceutical composition Further provided by the present disclosure is a pharmaceutical composition comprising any of the GLP-1 receptor antagonist peptides, dimers, multimers, or conjugates (or combinations thereof) disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition is preferably sterile and suitable for parenteral administration.

[0094] According to one embodiment, a pharmaceutical composition is provided comprising any of the novel GLP-1 receptor antagonists disclosed herein, preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient. Such a composition may contain the GLP-1 receptor antagonist disclosed herein at a concentration of at least 0.1 to 10 mg / ml, or more. In one embodiment, the pharmaceutical composition comprises an aqueous solution that has been sterilized and optionally stored in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition can be further packaged as part of a kit that includes a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at ambient room temperature or refrigerated temperature. In one embodiment, the pharmaceutical composition and / or kit comprises any of the GLP-1 receptor antagonists disclosed herein in combination with any existing therapeutic agent useful for treating hypoglycemia, including but not limited to glucose supplements (e.g., dextrose), hyperglycemic agents such as glucagon and glucagon analogs, and inhibitors of insulin secretion (e.g., diazoxide, octreotide).

[0095] According to one embodiment, the pharmaceutical composition disclosed herein is contemplated for use in a method of treating or preventing a state of hypoglycemia, more specifically, in a method of treating or preventing atypical hypoglycemia, or a medical condition associated with hypoglycemia.

[0096] The compositions of the present disclosure can be administered using any standard route of administration. Formulations suitable for parenteral administration can include aqueous and non-aqueous, isotonic sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that can include suspending, solubilizing, thickening, stabilizing, and preserving agents. The term "parenteral" means via a route other than through the gastrointestinal tract, such as subcutaneous, intramuscular, intraspinal, or intravenous. In one embodiment, the pharmaceutical composition comprising a GLP-1 receptor antagonist of the present disclosure is formulated for subcutaneous or intravenous administration. In one embodiment, the GLP-1 receptor antagonist of the present disclosure can be prepared as an aerosol formulation for administration via inhalation, either alone or in combination with other suitable components.

[0097] In one embodiment, the composition is formulated for oral delivery by co-formulating the GLP-1 receptor antagonist of the present disclosure with an absorption enhancer that can sufficiently enhance the absorption of the peptide antagonist. Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a delivery agent that has been reported to enhance the permeability of a diverse range of molecules, including peptides such as insulin, GLP-1, calcitonin, and other macromolecules such as heparin. According to one embodiment, a pharmaceutical composition for oral delivery is provided, the composition comprising the GLP-1 receptor antagonist of the present disclosure and SNAC, and optionally, the pharmaceutical composition is formulated as a tablet.

[0098] Acylation According to one embodiment, any of the peptides, dimers or multimers disclosed herein that exhibit GLP-1 antagonist activity can be further modified to have, for example, an improved therapeutic index and an extended duration of action in vivo when administered to a warm-blooded mammal including a human. More specifically, in one embodiment, the peptides and dimers disclosed herein are modified by covalent attachment of an alkyl or acyl group to the side chain of an amino acid of the antagonist peptide, optionally lysine, serine or cysteine, and the alkyl or acyl group is of a size sufficient to bind to serum albumin with high affinity. In one embodiment, the alkylated or acylated amino acid is located at the C-terminus of the GLP-1 antagonist peptide or dimer. In one embodiment, the GLP-1 antagonist peptide contains two acylated amino acids. In one embodiment, one or more of the amino acids of the GLP-1 receptor antagonist peptide are acylated with a fatty acid or a fatty diacid, optionally a C16-C18 fatty acid or a C16-C18 fatty diacid. According to one embodiment, one or more lysine residues of the GLP-1 antagonist peptide or dimer disclosed herein are modified by covalent attachment of a C16-C18 fatty acid or a C16-C18 fatty diacid to the side chain of lysine, optionally via a spacer. In one embodiment, the acylated lysine residue is the C-terminal amino acid of the GLP-1 antagonist peptide. In one embodiment, the acylated amino acid is present at position 40 of the GLP-1 antagonist peptide and at a second position selected from positions 7 or 12. In embodiments having two or more acylated amino acids, the acylated amino acids may be the same or different, and the attached acyl groups may be the same or different, provided that the acyl group is of a size sufficient to bind to serum albumin. In one embodiment, the acylated amino acid is lysine whose side chain is attached to a C16-C18 fatty acid or a C16-C18 fatty diacid, optionally via a spacer.

[0099] In one embodiment, the C16-C18 fatty acid or C16-C18 fatty diacid is attached to the side chain of an amino acid via a spacer, and the spacer comprises mini-PEG, γGlu, or any multimer or combination of mini-PEG and / or γGlu. In one embodiment, any of the GLP-1 receptor antagonist peptides disclosed herein can be optionally modified to include an acylated amino acid at positions optionally selected from 7, 12, and 40 with respect to the native sequence of exendin-4. In one embodiment, the acylated amino acid has the structure: -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p - and is a lysine residue having a C16-C18 fatty acid or C16-C18 fatty diacid attached to the lysine side chain via a spacer containing wherein k is an integer selected from 2, 4, 6, or 8, and p and q are independently integers selected from 1 or 2. In one embodiment, the spacer is -(γ-glutamic acid)-[COCH2(OCH2CH2) k NH]2-(γ-glutamic acid)-, or -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -, or -[COCH2(OCH2CH2) k NH]2-(γ-glutamic acid)2-, or -[COCH2(OCH2CH2) k NH]-(γ-glutamic acid)2-[COCH2(OCH2CH2) k NH]-, or -(γ-glutamic acid)2-[COCH2(OCH2CH2) k NH]2, or -(γ-glutamic acid)-[COCH2(OCH2CH2) k NH]-(γ-glutamic acid) p -[COCH2(OCH2CH2) kNH]-, or γ-glutamic acid, or γ-glutamic acid-γ-glutamic acid dipeptide, or γ-glutamic acid-[COCH2(OCH2CH2) k selected from the group consisting of NH]-γ-glutamic acid, wherein k is an integer selected from the range of 1 to 8, q and p are independently integers selected from the range of 1 to 8, optionally, k is 2, and q and p are independently 1 or 2. In one embodiment, the spacer is -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -, wherein k is an integer selected from the range of 1 to 4, q and p are independently integers selected from the range of 1 to 4, optionally, wherein k is 2, and q and p are independently 1 or 2, optionally, k is 2, and both q and p are 1.

[0100] In one embodiment, the GLP-1 receptor antagonist peptide optionally contains an acylated Lys located at the C-terminus of the peptide, and the side chain of Lys has the structure: -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p - and is acylated with a C16-C18 diacid via a spacer containing, wherein k is an integer selected from the range of 1 to 4, q and p are independently integers selected from the range of 1 to 4, optionally, k is 2, and q and p are independently 1 or 2, optionally, k is 2, and both q and p are 1. In one embodiment, the side chain of the acylated Lys has the structure -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -COC 16 H 32Containing CO2H, wherein k is 2, and p and q are, independently, 1 or 2. In one embodiment, DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 27) or DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 A GLP-1 receptor antagonist is provided that includes the sequence of (SEQ ID NO: 28), wherein R 20 is COOH or CONH2, and X 40 is -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -COC 14 H 28 CO2H or -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -COC 16 H 32 Lys acylated with CO2H, wherein k is 2, and p and q are independently 1 or 2.

[0101] Conjugate In some embodiments, any one of the GLP-1 antagonist peptides disclosed herein is conjugated to an immunoglobulin or a portion thereof (e.g., a variable region, CDR, or Fc region). Known types of immunoglobulins (Igs) include IgG, IgA, IgE, IgD, or IgM. The Fc region is the C-terminal region of the Ig heavy chain and is responsible for binding to Fc receptors that perform functions such as recycling (resulting in an extended half-life), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0102] In some embodiments, any one of the GLP-1 antagonist peptides disclosed herein is conjugated to a hydrophilic moiety. The hydrophilic moiety can covalently bind to the GLP-1 antagonist peptide under any suitable conditions used to react the protein with the activating polymer molecule. Activating groups that can be used to bind a water-soluble polymer to one or more proteins include, but are not limited to, sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, aziridine, oxirane, 5-pyridyl, and α-halogenated acyl groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). When conjugated to the peptide by reductive alkylation, the polymer selected should have a single reactive aldehyde such that the degree of polymerization is controlled. See, for example, Kinstler et al., Adv. Drug Delivery Rev. 54:477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54:459-476 (2002); and Zalipsky et al., Adv. Drug Delivery Rev. 16:157-182 (1995).

[0103] Suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyol (e.g., POG), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyoxyalkylene, polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymer, monomethoxy polyethylene glycol, mono (C1-C10) alkoxy or aryloxy polyethylene glycol, carboxymethyl cellulose, polyacetal, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, poly(β-amino acid) (either a homopolymer or a random copolymer), poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer (PPG) and other polyalkylene oxides, polypropylene oxide / ethylene oxide copolymer, curlanic acid or other polysaccharide polymers, ficoll or dextran and mixtures thereof. Dextran is a polysaccharide polymer of glucose subunits linked mainly by α1-6 bonds. Dextran is available in many molecular weight ranges, for example, from about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD to about 20, 30, 40, 50, 60, 70, 80 or 90 kD. In one embodiment, the hydrophilic moiety, such as a polyethylene glycol chain, has a molecular weight selected from the range of about 500 to about 40,000 daltons. In some embodiments, the hydrophilic moiety is a polyethylene glycol chain having a molecular weight selected from the range of about 500 to about 5,000 daltons, or from about 1,000 to about 5,000 daltons. In another embodiment, the hydrophilic moiety, such as a polyethylene glycol chain, has a molecular weight of about 10,000 to about 20,000 daltons.

[0104] In one embodiment, a conjugate derivative of the peptide of SEQ ID NO: 5 or SEQ ID NO: 23 is provided, wherein the dipeptide is covalently linked via a peptide bond to the N-terminus of the peptide of SEQ ID NO: 5 or SEQ ID NO: 23, and optionally, one of the amino acids of the dipeptide is an acylated amino acid. In one embodiment, the dipeptide has the structure X7X8, where X7 is an acylated amino acid, X8 is any amino acid, optionally, X7 is acylated Lys or dLys, X8 is Gly or C1-C4 N-alkylated Gly, optionally, X7 is Lys or dLys acylated with a C14-C20 fatty acid or fatty diacid, X8 is Gly or C1-C4 N-alkylated Gly (optionally Gly or sarcosine), optionally, X7 is Lys acylated with a C14-C20 fatty acid or fatty diacid via any of the spacers disclosed herein, and X8 is C1-C4 N-alkylated Gly (optionally Gly or sarcosine).

[0105] According to one embodiment, a conjugate derivative of any of the GLP-1 receptor antagonist peptides is provided, wherein a self-cleaving dipeptide is covalently linked via an amide bond to the amino acid side chain amine or N-terminal α-amine of the GLP-1 antagonist peptide disclosed herein. In one embodiment, the self-cleaving dipeptide is covalently linked to the N-terminal α-amine of the GLP-1 antagonist peptide.

[0106] In an exemplary embodiment, the self-cleaving dipeptide comprises the structure: A-B, where A is an amino acid or hydroxy acid, and B is an N-alkylated amino acid linked to the GLP-1 antagonist peptide via an amide bond between A-B and the amine of the GLP-1 antagonist peptide, and optionally, the chemical cleavage half-life (t 1 / 2 ) of A-B from the GLP-1 antagonist peptide is at least about 1 hour to about 1 week in PBS under physiological conditions. As used herein, the term "hydroxy acid" refers to an amino acid modified such that the α-carbon amino group is replaced with a hydroxyl group.

[0107] In some embodiments, the self-cleaving dipeptide has

[0108] [Chemical formula]

[0109] the general structure of wherein R1, R2, R4 and R8 are independently H, C1-C 18 alkyl, C2-C 18 alkenyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2 + )NH2, (C0-C4 alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C 12 alkyl(W1)C1-C 12 alkyl (wherein W1 is a heteroatom selected from the group consisting of N, S and O) selected from the group consisting of, R3 is C1-C 18 alkyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)NH2, (C1-C 18 alkyl)SH, (C0-C4 alkyl)(C3-C6) cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, and (C1-C4 alkyl)(C3-C9 heteroaryl) selected from the group consisting of, or R4 and R3 together with the atoms to which they are attached form a pyrrolidine ring, R5 is NHR6 or OH, R6 is H, C1-C8 alkyl, R7 is selected from the group consisting of H and OH, the chemical cleavage half-life (t 1 / 2 ) of A-B from the GLP-1 antagonist is at least about 1 hour to about 1 week in PBS under physiological conditions. In one embodiment, the dipeptide A-B covalently attaches to the N-terminal α amine of the GLP-1 antagonist amino acid sequence.

[0110] In one embodiment, the self-cleaving dipeptide

[0111]

Chemical formula

[0112] has the general structure of wherein R1 and R8 are independently H or C1-C8 alkyl, R2 and R4 are independently selected from the group consisting of H, C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, and (C1-C4 alkyl)(C6 aryl)R7, R3 is C1-C6 alkyl, R5 is NH2, R7 is selected from the group consisting of hydrogen and OH.

[0113] According to one embodiment, both the GLP-1 antagonist peptides and dimers disclosed herein can be further modified by attachment to a self-cleaving dipeptide, and the amino acids of the dipeptide are acylated with a fatty acyl group of a size sufficient to bind to serum albumin with high affinity.

[0114] In one embodiment, the amino acid "A" of the self-cleaving dipeptide "A-B" is a lysine residue acylated with a C16-C30 fatty acid or a C16-C30 diacid. In one embodiment, A and B are at least about 24 hours to about 240 hours, about 48 hours to about 168 hours, or about 48 to about 120 hours, or about 70 to about 100 hours, in a standard PBS solution under physiological conditions, of the GLP-1 antagonist peptides or dimers disclosed herein. The chemical cleavage half-life (t 1 / 2 ) of A-B is selected to provide.

[0115] In one embodiment, the self-cleaving dipeptide is

[0116]

Chemical formula

[0117] has the general structure of wherein R1 includes a side chain selected from the group consisting of C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally, a C16-C18 fatty acid or a C16-C18 diacid is optionally a spacer selected from the group consisting of γ-glutamic acid, γ-glutamic acid-γ-glutamic acid dipeptide, -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid)p, and γ-glutamic acid-[COCH2(OCH2CH2) k NH] q -γ-glutamic acid, and is covalently bonded to the side chain via any of the spacers disclosed herein, wherein k is an integer selected from the range of 1-8, q and p are independently integers selected from the range of 1-8, and optionally, k is 2, and q and p are independently 1 or 2, R2, R4 and R8 are independently H or C1-C4 alkyl, R3 is C1-C6 alkyl, R5 is NH2.

[0118] In one embodiment, the self-cleaving dipeptide is

[0119]

Chemical formula

[0120] has the general structure of wherein R1 is H, C1-C4 alkyl, (C1-C4 alkyl)OH or (C1-C4 alkyl)NH2, R3 is C1-C4 alkyl, R4 is H or C1-C4 alkyl, R5 is NH2, R8 is hydrogen.

[0121] According to one embodiment, the self-cleaving dipeptide A-B comprises an acylated amino acid residue as "A" and an N-alkylated Gly residue as "B", and the "B" amino acid is bonded to the N-terminal alpha amine of the GLP-1 receptor antagonist peptide via an amide bond, and optionally, the Lys residue is in the D-conformation. In one embodiment, the acylated amino acid "A" of the A-B dipeptide is

[0122]

Chemical formula

[0123] is an amino acid having the general structure of wherein n is an integer selected from the range of 1 to 4, and R 50 is NH-CO(CH2) 14~20 COOH, NH-[spacer]-CO(CH2) 14~20 COOH, S(CH2) 14~20 COOH and S-[spacer]-CO(CH2) 14~20Selected from the group consisting of COOH, and [Spacer] is any of the spacers disclosed herein. In one embodiment, the acylated amino acid of A is independently selected from lysine, d-lysine, ornithine, cysteine or homocysteine, and the side chain of the acylated amino acid is optionally covalently bonded to a C16-C22 fatty acid or a C16-C22 diacid via a spacer containing an amino acid or a dipeptide. In one embodiment, the spacer contains γ-glutamic acid. In one embodiment, the spacer contains multiple units of γ-glutamic acid and miniPeg polymer in any combination. In one embodiment, any spacer contains two γ-glutamic acids, and optionally, the two γ-glutamic acids are linked to each other via an intervening functionalized miniPEG polymer [COCH2(OCH2CH2)kHN]q, where k and q are each an integer independently selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0124] In one embodiment, the self-cleaving dipeptide is

[0125]

Chemical formula

[0126] has the general structure of wherein R1 is (C4 alkyl)NH2 or (C4 alkyl)NH(mPeg-γE-diacid)-C18, R2, R4 and R8 are each H, R3 is C1-C4 alkyl, R5 is NH2, and optionally, the first amino acid of the self-cleaving dipeptide is in the D-conformation.

[0127] In one embodiment, the GLP-1 antagonist peptides and dimers disclosed herein have the structure:

[0128]

Chemical formula

[0129] is covalently bound to the self-cleaving dipeptide, wherein, R1 is a side chain selected from the group consisting of C1-C 18 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally, a C16-C20 fatty acid or a C16-C20 diacid is covalently bound to the side chain, R2, R4 and R8 are independently H or C1-C4 alkyl, R3 is C1-C4 alkyl or R4 and R3 together with the atoms to which they are attached form a pyrrolidine ring, R5 is NH2, provided that when R4 and R3 together with the atoms to which they are attached form a pyrrolidine ring, R2 is not H.

[0130] In a further embodiment, the self-cleaving dipeptide has

[0131]

Chemical formula

[0132] the structure of, wherein R1 is (C1-C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1-C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1-C4 alkyl)NH-CO(CH2) 14~20 COOH or (C1-C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, R2 and R8 are each H, R4 is H or CH3, R3 is C1-C4 alkyl, R5 is NH2, and optionally, the first amino acid of the self-cleaving dipeptide is an amino acid in the D-configuration, and the spacer is selected from any of the spacers disclosed herein. In one embodiment, R2, R4 and R8 are each H.

[0133] In one embodiment, R1 is (C1-C4 alkyl)NH-CO(CH2) 16 COOH or (C1-C4 alkyl)NH-[spacer]-CO(CH2) 16 COOH, R2, R4 and R8 are each H, R3 is C1-C4 alkyl, optionally CH3, and optionally, the spacer has the structure: -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p - and wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0134] According to one embodiment, the self-cleaving dipeptide comprises an acylated amino acid as the first amino acid, the side chain of the acylated amino acid is acylated with a C16-C20 fatty acid or a C16-C20 diacid, optionally, the acylated amino acid is selected from C16-C20 acylated lysine, C16-C20 acylated ornithine, C16-C20 acylated cysteine and C16-C20 acylated homocysteine, optionally, the acylated amino acid of the dipeptide is C16-C20 acylated Lys, and optionally, the first amino acid of the self-cleaving dipeptide is an amino acid in D-configuration. Embodiment According to Embodiment 1, a GLP-1 receptor antagonist is provided, wherein the antagonist is R 10 -DVX 11 X 12 YLX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 (SEQ ID NO: 23) and wherein R 10is NH2 or the N-terminal extension of one or two amino acids, and one of the amino acids in the N-terminal extension is optionally acylated with a C14-C20 fatty acid or diacid via a spacer, and optionally, R 10 is a dipeptide of the structure: X7X8 (where X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1-C4 N-alkylated Gly), X 11 is Trp, dTrp or Ser, X 12 is Arg or an acylated amino acid, optionally acylated Lys, X 15 is Glu or dGlu, X 16 is Glu, dGlu, Asp, homoglutamic acid or homocysteic acid, X 19 is Val, cyclopropane, cyclopentane, cyclohexane or phenylglycine, X 20 is Arg, homolysine or citrulline, X 23 is Ile, or dIle, X 40 is an acylated amino acid, optionally acylated Lys, R 20 is COOH or CONH2, and optionally, one, two or three amino acids selected from positions 7, 10, 13 or 16 are substituted with Trp or dTrp, provided that X 10 or X 11 if one of them is Trp or dTrp, the other is not Trp or dTrp, and R 10 and X 12 are both not allowed to contain acylated amino acids, and optionally, one, two or three amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted with Aib.

[0135] According to Embodiment 2, the GLP-1 antagonist of Embodiment 1 is provided, wherein the antagonist is R 10 -DVX 11 X 12 YLX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 (including the amino acid sequence of SEQ ID NO: 23), wherein R 10 is NH2 or X7X8 (wherein X7 is an amino acid acylated with a C14-C20 fatty acid or a diacid, optionally via a spacer, and X8 is Gly or C1-C4 N-alkylated Gly), X 11 is Trp, dTrp or Ser, X 12 is Arg or an acylated amino acid, optionally an acylated Lys, X 15 is Glu or dGlu, X 16 is Glu, dGlu, Asp, homoglutamic acid or homocysteic acid, X 19 is Val, cyclopropane, cyclopentane, cyclohexane or phenylglycine, X 20 is Arg, homolysine or citrulline, X 23 is Ile, or dIle, X 40 is an acylated amino acid, optionally an acylated Lys, R 20 is COOH or CONH2, and optionally, one, two, or three of the amino acids at any of positions 16, 18, 19, 24, 26, or 28 are substituted with Aib.

[0136] According to Embodiment 3, the GLP-1 antagonist of Embodiment 1 or 2 is provided, wherein each acylated amino acid of the GLP-1 antagonist is acylated Lys. According to Embodiment 4, the GLP-1 antagonist according to any one of Embodiments 1 to 3 is provided, wherein X7 is acylated Lys, and X 12 is Arg.

[0137] According to Embodiment 5, the GLP-1 antagonist according to any one of Embodiments 1 to 3 is provided, wherein R 10 is NH2, and X 12 is acylated Lys. According to Embodiment 6, the GLP-1 antagonist according to any one of Embodiments 1 to 3 is provided, wherein X7 is NH2, and X 12 is Arg.

[0138] According to Embodiment 7, the GLP-1 antagonist of Embodiment 1 is provided, and this antagonist comprises an amino acid sequence selected from any one of SEQ ID NOs: 5 to 96, or any combination thereof.

[0139] According to Embodiment 8, the GLP-1 antagonist according to any one of Embodiments 1 to 7 is provided, wherein the acylated amino acids of the GLP-1 antagonist are independently Lys residues acylated with C16-C18 fatty acids or C16-C18 fatty diacids directly bonded to the Lys side chain or optionally bonded via a spacer, and the spacer is (i) γ-glutamic acid, (ii) minipegpolymer: -[COCH2(OCH2CH2) k NH]-(wherein k is 2, 4, 6 or 8), (iii) or any multiplicity or combination of i) and / or ii).

[0140] According to Embodiment 9, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 8, wherein the antagonist is DX 10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 5) and contains the amino acid sequence, where X 10 is Trp, dTrp or Val, X 11 is Trp, dTrp or Ser, X 15 is Glu or dGlu, X 16 is Trp, dTrp, dGlu or Glu, X 23 is Ile or dIle, X 40 is an acylated amino acid, R 20 is COOH or CONH2. Optionally, this peptide contains one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28, or optionally contains a substitution of acylated Lys at position 12, and the position numbers are relative to the native exendin-4 amino acid sequence.

[0141] According to Embodiment 10, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 9, where X 11 is Trp or dTrp. According to Embodiment 11, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 10, where the amino acid at any of positions 16, 18, 19, 24, 26 or 28 of SEQ ID NO: 5 is substituted with Aib, and optionally, Aib is substituted at position 18.

[0142] According to Embodiment 12, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 11, wherein the amino acid at position 12 is replaced with acylated Lys. According to Embodiment 13, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 11, wherein X 15 is dGlu, X 16 is Glu, X 23 is Ile.

[0143] According to Embodiment 14, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 11, wherein X 15 is Glu, X 16 is Glu, X 23 is Ile.

[0144] According to Embodiment 15, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 14, wherein X 40 is an amino acid having an acyl group bonded to the side chain of the amino acid, optionally via a spacer.

[0145] According to Embodiment 16, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 15, wherein X 40 is an amino acid containing the structure of Formula I (optionally Lys), Formula II (optionally Ser), or Formula III (optionally Cys), and each of Formulas I, II, and III is

[0146]

Chemical formula

[0147] as follows. According to Embodiment 17, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 16, wherein X 40is an acylated lysine.

[0148] According to Embodiment 18, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 17, wherein the acyl group of the acylated amino acid is (C1-C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1-C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1-C4 alkyl)NH-CO(CH2) 14~20 COOH or (C1-C4 alkyl)NH-[spacer]-CO(CH2) 14~20 is selected from COOH.

[0149] According to Embodiment 19, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 18, wherein the acyl group of the acylated amino acid is covalently bonded to the amino acid side chain of the acylated amino acid via a spacer.

[0150] According to Embodiment 20, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 19, wherein the spacer is an amino acid or a dipeptide. According to Embodiment 21, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 20, wherein the spacer is (i) γ-glutamic acid, (ii) minipeg polymer: -[COCH2(OCH2CH2) k NH]-(wherein k is 2, 4, 6 or 8), (iii) or includes any multiplicity or combination of i) and / or ii).

[0151] According to Embodiment 22, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 11, wherein the acylated amino acid is bonded to a C16-C18 fatty acid or a C16-C18 fatty diacid, and optionally, the acid or diacid has the structure: -(γ-glutamic acid) p -[COCH2(OCH2CH2) k NH]q -(γ-glutamic acid) n - or -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p -[COCH2(OCH2CH2) k NH] m - are linked via a spacer containing wherein k is an integer selected from 2, 4 or 8, m and n are independently integers selected from 0, 1 or 2, and p and q are independently integers selected from 1, 2, 4 or 8.

[0152] According to Embodiment 23, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 22, wherein the acylated amino acid is Lys having a C16 to C18 fatty acid bonded to the lysine side chain via a spacer, and the spacer has the structure: -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) p - and contains wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0153] According to Embodiment 24, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 23, wherein X 11 is Trp or dTrp, X 15 is Glu or dGlu, X 16 is Glu or dGlu, X 23 is Ile or dIle, X 40 is Lys acylated with a C16 or C18 diacid, and optionally, the diacid has the structure: -[COCH2(OCH2CH2) k NH] q -(γ-glutamic acid) plinked via a spacer containing - wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0154] According to Embodiment 25, there is provided a GLP-1 antagonist according to any one of Embodiments 1 to 24, wherein R 20 is CONH2. According to Embodiment 26, a dipeptide A-B linked to the GLP-1 antagonist via an amide bond:

[0155]

Chemical formula

[0156] There is provided a derivative of the GLP-1 antagonist according to any one of Claims 9 to 25, further comprising wherein R1, R2, R4 and R8 are independently H, C1-C 18 alkyl, C2-C 18 alkenyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2 + )NH2, (C0-C4 alkyl)(C3-C6 cycloalkyl), (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, (C1-C4 alkyl)(C3-C9 heteroaryl), and C1-C 12 alkyl(W1)C1-C 12 alkyl (wherein W1 is a heteroatom selected from the group consisting of N, S and O), and are selected from the group consisting of R3 is C1-C 18 alkyl, (C1-C 18 alkyl)OH, (C1-C 18 alkyl)NH2, (C1-C 18(alkyl)SH, (C0-C4 alkyl)(C3-C6) cycloalkyl, (C0-C4 alkyl)(C2-C5 heterocycle), (C0-C4 alkyl)(C6-C 10 aryl)R7, and are selected from the group consisting of (C1-C4 alkyl)(C3-C9 heteroaryl), or R4 and R3, together with the atoms to which they are attached, form a pyrrolidine ring, R5 is NHR6 or OH, R6 is H, C1-C8 alkyl, R7 is selected from the group consisting of H and OH, The chemical cleavage half-life (t 1 / 2 ) of A-B from the GLP-1 antagonist is at least about 1 hour to about 1 week in PBS under physiological conditions.

[0157] According to Embodiment 27, a GLP-1 antagonist of Embodiment 26 is provided, wherein the dipeptide A-B covalently binds to the N-terminal α amine of the GLP-1 antagonist amino acid sequence.

[0158] According to Embodiment 28, a GLP-1 antagonist according to any one of Embodiments 26 to 27 is provided, wherein R1 and R8 are independently H or C1-C8 alkyl, R2 and R4 are independently selected from the group consisting of H, C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, and (C1-C4 alkyl)(C6 aryl)R7, R3 is C1-C6 alkyl, R5 is NH2, R7 is selected from the group consisting of hydrogen and OH.

[0159] According to Embodiment 29, a GLP-1 antagonist according to any one of Embodiments 26 to 27 is provided, wherein R1 contains a side chain selected from the group consisting of C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally, C16-C30 fatty acid or C16-C30 diacid, and optionally, γ-glutamic acid, γ-glutamic acid-γ-glutamic acid dipeptide, and γ-glutamic acid-[COCH2(OCH2CH2) k NH] q -γ-glutamic acid is covalently bonded to the side chain via a spacer selected from the group consisting of, wherein k is an integer selected from the range of 1 to 8, q is an integer selected from the range of 1 to 8, optionally, k is 2, q is selected from the range of 1 to 8, R2, R4 and R8 are independently H or C1-C4 alkyl, R3 is C1-C6 alkyl, R5 is NH2.

[0160] According to Embodiment 30, there is provided a GLP-1 antagonist according to any one of Embodiments 26 to 29, wherein R1 is H, C1-C4 alkyl, (C1-C4 alkyl)OH or (C1-C4 alkyl)NH2, R2 is H, R3 is C1-C4 alkyl, R4 is H, or C1-C4 alkyl, R5 is NH2, R8 is hydrogen.

[0161] According to Embodiment 31, there is provided a GLP-1 antagonist according to any one of Embodiments 26 to 30, wherein the dipeptide A-B contains an acylated Lys residue and an N-alkylated Gly residue, and the Lys and N-alkylated Gly residues are bonded via a peptide bond, and optionally, the Lys residue is in the D-conformation.

[0162] According to Embodiment 32, there is provided a GLP-1 antagonist according to any one of Embodiments 26 to 30, wherein R1 is (C4 alkyl)NH2 or (C4 alkyl)NH(mPeg-γE-diacid)-C18, R2, R4, and R8 are each H, R3 is C1-C4 alkyl, R5 is NH2, R5 is an amine.

[0163] According to Embodiment 33, there is provided a pharmaceutical composition comprising the GLP-1 antagonist or derivative according to any one of Claims 1 to 32 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0164] According to Embodiment 34, there is provided a method for treating a patient suffering from atypical hypoglycemia, wherein the method comprises administering to a patient in need of treatment the pharmaceutical composition of Embodiment 33 in an amount effective to increase blood glucose levels.

[0165] Example 1 Ex-4(9-39)a (SEQ ID NO: 2) is an established antagonist of the GLP-1 receptor. However, its use as a therapeutic agent in humans is limited because the therapeutic agent is not of human origin and its in vivo duration of action is relatively short. When the N-terminus of human GLP-1 is similarly shortened, agonism decreases, but a high-potency antagonist cannot be obtained. Through a series of GLP-1 / Ex-4 hybrid peptides, it was confirmed that the minimal structural changes required to generate a pure antagonist derived from GLP-1 are Glu16, Val19, and Arg20, and an antagonist with approximately 3-fold higher potency in vitro was obtained compared to Ex-4(9-39)a. Site-specific acylation of the human-derived antagonist increased the potency as a GLP-1 receptor antagonist and increased the selectivity for the GIP receptor by 10-fold, resulting in the peptide "9-40Jant4-K" 40"(SEQ ID NO: 3)" was obtained (Patterson et al., ACS Chem. Biol. 2011, 6, 135 - 145).

[0166] As disclosed herein, variants of 9 - 40 Jant4 - K 40 were prepared to provide further improved GLP - 1 receptor antagonists. Peptide 9 - 40 Jant4 - K 40 is acylated at position 40 by direct attachment of a C16 acyl group to the Lys side chain. Variants were prepared by inserting a spacer between the Lys side chain and the C16 acyl group. Table 2 shows the GLP - 1 antagonist activity and solubility of these acylated variants.

[0167] 9 - 40 Jant4 - K 40 (DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK, SEQ ID NO: 3) was subjected to various amino acid substitutions, and the GLP - 1 antagonist activity and solubility of these variants are shown in Table 3.

[0168] Table 4 shows data regarding the effect of Aib substitution on the activity and solubility of the Jant4 - K40 (SEQ ID NO: 3) variant. Table 5 shows data regarding the effect of d - AA substitution on the solubility of the Jant4 - K40 (C16) (SEQ ID NO: 3) variant, and Table 6 shows data regarding Trp substitution at position 3 of Jant4 - K40 (SEQ ID NO: 3). Materials and Methods Fmoc Synthesis Peptides were prepared by an automated Fmoc / t-Bu solid-phase method using Wang resin (AAPPtec, Louisville, KY) and a Symphony peptide synthesizer (Peptide Technology, Tucson, AZ) starting with 6-Cl-HOBt / DIC activation. All conventional residues were purchased from Midwest Biotech (Fisher, IN), and 6-Cl-HOBt and DIC were obtained from AAPPtec (Louisville, KY). Peptides were cleaved from the resin and deprotected by treatment with TFA containing 2.5% TIS, 2.5% H2O, 1.5% methanol, 2.5% phenol, 0.5% DODT, and 0.5% dimethyl sulfoxide. Peptides were precipitated from the filtered TFA solution using cold ethyl ether according to standard procedures. Fatty acylation of the peptides was performed on resin using 10-fold excess Fmoc-Glu-OtBu / DEPBT / DIEA, repeating double coupling, followed by 10-fold excess palmitic acid or another fatty acid / DEPBT / DIEA.

[0169] Antagonist acylation Palmitic acid was introduced into the synthesized antagonist peptide using an orthogonal solid-phase protection scheme. Boc synthesis was utilized for peptide synthesis, enabling the selective introduction of base-sensitive side-chain protected Lys(Fmoc)-OH at Lys40. The fully protected peptide was treated on resin with 20% piperidine (v / v) in DMF for 30 minutes to remove the Lys40 side-chain Fmoc group. Amide bond formation was facilitated with an excess of fatty acid and 5 equivalents of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Fluka) in DMF / DIEA (4:1 v / v) for approximately 18 hours. The progress of the reaction was monitored using the ninhydrin test, and acylation was confirmed by ESI mass spectrometry after peptide cleavage.

[0170] Peptide purification Reverse-phase HPLC (RP-HPLC) was used for peptide purification. During preparative RP-HPLC purification, a C18 stationary phase (Vydac 218 TP, 250 mm_22 mm, 10 μm) was used with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. RP-HPLC using a C8 column (Zorbax 300 SB, 4.6 mm × 50 mm, 3.5 μm) was used to analyze the peak fractions. Peptide identity and purity were evaluated by analytical RP-HPLC and ESI- or MALDI-mass spectrometry. All peptides were found to have the correct molecular weight and were of approximately 95% purity. The lyophilized peptides were stored at 4°C.

[0171] GLP-1 receptor-mediated cAMP induction The ability of peptides to stimulate or block cAMP induction at the GLP-1 receptor was tested by a luciferase-based reporter gene assay. A cell construct capable of measuring receptor activation was constructed by co-transfecting HEK293 cells with the human GLP-1 receptor (Open Biosystems) and the cAMP-inducible (cAMP response element) luciferase gene. The bioassay was performed by first depleting the cells of serum in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 0.25% bovine growth serum (HyClone) for 16 h and then adding serial dilutions of the peptide over an appropriate concentration range in 96-well poly-D-lysine-coated plates (BD Biosciences). For the antagonism assay, a fixed concentration of GLP-1 (0.05 nM) was added to the assay plates after diluting the peptide. Incubation was continued at 37°C, 5% CO2 for 5 h, followed by the addition of an equal volume (100 μL) of LucLite luminescence substrate reagent (Perkin-Elmer).

[0172] MicroBeta 1450 liquid scintillation counting (Perkin-Elmer) was used to quantify the luminescence signal in counts per second (cps) after shaking the plate at 600 rpm for 3 minutes. Data were plotted using Origin software (OriginLab), and the effective concentration 50 (EC 50 ) or the inhibitory concentration 50 (IC 50 ) was determined by sigmoid fitting. Potency was determined by comparative analysis of relative EC 50 or IC 50 values. Each experiment was repeated at least 3 times, and each sample was assayed in duplicate.

[0173] Animals C57Bl / 6 mice were obtained from Jackson Laboratories. Mice were housed individually or in groups at 22 °C on a 12:12 hour light-dark cycle with free access to food and water. All studies were approved and conducted in accordance with the guidelines of the Animal Care and Use Committee of the University of Cincinnati.

[0174] Glucose tolerance test (GTT) To determine the glucose load, mice were fasted for 6 hours and then injected intraperitoneally with glucose. The injection consisted of 1.5 g of glucose per kg of body weight (25% w / v D-glucose (Sigma) in 0.9% w / v saline). Blood glucose levels (mg dL-1) in the tail were measured using a glucometer (FreeStyle Freedom Lite) at 0 minutes before injection and at 15, 30, 60, and 120 minutes after injection.

[0175] Acute in vivo test Before IP injection of dipeptidyl peptidase-IV protected Ex-4 (0.65 nmol kg-1), mice were injected subcutaneously with different doses of the peptide (in PBS) at either 1, 4, 8, 24, 48, 72, or 120. The GTT was performed 15 minutes after GLP-1 injection. Blood glucose values in the tail were obtained as described above.

[0176] [Table 2-1]

[0177]

Table 2-2

[0178]

Table 3

[0179]

Table 4

[0180]

Table 5

[0181]

Table 6

[0182] Example 2 Synthesis procedure of MBX-1416: The synthesis of MBX-1416 was carried out using the starting solid-phase resin of Fmoc-Ser(tBu)-Wang on a 0.1 mmol scale. The Fmoc-amino acids constituting residues 9 - 38 (Asp9 - Pro38) were sequentially coupled to the free N-terminal amine after removing the first Fmoc group from the Fmoc-Ser(tBu)-Wang resin. An ABI peptide synthesizer was used for automated assembly using a standard Fmoc / DIC / oxyma synthesis protocol as shown in the following table. Details of the Fmoc_DIC_oxyma_0.10 mmol protocol used with the ABI433A Synthesizer

[0183]

Table 7

[0184] C18 diacid coupling procedure: The N-terminal C18 diacid was coupled to the peptidyl resin after the complete peptide from Asp9 to SeR39 was constructed. This was introduced as tert-butyl protected C18 diacid (1 mmol) using DIC (1 mmol) and oxyma (1 mmol) in DMF. tert-Butyl protected C18 diacid (1 mmol) and oxyma (1 mmol) were dissolved in DMF (10 mL). DIC (1 mmol) was added to this mixture and pre-activated at room temperature for 10 - 15 minutes. This reaction mixture was added to the Asp9 - SeR39 peptidyl resin and coupling was allowed to proceed with stirring at room temperature for 4 hours. Purification of MBX-1416: MBX-1416 was purified using a Gemini (registered trademark) 10μm C8 100Å, LC 250×21.2mm reverse phase column (catalog number 00G-4763-PO-AX) with a linear gradient from 80%A and 20%B to 20%A and 80%B over 90 minutes at a flow rate of 15 ml / min by a Waters HPLC controller 600. The two buffers consisted of 0.1% TFA in 10% acetonitrile and 90% H2O (buffer A) and 0.1% TFA in 100% acetonitrile (buffer B).

Claims

1. R １０ -DVX １１ X １２ YLX １５ X １６ QAX １９ X ２０ EFX ２３ X ２４ WLVRGGPPSSGAPPPPS-R ２０ including the amino acid sequence of (SEQ ID NO: 97), R １０ However, it is a C14-C20 fatty acid or diacid covalently bonded to the N-terminal α-amine of the GLP-1 receptor antagonist amino acid sequence. X １１ However, it is Trp, dTrp or Ser, X １２ However, it is Arg or Ser, X １５ However, it is either Glu or dGlu. X １６ However, it is Glu, dGlu, Asp, homoglutamic acid, or homocysteic acid. X １９ However, these are Val, cyclopropane, cyclopentane, cyclohexane, or phenylglycine. X ２０ However, it is Arg, homolysine, or citrulline. X ２３ However, it is either Ile or dIle, X ２４ However, it is either Glu or Ala, R ２０ However, COOH or CONH ２ That is, GLP-1 receptor antagonist.

2. A GLP-1 receptor antagonist according to claim 1, wherein, based on the numbering of natural exendin 4 (SEQ ID NO: 1), one, two, or three amino acids at any of positions 16, 18, 19, 24, 26, or 28 are substituted with Aib.

3. The aforementioned antagonist, R １０ - DVX １１ X １２ YLX １５ X １６ QAX １９ X ２０ EFX ２３ EWLVRGGPSSGAPPPS-R ２０ (SEQ ID NO: 99) contains the amino acid sequence, R １０ However, it is a C14-C20 fatty acid or diacid covalently bonded to the N-terminal α-amine of the GLP-1 receptor antagonist amino acid sequence. X １１ However, it is Trp, dTrp or Ser, X １２ However, it is Arg or Ser, X １５ However, it is either Glu or dGlu. X １６ However, it is Glu, dGlu, Asp, homoglutamic acid, or homocysteic acid. X １９ However, it is Val, X ２０ However, it is Arg, homolysine, or citrulline. X ２３ However, it is either Ile or dIle, R ２０ However, COOH or CONH ２ That is, The GLP-1 receptor antagonist according to claim 1.

4. A GLP-1 receptor antagonist according to claim 3, wherein, based on the numbering of natural exendin 4 (SEQ ID NO: 1), one, two, or three amino acids at any of positions 16, 18, 19, 24, 26, or 28 are substituted with Aib.

5. R １０ However, -CO(CH ２ ) １４～２０ CH ３ or -CO(CH ２ ) １４～２０ COOH, X １１ However, it is Trp, dTrp or Ser, X １２ However, it is Ser, X １５ However, it is either Glu or dGlu. X １６ However, it is either Glu or dGlu. X １９ However, it is Val, X ２０ However, it is Arg, X ２３ However, it is Ile or dIle, R ２０ However, it is COOH. The GLP-1 receptor antagonist according to claim 1.

6. R １０ However, -CO(CH) is covalently bonded to the N-terminal α-amine of the GLP-1 receptor antagonist amino acid sequence. ２ ) １６～１８ COOH The GLP-1 receptor antagonist according to claim 1.

7. A GLP-1 receptor antagonist according to claim 1, wherein the amino acids at positions 16, 18, 19, 24, 26, or 28 are substituted with Aib, based on the numbering of natural exendin 4 (SEQ ID NO: 1).

8. The GLP-1 receptor antagonist according to claim 1, further comprising a C-terminal extension of 1 to 3 amino acids.

9. The GLP-1 receptor antagonist according to claim 8, wherein one of the amino acids in the C-terminal extension contains an acylated amino acid.

10. The GLP-1 receptor antagonist according to claim 8, wherein one of the amino acids in the C-terminal extension contains acylated Lys.

11. R １０ -DX １０ X １１ X １２ YLX １５ X １６ QAVREFX ２３ X ２４ WLVRGGPSSGAPPPS-R ２０ (SEQ ID NO: 98) contains the amino acid sequence, R １０ However, it is a C14-C20 fatty acid or diacid covalently bonded to the N-terminal α-amine of the GLP-1 receptor antagonist amino acid sequence. X １０ However, it is Trp, dTrp, or Val, X １１ However, it is Trp, dTrp or Ser, X １２ However, it is Arg, Lys or Ser, X １５ However, it is either Glu or dGlu. X １６ However, it is Trp, dTrp, dGlu, or Glu. X ２３ However, it is either Ile or dIle, X ２４ However, it is either Ala or Glu. R ２０ However, COOH or CONH ２ That is, GLP-1 receptor antagonist.

12. R １０ However, the -CO(CH) bonded to the N-terminal α-amine of the GLP-1 receptor antagonist ２ ) １４～２０ CH ３ or -CO(CH ２ ) １４～２０ COOH, X １０ However, it is Trp, dTrp, or Val, X １１ However, it is Trp, dTrp or Ser, X １２ However, it is Arg, Lys or Ser, X １５ However, it is either Glu or dGlu. X １６ However, it is Trp, dTrp, dGlu, or Glu. X ２３ However, it is either Ile or dIle, X ２４ However, it is either Ala or Glu. The GLP-1 receptor antagonist according to claim 11.

13. R １０ -DVSSYLEEQAVREFIAWLVKGGPPSSGAPPPS-R ２０ the amino acid sequence of (SEQ ID NO: 3), or an amino acid sequence that differs from SEQ ID NO: 3 by 1, 2, or 3 amino acid substitutions while retaining GLP-1 agonist activity, R １０ is -CO(CH ２ ), １４～２０ CH ３ or -CO(CH ２ ), １４～２０ COOH that binds to the N-terminal α -amine of a GLP-1 receptor antagonist, R ２０ However, COOH or CONH ２ That is, GLP-1 receptor antagonist.

14. The GLP-1 receptor antagonist according to claim 13, wherein the GLP-1 receptor antagonist comprises one to three substitutions of Aib in any combination of positions 16, 18, 19, 24, 26, or 28, the position numbers corresponding to the native exendin 4-amino acid sequence.

15. R １０ However, -CO(CH ２ ) １４～２０ COOH, R ２０ However, it is COOH. The GLP-1 receptor antagonist according to claim 13.

16. X １１ The GLP-1 receptor antagonist according to claim 11 or 13, wherein the antagonist is Trp or dTrp.

17. A GLP-1 receptor antagonist according to claim 11 or 13, wherein the amino acid at position 16, 18, 19, 24, 26, or 28 is substituted with Aib.

18. X １５ However, it is dGlu, X １６ However, it is Glu, X ２３ But it is Ile. The GLP-1 receptor antagonist according to claim 13.

19. X １５ However, it is Glu, X １６ However, it is Glu, X ２３ But it is Ile. The GLP-1 receptor antagonist according to claim 13.

20. The GLP-1 receptor antagonist according to claim 13, further comprising a C-terminal extension, wherein the amino acid in the C-terminal extension is an acylated amino acid having an acyl group attached to the side chain of the amino acid.

21. The GLP-1 receptor antagonist according to claim 20, wherein the acyl group is bonded to the side chain of an amino acid via a spacer.

22. The GLP-1 receptor antagonist according to claim 20, wherein the acylated amino acid is acylated lysine.

23. The GLP-1 receptor antagonist according to claim 22, wherein the acyl group of the acylated amino acid is covalently bonded to the amino acid side chain of the acylated amino acid via a spacer.

24. The spacer, i) γ-glutamic acid, ii) Mini Peg Polymer: - [COCH ２ (OCH ２ CH ２ ) ｋ NH]- (wherein k is 2, 4, 6 or 8), or Including any multiplicity or combination of iii)i) and / or ii), The GLP-1 receptor antagonist according to claim 23.

25. R ２０ However, CONH ２ The GLP-1 receptor antagonist according to claim 13.

26. A pharmaceutical composition comprising a GLP-1 receptor antagonist according to any one of claims 1, 11, and 13, and a pharmaceutically acceptable carrier, diluent, or excipient.

27. A method for treating a patient suffering from atypical hypoglycemia, comprising the step of administering to the patient in need of treatment an amount of the pharmaceutical composition according to claim 26 that is effective in raising blood glucose levels.

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