Glucagon-like peptide-1 receptor antagonists

Abstract

Glucagon-like peptide-1 (GLP-1) plays an important role in regulating the blood sugar level of a human body. The compound has the effects of stimulating insulin synthesis and secretion, inhibiting glucagon secretion and inhibiting food intake. Provided herein are GLP-1 receptor antagonist peptides and pharmaceutical compositions for the treatment of hypoglycemia. Further provided herein are methods of treating atypical hypoglycemia in patients who have changed to hyperinsulinemia, including patients who have changed to hyperinsulinemia following a weight loss surgery.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 395,783, filed on August 5, 2022, the disclosure of which is expressly incorporated herein.

[0003] Incorporation by Reference of Materials Submitted Electronically

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file and is hereby incorporated by reference in its entirety. The XML copy was created on August 1, 2023, is named 32993-392614.2_SL.xml, and is 281 kilobytes in size. Background Art

[0005] Glucagon-like peptide-1 (GLP-1) plays an important role in regulating the body's blood sugar levels. Its effects 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 a range of approximately 70 to 110 milligrams per deciliter (mg / dL) or 3.9 to 6.1 millimoles per liter (mmol / L). However, blood sugar levels may be too low, resulting in hypoglycemia. Hypoglycemia is most often caused by medications taken to control diabetes. Less common causes of hypoglycemia are referred to herein as "atypical hypoglycemia" and can occur independently of the administration of exogenous insulin.

[0006] Atypical hypoglycemia can occur in people who drink a lot of alcohol without eating because alcohol can prevent the formation of glucose in the liver. Also, people with advanced liver disease such as viral hepatitis, cirrhosis, or liver cancer may not be able to store and produce enough glucose. Atypical hypoglycemia can also occur in infants and children with congenital mutations that cause them to have hyperinsulinemia.

[0007] More recently, over the past decade, there has been increasing recognition that atypical hypoglycemia is a complication that may occur following surgical procedures performed to reverse extreme forms of obesity. Some patients (less than 10%) who undergo Roux-en-Y gastric bypass (RYGB) surgery subsequently develop hyperinsulinemic hypoglycemia, in which blood glucose concentrations may become low enough (20-40 mg / dL) to cause seizures, altered mental status, loss of consciousness, cognitive impairment, disability, and death.

[0008] One approach to treating hypoglycemia caused by hyperinsulinemia is to administer GLP-1 receptor antagonists. Such peptide antagonists block the ability of inappropriately elevated plasma GLP-1 concentrations to stimulate insulin secretion and act to normalize plasma glucose and reduce the risk of cognitive impairment, vascular disease, and potentially sudden death in patients experiencing atypical hypoglycemia.

[0009] 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-terminally truncated derivative of Exendin-4, which is known to function as a GLP-1 receptor antagonist. However, Ex-4(9-39)a has two significant limitations in its potential use for the treatment of chronic atypical hypoglycemia: its non-human amino acid sequence and its relatively short duration of action in vivo.

[0010] According to one embodiment of the present disclosure, a group of novel optimized GLP-1 antagonists are provided for use as candidate drugs for treating atypical hypoglycemia, including hyperinsulinemia-induced hypoglycemia caused by bariatric surgery or caused by congenital mutations. Summary of the invention

[0011] As disclosed herein, compositions and methods are provided for treating patients experiencing atypical hypoglycemia, and more particularly in one embodiment, for treating patients experiencing hyperinsulinemia-induced hypoglycemia. According to one embodiment, compositions and methods are provided for treating hyperinsulinemia-induced hypoglycemia resulting from bariatric surgery. In one embodiment, the method comprises administering an amount of a glucagon-like peptide-1 receptor antagonist (GLP1RA) effective to increase blood glucose levels and alleviate associated acute symptoms and chronic consequences associated with hypoglycemia.

[0012] According to one embodiment, a GLP-1 receptor antagonist peptide is provided, which comprises the following amino acid sequence

[0013] R 10 -DX 10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:5), wherein

[0014] X 10is Trp, dTrp or Val; X 11 is Trp, dTrp or Ser; X 15 Is Glu or dGlu

[0015] X 16 is Trp, dTrp, dGlu or Glu; X 23 It is Ile or dIle;

[0016] X 40 Absent or acylated amino acid,

[0017] R 10 NH2, -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH and

[0018] R 20 is COOH or CONH2, optionally wherein, relative to the native Exendin4 sequence (SEQ ID NO: 1), the peptide comprises one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28, or optionally, relative to the native Exendin4 sequence (SEQ ID NO: 1), a substitution of Lys at position 12, optionally with the proviso that when X 10 or X 11 one of them is Trp or dTrp, and the other is not Trp or dTrp, optionally provided that when X 40 When it does not exist, R 10 In one embodiment, a GLP-1 receptor antagonist of SEQ ID NO: 5 is provided, wherein X 40 is an acylated Lys, wherein the acyl group of the acylated Lys is a C16-C18 acid or diacid, which is optionally linked to the Lys side chain via a spacer.

[0019] In one embodiment, a GLP-1 receptor antagonist is provided, wherein the antagonist comprises the following amino acid sequence:

[0020] R 10 -DX 10 X 11 X 12 YXX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS(SEQ ID NO:98);

[0021] in,

[0022] X10 is Trp, dTrp or Val;

[0023] X 11 is Trp, dTrp or Ser; X 12 is Arg, Lys or Ser; X 15 Is Glu or dGlu

[0024] X 16 is Trp, dTrp, dGlu or Glu; X 23 It is Ile or dIle;

[0025] X 24 is Ala or Glu; and

[0026] R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH.

[0027] According to one embodiment, a GLP-1 receptor antagonist peptide is provided, which has the following amino acid sequence:

[0028] DVWRYL 15 EQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:96),

[0029] in,

[0030] X 15 It is dGlu;

[0031] X 40 is acylated Lys and R 20 It is COOH or CONH2, wherein the acyl group of acylated Lys is C16-C18 acid or diacid, which is optionally connected to the Lys side chain via a spacer. In one embodiment, the spacer includes any multiple or combination of minipeg or γ glutamate subunits or such minipeg or γGlu molecules. In one embodiment, the peptide of SEQ ID NO:96 is modified by 1, 2 or 3 amino acid substitutions, including, for example, aminoisobutyric acid (Aib) substitutions at one or more of positions 16, 18, 19, 24, 26 or 28 relative to the native Exendin4 sequence of SEQ ID NO:1; or the substitution of Lys at position 12.

[0032] According to one embodiment, a GLP-1 receptor antagonist peptide is provided, which comprises the following amino acid sequence

[0033] DX10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:5), wherein

[0034] X 10 is Trp, dTrp or Val;

[0035] X 11 is Trp, dTrp or Ser;

[0036] X 15 is Glu or dGlu;

[0037] X 16 is Trp, dTrp, dGlu or Glu;

[0038] X 23 It is Ile or dIle;

[0039] X 40 is an acylated amino acid, and R 20 is COOH or CONH2, optionally wherein, relative to the native Exendin4 sequence (SEQ ID NO: 1), the peptide includes one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28, or optionally, the peptide of SEQ ID NO: 5 is N-terminally extended by X7X8, wherein X7 is an acylated amino acid (e.g., Lys) and X8 is Gly or C1-C3 N-alkyl Gly, which has position numbering relative to the native Exendin4 sequence (SEQ ID NO: 1), optionally with the proviso that when X 10 or X 11 When one of them is Trp or dTrp, the other is not Trp or dTrp.

[0040] According to one embodiment, a GLP-1 receptor antagonist peptide is provided, which has the following amino acid sequence:

[0041] DVWRYL 15 EQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:96),

[0042] in,

[0043] X 15 It is dGlu;

[0044] X 40 is acylated Lys and R 20 It is COOH or CONH2, wherein the acyl group of acylated Lys is C16-C18 acid or diacid, which is optionally connected to the Lys side chain via a spacer. In one embodiment, the spacer includes any multiple or combination of minipeg or γ glutamate subunits or such minipeg or γGlu molecules. In one embodiment, the peptide of SEQ ID NO:96 is modified by 1, 2 or 3 amino acid substitutions, including aminoisobutyric acid (Aib) substitutions at one or more of positions 16, 18, 19, 24, 26 or 28 relative to the native Exendin4 sequence of SEQ ID NO:1; or by adding a dipeptide X7X8 to the N-terminus of SEQ ID NO:96 and modifying, wherein X7 is an acylated amino acid (e.g., Lys) and X8 is Gly or C1-C3 N- alkyl Gly, which has a position number relative to the native Exendin4 sequence (SEQ ID NO:1).

[0045] 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, wherein X7 is an acylated lysine, optionally wherein the lysine is in a D-conformation and X8 is any amino acid. In one embodiment, the N-terminal extension is a self-cleaving dipeptide linked to the N-terminal alpha amine of a 9-29exendin4 analog (e.g., a peptide of SEQ ID NO:5) to form a prodrug of any GLP-1 antagonist disclosed herein. One advantage of using a prodrug derivative of a GLP-1 antagonist is that this approach extends the biological half-life of the peptide based on a strategy of inhibiting recognition of the prodrug by the GLP-1 receptor. In one embodiment, the prodrug derivative comprises a self-cleaving dipeptide (AB) covalently linked to a GLP-1 antagonist, wherein the dipeptide is cleaved under physiological conditions and in the absence of enzymatic activity to restore the full activity of the GLP-1 antagonist. In one embodiment, the GLP-1 antagonist is modified by covalently linking one or more dipeptides (AB) to the amine of the GLP-1 antagonist, wherein A is an amino acid or hydroxy acid, and B is an N-alkylated amino acid linked 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, AB comprises the following structure:

[0046]

[0047] The GLP-1 antagonist is linked via an amide bond between the carboxyl group of AB and the amine of the GLP-1 antagonist, wherein

[0048] R1, R2, R4 and R8 are independently selected from the group consisting of: H, C1-C 18 Alkyl, C2-C 18 Alkenyl, (C1-C 18 Alkyl)OH, (C1-C 18 (C2-C3 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH 2+ )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;

[0049] R3 is selected from the group consisting of: 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, (C 0- 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 atoms to which they are attached form a pyrrolidine ring;

[0050] R5 is NHR6 or OH;

[0051] R6 is H, C1-C8 alkyl; and

[0052] R7 is selected from the group consisting of H and OH, wherein the chemical cleavage half-life (t 1 / 2) is at least about 1 hour to about 1 week. In one embodiment, the dipeptide AB is covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence. In one embodiment, wherein 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.

[0053] The present disclosure also provides a pharmaceutical composition comprising the GLP-1 antagonist peptide and variant peptide described herein and any one of a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, a method for treating a patient suffering from atypical hypoglycemia is provided, wherein the method comprises the step of administering to a patient in need thereof an amount of a pharmaceutical composition effective to increase blood glucose levels, wherein the pharmaceutical composition comprises the GLP-1 antagonist peptide or variant peptide described herein.

[0054] According to one embodiment, a method for treating atypical hypoglycemia is provided, wherein the method comprises the step of administering a therapeutically effective amount of any GLP-1 receptor antagonist peptide disclosed herein that increases blood glucose levels. In one embodiment, the GLP-1 receptor antagonist is acylated with a fatty acid or diacid group of sufficient size to bind serum albumin with high affinity. 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 sufficient size to bind serum albumin with high affinity. In one embodiment, the N-terminal alpha amine of the GLP-1 receptor antagonist is acylated, optionally with a second acylation at the C-terminus. In one embodiment, the GLP-1 receptor antagonist is acylated at the C-terminus, optionally wherein the acylated amino acid is the C-terminal amino acid, and optionally, the GLP-1 antagonist is further modified by attaching a self-cleavable dipeptide via an amide bond, optionally wherein the amino acids of the dipeptide are acylated with a fatty acyl group of sufficient size to bind serum albumin with high affinity. In one embodiment, an acylated amino acid is added to the C-terminal position 40 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 linked to the amino acid side chain via any spacer disclosed herein.

[0055] In one embodiment, a pharmaceutical composition for treating atypical hypoglycemia includes any GLP-1 receptor antagonist disclosed herein in combination with any existing therapeutic agent that can be used to treat hypoglycemia. For example, a pharmaceutical composition may include a GLP-1 receptor antagonist of the present invention and one or more of the following: a glucose supplement (e.g., dextrose); a blood glucose raising agent such as glucagon and glucagon analogs and an insulin secretion inhibitor (e.g., diazoxide, octreotide). BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The mice were subcutaneously administered with vehicle control, Ex-4 (a GLP-1 agonist having the amino acid sequence of SEQ ID NO: 1), or a GLP-1 antagonist peptide (DVSRYLEEQAVREFIEWLVRGGPSSGAPPPSK 40 [mPEG-γE-C16] acid; SEQ ID NO: 16, a graph showing the dose-dependent changes in blood glucose levels over time following intraperitoneal administration of glucose (1.5 g glucose / kg body weight), wherein glucose was administered subcutaneously 4 hr after the GLP-1 antagonist.

[0057] Figure 2A and 2B Data from a glucose tolerance test are presented, in which mice were given a subcutaneous dose of a GLP-1 antagonist followed by intraperitoneal administration of glucose (1.5 g glucose / kg body weight) four hours later. The results showed that SEQ ID NO: 16 ( Figure 2A ) is a complete antagonist with much higher potency than the GLP-1 antagonist Ex-9-40 (DVSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; SEQ ID NO: 2); Figure 2B ).

[0058] Figure 3 Data from a glucose tolerance test investigating the effect of Aib displacement on the efficacy of a GLP-1 antagonist are presented. Mice were administered a GLP-1 antagonist subcutaneously followed by intraperitoneal administration of glucose (1.5 g glucose / kg body weight) four hours later. Figure 3 The following activities were exhibited: 9-40Jant4-K comprising the sequence of (SEQ ID NO: 47) 40(DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK; SEQ ID NO:3), and its derivatives including a series of Aib substitutions: SEQ ID NO:43 (Aib at position 20), SEQ ID NO:44 (Aib at position 18), and SEQ ID NO:45 (Aib at position 11), SEQ ID NO:46 (Aib at position 10).

[0059] Figure 4 Data from a glucose tolerance test investigating the effects of D-amino acid substitutions on the efficacy of GLP-1 antagonists are presented. GLP-1 antagonists were administered subcutaneously to mice followed by intraperitoneal administration of glucose (1.5 g glucose / kg body weight) four hours later. (miniPEG)2-γE-C16 acylated 9-40Jant4-K 40 (DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSX 40 (SEQ ID NO: 12)) GLP-1 antagonist activity of the peptide SEQ ID NO: 48, wherein X 40 is Lys acylated with (miniPEG)2-γGlu-C16 and has a C-terminal amide) relative to vehicle control and variants comprising substitutions, wherein X 40 is Lys acylated by C16 and C-terminal amide, with the corresponding amino acid in the following D-conformation: SEQ ID NO: 49 (d-Glu 15 )、SEQ ID NO:50(d-Val 19 )、SEQ ID NO:51(d-Ile 23 ).

[0060] Figures 5A-5D Data are provided for prodrug derivatives of the GLP-1 receptor antagonists of the present invention. Figure 5A The prodrug dK7(mPeg-γE-diacid C18)N-Me-Gly incubated in PBS at 37°C is presented. 8 Mass spectrophotometric data of peptide SEQ ID NO: 19 over time. Figures 5B-5D Is presented in the glucose tolerance test, DVSRYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 ; A graph showing the GLP-1 antagonist activity of a dipeptide prodrug of SEQ ID NO: 18 to which the peptide has been covalently linked dK7(mPeg-γE-diacid C18)N-iPr-Gly 8 Dipeptide modification, in which 24h after antagonist administration ( Figure 5B)、48h( Figure 5C ) or 120h( Figure 5D )Administer a glucose load.

[0061] Fig. 6A and 6B Data are provided for GLP-1 receptor antagonist derivatives of the invention comprising two acylated amino acids. Fig. 6A and 6B is a graph presenting the results of a glucose tolerance test, wherein 24 h after administration of the antagonist ( Fig. 6A ) or 48h( Figure 6B ) administers a glucose load, wherein

[0062] SEQ ID NO: 16 is R 12 ,E 24 ,R 28, K 40 [mPEG-γE-C16]-(Jant4,9-40) acid (SEQ ID NO:16);

[0063] SEQ ID NO: 17 is dK7 (mPeg-γE (C18-diacid) G 8 ,R 12 ,E 24 ,R 28 ,K 40 [mPEG-γE-C16]-(Jant4,9-40) acid (SEQ ID NO:17);

[0064] SEQ ID NO:18dK 7 (mPeg-γE(C18-diacid)i-Pr,G 8 ,R 12 ,E 24 ,R 28 ,K 40 [mPEG-

[0065] γE-C16]-(Jant4,9-40) acid (SEQ ID NO:18);

[0066] SEQ ID NO:19dK7(mPeg-γE(C18-diacid)N-Me,G8,R 12, E 24 ,R 28 ,K 40 [mPEG-γE-C16]-(Jant4,9-40) acid (SEQ ID NO:19);

[0067] SEQ ID NO:20dK 7 (K(mPeg-γE-C18 diacid)2G8 ,R 12 ,E 24 ,R 28 K 40 [mPEG-γE-

[0068] C18 diacid](Jant4,9-40)amide (SEQ ID NO:20);

[0069] SEQ ID NO:21dK 7 (K(mPeg-γE-C18 diacid)2G 8 ,R 12 ,E 24 ,R 28 K 40 [mPEG-γE-C16](Jant4,9-40)amide (SEQ ID NO:21);

[0070] SEQ ID NO:22dK 7 (K(mPeg-γE-C18 diacid)2N-Me,G 8 ,R 12 ,E 24 ,R 28

[0071] K 40 [mPEG-γE-C16](Jant4,9-40)amide (SEQ ID NO:22).

[0072] Figure 7 A table of various N-terminally acylated GLP-1 antagonists prepared according to the present disclosure (MBX 1391; SEQ ID NO: 118; MBX 1401; SEQ ID NO: 119; MBX 1402; SEQ ID NO: 120; MBX 1403; SEQ ID NO: 121; MBX 1404; SEQ ID NO: 122; MBX 1406; SEQ ID NO: 123; MBX 1407; SEQ ID NO: 124; MBX 1408; SEQ ID NO: 125; MBX 1416; SEQ ID NO: 126) is provided. All of these GLP-1 analogs are also acylated at the carboxyl terminus, with the exception of the final compound MBX 1416, which is acylated only at the N terminus.

[0073] Figure 8The graph presents that mice were subcutaneously injected with three different doses of 10 nmol / kg, 30 nmol / kg, 100 nmol / kg and 300 nmol / kg of a di-acylated GLP-1 antagonist (MBX 1407; SEQ ID NO: 116), and blood glucose levels were measured at 24 hr, 48 hr and 72 hr, showing a dose-dependent response.

[0074] Fig. 9 The following graph is presented: Blood glucose levels over time in mice injected subcutaneously with 300 nmol / kg of a diacylated GLP-1 antagonist (MBX 1407; X-DVSSYLEEQAVREFIAWLVRGGPSSGAPPPSO-acid; SEQ ID NO: 116) and a monoacylated GLP-1 antagonist (MBX1342; DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSO-amide; SEQ ID NO: 117), wherein X = C18-diacid and O = Lys (C18-diacid). The single-site lipidated GLP-1 antagonist is more potent than the dual-site lipidated GLP-1 antagonist.

[0075] Fig.10 The IC values ​​of various GLP-1 antagonist analogs (MBX 1342; SEQ ID NO: 127; MBX 1373; SEQ ID NO: 128; MBX 1416; SEQ ID NO: 126; MBX 1417; SEQ ID NO: 129; MBX 1418; SEQ ID NO: 130) are presented. 50 (nMoles) measured in an in vitro assay using GLP-1R overexpressing 293 cells.

[0076] Fig.11A and 11B The patients were given a subcutaneous injection of 300 nmol / kg of a diacylated GLP-1 antagonist (MBX 1407; Fig.11A ) and monoacylated GLP-1 antagonists (MBX 1342; Fig. 11B ) over time. The single-site lipidated GLP-1 antagonist is more effective than the dual-site lipidated GLP-1 antagonist.

[0077] Fig.12 Data are presented on the time course of blood glucose in mice injected subcutaneously with 100 or 300 nmol / kg of a non-acylated GLP-1 antagonist (MBX 1118 (Ex-9)), a di-acylated GLP-1 antagonist (MBX 1407), and a mono-acylated GLP-1 antagonist (MBX 1416), measured on days 2, 4, 7, 9, 11, and 14, respectively.

[0078] Fig.13 Shown are the results of a glucose tolerance test performed on day 14 after subcutaneous injection of 100 or 300 nmol / kg of a non-acylated GLP-1 antagonist (MBX 1118 (Ex-9)), a di-acylated GLP-1 antagonist (MBX 1407), and a mono-acylated GLP-1 antagonist (MBX 1416). DETAILED DESCRIPTION

[0079] definition

[0080] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions given below.

[0081] As used herein, the term "about" refers to greater than or less than 10% of the value or range of values, but is not intended to specify any value or range of values ​​as only this broader definition. Each value or range of values ​​beginning with the term "about" is also intended to cover the embodiments of the absolute value or range of values.

[0082] As used herein, the term "amino acid" encompasses any molecule comprising both an amino and carboxyl functional group, wherein the amino and carboxyl groups are attached to the same carbon (α carbon). The α carbon optionally may have one or two additional organic substituents. Amino acid can be specified by its three-letter code, single-letter code, or in some cases specified by the name of its side chain. For example, the unconventional amino acid including the cyclohexane group connected to the α carbon is referred to as "cyclohexane" or "cyclohexyl". For purposes of this disclosure, in the case of not specifying its stereochemistry, the naming of amino acid is intended to encompass the L or D form of amino acid, or a racemic mixture. However, in the case where amino acid is specified by its three-letter code (i.e. Lys), this naming is intended to specify the natural L form of amino acid, and the D form will be specified by including lowercase d before the three-letter code or single code (i.e. dLys or dK).

[0083] The term "hydroxy acid" as used herein refers to an amino acid modified to replace the alpha carbon amino group with a hydroxy group.

[0084] As used herein, the term "non-coded amino acid" encompasses any amino acid that is not the L-isomer of any 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.

[0085] A "biologically active polypeptide" refers to a polypeptide that is capable of exerting a biological effect in vitro and / or in vivo.

[0086] As used herein, general references to peptides are intended to encompass peptides with modified amino and carboxyl termini. For example, an amino acid sequence specifying standard amino acids is intended to encompass the standard amino acids at the N-terminus and C-terminus and the corresponding hydroxy acids at the N-terminus and / or the corresponding C-terminal amino acid modified to include an amide group replacing the terminal carboxylic acid.

[0087] As used herein, an "acylated" amino acid is an amino acid that includes an acyl group that is non-natural to a naturally occurring amino acid, regardless of the manner in which it is produced. Exemplary methods for producing acylated amino acids and acylated peptides are known in the art and include acylation of the amino acid prior to inclusion in a peptide, or subsequent chemical acylation of the peptide following peptide synthesis. In some embodiments, the acyl group imparts to the peptide one or more of: (i) an increased circulating half-life, (ii) a delayed onset of action, (iii) an increased duration of action, (iv) improved resistance to proteases, and (v) increased potency at the GLP-1 receptor.

[0088] As used herein, an "alkylated" amino acid is an amino acid that includes an alkyl group that is non-natural to a naturally occurring amino acid, regardless of the manner in 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 inclusion in a peptide, or performing peptide synthesis followed by chemical alkylation of the peptide. Without being bound by any particular theory, it is believed that alkylation of a peptide will achieve similar, if not identical, effects as acylation of a peptide, such as increased circulating half-life, delayed onset of action, increased duration of action, improved resistance to proteases.

[0089] As used herein, the term "pharmaceutically acceptable carrier" includes any of the following: any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. The term also includes any agent approved by the U.S. federal government regulatory agency or listed in the U.S. Pharmacopoeia for use in animals (including humans).

[0090] The term "pharmaceutically acceptable salt" as used herein refers to salts that retain the biological activity of the parent compound and are not biologically or otherwise undesirable compounds. Many compounds disclosed herein are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0091] As used herein, the term "hydrophilic moiety" refers to any compound that is readily soluble in water or readily absorbs water and is tolerated in vivo in mammalian species without toxic effects (i.e., biocompatibility). 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 cellulose such as hydroxymethyl cellulose or hydroxyethyl cellulose and copolymers thereof, and natural polymers, including, for example, albumin, heparin, and dextran.

[0092] As used herein, the term "treating" includes alleviating the symptoms associated with a particular disorder or condition and / or preventing or eliminating the symptoms. For example, as used herein, the term "treating hypoglycemia" generally refers to maintaining or increasing blood glucose levels to near normal levels.

[0093] As used herein, an "effective" amount or a "therapeutically effective amount" of a GLP-1 receptor antagonist refers to an amount of a 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. An "effective" amount will vary from subject to subject, depending on the age and general condition of the individual, the mode of administration, etc. Therefore, it is not always possible to specify an exact "effective amount". However, an appropriate "effective" amount in any individual case can be determined by a person of ordinary skill in the art using routine experimentation.

[0094] The term "parenteral" means not through the digestive tract but by some other route, such as intranasal, inhalation, subcutaneous, intramuscular, intraspinal, or intravenous.

[0095] As used herein, the term "derivative" is intended to encompass chemical modifications of compounds (e.g., amino acids), including in vitro chemical modifications, such as by introducing groups into the side chains at one or more positions of the polypeptide, such as nitro groups in tyrosine residues, or iodine in tyrosine residues, or by converting free carboxyl groups into ester groups or amide groups, or converting amino groups into amides by acylation, or by acylation of hydroxyl groups to produce esters, or by alkylation of primary amines to produce secondary amines, or by attaching hydrophilic moieties to amino acid side chains. Other derivatives are obtained by oxidation or reduction of the side chains of amino acid residues in the polypeptide.

[0096] As used herein, the term "identity" refers 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 an identical sequence have 100% identity, while two sequences that have amino acid deletions, additions or substitutions relative to each other have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as computer programs that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215: 403-410) can be used to determine sequence identity.

[0097] As used herein, the term "selectivity" of a molecule for a first receptor relative to a second receptor refers to the ratio of the EC of the molecule at the second receptor to the 50 Divide by the EC of the molecule at the first receptor 50 For example, at the first receptor EC 50 is 1 nM and the EC at the second receptor 50 A molecule at 100 nM is 100-fold selective for the first receptor relative to the second receptor.

[0098] As used herein, amino acid "modification" refers to the replacement of an amino acid, or the derivatization of an amino acid by adding a chemical group to an amino acid and / or removing a chemical group from an amino acid, and includes replacement with any of the 20 amino acids commonly found in human proteins and atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Atypical amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derived from naturally occurring amino acids.

[0099] As used herein, an amino acid "substitution" refers to the replacement of one amino acid residue with a different amino acid residue.

[0100] As used herein, the term "conservative amino acid substitution" is defined herein as an exchange within one of the following five groups:

[0101] I. Small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, Gly;

[0102] II. Polar, negatively charged residues and their amides:

[0103] Asp, Asn, Glu, Gln, cysteine ​​and homocysteic acid;

[0104] III. Polar, positively charged residues:

[0105] His, Arg, Lys; Ornithine (Orn)

[0106] IV. Large aliphatic nonpolar residues:

[0107] Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine

[0108] V. Large aromatic residues:

[0109] Phe, Tyr, Trp, acetylphenylalanine.

[0110] Throughout the application, all references to specific amino acid positions by number (e.g., position 28) refer to the amino acid at that position in natural Exendin4 (SEQ ID NO: 1) or the corresponding amino acid position in any of its analogs. For example, "position 28" mentioned herein will mean the corresponding position 27 of the Exendin4 analog, in which the first amino acid of SEQ ID NO: 1 is deleted. Therefore, 9-39Exendin4 represents an N-terminally truncated Exendin4 peptide in which the first 8 amino acids are deleted. In addition, reference to positions greater than 39 (natural Exendin4 has only 39 amino acids) is intended to refer to amino acid positions in analogs having a C-terminal amino acid extension after the corresponding position 39 of SEQ ID NO: 1.

[0111] As used herein, the general term "polyethylene glycol chain" or "PEG chain" refers to a mixture of condensation products of ethylene oxide and water in a branched or linear chain, having the general formula H(OCH2CH2) n OH represents, wherein n is at least 2. "Polyethylene glycol chain" (or "PEG chain") is used in combination with a numerical suffix to indicate its approximate average molecular weight. For example, PEG-5,000 refers to polyethylene glycol chains having an overall average molecular weight of about 5,000 Daltons.

[0112] As used herein, the term "PEGylated" and like terms refer to a compound that has been modified from its native state by attaching a polyethylene glycol chain to the compound. A "PEGylated polypeptide" is a polypeptide that has a PEG chain covalently attached to the polypeptide.

[0113] As used herein, the term "miniPEG" or "OEG" defines a functionalized polyethylene compound comprising the following structure:

[0114]

[0115] 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 optimal spacing of the two entities, or can further provide an unstable connection that allows the two entities to be separated from each other. Unstable connections include photocleavable groups, acid-labile moieties, base-labile moieties, and enzymatically cleavable groups.

[0116] As used herein, a "dimer" is a complex comprising two subunits covalently bound to each other via a linker. The term dimer, when used without any qualifying language, encompasses both homodimers and heterodimers. A homodimer comprises two identical subunits, while a heterodimer comprises two different subunits, although the two subunits are substantially similar to each other.

[0117] As used herein, the term C16-C20 fatty acid refers to the structure:

[0118] CO(CH2) 14-20 CH3 and the term C16-C20 diacids represent the structure: -CO(CH2) 14-20 COOH, where the prefix "C16-C20" indicates the variable total number of carbons in the compound covered by the name. For example, the C18 diacid represents the structure: -CO(CH2) 16 COOH. As used herein, a general reference to an acylated amino acid encompasses both amino acids whose side chains are acylated with fatty acids and amino acids whose side chains are acylated with diacids.

[0119] Physiological conditions as disclosed herein are intended to include a temperature of about 35° C. to 40° C. and a pH of about 7.0 to about 7.4, and more typically include a pH of 7.2 to 7.4 and a temperature of 36° C. to 38° C. Since the physiological pH and temperature of the human body are strictly regulated within a highly defined range, the rate of conversion from the dipeptide / drug complex (prodrug) to the drug will show high intra- and inter-patient reproducibility.

[0120] As used herein, the term "C1-C n "Alkyl" (wherein n can be 1 to 6) means a branched or straight chain alkyl group having one 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.

[0121] As used herein, the term "C2-C n"Alkenyl" (wherein n may be 2 to 6) represents an ethylenically unsaturated branched or straight chain group having 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.

[0122] The term "C2-C n "Alkynyl" (wherein n may be 2 to 6) refers to an unsaturated branched or straight chain group having 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.

[0123] As used herein, the term "aryl" refers to a monocyclic or bicyclic carbocyclic ring system having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. The size of the aromatic rings 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 group attached to the parent moiety through a one to three membered alkyl chain.

[0124] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and at least one nitrogen, oxygen or sulfur atom in the aromatic rings. The size of the heteroaromatic rings and the presence of substituents or linking groups are indicated by specifying the number of carbons present. For example, the term "(C1-C n "C5-C6 heteroaryl" refers to a 5 or 6 membered heteroaryl attached to the parent moiety through a one to "n" membered alkyl chain.

[0125] As used herein, the term "halogen" refers to one or more members selected from the group consisting of fluorine, chlorine, bromine and iodine.

[0126] As used herein, the term "patient" is intended to encompass any warm-blooded vertebrate domesticated animal (including, for example, but not limited to, livestock, horses, cats, dogs and other pets) and humans without further designation, and is not limited to individuals under the direct care of a physician.

[0127] As used herein, the term "isolated" means having been removed from its natural environment.

[0128] As used herein, the term "purified" relates to the isolation of a molecule or compound in a form substantially free of contaminants normally associated with the molecule or compound in nature or native environment, and means increased purity resulting from separation from other components of the original composition. The term "purified peptide" is used herein to describe a peptide that has been separated from other compounds, including but not limited to nucleic acid molecules, lipids and carbohydrates.

[0129] As used herein, the term "peptide" encompasses sequences of 2 or more amino acids and generally less than 50 amino acids, wherein the amino acids are naturally occurring or encoded or non-naturally occurring or non-encoded amino acids. Non-naturally occurring amino acids refer to amino acids that do not naturally occur in vivo but can be incorporated into the peptide structures described herein. As used herein, "non-encoded" refers to amino acids that are not L-isomers of any 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.

[0130] As used herein, "partial non-peptide" refers to a molecule in which a portion of the molecule is a biologically active chemical compound or substituent and does not include an amino acid sequence.

[0131] "Peptide mimics" refers to compounds that have a structure different from the general structure of existing peptides, but function in a manner similar to existing peptides, such as by simulating the biological activity of the peptide. Peptide mimics generally include naturally occurring amino acids and / or non-natural amino acids, but may also include modifications to the peptide backbone. For example, a peptide mimetic may include a naturally occurring amino acid sequence in which a non-peptide portion is inserted or replaced, such as a reverse inversion fragment, or a non-peptide bond is incorporated, such as an aza-peptide bond (CO is replaced by NH) or a pseudo-peptide bond (e.g., NH is replaced by CH2), or an ester bond (e.g., a decapeptide in which one or more amide (-CONHR-) bonds are replaced by an ester (COOR) bond). Alternatively, a peptide mimetic may not contain any naturally occurring amino acids.

[0132] As used herein, the term "charged amino acid" or "charged residue" refers to an amino acid that includes a side chain that is negatively charged (i.e., deprotonated) or positively charged (i.e., protonated) in aqueous solution at physiological pH. For example, negatively charged amino acids include aspartic acid, glutamic acid, cysteine, homocysteine, and homoglutamic acid, while positively charged amino acids include arginine, lysine, and histidine. Charged amino acids include the charged amino acids of the 20 common amino acids in human proteins, as well as atypical or non-naturally occurring amino acids.

[0133] As used herein, the term "acidic amino acid" refers to an amino acid that includes a second acidic moiety (in addition to the alpha carboxylic acid of the amino acid), including, for example, a side chain carboxylic acid or sulfonic acid group.

[0134] As used herein, the term "prodrug" is defined as any compound that undergoes chemical modification before exhibiting its full pharmacological effect.

[0135] As used herein, a "dipeptide" is an α-amino acid

[0136] Or the result of an α-carboxylic acid being linked to another amino acid via a peptide bond.

[0137] As used herein, the term "chemical cleavage" without any further designation encompasses non-enzymatic reactions that result in the breaking of covalent chemical bonds.

[0138] As used herein, the term "atypical hypoglycemia" is defined as a condition of hypoglycemia that occurs in a patient independent of exogenous insulin administration.

[0139] abbreviation:

[0140] Lowercase k = D isomer of lysine

[0141] Capital K = L isomer of lysine

[0142] γE=L isomer of γ-glutamic acid,

[0143] (miniPEG)2=COCH2OCH2CH2OCH2CH2NH

[0144] COC 16 H 32 CO2H=(C18 diacid)

[0145] (N-Me)G = Sarcosine

[0146] Implementation

[0147] According to one embodiment of the present disclosure, compositions and methods for treating patients with hypoglycemia independent of exogenous insulin administration (i.e., atypical hypoglycemia) are provided. According to the present disclosure, a composition comprising a GLP-1 antagonist is administered to a patient with atypical hypoglycemia in an amount sufficient to increase blood glucose levels and / or alleviate the associated acute symptoms and chronic consequences associated with hypoglycemia. In one embodiment, the patient experiencing atypical hypoglycemia also suffers from a hyperinsulinemia condition. In one embodiment, the hyperinsulinemia condition occurs after the patient undergoes bariatric surgery.

[0148] According to one embodiment, a method of treating a patient suffering from atypical hypoglycemia comprises 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 sufficient size to bind to serum albumin with high affinity. In one embodiment, the C14-C20 acyl group is covalently bound to the N-terminal alpha amine of the GLP-1 receptor antagonist peptide.

[0149] In one embodiment, the GLP-1 receptor antagonist is acylated at the N-terminus of the GLP-1 receptor antagonist, optionally wherein an acetyl group is covalently linked to the N-terminal alpha amine of the glucagon peptide. The acyl group of the acylated glucagon peptide can be of any size, such as a carbon chain of any length, and can be linear or branched. In some specific embodiments of the invention, the acyl group is a C12 to C30 fatty acid or a fatty diacid. For example, the acyl group can be any of the following: C12 fatty acid / diacid, C14 fatty acid / diacid, C16 fatty acid / diacid, C18 fatty acid / diacid, C20 fatty acid / diacid, C22 fatty acid / diacid, C24 fatty acid / diacid, C26 fatty acid / diacid, C28 fatty acid / diacid, or C30 fatty acid / diacid. In some embodiments, the acyl group is a C14 to C20 fatty acid, such as a C14 fatty acid or a fatty diacid, a C16 fatty acid or a fatty diacid, or a C16 fatty acid or a fatty diacid.

[0150] According to one embodiment, the acylated GLP-1 receptor antagonist peptide comprises the amino acid sequence DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK (SEQ ID NO: 3) or an analog thereof having at least 95% sequence identity to SEQ ID NO: 3 while retaining GLP-1 receptor antagonist activity.

[0151] According to one embodiment, the acylated GLP-1 receptor antagonist peptides disclosed herein include analogs of the peptide DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK (SEQ ID NO: 3), wherein 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, wherein the modifications are selected from amino acid substitutions, additions or modifications to 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 replacement with a d-isomer at one or more of positions 15, 16, 17, 19, 20, 21 and 23 (numbering relative to native Exendin4 (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.

[0152] According to one embodiment, a GLP-1 receptor antagonist is provided, which includes an 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, a peptide is provided that differs from the peptide of SEQ ID NO: 4 by one or more of the following:

[0153] i. replacing 1, 2 or 3 amino acids at any of positions 7, 10, 11, 13 or 16 with Trp or dTrp;

[0154] i. replacing 1, 2 or 3 amino acids at any of positions 15, 16 or 23 with the corresponding amino acids in the D conformation;

[0155] ii. replacing 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 with Aib;

[0156] iii. substitution at position 12 with an acylated amino acid, optionally acylated lysine;

[0157] iv. substitution at position 16 with dGlu, Asp, homoglutamic acid or homocysteine;

[0158] v. substitution at position 19 with cyclopropane, cyclopentane, cyclohexane or phenylglycine;

[0159] vi. substitution at position 20 with dArg, homolysine or citrulline;

[0160] vii. replacing the native C-terminal carboxyl group with an amide;

[0161] viii. adding an N-terminal extension of 1 to 3 amino acids, optionally wherein one of the N-terminal extension amino acids is acylated;

[0162] ix. replacing the C-terminal amino acid with an acylated amino acid;

[0163] Any combination of xi) to x).

[0164] According to one embodiment, a GLP-1 receptor antagonist is provided, which comprises the following amino acid sequence:

[0165] R 10 -DVX 11 X 12 YXX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 SEQ ID NO:23), wherein

[0166] R 10 NH2, -CO(CH2) 14-20 CH3, -CO(CH2) 14-20 COOH or an N-terminal extension of 1, 2 or 3 amino acids, wherein one of the amino acids of the N-terminal extension is acylated with a C16-C18 fatty acid or diacid, optionally via a spacer, optionally wherein R 10 is a dipeptide having the following structure: X7X8, wherein X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1-C4 N-alkylated Gly;

[0167] X 11 is Trp, dTrp or Ser;

[0168] X 12 is Arg or an acylated amino acid, optionally acylated Lys;

[0169] X 15 is Glu or dGlu;

[0170] X 16is Glu, dGlu, Asp, homoglutamate, or homocysteine;

[0171] X 19 is Val, cyclopropane, cyclopentane, cyclohexane, or phenylglycine;

[0172] X 20 is Arg, homolysine, or citrulline;

[0173] X 23 It is Ile or dIle;

[0174] X 40 is an acylated amino acid, optionally acylated Lys; and

[0175] R 20 is COOH or CONH2, optionally wherein 1, 2 or 3 amino acids selected from positions 7, 10, 13 or 16 are replaced by Trp or dTrp, optionally with the proviso that positions 10 and 11 cannot be Trp or dTrp at the same time; further optionally with the proviso that R 10 and X 12 Acylated amino acids cannot be included simultaneously; optionally wherein 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted with Aib.

[0176] In one embodiment, only one of positions 7, 12, and 40 of the peptide of SEQ ID NO: 23 comprises an acylated amino acid. In one embodiment, two of positions 7, 12, and 40 of the peptide of SEQ ID NO: 23 comprise an acylated amino acid. In one embodiment, the acylated amino acids at positions 7, 12, and 40 are independently amino acids comprising a structure of Formula I (optionally Lys) or Formula II (optionally Ser), wherein each of Formula I and Formula II is:

[0177]

[0178] In one embodiment, a peptide of SEQ ID NO: 23 is provided, wherein R 10 is X7X8, wherein X7 is acylated Lys or acylated dLys, X 12 is Arg and X 40 In one embodiment, a peptide of SEQ ID NO: 23 is provided, wherein R 10 is NH2, X 12 is acylated Lys and X 40 In one embodiment, a peptide of SEQ ID NO: 23 is provided, wherein R 10 is NH2, X 12is Arg and X 40 In one embodiment, the acylated amino acid of the peptide of SEQ ID NO: 23 is an acylated Lys residue, optionally wherein 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, optionally wherein the fatty acid or fatty diacid is linked to the side chain of the Lys residue via any spacer molecule disclosed herein.

[0179] According to one embodiment, a GLP-1 receptor antagonist is provided, which comprises the following amino acid sequence:

[0180] R 10 -DVX 11 X 12 YLEX 16 QAVREFIEWLVRGGPSSGAPPPSX 40 -R 20 SEQ ID NO:24),

[0181] in,

[0182] R 10 is NH2-CO(CH2) 14-20 CH3, -CO(CH2) 14-20 COOH or a dipeptide having the following structure: X7X8, wherein X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1-C4 N-alkylated Gly;

[0183] X 11 is Trp, dTrp or Ser;

[0184] X 12 is Arg or an acylated amino acid, optionally acylated Lys;

[0185] X 16 is Glu or Asp;

[0186] X 40 is an acylated amino acid, optionally acylated Lys; and

[0187] R 20 is COOH or CONH2, optionally wherein 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.

[0188] According to one embodiment, a GLP-1 receptor antagonist is provided, which comprises the following amino acid sequence:

[0189] R 10 -DVX 11 X 12 YLEX 16 QAVREFIEWLVRGGPSSGAPPPSX 40 -R 20 SEQ ID NO:24),

[0190] in,

[0191] R 10 is a dipeptide of the structure: X7X8, wherein X7 is acylated Lys or acylated dLys, and X8 is Gly or sarcosine;

[0192] X 11 is Trp or dTrp;

[0193] X 12 is Arg;

[0194] X 16 It is Glu;

[0195] X 40 is acylated Lys; and

[0196] R 20 is COOH or CONH2, wherein the acylated Lys residues of the peptide are independently acylated with a C16-C18 fatty acid or fatty diacid, optionally via a spacer as disclosed herein, optionally wherein 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted with Aib.

[0197] In one embodiment, a GLP-1 receptor antagonist is provided, wherein the antagonist comprises the following amino acid sequence:

[0198] DX 10 X 11 X 12 YXX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS(SEQ ID NO:98);

[0199] in,

[0200] X 10 is Trp, dTrp or Val;

[0201] X 11 is Trp, dTrp or Ser;

[0202] X 12is Arg, Lys or Ser;

[0203] X 15 is Glu or dGlu;

[0204] X 16 is Trp, dTrp, dGlu or Glu;

[0205] X 23 It is Ile or dIle;

[0206] X 24 is Ala or Glu;

[0207] as well as

[0208] The GLP-1 receptor antagonist is acylated with a fatty acid or diacid group of sufficient size to bind serum albumin with high affinity, optionally wherein the amino acid of the GLP-1 receptor antagonist is acylated with a C16-C18 fatty acid or a C16-C18 fatty diacid.

[0209] In one embodiment, a GLP-1 receptor antagonist is provided, wherein the antagonist comprises the following amino acid sequence:

[0210] R 10 -DX 10 X 11 X 12 YXX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS-R 20 (SEQ ID NO:98), or a sequence that differs from SEQ ID NO:98 by 1 or 2 amino acid substitutions;

[0211] in,

[0212] X 10 is Trp, dTrp or Val;

[0213] X 11 is Trp, dTrp or Ser; X 12 is Arg, Lys or Ser; X 15 Is Glu or dGlu

[0214] X 16 is Trp, dTrp, dGlu or Glu; X 23 It is Ile or dIle;

[0215] X 24 is Ala or Glu;

[0216] R10 NH2, -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH; and R 20 yes

[0217] COOH or CONH2. In one embodiment, a sequence of SEQ ID NO: 98 is provided, wherein

[0218] X 10 It's Val;

[0219] X 11 is Trp, dTrp or Ser; X 12 is Arg, Lys or Ser; X 15 Is Glu or dGlu

[0220] X 16 is dGlu or Glu; X 23 It is Ile;

[0221] X 24 is Ala or Glu;

[0222] R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH and

[0223] R 20 is COOH, optionally wherein the peptide comprises one or more Aib substitutions at any of positions 16, 18, 19, 24, 26 or 28 relative to the native Exendin4 sequence (SEQ ID NO: 1).

[0224] In one embodiment, a GLP-1 receptor antagonist is provided, wherein the antagonist comprises the following amino acid sequence:

[0225] R 10 -DX 10 X 11 X 12 YXX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS-R 20 (SEQ ID NO:98);

[0226] in,

[0227] X 10 It's Val;

[0228] X11 It is Ser;

[0229] X 12 is Arg;

[0230] X 15 is Glu or dGlu;

[0231] X 16 is dGlu or Glu;

[0232] X 23 It is Ile;

[0233] X 24 is Glu; and

[0234] R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH and

[0235] R 20 It is COOH or CONH2.

[0236] In one embodiment, the GLP-1 antagonist peptide comprises the following amino acid sequence: 10 -DVSSYLEEQAVREFIAWLVKGGPSSGAPPPS(SEQ ID NO:3) or

[0237] 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, wherein R 10 is -CO(CH2) linked to the N-terminal alpha amine of the amino acid sequence of SEQ ID NO:3 14-20 CH3 or -CO(CH2) 14-20 COOH, and R 20 is COOH or CONH2, optionally wherein R 20 It's COOH.

[0238] In one embodiment, the GLP-1 antagonist peptide comprises the following amino acid sequence: DVX 11 RYLQ 15 X 16 AVREFX23EWLVRGGPSSGAPPPSX 40 -R 20 (SEQ ID NO:25), wherein

[0239] X 11 is Trp, dTrp or Ser;

[0240] X15 is Glu or dGlu;

[0241] X 16 is Glu or dGlu;

[0242] X 23 It is Ile or dIle;

[0243] X 40 is an acylated amino acid, optionally Lys acylated with a C16-C18 fatty acid or diacid, optionally via a spacer; and

[0244] R 20 is COOH or CONH2. Optionally, the GLP-1 antagonist of SEQ ID NO: 25 is further modified by substitution with one or more Aib at any of positions 16, 18, 19, 24, 26 or 28, or optionally substitution with acylated Lys at position 12, relative to the numbering of the native Exendin4 sequence of SEQ ID NO: 1. In one embodiment, a peptide of SEQ ID NO: 25 is provided, further comprising substitution of acylated Lys at position 12 and optionally substitution of Aib at position 27.

[0245] In one embodiment, the GLP-1 antagonist peptide comprises the following amino acid sequence:

[0246] DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R2 (SEQ ID NO: 6) or

[0247] X7X8DV 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID

[0248] NO:26), wherein X7 is acylated Lys or acylated dLys, and X8 is Gly or sarcosine, X 11 is Trp or dTrp, X 40 is an acylated amino acid, optionally acylated Lys, wherein the acylated Lys residue comprises a C16-C18 fatty acid or diacid covalently linked to the Lys side chain, optionally via a spacer, and R 20is COOH or CONH2. Optionally, the peptides of SEQ ID NO:6 and SEQ ID NO:26 can be further modified by substitution with Aib at any of positions 16, 18, 19, 24, 26 or 28 based on the numbering of native Exendin4 (SEQ ID NO:1), or the peptide of SEQ ID NO:6 is optionally substituted with acylated Lys at position 12.

[0249] In one embodiment, the GLP-1 antagonist peptide comprises the following amino acid sequence: DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:6 or DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO: 7) or an amino acid that differs from SEQ ID NO: 6 or SEQ ID NO: 7 by 1 or 2 amino acid substitutions, wherein X 11 is Trp or dTrp, X 40 is an amino acid having an acyl group of sufficient size to bind with high affinity to serum albumin attached to the side chain of the amino acid, optionally wherein the acyl group is attached via a spacer, and R 20 is COOH or CONH2, optionally with an Aib substitution at any one of positions 16, 18, 19, 24, 26 or 28 relative to the numbering of native Exendin4 (SEQ ID NO: 1), and optionally wherein the carboxyl group of the C-terminal amino acid is replaced by an amide (i.e., R 20 is CONH2). In one embodiment, X 40 is an amino acid comprising a structure of Formula I (optionally Lys), Formula II (optionally Ser), or Formula III (optionally Cys), wherein each of Formulas I, II, and III is:

[0250]

[0251] In one embodiment, the GLP-1 antagonist peptide comprises the following amino acid sequence:

[0252] DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 -COOH (SEQ ID NO: 9) or

[0253] DVX 11RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 -NH2 (SEQ ID NO: 10), wherein X 11 is Trp or dTrp, and X 40 X is an amino acid having an acyl group optionally attached to the amino acid side chain via a spacer. 40 It is acylated Lys.

[0254] According to one embodiment, a GLP-1 receptor antagonist peptide is provided, which has the following amino acid sequence:

[0255] XV 12 YLEEQAVREFIEWLVRGGPSSGAPPPSX 40 -NH2 (SEQ ID NO: 8),

[0256] in,

[0257] X 12 is acylated Lys; and

[0258] X 40 is an acylated Lys with an amide substituted for the C-terminal carboxylic acid, wherein the acyl group of the acylated Lys is a C16-C18 acid or diacid, optionally linked to the Lys side chain via a spacer.

[0259] In yet another aspect, the amino acid at position 1 of any GLP-1 receptor antagonist disclosed herein is modified to inhibit protease degradation of the peptide. In one embodiment, inhibition of the protease is achieved by:

[0260] i. acylate the side chain of the N-terminal amino acid;

[0261] ii. replacing the N-terminal amino acid with its D-stereoisomer;

[0262] iii. modifying the N-terminal alpha amine, including, for example, covalently linking an acetyl group to the alpha amine, or removing the alpha amine group; or

[0263] iv. Any combination of i)-iii).

[0264] In one embodiment, a DVX 11 RYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R2 (SEQ ID NO: 6), which has up to 3 amino acid modifications relative to SEQ ID NO: 6, wherein X 11 is Trp or dTrp, X 40 is an acylated amino acid, and R20 is COOH or CONH2, wherein the peptide exhibits antagonist activity against human GLP-1.

[0265] In one embodiment, the GLP-1 receptor antagonist comprises a peptide selected from the group consisting of:

[0266] DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:27)

[0267] DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:28)

[0268] DV(dW)RYLE(Aib)QAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:29)

[0269] DVWRYLE(Aib)QAVREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:30

[0270] DV(dW)RYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:31)

[0271] DVWRYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:32)

[0272] DV(dW)RYLEEQA(Aib)REFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:33)

[0273] DVWRYLEEQA(Aib)REFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:34)

[0274] DV(dW)RYLEEQAVREFI(Aib)WLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:35)

[0275] DVWRYLEEQAVREFI(Aib)WLVRGGGPSSGAPPPSX40 R20, (SEQ ID NO:36)

[0276] DV(dW)RYLEEQAVREFIEW(Aib)VRGGPSSGAPPPSX40 R20, (SEQ ID NO:37)

[0277] DVWRYLEEQAVREFIEW(Aib)VRGGPSSGAPPPSX40 R20, (SEQ ID NO:38)

[0278] DV(dW)RYLEEQAVREFIEWLV(Aib)GGPSSGAPPPSX40 R20, (SEQ ID NO:39)

[0279] DVWRYLEEQAVREFIEWLV(Aib)GGPSSGAPPPSX40 R20, (SEQ ID NO:40)

[0280] DV(dW)RYLEEQAV(dR)EFIEWLVRGGPSSGAPPPSX40 R20, (SEQ ID NO:41)

[0281] DVWRYLEEQAV(dR)EFIEWLVRGGPSSGAPPPSX 40 R 20 , (SEQ ID NO:42),

[0282] Among them, X 40 is an acylated amino acid, optionally a Lys acylated with a C16-C18 fatty acid or diacid, and R 20 is COOH or CONH2, wherein the peptide exhibits antagonist activity against human GLP-1.

[0283] In one embodiment, the GLP-1 receptor antagonist comprises a peptide selected from the group consisting of:

[0284] R 10 -DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPS-R 20 , (SEQ ID NO: 100)

[0285] R 10 -DVWRYLEEQAVREFIEWLVRGGPSSGAPPPS-R 20 , (SEQ ID NO: 101)

[0286] R 10 -DV(dW)RYLE(Aib)QAVREFIEWLVRGGPSSGAPPPS-R 20，(SEQ ID NO:102)

[0287] R 10 -DVWRYLE(Aib)QAVREFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:103)

[0288] R 10 -DV(dW)RYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:104)

[0289] R 10 -DVWRYLEEQ(Aib)VREFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:105)

[0290] R 10 -DV(dW)RYLEEQA(Aib)REFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:106)

[0291] R 10 -DVWRYLEEQA(Aib)REFIEWLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:107)

[0292] R 10 -DV(dW)RYLEEQAVREFI(Aib)WLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:108)

[0293] R 10 -DVWRYLEEQAVREFI(Aib)WLVRGGPSSGAPPPS-R 20 ,(SEQ ID NO:109)

[0294] R 10 -DV(dW)RYLEEQAVREFIEW(Aib)VRGGPSSGAPPPS-R 20， (SEQ ID NO:110)

[0295] R 10 -DVWRYLEEQAVREFIEW(Aib)VRGGPSSGAPPPS-R 20 ,(SEQ ID NO:111)

[0296] R 10 -DV(dW)RYLEEQAVREFIEWLV(Aib)GGPSSGAPPPS-R 20， (SEQ ID NO:112)

[0297] R 10 DVWRYLEEQAVREFIEWLV(Aib)GGPSSGAPPPS-R 20 , (SEQ ID NO: 113)

[0298] R 10 -DV(dW)RYLEEQAV(dR)EFIEWLVRGGPSSGAPPPS-R 20， (SEQ ID NO:114)

[0299] R 10 -DVWRYLEEQAV(dR)EFIEWLVRGGPSSGAPPPS-R 20 , (SEQ ID NO: 115),

[0300] Among them, R 10 It is -CO(CH2) connected to the N-terminal alpha amine of the peptide 14-20 CH3 or -CO(CH2) 14-20 COOH, and R 20 is COOH or CONH2, wherein the peptide exhibits antagonist activity against human GLP-1.

[0301] Additional exemplary species of the present disclosure include those listed in Table 1:

[0302] Table 1

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] In one embodiment, dimers and multimers including two or more GLP-1 receptor antagonist peptides of the present disclosure are prepared, including homo or hetero multimers or homo or hetero dimers. Two or more GLP-1 receptor antagonist peptides can be linked together using standard linkers and procedures known to those skilled in the art. For example, by using a bifunctional thiol cross-linker and a bifunctional amine cross-linker, a dimer can be formed between two peptides, especially for GLP-1 receptor antagonist peptides including or substituted with the following: cysteine, lysine, ornithine, homocysteine ​​or acetylphenylalanine residues. The dimer can be a homodimer or can be a heterodimer. In an exemplary embodiment, the linker connecting two (or more) analogs is PEG, such as 5kDa PEG, 20kDa PEG. In some embodiments, the linker is a disulfide bond. For example, each monomer of the dimer can include a Cys residue (e.g., a terminal or internally positioned Cys) and the sulfur atom of each Cys residue participates in the formation of a disulfide bond. In exemplary aspects, each monomer of the dimer is linked via a thioether bond.In exemplary aspects, the epsilon amine of a Lys residue of one monomer is bonded to a Cys residue, which in turn is linked to the epsilon amine of a Lys residue of another monomer via a chemical moiety.

[0311] In some embodiments, monomers are linked via terminal amino acids (e.g., N-terminal or C-terminal, optionally wherein amino acids are added to the end of the peptide to be dimerized), via internal amino acids, or via terminal amino acids of at least one monomer and internal amino acids of at least one other monomer. In some embodiments, the monomers of a multimer are linked together in a "tail-to-tail" orientation, wherein the C-terminal amino acids of each monomer are linked together. Alternatively, in one embodiment, a multimer is linked together in a "head-to-head" orientation, wherein the N-terminal amino acids of each monomer are linked together.

[0312] In one embodiment, the C-terminal amino acid of any GLP-1 antagonist peptide disclosed herein may be modified to replace the native carboxyl group with an amide. In one embodiment, the C-terminal amino acid of any GLP-1 antagonist peptide disclosed herein includes a native amino acid carboxyl group.

[0313] Pharmaceutical composition

[0314] The present disclosure further provides a pharmaceutical composition comprising any of the GLP-1 receptor antagonist peptides, dimers, multimers or conjugates (or combinations thereof) of the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition is preferably sterile and suitable for parenteral administration.

[0315] According to one embodiment, a pharmaceutical composition is provided, which includes any novel GLP-1 receptor antagonist 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 a GLP-1 receptor antagonist disclosed herein at a concentration of at least 0.1-10 mg / ml or higher. In one embodiment, the pharmaceutical composition includes an aqueous solution that is sterilized and optionally stored in various packaging containers. In other embodiments, the pharmaceutical composition includes 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 can be labeled for storage at ambient room temperature or refrigerated temperature. In one embodiment, the pharmaceutical composition and / or kit includes any GLP-1 receptor antagonist disclosed herein in combination with any existing therapeutic agent that can be used to treat hypoglycemia, including but not limited to glucose supplements (e.g., dextrose); glucose-raising agents such as glucagon and glucagon analogs, and insulin secretion inhibitors (e.g., diazoxide, octreotide).

[0316] According to one embodiment, the pharmaceutical compositions disclosed herein are contemplated for use in a method for treating or preventing a hypoglycemic condition, and more specifically, treating or preventing atypical hypoglycemia or a medical condition associated with hypoglycemia.

[0317] The compositions of the present disclosure can be administered using any standard route of administration. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but through some other route such as subcutaneous, intramuscular, intraspinal or intravenous. In one embodiment, a 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 preparation for administration via inhalation alone or in combination with other suitable components.

[0318] 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 substantially 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 variety 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, wherein the composition comprises a GLP-1 receptor antagonist of the present disclosure and SNAC, optionally wherein the pharmaceutical composition is formulated as a tablet.

[0319] Acylation

[0320] According to one embodiment, any of the peptides, dimers or multimers disclosed herein that exhibit GLP-1 antagonist activity may be further modified to have an improved therapeutic index and a prolonged in vivo duration of action when administered to warm-blooded mammals (including, for example, homo sapiens). More particularly, in one embodiment, the peptides and dimers disclosed herein are modified by covalently linking an alkyl or acyl group to the side chain of an amino acid (optionally lysine, serine or cysteine) of the antagonist peptide, wherein the alkyl or acyl group is of sufficient size to bind 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 includes two acylated amino acids. In one embodiment, one or more amino acids of the GLP-1 receptor antagonist peptide are acylated by fatty acids or fatty diacids, optionally C16-C18 fatty acids or C16-C18 fatty diacids. 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 a lysine side chain, 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 position 7 or 12. In embodiments with 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, as long as the acyl groups are of sufficient size to bind serum albumin. In one embodiment, the acylated amino acid is lysine, wherein the side chain is optionally attached to a C16-C18 fatty acid or a C16-C18 fatty diacid via a spacer.

[0321] In one embodiment, a C16-C18 fatty acid or a C16-C18 fatty diacid is linked to the side chain of an amino acid via a spacer, wherein the spacer comprises miniPEG, γGlu, or any polymer or combination of miniPEG and / or γGlu. In one embodiment, any GLP-1 receptor antagonist peptide disclosed herein may be modified to include an acylated amino acid, optionally at a position selected from 7, 12, and 40 with reference to the native sequence of exendin4. In one embodiment, the acylated amino acid is a lysine residue having a C16 to C18 fatty acid or a C16 to C18 fatty diacid linked to the side chain of lysine via a spacer comprising the following structure:

[0322] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -

[0323] wherein k is an integer selected from 2, 4, 6 or 8, and p and q are independently an integer selected from 1 or 2. In one embodiment, the spacer is selected from the group consisting of:

[0324] -(γ-Glutamic acid)-[COCH2(OCH2CH2) k NH]2-(γ-glutamic acid)-, or

[0325] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -[COCH2(OCH2CH2) k NH] q -

[0326] (γ-Glutamate) p -,or

[0327] -[COCH2(OCH2CH2) k NH]2-(γ-glutamic acid)2-, or

[0328] -[COCH2(OCH2CH2) k NH]-(γ-glutamic acid)2-[COCH2(OCH2CH2) k NH]-, or

[0329] -(γ-Glutamic Acid)2-[COCH2(OCH2CH2) k NH]2, or

[0330] -(γ-Glutamic acid)-[COCH2(OCH2CH2) k NH]-(γ-glutamic acid) p -

[0331] [COCH2(OCH2CH2) k NH]- or γ-glutamic acid, or γ-glutamic acid-γ-glutamic acid dipeptide, or γ-glutamic acid-[COCH2(OCH2CH2) k NH]-γ-glutamate, where

[0332] k is an integer selected from the range of 1-8; and

[0333] q and p are independently integers selected from the range of 1-8, optionally wherein k is 2 and q and p are independently 1 or 2. In one embodiment, the spacer is

[0334] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -,in

[0335] k is an integer selected from the range of 1-4; and

[0336] q and p are independently selected from integers in the range of 1-4, optionally

[0337] wherein k is 2 and q and p are independently 1 or 2, optionally wherein k is 2 and q and p are both 1.

[0338] In one embodiment, the GLP-1 receptor antagonist peptide comprises an acylated Lys, optionally at the C-terminus of the peptide, wherein the side chain of the Lys is acylated with a C16-C18 diacid via a spacer comprising the following structure: -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -,in,

[0339] k is an integer selected from the range of 1-4; and

[0340] q and p are independently integers selected from the range of 1-4, optionally wherein k is 2 and q and p are independently 1 or 2, optionally wherein k is 2 and q and p are both 1. In one embodiment, the side chain of the acylated Lys comprises the structure -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -COC 16 H 32 CO2H, wherein k is 2 and p and q are independently 1 or 2. In one embodiment, a GLP-1 receptor antagonist is provided, comprising the following sequence: DV(dW)RYLEEQAVREFIEWLVRGGPSSGAPPPSX40 R 20 , (SEQ ID NO: 27) or

[0341] DVWRYLEEQAVREFIEWLVRGGPSSGAPPPSX 40 R 20 , (SEQ ID NO: 28), wherein,

[0342] R 20 is COOH or CONH2 and X 40 is -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -COC 14 H 28 CO2H or -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -COC 16 H 32 CO2H acylated Lys, wherein k is 2 and p and q are independently 1 or 2.

[0343] Conjugate

[0344] In some embodiments, any 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 immunoglobulin (Ig) types include IgG, IgA, IgE, IgD, or IgM. The Fc region is the C-terminal region of the Ig heavy chain, responsible for binding to the Fc receptor, performing activities such as recycling (resulting in half-life extension), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC).

[0345] In some embodiments, any of the GLP-1 antagonist peptides disclosed herein is conjugated to a hydrophilic portion. The hydrophilic portion can be covalently linked to the GLP-1 antagonist peptide under any suitable conditions for reacting the protein with an activated polymer molecule. Activated groups that can be used to connect water-soluble polymers to one or more proteins include, but are not limited to, sulfones, maleimides, sulfhydryls, thiols, trifluoromethanesulfonate groups, trifluoroethylsulfonic acid monomethoxy groups (tresylate), azidirine, oxirane, 5-pyridyl, and α-haloacyl groups (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). If connected to a peptide by reductive alkylation, the selected polymer should have a single reactive aldehyde in order to control the degree of polymerization. See, e.g., 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).

[0346] Suitable hydrophilic moieties include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene polyols (e.g., POG), polyoxyethylene sorbitol, polyoxyethylene glucose, polyoxyethylene glycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde, copolymers of ethylene glycol / propylene glycol, 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 copolymers, poly(β-amino acids) (homopolymers or random copolymers), poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer (PPG) and other polyakylene oxides, polypropylene oxide / ethylene oxide copolymers, colonic acid or other polysaccharide polymers, polysucrose or dextran, and mixtures thereof. Dextran is a polysaccharide polymer of glucose subunits, mainly connected by α1-6 keys. Dextran can be obtained in a variety of molecular weight ranges, such as about 1kD to about 100kD, or about 5, 10, 15 or 20kD to about 20, 30, 40, 50, 60, 70, 80 or 90kD. In one embodiment, the hydrophilic part such as polyethylene glycol chain has a molecular weight selected from about 500 to about 40,000 daltons. In some embodiments, the hydrophilic part is a polyethylene glycol chain with a molecular weight selected from about 500 to about 5,000 daltons or about 1,000 to about 5,000 daltons. In another embodiment, the hydrophilic part such as polyethylene glycol chain has a molecular weight of about 10,000 to about 20,000 daltons.

[0347] In one embodiment, a conjugate derivative of a peptide of SEQ ID NO: 5 or SEQ ID NO: 23 is provided, wherein the dipeptide is covalently linked to the N-terminus of the peptide of SEQ ID NO: 5 or SEQ ID NO: 23 via a peptide bond, optionally wherein one of the amino acids of the dipeptide is an acylated amino acid. In one embodiment, the dipeptide has a structure of X7X8, wherein X7 is an acylated amino acid and X8 is any amino acid, optionally wherein X7 is acylated Lys or dLys and X8 is Gly or C1-C4 N-alkylated Gly, optionally wherein X7 is Lys or dLys acylated with C14-C20 fatty acids or fatty diacids and X8 is Gly or C1-C4N-alkylated Gly (optionally Gly or sarcosine), optionally wherein X7 is Lys acylated with C14-C20 fatty acids or fatty diacids via any spacer disclosed herein, and X8 is C1-C4 N-alkylated Gly (optionally Gly or sarcosine).

[0348] According to one embodiment, a conjugate derivative of any GLP-1 receptor antagonist peptide is provided, wherein the self-cleaving dipeptide is covalently bound to an amino acid side chain amine or N-terminal alpha amine of a GLP-1 antagonist peptide disclosed herein via an amide bond. In one embodiment, the self-cleaving dipeptide is covalently bound to the N-terminal alpha amine of the GLP-1 antagonist peptide.

[0349] In exemplary aspects, the self-cleaving dipeptide comprises the following structure: AB, wherein A is an amino acid or a hydroxy acid; and B is an N-alkylated amino acid attached to a GLP-1 antagonist peptide via an amide bond between AB and an amine of the GLP-1 antagonist peptide, optionally wherein the chemical cleavage half-life (t1 / 2) of AB from the GLP-1 antagonist peptide in PBS under physiological conditions is at least about 1 hour to about 1 week. As used herein, the term "hydroxy acid" refers to an amino acid that has been modified to replace the alpha carbon amino group with a hydroxyl group.

[0350] In some embodiments, the self-cleaving dipeptide has the following general structure:

[0351]

[0352] in,

[0353] R1, R2, R4 and R8 are independently selected from the group consisting of: H, C1-C 18 Alkyl, C2-C 18 Alkenyl, (C1-C 18 Alkyl)OH, (C1-C 18 (C2-C3 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH 2+ )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;

[0354] R3 is selected from the group consisting of: 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, (C 0-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 atoms to which they are attached form a pyrrolidine ring;

[0355] R5 is NHR6 or OH;

[0356] R6 is H, C1-C8 alkyl; and

[0357] R7 is selected from the group consisting of H and OH

[0358] Wherein, under physiological conditions in PBS, the chemical cleavage half-life (t 1 / 2 ) is at least about 1 hour to about 1 week. In one embodiment, the dipeptide AB is covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence.

[0359] In one embodiment, the self-cleaving dipeptide has the following general structure:

[0360]

[0361] in,

[0362] R1 and R8 are independently H or C1-C8 alkyl;

[0363] 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;

[0364] R3 is (C1-C6) alkyl;

[0365] R5 is NH2; and

[0366] R7 is selected from the group consisting of hydrogen and OH.

[0367] According to one embodiment, any of the GLP-1 antagonist peptides and dimers disclosed herein can be further modified by attachment to a self-cleaving dipeptide, wherein the amino acids of the dipeptide are acylated with a fatty acyl group of sufficient size to bind serum albumin with high affinity.

[0368] In one embodiment, the amino acid "A" of the self-cleaving dipeptide "AB" is a lysine residue acylated with a C16-C30 fatty acid or a C16-C30 diacid. In one embodiment, A and B are selected so that the chemical cleavage half-life (t1 / 2) of AB from the GLP-1 antagonist peptide or dimer disclosed herein in a standard PBS solution under physiological conditions is 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.

[0369] In one embodiment, the self-cleaving dipeptide has the following general structure:

[0370]

[0371] in,

[0372] 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, optionally wherein a C16-C18 fatty acid or a C16-C18 diacid is covalently attached to the side chain, optionally via any spacer disclosed herein, including a spacer selected from the group consisting of: gamma glutamic acid, gamma glutamic acid-gamma glutamic acid dipeptide, -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p and γ-glutamic acid-[COCH2(OCH2CH2) k NH] q -γ-glutamic acid, of which

[0373] k is an integer selected from the range of 1-8; and

[0374] q and p are independently integers selected from the range of 1-8, optionally wherein k is 2 and q and p are independently 1 or 2;

[0375] R2, R4 and R8 are independently H or C1-C4 alkyl;

[0376] R3 is C1-C6 alkyl; and

[0377] R5 is NH2.

[0378] In one embodiment, the self-cleaving dipeptide has the following general structure:

[0379]

[0380] in,

[0381] R1 is H, C1-C4 alkyl, (C1-C4 alkyl)OH or (C1-C4 alkyl)NH2; R2 is H,

[0382] R3 is (C1-C4)alkyl;

[0383] R4 is H or C1-C4 alkyl; R5 is NH2; and

[0384] R8 is hydrogen.

[0385] According to one embodiment, the self-cleaving dipeptide AB comprises an acylated amino acid residue as "A" and an N-alkylated Gly residue as "B", wherein the "B" amino acid is linked to the N-terminal alpha amine of the GLP-1 receptor antagonist peptide via an amide bond, optionally wherein the Lys residue is in a D-conformation. In one embodiment, the acylated amino acid "A" of the AB dipeptide is an amino acid having the following general structure:

[0386]

[0387] Wherein n is an integer selected from the range of 1-4 and R 50 Selected from the group consisting of: NH-CO(CH2) 14- 20 COOH, NH-[Spacer]-CO(CH2) 14-20 COOH, S(CH2) 14-20 COOH and S-[Spacer]-CO(CH2) 14-20 COOH, wherein [spacer] is any spacer disclosed herein. In one embodiment, the acylated amino acid of A is independently selected from lysine, d-lysine, ornithine, cysteine ​​or homocysteine, wherein the side chain of the acylated amino acid is covalently linked to a C16-C22 fatty acid or a C16-C22 diacid, optionally via a spacer comprising an amino acid or a dipeptide. In one embodiment, the spacer comprises gamma glutamic acid. In one embodiment, the spacer comprises a plurality of gamma glutamic acid units and a miniPeg polymer in any combination. In one embodiment, the optional spacer comprises two gamma glutamic acids, optionally wherein the two gamma glutamic acids are linked via an inserted functionalized miniPEG polymer [COCH2(OCH2CH2) k HN] q are connected to each other, wherein k and q are each an integer independently selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0388] In one embodiment, the self-cleaving dipeptide has the following general structure:

[0389]

[0390] in

[0391] R1 is (C4 alkyl)NH2 or (C4 alkyl)NH(mPeg-γE-diacid)-C18;

[0392] R2, R4 and R8 are each H;

[0393] R3 is C1-C4 alkyl; and

[0394] R5 is NH2, optionally wherein the first amino acid of the self-cleavable dipeptide is in the D-conformation.

[0395] In one embodiment, the GLP-1 antagonist peptides and dimers disclosed herein are covalently linked to a self-cleaving dipeptide having the following structure:

[0396]

[0397] in

[0398] R1 is selected from C1-C 18 A side chain selected from the group consisting of alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH and (C1-C4 alkyl)NH2, optionally wherein a C16-C20 fatty acid or a C16-C20 diacid is covalently attached to the side chain;

[0399] R2, R4 and R8 are independently H or C1-C4 alkyl;

[0400] R3 is C1-C4 alkyl, or R4 and R3 together with the atoms to which they are attached form a pyrrolidine ring; and

[0401] 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.

[0402] In other embodiments, the self-cleaving dipeptide has the structure:

[0403]

[0404] 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) 14-20COOH; R2 and R8 are each H; R4 is H or CH3; R3 is C1-C4 alkyl and R5 is NH2, optionally wherein the first amino acid of the self-cleaving dipeptide is an amino acid in a D-stereochemical conformation and the spacer is selected from any spacer disclosed herein. In one embodiment, R2, R4 and R8 are each H. 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 and R3 is C1-C4 alkyl, optionally CH3, optionally wherein the spacer comprises the following structure:

[0405] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -

[0406] wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0407] According to one embodiment, the self-cleaving dipeptide comprises an acylated amino acid as the first amino acid, wherein the side chain of the acylated amino acid is acylated by a C16-C20 fatty acid or a C16-C20 diacid, optionally wherein 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 wherein the acylated amino acid of the dipeptide is C16-C20 acylated Lys, optionally wherein the first amino acid of the self-cleaving dipeptide is an amino acid in a D-stereochemical conformation.

[0408] Implementation

[0409] According to embodiment 1, a GLP-1 receptor antagonist is provided, wherein the antagonist comprises the following amino acid sequence:

[0410] R 10 -DVX 11 X 12 YXX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 SEQ ID NO:23), wherein

[0411] R 10is NH2 or an N-terminal extension of 1 or 2 amino acids, wherein one of the amino acids of the N-terminal extension is acylated with a C14-C20 fatty acid or diacid, optionally via a spacer, and optionally wherein R 10 is a dipeptide of the following structure: X7X8, wherein X7 is an acylated amino acid, optionally acylated Lys or acylated dLys, and X8 is Gly or C1-C4 N-alkylated Gly;

[0412] X 11 is Trp, dTrp or Ser;

[0413] X 12 is Arg or an acylated amino acid, optionally acylated Lys;

[0414] X 15 is Glu or dGlu;

[0415] X 16 is Glu, dGlu, Asp, homoglutamate, or homocysteine;

[0416] X 19 is Val, cyclopropane, cyclopentane, cyclohexane, or phenylglycine;

[0417] X 20 is Arg, homolysine, or citrulline;

[0418] X 23 is Ile or dIle; and

[0419] X 40 is an acylated amino acid, optionally acylated Lys; and

[0420] R 20 is COOH or CONH2, optionally wherein 1, 2 or 3 amino acids selected from positions 7, 10, 13 or 16 are replaced by Trp or dTrp, provided that when X 10 or X 11 When one of them is Trp or dTrp, the other is not Trp or dTrp, and R 10 and X 12 Both cannot simultaneously include acylated amino acids; optionally wherein 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted with Aib.

[0421] According to embodiment 2, the GLP-1 antagonist of embodiment 1 is provided, wherein the antagonist comprises the following amino acid sequence

[0422] R 10 -DVX 11 X 12 YXX15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPSX 40 -R 20 SEQ ID NO:23), wherein

[0423] R 10 is NH2 or X7X8, wherein X7 is an amino acid optionally acylated with a C14-C20 fatty acid or diacid via a spacer, and X8 is Gly or C1-C4 N-alkylated Gly;

[0424] X 11 is Trp, dTrp or Ser;

[0425] X 12 is Arg or an acylated amino acid, optionally acylated Lys;

[0426] X 15 is Glu or dGlu;

[0427] X 16 is Glu, dGlu, Asp, homoglutamate, or homocysteine;

[0428] X 19 is Val, cyclopropane, cyclopentane, cyclohexane, or phenylglycine;

[0429] X 20 is Arg, homolysine, or citrulline;

[0430] X 23 is Ile or dIle; and

[0431] X 40 is an acylated amino acid, optionally acylated Lys; and

[0432] R 20 is COOH or CONH2, optionally wherein 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted with Aib.

[0433] According to embodiment 3, there is provided the GLP-1 antagonist of embodiment 1 or 2, wherein each acylated amino acid of the GLP-1 antagonist is acylated Lys.

[0434] According to embodiment 4, a GLP-1 antagonist according to any one of embodiments 1 to 3 is provided, wherein X7 is acylated Lys and X 12 It is Arg.

[0435] According to embodiment 5, a GLP-1 antagonist according to any one of embodiments 1 to 3 is provided, wherein R 10 is NH2 and X 12 It is acylated Lys.

[0436] According to embodiment 6, there is provided a GLP-1 antagonist of any one of embodiments 1-3, wherein X7 is NH2 and X 12 It is Arg.

[0437] According to embodiment 7, a GLP-1 antagonist as described in embodiment 1 is provided, wherein the antagonist comprises an amino acid sequence selected from any one of SEQ ID NO: 5 to SEQ ID NO: 96 or any combination thereof.

[0438] According to embodiment 8, a GLP-1 antagonist according to any one of embodiments 1 to 7 is provided, wherein the acylated amino acid of the GLP-1 antagonist is independently a Lys residue acylated with a C16-C18 fatty acid or a C16-C18 fatty diacid, wherein the C16-C18 fatty acid or the C16-C18 fatty diacid is directly linked to the Lys side chain or optionally linked via a spacer, wherein the spacer comprises

[0439] (i) gamma-glutamate,

[0440] (ii) Minipeg polymer: -[COCH2(OCH2CH2) k NH]-, wherein k is 2, 4, 6 or 8,

[0441] (iii) or any multiple or combination of i) and / or ii).

[0442] According to embodiment 9, a GLP-1 antagonist according to any one of embodiments 1 to 8 is provided, wherein the antagonist comprises the following amino acid sequence

[0443] DX 10 X 11 RYLX 15 X 16 QAVREFX 23 EWLVRGGPSSGAPPPSX 40 R 20 (SEQ ID NO:5), wherein

[0444] X 10 is Trp, dTrp or Val;

[0445] X 11 is Trp, dTrp or Ser;

[0446] X15 is Glu or dGlu;

[0447] X 16 is Trp, dTrp, dGlu or Glu;

[0448] X 23 It is Ile or dIle;

[0449] X 40 is an acylated amino acid; and

[0450] R 20 is COOH or CONH2, optionally wherein the peptide comprises one or more substitutions of Aib at any of positions 16, 18, 19, 24, 26 or 28, or optionally a substitution of an acylated Lys at position 12, wherein the position numbering is relative to the native Exendin4 amino acid sequence.

[0451] According to embodiment 10, a GLP-1 antagonist according to any one of embodiments 1 to 9 is provided, wherein X 11 It is Trp or dTrp.

[0452] According to embodiment 11, a GLP-1 antagonist of any one of embodiments 1-10 is provided, wherein the amino acid at any of positions 16, 18, 19, 24, 26 or 28 of SEQ ID NO:5 is substituted with Aib, optionally wherein Aib is substituted at position 18.

[0453] According to embodiment 12, there is provided the GLP-1 antagonist of any one of embodiments 1 to 11, wherein the amino acid at position 12 is substituted with acylated Lys.

[0454] According to embodiment 13, there is provided a GLP-1 antagonist according to any one of embodiments 1 to 11, wherein

[0455] X 15 It is dGlu;

[0456] X 16 is Glu; and

[0457] X 23 It's Ile.

[0458] According to embodiment 14, a GLP-1 antagonist according to any one of embodiments 1 to 11 is provided, wherein

[0459] X 15 It is Glu;

[0460] X 16 is Glu; and

[0461] X 23 It's Ile.

[0462] According to embodiment 15, there is provided the GLP-1 antagonist of any one of embodiments 1-14, wherein X40 is an amino acid having an acyl group optionally linked to the side chain of the amino acid via a spacer.

[0463] According to embodiment 16, a GLP-1 antagonist according to any one of embodiments 1 to 15 is provided, wherein X 40 is an amino acid comprising a structure of Formula I (optionally Lys), Formula II (optionally Cys), or Formula III (optionally Ser), wherein each of Formulas I, II, and III is:

[0464]

[0465]

[0466] According to embodiment 17, a GLP-1 antagonist according to any one of embodiments 1 to 16 is provided, wherein X 40 It is acylated lysine.

[0467] According to embodiment 18, a GLP-1 antagonist according to any one of embodiments 1 to 17 is provided, wherein the acyl group of the acylated amino acid is selected from (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.

[0468] According to embodiment 19, there is provided the GLP-1 antagonist of any one of embodiments 1 to 18, wherein the acyl group of the acylated amino acid is covalently linked to the amino acid side chain of the acylated amino acid via a spacer.

[0469] According to embodiment 20, there is provided the GLP-1 antagonist of any one of embodiments 1-19, wherein the spacer is an amino acid or a dipeptide.

[0470] According to embodiment 21, a GLP-1 antagonist according to any one of embodiments 1 to 20 is provided, wherein the spacer comprises

[0471] (i) gamma-glutamate,

[0472] (ii) Minipeg polymer: -[COCH2(OCH2CH2) k NH]-, wherein k is 2, 4, 6 or 8,

[0473] (iii) or any multiple or combination of i) and / or ii).

[0474] 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 linked to a C16 to C18 fatty acid or a C16 to C18 fatty diacid, optionally wherein the acid or diacid is linked via a spacer comprising the following structure:

[0475] -(gamma glutamate) p -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) n -

[0476] or

[0477] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -[COCH2(OCH2CH2) k NH] m -;

[0478] 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.

[0479] According to embodiment 23, there is provided a GLP-1 antagonist of any one of embodiments 1-22, wherein the acylated amino acid is Lys having a C16 to C18 fatty acid attached to the lysine side chain via a spacer comprising the structure:

[0480] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -

[0481] wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0482] According to embodiment 24, a GLP-1 antagonist according to any one of embodiments 1 to 23 is provided, wherein

[0483] X 11 is Trp or dTrp;

[0484] X15 is Glu or dGlu;

[0485] X 16 is Glu or dGlu;

[0486] X 23 is Ile or dIle; and

[0487] X 40 is Lys acylated with a C16 or C18 diacid, optionally wherein the diacid is linked via a spacer comprising the structure:

[0488] -[COCH2(OCH2CH2) k NH] q -(gamma glutamate) p -

[0489] wherein k is 2, and p and q are independently integers selected from 1 or 2.

[0490] According to embodiment 25, a GLP-1 antagonist according to any one of embodiments 1 to 24 is provided, wherein R 20 It is CONH2.

[0491] According to embodiment 26, there is provided a derivative of the GLP-1 antagonist of any one of claims 9 to 25, which further comprises a dipeptide AB:

[0492]

[0493] It is connected to the GLP-1 antagonist via an amide bond, wherein

[0494] R1, R2, R4 and R8 are independently selected from the group consisting of: H, C1-C 18 Alkyl, C2-C 18 Alkenyl, (C1-C 18 Alkyl)OH, (C1-C 18 (C2-C3 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH 2+ )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;

[0495] R3 is selected from the group consisting of: 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, (C 0- 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 atoms to which they are attached form a pyrrolidine ring;

[0496] R5 is NHR6 or OH;

[0497] R6 is H, C1-C8 alkyl; and

[0498] R7 is selected from the group consisting of H and OH

[0499] Wherein, under physiological conditions in PBS, the chemical cleavage half-life (t 1 / 2 ) is at least about 1 hour to about 1 week.

[0500] According to embodiment 27, a GLP-1 antagonist as described in embodiment 26 is provided, wherein the dipeptide AB is covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence.

[0501] According to embodiment 28, a GLP-1 antagonist according to any one of embodiments 26-27 is provided, wherein

[0502] R1 and R8 are independently H or C1-C8 alkyl;

[0503] 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;

[0504] R3 is (C1-C6) alkyl;

[0505] R5 is NH2; and

[0506] R7 is selected from the group consisting of hydrogen and OH.

[0507] According to embodiment 29, a GLP-1 antagonist according to any one of embodiments 26-27 is provided, wherein

[0508] R1, comprises 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, optionally wherein a C16-C30 fatty acid or a C16-C30 diacid is covalently attached to the side chain, optionally via a spacer selected from the group consisting of γ-glutamic acid, γ-glutamic acid-γ-glutamic acid dipeptide and γ-glutamic acid-[COCH2(OCH2CH2) k NH] q -gamma glutamic acid group, wherein

[0509] k is an integer selected from the range of 1-8; and

[0510] q is an integer selected from the range of 1-8, optionally wherein k is 2 and q is selected from the range of 1-8;

[0511] R2, R4 and R8 are independently H or C1-C4 alkyl;

[0512] R3 is C1-C6 alkyl; and

[0513] R5 is NH2.

[0514] According to embodiment 30, a GLP-1 antagonist according to any one of embodiments 26 to 29 is provided, wherein

[0515] R1 is H, C1-C4 alkyl, (C1-C4 alkyl)OH or (C1-C4 alkyl)NH2;

[0516] R2 is H;

[0517] R3 is (C1-C4)alkyl;

[0518] R4 is H or C1-C4 alkyl;

[0519] R5 is NH2; and

[0520] R8 is hydrogen.

[0521] According to embodiment 31, a GLP-1 antagonist according to any one of embodiments 26-30 is provided, wherein the dipeptide AB comprises an acylated Lys residue and an N-alkylated Gly residue, wherein the Lys and N-alkylated Gly residues are linked via a peptide bond, optionally wherein the Lys residue is in a D-conformation.

[0522] According to embodiment 32, a GLP-1 antagonist according to any one of embodiments 26-30 is provided, wherein

[0523] R1 is (C4 alkyl)NH2 or (C4 alkyl)NH(mPeg-γE-diacid)-C18;

[0524] R2, R4 and R8 are each H;

[0525] R3 is C1-C4 alkyl; and R5 is NH2;

[0526] R5 is an amine.

[0527] According to embodiment 33, a pharmaceutical composition is provided, which comprises the GLP-1 antagonist or derivative according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier, diluent or excipient.

[0528] According to embodiment 34, a method for treating a patient suffering from atypical hypoglycemia is provided, wherein the method comprises the step of administering to a patient in need thereof an amount of the pharmaceutical composition of embodiment 33 effective to increase blood glucose levels.

[0529] Example 1

[0530] Ex-4(9-39)a (SEQ ID NO:2) is an established GLP-1 receptor antagonist. However, its use as a therapeutic in humans is limited by its non-human origin and its relatively short duration of action in vivo. Comparable N-terminal truncation of human GLP-1 reduces agonism but does not provide a highly potent antagonist. Through a series of GLP-1 / Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1 based antagonist were identified as Glu16, Val19, and Arg20, and the resulting antagonist was approximately 3-fold more potent in vitro compared to Ex-4(9-39)a. Site-specific acylation of the human based antagonist generated the peptide "9-40Jant4 K 40 [C16] (SEQ ID NO: 3), which has increased potency as a GLP-1 receptor antagonist and is 10-fold more selective over the GIP receptor (Patterson, et al, ACS Chem. Biol. 2011, 6, 135-145).

[0531] As disclosed herein, 9-40Jant4-K has been prepared 40 Variants of to provide further improved GLP-1 receptor antagonists. Peptide 9-40Jant4-K 40 It is acylated at position 40 via direct attachment of the C16 acyl group to the Lys side chain. Variants with a spacer inserted between the Lys side chain and the C16 acyl group have been prepared. Table 2 lists the GLP-1 antagonist activity and solubility of these acylated variants.

[0532] 9-40Jant4-K 40 Various amino acid substitutions were made to the primary sequence of (DVSSYLEEQAVREFIAWLVKGGPSSGAPPPSK; SEQ ID NO: 3), and the GLP-1 antagonist activity and solubility of these variants are provided in Table 3.

[0533] Table 4 provides information on the Aib substitutions for Jant4-K 40 Table 5 provides data on the effects of d-AA substitutions on the activity and solubility of Jant4-K 40 Table 6 provides data on the effect of the solubility of the Jant4-K 40 (SEQ ID NO:3) Data for Trp substitution at position 3.

[0534] Materials and methods

[0535] Fmoc Synthesis

[0536] Using Symphony peptide synthesizer (Peptide Technology, Tucson, AZ) with Wang resin (AAPPtec, Louisville, KY) and 6-Cl-HOBt / DIC activation, peptides were prepared by automated Fmoc / t-Bu solid phase method. All conventional residues were purchased from Midwest Biotech (Fisher, IN), and 6-Cl-HOBt and DIC were obtained from AAPPtec (Louisville, KY). By using TFA treatment containing 2.5% TIS, 2.5% H2O, 1.5% methanol, 2.5% phenol, 0.5% DODT and 0.5% dimethyl sulfoxide, peptides were cut off from resin and deprotected. According to standard procedures, peptides were precipitated from filtered TFA solutions with cold ether. Fatty acylation of the peptide was performed on the resin with a ten-fold excess of Fmoc-Glu-OtBu / DEPBT / DIEA, repeated for double coupling, followed by a ten-fold excess of palmitic acid or another fatty acid / DEPBT / DIEA.

[0537] Antagonist acylation

[0538] Palmitic acid was introduced into the synthesized antagonist peptide using an orthogonal solid phase protection scheme. Boc synthesis was used for peptide synthesis, allowing the selective introduction of base-sensitive side chain protected Lys(Fmoc)-OH at Lys40. The fully protected peptide was treated with 20% piperidine in DMF (v / v) on the resin for 30 min to remove the Lys40 side chain Fmoc group.

[0539] Excess fatty acid and 5 equivalents of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP) (Fluka) were added in DMF / DIEA (4:1 v / v) for approximately 18 h to promote amide bond formation. The progress of the reaction was monitored using the ninhydrin test, and acylation was confirmed by ESI mass spectrometry after peptide cleavage.

[0540] Peptide purification

[0541] Peptide purification was performed using reverse phase HPLC (RP-HPLC). In the preparative RP-HPLC purification process, a linear acetonitrile gradient in a C18 stationary phase (Vydac 218TP, 250mm-22mm, 10 μm) and 0.1% trifluoroacetic acid was used. RP-HPLC with a C8 column (Zorbax 300SB, 4.6mm X 50mm, 3.5 μm) was used to analyze the peak fractions. The identity and purity of the peptides were assessed by analytical RP-HPLC and ESI or MALDI mass spectrometry. All peptides were found to have the correct molecular weight and a purity of approximately 95% pure. Lyophilized peptides were stored at 4 ° C.

[0542] GLP-1 receptor-mediated cAMP induction

[0543] The ability of peptides to stimulate or block cAMP induction at the GLP-1 receptor was examined by a luciferase-based reporter gene assay. HEK 293 cells co-transfected with human GLP-1 receptor (Open Biosystems) and cAMP-inducible (cAMP response element) luciferase genes constituted a cell construct that can measure receptor activation. The bioassay was performed by first depriving cells of serum for 16 h in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 0.25% bovine growth serum (HyClone), and then adding serial dilutions of peptides in an appropriate concentration range in 96-well poly-D-lysine coated plates (BD Biosciences). For antagonist assays, a constant concentration of GLP-1 (0.05 nM) was added to the assay plate after dilution of the peptide. Incubation was continued for 5 h at 37 ° C, 5% CO2, and then an equal volume (100 μL) of LucLite luminescent substrate reagent (Perkin-Elmer) was added.

[0544] After shaking the plate at 600 rpm for 3 min, the luminescent signal was quantified in counts per second (cps) using a MicroBeta 1450 liquid scintillation counter (Perkin-Elmer). Data were plotted using Origin software (OriginLab) and the effective concentration 50 (EC 50 ) or inhibitory concentration 50 (IC 50 ). By relative EC50 or IC 50 The potency was determined by comparative analysis of the values. Each experiment was repeated at least three times and each sample was assayed in duplicate.

[0545] animal

[0546] C57Bl / 6 mice were obtained from Jackson Laboratories. Mice were housed individually or in groups in a 12:12 h light-dark cycle at 22°C with free access to food and water. All studies were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Cincinnati.

[0547] Glucose tolerance test (GTT)

[0548] To measure glucose tolerance, mice were fasted for 6 h and injected intraperitoneally with glucose. The injection consisted of 1.5 g glucose per kg body weight (25% w / v D-glucose (Sigma) in 0.9% w / v saline). Tail blood glucose levels (mg dL-1) were measured at 0 min before injection and 15, 30, 60 and 120 min after injection using a handheld blood glucose meter (FreeStyle Freedom Lite).

[0549] Acute in vivo studies

[0550] Mice received subcutaneous injections of different doses of peptide (in PBS) 1, 4, 8, 24, 48, 72 or 120 and then challenged with IP injection of dipeptidyl peptidase-IV protected Ex-4 (0.65 nmol kg-1). GTT was performed fifteen minutes after GLP-1 injection. Tail blood glucose values ​​were obtained as described above.

[0551] Table 2: Jant 4-K 40 C16 linker optimization / analysis of sequence (SEQ ID NO:3)#

[0552]

[0553]

[0554] Table 3: Jant4-K 40 Amino acid substitution variants of (SEQ ID NO: 3)

[0555]

[0556] ￥K40[miniPEG-gE-C16]

[0557] High viscosity solution

[0558] Table 4: Effects of Aib on Jant4-K 40 (SEQ ID NO:3) Effect of activity and solubility

[0559]

[0560] Table 5: Improvement of Jant4-K with d-AA 40 Solubility of (C16) (SEQ ID NO: 3)

[0561]

[0562] Table 6: Trp substitution at position 3 of Jant4-K40 (SEQ ID NO: 3)

[0563]

[0564]

[0565] Example 2

[0566] Synthesis procedure of MBX-1416:

[0567] The synthesis of MBX-1416 was carried out on a 0.1 mmol scale using a starting solid phase resin of Fmoc-Ser(tBu)-Wang. Once the first Fmoc-group was removed from the Fmoc-Ser(tBu)-Wang resin, the Fmoc-amino acids constituting residues 9 to 38 (Asp9-Pro38) were coupled sequentially to the free N-terminal amine. As shown in the table below, standard Fmoc / DIC / Oxyma synthesis protocols were used and automated assembly was performed using an ABI peptide synthesizer.

[0568] Details of the Fmoc_DIC_Oxyma_0.10mmol protocol used on the ABI433A synthesizer

[0569]

[0570] C18 Diacid Coupling Procedure:

[0571] Once the complete Asp9 to Ser39 peptide is assembled, the N-terminal C18 diacid is coupled to the peptidyl resin. It is introduced as a tert-butyl protected C18 diacid (1 mmol) using DIC (1 mmol) and Oxyma (1 mmol) in DMF. The tert-butyl protected C18 diacid (1 mmol) and Oxyma (1 mmol) are dissolved in DMF (10 mL). DIC (1 mmol) is added to the mixture and pre-activated for 10-15 min at room temperature. The reaction mixture is added to the Asp9-Ser39 peptidyl resin and stirred at room temperature for 4 h for coupling.

[0572] MBX-1416 Purification:

[0573] MBX-1416 was purified by Waters HPLC Controller 600 using 10μm C8 LC250X21.2 mm reverse phase column (Cat. No. 00G-4763-PO-AX) with a linear gradient in 90 min from 80% A and 20% B to 20% A and 80% B at a flow rate of 15 ml / min. 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. A GLP-1 receptor antagonist, comprising the following amino acid sequence: R 10 -DVX 11 X 12 YLX 15 X 16 QAX 19 X 20 EFX 23 X 24 WLVRGGPSSGAPPPS- R 20 SEQ ID NO:97), wherein R 10 is a C14-C20 fatty acid or diacid covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence; X 11 is Trp, dTrp or Ser; X 12 is Arg or Ser; X 15 is Glu or dGlu; X 16 is Glu, dGlu, Asp, homoglutamate, or homocysteine; X 19 is Val, cyclopropane, cyclopentane, cyclohexane, or phenylglycine; X 20 is Arg, homolysine, or citrulline; X 23 It is Ile or dIle; X 24 is Glu or Ala; and R 20 is COOH or CONH2, optionally wherein, based on the numbering of native Exendin4 (SEQ ID NO: 1), 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted by Aib.

2. The GLP-1 antagonist according to claim 1, wherein The antagonist comprises the following amino acid sequence: R 10 -DVX 11 X 12 YLX 15 X 16 QAX 19 X 20 EFX 23 EWLVRGGPSSGAPPPS-R 20 SEQ ID NO:99), wherein, R 10 is a C14-C20 fatty acid or diacid covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence; X 11 is Trp, dTrp or Ser; X 12 is Arg or Ser; X 15 is Glu or dGlu; X 16 is Glu, dGlu, Asp, homoglutamate, or homocysteine; X 19 It's Val; X 20 is Arg, homolysine, or citrulline; X 23 is Ile or dIle; and R 20 is COOH or CONH2, optionally wherein, based on the numbering of native Exendin4 (SEQ ID NO: 1), 1, 2 or 3 amino acids at any of positions 16, 18, 19, 24, 26 or 28 are substituted by Aib.

3. The GLP-1 antagonist according to claim 1 or 2, wherein R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH; X 11 is Trp, dTrp or Ser; X 12 It is Ser; X 15 is Glu or dGlu; X 16 is Glu or dGlu; X 19 It's Val; X 20 is Arg; X 23 is Ile or dIle; and R 20 It's COOH.

4. The GLP-1 antagonist according to any one of claims 1 to 3, wherein R 10 yes -CO(CH2) 16-18 COOH, covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence.

5. The GLP-1 antagonist analog according to any one of claims 1 to 4, wherein Based on the numbering of native Exendin4 (SEQ ID NO: 1), the amino acid at position 16, 18, 19, 24, 26 or 28 is substituted by Aib.

6. The GLP-1 antagonist analogue according to any one of claims 1 to 6, further comprising a C-terminal extension of 1 to 3 amino acids, optionally wherein, One of the amino acids of the C-terminal extension comprises an acylated amino acid, optionally acylated Lys.

7. A GLP-1 receptor antagonist, comprising the following amino acid sequence: R 10 -DX 10 X 11 X 12 YXX 15 X 16 QAVREFX 23 X 24 WLVRGGPSSGAPPPS-R 20 (SEQ ID NO:98); wherein, R 10 is a C14-C20 fatty acid or diacid covalently linked to the N-terminal alpha amine of the GLP-1 antagonist amino acid sequence; 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 It is Ile or dIle; X 24 is Ala or Glu; and R 20 It is COOH or CONH2.

8. The GLP-1 receptor antagonist according to claim 7, wherein R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH, linked to the N-terminal alpha amine of the GLP-1 receptor antagonist; 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; and X 24 It is Ala or Glu.

9. A GLP-1 receptor antagonist, comprising R 10 -DVSSYLEEQAVREFIAWLVKGGPSSGAPPPS-R 20 (SEQ ID NO:3) or an amino acid sequence that differs from SEQ ID NO:3 by 1, 2 or 3 amino acid substitutions but retains GLP-1 agonist activity, wherein R 10 Yes -CO(CH2) 14-20 CH3 or -CO(CH2) 14-20 COOH, linked to the N-terminal alpha amine of the GLP-1 receptor antagonist; and R 20 It is COOH or CONH2.

10. The GLP-1 receptor antagonist according to claim 9, wherein The GLP-1 receptor antagonist comprises one to three Aib substitutions at any combination of positions 16, 18, 19, 24, 26 or 28, wherein the position numbers are relative to the native Exendin4 amino acid sequence.

11. The GLP-1 receptor antagonist according to claim 9 or 10, wherein R 10 Yes -CO(CH2) 14-20 COOH; and R 20 It's COOH.

12. The GLP-1 antagonist according to any one of claims 7 to 11, wherein X 11 It is Trp or dTrp.

13. The GLP-1 antagonist according to any one of claims 7 to 12, wherein The amino acid at position 16, 18, 19, 24, 26 or 28 is substituted with Aib.

14. The GLP-1 antagonist according to any one of claims 9 to 13, wherein X 15 It is dGlu; X 16 is Glu; and X 23 It's Ile.

15. The GLP-1 antagonist according to any one of claims 9 to 13, wherein X 15 It is Glu; X 16 is Glu; and X 23 It's Ile. 16 . The GLP-1 antagonist according to claim 9 , further comprising a C-terminal extension of an amino acid having an acyl group, the acyl group being optionally linked to the side chain of the amino acid via a spacer.

17. The GLP-1 antagonist according to claim 16, wherein The acylated amino acid is acylated lysine.

18. The GLP-1 antagonist according to claim 16 or 17, wherein The acyl group of the acylated amino acid is covalently linked to the amino acid side chain of the acylated amino acid via a spacer, optionally wherein, The spacer group includes i) gamma glutamate, ii) Minipeg polymer: -[COCH2(OCH2CH2) k NH]-, wherein k is 2, 4, 6 or 8, iii) or any multiple or combination of i) and / or ii).

19. The GLP-1 antagonist according to any one of claims 9 to 18, wherein R 20 It is CONH2.

20. A pharmaceutical composition comprising the GLP-1 antagonist according to any one of claims 1 to 19 and a pharmaceutically acceptable carrier, diluent or excipient.

21. A method for treating a patient suffering from atypical hypoglycemia, the method comprising the step of administering to a patient in need thereof an amount of the pharmaceutical composition according to claim 20 effective to increase blood glucose levels.

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