Optimized GIP peptide analogues

Abstract

To provide glucose-dependent insulinotropic peptide (GIP)-derived peptide analogues which are antagonists of the GIP receptor.SOLUTION: The invention provides GIP-derived peptide analogues having specific sequences. The GIP peptide analogues are optimized by comprising amino acid substitutions A13Aib and / or N24E, and are fatty acid conjugated with / without a linker, thereby having improved solubility and / or physical stability.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to glucose-dependent insulinotropic peptide (GIP)-derived peptide analogs that are antagonists of the GIP receptor. These GIP peptide analogs are optimized by including the amino acid substitutions A13Aib and / or N24E and are fatty acid conjugated with or without a linker, thereby having improved solubility and / or physical stability while retaining or even improving their antagonistic effect at the GIP receptor. [Background technology]

[0002] Glucose-dependent insulinotropic peptide (GIP) is a hormone secreted from intestinal K cells after a meal. 1 Like its sister hormone glucagon-like peptide 1 (GLP-1), GIP is a potent insulin secretagogue. 2 Glucagon-suppressing effect of GLP-1 3,4 In contrast, GIP has been shown to exhibit glucagon-releasing properties under certain conditions ( 3,5~13 Interest in understanding the biology of GIP is fueled by the association of rodent GIPR (GIP receptor) with adiposity. 14~21 In humans, GIPR expression in adipose tissue is also less clear. 22 , Association between high BMI and increased GIP levels 22,23 Increased adipose tissue blood flow and triacylglycerol (TAG) deposition following GIP administration under conditions of high insulin and glucose levels 24 Decreased basal and postprandial GIP levels observed in diet-treated obese children. 25 , as well as increased fasting GIP levels observed in healthy young men consuming a high-fat diet. 26 There is evidence for a role for GIP in fat metabolism, with the demonstration of

[0003] Hence the discovery of exendin(9-39), a GLP-1 receptor antagonist. 27,28 Following the advances in our understanding of GLP-1, its potential as an anti-obesity drug, coupled with a general demand from researchers, has attracted further attention for the development of potent GIPR antagonists. Many different strategies have been used to antagonize GIP function, e.g., small molecule receptor antagonists. 29 , immunization against GIP 30~32 , various truncations and mutations of the GIP molecule with antagonistic properties 33~39 , and more recently, potent antagonist antibodies against GIPR 40 is being undertaken.

[0004] Under physiological conditions, the 42-amino acid hormone GIP is degraded by the enzyme dipeptidyl peptidase 4 (DPP-4), which cleaves the GIP molecule at the third position to yield GIP(3-42). Synthetic porcine GIP3-42 did not exhibit antagonistic properties in porcine or perfused rat pancreas at physiological concentrations, but did antagonize the human GIPR in vitro. 41 Many peptide hormones are post-translationally modified, resulting in a variety of biological forms with different lengths and amino acid modifications. 42,43 Therefore, GIP(1-30) is generated as a result of post-translational processing. 44 and that it is an agonist for GIPR 33,45 When GIP(1-30) is secreted into the circulation in humans, DPP-4-catalyzed cleavage would result in GIP(3-30). The sequence of native GIP(3-30) is EGTFISDYSIAMDKIHQQDFVNWLLAQK (SEQ ID NO: 68).

[0005] However, GIP(3-30) is poorly soluble at neutral pH of about 7.5, making it unsuitable for pharmaceutical administration.

[0006] Based on this, in addition to having sufficiently high antagonist activity at the GIP receptor, it is necessary for GIP peptide analogs to be sufficiently soluble in aqueous liquid media (especially at physiological pH, such as pH about 7.5 in the absence of GIP(3-30)) and stable, e.g., physically stable. These analogs may be conveniently provided in the form of ready-to-use liquid pharmaceutical formulations suitable for immediate injection, and may be capable of being stored for a sufficiently long period before use. Summary of the Invention

[0007] The present inventors have identified acylated GIP peptides that are antagonists of GIPR, containing the amino acid substitutions A13Aib and / or N24E, which surprisingly result in optimized properties such as improved solubility and / or physical stability, as well as retained or even improved antagonistic properties, making them potentially useful in a number of therapeutic applications.

[0008] The GIP peptides of the present disclosure are N-terminally truncated relative to native GIP(1-42) and do not contain at least the first two amino acids at positions 1 and 2 of GIP(1-42).

[0009] In one aspect, the present disclosure provides the amino acid sequence SEQ ID NO:1:

[0010] [ka]

[0011] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO: 1; N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and The peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0012] An important advantage of the above embodiments, in which the GIP peptide analog comprises the amino acid substitutions A13Aib and / or N24E, is that the solubility and / or stability is improved compared to, for example, native GIP(3-30).

[0013] Improved solubility may include or consist of improved solubility compared to GIP(3-30), for example, at pH 7 (e.g., in 50 mM phosphate buffer at pH 7), pH 7.5 (e.g., in 50 mM phosphate buffer at pH 7.5), pH 8 (e.g., in MilliQ water at pH 8), and / or pH 8.5 (e.g., in MilliQ water at pH 8.5). Measurements may be performed under the conditions indicated in "Evaluation of Solubility." A solubility of greater than 1 mg / ml, or 5 mg / ml, or greater than 7.5 mg / ml, or greater than 10 mg / ml, or even greater than 15 mg / ml may be desired.

[0014] Improved stability may include or consist of, for example, improved physical stability and / or improved chemical stability compared to GIP(3-30).

[0015] Improved physical stability may include or consist of, for example, a reduced tendency to aggregate, either to form soluble or insoluble aggregates, e.g., fibrils. Aggregation (e.g., fibril formation) may be measured, for example, at a starting concentration of 1 mg / ml of dissolved peptide at pH 7.5 and 25 degrees Celsius. An appropriate period of time, e.g., 24 hours, 50 hours, or 96 hours, may be used. Aggregation may be measured under the conditions set forth in "Assessing Physical Stability," with or without stirring. It may be desirable for no fibrils to be detected within 96 hours with stirring.

[0016] A further important advantage of the above embodiment is that the antagonistic effect of the GIP peptide analog is preferably maintained or even improved, which may be particularly true when the GIP peptide analog contains the amino acid substitution A13Aib.

[0017] In another aspect, the invention relates to the use of such GIP peptide analogs as pharmaceuticals.

[0018] In yet another aspect, the invention relates to the use of such GIP peptide analogs in a method for treating a condition selected from the group consisting of metabolic syndrome, obesity, pre-diabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low density lipoprotein (VLDL), low high density lipoprotein (HDL) levels, dyslipidemia, elevated / depressed low density lipoprotein (LDL), high cholesterol levels, abnormal deposition of lipids, cardiovascular disease, elevated blood pressure and atherosclerosis. [Brief explanation of the drawings]

[0019] [Figure 1]Comparison of a reference GIP analog with low physical stability that forms fibrils (AT364 in phosphate buffer—Figure 1A) with a GIP peptide analog with high physical stability that does not form fibrils (AT763 in phosphate buffer—Figure 1B). Note that both curves start at 0–40 absorbance units (AU), indicating the absence of fibrils; however, only in Figure 1A is an increase in absorbance observed due to fibril formation. Also note the different scales on the y-axis. The gap in the curves is due to an unfortunate result related to a restart of the plate reader software approximately 16 hours after the start of the measurement. The plate reader subjected the sample to orbital rotation during the entire measurement, even during the 16-hour period when data was lost. The first 13 cycles were recorded and could be used to determine the pre-transfer baseline (see starting point). Measurement data collection was resumed, and another 764 cycles were performed, totaling approximately 94 hours. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition The term "affinity" refers to the strength of binding between a receptor and its ligand(s). In the present context, the affinity (Ki) of a peptide antagonist for its binding site will determine the duration of inhibition of agonist activity. Antagonist affinity can be determined experimentally using Schild regression for functional studies, or by 1) competitive binding experiments using the Cheng-Prusoff equation, 2) saturation binding experiments using the Scatchard equation, or 3) kinetic studies using on-rate and off-rate determinations (K, respectively). on and K. off This can be determined by radioligand binding studies such as

[0021] The term "IC50" stands for half-maximal inhibitory concentration (IC50) and is a measure of a substance's effectiveness in inhibiting a specific biological or biochemical function. This quantitative measurement indicates the amount of a particular drug or other substance (e.g., an antagonist) required to half-inhibit a given biological process (or a component of that process, i.e., an enzyme, cell, cellular receptor, or microorganism). It is commonly used as a measure of antagonist drug potency in pharmacological studies. IC50 represents the concentration of drug required for 50% inhibition in vitro. In this context, the IC50 value can also refer to the concentration of drug that displaces 50% of a radiolabeled ligand from its receptor, a characterization of drug affinity performed in competitive binding experiments.

[0022] The term "agonist" in this context refers to a peptide, or analog thereof, that is capable of binding to and activating a downstream signaling cascade from a receptor.

[0023] The term "antagonist" in this context refers to a GIP peptide analogue, as defined herein, that can bind to a receptor and block or reduce the agonist-mediated response of the receptor. Antagonists usually do not induce a biological response by themselves when they bind to a receptor. Antagonists have affinity for their cognate receptors but lack potency; their binding to the receptor will inhibit the function of an agonist or inverse agonist at the receptor. Antagonists mediate their effectiveness by binding to the active (orthosteric) or allosteric site on the receptor, or they may interact with a unique binding site that is not normally involved in the biological regulation of receptor activity. Antagonist activity can be reversible or irreversible, depending on the lifetime of the antagonist-receptor complex, which in turn depends on the nature of the antagonist-receptor binding. Most drug antagonists typically achieve their efficacy by competing with endogenous ligands or substrates for structurally defined binding sites on receptors. Antagonists can be competitive, non-competitive, uncompetitive, silent antagonists, partial agonists, or inverse agonists.

[0024] A competitive antagonist (also known as a surmountable antagonist) reversibly binds to the receptor at the same binding site (i.e., the active site) as the endogenous ligand or agonist, but does not activate the receptor. The agonist and antagonist thus "compete" for the same binding site on the receptor. Once bound, the antagonist blocks agonist binding. The level of receptor activation is determined by the relative affinity of each molecule for the site and their relative concentrations. A high concentration of a competitive antagonist will increase the proportion of receptors occupied by the antagonist.

[0025] The term "noncompetitive antagonism" (also called insurmountable or insurmountable antagonism) describes two distinct phenomena with functionally similar results: one in which an antagonist binds to the active site of a receptor, and one in which an antagonist binds to an allosteric site of a receptor. Unlike competitive antagonists, which affect the amount of agonist required to achieve a maximal response but not the magnitude of the maximal response, noncompetitive antagonists reduce the magnitude of the maximal response that can be achieved with a given amount of agonist.

[0026] The term "silent antagonist" refers to a competitive receptor antagonist that has no intrinsic activity to activate the receptor.

[0027] The term "partial agonist" refers to an agonist that, at a given receptor, may differ in the amplitude of the functional response elicited after maximal receptor occupancy. A partial agonist can act as a competitive antagonist in the presence of a full agonist (or a more potent agonist) to compete with the full agonist for receptor occupancy, thereby resulting in a net decrease in receptor activation compared to that observed with the full agonist alone.

[0028] The term "inverse agonist" refers to a ligand, such as a GIP peptide analog, that can bind to the same receptor binding site as an agonist and antagonize its effect. Furthermore, inverse agonists can also inhibit the basal activity of constitutively active receptors.

[0029] The term "glucose-dependent insulinotropic polypeptide receptor (GIPR) antagonist" as used herein refers to a compound, such as a peptide, that is capable of binding to the GIPR and blocking or reducing the agonist-mediated response of the GIPR.

[0030] The term "individual" refers to a vertebrate, a particular member of a mammalian species, preferably a primate, including a human. As used herein, "subject" and "individual" can be used interchangeably.

[0031] An "isolated peptide" is a peptide that has been separated and / or recovered from its natural components, typically a cell, or environment, essentially free from contaminating cellular components such as carbohydrates, lipids, or other proteinaceous impurities inherently associated with the polypeptide. Typically, a preparation of isolated peptide contains the peptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. The term "isolated" does not exclude the presence of the same peptide in alternative physical forms, such as dimers, tetramers, or glycosylated or derivatized forms.

[0032] An "amino acid residue" can be a natural or unnatural amino acid residue linked by a peptide bond or a bond other than a peptide bond. Amino acid residues can be in the D- or L-configuration. An amino acid residue comprises an amino-terminal portion (NH2) and a carboxy-terminal portion (COOH) separated by a central portion comprising a carbon atom or a chain of carbon atoms, at least one of which contains at least one functional side chain. NH2 refers to the amino group present at the amino terminus of an amino acid or peptide, and COOH refers to the carboxy group present at the carboxy terminus of an amino acid or peptide. The common term amino acid includes both natural and unnatural amino acids. Natural amino acids according to standard nomenclature, as listed in J. Biol. Chem., 243:3552-59 (1969) and adopted in 37 CFR, section 1.822(b)(2), belong to the amino acid groups listed therein: Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N, and C. Unnatural amino acids are those not listed immediately above and include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues.

[0033] An "equivalent amino acid residue" refers to an amino acid residue that can replace another amino acid residue in a polypeptide without substantially altering the structure and / or functionality of the polypeptide. Thus, equivalent amino acids have similar properties, such as side chain bulk, side chain polarity (polar or nonpolar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral, or basic), and side chain organization of the carbon molecule (aromatic / aliphatic). Therefore, an "equivalent amino acid residue" can be considered a "conservative amino acid substitution," a substitution of an amino acid whose side chain has similar biochemical properties and therefore does not affect the function of the peptide.

[0034] Among common amino acids, for example, "conservative amino acid substitutions" can also be indicated by substitutions between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine; (2) phenylalanine, tyrosine, and tryptophan; (3) serine and threonine; (4) aspartic acid and glutamic acid; (5) glutamine and asparagine; and (6) lysine, arginine, and histidine.

[0035] Within the meaning of the term "equivalent amino acid substitution" as applied herein, one amino acid can in one embodiment be substituted for another amino acid within the group of amino acids set forth herein below: i) Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys) ii) amino acids with nonpolar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met) iii) Amino acids with aliphatic side chains (Gly, Ala, Val, Leu, Ile) iv) Amino acids with cyclic side chains (Phe, Tyr, Trp, His, Pro) v) Amino acids with aromatic side chains (Phe, Tyr, Trp) vi) Amino acids with acidic side chains (Asp, Glu) vii) Amino acids with basic side chains (Lys, Arg, His) viii) Amino acids with amide side chains (Asn, Gln) ix) Amino acids with hydroxy side chains (Ser, Thr, Tyr) x) amino acids with sulfur-containing side chains (Cys, Met), xi) Neutral and slightly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr) xii) hydrophilic and acidic amino acids (Gln, Asn, Glu, Asp), and xiii) Hydrophobic amino acids (Leu, Ile, Val)

[0036] Additionally, serine residues of the peptides of the present disclosure can be substituted with an amino acid selected from the group consisting of Gln, Asn, and Thr (all amino acids having polar, uncharged side chains); independently, glycine residues (Gly) are substituted with an amino acid selected from the group consisting of Ala, Val, Leu, and Ile; independently, arginine residues (Arg) are substituted with an amino acid selected from the group consisting of Lys and His (all having positively charged side chains); and independently, lysine residues (Lys) are substituted with an amino acid selected from the group consisting of Arg and His. independently, a methionine residue (Met) can be substituted with an amino acid selected from the group consisting of Leu, Pro, Ile, Val, Phe, Tyr and Trp (all having hydrophobic side chains); independently, a glutamine residue (Gln) can be substituted with an amino acid selected from the group consisting of Asp, Glu, and Asn; and independently, an alanine residue (Ala) can be substituted with an amino acid selected from the group consisting of Gly, Val, Leu, and Ile.

[0037] When the L or D configuration (optical isomer) is not specified, the amino acid in question is understood to have the naturally occurring L configuration (see Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984)) or D configuration, and therefore the peptide formed may be composed of L, D amino acids, or mixed L and D sequences.

[0038] As used herein, a glutamic acid (Glu) mimetic is a moiety having two carboxy functional groups separated by three carbon atoms. Examples include beta-Glu, gamma-Glu, or glutaric acid. Glutaric acid is also known as pentanedioic acid.

[0039] A "functional variant" of a peptide is a peptide that can essentially perform the same function as the peptide of which it is a functional variant. In particular, a functional variant can essentially bind to the same molecule, such as a receptor, or can perform the same receptor-mediated response as the peptide of which it is a functional variant. A functional variant of a "glucose-dependent insulinotropic peptide (GIP) analog" is a peptide that can bind to GIPR and activate or inhibit downstream GIPR signaling, such as cAMP production. A functional variant of a glucose-dependent insulinotropic peptide receptor (GIPR) antagonist is a peptide that can bind to GIPR and inhibit or reduce agonist-mediated GIPR signaling, such as cAMP production.

[0040] A "bioactive agent" (i.e., biologically active substance / drug) is any agent, drug, compound, composition, or mixture that provides some pharmacological, often beneficial, effect that can be demonstrated in vitro or in vivo. It refers to GIP peptide analogs and compounds as defined herein or compositions containing them. As used herein, the term further includes any physiologically or pharmacologically active substance that produces a local or systemic effect in an individual.

[0041] The terms "drug" and "pharmaceutical product" as used herein include biologically, physiologically, or pharmacologically active substances that act locally or systemically in the human or animal body.

[0042] The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full range of treatments for a given condition that a patient is suffering from, and refers equally to curative, prophylactic or preventative therapy and ameliorative or palliative therapy, such as administering a peptide or composition to prevent or reduce the risk of acquiring the condition, disease or disorder, for the following purposes: alleviating or alleviating symptoms or complications; delaying the progression of the condition and partially arresting clinical symptoms, disease or disorder; curing or eliminating the condition, disease or disorder; improving or alleviating the condition or symptoms, and remission, whether detectable or undetectable (whether partial or total); and / or preventing or reducing the risk of acquiring the condition, disease or disorder, and "preventing" or "prevention" should be understood to refer to the management and care of a patient for the purpose of preventing the onset of the condition, disease or disorder, including the administration of an active compound to prevent or reduce the risk of developing symptoms or complications. As used herein, the term "amelioration," and variations thereof, means a decrease in the severity and / or undesirable symptoms of a physiological condition or symptom, and / or a slower or longer time course of progression, compared to when the compositions of the invention are not administered.

[0043] The individual to be treated is preferably a mammal, especially a human being, although treatment of animals such as mice, rats, dogs, cats, cows, horses, sheep and pigs is also encompassed herein.

[0044] An "individual in need thereof" refers to an individual who may benefit from the present disclosure. In one embodiment, the individual in need thereof is an individual suffering from a disease, and the disease may be a metabolic disease or disorder such as obesity or diabetes, a bone density disorder, or cancer.

[0045] Treatment according to the present invention may be prophylactic, ameliorative and / or curative.

[0046] A "pharmacologically effective amount," "pharmaceutically effective amount," or "physiologically effective amount" of a bioactive agent is the quantity of bioactive agent present in a pharmaceutical composition, as described herein, that is necessary to provide a desired level of active agent in the bloodstream or at the site of action in the treated individual (e.g., lung, gastric system, colorectal system, prostate, etc.) to provide the expected physiological response when such composition is administered. Bioactive agent in this context refers to a GIP peptide analog as disclosed herein.

[0047] "Co-administering" or "co-administration" as used herein refers to the administration of one or more GIP peptide analogs of the present invention and a state-of-the-art pharmaceutical composition. The at least two components can be administered separately, sequentially, or simultaneously.

[0048] As used herein, "physical stability" refers to a measure of the tendency of a peptide (e.g., a GIP peptide analog of the present invention) to form soluble or insoluble aggregates, e.g., as a result of stress and / or interactions with destabilizing surfaces, such as hydrophobic surfaces and interfaces. The physical stability of an aqueous peptide solution can be assessed by visual inspection and / or turbidity measurement after exposing the composition, packed in an appropriate cartridge (e.g., cartridge or vial), to mechanical / physical stress (e.g., agitation) for various periods of time. A composition can be classified as physically unstable with respect to peptide aggregation if it exhibits visual turbidity. Alternatively, the turbidity of a composition can be assessed by simple turbidity measurements, which are well known to those skilled in the art. The physical stability of an aqueous peptide composition can also be assessed by using an agent that functions as a spectroscopic probe of the conformational state of the peptide. The probe is preferably a small molecule that preferentially binds to non-native conformers of the peptide. One example of such a small molecule spectroscopic probe is Thioflavin T, a fluorescent dye widely used for the detection of amyloid fibrils. In the presence of fibrils, and possibly other peptide structures, Thioflavin T, when bound to the fibril form of the peptide, gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm. Unbound Thioflavin T is essentially non-fluorescent at the wavelengths of interest.

[0049] Detailed Description GIP refers to glucose-dependent insulinotropic polypeptide, also known as gastrointestinal inhibitory peptide (or polypeptide). As used herein, the abbreviation GIP or hGIP refers to human GIP (Uniprot accession number P09681). GIP is derived from a 153-amino acid proprotein and circulates as a biologically active 42-amino acid peptide. It is synthesized by K cells in the duodenal mucosa and jejunum of the gastrointestinal tract.

[0050] GIPR (or GIP receptor) refers to the gastric inhibitory polypeptide receptor. These seven transmembrane proteins are found at least in beta cells in the pancreas. As used herein, the abbreviation GIPR or hGIPR refers to human GIPR (Uniprot accession number P48546).

[0051] In one embodiment, exendin-4 is a peptide having the amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 69).

[0052] In one embodiment, GIP(3-30) is a peptide having the amino acid sequence EGTFISDYSIAMDKIHQQDFVNWLLAQK (SEQ ID NO: 68; GIP3-30).

[0053] The present inventors have identified acylated GIP peptide analogs that are antagonists of GIPR, containing the amino acid substitutions A13Aib and / or N24E, which surprisingly result in improved solubility and / or physical stability, as well as retained or improved antagonistic properties, making them potentially useful in a number of therapeutic applications.

[0054] GIP peptides Substituted GIP peptide analogs Amino acid sequence SEQ ID NO: 1:

[0055] [ka]

[0056] (wherein X1 is any amino acid or is omitted) It is one aspect of the present disclosure to provide a glucose-dependent insulinotropic peptide (GIP) analog, or a functional variant thereof, consisting of: N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and The peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] (wherein X1 is any amino acid or is omitted) It is also an aspect of the present disclosure to provide a GIP analog or functional variant thereof consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:2 (excluding E at position 24); SEQ ID NO:3 (excluding Aib at position 13); and SEQ ID NO:4 (excluding Aib at position 13 and E at position 24); Z is a peptide comprising one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39), and The peptide, or the functional variant thereof, is modified by attaching one fatty acid molecule to one amino acid residue at any position of SEQ ID NO: 2-4, or to one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0061] An important advantage of the above embodiments in which position 13 is substituted with Aib and / or position 24 is substituted with E is that solubility and / or physical stability appear to be improved. In both cases where position 13 is substituted with Aib and position 24 is substituted with E, a significant or even synergistic improvement in solubility and / or physical stability can be obtained.

[0062] In one embodiment, the present disclosure provides a GIP peptide analogue as defined herein above, wherein said GIP peptide analogue is an antagonist of GIPR.

[0063] GIP peptides that are modified relative to the native GIP peptide are called GIP peptide analogs. GIP peptide analogs according to the present disclosure are preferably GIPR antagonists.

[0064] In one embodiment, a GIP peptide analog, or functional variant thereof, according to the present disclosure is an isolated peptide.

[0065] In one embodiment of the disclosure, the GIP peptide analogs have improved solubility compared to the corresponding sequences, for example, lacking Aib at position 13 and / or lacking E at position 24.

[0066] In one embodiment of the present disclosure, the GIP peptide analog has improved solubility compared to native GIP(3-30) and / or compared to AT364 (SEQ ID NO: 6; GIP(3-30)[H18K]C16 diacid+Cex(31-39).

[0067] In one embodiment of the disclosure, the GIP peptide analog has improved solubility compared to GIP(3-30) and / or compared to AT364 (SEQ ID NO: 6) at pH 7 to 9, e.g., pH 7 to 8.5, e.g., pH 7.0 to 8.0, or pH 7.5 to 8.5, as measured, e.g., by visual inspection or as measured, e.g., by UV microplate, and the turbidity absorbance criterion for peptide solubility of ≥ 1 mg / ml can be set as an absorbance at 325 nm of ≤ 0.02 absorbance units (e.g., 5 to 6 times the standard deviation of 8 buffer samples in a plate).

[0068] In one embodiment, the GIP peptide analogue has a water solubility of at least 1 mg / ml, such as at least 5 mg / ml, such as at least 7.5 mg / ml, such as at least 10 mg / ml, such as at least 15 mg / ml.

[0069] In one embodiment, the GIP peptide analog has an aqueous solubility of at least 1 mg / ml, e.g., at least 5 mg / ml, e.g., at least 7.5 mg / ml, e.g., at least 10 mg / ml, e.g., at least 15 mg / ml, at a pH of 7 to 9, e.g., a pH of about 7.5 or about 8.

[0070] In one embodiment, the GIP peptide analog has improved physical stability as measured by a fibrillation lag time in a ThT assay of greater than about 24 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 50 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 96 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 168 hours.

[0071] In one embodiment, the GIP peptide analog does not form fibrils when stirred within about 24 hours, eg, within about 50 hours, eg, within about 96 hours.

[0072] In one embodiment, the GIP peptide analog has improved physical stability as measured by a fibrillation lag time in a ThT assay of greater than about 24 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 50 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 96 hours, e.g., a fibrillation lag time in a ThT assay of greater than about 168 hours, at a pH of 7 to 8.5, e.g., about pH 7.5.

[0073] In one embodiment of the disclosure, the GIP peptide analog has improved physical stability compared to GIP(3-30) and / or compared to AT364 (SEQ ID NO: 6) at pH 7 to 9, e.g., pH 7 to 8.5, e.g., pH 7.0 to 8.0, or pH 7.5 to 8.5, as measured in an assay that measures aggregation, e.g., via a Thioflavin T (ThT) assay, examples of which are described in the "Evaluating Physical Stability" section.

[0074] In one embodiment of the present disclosure, the GIP peptide analog, or said functional variant thereof, is modified by attaching one fatty acid molecule at one amino acid residue at positions 3 to 29 of SEQ ID NO:1.

[0075] In one embodiment of the disclosure, there is provided a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, wherein: The amino acid at position 3 is selected from E, glutaric acid, succinic acid and adipic acid; The amino acid at position 9 is selected from D and E; The amino acid at position 11 is selected from S, K and A: the amino acid at position 12 is selected from I and K; The amino acid at position 13 is selected from A, 2-aminoisobutyric acid (Aib), and K; the amino acid at position 14 is selected from M, L and Nle; the amino acid at position 15 is selected from D and E; The amino acid at position 16 is selected from K and R; the amino acid at position 17 is selected from I and K; The amino acid at position 18 is selected from H and K; The amino acid at position 20 is selected from Q and K; the amino acid at position 21 is selected from D and E; the amino acid at position 24 is selected from N, Q and E; the amino acid at position 34, if present, is selected from P and K; and / or The amino acid at position 40, if present, is K or absent.

[0076] In one embodiment of the disclosure, there is provided a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, wherein: The amino acid at position 4 is G; The amino acid at position 5 is T; The amino acid at position 6 is F; The amino acid at position 7 is I; The amino acid at position 22 is F; The amino acid at position 23 is V; The amino acid at position 25 is W; the amino acid at position 26 is L; and / or The amino acid at position 27 is L.

[0077] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 4 is G, is provided.

[0078] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 5 is T, is provided.

[0079] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 6 is F, is provided.

[0080] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 7 is I is provided.

[0081] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 10 is Y, is provided.

[0082] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 22 is F, is provided.

[0083] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, is provided, wherein the amino acid at position 23 is V.

[0084] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 25 is W, is provided.

[0085] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 26 is L, is provided.

[0086] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, wherein the amino acid at position 27 is L, is provided.

[0087] In one embodiment of the present disclosure, there is provided a GIP peptide analog as defined herein above, wherein the functional variant has one distinct amino acid substitution at any amino acid residue in any one of SEQ ID NOs: 1 to 4, for example, two distinct amino acid substitutions, for example, three distinct amino acid substitutions, for example, four distinct amino acid substitutions, or, for example, one to four distinct amino acid substitutions at any amino acid residue in any one of SEQ ID NOs: 1 to 4.

[0088] In one embodiment, the functional variant has 1 to 2 distinct amino acid substitutions, for example, 2 to 3 distinct amino acid substitutions, for example, 3 to 4 distinct amino acid substitutions, for example, 4 to 5 distinct amino acid substitutions, for example, 5 to 6 distinct amino acid substitutions, for example, 6 to 7 distinct amino acid substitutions, for example, 7 to 8 distinct amino acid substitutions, at any amino acid residue in any one of SEQ ID NOs: 1 to 4.

[0089] In one embodiment, the functional variant has one distinct amino acid substitution, for example, two distinct amino acid substitutions, for example, three distinct amino acid substitutions, for example, four distinct amino acid substitutions at any amino acid residue in any one of SEQ ID NOs: 1 to 4, and the substitutions are conservative amino acid substitutions.

[0090] In one embodiment, the functional variant has 1 to 7 distinct amino acid substitutions at any one of amino acid residues 3 to 30 of any one of SEQ ID NOs: 1 to 4, such as 1 distinct amino acid substitution, for example, 2 distinct amino acid substitutions, for example, 3 distinct amino acid substitutions, for example, 4 distinct amino acid substitutions, for example, 5 distinct amino acid substitutions, for example, 6 distinct amino acid substitutions, for example, 7 distinct amino acid substitutions.

[0091] In one embodiment there is provided a GIP peptide analog as defined herein above, wherein at least one amino acid residue of the GIP peptide analog of any one of SEQ ID NOs: 1-4 is substituted with E, for example at least one amino acid residue at any one of positions 9, 15 and 21 of any one of SEQ ID NOs: 1-4 is substituted with E.

[0092] Substitution of one or more amino acid residues at any one of positions 9, 15, and 21 of the peptides of any one of SEQ ID NOs: 1 to 4 as defined herein with E may result in improved antagonistic effect, improved solubility, and / or improved stability of the substituted peptide.

[0093] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein X1 is an amino acid residue selected from the group consisting of E, glutaric acid, succinic acid and adipic acid.

[0094] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein X 1 is E.

[0095] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein X1 is glutaric acid.

[0096] In one embodiment there is provided a GIP peptide analogue according to any one of the preceding claims, wherein X1 is succinic acid.

[0097] In one embodiment there is provided a GIP peptide analogue according to any one of the preceding claims, wherein X1 is adipic acid.

[0098] The GIP peptide analogs according to the present disclosure having E at position 3 are very potent antagonists at GIPR. However, having E at position 3 may also result in an unstable compound. Without wishing to be bound by theory, E at position 3 may form pyroGlu by cyclization between the amino group at the N-terminus and the side chain carboxylic acid of E. Therefore, it may be advantageous to substitute E at position 3. The inventors recognize that an amino group at the N-terminus may not be necessary to obtain a potent antagonist.

[0099] Substituting glutaric acid for E at position 3 (i.e., the first amino acid from the N-terminus) may be advantageous because glutaric acid does not have an amino group, making N-terminal pyroGlu formation impossible. PyroGlu formation may be an undesirable side reaction for glutamic acid. Substitution with glutaric acid at position 3 may also improve efficacy. Glutaric acid is naturally produced in the body during the metabolism of some amino acids, including lysine and tryptophan. Succinic acid and adipic acid can be used in place of glutaric acid.

[0100] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein D at position 9 of any one of SEQ ID NOs: 1-4, or functional variants thereof, is substituted with any amino acid, for example with E.

[0101] The advantage of having an E at position 9 is that it may improve potency and / or stability and / or solubility.

[0102] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the S at position 11 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, or for example with an amino acid residue selected from the group consisting of A and K.

[0103] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the I at position 12 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, or for example with K.

[0104] In one embodiment, there is provided a GIP peptide analog as defined herein above, wherein the A at position 13 of SEQ ID NO:1 and SEQ ID NO:2, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, for example, substituted with 2-aminoisobutyric acid (Aib) or K.

[0105] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein A at position 13 of SEQ ID NO:1 and SEQ ID NO:2, or a functional variant thereof, is replaced with 2-aminoisobutyric acid (Aib).

[0106] The advantage of having Aib at position 13 is that it may significantly improve GIPR antagonistic effect. In addition, Aib at position 13 may also improve the stability, e.g., in vivo stability or physical stability, of the peptide.

[0107] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the M at position 14 of any one of SEQ ID NOs: 1-4, or functional variants thereof, is substituted with any amino acid, such as a conservative amino acid substitution, for example, with an amino acid residue selected from the group consisting of L and norleucine (Nle).

[0108] Because M is susceptible to oxidation, it may be advantageous to substitute it with another amino acid such as L or Nle, which may also retain potency.

[0109] In one embodiment, there is provided a GIP peptide analog as defined herein above, wherein the D at position 15 of any one of SEQ ID NOs: 1-4, or functional variants thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., with E. The advantage of having E at position 15 is that it may improve potency and / or stability and / or solubility.

[0110] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein K at position 16 of any one of SEQ ID NOs: 1-4, or functional variants thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., substituted with R.

[0111] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the I at position 17 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., with K.

[0112] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the H at position 18 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., with K.

[0113] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein Q at position 20 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, or for example with K.

[0114] In one embodiment, there is provided a GIP peptide analog as defined herein above, wherein the D at position 21 of any one of SEQ ID NOs: 1-4, or functional variants thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., with E. The advantage of having E at position 21 is that it may improve potency and / or stability and / or solubility.

[0115] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein the N at position 24 of SEQ ID NO:1 and SEQ ID NO:3, or a functional variant thereof, is substituted with any amino acid, such as a conservative amino acid substitution, e.g., substituted with Q, or e.g., substituted with E.

[0116] In one embodiment, the GIP peptide analog comprises at least one substitution to K and one substitution to E or Aib at any one of amino acid residues 3 to 30 of any one of SEQ ID NOs: 1-4.

[0117] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, is provided, wherein: the amino acid residue at position 3 is E, glutaric acid, succinic acid or adipic acid; The amino acid residue at position 4 is G. The amino acid residue at position 5 is T. The amino acid residue at position 6 is F. The amino acid residue at position 7 is I, The amino acid residue at position 8 is S. the amino acid residue at position 9 is D or E; The amino acid residue at position 10 is Y. the amino acid residue at position 11 is S, K or A; the amino acid residue at position 12 is I or K; the amino acid residue at position 13 is A, Aib, or K; the amino acid residue at position 14 is M, L or Nle; the amino acid residue at position 15 is D or E; the amino acid residue at position 16 is K or R; the amino acid residue at position 17 is I or K; the amino acid residue at position 18 is H or K; The amino acid residue at position 19 is Q. the amino acid residue at position 20 is Q or K; the amino acid residue at position 21 is D or E; The amino acid residue at position 22 is F. The amino acid residue at position 23 is V. the amino acid residue at position 24 is N, Q or E; The amino acid residue at position 25 is W. the amino acid residue at position 26 is L; the amino acid residue at position 27 is L; The amino acid residue at position 28 is A. the amino acid residue at position 29 is Q, and / or The amino acid residue at position 30 is K.

[0118] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOS: 1-4, or a functional variant thereof, is provided, wherein: the amino acid residue at position 3 is E, glutaric acid, succinic acid or adipic acid; The amino acid residue at position 4 is G. The amino acid residue at position 5 is T. The amino acid residue at position 6 is F. the amino acid residue at position 9 is D or E; The amino acid residue at position 10 is Y. the amino acid residue at position 11 is S, K or A; the amino acid residue at position 12 is I or K; the amino acid residue at position 13 is A, Aib, or K; the amino acid residue at position 14 is M, L or Nle; the amino acid residue at position 15 is D or E; the amino acid residue at position 16 is K or R; the amino acid residue at position 18 is H or K; The amino acid residue at position 19 is Q. the amino acid residue at position 20 is Q or K; the amino acid residue at position 21 is D or E; The amino acid residue at position 22 is F. The amino acid residue at position 23 is V. the amino acid residue at position 24 is N, Q or E; The amino acid residue at position 25 is W. the amino acid residue at position 26 is L, and / or The amino acid residue at position 27 is L.

[0119] In one embodiment, a GIP peptide analog selected from any one of SEQ ID NOs: 1-4, or a functional variant thereof, is provided, wherein: the amino acid residue at position 3 is E, glutaric acid, succinic acid or adipic acid; The amino acid residue at position 4 is G. The amino acid residue at position 5 is T. The amino acid residue at position 6 is F. the amino acid residue at position 9 is D or E; the amino acid residue at position 13 is A, Aib, or K; the amino acid residue at position 14 is M, L or Nle; the amino acid residue at position 15 is D or E; the amino acid residue at position 18 is H or K; the amino acid residue at position 21 is D or E; The amino acid residue at position 22 is F. The amino acid residue at position 23 is V. the amino acid residue at position 24 is N, Q or E; The amino acid residue at position 25 is W. the amino acid residue at position 26 is L, and / or the amino acid residue at position 27 is L; The functional variant has one distinct amino acid substitution at any amino acid residue in any one of SEQ ID NOs: 1 to 4, for example, two distinct amino acid substitutions, for example, three distinct amino acid substitutions, for example, four distinct amino acid substitutions, or, for example, one to four distinct amino acid substitutions at any amino acid residue in any one of SEQ ID NOs: 1 to 4.

[0120] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein Z comprises one or more consecutive amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39).

[0121] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein Z consists of one or more consecutive amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39).

[0122] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein Z consists of one or more consecutive amino acid residues at the C-terminus of exendin-4(30-39) (GPSSGAPPPS; SEQ ID NO: 61; CE30-39).

[0123] In some embodiments, Z includes at least one G or one P. In some embodiments, Z includes at least two Ps.

[0124] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein Z is -glycine or proline, -GP, GPS, GPSS, GPSSG, GPSSGA, GPSSGAP, GPSSGAPP, GPSSGAPPP and GPSSGAPPPS, -PS, PSS, PSSG, PSSGA, PSSGAP, PSSGAPP, PSSGAPPP and PSSGAPPPS, GPSSGA, GPSSGAP, GPSSGAPP, GPSSGAPPP, GPSSGAPPPS, or a variant thereof containing one or two distinct amino acid substitutions at any one of the amino acid residues, or - PSSG, PSSGA, PSSGAP, PSSGAPP, PSSGAPPP and PSSGAPPPS, or variants thereof containing one or two distinct amino acid substitutions at any one of the amino acid residues The peptide is selected from the group consisting of:

[0125] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein a fatty acid molecule is not attached at the amino acid residue at position 3 or the N-terminal amino group of the amino acid residue at position 3 of any one of SEQ ID NOs: 1-4 or functional variants thereof.

[0126] In one embodiment, a GIP peptide analog is provided as defined herein above, wherein the GIP peptide analog has a free N-terminus. Thus, the N-terminus of the GIP peptide analog comprises an unsubstituted amino (-NH2) moiety, such as an unacetylated, unacylated, or unalkylated one. Thus, the N-terminus of the GIP peptide analog may comprise a free amino (-NH2) moiety.

[0127] In one embodiment there is provided a GIP peptide analog as defined herein above, wherein a fatty acid molecule is attached to the side chain of an amino acid residue at position 11, 12, 13, 16, 17, 18, 20, if present, position 34 or, if present, position 40 of said GIP peptide analog, such as any one of SEQ ID NOs: 1-4, or functional variants thereof.

[0128] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein said fatty acid molecule is attached to an amino acid residue at any one of positions 12, 13, 16, 17, 18, position 34, if present, or position 40, if present, of any one of SEQ ID NOs: 1-4, or a functional variant thereof.

[0129] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein said fatty acid molecule is attached to an amino acid residue at position 18 of any one of SEQ ID NOs: 1 to 4, or a functional variant thereof.

[0130] In one embodiment there is provided a GIP peptide analog as defined herein above, wherein a fatty acid molecule is attached to the epsilon-amino group of a K residue of said GIP peptide analog such as any one of SEQ ID NOs: 1-4, or a functional variant thereof comprising at least one K residue.

[0131] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein a fatty acid molecule is attached to the side chain amino group of the amino acid residue at position 18 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, and wherein H at position 18 is replaced with K or Orn in any one of SEQ ID NOs: 1-4.

[0132] Attachment of a fatty acid, with or without a linker, to the side chain amino group of the amino acid residue at position 18 may result in GIP peptide analogs with particularly high antagonistic potency.

[0133] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein a fatty acid molecule is attached to the side chain amino group of the amino acid residue at position 11 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, and wherein S at position 11 is replaced with K or Orn in any one of SEQ ID NOs: 1-4.

[0134] In one embodiment there is provided a GIP peptide analogue as defined herein above, wherein a fatty acid molecule is attached to the side chain amino group of the amino acid residue at position 12 of any one of SEQ ID NOs: 1-4, or a functional variant thereof, and wherein I at position 12 is replaced with K or Orn in any one of SEQ ID NOs: 1-4.

[0135] In one embodiment there is provided a GIP peptide analogue as defined herein above, said GIP peptide analogue comprising:

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] [ka]

[0144]

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[0145]

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[0146]

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[0147]

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[0148]

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[0149]

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[0150]

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[0151]

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[0152]

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[0153]

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[0154]

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[0155]

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[0156]

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[0157]

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[0158]

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[0159]

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[0160]

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[0161]

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[0162]

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[0163]

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[0164]

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[0165] [ka]

[0166] [ka]

[0167] [ka]

[0168] [ka]

[0169] [ka]

[0170] [ka]

[0171] having an amino acid sequence selected from the group consisting of: The peptides may be modified by attaching a fatty acid molecule at any position in any one of the above sequences, and the peptides may be carboxylated at the C-terminus.

[0172] In one embodiment, the GIP peptide analog is C-terminally amidated (-NH2) or C-terminally carboxylated (-COOH).

[0173] In one embodiment, the GIP peptide analog is carboxylated (—COOH) at the C-terminus. Without being bound by theory, it is possible that a free C-terminal carboxylic acid can aid in improving solubility.

[0174] Functional variants - mutants In one embodiment, one or more, or all, of the amino acid substitutions are conservative amino acid substitutions (or synonymous substitutions). Conservative substitutions are substitutions of amino acids whose side chains have similar biochemical properties and therefore do not affect the function of the peptide.

[0175] Specific amino acid substitutions as disclosed herein are K to R; E to D, glutaric acid; M to L; Q to E; I to V; I to L, Aib; A to Aib; Y to W; S to T; N to S; M to Nle; H to K; D to E; N to Q.

[0176] In another embodiment, a functional variant as defined herein comprises a sequence in which an alkyl amino acid is substituted with an alkyl amino acid, an aromatic amino acid is substituted with an aromatic amino acid, a sulfur-containing amino acid is substituted with a sulfur-containing amino acid, a hydroxy-containing amino acid is substituted with a hydroxy-containing amino acid, an acidic amino acid is substituted with an acidic amino acid, a basic amino acid is substituted with a basic amino acid, and / or a dibasic monocarboxylic amino acid is substituted with a dibasic monocarboxylic amino acid.

[0177] Conservative substitutions may be introduced at any one or more of the above specified positions of a GIP peptide analog selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, so long as the resulting variant maintains functionality. However, it may also be desirable to introduce non-conservative substitutions (non-synonymous substitutions) at one or more positions.

[0178] In one embodiment, the non-conservative substitution that results in the formation of a variant of a GIP peptide analog selected from any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 is i) a substitution of an amino acid residue having a substantially different polarity, e.g., a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln, or a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe, or Met) with a residue having a charged amino acid such as Asp, Glu, Arg, or Lys, or a charged or polar residue with a non-polar group; and / or ii) These include substitutions of amino acid residues whose effect on peptide backbone orientation is substantially different, such as substitution of Pro or Gly by or for another residue, and / or iii) substitutions of amino acid residues whose effect on peptide backbone orientation is substantially different, such as substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and / or iv) substitutions of amino acid residues whose steric bulk is substantially different, such as substitution of a bulky residue such as His, Trp, Phe or Tyr for one with a minor side chain, e.g., Ala, Gly or Ser (and vice versa).

[0179] In one embodiment, amino acid substitutions can be made based on their hydrophobicity and hydrophilicity values ​​as well as the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.

[0180] GIP peptide analogs or functional variant counterparts thereof as defined herein comprise proteinogenic or natural amino acids, i.e., the 22 amino acids naturally incorporated into polypeptides. Of these, 20 are encoded by the universal genetic code, and the remaining two, selenocysteine ​​(Sec, U) and pyrrolysine (Pyl, O), are incorporated into proteins by unique synthetic mechanisms.

[0181] In one embodiment, a GIP peptide analog as defined herein comprises one or more non-naturally occurring amino acid residues (non-naturally occurring, non-proteinogenic or non-standard amino acids) or amino acid mimetics such as glutaric acid. Non-naturally occurring amino acid residues include, for example, but are not limited to, Aib, beta-2-naphthyl-alanine, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, ornithine (Orn), trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinecarboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norleucine (Nle), methoxynine (Mox), norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.

[0182] In one embodiment, the amino acid Met is substituted with an oxidation-resistant amino acid analog, such as norleucine (Nle) or Leu, which preserve the amino acid side chain length important for hydrophobic interactions but do not preserve hydrogen-bonding properties; or methoximine (Mox), a non-standard amino acid that more closely resembles the electronic properties of Met compared to Nle; or Lys.

[0183] Standard and / or non-standard amino acids can be linked by peptide bonds (to form a linear peptide chain) or non-peptide bonds (e.g., via the variable side chains of the amino acids). Preferably, the amino acids of the peptides defined herein are linked by peptide bonds.

[0184] The term peptide also encompasses post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as known in the art, including acetylation, phosphorylation, methylation, glucosylation, glycation, amidation, hydroxylation, deimination, deamidation, carbamoylation, and sulfation of one or more amino acid residues, and also proteolytic modifications by lysosomal cathepsins, and known proteinases, including calpains, secretases, and matrix metalloproteinases.

[0185] Functional equivalents of peptides may also include chemical modifications such as by ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids such as ornithine that do not normally occur in human proteins (non-proteinogenic).

[0186] To mimic the important parts of peptide structure, sterically similar compounds can be formulated.This can be achieved by modeling and chemical design techniques known to those skilled in the art.For example, esterification and other alkylation can be used to modify the amino terminus of the di-arginine peptide backbone, for example, to mimic tetrapeptide structure.It will be understood that all such sterically similar constructs are within the scope of the present invention.Peptides with N- and C-terminal alkylation and esterification are also included within the scope of the present invention.For example, glutaric acid is a sterically similar compound that mimics glutamic acid.

[0187] In one embodiment, the N-terminal amino acid of the GIP peptide analogs of the present disclosure does not have any chemical modifications. It may be advantageous for the amino group at the N-terminus of the GIP peptide analog to be free, i.e., unsubstituted, since substitutions may result in agonistic effects at the GIPR.

[0188] In one embodiment, the N-terminus, ie, the NH2 group at the N-terminus, is absent, for example, when position 3 is substituted with glutaric acid, which does not contain an amino group.

[0189] When present, increasing the length of the fatty acid or linker appears to potentially reduce antagonistic potency, but simultaneous incorporation of an Aib residue at position 13, particularly in combination with an E at one or more of positions 9, 15, 21, and 24, e.g., in combination with an E at position 24, appears to compensate for some or all of the reduced potency.

[0190] Bonding of fatty acid molecules In one embodiment, the fatty acid molecule has a side chain amino alkyl group (-C n H 2n It binds to one or more amino acid residues having a hydroxyl group (NH2).

[0191] In one embodiment, the fatty acid molecule is attached to one or more amino acid residues having a side chain amino group (NH2).

[0192] In one embodiment, the fatty acid molecule is attached to the amino group (NH2) of the amino acid residue.

[0193] In one embodiment, a fatty acid molecule is attached to the side chain amino group of the amino residue.

[0194] In one embodiment, the fatty acid molecule is attached to the ε (epsilon) side chain amino group of a lysine residue (Lys, K).

[0195] In one embodiment, a fatty acid molecule is attached to the δ (delta) side chain amino group of an ornithine residue (Orn).

[0196] In one embodiment, the amino acid residue having an attached fatty acid molecule is selected from the group consisting of Lys and Orn.

[0197] In one embodiment, the amino acid residue having an attached fatty acid molecule is Lys.

[0198] In one embodiment, a fatty acid molecule is attached to the delta amino group of the Orn residue of said GIP peptide analog, such as any one of SEQ ID NOs: 1-4, or a functional variant comprising an Orn amino acid residue.

[0199] In one embodiment, a fatty acid molecule is attached to the epsilon amino group of the K residue of said GIP peptide analog, such as any one of SEQ ID NOs: 1-4, or a functional variant thereof.

[0200] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of one fatty acid molecule, wherein the fatty acid molecule is a straight chain fatty acid.

[0201] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of one fatty acid molecule, wherein the fatty acid molecule is a branched fatty acid.

[0202] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of one fatty acid molecule, the fatty acid molecule being a monoacyl fatty acid molecule containing one acyl group, preferably a carboxyl group located at one end of the fatty acid molecule.

[0203] For example, the GIP peptide may be represented by Formula I:

[0204] [ka]

[0205] As depicted in Figure 1, it may be conjugated to a monoacyl fatty acid (e.g., hexadecanoyl) via a linker, L.

[0206] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of a single fatty acid molecule, wherein the fatty acid molecule is a diacyl fatty acid molecule. A diacyl fatty acid molecule is a fatty acid molecule that contains two carboxyl groups. Preferably, one or both carboxyl groups are located at one or each end of the fatty acid molecule.

[0207] For example, the GIP peptide may be represented by Formula II:

[0208] [ka]

[0209] As depicted in Figure 1, the carboxy-pentadecanoyl group can be conjugated via a linker, L, to a diacyl fatty acid (e.g., 15-carboxy-pentadecanoyl), also called a "diacid."

[0210] In one embodiment of the disclosure, a GIP peptide analog is provided that is modified by the attachment of one fatty acid molecule, said fatty acid molecule having the formula CH3(CH2) n It contains an acyl group of the formula CO-, where n is an integer from 4 to 24.

[0211] In one embodiment, the fatty acid molecule is CH3(CH2)6CO-, CH3(CH2)8CO-, CH3(CH2) 10 CO-, CH3(CH2) 12 CO-, CH3(CH2) 14 CO-, CH3(CH2) 16 CO-, CH3(CH2) 18 CO-, CH3(CH2) 20 CO- and CH3(CH2) 22 CO—.

[0212] In one embodiment, the fatty acid molecule is CH3(CH2) 10 CO-(Lauryl, C12), CH3(CH2) 12 CO-(myristoyl, C14), CH3(CH2)14 CO-(palmitoyl, C16), CH3(CH2) 16 CO-(Stearyl, C18), CH3(CH2) 18 CO-(arachidyl, C20) and CH3(CH2) 20 CO-(behenyl, C22).

[0213] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by attaching one fatty acid molecule, said fatty acid molecule containing two acyl groups, each of which is represented by HOOC-CH3(CH2) 10 CO-(dodecanoyl, C12), HOOC-CH3(CH2) 12 CO-(1-tetradecanoyl, C14), HOOC-CH3(CH2) 14 CO-(hexadecanoyl, C16), HOOC-CH3(CH2) 15 CO-(15-carboxy-pentadecanoyl, C17), HOOC-CH3(CH2) 16 CO-(octadecanoyl, C18), HOOC-CH3(CH2) 17 CO-(17-carboxy-heptadecanoyl, C19), HOOC-CH3(CH2) 18 CO-(eicosanoyl, C20), HOOC-CH3(CH2) 19 CO-(19-carboxy-nonadecanoyl, C21) and HOOC-CH3(CH2) 20 CO-(behenyl, C22).

[0214] In one embodiment, the fatty acid molecule has the formula COOH(CH) n It contains an acyl group of CO-(dicarboxylic acid), where n is an integer from 4 to 24.

[0215] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of one fatty acid molecule, said fatty acid molecule being COOH(CH2) 14 CO-, COOH(CH2) 16 CO-, COOH(CH2) 18CO- and COOH(CH2) 20 CO—.

[0216] In one embodiment, the fatty acid molecule is COOH(CH) 14 Containing or consisting of CO-

[0217] In one embodiment, the fatty acid molecule is COOH(CH) 16 Containing or consisting of CO-

[0218] In one embodiment, the fatty acid molecule is COOH(CH) 18 Containing or consisting of CO-

[0219] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of a fatty acid molecule, wherein the fatty acid molecule is directly attached to the epsilon amino group of the side chain of an amino acid residue of the GIP peptide analog.

[0220] Linking fatty acid molecules via a linker The attachment of the fatty acid molecule to the peptide herein can occur directly or indirectly, i.e. via a linker or spacer.

[0221] In one embodiment of the present disclosure, a GIP peptide analog is provided that is modified by the attachment of a single fatty acid molecule, wherein the fatty acid molecule is attached to an amino acid residue via a linker.

[0222] In one embodiment, a fatty acid molecule according to the present disclosure has formula III:

[0223] [ka]

[0224] As depicted in Figure 1, the nucleotides are linked to amino acid residues via a linker or spacer.

[0225] In one embodiment, the fatty acid molecule is attached to the amino acid residue via a linker in such a way that the carboxyl group of the fatty acid molecule forms an amide bond with the amino group of the linker.

[0226] In some embodiments, the linker is: a. one or more α,ω-amino acids; b. one or more amino acids selected from the group consisting of succinic acid, Lys, Glu, and Asp; c.4-Abu, dy-aminobutyric acid e. Dipeptides, for example, wherein the C-terminal amino acid residue is Lys, His or Trp, preferably Lys, and the N-terminal amino acid residue is selected from the group including Ala, Arg, Asp, Asn, Gly, Glu, Gln, Ile, Leu, Val, Phe and Pro, for example, Gly-Lys; f. one or more of gamma-aminobutanoyl (gamma-aminobutyric acid), gamma-glutamyl (gamma-glutamic acid), beta-asparagyl, beta-alanyl, and glycyl; g. γ-Glutamic acid-[8-amino-3,6-dioxaoctanoic acid] n (γGlu-AEEAc n ) (wherein n is an integer from 1 to 50, for example, an integer from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50) and wherein the amino acid sequence is a nucleotide sequence selected from the group consisting of:

[0227] In some embodiments, the linker is: a. α-amino acid, γ-amino acid or ω-amino acid; b. one or more amino acids selected from the group consisting of succinic acid, Lys, Glu, and Asp; c. one or more amino acids selected from the group consisting of Gly and Ser; d. one or more amino acids selected from the group consisting of Ala, Glu, Lys, and Leu; e. one or more of γ-aminobutanoyl (γ-aminobutyric acid), γ-Glu (γ-glutamic acid), β-Asp (β-asparagyl), β-Ala (β-alanyl), 2-aminoisobutyric acid (Aib), and GIy; f.[8-Amino-3,6-dioxaoctanoic acid] n (AEEAc n ) (wherein n is an integer of 1 to 50, for example, an integer of 1 to 4, 1 to 3, or 1 to 2). and wherein the amino acid sequence is a nucleotide sequence selected from the group consisting of:

[0228] In one embodiment, the linker comprises γ-Glu, one or more 8-amino-3,6-dioxaoctanoic acids (AEEAc), or a combination thereof.

[0229] In one embodiment, the linker comprises or consists of GGGS or SGGG.

[0230] In one embodiment, the linker comprises or consists of ALEA or AELA.

[0231] In one embodiment, the linker comprises or consists of 2-aminoisobutyric acid (Aib).

[0232] In one embodiment, the linker comprises or consists of yGlu.

[0233] In one embodiment, the linker comprises or consists of KAAAEKAAAEKAAAE.

[0234] In one embodiment of the present disclosure, the linker is [8-amino-3,6-dioxaoctanoic acid] n (AEEAc) nIn the formula, n is an integer from 1 to 50, for example, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, and preferably n is 1, 2, or 3.

[0235] In one embodiment of the present disclosure, the linker comprises or consists of γ-Glu and one AEEAc, such as γ-Glu and two AEEAc, such as γ-Glu and three AEEAc.

[0236] Exemplary linkers disclosed herein are those that can be attached to an amino acid residue of a GIP peptide analog via either one of the linker's termini. Thus, for example, a linker can be γ-glutamic acid-8-amino-3,6-dioxaoctanoic acid (γ-Glu)-(AEEAc). n ), the linker is preferably connected via γ-Glu or AEEAc n can be linked to an amino acid residue of a GIP peptide analog via

[0237] In one embodiment, the linker is [γ-glutamic acid]-[8-amino-3,6-dioxaoctanoic acid] (γ-Glu)-AEEAc or [8-amino-3,6-dioxaoctanoic acid]-[γ-glutamic acid] (AEEAc-γ-Glu). For example, a GIP peptide can be conjugated to a fatty acid (e.g., C16 or palmitic / palmitoyl in Formula IV, although any other fatty acid can be used) via [γ-glutamic acid]-[8-amino-3,6-dioxaoctanoic acid], as depicted in Formula IV:

[0238] [ka]

[0239] For example, a GIP peptide can be conjugated to a fatty acid (e.g., C16 or palmitic / palmitoyl in Formula IV, although any other fatty acid can be used) via [8-amino-3,6-dioxaoctanoic acid]-[γ-glutamic acid], as depicted in Formula V:

[0240] [ka]

[0241] In one embodiment of the present disclosure, the fatty acid molecule is attached to the amino acid residue via a linker, and the combination of the linker and the fatty acid is: i. Hexadecanoyl-γ-Glu- ii. Hexadecanoyl-γ-Glu-γ-Glu- iii.Hexadecanoyl-γ-Glu-AEEAc- iv. Hexadecanoyl-γ-Glu-AEEAc-AEEAc- v.Hexadecanoyl-γ-Glu-AEEAc-AEEAc-AEEAc- vi.[15-Carboxy-pentadecanoyl]-γ-Glu- vii. [15-Carboxy-pentadecanoyl]-γ-Glu-γ-Glu- viii. [15-Carboxy-pentadecanoyl]-γ-Glu-AEEAc- ix. [15-Carboxy-pentadecanoyl]-γ-Glu-AEEAc-AEEAc- x.[15-Carboxy-pentadecanoyl]-γ-Glu-AEEAc-AEEAc-AEEAc- xi. Octadecanoyl-γ-Glu- xii. Octadecanoyl-γ-Glu-γ-Glu- xiii. Octadecanoyl-γ-Glu-AEEAc- xiv. Octadecanoyl-γ-Glu-AEEAc-AEEAc- xv. Octadecanoyl-γ-Glu-AEEAc-AEEAc-AEEAc- xvi.[17-Carboxy-heptadecanoyl]-γ-Glu- xvii. [17-Carboxy-heptadecanoyl]-γ-Glu-γ-Glu- xviii.[17-Carboxy-heptadecanoyl]-γ-Glu-AEEAc- xix.[17-Carboxy-heptadecanoyl]-γ-Glu-AEEAc-AEEAc- xx.[17-Carboxy-heptadecanoyl]-γ-Glu-AEEAc-AEEAc-AEEAc- xxi. Eicosanoyl-γ-Glu- xxii.Eicosanoyl-γ-Glu-γ-Glu- xxiii.Eicosanoyl-γ-Glu-AEEAc- xxiv. Eicosanoyl-γ-Glu-AEEAc-AEEAc- xxv.Eicosanoyl-γ-Glu-AEEAc-AEEAc-AEEAc- xxvi.[19-Carboxy-nonadecanoyl]-γ-Glu- xxvii. [19-Carboxy-nonadecanoyl]-γ-Glu-γ-Glu- xxviii.[19-Carboxy-nonadecanoyl]-γ-Glu-AEEAc- xxix.[19-Carboxy-nonadecanoyl]-γ-Glu-AEEAc-AEEAc- xxx.[19-Carboxy-nonadecanoyl]-γ-Glu-AEEAc-AEEAc-AEEAc- is selected from the group consisting of:

[0242] In one embodiment of the present disclosure, the fatty acid molecule is attached to the amino acid residue via a linker, and the combination of the linker and the fatty acid is: i.[15-carboxypentadecanoyl]-yGlu ii. [17-carboxy-heptadecanoyl]-γ-Glu-AEEAc-AEEAc-, and iii. [17-carboxy-heptadecanoyl]-yGlu-yGlu is selected from the group consisting of:

[0243] GIP peptides with fatty acids In one embodiment of the disclosure there is provided a GIP peptide analog as defined herein, wherein the GIP peptide analog or functional variant thereof comprises:

[0244] [ka]

[0245] [ka]

[0246] [ka]

[0247] [ka]

[0248] [ka]

[0249] [ka]

[0250] [ka]

[0251] [ka]

[0252] [ka]

[0253]

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[0254]

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[0255]

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[0256]

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[0257]

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[0258]

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[0259]

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[0260]

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[0261]

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[0262]

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[0263]

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[0264]

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[0265]

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[0266]

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[0267]

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[0268]

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[0269]

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[0270]

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[0271]

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[0272]

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[0273]

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[0274]

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[0275]

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[0276]

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[0277]

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[0278]

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[0279]

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[0280]

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[0281]

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[0282]

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[0283]

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[0284]

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[0285]

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[0286]

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[0287]

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[0288]

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[0289]

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[0290]

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[0291]

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[0292]

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[0293]

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[0294]

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[0295]

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[0296] [ka]

[0297] [ka]

[0298] [ka]

[0299] [ka]

[0300] [ka]

[0301] wherein said fatty acid is attached directly or via a linker as defined herein.

[0302] Therefore, C16 is fatty acid CH3(CH2) 14 CO-(palmitoyl), C18 is fatty acid CH3(CH2) 16 CO-(stearyl). The suffix "-diacid" means that the fatty acid molecule is a diacyl fatty acid molecule. Such a suffix does not refer to a monoacyl fatty acid molecule.

[0303] Therefore, C20 is fatty acid CH3(CH2) 18 CO-(arachidyl). The suffix "-diacid" means that the fatty acid molecule is a diacyl fatty acid molecule. Such a suffix does not refer to a monoacyl fatty acid molecule.

[0304] Therefore, C22 is fatty acid CH3(CH2)20 CO-(behenyl). The suffix "-diacid" means that the fatty acid molecule is a diacyl fatty acid molecule. Such a suffix does not refer to a monoacyl fatty acid molecule.

[0305] Determination of antagonist properties and affinity To determine whether a peptide is an antagonist of GIPR, methods known in the art can be used, for example, by determining the IC50 of the peptide. This can be done by generating a dose-response curve and examining the effect of different concentrations of the peptide on reversing agonist activity. The agonist can be GIP1-42, e.g., hGIP-1-42 or hGIP1-30. The GIPR can be hGIPR, rGIPR, mGIPR, canine GIPR, porcine GIPR, or rhesus GIPR. The IC50 value can be calculated for a given antagonist by determining the concentration required to inhibit half of the maximal biological response of the agonist. A method for determining whether a peptide is an antagonist is described in Example 4, although other methods known in the art can also be used. For example, a Schilt plot analysis can be performed on an hGIP1-42 cAMP dose-response curve with increasing concentrations of a GIP-derived peptide. In this way, the type of antagonist activity can also be determined.

[0306] The GIP peptide analogs of the present disclosure are characterized by having antagonistic activity against the GIPR, and in particular, the GIP peptide analogs of the present disclosure are potent antagonists of the GIPR.

[0307] In one embodiment of the present disclosure, the GIP peptide analog inhibits GIPR activity by at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, e.g., about 100%, as measured via an assay that determines the decrease in intracellular cAMP, as measured via the CisBio cAMP assay (Alternative 1), and / or via the "Ghadam" assay (Alternative 2), as described in Materials and Methods.

[0308] In one embodiment of the disclosure, the GIP peptide analog inhibits GIPR activity by at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., about 100%, where inhibition of GIPR activity is determined as a decrease in intracellular cAMP as measured via an assay that determines a decrease in intracellular cAMP, as described in Materials and Methods, e.g., via the CisBio cAMP assay (Alternative 1) and / or via the "Ghadam" assay (Alternative 2). % inhibition is % inhibition of Emax; if a peptide inhibits 85% Emax, it means that there is the remaining 15% activity of GIPR.

[0309] In one embodiment of the present disclosure, the GIP peptide analog has a GIPR antagonizing potency corresponding to an IC50 value of less than 50 nM, e.g., an IC50 value of less than 10 nM, e.g., an IC50 value of less than 5 nM, e.g., an IC50 value of less than 1 nM, e.g., an IC50 value of 0.001 nM to 1 nM, wherein the antagonizing activity (also referred to as "potency") is measured via an assay that determines the decrease in intracellular cAMP, e.g., via the CisBio cAMP assay and / or via the "Ghadam" assay, as described in "Materials and Methods."

[0310] Methods for determining the antagonist activity of a compound such as a GIP peptide analog are known to those skilled in the art. Representative methods that can be used to determine the antagonist activity of a compound such as a GIP peptide analog can be found in the "Examples" section of this specification, for example, these methods include measuring intracellular cAMP and determining the decrease in intracellular cAMP that results from treating cells with a GIP peptide analog.

[0311] The GIP peptide analogs of the present disclosure are also characterized by low or no agonist activity at GIPR. GIP peptide analogs having low or no agonist activity at GIPR, for example, 20% or less, preferably 10% or less, and even more preferably 5% or less agonist activity, are also referred to as "silent agonists."

[0312] In one embodiment, the GIP peptide analogs of the present disclosure can stimulate GIPR activity by up to 30%, such as up to 25%, for example, up to 20%, for example, up to 15%, for example, up to 10%, for example, up to 5%, and in one embodiment, the GIPR peptide analogs of the present disclosure have no agonist activity towards GIPR, i.e., stimulate about 0% GIPR activity.

[0313] Agonist activity of GIP peptide analogs for GIPR can be determined in the same manner as antagonist activity, except that an increase in intracellular cAMP is measured instead of a decrease, as described in Materials and Methods.

[0314] Treatment method It is also an aspect to provide a GIP peptide analogue as defined herein, or a composition comprising a GIP peptide analogue, for use as a pharmaceutical.

[0315] In one embodiment, the amino acid sequence SEQ ID NO:1:

[0316] [ka]

[0317] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO: 1; N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and For use as a pharmaceutical, the peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0318] In one embodiment,

[0319] [ka]

[0320] [ka]

[0321] [ka]

[0322] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO:2 (excluding E at position 24); SEQ ID NO:3 (excluding Aib at position 13); and SEQ ID NO:4 (excluding Aib at position 13 and E at position 24); Z is a peptide comprising one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39), and For use as a pharmaceutical, the peptide, or the functional variant thereof, is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 2 to 4, or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0323] In one embodiment, the amino acid sequence SEQ ID NO:1:

[0324] [ka]

[0325] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO: 1; N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and For use in a method for inhibiting or reducing one or more of: i) GIP-induced glucagon secretion, ii) GIP-induced insulin secretion, iii) GIP-induced somatostatin secretion, iv) GIP-induced glucose uptake, v) GIP-induced fatty acid synthesis and / or fatty acid uptake, vi) high or increased GIPR expression or activity, vii) postprandial GIP release, viii) serum levels of free fatty acids and / or triglycerides, ix) GIP-induced appetite stimulation, x) GIP-induced reduction in energy expenditure, xi) GIP-induced increase in absorption of nutrients from the intestine, xii) GIP-induced reduction in the appetite-suppressing effect of GLP-1, xiii) GIP-induced leptin resistance, said peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0326] In one embodiment:

[0327] [ka]

[0328] [ka]

[0329] [ka]

[0330] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO:2 (excluding E at position 24); SEQ ID NO:3 (excluding Aib at position 13); and SEQ ID NO:4 (excluding Aib at position 13 and E at position 24); Z is a peptide comprising one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39), and For use in a method for inhibiting or reducing one or more of: i) GIP-induced glucagon secretion, ii) GIP-induced insulin secretion, iii) GIP-induced somatostatin secretion, iv) GIP-induced glucose uptake, v) GIP-induced fatty acid synthesis and / or fatty acid uptake, vi) high or increased GIPR expression or activity, vii) postprandial GIP release, viii) serum levels of free fatty acids and / or triglycerides, ix) GIP-induced appetite stimulation, x) GIP-induced reduction in energy expenditure, xi) GIP-induced increase in absorption of nutrients from the intestine, xii) GIP-induced reduction in the appetite-suppressing effect of GLP-1, xiii) GIP-induced leptin resistance, the peptide, or said functional variant thereof, is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NOs: 2-4, or at one amino acid residue at any position of Z SEQ ID NOs: 67; CE31-39.

[0331] In one embodiment, the amino acid sequence SEQ ID NO:1:

[0332] [ka]

[0333] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO: 1; N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and For use in a method for treating a condition selected from the group consisting of metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low density lipoprotein (VLDL), low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, abnormal lipid deposition, cardiovascular disease, elevated blood pressure and atherosclerosis, the peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0334] In one embodiment:

[0335] [ka]

[0336] [ka]

[0337] [ka]

[0338] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO:2 (excluding E at position 24); SEQ ID NO:3 (excluding Aib at position 13); and SEQ ID NO:4 (excluding Aib at position 13 and E at position 24); Z is a peptide comprising one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39), and For use in a method for treating a condition selected from the group consisting of metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low density lipoprotein (VLDL), low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, abnormal deposition of lipids, cardiovascular disease, elevated blood pressure and atherosclerosis, the peptide, or the functional variant thereof, is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NOs: 2-4, or at one amino acid residue at any position of Z SEQ ID NOs: 67; CE31-39.

[0339] In one embodiment, the amino acid sequence SEQ ID NO:1:

[0340] [ka]

[0341] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO: 1; N at position 24 of SEQ ID NO: 1, or a functional variant thereof, is substituted with E, and / or A at position 13 of SEQ ID NO: 1, or a functional variant thereof, is substituted with 2-aminoisobutyric acid (Aib); Z is a peptide containing one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39) or is omitted; and - Treating a condition selected from the group consisting of metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low density lipoprotein (VLDL), low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, abnormal deposition of lipids, cardiovascular disease, elevated blood pressure and atherosclerosis, or -Induces weight loss For use in the manufacture of a medicament for the treatment of a disease, the peptide or said functional variant thereof is modified by attaching one fatty acid molecule at one amino acid residue at any position of SEQ ID NO: 1 or at one amino acid residue at any position of Z SEQ ID NO: 67; CE31-39.

[0342] In one embodiment:

[0343] [ka]

[0344] [ka]

[0345] [ka]

[0346] (wherein X1 is any amino acid or is omitted) or a functional variant thereof, wherein the variant has 1 to 8 distinct amino acid substitutions at any amino acid of SEQ ID NO:2 (excluding E at position 24); SEQ ID NO:3 (excluding Aib at position 13); and SEQ ID NO:4 (excluding Aib at position 13 and E at position 24); Z is a peptide comprising one or more amino acid residues at the C-terminus of exendin-4(31-39) (PSSGAPPPS; SEQ ID NO: 67; CE31-39), and - Treating a condition selected from the group consisting of metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low density lipoprotein (VLDL), low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, abnormal deposition of lipids, cardiovascular disease, elevated blood pressure and atherosclerosis, or -Induces weight loss For use in the manufacture of a medicament for the treatment of a disease characterized in that the peptide, or the functional variant thereof, is modified by attaching one fatty acid molecule to one amino acid residue at any position of SEQ ID NOs: 2-4, or to one amino acid residue at any position of Z SEQ ID NOs: 67; CE31-39.

[0347] In one particular embodiment, there is provided a GIP peptide analogue as defined herein for use in a method of treating obesity.

[0348] In one particular embodiment, there is provided a GIP peptide analogue as defined herein for use in a method of treating diabetes, including type I diabetes and type II diabetes.

[0349] In one particular embodiment, there is provided a GIP peptide analogue as defined herein for use in a method of treating insulin resistance.

[0350] The obesity-related disorder can be any one of: increased food intake, increased appetite, bulimia, bulimia nervosa, antipsychotic or steroid-induced obesity, decreased / increased gastric motility, delayed / increased gastric emptying, decreased physical mobility, osteoarthritis, dyslipidemia, elevated / decreased low-density lipoprotein (LDL), high cholesterol levels, and abnormal deposition of lipids.

[0351] In some embodiments, dyslipidemia, elevated / decreased low density lipoprotein (LDL), cholesterol, and abnormal deposition of lipids, is referred to as a fatty acid metabolism disorder.

[0352] The diabetes-related disorder can be any one of: impaired glucose tolerance (IGT), progression of IGT to type 2 diabetes, progression of non-insulin-dependent type 2 diabetes to insulin-dependent type 2 diabetes, decreased beta cell function, decreased beta cell mass, increased beta cell apoptosis, and decreased glucose sensitivity of beta cells.

[0353] The cardiovascular disease can be any one of coronary heart disease, myocardial infarction, reperfusion injury, stroke, cerebral ischemia, left ventricular hypertrophy, coronary artery disease, hypertension, essential hypertension, acute hypertensive emergency, cardiomyopathy, cardiac dysfunction, exercise intolerance, acute and / or chronic heart failure, cardiac arrhythmia, cardiac arrhythmia, syncopy, angina pectoris, cardiac bypass and / or stent reocclusion, intermittent claudication (also called atherosclerosis oblitterens), diastolic dysfunction, and systolic dysfunction, and combinations thereof.

[0354] Also provided is a method for treating metabolic syndrome, obesity, overweight, diabetes, insulin resistance, an obesity-related disorder as defined herein, or a diabetes-related disorder as defined herein; said method comprising the step of administering to an individual in need thereof an effective amount of a peptide as defined herein.

[0355] An individual in need, as referred to herein, is an individual who can benefit from the administration of a peptide or pharmaceutical composition according to the present disclosure. Such an individual suffers from or is at risk of suffering from metabolic syndrome and / or a metabolic disorder, such as obesity, overweight, diabetes, insulin resistance, an obesity-related disorder as defined herein, or a diabetes-related disorder as defined herein. The individual may be a human being, male or female, young, middle-aged, or elderly. The disorder to be treated or prevented in an individual may be related to the individual's age, the individual's overall health, the medication used to treat the individual, and whether the individual has a past history of a disease or disorder that may cause or induce metabolic syndrome, and / or a metabolic disorder, such as obesity, overweight, diabetes, insulin resistance, an obesity-related disorder as defined herein, or a diabetes-related disorder as defined herein. In some embodiments, the disorder to be treated is associated with GIP-induced glucagon secretion, GIP-induced insulin secretion, GIP-induced somatostatin secretion, GIP-induced glucose uptake, GIP-induced fatty acid synthesis and / or fatty acid uptake, high expression and / or activity of GIPR, postprandial GIP release; the term "high" is understood to refer to a level higher than the corresponding level observed in an individual not requiring treatment.

[0356] Preparation method (peptide) The peptides according to the present disclosure can be prepared by any method known in the art. Thus, the GIP-derived peptides can be prepared by standard peptide preparation techniques such as solution synthesis or Merrifield-type solid phase synthesis.

[0357] In one embodiment, a peptide as defined herein is a non-naturally occurring peptide derived from a naturally occurring native GIP, such as GIP(1-42).

[0358] In one embodiment, a peptide according to the present disclosure is synthetically manufactured or produced.

[0359] Methods for the synthetic production of peptides are well known in the art. Detailed descriptions and practical advice for producing synthetic peptides can be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant GA ed., Oxford University Press, 2002, or: Pharmaceutical Formulation: Development of Peptides and Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

[0360] In one embodiment, the peptide or peptide sequence of the invention is produced synthetically, in particular by the sequence-assisted peptide synthesis (SAPS) method, by solution synthesis, by solid phase peptide synthesis (SPPS) such as Merrifield solid phase synthesis, by recombinant techniques (production by a host cell comprising a first nucleic acid sequence encoding the peptide operatively associated with a second nucleic acid capable of directing expression in said host cell) or by enzymatic synthesis, which are well known to those skilled in the art.

[0361] Peptides can be synthesized batchwise on a fully automated peptide synthesizer using 9-fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc) as the Na-amino protecting group and suitable common protecting groups for side chain functionalities.

[0362] After purification, such as by reverse-phase HPLC, the peptides can be further processed, for example, to obtain cyclic or C- or N-terminally modified isoforms. Methods for cyclization and terminal modifications are well known in the art.

[0363] The peptides according to the invention can be synthesized as monomers or multimers such as dimers or tetramers.

[0364] Pharmaceutical Compositions and Formulations While it is possible for the bioactive agents of the present disclosure to be administered as raw chemicals (peptides), it may be preferable to present them in the form of a pharmaceutical formulation, which may be referred to as a pharmaceutical composition, a pharmaceutically acceptable composition, or a pharmaceutically safe composition.

[0365] Thus, there is further provided a pharmaceutical formulation comprising a bioactive agent of the invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient, and / or diluent. The pharmaceutical formulation can be prepared by conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

[0366] Pharmaceutically acceptable salts of the present peptide compounds that can be prepared are also intended to be covered by the present invention. These salts will be acceptable for their application in pharmaceutical use. That is, it means that the salts retain the biological activity of the parent compound and that the salts have no undesired or adverse effects in their application and use in the treatment of disease.

[0367] Pharmaceutically acceptable salts are prepared by standard methods. If the parent compound is a base, it can be treated, for example, with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it can be treated, for example, with an inorganic or organic base in a suitable solvent.

[0368] The peptide compounds as disclosed herein can be administered in effective amounts in the form of their alkali metal salts or alkaline earth metal salts in parallel, simultaneously or together with pharmaceutically acceptable carriers or diluents, particularly preferably in the form of pharmaceutical compositions thereof, whether by oral, rectal or parenteral (including subcutaneous) route.

[0369] Examples of pharmaceutically acceptable acid addition salts for use in the pharmaceutical compositions of the present invention include those derived from mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, p-toluenesulfonic acid, and arylsulfonic acids.

[0370] In certain embodiments, peptides according to the present disclosure are formulated as acetate, Cl − (chloride) or Na + (sodium) salts.

[0371] In certain embodiments of the present invention (e.g., liquid compositions), the compositions are stable, e.g., physically stable, for at least 4 days of use. In further embodiments, the compositions are stable for at least 2 weeks of use and at least 6 months of storage. In still further embodiments, the compositions are stable for at least 2 weeks of use and at least 1 year of storage. In yet further embodiments, the compositions are stable for at least 4 weeks of use and at least 2 years of storage.

[0372] In this regard, the term "use" for purposes of this paragraph refers to removing a pharmaceutical composition from storage and thereby subjecting it to ambient conditions (conditions of light / dark, temperature, agitation, etc.) for the purpose of using the composition for therapeutic purposes, and the term "storage" for purposes of this paragraph refers to storage under non-agitated conditions in a refrigerator or freezer at a temperature not exceeding about 5 degrees Celsius. The skilled worker will understand the typical range of uses and storage conditions to which these pharmaceutical compositions may be subjected.

[0373] Administration and Dosage According to the present disclosure, a peptide as defined herein, or a composition containing a peptide, is administered to an individual in need of treatment at a pharmaceutically or therapeutically effective dose. Dosage requirements will vary depending on the specific drug composition used, the route of administration, and the specific subject being treated, as well as the severity and type of the disorder and the subject's weight and general condition. Those skilled in the art will recognize that the optimal amount and interval of individual doses of the peptide compound are determined by the nature and extent of the condition being treated, the form, route, and site of administration, and the specific patient being treated, and that such optimal values ​​can be determined by conventional techniques. Those skilled in the art will also understand that the optimal course of treatment, i.e., the number of doses of the compound given per day for a defined number of days, can be ascertained using a conventional course of treatment determination testing.

[0374] In one embodiment, the bioactive agent is administered at least once a day, such as once a day, for example, twice a day, for example, three times a day, for example, four times a day, for example, five times a day.

[0375] Doses can also be administered at intermittent or intervals, whereby a dose is not administered daily. Rather, one or more doses can be administered every 2 days, 3 days, 4 days, 5 days, 6 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, or intervals within these ranges (e.g., every 2 to 4 weeks, or every 4 to 6 weeks).

[0376] In one embodiment, the dosage is administered once weekly, for example, once a week, for example, one dosage per week.

[0377] Administration route It will be understood that the preferred route of administration will depend on the general condition and age of the subject being treated, the nature of the condition being treated, the location of the tissue to be treated within the body and the active ingredient selected, but may be, for example, subcutaneous.

[0378] Systemic treatment For systemic treatment according to the present disclosure, the route of administration can introduce the bioactive agent into the bloodstream and ultimately target the desired site of action.

[0379] Such routes of administration may be any suitable route, such as enteral routes (including oral, rectal, nasal, pulmonary, buccal, sublingual, transdermal, intracisternal and intraperitoneal administration), and / or parenteral routes (including subcutaneous, intramuscular, intraspinal, intracerebral, intravenous and intradermal administration).

[0380] Parenteral administration Parenteral administration is any administration route that is not oral / enteral, thereby avoiding the first-pass degradation of pharmaceuticals in the liver.Therefore, parenteral administration includes injection and perfusion, for example, bolus injection or continuous infusion, for example, intravenous administration, intramuscular administration or subcutaneous administration.In addition, parenteral administration includes inhalation and topical administration.

[0381] Thus, bioactive agents can be administered topically through any mucous membrane of an animal, such as by giving a biologically active substance to the nose, vagina, eye, mouth, genitals, lungs, gastrointestinal tract, or rectum, preferably the mucous membranes of the nose or mouth; thus, parenteral administration can also include buccal, sublingual, nasal, rectal, vaginal, and intraperitoneal administration, as well as pulmonary and bronchial administration by inhalation or device. Agents can also be administered topically through the skin.

[0382] According to an advantageous embodiment of the invention, the GIP analogue is administered subcutaneously.

[0383] Local treatment In one embodiment, the bioactive agent according to the present invention can be used as a local treatment, i.e., can be introduced directly to the site(s) of action. Thus, the bioactive agent can be applied directly to the skin or mucosa, or the bioactive agent can be injected into the site of action, e.g., the affected tissue or a peripheral artery directly connected to the affected tissue. These administration forms preferably avoid the blood-brain barrier.

[0384] Kit of Parts The present disclosure also relates to kits of parts comprising one or more of the above-described bioactive agents and at least one additional or further component, eg, one or more second active ingredients.

[0385] References 1. Baggio LL, Drucker DJ. Biology of Incretins: GLP-1 and GIP. Gastroenterology 2007;132(6):2131-2157. 2. Holst JJ. On the Physiology of GIP and GLP-1. Horm Metab Res 2004;36(11 / 12):747-754. 3. Heer J, Rasmussen C, Coy DH, Holst JJ. Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide, inhibits glucagon secretion via somatostatin (receptor subtype 2) in the perfused rat pancreas. Diabetologia 2008;51(12):2263-2270. 4. Gutniak M, Orskov C, Holst JJ, Ahren B, Efendic S. Antidiabetogenic Effect of Glucagon-like Peptide-1 (7-36)amide in Normal Subjects and Patients with Diabetes Mellitus. N Engl J Med 1992;326(20):1316-1322. 5. Christensen M, Vedtofte L, Holst JJ, Vilsboell T, Knop FK. Glucose-Dependent Insulinotropic Polypeptide: A Bifunctional Glucose-Dependent Regulator of Glucagon and Insulin Secretion in Humans. Diabetes 2011;60(12):3103-3109. 6. Pederson R, Brown J. Interaction of Gastric Inhibitory Polypeptide, Glucose, and Arginine on Insulin and Glucagon Secretion from the Perfused Rat Pancreas. Endocrinology 1978;103(2):610-615. 7. Adrian TE, Bloom SR, Hermansen K, Iversen J. Pancreatic polypeptide, glucagon and insulin secretion from the isolated perfused canine pancreas. Diabetologia 1978;14(6):413-417. 8. Brunicardi FC, Druck P, Seymour NE, Sun YS, Elahi D, Andersen DK. Selective neurohormonal interactions in islet cell secretion in the isolated perfused human pancreas. Journal of Surgical Research 1990;48(4):273-278. 9. Dupre J, Caussignac Y, McDonald TJ, Van Vliet S. Stimulation of Glucagon Secretion by Gastric Inhibitory Polypeptide in Patients with Hepatic Cirrhosis and Hyperglucagonemia. The Journal of Clinical Endocrinology & Metabolism 1991;72(1):125-129. 10. Ding WG, Renstrom E, Rorsman P, Buschard K, Gromada J. Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism. Diabetes 1997;46(5):792-800. 11. Meier JJ, Gallwitz B, Siepmann N et al. Gastric inhibitory polypeptide (GIP) dose-dependently stimulates glucagon secretion in healthy human subjects at euglycaemia. Diabetologia 2003;46(6):798-801. 12. Christensen MB, Calanna S, Holst JJ, Vilsboell T, Knop FK. Glucose-dependent Insulinotropic Polypeptide: Blood Glucose Stabilizing Effects in Patients With Type 2 Diabetes. The Journal of Clinical Endocrinology & Metabolism 2013;99(3):E418-E426. 13. Christensen M, Calanna S, Sparre-Ulrich AH et al. Glucose-Dependent Insulinotropic Polypeptide Augments Glucagon Responses to Hypoglycemia in Type 1 Diabetes. Diabetes 2014. 14. Song DH, Getty-Kaushik L, Tseng E, Simon J, Corkey BE, Wolfe MM. Glucose-Dependent Insulinotropic Polypeptide Enhances Adipocyte Development and Glucose Uptake in Part Through Akt Activation. Gastroenterology 2007;133(6):1796-1805. 15. Miyawaki K, Yamada Y, Ban N et al. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med 2002;8(7):738-742. 16. Starich GH, Bar RS, Mazzaferri EL. GIP increases insulin receptor affinity and cellular sensitivity in adipocytes. Am J Physiol 1985;249(6 Pt 1):E603-E607. 17. Getty-Kaushik L, Song DH, Boylan MO, Corkey BE, Wolfe MM. Glucose-Dependent Insulinotropic Polypeptide Modulates Adipocyte Lipolysis and Reesterification. Obesity 2006;14(7):1124-1131. 18. Hauner H, Glatting G, Kaminska D, Pfeiffer EF. Effects of gastric inhibitory polypeptide on glucose and lipid metabolism of isolated rat adipocytes. Ann Nutr Metab 1988;32(5-6):282-288. 19. Kim SJ, Nian C, Karunakaran S, Clee SM, Isales CM, McIntosh CHS. GIP-Overexpressing Mice Demonstrate Reduced Diet-Induced Obesity and Steatosis, and Improved Glucose Homeostasis. PLoS ONE 2012;7(7):e40156. 20. Nasteska D, Harada N, Suzuki K et al. Chronic Reduction of GIP Secretion Alleviates Obesity and Insulin Resistance Under High-Fat Diet Conditions. Diabetes 2014;63(7):2332-2343. 21. Miyawaki K, Yamada Y, Yano H et al. Glucose intolerance caused by a defect in the entero-insular axis: A study in gastric inhibitory polypeptide receptor knockout mice. Proceedings of the National Academy of Sciences 1999;96(26):14843-14847. 22. Ahlqvist E, Osmark P, Kuulasmaa T et al. Link Between GIP and Osteopontin in Adipose Tissue and Insulin Resistance. Diabetes 2013;62(6):2088-2094. 23. Calanna S, Christensen M, Holst JJ et al. Secretion of Glucose-Dependent Insulinotropic Polypeptide in Patients With Type 2 Diabetes: Systematic review and meta-analysis of clinical studies. Diabetes Care 2013;36(10):3346-3352. 24. Asmar M, Simonsen L, Madsbad S, Stallknecht B, Holst JJ, Bulow J. Glucose-Dependent Insulinotropic Polypeptide May Enhance Fatty Acid Re-esterification in Subcutaneous Abdominal Adipose Tissue in Lean Humans. Diabetes 2010;59(9):2160-2163. 25. Deschamps I, Heptner W, Desjeux JF, Baltakse V, Machinot S, Lestradet H. Effects of diet on insulin and gastric inhibitory polypeptide levels in obese children. Pediatr Res 1980;14(4 Pt 1):300-303. 26. Brons C, Jensen CB, Storgaard H et al. Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men. The Journal of Physiology 2009;587(10):2387-2397. 27. Raufman JP, Singh L, Eng J. Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Description of exendin-3(9-39) amide, a specific exendin receptor antagonist. Journal of Biological Chemistry 1991;266(5):2897-2902. 28. Jorgensen NB, Dirksen C, Bojsen-Moller KN et al. Exaggerated Glucagon-Like Peptide 1 Response Is Important for Improved β-Cell Function and Glucose Tolerance After Roux-en-Y Gastric Bypass in Patients With Type 2 Diabetes. Diabetes 2013;62(9):3044-3052. 29. Nakamura T, Tanimoto H, Mizuno Y, Tsubamoto Y, Noda H. Biological and functional characteristics of a novel lowGComolecular weight antagonist of glucose-dependent insulinotropic polypeptide receptor, SKL-14959, in vitro and in vivo. Diabetes, Obesity and Metabolism 2012;14(6):511-517. 30. Ebert R, Illmer K, Creutzfeldt W. Release of gastric inhibitory polypeptide (GIP) by intraduodenal acidification in rats and humans and abolishment of the incretin effect of acid by GIP-antiserum in rats. Gastroenterology 1979;76(3):515-523. 31. Fulurija A, Lutz TA, Sladko K et al. Vaccination against GIP for the Treatment of Obesity. PLoS ONE 2008;3(9):e3163. 32. Irwin N, McClean PL, Patterson S, Hunter K, Flatt PR. Active immunisation against gastric inhibitory polypeptide (GIP) improves blood glucose control in an animal model of obesity-diabetes. Biological Chemistry. bchm 390, 75. 2009. 16-7-2014. 33. Hinke SA, Manhart S, Pamir N et al. Identification of a bioactive domain in the amino-terminus of glucose-dependent insulinotropic polypeptide (GIP). Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 2001;1547(1):143-155. 34. Tseng CC, Kieffer TJ, Jarboe LA, Usdin TB, Wolfe MM. Postprandial stimulation of insulin release by glucose-dependent insulinotropic polypeptide (GIP). Effect of a specific glucose-dependent insulinotropic polypeptide receptor antagonist in the rat. J Clin Invest 1996;98(11):2440-2445. 35. Irwin N, Green BD, Parker JC, Gault VA, O’Harte FPM, Flatt PR. Biological activity and antidiabetic potential of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide, GIP(1-16) and (Pro3)GIP(1-16). Regulatory Peptides 2006;135(1GCo2):45-53. 36. Kerr BD, Flatt AJS, Flatt PR, Gault VA. Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide. Biochemical and Biophysical Research Communications 2011;404(3):870-876. 37. Gelling RW, Coy DH, Pederson RA et al. GIP(6-30amide) contains the high affinity binding region of GIP and is a potent inhibitor of GIP1-42 action in vitro. Regulatory Peptides 1997;69(3):151-154. 38. Deacon CFP. GIP-(3-42) does not antagonize insulinotropic effects of GIP at physiological concentrations. American Journal of Physiology - Endocrinology and Metabolism 2006;291(3):E468-E475. 39. Gault VA, O’Harte FPM, Harriott P, Flatt PR. Characterization of the Cellular and Metabolic Effects of a Novel Enzyme-Resistant Antagonist of Glucose-Dependent Insulinotropic Polypeptide. Biochemical and Biophysical Research Communications 2002;290(5):1420-1426. 40. Ravn P, Madhurantakam C, Kunze S et al. Structural and Pharmacological Characterization of Novel Potent and Selective Monoclonal Antibody Antagonists of Glucose-dependent Insulinotropic Polypeptide Receptor. Journal of Biological Chemistry 2013;288(27):19760-19772. 41. Deacon CF, Plamboeck A, Rosenkilde MM, de Heer J, Holst JJ. GIP-(3-42) does not antagonize insulinotropic effects of GIP at physiological concentrations. American Journal of Physiology - Endocrinology and Metabolism 2006;291(3):E468-E475. 42. Goetze JP, Hunter I, Lippert SK, Bardram L, Rehfeld JF. Processing-independent analysis of peptide hormones and prohormones in plasma. Front Biosci 2012;17:1804-1815. 43. Goetze JP, Rehfeld JF. Peptide hormones and their prohormones as biomarkers. Biomarkers Med 2009;3(4):335-338. 44. Fujita Y, Asadi A, Yang GK, Kwok YN, Kieffer TJ. Differential processing of pro-glucose-dependent insulinotropic polypeptide in gut. American Journal of Physiology - Gastrointestinal and Liver Physiology 2010;298(5):G608-G614. 45. Widenmaier SB, Kim SJ, Yang GK et al. A GIP Receptor Agonist Exhibits beta-Cell Anti-Apoptotic Actions in Rat Models of Diabetes Resulting in Improved beta-Cell Function and Glycemic Control. PLoS ONE 2010;5(3):e9590. 46. Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52(2):456-467. 47. Kissow H, Hartmann B, Holst JJ et al. Glucagon-like peptide-1 (GLP-1) receptor agonism or DPP-4 inhibition does not accelerate neoplasia in carcinogen treated mice. Regulatory Peptides 2012;179(1-3):91-100.

[0386] Examples This example supports the following conclusions: 1) GIP peptide analogs according to embodiments of the present disclosure that include substitutions A13Aib and / or N24E have improved solubility and / or stability, eg, physical stability. 2) Individual amino acid substitutions at specific sites, for example, Aib at position 13, result in improved antagonism at the GIP receptor. 3) Some acylation sites, such as position 18, show great potential for GIP(3-30)+Z with substitutions A13Aib and / or N24E.

[0387] material and method The production and action of the GIP(3-30) peptide itself are disclosed in International Publication WO2016 / 034186.

[0388] material Human GIP(1-42) was purchased from Phoenix Pharmaceuticals Inc., and the remaining GIP peptide analogs were synthesized by Caslo™, Lyngby, Denmark, Almac Group, Craigavon, UK, Peptides & Elephants GmbH, Hennigsdorf, Germany, and WuXi AppTec, China. Human GIP receptor cDNA (SC110906) was purchased from Origene, Rockville, MD, USA, and cloned into the pCMV-Script vector.

[0389] Transfection and cell culture COS-7 cells were cultured in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal bovine serum, 2 mM glutamine, 180 units / ml penicillin, and 45 μg / ml streptomycin at 10% CO and 37°C. Transient transfection of COS-7 cells for cAMP accumulation was performed using the calcium phosphate precipitation method supplemented with chloroquine. 46~47 .

[0390] HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 180 units / ml penicillin, and 45 μg / ml streptomycin at 10% CO and 37° C. Transient transfection with hGIPR for the CisBio assay from Table 2C was performed by using Lipofectamine 2000.

[0391] cAMP assay Alternative 1 (also called CisBio assay): The in vitro functional activity of compounds against the human GIP receptor can also be measured using HEK-293 cells transiently expressing the receptor. On the day of the assay, cells were resuspended in HBSS buffer (Gibco, 14025-50) supplemented with 20 mM HEPES (Gibco, 15630-106), 0.1% Pluronic F-68 (Gibco, 24040-032), and 0.1% casein (Sigma, C4765), and seeded at a density of 5,000 cells / well in a 384-well plate. The GIP peptide analogs of the present disclosure were diluted in HBSS buffer supplemented with 20 mM HEPES, 0.1% Pluronic, 0.1% casein, and 500 μM IBMX. To test for antagonistic properties, the GIP peptide analogs to be tested were each added independently to cells and incubated at 37°C for 20 minutes before the addition of an agonist (GIP1-42) at an EC50 concentration, followed by a 30-minute incubation at 37°C. The resulting decrease in intracellular cAMP was quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit. The assay is based on the competition between natural cAMP produced by cells and cAMP labeled with dye d2 for binding to a cryptate-labeled antibody. The specific signal (i.e., the energy transfer signal) is inversely proportional to the concentration of cAMP in the sample.

[0392] The cAMP-d2 conjugate and the antibody anti-cAMP-cryptate were both diluted in the lysis buffer provided in the kit and added to the cells according to the manufacturer's protocol. The resulting competitive assay was incubated at room temperature for 60 minutes, and the signal was detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The HTRF ratio (emission at 665 nm / 620 nm x 10,000) is inversely proportional to the amount of cAMP present and is converted to nM cAMP per well using a cAMP standard curve. Dose-response curves were fitted using nonlinear regression analysis (four-parameter logistic equation) in GraphPad Prism, thereby estimating pIC50 values.

[0393] To test for agonist properties at the GIP receptor, compounds were diluted and added to cells as described above and incubated for 30 minutes at 37° C. The resulting increase in intracellular cAMP was determined using the CisBio cAMP Dynamic 2 HTRF assay kit as described above.

[0394] Dose-response curves were fitted using nonlinear regression analysis (four-parameter logistic equation) in GraphPad Prism, thereby estimating pIC50 values.

[0395] Alternative 2 (also called Gaddam assay): Antagonist potency (pKb) was estimated in a functional setting. Estimated pKb values ​​were calculated from the shift in the agonist concentration-response curve in the presence of a single dose of GIP peptide antagonist using the Gaddam equation: pKb = log(DR-1) - log(B), where DR(EC50' / EC50) is the dose ratio calculated from the EC50 of GIP1-42 obtained in the presence and absence of antagonist (EC50), and B is the antagonist concentration used.

[0396] The in vitro functional evaluation of compounds against the human GIP receptor was measured using HEK-293 cells transiently expressing the receptor. On the day of the assay, cells were resuspended in HBSS buffer (Gibco, 14025-50) supplemented with 20 mM HEPES (Gibco, 15630-106), 0.1% Pluronic F-68 (Gibco, 24040-032), and 0.1% casein (Sigma, C4765), and seeded at a density of 3500 cells / well in a 384-well plate. The GIP peptide analogs of the present disclosure were diluted in HBSS buffer supplemented with 20 mM HEPES, 0.1% Pluronic, 0.1% casein, and 500 μM IBMX. The GIP peptide analogs to be tested were added to the cells independently at a concentration of 3.16 nM for compounds AT705-AT718, 31.6 nM for compounds AT719-AT725 and AT745-AT755, and 100 nM for compounds AT739-AT744, and incubated at 37°C for 20 min. Subsequently, increasing doses of the agonist (GIP1-42) were added to the cells and incubated for an additional 30 min at 37°C. Intracellular cAMP was quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit. The assay is based on the competition between natural cAMP produced by the cells and cAMP labeled with dye d2 for binding to a cryptate-labeled antibody. The specific signal (i.e., energy transfer signal) is inversely proportional to the concentration of cAMP in the sample.

[0397] The cAMP-d2 conjugate and the antibody anti-cAMP-cryptate were both diluted in the lysis buffer provided with the kit and added to the cells according to the manufacturer's protocol. The resulting competitive assay was incubated at room temperature for 60 minutes, and the signal was detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The HTRF ratio (emission at 665 nm / 620 nm x 10,000) is inversely proportional to the amount of cAMP present and is converted to nM cAMP per well using a cAMP standard curve.

[0398] Alternative 3 (also called Silt assay) Antagonist potency was also determined by Schild assay. GIP1-42 EC50 was measured in the absence of antagonist, and EC50' was measured in the presence of increasing concentrations of antagonist. These values ​​were used to calculate the dose ratio (DR = EC50' / EC50) for each antagonist concentration, and log(DR-1) was plotted against log(antagonist concentration). The slope of the resulting line was fixed at 1, and pKb was determined as the intercept with the horizontal axis.

[0399] The in vitro functional evaluation of compounds against the human GIP receptor was measured using HEK-293 cells transiently expressing the receptor. On the day of the assay, cells were resuspended in HBSS buffer (Gibco, 14025-50) supplemented with 20 mM HEPES (Gibco, 15630-106), 0.1% Pluronic F-68 (Gibco, 24040-032), and 0.1% casein (Sigma, C4765), and seeded at a density of 3500 cells / well in a 384-well plate. The GIP peptide analogs of the present disclosure were diluted in HBSS buffer supplemented with 20 mM HEPES, 0.1% Pluronic, 0.1% casein, and 500 μM IBMX. The GIP peptide analogs to be tested were added independently to the cells at concentrations of 10 nM, 100 nM, and 1000 nM for GIP3-30 and AT759, 3.16 nM, 31.6 nM, and 316 nM for AT158, AT364, AT760, and AT761, 1 nM, 10 nM, and 100 nM for compounds AT762 and AT763, and 31.6 nM, 316 nM, and 3160 nM for AT758, followed by incubation at 37°C for 20 minutes. Subsequently, increasing doses of the agonist (GIP1-42) were added to the cells and incubated for an additional 30 minutes at 37°C. Intracellular cAMP was quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit. The assay is based on the competition between natural cAMP produced by cells and cAMP labeled with dye d2 for binding to a cryptate-labeled antibody. The specific signal (i.e., the energy transfer signal) is inversely proportional to the concentration of cAMP in the sample.

[0400] The cAMP-d2 conjugate and the antibody anti-cAMP-cryptate were both diluted in the lysis buffer provided in the kit and added to the cells according to the manufacturer's protocol. The resulting competitive assay was incubated at room temperature for 60 minutes, and the signal was detected using a PerkinElmer Envision® instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The HTRF ratio (emission at 665 nm / 620 nm x 10,000) is inversely proportional to the amount of cAMP present and is converted to nM cAMP per well using a cAMP standard curve. Dose-response curves were fitted using nonlinear regression analysis (four-parameter logistic equation) in GraphPad Prism, thereby estimating pIC50 values.

[0401] GIP peptide analogs were also functionally evaluated, and potency was determined using the same assay described above, with some modifications. Functionality was measured in CHO cells stably transfected with GIPR. Cells were resuspended in HBSS buffer containing 5 mM HEPES, 0.1% casein, and 500 μM IBMX. To test, increasing doses of GIP analogs were added independently to the cells and incubated for 20 minutes at 37°C before addition of an agonist (GIP1-42) at EC50-EC80 concentrations, followed by a 30-minute incubation at 37°C. The resulting decrease in cAMP was quantified as described in the previous section.

[0402] [Table 1] TIFF2025172864000120.tif242162TIFF2025172864000121.tif242162TIFF2025172864000122.tif242162TIFF2025172864000123.tif44162

[0403] [Table 2]

[0404] [Table 3]

[0405] [Table 4] TIFF2025172864000127.tif8162

[0406] [Table 5]

[0407] result It can be seen from Tables 2B and 2C that the A13Aib substitution in GIP peptide analogs can improve the antagonistic effect at the GIP receptor.

[0408] Solubility and physical stability Physical stability assessment The amyloid-specific dye thioflavin T (ThT), which is frequently used to indicate the presence of fibrils in solution, was used to detect aggregation in the form of fibril formation.

[0409] ThT fluoresces weakly at approximately 527 nm, but upon binding to beta-sheet-rich structures, it exhibits a red-shift in its emission spectrum to approximately 486 nm and an increase in emission intensity. Continuous measurement of the fluorescence emission at 486 nm can be used as a measure of the fibrillation behavior of peptides and proteins. The time to the onset of fibrillation, or fibrillation lag time, is estimated here by defining the lag time (Tlag) as the point at which the signal relative to the pre-transfer baseline reaches 10% of the post-transfer baseline.

[0410] chemical products Sodium phosphate dibasic (NaHPO, anhydrous, Sigma, lot: SLBL9126V) Sodium phosphate monobasic (NaHPO, anhydrous, Sigma, lot: SLBP1516V)

[0411] A 50 mM sodium phosphate buffer solution for dissolving some samples was prepared in ultrapure water with a resistivity of 18.2 MΩ cm (Milli-Q® Reference A+ system, Merck). The buffer was adjusted to pH 7.4 and filtered.

[0412] Ultrapure water (MilliQ) for dissolving some samples was adjusted to pH 7.4 with NaOH and filtered before sample preparation.

[0413] Sample preparation Peptides were dissolved in sodium phosphate buffer (50 mM, pH 7.4, filtered) or ultrapure water (MilliQ) (adjusted to pH 7.4 with NaOH, filtered) (see Table 3). All peptide samples were prepared at concentrations of 1 mg / ml, 5 mg / ml, 7.5 mg / ml, or 15 mg / ml. All samples, except for the reference peptides GIP(3-30), AT158, and AT482, dissolved readily and produced clear, colorless solutions upon gentle mixing.

[0414] The peptide samples were then filtered through a 0.22 μm nylon filter (Q-Max® RR syringe filter, 13 mm, Frisenette, Denmark) to produce a particle-free solution. All samples were added at t=0 to a 96-well plate reader for the ThT assay.

[0415] For each sample, 22 μL of ThT (1 mM) was added to 1.2 mL of peptide solution. From this sample mixture, 200 μL / well was pipetted into 4 or 5 wells (n = 4 or 5). A blank sample (buffer + ThT) was also included. One 3 mm silica bead was added to each well containing a sample, and the plate was subjected to orbital rotation at 300 rpm to agitate and stress the sample. The temperature was maintained at 25°C during the measurements.

[0416] Plate reader settings: Excitation wavelength: 450 nm Dichroic filter: 465nm Emission wavelength: 486nm Focal height: 3.5mm Gain: 1000 Number of cycles: 960 Cycle time: 360 seconds Number of flushes per well: 20

[0417] result A summary of the results can be found in Table 3. In addition, Figure 1 shows a comparison between a highly stable GIP peptide analog that does not form fibrils (AT763 in phosphate buffer - Figure 1B) and a reference GIP analog that does form fibrils and has poor physical stability (AT364 in phosphate buffer - Figure 1A).

[0418] Table 3 confirms that the A13Aib and / or N24E substitutions in GIP peptide analogs improve the physical stability of the GIP peptide analogs, as measured by a reduced tendency to form fibrils in the ThT assay. See, e.g., AT760 vs. AT364, AT762 vs. AT677, and AT763 vs. AT677. GIP peptide analogs according to embodiments of the present invention did not exhibit an increase in absorbance intensity over the 96-hour measurement period, suggesting that the samples did not fibrillate and that the peptides were physically stable in aqueous solution. See, e.g., AT673, AT695, and AT696 vs. AT364, and e.g., AT749 vs. AT158 and AT719. See also, AT677 vs. AT717 and AT755.

[0419] It can also be seen from Table 3 that fatty acids can be attached at different positions, e.g., positions 12, 13, 16, 17, 18, 34, and 40, and retain improved physical stability. See, for example, AT739, AT740, AT741, AT742, AT743, AT744, and AT668, which did not fibrillate within 96 hours.

[0420] Solubility evaluation The clear visual appearance can be considered as an indicator of the immediate solubility of the peptide. Therefore, it can be confirmed that all peptides according to embodiments of the present invention showed improved solubility compared to GIP(3-30), AT158, and AT482. Therefore, the A13Aib and / or N24E substitutions appear to improve solubility.

[0421] [Table 6] TIFF2025172864000130.tif242162TIFF2025172864000131.tif242162TIFF2025172864000132.tif194162Fnd=No fibrils detected during the experimental period with stirring (96 hours). # Most of the peptide precipitated and was trapped on the filter during filtration.

Claims

1. Amino acid sequence number 18: 【Chemistry 1】 Alternatively, a glucose-dependent insulin secretion-stimulating peptide (GIP) analog comprising a functional variant of SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof, wherein the functional variant has one or two distinct amino acid substitutions in any of the amino acids of SEQ ID NO:

18. One or more amino acid residues at positions 9, 15, 21, and / or 24 of the functional mutant are E, and The amino acid residue at position 13 of the functional mutant is 2-aminoisobutyric acid (Aib), and The amino acid residue at position 14 of the functional mutant is L or norleucine (Nle), and A single fatty acid molecule is bound directly or via a linker to any amino acid residue at any position in SEQ ID NO: 18 or its functional variant. The aforementioned GIP analog is an antagonist of GIPR. Glucose-dependent insulinotropic peptide (GIP) analogues, or pharmaceutically acceptable salts thereof.

2. The GIP analog or its functional variant is a. Having a pH of 7 to 9 and a water solubility of at least 1 mg / ml, b. Having improved physical stability as measured by the fibrillation delay time in a ThT assay exceeding 24 hours. c. Inhibits at least 80% of GIPR activity, and the inhibition of GIPR activity is determined as a decrease in intracellular cAMP, and / or d. A GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, having GIPR antagonistic activity corresponding to an IC50 value of less than 50 nM.

3. The functional variant has one amino acid substitution at any amino acid residue of the amino acid sequence, the GIP analog according to claim 1 or a pharmaceutically acceptable salt thereof.

4. The GIP analog or a pharmaceutically acceptable salt thereof according to Claim 1, wherein in the functional mutant, the amino acid residues at positions 9 and 24 are E, the amino acid residues at positions 9 and 21 are E, the amino acid residues at positions 9 and 15 are E, the amino acid residues at positions 21 and 24 are E, the amino acid residues at positions 15 and 24 are E, or the amino acid residues at positions 15 and 21 are E.

5. The GIP analog or a pharmaceutically acceptable salt thereof according to Claim 1, wherein in the functional mutant, the amino acid residues at positions 9, 15 and 24 are E; the amino acid residues at positions 9, 21 and 24 are E; the amino acid residues at positions 9, 15 and 21 are E; or the amino acid residues at positions 15, 21 and 24 are E.

6. The GIP analog or a pharmaceutically acceptable salt thereof according to Claim 1, wherein in the functional mutant, the amino acid residues at positions 9, 15, 21, and 24 are E.

7. The GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the functional mutant has a K amino acid residue at position 18.

8. In the functional variant, The amino acid residue at position 3 is selected from E, S, G, V, 2-aminoisobutyric acid (Aib), P, D, γ-glutamic acid (γGlu), D-γ-glutamic acid (D-γGlu), β-glutamic acid (βGlu), pyroE (pyroglutamic acid), and glutaric acid; The amino acid residue at position 11 is selected from S, K, Orn, and A; The amino acid residue at position 12 is selected from I, K, Orn, and 2-aminoisobutyric acid (Aib); The amino acid residue at position 16 is selected from K and R; The amino acid residue at position 17 is selected from I, Orn, and K; The amino acid residue at position 20 is selected from Q, Orn, and K; The amino acid residue at position 28 is selected from A and E; The amino acid residue at position 29 is selected from Q and G; The amino acid residue at position 30 is selected from K, R, G, and A; The amino acid residue at position 32 is selected from K and R; and / or The amino acid residue at position 33 is selected from K and R. A GIP analog or a pharmaceutically acceptable salt thereof as described in claim 1.

9. The GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the fatty acid molecule is directly or via a linker bound to any amino acid residue from position 3 to 29 of the amino acid sequence or a functional variant thereof.

10. The GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the fatty acid molecule is directly or via a linker bonded to the side chain amino group of any amino acid residue at position 11, 12, 13, 16, 17, 18, or 20 of the amino acid sequence or a functional variant thereof.

11. The GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the fatty acid molecule is bonded to the side chain amino group of the lysine amino acid residue at position 18 of the amino acid sequence or a functional variant thereof.

12. The fatty acid molecule is a straight-chain fatty acid or a branched fatty acid, and / or The fatty acid molecule is a monoacyl fatty acid molecule or a diacyl fatty acid molecule, and / or The fatty acid molecule comprises an acyl group of the formula CH3(CH2)nCO-, where n is an integer from 4 to 24, and / or The fatty acid molecule contains one or more acyl groups selected from CH3(CH2)6CO-, CH3(CH2)8CO-, CH3(CH2)10CO-, CH3(CH2)12CO-, CH3(CH2)14CO-, CH3(CH2)16CO-, CH3(CH2)18CO-, CH3(CH2)20CO-, and CH3(CH2)22CO-, and / or The fatty acid molecule comprises an acyl group of the formula COOH(CH₂)nCO-, where n is an integer from 4 to 24, and / or The aforementioned fatty acid molecules are HOOC-CH3(CH2)10CO-(dodecanoyl, C12), HOOC-CH3(CH2)12CO-(1-tetradecanoyl, C14), HOOC-CH3(CH2)14CO-(hexadecanoyl, C16), HOOC-CH3(CH2)15CO-(15-carboxypentadecanoyl, C17), HOOC-CH3(CH2)16CO-(octadecanoyl, C18), HOOC-CH3(CH2)17CO-(17-carboxyheptadecanoyl, C19), and HOOC-CH3(CH2)18 comprising one or more acyl groups selected from CO-(eicosanoyl, C20), HOOC-CH3(CH2)19CO-(19-carboxynonadecanoyl, C21), and HOOC-CH3(CH2)20CO-(behenyl, C22), and / or The fatty acid molecule contains an acyl group selected from COOH(CH₂)₄CO-, COOH(CH₂)₆CO-, COOH(CH₂)₁₆CO-, and / or COOH(CH₂)₆CO-. The aforementioned fatty acid molecule is COOH(CH₂)₄CO. A GIP analog or a pharmaceutically acceptable salt thereof as described in claim 1.

13. The fatty acid molecule is bound to an amino acid residue via a linker, and the linker is: a. α-amino acids, γ-amino acids, or ω-amino acids, b. Lys, c. One or more amino acids selected from succinic acid, Lys, Glu, and Asp, d. One or more amino acids selected from Gly and Ser, e. One or more amino acids selected from Ala, Glu, Lys, and Leu, f. One or more γ-aminobutanoyl (γ-aminobutyric acid), γ-Glu (γ-glutamic acid), β-Asp (β-asparagyl), β-Ala (β-alanyl), 2-aminoisobutyric acid (Aib), and Gly, g. [8-amino-3,6-dioxaoctanoic acid] n (AEEAc n) (wherein n is an integer from 1 to 50, for example, an integer from 1 to 4, 1 to 3, or 1 to 2) Including one or more parts individually selected from, A GIP analog or a pharmaceutically acceptable salt thereof as described in claim 1.

14. The combination of the linker and the fatty acid molecule is i. [15-carboxypentadecanoyl]-yGlu ii. [17-carboxyheptadecanoyl]-γ-Glu-AEEAc-AEEAc-, and iii. [17-carboxy-heptadecanoyl]-yGlu-yGlu A GIP analog or a pharmaceutically acceptable salt thereof as described in claim 13, selected from the above.

15. The GIP analog is the GIP analog according to claim 1, having a free C-terminal carboxylic acid, or a pharmaceutically acceptable salt thereof.

16. The GIP analog is A GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the GIP analog is selected from and the GIP analog has a free C-terminal carboxylic acid.

17. The GIP analog is A pharmaceutically acceptable salt of a GIP analog selected from, wherein the pharmaceutically acceptable salt of the GIP analog is selected from a sodium salt, a chloride salt, an acetate salt, and a trifluoroacetate (TFA), as described in claim 1.

18. GIP analogs are The GIP analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein one fatty acid molecule is directly or via a linker bonded to one amino acid residue at any position in SEQ ID NO: 18, and the GIP analog has a free C-terminal carboxylic acid.

19. GIP analogs are The GIP analog according to claim 1, wherein the GIP analog has a free C-terminal carboxylic acid, or a pharmaceutically acceptable salt thereof.

20. The GIP analog is The GIP analog according to claim 1, wherein the pharmaceutically acceptable salt is a pharmaceutically acceptable salt of which one fatty acid molecule is bonded directly or via a linker to one amino acid residue at any position in SEQ ID NO: 18, and the pharmaceutically acceptable salt is selected from sodium salt, chloride salt, acetate salt and trifluoroacetate (TFA).

21. The GIP analog is The GIP analog according to claim 1, wherein the pharmaceutically acceptable salt is selected from sodium salt, chloride salt, acetate salt and trifluoroacetate (TFA).

22. A pharmaceutical product comprising a GIP analogue according to claim 1 for use in the treatment of a condition selected from metabolic syndromes, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low levels of very low-density lipoprotein (VLDL), low high-density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low-density lipoprotein (LDL), high cholesterol levels, abnormal lipid deposition, cardiovascular disease, elevated blood pressure, and atherosclerosis.

23. A pharmaceutical product comprising the GIP analog described in Claim 1 for use in the treatment of obesity or type 2 diabetes.

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