Optimized gip peptide analogs

Abstract

Disclosed are glucose-dependent insulinotropic peptide (GIP) derived peptide analogs that are antagonists of the GIP receptor. These GIP peptide analogs are modified by inclusion of the amino acid substitutions A13Aib and / or N24E and conjugated with a fatty acid with or without a linker, resulting in improved solubility and / or physical stability.

Description

Technical Field

[0001] This invention relates to peptide analogs derived from glucose-dependent insulinotropic peptide (GIP), which are antagonists of the GIP receptor. These GIP peptide analogs are optimized by incorporating amino acid substitutions of A13Aib and / or N24E, and by fatty acid conjugation with or without a linker, thereby achieving improved solubility and / or physical stability while retaining or even improving antagonistic activity against the GIP receptor. Background Technology

[0002] Glucose-dependent insulinotropic peptide (GIP) is a hormone secreted by K cells in the intestine after a meal. 1 Like its sister hormone glucagon-like peptide-1 (GLP-1), GIP is a potent insulin secretagogue. 2 . Glucagon inhibitory effect of GLP-1 3，4 Conversely, it has been shown that GIP exhibits glucagon-releasing properties under certain conditions. 3，5-13 The association between rodent GIPR (GIP receptor) and obesity has heightened interest in understanding the biology of GIP. 14-21 While the exact role of GIP in human lipid metabolism is not yet fully understood, there is evidence suggesting its expression in adipose tissue. 22 The association between high BMI and elevated GIP levels 22，23 In a state of high insulin and high glucose, GIP administration increases blood flow to adipose tissue and TAG (triglyceride) deposition. 24 Decreased basal and postprandial GIP levels were observed in obese children on diets. 25 Elevated fasting GIP levels were observed in healthy young men on a high-fat diet. 26 .

[0003] Therefore, in addition to the discovery of GLP-1 receptor antagonist agonist peptide (9-39) 27，28 Beyond the general need for GLP-1, which has been a subject of advancements in researchers' understanding, its potential as an anti-obesity agent has also spurred increased interest in developing potent GIPR antagonists. Many different strategies have been employed to antagonize GLP-1 function, such as small molecule receptor antagonists. 29 Immunity against GIP 30-32 Various truncations and mutations of GIP molecules with antagonistic properties 33-39 And recently, potent antagonist antibodies against GIPR. 40 .

[0004] Under physiological conditions, the 42-amino acid hormone GIP is degraded by dipeptidyl peptidase 4 (DPP-4), which cleaves the GIP molecule at the third position to yield GIP(3-42). Synthetically produced porcine GIP3-42 did not exhibit antagonistic properties in pigs or perfused rat pancreas at physiological concentrations, but it did antagonize human GIPR in vitro. 41 Many peptide hormones undergo post-translational modification, resulting in various biological forms with different lengths and amino acid modifications. 42，43 Therefore, it has been shown that GIP(1-30) is generated due to post-translation processing. 44 And it is an agonist of GIPR. 33，45 If GIP(1-30) is secreted into the human circulation, it will be cleaved by DPP-4 to produce GIP(3-30). The sequence of natural GIP(3-30) is EGTFISDYSIAMDKIHQQDFVNWLLAQK (SEQ ID NO:68).

[0005] However, GIP(3-30) is poorly soluble at a neutral pH of about 7.5 and is therefore unsuitable for drug administration.

[0006] Building on this, there is a need for such GIP peptide analogs that, in addition to exhibiting satisfactory high antagonistic activity against the GIP receptor, are also sufficiently soluble in aqueous liquid media (especially at physiological pH values, such as approximately 7.5, in which case GIP(3-30) is insoluble) and stable, such as physically stable. These analogs can advantageously be provided in the form of ready-to-use liquid pharmaceutical formulations suitable for immediate injection, and can be stored for a satisfactory long period prior to use. Summary of the Invention

[0007] The inventors have identified acylated GIP peptides comprising amino acid substitutions for A13Aib and / or N24E and acting as GIPR antagonists, which unexpectedly result in optimized properties such as improved solubility and / or physical stability, as well as retained or even improved antagonistic properties. This makes them potentially useful in a range of therapeutic applications.

[0008] The GIP peptide disclosed herein is N-terminally truncated compared to natural GIP (1-42) and at least does not contain the first two amino acids at positions 1 and 2 of GIP (1-42).

[0009] In one respect, this disclosure relates to a glucose-dependent insulinotropic peptide (GIP) analogue, which consists of the amino acid sequence SEQ ID NO:1:

[0010]

[0011] Where X1 is any amino acid or is omitted;

[0012] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0013] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0014] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0015] The peptide is modified by attaching a fatty acid molecule to an amino acid residue at any position of SEQ ID NO 1 or any position of the functional variant thereof, or to an amino acid residue at any position of Z SEQ ID NO:67; CE31-39.

[0016] In the case of GIP peptide analogs containing amino acid substitutions for A13Aib and / or N24E, an important advantage of the above aspects is that the solubility and / or stability are improved compared to, for example, natural GIP (3-30).

[0017] Improved solubility may include or constitute improved solubility of GIP(3-30) compared to, 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). This can be determined under the conditions shown in “Evaluation of Solubility”. Solubility greater than 1 mg / ml or 5 mg / ml or greater than 7.5 mg / ml, greater than 10 mg / ml, or even greater than 15 mg / ml may be desired.

[0018] Improved stability may include or constitute, for example, improved physical stability and / or improved chemical stability compared to GIP (3-30).

[0019] Improved physical stability can include or constitute a reduced tendency to aggregate (e.g., form soluble or insoluble aggregates, such as fibrils). For example, aggregation (e.g., fibril formation) can be determined at an initial concentration of 1 mg / ml of soluble peptide at pH 7.5 and 25°C. Appropriate time periods, such as 24 hours, 50 hours, or 96 hours, can be used. Aggregation can be determined with or without agitation under the conditions shown in “Assessment of Physical Stability”. No fibrils may be detected within 96 hours under agitation.

[0020] Another important advantage of the above aspects is that the antagonistic effect of GIP peptide analogs is preferably preserved or even improved. This is especially likely when the GIP peptide analog contains amino acid substitutions for A13Aib.

[0021] In another respect, the present invention relates to the use of such GIP peptide analogs as pharmaceutical agents.

[0022] In another aspect, the present invention relates to the use of such GIP peptide analogs in methods of treating conditions selected from: 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 levels of high-density lipoprotein (HDL), dyslipidemia, elevated / decreased low-density lipoprotein (LDL), high cholesterol levels, abnormal lipid deposition, cardiovascular disease, high blood pressure, and atherosclerosis. Attached Figure Description

[0023] Figure 1 The reference GIP analog (AT364 in phosphate buffer) has lower physical stability in forming fibrils. Figure 1 A) With GIP peptide analogs that do not form fibrils and have high physical stability (AT763 in phosphate buffer). Figure 1 Compare between B). Note that both curves begin at 0-40 absorbance units (AU), indicating the absence of fibrils, but only when... Figure 1 An increase in absorbance was observed in A, which is due to the formation of fibrils. Also note the different scales on the Y-axis. The gap in the curve is due to an unfortunate issue related to a restart of the plate reader software approximately 16 hours after the start of the measurement. Throughout the measurement, even during the 16 hours of data loss, the plate reader caused the sample to undergo orbital rotation. The first 13 cycles were recorded and can be used to determine the baseline before transition (see Start Point). Measurement data collection was restarted, and 764 more cycles were run, for a total of approximately 94 hours.

[0024] definition

[0025] The term "affinity" refers to the strength of binding between a receptor and one or more ligands. In this context, the affinity (Ki) of a peptide antagonist for its binding site will determine the duration of agonist activity inhibition. Antagonist affinity can be determined experimentally using Schild regression in functional studies or through radioligand binding studies, such as: 1) competitive binding experiments using the Cheng-Prusoff equation, 2) saturation binding experiments using the Scatchard equation, or 3) determining the binding and dissociation rates (Ki and Ki, respectively). on and K off The dynamics of ).

[0026] The term "IC50" stands for half-maximal inhibitory concentration (IC50), which is a measure of a substance's effectiveness in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (e.g., an antagonist) is required to inhibit a given biological process (or a component of that process, i.e., an enzyme, cell, cell receptor, or microorganism) by half. In pharmacological studies, it is commonly used as a measure of the efficacy of antagonists. IC50 represents the drug concentration required for 50% inhibition in vitro. In this context, the IC50 value can also refer to the drug concentration at which 50% of the radiolabeled ligand is transferred from the receptor, a characterization of drug affinity performed in a competitive binding experiment.

[0027] In this context, the term "agonist" refers to a peptide or analogue that can bind to a receptor and activate downstream signaling cascades of the receptor.

[0028] In this context, the term "antagonist" refers to a GIP peptide analog as defined herein, which binds to a receptor and blocks or reduces the agonist-mediated response of said receptor. After binding to the receptor, antagonists typically do not elicit a biological response on their own. Antagonists have an affinity for their homologous receptors but are ineffective, and the binding of an antagonist to its receptor inhibits the function of the receptor by either an agonist or an inverse agonist. Antagonists mediate their action by binding to the active (ortho-)site or allosteric site of the receptor, or they may interact at unique binding sites that are 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 exert their potency by competing with endogenous ligands or substrates at structurally defined binding sites on the receptor. Antagonists can be competitive, non-competitive, non-competitive, silencing antagonists, partial agonists, or inverse agonists.

[0029] Competitive antagonists (also known as overcomeable antagonists) reversibly bind to the receptor at the same binding site (i.e., the active site) as endogenous ligands or agonists, but do not activate the receptor. Agonists and antagonists thus "compete" for the same binding site on the receptor. Once bound, the antagonist blocks agonist binding. The receptor's activity level is determined by the relative affinity of each molecule for the site and its relative concentration. High concentrations of competitive antagonists increase the proportion of the receptor occupied by the antagonist.

[0030] The term "non-competitive antagonism" (also known as insurmountable or insurmountable antagonism) describes two distinct phenomena with functionally similar outcomes: one in which the antagonist binds to the active site of the receptor, and the other in which the antagonist binds to the allosteric site of the receptor. Unlike competitive antagonists, which affect the amount of agonist required to achieve a maximal response but not the strength of that maximal response, non-competitive antagonists reduce the strength of a maximal response that can be achieved with any amount of agonist.

[0031] The term "silencing antagonist" refers to a competitive receptor antagonist that has absolutely no intrinsic activity in activating the receptor.

[0032] The term "partial agonist" refers to an agonist whose amplitude of the functional response induced after maximum receptor occupancy may differ in a given receptor. In the presence of a full agonist (or a more effective agonist), a partial agonist can act as a competitive antagonist because it competes with the full agonist for receptor occupancy, thus producing a net reduction in receptor activation compared to that observed with the full agonist alone.

[0033] The term "inverse agonist" refers to a ligand that can bind to the same receptor binding site as an agonist and antagonize its effect, such as GIP peptide analogs. Furthermore, inverse agonists can also inhibit the basal activity of constitutively active receptors.

[0034] As used herein, the term “glucose-dependent insulinotropic peptide receptor (GIPR) antagonist” refers to a compound, such as a peptide, that can bind to the GIPR and block or reduce GIPR agonist-mediated responses.

[0035] The term "individual" refers to a specific member of a vertebrate, mammalian species, preferably a primate including humans. As used herein, "subject" and "individual" may be used interchangeably.

[0036] "Isolated peptides" are peptides isolated and / or recovered from components of their natural environment (typically the cellular environment) that are substantially free of contaminating cellular components, such as carbohydrates, lipids, or other protein impurities, that are associated with the peptide in nature. Typically, formulations of isolated peptides contain peptides in highly purified forms (i.e., at least about 80%, at least about 90%, at least about 95%, greater than 95%, or greater than 99%). The term "isolated" does not exclude the presence of the same peptide in alternative physical forms (such as dimers, tetramers, or alternatively glycosylated or derived forms).

[0037] "Amino acid residue" can be a natural or non-natural amino acid residue linked by a peptide bond or a bond other than a peptide bond. Amino acid residues can be D-configured or L-configured. An amino acid residue comprises an amino-terminal portion (NH2) and a carboxyl-terminal portion (COOH) separated by a central portion containing a carbon atom or a chain of carbon atoms, at least one of which contains at least one side chain or functional group. NH2 refers to the amino group present at the amino terminus of an amino acid or peptide, and COOH refers to the carboxyl group present at the carboxyl terminus of an amino acid or peptide. The general term amino acid includes both natural and non-natural amino acids. Natural amino acids, as listed in J. Biol. Chem., 243:3552-59 (1969) and in the standard nomenclature adopted in 37 C. FR, section 1.822(b)(2), belong to the group of amino acids listed herein: Y, G, F, M, A, S, I, L, T, V, P, K, H, Q, E, W, R, D, N, and C. Non-natural amino acids are those immediately following those not listed above. Furthermore, non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues.

[0038] "Equivalent amino acid residues" refer to amino acid residues that can replace another amino acid residue in a polypeptide without substantially altering the polypeptide's structure and / or function. Therefore, equivalent amino acids possess similar properties, such as side chain volume, side chain polarity (polar or nonpolar), hydrophobicity (hydrophobic or hydrophilic), pH (acidic, neutral, or basic), and the side chain configuration of the carbon molecule (aromatic / aliphatic). Thus, "equivalent amino acid residues" can be considered "conserved amino acid substitutions," and are substitutions of amino acids with similar biochemical properties in their side chains that do not affect the peptide's function.

[0039] Among common amino acids, for example, “conservative amino acid substitutions” can also be described 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.

[0040] Within the meaning of the term "equivalent amino acid substitution" as used herein, one amino acid can substitute for another amino acid (in one embodiment, within the group of amino acids shown below):

[0041] i) Amino acids with polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys)

[0042] ii) Amino acids with nonpolar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met)

[0043] iii) Amino acids with aliphatic side chains (Gly, Ala, Val, Leu, Ile)

[0044] iv) Amino acids with cyclic side chains (Phe, Tyr, Trp, His, Pro)

[0045] v) Amino acids with aromatic side chains (Phe, Tyr, Trp)

[0046] vi) Amino acids with acidic side chains (Asp, Glu)

[0047] vii) Amino acids with basic side chains (Lys, Arg, His)

[0048] viii) Amino acids with amide side chains (Asn, Gln)

[0049] ix) Amino acids with hydroxyl side chains (Ser, Thr, Tyr)

[0050] x) Amino acids with sulfur-containing side chains (Cys, Met)

[0051] xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr)

[0052] xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and

[0053] xiii) Hydrophobic amino acids (Leu, Ile, Val)

[0054] Furthermore, the serine residues of the peptides in this disclosure may be substituted with amino acids selected from Gln, Asn, and Thr (all amino acids having polar, uncharged side chains); and independently, glycine residues (Gly) may be substituted with amino acids selected from Ala, Val, Leu, and Ile; and independently, arginine residues (Arg) may be substituted with amino acids selected from Lys and His (both having positively charged side chains); and independently, lysine residues (Lys) may be substituted with amino acids selected from Arg and His; and independently, methionine residues (Met) may be substituted with amino acids selected from Leu, Pro, Ile, Val, Phe, Tyr, and Trp (all having hydrophobic side chains); and independently, glutamine residues (Gln) may be substituted with amino acids selected from Asp, Glu, and Asn; and independently, alanine residues (Ala) may be substituted with amino acids selected from Gly, Val, Leu, and Ile.

[0055] Unless otherwise specified, the amino acid in question is understood to have a natural L form (see Pure & Appl. Chem. Vol. 56(5), pp. 595-624 (1984)) or D form, such that the peptide formed may consist of amino acids in L form, D form, or a mixture of L and D forms.

[0056] As used in this article, glutamate (Glu) analogs are moieties having two carboxyl functional groups separated by three carbon atoms. Examples are β-Glu, γ-Glu, or glutaric acid. Glutaric acid is also known as pentanedioic acid.

[0057] A "functional variant" of a peptide is a peptide that performs substantially the same function as its functional variant counterpart. Specifically, a functional variant may bind to substantially the same molecules (such as receptors) or perform the same receptor-mediated responses as its functional variant counterpart. Functional variants of "glucose-dependent insulinotropic peptide (GIP) analogs" are peptides that can bind to GIPR and activate or inhibit downstream GIPR signaling (such as cAMP production). Functional variants of glucose-dependent insulinotropic peptide receptor (GIPR) antagonists are peptides that can bind to GIPR and inhibit or reduce agonist-mediated GIPR signaling (such as cAMP production).

[0058] "Bioactive agent" (i.e., biologically active substance / pharmaceutical) is any agent, drug, compound, composition of substance, or mixture that provides some pharmacological action (usually a beneficial action) that can be demonstrated in vivo or in vitro. It refers to GIP peptide analogs as defined herein and compounds or compositions containing them. As used herein, this term also includes any physiologically or pharmacologically active substance that produces local or systemic effects in an individual.

[0059] As used herein, the terms “drug” or “pharmaceutical” include biologically, physiologically, or pharmacologically active substances that act locally or systemically in humans or animals.

[0060] As used herein, the term "treatment" refers to the management and care of a patient for the purpose of combating a symptom, disease, or disorder. The term is intended to encompass the full range of treatments for a given symptom in a patient, equally including curative, prophylactic / preventative, and ameliorative or palliative therapies, such as the administration of peptides or compositions for the purpose of: reducing or alleviating symptoms or complications; delaying the progression of the symptom, partially halting clinical manifestations, disease, or disorder; curing or eliminating the symptom, disease, or disorder; reducing or alleviating, and resolving (whether partially or entirely), whether detectable or undetectable, symptoms or symptom; and / or preventing the symptom, disease, or disorder or reducing the risk of acquiring the symptom, disease, or disorder. "Preventing" or "prevention" should be understood as the management and care of a patient for the purpose of hindering the development of a symptom, disease, or disorder, and includes the administration of active compounds to prevent symptoms or complications or reduce the risk of their onset. As used herein, the term “mitigation” and variations thereof mean a reduction or prolongation in the degree and / or undesirable manifestation of a physiological condition or symptom and / or the time course of its progression compared to the absence of the composition of the present invention.

[0061] The individuals to be treated are preferably mammals, specifically humans. However, treatment of animals such as mice, rats, dogs, cats, cattle, horses, sheep, and pigs is also included in this article.

[0062] "Individuals in need" refers to individuals who can benefit from this disclosure. In one embodiment, the individual in need is an individual with a disease, which may be a metabolic disease or disorder (such as obesity or diabetes), bone density disorder, or cancer.

[0063] The treatment according to the present invention can be preventive, alleviating, and / or curative.

[0064] The “pharmacologically effective amount,” “pharmacologically effective amount,” or “physiologically effective amount” of a bioactive agent is the amount of active agent present in such a composition required to deliver a desired level of the active agent in the bloodstream or at the site of action (e.g., lungs, gastrointestinal system, colorectal system, prostate, etc.) of an individual to produce an expected physiological response when the pharmaceutical composition as described herein is administered. In this context, a bioactive agent refers to the GIP peptide analogs disclosed herein.

[0065] As used herein, “co-administering” refers to the administration of one or more GIP peptide analogs of the present invention and state-of-the-art pharmaceutical compositions. The at least two components may be administered separately, sequentially, or simultaneously.

[0066] As used herein, “physical stability” refers to a measure of the tendency of a peptide (e.g., the GIP peptide analogue of the present invention) to form soluble or insoluble aggregates of the peptide, for example due to the peptide’s interaction with pressure and / or with destabilizing interfaces and surfaces (e.g., hydrophobic surfaces and interfaces). The physical stability of an aqueous peptide solution can be evaluated by visual inspection and / or turbidity measurement after the composition, filled in a suitable cartridge (e.g., a tube or vial), has been exposed to mechanical / physical pressure (e.g., agitation) for different time periods. When the composition exhibits visible turbidity, it can be classified as physically unstable in terms of peptide aggregation. Alternatively, the turbidity of the composition can be evaluated by simple turbidity measurements well known to those skilled in the art. The physical stability of an aqueous peptide composition can also be evaluated by using a reagent that acts as a spectroscopic probe of the peptide’s conformational state. The probe is preferably a small molecule that preferentially binds to non-native conformational isomers of the peptide. An example of such a small molecule spectroscopic probe is thiosulfate T, a fluorescent dye widely used for the detection of amyloid fibrils. In the presence of protofibrils and possibly other peptide configurations, thiosulfate T exhibits a new excitation maxima at approximately 450 nm and enhanced emission at approximately 482 nm when bound to the protofibril form of the peptide. Unbound thiosulfate T is essentially nonfluorescent at the wavelengths in question. Detailed Implementation

[0067] GIP stands for glucose-dependent insulinotropic peptide, also known as gastric inhibitory peptide (or gastric inhibitory 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 proteomic protein and cycles as a biologically active 42-amino acid peptide. It is synthesized by K cells in the mucosa of the duodenum and jejunum in the gastrointestinal tract.

[0068] GIPR (or GIP receptor) refers to the gastric inhibitory polypeptide receptor. These seven-transmembrane proteins are found on at least the β cells of the pancreas. As used herein, the abbreviation GIPR or hGIPR refers to the human GIPR (Uniprot accession number P48546).

[0069] In one embodiment, agonist-4 is a peptide with an amino acid sequence.

[0070] Peptide of HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 69).

[0071] In one embodiment, GIP(3-30) is a protein with an amino acid sequence.

[0072] Peptide of EGTFISDYSIAMDKIHQQDFVNWLLAQK (SEQ ID NO:68; GIP3-30).

[0073] The inventors have identified such acylated GIP peptide analogs comprising amino acid substitutions of A13Aib and / or N24E and acting as GIPR antagonists, which unexpectedly resulted in improved solubility and / or physical stability, as well as retained or improved antagonistic properties. This makes them potentially useful in a range of therapeutic applications.

[0074] GIP peptides

[0075] Substituted GIP peptide analogs

[0076] One aspect of this disclosure provides a glucose-dependent insulinotropic peptide (GIP) analogue, which consists of the amino acid sequence SEQ ID NO:1:

[0077]

[0078] Where X1 is any amino acid or is omitted;

[0079] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0080] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0081] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0082] The peptide is modified by attaching a fatty acid molecule to an amino acid residue at any position of SEQ ID NO 1 or any position of the functional variant thereof, or to an amino acid residue at any position of Z SEQ ID NO:67; CE31-39.

[0083] One aspect of this disclosure also provides a GIP analogue composed of an amino acid sequence selected from the following:

[0084]

[0085] (SEQ ID NO:2)

[0086]

[0087] (SEQ ID NO:3), and

[0088]

[0089] (SEQ ID NO:4),

[0090] Where X1 is any amino acid or is omitted;

[0091] or its functional variants, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid position in 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);

[0092] Wherein Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39), and

[0093] The peptide is modified by attaching a fatty acid molecule to an amino acid residue at any position of SEQ ID NO:2-4 or any position of the functional variant thereof, or to an amino acid residue at any position of Z SEQ ID NO:67; CE31-39.

[0094] When position 13 is replaced by Aib and / or position 24 is replaced by E, a significant advantage of the above aspects is that solubility and / or physical stability appear to be improved. Excellent or even synergistic improvements in solubility and / or physical stability can be obtained when position 13 is replaced by Aib and position 24 is replaced by E.

[0095] In one embodiment, this disclosure provides a GIP peptide analog as defined above, wherein the GIP peptide analog is a GIPR antagonist.

[0096] GIP peptides that have been modified compared to natural GIP peptides are referred to as GIP peptide analogs. According to this disclosure, the preferred GIP peptide analogs are GIPR antagonists.

[0097] In one embodiment, the GIP peptide analog or a functional variant thereof according to this disclosure is a separate peptide.

[0098] In one embodiment of this disclosure, the GIP peptide analog has improved solubility, for example, compared to a corresponding sequence that does not have Aib at position 13 and / or E at position 24.

[0099] In one embodiment of this disclosure, the GIP peptide analog has improved solubility compared to natural GIP(3-30) and / or compared to AT364 (SEQ ID NO: 6; GIP(3-30)[H18K]C16 diacid + Cex(31-39)).

[0100] In one embodiment of this disclosure, the GIP peptide analog has improved solubility at pH 7 to 9, such as at pH 7 to 8.5, such as at pH 7.0 to 8.0, or at pH 7.5 to 8.5, compared to GIP (3-30) and / or compared to AT364 (SEQ ID NO: 6), as measured by visual inspection or, for example, in a UV microplate, wherein the turbidity absorbance standard for peptide solubility ≥1 mg / ml can be set as absorbance at 325 nm ≤ 0.02 absorbance units (e.g., 5 to 6 times the standard deviation of 8 buffer samples in the plate).

[0101] In one embodiment, the GIP peptide analog 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.

[0102] In one embodiment, the GIP peptide analog has a water solubility of at least 1 mg / ml, at least 5 mg / ml, at least 7.5 mg / ml, at least 10 mg / ml, or at least 15 mg / ml at a pH between 7 and 9, such as about pH 7.5 or about 8.

[0103] In one embodiment, the GIP peptide analog has improved physical stability, such as as measured by fibrillation delay time in a ThT assay of more than about 24 hours, such as fibrillation delay time in a ThT assay of more than about 50 hours, such as fibrillation delay time in a ThT assay of more than about 96 hours, such as fibrillation delay time in a ThT assay of more than 168 hours.

[0104] In one embodiment, the GIP peptide analog does not form fibrils even when agitated for about 24 hours, such as about 50 hours or about 96 hours.

[0105] In one embodiment, the GIP peptide analog has improved physical stability at pH 7 to 8.5, such as at about pH 7.5, as measured by fibrillation delay time in a ThT assay greater than about 24 hours, such as a fibrillation delay time in a ThT assay greater than about 50 hours, such as a fibrillation delay time in a ThT assay greater than about 96 hours, such as a fibrillation delay time in a ThT assay greater than about 168 hours.

[0106] In one embodiment of this disclosure, the GIP peptide analogues have improved physical stability at pH 7 to 9, such as pH 7 to 8.5, such as pH 7.0 to 8.0, or pH 7.5 to 8.5, compared to GIP (3-30) and / or compared to AT364 (SEQ ID NO:6), as in assays for determining aggregation, such as those measured via thiosulfate T (ThT), examples of which are described in the “Assessment of Physical Stability” section.

[0107] In one embodiment of this disclosure, the GIP peptide analog is modified by attaching a fatty acid molecule to an amino acid residue at positions 3 to 29 of SEQ ID NO 1 or its functional variants thereof.

[0108] In one embodiment of this disclosure, a GIP peptide analog selected from any one of SEQ ID NO:1-4 is provided, wherein:

[0109] The third amino acid is selected from E, glutaric acid, succinic acid, and adipic acid;

[0110] The amino acid at position 9 is selected from D and E;

[0111] The 11th amino acid is selected from S, K, and A;

[0112] The amino acid at position 12 is selected from I and K;

[0113] The amino acid at position 13 is selected from A, 2-aminoisobutyric acid (Aib), and K;

[0114] The amino acid at position 14 is selected from M, L, and Nle;

[0115] The amino acid at position 15 is selected from D and E;

[0116] The amino acid at position 16 is selected from K and R;

[0117] The amino acid at position 17 is selected from I and K;

[0118] The amino acid at position 18 is selected from H and K;

[0119] The 20th amino acid is selected from Q and K;

[0120] The amino acid at position 21 is selected from D and E;

[0121] The amino acid at position 24 is selected from N, Q, and E;

[0122] The amino acid at position 34, if present, is selected from P and K; and / or

[0123] The 40th amino acid is either K or absent.

[0124] In one embodiment of this disclosure, a GIP peptide analog selected from any one of SEQ ID NO:1-4 is provided, wherein:

[0125] The fourth amino acid is G;

[0126] The fifth amino acid is T;

[0127] The sixth amino acid is F;

[0128] The 7th amino acid is I;

[0129] The 22nd amino acid is F;

[0130] The 23rd amino acid is V;

[0131] The 25th amino acid is W;

[0132] The 26th amino acid is L; and / or

[0133] The 27th amino acid is L.

[0134] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 4 is G.

[0135] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 5 is T.

[0136] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 6 is F.

[0137] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 7 is I.

[0138] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 10 is Y.

[0139] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 22 is F.

[0140] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 23 is V.

[0141] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 25 is W.

[0142] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 26 is L.

[0143] In one embodiment, a GIP peptide analog or a functional variant thereof selected from any one of SEQ ID NO:1-4 is provided, wherein the amino acid at position 27 is L.

[0144] In one embodiment of this disclosure, a GIP peptide analog as defined above is provided, wherein the functional variant has one single amino acid substitution at any amino acid residue in any of SEQ ID NO:1-4, such as two single amino acid substitutions, such as three single amino acid substitutions, such as four single amino acid substitutions, or one to four single amino acid substitutions at any amino acid residue in any of SEQ ID NO:1-4.

[0145] In one embodiment, the functional variant has 1 to 2 individual amino acid substitutions at any amino acid residue in any of SEQ ID NO:1-4, such as 2 to 3 individual amino acid substitutions, such as 3 to 4 individual amino acid substitutions, such as 4 to 5 individual amino acid substitutions, such as 5 to 6 individual amino acid substitutions, such as 6 to 7 individual amino acid substitutions, such as 7 to 8 individual amino acid substitutions.

[0146] In one embodiment, the functional variant has one single amino acid substitution at any amino acid residue in any of SEQ ID NO:1-4, such as two single amino acid substitutions, such as three single amino acid substitutions, such as four single amino acid substitutions, wherein the substitution is a conserved amino acid substitution.

[0147] In one embodiment, the functional variant has 1 to 7 individual amino acid substitutions at any one of amino acid residues 3 to 30 in any one of SEQ ID NO:1-4, such as 1 individual amino acid substitution, such as 2 individual amino acid substitutions, such as 3 individual amino acid substitutions, such as 4 individual amino acid substitutions, such as 5 individual amino acid substitutions, such as 6 individual amino acid substitutions, such as 7 individual amino acid substitutions.

[0148] In one embodiment, a GIP peptide analog as defined above is provided, wherein at least one amino acid residue of any of the GIP peptide analogs in SEQ ID NO:1-4 is substituted with E, such that at least one amino acid residue at any of the 9th, 15th and 21st positions in any of the GIP peptide analogs in SEQ ID NO:1-4 is substituted with E.

[0149] E substitution of one or more amino acid residues at any of the 9th, 15th, and 21st positions of any peptide in any of the SEQ ID NO:1-4 as defined herein can result in increased antagonism, increased solubility, and / or increased stability of the substituted peptide.

[0150] In one embodiment, a GIP peptide analog as defined above is provided, wherein X1 is an amino acid residue selected from E, glutaric acid, succinic acid, and adipic acid.

[0151] In one implementation, a GIP peptide analog as defined above is provided, wherein X1 is E.

[0152] In one implementation, a GIP peptide analog as defined above is provided, wherein X1 is glutaric acid.

[0153] In one embodiment, a GIP peptide analog according to any one of the preceding claims is provided, wherein X1 is succinic acid.

[0154] In one embodiment, a GIP peptide analog according to any one of the preceding claims is provided, wherein X1 is adipic acid.

[0155] GIP peptide analogs according to this disclosure having an E at the 3rd position can be very potent antagonists of GIPR. However, having an E at the 3rd position can also lead to instability of the compound. Not wishing to be bound by theory, the E at the 3rd position can form pyroGlu through cyclization between the N-terminal amino group and the carboxylic acid side chain of E. Therefore, substituting the E at the 3rd position may be advantageous. The inventors have discovered that the N-terminal amino group may not be necessary to obtain a potent antagonist.

[0156] Replacing the E at position 3 (i.e., the first amino acid from the N-terminus) with glutaric acid may be advantageous because glutaric acid lacks an amino group and therefore cannot form the N-terminal pyroGlu. The formation of pyroGlu might be an unwanted side effect for glutamate. Replacing the E at position 3 with glutaric acid may also increase potency. 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 instead of glutaric acid.

[0157] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 9th D of any of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as E.

[0158] The advantage of having E in position 9 is that the potency and / or stability and / or solubility may be improved.

[0159] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 11th S of any one of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid substitution or such as a substitution with an amino acid residue selected from A and K.

[0160] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 12th position I of any one of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid substitution, or such as being substituted with K.

[0161] In one embodiment, a GIP peptide analog as defined above is provided, wherein position 13A of SEQ ID NO:1 and SEQ ID NO:2 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as substituted with 2-aminoisobutyric acid (Aib) or K.

[0162] In one embodiment, a GIP peptide analog as defined above is provided, wherein position 13A of SEQ ID NO:1 and SEQ ID NO:2 or a functional variant thereof is replaced by 2-aminoisobutyric acid (Aib).

[0163] The advantage of having Aib at position 13 is that GIPR antagonism may be significantly enhanced. Furthermore, Aib at position 13 can increase peptide stability, such as in vivo stability or physical stability.

[0164] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 14th position M of any one of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid substitution, such as substitution with an amino acid residue selected from L and ortholeucine (Nle).

[0165] Since M is easily oxidized, it may be advantageous to replace it with another amino acid (such as L or Nle), which may also preserve its potency.

[0166] In one embodiment, a GIP peptide analog as defined above is provided, wherein the D at position 15 of any of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as E. The advantage of having E at position 15 is that potency and / or stability and / or solubility may be improved.

[0167] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 16th K position of any one of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as R.

[0168] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 17th position I of any one of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as K.

[0169] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 18th H of any of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as K.

[0170] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 20th position Q of any of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid substitution or such as being substituted with K.

[0171] In one embodiment, a GIP peptide analog as defined above is provided, wherein the D at position 21 of any of SEQ ID NO:1-4 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as E. The advantage of having E at position 21 is that potency and / or stability and / or solubility may be improved.

[0172] In one embodiment, a GIP peptide analog as defined above is provided, wherein the 24th N of SEQ ID NO:1 and SEQ ID NO:3 or a functional variant thereof is substituted with any amino acid, such as a conserved amino acid, such as being substituted with Q or such as being substituted with E.

[0173] In one embodiment, the GIP peptide analog contains at least one substitution for K and one substitution for E or Aib at any one of amino acid residues 3 to 30 of any one of SEQ ID NO:1-4.

[0174] In one embodiment, a GIP peptide analog is provided, selected from any one of SEQ ID NO:1-4, wherein:

[0175] The third amino acid residue is E, glutaric acid, succinic acid, or adipic acid.

[0176] The fourth amino acid residue is G.

[0177] The fifth amino acid residue is T.

[0178] The 6th amino acid residue is F.

[0179] The 7th amino acid residue is I.

[0180] The 8th amino acid residue is S.

[0181] The 9th amino acid residue is either D or E.

[0182] The 10th amino acid residue is Y.

[0183] The 11th amino acid residue is S, K, or A.

[0184] The 12th amino acid residue is either I or K.

[0185] The 13th amino acid residue is A, Aib, or K.

[0186] The 14th amino acid residue is M, L, or Nle.

[0187] The 15th amino acid residue is either D or E.

[0188] The 16th amino acid residue is K or R.

[0189] The 17th amino acid residue is either I or K.

[0190] The 18th amino acid residue is H or K.

[0191] The 19th amino acid residue is Q.

[0192] The 20th amino acid residue is either Q or K.

[0193] The amino acid residue at position 21 is either D or E.

[0194] The 22nd amino acid residue is F.

[0195] The 23rd amino acid residue is V.

[0196] The 24th amino acid residue is N, Q, or E.

[0197] The 25th amino acid residue is W.

[0198] The 26th amino acid residue is L.

[0199] The 27th amino acid residue is L.

[0200] The 28th amino acid residue is A.

[0201] The 29th amino acid residue is Q, and / or

[0202] The 30th amino acid residue is K.

[0203] Or its functional variants.

[0204] In one embodiment, a GIP peptide analog is provided, selected from any one of SEQ ID NO 1-4, wherein:

[0205] The third amino acid residue is E, glutaric acid, succinic acid, or adipic acid.

[0206] The fourth amino acid residue is G.

[0207] The fifth amino acid residue is T.

[0208] The 6th amino acid residue is F.

[0209] The 9th amino acid residue is either D or E.

[0210] The 10th amino acid residue is Y.

[0211] The 11th amino acid residue is S, K, or A.

[0212] The 12th amino acid residue is either I or K.

[0213] The 13th amino acid residue is A, Aib, or K.

[0214] The 14th amino acid residue is M, L, or Nle.

[0215] The 15th amino acid residue is either D or E.

[0216] The 16th amino acid residue is K or R.

[0217] The 18th amino acid residue is H or K.

[0218] The 19th amino acid residue is Q.

[0219] The 20th amino acid residue is either Q or K.

[0220] The amino acid residue at position 21 is either D or E.

[0221] The 22nd amino acid residue is F.

[0222] The 23rd amino acid residue is V.

[0223] The 24th amino acid residue is N, Q, or E.

[0224] The 25th amino acid residue is W.

[0225] The 26th amino acid residue is L, and / or

[0226] The 27th amino acid residue is L.

[0227] Or its functional variants.

[0228] In one embodiment, a GIP peptide analogue is provided, selected from any one of SEQ ID NO:1-4, wherein

[0229] The third amino acid residue is E, glutaric acid, succinic acid, or adipic acid.

[0230] The fourth amino acid residue is G.

[0231] The fifth amino acid residue is T.

[0232] The 6th amino acid residue is F.

[0233] The 9th amino acid residue is either D or E.

[0234] The 13th amino acid residue is A, Aib, or K.

[0235] The 14th amino acid residue is M, L, or Nle.

[0236] The 15th amino acid residue is either D or E.

[0237] The 18th amino acid residue is H or K.

[0238] The amino acid residue at position 21 is either D or E.

[0239] The 22nd amino acid residue is F.

[0240] The 23rd amino acid residue is V.

[0241] The 24th amino acid residue is N, Q, or E.

[0242] The 25th amino acid residue is W.

[0243] The 26th amino acid residue is L, and / or

[0244] The 27th amino acid residue is L.

[0245] or functional variants thereof, wherein the functional variant has one single amino acid substitution at any amino acid residue in any of SEQ ID NO:1-4, such as two single amino acid substitutions, such as three single amino acid substitutions, such as four single amino acid substitutions, or one to four single amino acid substitutions at any amino acid residue in any of SEQ ID NO:1-4.

[0246] In one embodiment, a GIP peptide analog as defined above is provided, wherein Z comprises one or more consecutive amino acid residues at the C-terminus of agonist peptide-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39).

[0247] In one embodiment, a GIP peptide analog as defined above is provided, wherein Z consists of one or more consecutive amino acid residues at the C-terminus of agonist peptide-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39).

[0248] In one embodiment, a GIP peptide analog as defined above is provided, wherein Z consists of one or more consecutive amino acid residues at the C-terminus of agonist peptide-4(30-39) (GPSSGAPPPS; SEQ ID NO:61; CE30-39).

[0249] In some embodiments, Z includes at least one G or one P. In some embodiments, Z includes at least two Ps.

[0250] In one implementation, a GIP peptide analog as defined above is provided, wherein Z is a peptide selected from:

[0251] - Glycine or proline,

[0252] -GP, GPS, GPSS, GPSSG, GPSSGA, GPSSGAP, GPSSGAPP, GPSSGAPPP and GPSSGAPPPS,

[0253] -PS, PSS, PSSG, PSSGA, PSSGAP, PSSGAPP, PSSGAPPP and PSSGAPPPS,

[0254] -GPSSGA, GPSSGAP, GPSSGAPP, GPSSGAPPP, GPSSGAPPPS, or variants thereof containing one or two separate amino acid substitutions at any one of the amino acid residues, or

[0255] -PSSG, PSSGA, PSSGAP, PSSGAPP, PSSGAPPP and PSSGAPPPS,

[0256] Or a variant thereof containing one or two separate amino acid substitutions at any of the amino acid residues.

[0257] In one embodiment, a GIP peptide analog as defined above is provided, wherein the fatty acid molecule is not attached to the third amino acid residue or the N-terminal amino group of any of SEQ ID NO:1-4 or a functional variant thereof.

[0258] In one embodiment, a GIP peptide analog as defined above is provided, wherein the GIP peptide analog has a free N-terminus. Therefore, the N-terminus of the GIP peptide analog comprises an unsubstituted (e.g., unacetylated, unacylated, or unalkylated) amino (-NH2) moiety. Thus, the N-terminus of the GIP peptide analog may comprise a free amino (-NH2) moiety.

[0259] In one embodiment, a GIP peptide analog as defined above is provided, wherein the fatty acid molecule is attached to the side chain of an amino acid residue at position 11, 12, 13, 16, 17, 18, 20, position 34 (if present), or position 40 (if present) of the GIP peptide analog (such as any one of SEQ ID NO: 1-4 or a functional variant thereof).

[0260] In one embodiment, a GIP peptide analog as defined above is provided, wherein the fatty acid molecule is attached to an amino acid residue at any of the 12th, 13th, 16th, 17th, 18th, 34th (if present), or 40th (if present) positions of any of the SEQ ID NO:1-4 or their functional variants thereof.

[0261] In one embodiment, a GIP peptide analog as defined above is provided, wherein the fatty acid molecule is attached to the 18th amino acid residue of any one of SEQ ID NO:1-4 or a functional variant thereof.

[0262] In one embodiment, a GIP peptide analog as defined above is provided, wherein a fatty acid molecule is attached to the ε-amino group of the K residue of the GIP peptide analog (such as any one of SEQ ID NO: 1-4 or a functional variant thereof containing at least one K residue).

[0263] In one embodiment, a GIP peptide analog as defined above is provided, wherein a fatty acid molecule is attached to the side chain amino group of the 18th amino acid residue of any of SEQ ID NO:1-4 or a functional variant thereof, wherein in any of SEQ ID NO:1-4, the 18th H has been substituted with K or Orn.

[0264] With or without a linker, the attachment of fatty acids to the side chain amino group of the amino acid residue at position 18 may result in GIP peptide analogs having particularly high antagonistic potency.

[0265] In one embodiment, a GIP peptide analog as defined above is provided, wherein a fatty acid molecule is attached to the side chain amino group of the 11th amino acid residue of any of SEQ ID NO:1-4 or a functional variant thereof, wherein in any of SEQ ID NO:1-4, the 11th S has been substituted with K or Orn.

[0266] In one embodiment, a GIP peptide analog as defined above is provided, wherein a fatty acid molecule is attached to the side chain amino group of the 12th amino acid residue of any of SEQ ID NO:1-4 or a functional variant thereof, wherein in any of SEQ ID NO:1-4, the 12th I has been substituted with K or Orn.

[0267] In one embodiment, a GIP peptide analog as defined above is provided, wherein the GIP peptide analog has an amino acid sequence selected from the following:

[0268] EGTFISEYSAibANleEKIKQQDFVEWLLAQK-Z; SEQ ID NO:70; GIP(3-30)[D9E; I12Aib; M14Nle; D15E; H18K; N24E]

[0269] EGTFISEYSIAibMEKIKQQDFVEWLLAQK-Z; SEQ ID NO:72; GIP(3-30)[D9E; A13Aib; D15E; H18K; N24E]

[0270] EGTFISDYSIAMDKIKQQDFVEWLLAQK-Z; SEQ ID NO:46; GIP(3-30)[H18K; N24E]

[0271] EGTFISDYSIAibMDKIKQQDFVEWLLAQK-Z; SEQ ID NO73; GIP(3-30)[A13Aib; H18K; N24E]

[0272] EGTFISDYSIAibLDKIKQQDFVEWLLAQK-Z; SEQ ID NO:74; GIP(3-30)[A13Aib; M14L; H18K; N24E]

[0273] EGTFISDYSIAibNleDKIKQQDFVEWLLAQK-Z; SEQ ID NO:75; GIP(3-30)[A13Aib; M14Nle; H18K; N24E]

[0274] EGTFISDYSIALDKIKQQDFVEWLLAQK-Z; SEQ ID NO:76; GIP(3-30)[M14L; H18K; N24E]

[0275] EGTFISDYSIANleDKIKQQDFVEWLLAQK-Z;SEQ ID NO:77;GIP(3-30)[M14Nle;H18K;N24E],

[0276] EGTFISDYSIAKDKIKQQDFVEWLLAQK-Z; SEQ ID NO:78; GIP(3-30)[M14K; H18K; N24E]

[0277] EGTFISEYSIAibLEKIKQQEFVEWLLAQK-Z; SEQ ID NO:79; GIP(3-30)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0278] EGTFISEYSIAibNleEKIKQQEFVEWLLAQK-Z; SEQ ID NO:80; GIP(3-30)[D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0279] EGTFISEYSIALEKIKQQEFVEWLLAQK-Z; SEQ ID NO:81; GIP(3-30)[D9E; M14L; D15E; H18K; D21E; N24E],

[0280] EGTFISEYSIANleEKIKQQEFVEWLLAQK-Z; SEQ ID NO:82; GIP(3-30)[D9E; M14Nle; D15E; H18K; D21E; N24E],

[0281] XGTFISDYSIANleDKIKQQDFVEWLLAQK-Z; SEQ ID NO:83; GIP(3-30)[E3 Glutaric acid (X); M14Nle; H18K; N24E],

[0282] EGTFISEYSIAibLEKIKQQDFVEWLLAQK-Z; SEQ ID NO:84; GIP(3-30)

[0283] [D9E; A13Aib; M14L; D15E; H18K; N24E]XGTFISEYSIAibLEKIKQQEFVEWLLAQK-Z; SEQID NO:85; GIP(3-30)[E3 Glutaric acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0284] XGTFISEYSIAibNleEKIKQQEFVEWLLAQK-Z; SEQ ID NO:86; GIP(3-30)[E3 Glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0285] XGTFISEYSIALEKIKQQEFVEWLLAQK-Z; SEQ ID NO:87; GIP(3-30)[E3 Glutaric acid; D9E; M14L; D15E; H18K; D21E; N24E],

[0286] XGTFISEYSIAibMEKIKQQEFVEWLLAQK-Z; SEQ ID NO:88; GIP(3-30)[E3 Glutaric acid; D9E; A13Aib; D15E; H18K; D21E; N24E],

[0287] EGTFISEYSIAibMEKIKQQEFVEWLLAQK-Z; SEQ ID NO:89; GIP(3-30)[D9E; A13Aib; D15E; H18K; D21E; N24E], EGTFISEYSIAMEKIKQQEFVEWLLAQK-Z; SEQ ID NO:90; GIP(3-30)[D9E; D15E; H18K; D21E; N24E],

[0288] EGTFISEYSIAibLDKIKQQDFVEWLLAQK-Z; SEQ ID NO:91; GIP(3-30)[D9E; A13Aib; M14L; H18K; N24E]

[0289] XGTFISDYSIAibMDKIKQQDFVEWLLAQK-Z; SEQ ID NO:92; GIP(3-30)[E3 Glutaric acid; A13Aib; H18K; N24E],

[0290] EGTFISDYSKAMDKIHQQDFVEWLLAQK-Z; SEQ ID NO:93; GIP(3-30)[I12K; N24E],

[0291] EGTFISDYSIKMDKIHQQDFVEWLLAQK-Z; SEQ ID NO:94; GIP(3-30)[A13K; N24E],

[0292] EGTFISDYSIAMDKIHQQDFVEWLLAQK-Z; SEQ ID NO:95; GIP(3-30)[N24E],

[0293] EGTFISDYSIAMDKKHQQDFVEWLLAQK-Z; SEQ ID NO:96; GIP(3-30)[I17K; N24E],

[0294] EGTFISDYSIAMDKIHQQDFVEWLLAQKPSSKAPPPS;SEQ ID NO:40;GIP(3-30)[N24E]CEX(31-39)[34K],

[0295] EGTFISDYSIAMDKIHQQDFVEWLLAQKPSSGAPPPS;SEQ ID NO:41;GIP(3-30)[N24E]CEX(31-39)[40K],

[0296] EGTFISDYAIAMDKKHQQDFVEWLLAQK-Z; SEQ ID NO:97; GIP(3-30)[S11A; H18K; N24E],

[0297] EGTFISEYSIAibMEKIKQQDFVEWLLAQK-Z; SEQ ID NO:72; GIP(3-30)[D9E, A13Aib; D15E; H18K; N24E],

[0298] XGTFISEYSIAiBLEKIKQQEFVEWLLAQK-Z; SEQ ID NO: 98; GIP (3-30) [E3 succinate; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0299] XGTFISEYSIAiBLEKIKQQEFVEWLLAQK-Z; SEQ ID NO: 99; GIP (3-30) [E3 adipic acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0300] EGTFISDYSIAibMDKIKQQDFVNWLLAQK-Z; SEQ ID NO:100; GIP(3-30)[A13Aib;H18K],

[0301] XGTFISDYSIAMDKIKQQDFVEWLLAQK-Z;SEQ ID NO:101;GIP(3-30)[E3glutaric acid;H18K;N24E], and

[0302] XGTFISDYSIAibMDKIKQQDFVNWLLAQK-Z; SEQ ID NO:102; GIP(3-30)[E3 glutaric acid; A13Aib; H18K],

[0303] The peptide is modified by attaching a fatty acid molecule at any position in any of the sequences above, and the peptide may be C-terminated with carboxylation.

[0304] In one embodiment, the GIP peptide analog is C-terminally amidated (-NH2) or C-terminally carboxylated (-COOH).

[0305] In one embodiment, the GIP peptide analog is C-terminally carboxylated (-COOH).

[0306] Unbound by any theory, free C-terminal carboxylic acids may help increase solubility.

[0307] Functional variants - mutants

[0308] In one embodiment, one or more or all of the amino acid substitutions are conserved amino acid substitutions (or synonymous substitutions). A conserved substitution is the substitution of an amino acid whose side chain has similar biochemical properties and therefore does not affect the function of the peptide.

[0309] The specific amino acid substitutions disclosed in this article 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.

[0310] In another embodiment, the functional variants as defined herein include the following sequences: wherein an alkyl amino acid replaces an alkyl amino acid, wherein an aromatic amino acid replaces an aromatic amino acid, wherein a sulfur-containing amino acid replaces a sulfur-containing amino acid, wherein a hydroxyl-containing amino acid replaces a hydroxyl-containing amino acid, wherein an acidic amino acid replaces an acidic amino acid, wherein a basic amino acid replaces a basic amino acid, and / or wherein a dibasic carboxylic acid amino acid replaces a dibasic carboxylic acid amino acid.

[0311] Conservative substitutions may be introduced into one or more of the specified positions described above in GIP peptide analogs selected from any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, provided that the resulting variant retains functionality. However, it may also be desirable to introduce non-conservative substitutions (non-synonymous substitutions) at one or more positions.

[0312] In one embodiment, the nonconservative substitutions resulting in 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 include substitutions of amino acid residues that are substantially different in polarity, such as residues with nonpolar side chains (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe, or Met) replacing residues with polar side chains (such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln) or charged amino acids (such as Asp, Glu, Arg, or Lys), or charged or polar residues replacing nonpolar residues; and / or Or ii) have substantially different effects on peptide backbone orientation, such as Pro or Gly being replaced by another residue or replacing another residue; and / or iii) have substantially different charges, such as a negatively charged residue (e.g., Glu or Asp) replacing a positively charged residue (e.g., Lys, His, or Arg) (and vice versa); and / or iv) have substantially different spatial volumes, such as a bulky residue (e.g., His, Trp, Phe, or Tyr) replacing a residue with a smaller side chain (e.g., Ala, Gly, or Ser) (and vice versa).

[0313] In one embodiment, the substitution of amino acids can be based on their hydrophobic and hydrophilic values, as well as the relative similarity of the substituents in the amino acid side chains, including charge, size, etc.

[0314] As defined in this paper, GIP peptide analogs or their functional variant counterparts contain proteogenous or native amino acids, namely the 22 amino acids naturally incorporated into the polypeptide. Of these amino acids, 20 are encoded by the universal genetic code, and the remaining two (selenocysteine ​​(Sec, U) and pyrrolidone (Pyl, O)) are incorporated into the protein through a unique synthetic mechanism.

[0315] In one embodiment, a GIP peptide analog as defined herein comprises one or more non-naturally occurring amino acid residues (non-natural, non-proteogenous, or non-standard amino acids) or amino acid mimics (such as glutaric acid). Non-naturally occurring amino acids include, for example, but not limited to, Aib, β-2-naphthyl-alanine, trans-3-methylproline, 2,4-methyleneproline, cis-4-hydroxyproline, ornithine (Orn), trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethyl homocysteine, nitroglutamine, homoglutamine, pipercoic acid, thiazolidinylcarboxylic acid, dehydroproline, 3-methylproline and 4-methylproline, 3,3-dimethylproline, tert-leucine, ortholeucine (Nle), methoxinine (Mox), valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.

[0316] In one embodiment, the amino acid Met is replaced by an antioxidant amino acid analogue, such as ortholeucine (Nle) or Leu, which retains the length of the amino acid side chain that is important for hydrophobic interactions but does not retain its hydrogen bonding properties; or methoxyamine (Mox), a non-canonical amino acid that is more similar to Met in electronic properties than Nle; or Lys.

[0317] Standard and / or non-standard amino acids can be linked by peptide bonds (to form a linear peptide chain) or by non-peptide bonds (e.g., by variable side chains of the amino acids). Preferably, the amino acids of the peptide as defined herein are linked by peptide bonds.

[0318] As is known in the art, the term peptide also includes post-translational modifications introduced by chemical or enzymatic catalytic reactions. These post-translational modifications include acetylation, phosphorylation, methylation, glycosylation, amylation, hydroxylation, deiminolation, deamidation, carbamylation, and sulfation of one or more amino acid residues, as well as proteolytic modifications by known proteases, including lysosomal cathepsins, calpapsins, secretases, and matrix metalloproteinases.

[0319] Similarly, functional equivalents of peptides can contain chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), polyethylene glycolation (derivation with polyethylene glycol), or insertion (or chemical substitution) of amino acids that are not normally present in human proteins (non-proteinogens) (such as ornithine).

[0320] Spatially similar compounds can be formulated to mimic key portions of peptide structures. This can be achieved using modeling and chemical design techniques known to those skilled in the art. For example, esterification and other alkylations can be used to modify the amino terminus of, for example, a diarginine peptide backbone to mimic a tetrapeptide structure. It should be understood that all such spatially similar constructs fall within the scope of this invention. Peptides with N-terminal and C-terminal alkylations and esterifications are also included in this invention. For example, glutaric acid is a spatially similar compound that mimics glutamic acid.

[0321] In one embodiment, the N-terminal amino acid of the GIP peptide analog of this disclosure is not chemically modified. It may be advantageous that the N-terminal amino group of the GIP peptide analog is free, i.e., unsubstituted, as substitution could lead to stimulatory activity towards GIPR.

[0322] In one embodiment, the N-terminus (i.e., the NH2 group at the N-terminus) is absent, for example, when the 3rd position is replaced by glutaric acid without an amino group.

[0323] It appears that lengthening the fatty acid or linker (if present) may reduce antagonistic efficacy. However, simultaneous incorporation of an Aib residue at position 13 appears to compensate for some or all of the reduced efficacy, especially with E combinations at one or more of positions 9, 15, 21, and 24, such as the E combination at position 24.

[0324] Attachment of fatty acid molecules

[0325] In one embodiment, a fatty acid molecule is attached to an amino-alkyl (-C) side chain. n H 2n On one or more amino acid residues of NH2).

[0326] In one embodiment, a fatty acid molecule is attached to one or more amino acid residues having a side chain amino group (NH2).

[0327] In one embodiment, a fatty acid molecule is attached to the amino group (NH2) of an amino acid residue.

[0328] In one embodiment, a fatty acid molecule is attached to the side chain amino group of an amino acid residue.

[0329] In one embodiment, a fatty acid molecule is attached to the ε (epsilon) side chain amino group of a lysine residue (Lys, K).

[0330] In one embodiment, the fatty acid molecule is attached to the δ (delta) side chain amino group of the ornithine residue (Orn).

[0331] In one embodiment, the amino acid residues attached to the fatty acid molecule are selected from Lys and Orn.

[0332] In one embodiment, the amino acid residue attached to the fatty acid molecule is Lys.

[0333] In one embodiment, a fatty acid molecule is attached to the δ-amino group of the GIP peptide analog (such as any one of SEQ ID NO: 1-4) or a functional variant of the Orn amino acid residue.

[0334] In one embodiment, a fatty acid molecule is attached to the ε-amino group of the K residue of the GIP peptide analog (such as any one of SEQ ID NO: 1-4) or a functional variant thereof.

[0335] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule, wherein the fatty acid molecule is a straight-chain fatty acid.

[0336] In one embodiment of this disclosure, a GIP peptide analog is provided which is modified by attaching a fatty acid molecule, wherein the fatty acid molecule is a branched-chain fatty acid.

[0337] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule, wherein the fatty acid molecule is a monoacyl fatty acid molecule containing an acyl group. Preferably, a carboxyl group is located at one end of the fatty acid molecule.

[0338] For example, GIP peptides can be conjugated to monoacyl fatty acids (such as hexadecyl) via linker L, as described in Formula I:

[0339]

[0340] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule, said fatty acid molecule being a diacyl fatty acid molecule. A diacyl fatty acid molecule is a fatty acid molecule containing two carboxyl groups. Preferably, one or both carboxyl groups are located at one or each end of the fatty acid molecule.

[0341] For example, GIP peptides can be conjugated to diacid fatty acids (also known as "diacids") (such as 15-carboxy-pentadecanoyl) via linker L, as depicted in Formula II:

[0342]

[0343] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule, wherein the fatty acid molecule comprises the formula CH3(CH2). n The acyl group of CO-, where n is an integer from 4 to 24.

[0344] In one embodiment, the fatty acid molecule comprises one or more acyl groups selected from the following: 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-.

[0345] In one embodiment, the fatty acid molecule contains an acyl group selected from CH3(CH2). 10 CO-(lauryl, C12), CH3(CH2) 12 CO-(myristoyl, C14), CH3(CH2) 14 CO-(palmitoyl, C16), CH3(CH2) 16 CO-(stearoyl, C18), CH3(CH2) 18 CO-(arachidoyl, C20) and CH3(CH2) 20 CO-(Yamyuki, C22).

[0346] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule containing two acyl groups individually selected from HOOC-CH3(CH2). 10 CO-(dodecanoyl, C12), HOOC-CH3(CH2) 12 CO-(1-Tetradecanoyl, C14), HOOC-CH3(CH2) 14 CO-(hexadecyl, C16), HOOC-CH3(CH2) 15 CO-(15-carboxy-pentadecanoyl, C17), HOOC-CH3(CH2) 16 CO-(octadecyl, C18), HOOC-CH3(CH2) 17 CO-(17-carboxy-heptadecanoyl, C19), HOOC-CH3(CH2) 18CO-(eicosyl, C20), HOOC-CH3(CH2) 19 CO-(19-carboxy-nonadecanoyl, C21) and HOOC-CH3(CH2) 20 CO-(Yamyuki, C22).

[0347] In one embodiment, the fatty acid molecule contains the formula COOH(CH2). n The acyl group of CO-(dicarboxylic acid), where n is an integer from 4 to 24.

[0348] In one embodiment of this disclosure, a GIP peptide analog is provided, which is modified by attaching a fatty acid molecule containing an acyl group selected from COOH(CH2). 14 CO-, COOH(CH2) 16 CO-, COOH(CH2) 18 CO- and COOH(CH2) 20 CO-.

[0349] In one embodiment, the fatty acid molecule comprises COOH(CH2). 14 CO- or composed of it.

[0350] In one embodiment, the fatty acid molecule comprises COOH(CH2). 16 CO- or composed of it.

[0351] In one embodiment, the fatty acid molecule comprises COOH(CH2). 18 CO- or composed of it.

[0352] In one embodiment of this disclosure, a GIP peptide analog is provided which is modified by attaching a fatty acid molecule, wherein the fatty acid molecule is directly attached to the ε-amino group of the side chain of an amino acid residue of the GIP peptide analog.

[0353] Fatty acid molecules attach via a linker

[0354] Fatty acid molecules can attach to peptides in this article directly or indirectly (i.e., via linkers or spacers).

[0355] In one embodiment of this disclosure, a GIP peptide analog is provided that is modified by attaching a fatty acid molecule, wherein the fatty acid molecule is attached to an amino acid residue via a linker.

[0356] In one embodiment, the fatty acid molecule according to this disclosure is attached to an amino acid residue via a linker or spacer, as depicted in Formula III:

[0357]

[0358] In one embodiment, a fatty acid molecule is attached to an 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.

[0359] In some embodiments, the connector comprises one or more portions, said one or more portions being independently selected from:

[0360] a. One or more α,ω-amino acids,

[0361] b. One or more amino acids selected from succinic acid, Lys, Glu, and Asp.

[0362] c.4-Abu,

[0363] dy-aminobutyric acid

[0364] e. Dipeptides, such as those described below, wherein the C-terminal amino acid residue is Lys, His, or Trp, preferably Lys, and wherein the N-terminal amino acid residue is selected from Ala, Arg, Asp, Asn, Gly, Glu, Gln, Ile, Leu, Val, Phe, and Pro, such as Gly-Lys.

[0365] f. One or more of γ-aminobutyryl (γ-aminobutyric acid), γ-glutamyl (γ-glutamic acid), β-asparagine acyl, β-alanyl, and glycyl, and

[0366] g. γ-Glutamic acid-[8-amino-3,6-dioxanoic acid] n (γGlu-AEEAc n ), where n is an integer between 1 and 50, such as an integer between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, and 45-50.

[0367] In some embodiments, the connector comprises one or more portions, said one or more portions being independently selected from:

[0368] a. α-amino acids, γ-amino acids, or ω-amino acids,

[0369] b. One or more amino acids selected from succinic acid, Lys, Glu, and Asp.

[0370] c. One or more amino acids selected from Gly and Ser.

[0371] d. One or more amino acids selected from Ala, Glu, Lys, and Leu.

[0372] e. one or more of γ-aminobutyryl (γ-aminobutyric acid), γ-Glu (γ-glutamic acid), β-Asp (β-asparagine acyl), β-Ala (β-alanyl), 2-aminoisobutyric acid (Aib), and Gly, and

[0373] f. [8-Amino-3,6-dioxanoic acid] n (AEEAc n ), where n is an integer between 1 and 50, such as an integer between 1-4, 1-3, or 1-2.

[0374] In one embodiment, the connector comprises γ-Glu, one or more 8-amino-3,6-dioxanoic acid (AEEAc), or a combination thereof.

[0375] In one embodiment, the connector comprises or is composed of GGGS or SGGG.

[0376] In one embodiment, the connector comprises or is composed of ALEA or AELA.

[0377] In one embodiment, the connector comprises or is composed of 2-aminoisobutyric acid (Aib).

[0378] In one embodiment, the connector comprises or is composed of yGlu.

[0379] In one embodiment, the connector comprises or is composed of KAAAEKAAAEKAAAE.

[0380] In one embodiment of this disclosure, the connector comprises [8-amino-3,6-dioxanoic acid]. n (AEEAc) n Or composed of, where n is an integer between 1 and 50, such as an integer between 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, preferably where n is 1, 2 or 3.

[0381] In one embodiment of this disclosure, the connector comprises a γ-Glu and an AEEAc, such as a γ-Glu and two AEEAc, for example a γ-Glu and three AEEAc, or consists of the same.

[0382] The examples of linkers disclosed herein allow them to be attached to amino acid residues of GIP peptide analogs via either end of the linker. Thus, if, for example, the linker contains γ-glutamic acid-8-amino-3,6-dioxanoic acid (γ-Glu)-(AEEAc) n If one or more repetitions of ) are made, the connector may be via γ-Glu or via AEEAc n It is attached to the amino acid residues of GIP peptide analogs.

[0383] In one embodiment, the linker is [γ-glutamic acid]-[8-amino-3,6-dioxanoic acid](γ-Glu)-AEEAc or [8-amino-3,6-dioxanoic acid]-[γ-glutamic acid](AEEAc-γ-Glu). For example, the GIP peptide can be conjugated via [γ-glutamic acid]-[8-amino-3,6-dioxanoic acid] to a fatty acid (such as C16 or palmitic acid / palmitoyl group in Formula IV, but any other fatty acid can be used) as depicted in Formula IV:

[0384]

[0385] Formula IV: The formula described does not depict stereochemistry and is usually in the natural L-form unless otherwise stated.

[0386] For example, GIP peptides can be conjugated via [8-amino-3,6-dioxanoic acid]-[γ-glutamic acid] to a fatty acid (such as C16 or palmitic acid / palmitoyl group in Formula IV, but any other fatty acid can be used), as depicted in Formula V:

[0387]

[0388] Formula V: The formula does not describe stereochemistry and is usually used in the natural L- form unless otherwise stated.

[0389] In one embodiment of this disclosure, a fatty acid molecule is attached to an amino acid residue via a linker, and wherein the combination of the linker and the fatty acid is selected from:

[0390] i. hexadecanoyl-Y-Glu-

[0391] ii. Hexadecyl-Y-Glu-Y-Glu-

[0392] iii. Hexadecyl-Y-Glu-AEEAc-

[0393] iv. Hexadecyl-Y-Glu-AEEAc-AEEAc-

[0394] v. Hexadecanoyl-Y-Glu-AEEAc-AEEAc-AEEAc-

[0395] vi. [15-Carboxy-pentadecanoyl]-Y-Glu-

[0396] vii. [15-Carboxy-pentadecanoyl]-Y-Glu-Y-Glu-

[0397] viii. [15-Carboxy-pentadecanoyl]-Y-Glu-AEEAc-

[0398] ix.[15-Carboxy-pentadecanoyl]-Y-Glu-AEEAc-AEEAc-

[0399] x.[15-Carboxyl-pentadecanyl]-Y-Glu-AEEAc-AEEAc-AEEAc-

[0400] xi.octadecyl-Y-Glu-

[0401] xii. Octyl-Y-Glu-Y-Glu-

[0402] xiii. Octyl-Y-Glu-AEEAc-

[0403] xiv. Octyl-Y-Glu-AEEAc-AEEAc-

[0404] xv.octadecyl-Y-Glu-AEEAc-AEEAc-AEEAc-

[0405] xvi.[17-Carboxy-heptadecanoyl]-Y-Glu-

[0406] xvii.[17-Carboxy-heptadecanoyl]-Y-Glu-Y-Glu-

[0407] xviii.[17-Carboxy-heptadecanoyl]-Y-Glu-AEEAc-

[0408] xix.[17-Carboxy-heptadecanoyl]-Y-Glu-AEEAc-AEEAc-

[0409] xx.[17-Carboxy-Heptadecanyl]-Y-Glu-AEEAc-AEEAc-AEEAc-

[0410] xxi. Eicosyl-Y-Glu-

[0411] xxii. Eicosyl-Y-Glu-Y-Glu-

[0412] xxiii. Eicosyl-Y-Glu-AEEAc-

[0413] xxiv. Eicosyl-Y-Glu-AEEAc-AEEAc-

[0414] xxv. icosanoyl-Y-Glu-AEEAc-AEEAc-AEEAc-

[0415] xxvi.[19-carboxy-nonadecanoyl]-Y-Glu-

[0416] xxvii.[19-Carboxy-Ninedecyl]-Y-Glu-γ-Glu-

[0417] xxviii.[19-carboxy-nonadecanoyl]-γ-Glu-AEEAc-

[0418] xxix.[19-Carboxy-Ninedecyl]-γ-Glu-AEEAc-AEEAc-

[0419] xxx.[19-Carboxyl-Ninedecyl]-γ-Glu-AEEAc-AEEAc-AEEAc-.

[0420] In one embodiment of this disclosure, a fatty acid molecule is attached to an amino acid residue via a linker, and wherein the combination of the linker and the fatty acid is selected from:

[0421] i.[15-Carboxypentadecanyl]-yGlu

[0422] ii. [17-Carboxy-heptadecanoyl]-γ-Glu-AEEAc-AEEAc-, and

[0423] iii.[17-Carboxy-heptadecanoyl]-yGlu-yGlu.

[0424] GIP peptides containing fatty acids

[0425] In one embodiment of this disclosure, a GIP peptide analog as defined herein is provided, wherein the GIP peptide analog is selected from:

[0426] EGTFISEYSIAMEKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:10; GIP(3-30)+Cex(31-39)[D9E; D15E; H18K; N24E],

[0427] EGTFISEYSAibANleEKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:71; GIP(3-30)+Cex(31-39)[D9E; I12Aib; M14Nle; D15E; H18K; N24E],

[0428] EGTFISEYSIAibMEKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:33; GIP(3-30)+Cex(31-39)[D9E; A13Aib; D15E; H18K; N24E],

[0429] EGTFISDYSIAMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:47; GIP(3-30)+Cex(31-39)[H18K;N24E],

[0430] EGTFISDYSIAibMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:12; GIP(3-30)+Cex(31-39)[A13Aib; H18K; N24E],

[0431] EGTFISDYSIAibLDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K;SEQ ID NO:13;GIP(3-30)+Cex(31-39)[A13Aib;M14L;H18K;N24E],

[0432] EGTFISDYSIAibLDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / 18K;SEQID NO:13;GIP(3-30)+Cex(31-39)[A13Aib;M14L;H18K;N24E],

[0433] EGTFISDYSIAibNleDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:14; GIP(3-30)+Cex(31-39)[A13Aib;M14Nle;H18K;N24E],

[0434] EGTFISDYSIAibNleDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:14; GIP(3-30)+Cex(31-39)[A13Aib; M14Nle; H18K; N24E],

[0435] EGTFISDYSIALDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:15; GIP(3-30)+Cex(31-39)[M14L; H18K; N24E],

[0436] EGTFISDYSIALDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQID NO:15; GIP(3-30)+Cex(31-39)[M14L; H18K; N24E],

[0437] EGTFISDYSIANleDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:16; GIP(3-30)+Cex(31-39)[M14Nle; H18K; N24E],

[0438] EGTFISDYSIANleDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQID NO:16; GIP(3-30)+Cex(31-39)[M14Nle; H18K; N24E],

[0439] EGTFISDYSIAKDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:17; GIP(3-30)+Cex(31-39)[M14K; H18K; N24E],

[0440] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:18; GIP(3-30)+Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0441] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:18; GIP(3-30)+Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0442] EGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:19; GIP(3-30)+Cex(31-39)[D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0443] EGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:19; GIP(3-30)+Cex(31-39)[D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0444] XGTFISDYSIAMDKIKQQDFVNWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:20; GIP(3-30)+Cex(31-39)[E3 glutamic acid(X); H18K], EGTFISEYSIALEKIKQQEFVEWL LAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:21; GIP(3-30)+Cex(31-39)[D9E; M14L; D15E; H18K; D21E; N24E],

[0445] EGTFISEYSIANleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQID NO:22; GIP(3-30)+Cex(31-39)[D9E; M14Nle; D15E; H18K; D21EN24E],

[0446] XGTFISDYSIANleDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:24; GIP(3-30)+Cex(31-39)[E3 glutamic acid(X); M14Nle; H18K; N24E],

[0447] XGTFISDYSIANleDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:24; GIP(3-30)+Cex(31-39)[E3 glutaric acid (X); M14Nle; H18K; N24E],

[0448] XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:25; GIP(3-30)+Cex(31-39)[E3 glutaric acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0449] XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:26; GIP(3-30)-Cex(31-39)[E3 glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0450] XGTFISEYSIALEKIKQQEFVEWLLAQKPSSGAPPPS-OH-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:27; GIP(3-30)Cex(31-39)[E3 glutaric acid; D9E; M14L; D15E; H18K; D21E; N24E],

[0451] XGTFISEYSIAibMEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQID NO:28; GIP(3-30-Cex(31-39)[E3 glutaric acid; D9E; A13Aib; D15E; H18K; D21E; N24E],

[0452] EGTFISEYSIAibMEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQID NO:29; GIP(3-30)-Cex(31-39)[D9E; A13Aib; D15E; H18K; D21E; N24E],

[0453] EGTFISEYSIAMEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:9; GIP(3-30)Cex(31-39)[D9E; D15E; H18K; D21E; N24E],

[0454] EGTFISEYSIAibLDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:30; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; H18K; N24E],

[0455] XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:25; GIP(3-30)Cex(31-39)[E3 glutaric acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0456] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-dioic acid / 18K; SEQ ID NO:18; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0457] XGTFISDYSIAibMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:32; GIP(3-30)Cex(31-39)[E3 glutaric acid; A13Aib; H18K; N24E],

[0458] EGTFISEYSIAibMEKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioic acid / 18K; SEQ ID NO:33; GIP(3-30)Cex(31-39)[D9E; A13Aib; D15E; H18K; N24E],

[0459] XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:25; GIP(3-30)Cex(31-39)[E3 glutaryl acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0460] EGTFISEYSIALEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:21; GIP(3-30)Cex(31-39)[D9E; M14L; D15E; H18K; N24E],

[0461] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-(GGGS-C16-diacid / 18K); SEQ IDNO:18; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0462] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-(ALEA-C16-diacid / 18K); SEQ IDNO:18; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0463] EGTFISDYSKAMDKIHQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 12K; SEQ ID NO:36; GIP(3-30)Cex(31-39)[I12K; N24E],

[0464] EGTFISDYSIKMDKIHQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 13K; SEQ ID NO:37; GIP(3-30)Cex(31-39)[A13K; N24E],

[0465] EGTFISDYSIAMDKIHQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 16K; SEQ ID NO:38; GIP(3-30)Cex(31-39)[N24E],

[0466] EGTFISDYSIAMDKKHQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 17K;SEQ ID NO:39;GIP(3-30)Cex(31-39)[I17K;N24E],

[0467] EGTFISDYSIAMDKIHQQDFVEWLLAQKPSSKAPPPS C16-diacid / 34K; SEQ ID NO:40; GIP(3-30)Cex(31-39)[N24E;34K],

[0468] EGTFISDYSIAMDKIHQQDFVEWLLAQKPSSGAPPPSK-C16-diacid / 40K; SEQ ID NO:41; GIP(3-30)Cex(31-39)[N24E;40K],

[0469] EGTFISDYAIAMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-dioxide / 18K; SEQ ID NO:43; GIP(3-30)Cex(31-39)[S11A;H18K;N24E],

[0470] EGTFISDYSIAMDKIKQQDFVEWLLAQK-C16-dioxide / 18K; SEQ ID NO:46; GIP(3-30)[H18K;N24E],

[0471] EGTFISDYSIAMDKIKQQDFVEWLLAQKPSSGAPPPS-C20-Dioxide / 18K; SEQ ID NO:47; GIP(3-30)Cex(31-39)-OH[H18K;N24E],

[0472] EGTFISDYSIAMDKIKQQDFVEWLLAQKPSSGAPPPS-C16 / 18K;SEQ ID NO:47;GIP(3-30)Cex(31-39)[H18K;N24E],

[0473] EGTFISEYSIAibLEKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-Dioxide / 18K;SEQID NO:50;GIP(3-30)Cex(31-39)[D9E;A13Aib;M14L;D15E;H18K;N24E],

[0474] XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-(C16-diacid / 18K); SEQ ID NO:26; GIP(3-30)Cex(31-39)[E3 glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K; D21E; N24E],

[0475] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-(Aib-C16-diacid / 18K); SEQ ID NO:18; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0476] EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-(KAAAEKAAAEKAAAE-C16-diacid / 18K); SEQ ID NO:18; GIP(3-30)Cex(31-39)[D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0477] XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / 18K; SEQID NO:48; GIP(3-30)Cex(31-39)[E3 succinic acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0478] XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / 18K; SEQID NO:49; GIP(3-30)Cex(31-39)[E3 adipic acid; D9E; A13Aib; M14L; D15E; H18K; D21E; N24E],

[0479] EGTFISDYSIAibMDKIKQQDFVNWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:52; GIP(3-30)Cex(31-39)[A13Aib; H18K],

[0480] XGTFISDYSIAMDKIKQQDFVEWLLAQKPSSGAPPPS-C16- glutaric acid / 18K; SEQ ID NO:53; GIP(3-30)Cex(31-39)[E3 glutaric acid; H18K; N24E], and

[0481] XGTFISDYSIAibMDKIKQQDFVNWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO:54; GIP(3-30)Cex(31-39)[E3 glutaric acid; A13Aib; H18K],

[0482] or its functional variants,

[0483] The fatty acids thereon are attached directly or via a connector as defined herein.

[0484] Therefore, C16 is the fatty acid CH3(CH2). 14 CO- (palmitoyl) and C18 is a fatty acid CH3 (CH2). 16 CO- (stearoyl). The suffix "-diacid" indicates that the fatty acid molecule is a diacyl fatty acid molecule. The absence of such a suffix indicates a monoacyl fatty acid molecule.

[0485] Therefore, C20 is the fatty acid CH3(CH2). 18 CO-(arachidoyl). The suffix "-diacid" indicates that the fatty acid molecule is a diacyl fatty acid molecule. The absence of such a suffix indicates a monoacyl fatty acid molecule.

[0486] Therefore, C22 is the fatty acid CH3(CH2). 20 CO-(behenyl). The suffix "-diacid" indicates that the fatty acid molecule is a diacyl fatty acid molecule. The absence of such a suffix indicates a monoacyl fatty acid molecule.

[0487] Determining antagonist properties and affinity

[0488] To determine whether a peptide is an antagonist of a GIPR, methods known in the art can be used, such as by determining the IC50 of the peptide. This can be done by constructing a dose-response curve and examining the effect of different concentrations of the peptide on reversing the agonist activity. The agonist can be GIP1-42, such as hGIP-1-42 or hGIP1-30. The GIPR can be hGIPR, rGIPR, mGIPR, canine GIPR, porcine GIPR, or rhesus monkey (Macaca mulatta) GIPR. The IC50 value of a given antagonist can be calculated by determining the concentration required to inhibit half of the maximum biological response of the agonist. A method for determining whether a peptide is an antagonist has been described in Example 4, but other methods known in the art can also be used. For example, a Schild plot analysis can be performed on the dose-response curve of hGIP1-42 cAMP at progressively increasing concentrations of the GIP-derived peptide. In this way, the type of antagonist activity can also be determined.

[0489] The GIP peptide analogues of this disclosure are characterized by antagonistic activity against GIPR. Specifically, the GIP peptide analogues of this disclosure are potent GIPR antagonists.

[0490] In one embodiment of this disclosure, the GIP peptide analog inhibits at least 80%, such as at least 85%, at least 90%, at least 95%, or about 100% of GIPR activity, as measured by an assay that determines a reduction in intracellular cAMP, such as by a CisBio cAMP assay (Alternative 1) and / or by a “Gaddum” assay (Alternative 2), said assays are described in “Materials and Methods”.

[0491] In one embodiment of this disclosure, the GIP peptide analog inhibits at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as about 100%, of GIPR activity, wherein the inhibition of GIPR activity is determined by a reduction in intracellular cAMP, such as measured by an assay that determines a reduction in intracellular cAMP, such as by a CisBio cAMP assay (Alternative 1) and / or by a “Gaddum” assay (Alternative 2), said assays are described in “Materials and Methods”. Inhibition % is the inhibition % of Emax, meaning that if the peptide inhibits 85% of Emax, then 15% of the GIPR activity remains.

[0492] In one embodiment of this disclosure, the GIP peptide analog has GIPR antagonistic potency corresponding to an IC50 value of less than 50 nM, such as an IC50 value of less than 10 nM, such as an IC50 value of less than 5 nM, such as an IC50 value of less than 1 nM, such as an IC50 value of less than 0.001 nM to 1 nM, wherein the antagonistic activity (also referred to as “potency”) is measured by an assay that determines a reduction in intracellular cAMP, such as by a CisBio cAMP assay and / or by a “gaddum” assay, which is described in “Materials and Methods”.

[0493] Methods for determining the antagonistic activity of compounds (such as GIP peptide analogs) are known to those skilled in the art. Exemplary methods for determining the antagonistic activity of compounds (such as GIP peptide analogs) can be found in the “Examples” herein, for example, these methods include measuring intracellular cAMP and determining the reduction of intracellular cAMP due to treatment of cells with GIP peptide analogs.

[0494] The GIP peptide analogs disclosed herein are further characterized by having low or no agonistic activity toward GIPR. GIP peptide analogs having low or no agonistic activity toward GIPR (e.g., 20% or less, preferably 10% or less, or even more preferably 5% or less agonistic activity) are also referred to as “silencing antagonists”.

[0495] In one embodiment, the GIP peptide analogue of this disclosure is capable of stimulating up to 30%, such as up to 25%, such as up to 20%, such as up to 15%, such as up to 10%, such as up to 5% of GIPR activity; in another embodiment, the GIP peptide analogue of this disclosure has no GIPR stimulating activity, i.e., it stimulates approximately 0% of GIPR activity.

[0496] The agonistic activity of GIP peptide analogs to GIPR can be determined in the same manner as antagonistic activity, but by measuring the increase rather than the decrease in intracellular cAMP, as described in "Materials and Methods".

[0497] Treatment

[0498] In one aspect, GIP peptide analogs or compositions comprising said GIP peptide analogs as defined herein are also provided for use as pharmaceutical agents.

[0499] In one embodiment, a glucose-dependent insulinotropic peptide (GIP) analogue is provided, which consists of the amino acid sequence SEQ ID NO:1:

[0500]

[0501] Where X1 is any amino acid or is omitted;

[0502] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0503] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0504] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0505] The peptide is modified by attaching a fatty acid molecule to an amino acid residue at any position of SEQ ID NO 1 or any position of the functional variant thereof, or to an amino acid residue at any position of Z SEQ ID NO:67; CE31-39, and is used as a pharmaceutical agent.

[0506] In one implementation, a GIP analogue selected from the following is provided:

[0507]

[0508]

[0509] (SEQ ID NO:2)

[0510]

[0511] (SEQ ID NO:3), and

[0512]

[0513] (SEQ ID NO:4),

[0514] Where X1 is any amino acid or is omitted;

[0515] or its functional variants, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid position in 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);

[0516] Wherein Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39), and

[0517] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO:2-4 or any position of the functional variant thereof, or to any amino acid residue at any position of Z SEQ ID NO:67; CE31-39, and is used as a pharmaceutical agent.

[0518] In one embodiment, a glucose-dependent insulinotropic peptide (GIP) analogue is provided, which consists of the amino acid sequence SEQ ID NO:1:

[0519]

[0520] Where X1 is any amino acid or is omitted;

[0521] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0522] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0523] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0524] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO:1 or any position of the functional variant thereof, or to any amino acid residue at any position of Z SEQ ID NO:67; CE31-39, for use in inhibiting or reducing one or more of the following: 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 incorporation, 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 increased appetite, x) GIP-induced decreased energy expenditure, xi) GIP-induced increased intestinal nutrient absorption, xii) GIP-induced decreased appetite-suppressing effect of GLP-1, xiii) GIP-induced leptin resistance.

[0525] In one implementation, a GIP analogue selected from the following is provided:

[0526]

[0527] (SEQ ID NO:2)

[0528]

[0529] (SEQ ID NO:3), and

[0530]

[0531] (SEQ ID NO:4),

[0532] Where X1 is any amino acid or is omitted;

[0533] or its functional variants, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid position in 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);

[0534] Wherein Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39), and

[0535] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO:2-4 or any position of the functional variant thereof, or to any amino acid residue at any position of Z SEQ ID NO:67; CE31-39, for use in inhibiting or reducing one or more of the following: 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 incorporation, 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 increased appetite, x) GIP-induced decreased energy expenditure, xi) GIP-induced increased intestinal nutrient absorption, xii) GIP-induced decreased appetite suppression by GLP-1, xiii) GIP-induced leptin resistance.

[0536] In one embodiment, a glucose-dependent insulinotropic peptide (GIP) analogue is provided, which consists of the amino acid sequence SEQ ID NO:1:

[0537]

[0538]

[0539] Where X1 is any amino acid or is omitted;

[0540] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0541] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0542] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0543] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO:1 or any position of Z SEQ ID NO:67; CE31-39, for use in treating a condition selected from: metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low very low density lipoprotein (VLDL) levels, low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, dyslipidemia, cardiovascular disease, high blood pressure, and atherosclerosis.

[0544] In one implementation, a GIP analogue selected from the following is provided:

[0545]

[0546] (SEQ ID NO:2)

[0547]

[0548] (SEQ ID NO:3), and

[0549]

[0550] (SEQ ID NO:4),

[0551] Where X1 is any amino acid or is omitted;

[0552] or its functional variants, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid position in 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);

[0553] Wherein Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39), and

[0554] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO:2-4 or any position of the functional variant thereof, or to any amino acid residue at any position of Z SEQ ID NO:67; CE31-39, for use in treating a condition selected from the following: metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low very low density lipoprotein (VLDL) levels, low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, dyslipidemia, cardiovascular disease, high blood pressure, and atherosclerosis.

[0555] In one embodiment, a glucose-dependent insulinotropic peptide (GIP) analogue is provided, which consists of the amino acid sequence SEQ ID NO:1:

[0556]

[0557] Where X1 is any amino acid or is omitted;

[0558] Or functional variants thereof, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid in SEQ ID NO:1,

[0559] Wherein, in SEQ ID NO:1 or a functional variant thereof, the N at position 24 is replaced by E and / or where, in SEQ ID NO:1 or a functional variant thereof, the A at position 13 is replaced by 2-aminoisobutyric acid (Aib),

[0560] Where Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39) or is omitted, and

[0561] The peptide is modified by attaching a fatty acid molecule to an amino acid residue at any position of SEQ ID NO 1 or any position of the functional variant thereof, or to an amino acid residue at any position of Z SEQ ID NO:67; CE31-39, for the manufacture of pharmaceutical agents for:

[0562] Treatment is selected from the following conditions: metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low very low density lipoprotein (VLDL) levels, low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, dyslipidemia, cardiovascular disease, high blood pressure, and atherosclerosis, or

[0563] -Induces weight loss.

[0564] In one implementation, a GIP analogue selected from the following is provided:

[0565]

[0566] (SEQ ID NO:2)

[0567]

[0568] (SEQ ID NO:3), and

[0569]

[0570] (SEQ ID NO:4),

[0571] Where X1 is any amino acid or is omitted;

[0572] or its functional variants, wherein the variants have 1 to 8 individual amino acid substitutions at any amino acid position in 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);

[0573] Wherein Z is a peptide containing one or more amino acid residues at the C-terminus of agonistin-4(31-39) (PSSGAPPPS; SEQ ID NO:67; CE31-39), and

[0574] The peptide is modified by attaching a fatty acid molecule to any amino acid residue at any position of SEQ ID NO 2-4 or any position of the functional variant thereof, or to any amino acid residue at any position of Z SEQ ID NO:67; CE31-39, for the manufacture of pharmaceutical agents for:

[0575] Treatment is selected from the following conditions: metabolic syndrome, obesity, prediabetes, type 1 diabetes, type 2 diabetes, insulin resistance, elevated fasting blood glucose, hyperglycemia, elevated fasting serum triglyceride levels, low very low density lipoprotein (VLDL) levels, low high density lipoprotein (HDL) levels, dyslipidemia, elevated / decreased low density lipoprotein (LDL), high cholesterol levels, dyslipidemia, cardiovascular disease, high blood pressure, and atherosclerosis, or

[0576] -Induces weight loss.

[0577] In one particular implementation, a GIP peptide analog as defined herein is provided in a method of treating obesity.

[0578] In one particular implementation, a GIP peptide analog as defined herein is provided in a method of treating diabetes (including type I and type II diabetes).

[0579] In one particular implementation, a GIP peptide analog as defined herein is provided in a method of treating insulin resistance.

[0580] Obesity-related disorders can be any of the following: increased food intake, increased appetite, binge eating, bulimia nervosa, obesity caused by the use of antipsychotic drugs or steroids, decreased / increased gastric motility, delayed / increased gastric emptying, decreased physical activity, osteoarthritis, dyslipidemia, increased / decreased low-density lipoprotein (LDL), high cholesterol levels, and abnormal lipid deposition.

[0581] In some implementations, dyslipidemia, elevated / decreased low-density lipoprotein (LDL), cholesterol, and abnormal lipid deposition are referred to as fatty acid metabolism disorders.

[0582] Diabetes-related disorders can be any of the following: impaired glucose tolerance (IGT), progression from IGT to type 2 diabetes, progression from insulin-independent type 2 diabetes to insulin-independent type 2 diabetes, decreased β-cell function, decreased β-cell mass, increased β-cell apoptosis, or decreased glucose sensitivity to β-cells.

[0583] Cardiovascular disease can be any of the following: coronary artery disease, myocardial infarction, reperfusion injury, stroke, cerebral ischemia, left ventricular hypertrophy, coronary artery disease, hypertension, essential hypertension, acute hypertensive emergency, cardiomyopathy, heart failure, exercise intolerance, acute and / or chronic heart failure, arrhythmia, cardiac arrhythmia, syncope, angina pectoris, cardiac bypass and / or stent re-occlusion, intermittent claudication (also known as atherosclerotic occlusion), diastolic dysfunction and systolic dysfunction, and combinations thereof.

[0584] A method for treating metabolic syndrome, obesity, overweight, diabetes, insulin resistance, obesity-related disorders as defined herein, or diabetes-related disorders as defined herein is also provided; the method includes the step of administering an effective amount of a peptide as defined herein to an individual in need.

[0585] The individuals referred to herein as having a need are those who can benefit from the administration of the peptide or pharmaceutical composition according to this disclosure. Such individuals may have metabolic syndrome and / or metabolic disorders (such as obesity, overweight, diabetes, insulin resistance, obesity-related disorders as defined herein, or diabetes-related disorders as defined herein), or be at risk of developing these. The individuals may be any person, male or female, infant, middle-aged, or older. The individual's disorder to be treated or prevented may be related to: the individual's age, the individual's overall health status, the medication used to treat the individual, and whether the individual has a history of a disease or disorder that may induce or has induced metabolic syndrome and / or metabolic disorders (such as obesity, overweight, diabetes, insulin resistance, obesity-related disorders as defined herein, or diabetes-related disorders as defined herein). In some implementations, the barrier to be treated is associated with the following: 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 incorporation, high expression or activity of GIPR, and postprandial GIP release; wherein the term “high” should be interpreted as a level higher than the corresponding level observed in individuals who do not require treatment.

[0586] Preparation method (peptide)

[0587] The peptides according to this disclosure can be prepared by any method known in the art. Therefore, GIP-derived peptides can be prepared by standard peptide preparation techniques, such as solution synthesis or Merrifield-type solid-phase synthesis.

[0588] In one implementation, the peptides defined herein are non-naturally occurring peptides; derived from naturally occurring GIPs, such as GIP(1-42).

[0589] In one embodiment, the peptide according to this disclosure is synthesized or produced.

[0590] Methods for synthesizing peptides are well known in the art. Detailed instructions and practical recommendations for producing synthetic peptides can be found in *Synthetic Peptides: A User's Guide (Advances in Molecular Biology), edited by Grant GA, Oxford University Press, 2002, or in *Pharmaceutical Formulation: Development of Peptides and Proteins*, edited by Frokjaer and Hovgaard, Taylor and Francis, 1999.

[0591] In one embodiment, the peptide or peptide sequence of the present invention is synthesized, particularly by sequence-assisted peptide synthesis (SAPS) methods, by solution synthesis, by solid-phase peptide synthesis (SPPS) such as Merrifield-type solid-phase synthesis, by recombinant technology (produced by a host cell containing a first nucleic acid sequence encoding a peptide, the first nucleic acid sequence being operatively associated with a second nucleic acid capable of being directed to expression in the host cell), or by enzymatic synthesis. These are well known to those skilled in the art.

[0592] Peptides can be synthesized in batches on a fully automated peptide synthesizer using 9-fluorenylmethoxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc) as suitable common protecting groups for Na-amino and side chain functional groups.

[0593] After purification, such as by reversed-phase HPLC, the peptide can be further processed to obtain isoforms modified, for example, by rings, C-terminus, or N-terminus. Methods for cyclization and terminal modification are well known in the art.

[0594] The peptides according to the present invention can be synthesized as monomers or polymers (such as dimers or tetramers).

[0595] Pharmaceutical compositions and formulations

[0596] Although the bioactive agents described in this disclosure can be administered as chemical raw materials (peptides), they are sometimes preferably presented as pharmaceutical formulations. Such pharmaceutical formulations may be referred to as pharmaceutical compositions, pharmaceutically acceptable compositions, or pharmaceutically safe compositions.

[0597] Therefore, a pharmaceutical formulation is further provided, comprising the bioactive agent of the present invention, or a pharmaceutically acceptable salt or ester thereof, and a pharmaceutically acceptable carrier, excipient, and / or diluent. The pharmaceutical formulation may be prepared using conventional techniques, such as those described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

[0598] The present invention also intends to include pharmaceutically acceptable salts of the peptide compounds of the invention, where they can be prepared. These salts will be acceptable for use in their pharmaceutical applications. This means that the salts will retain the biological activity of the parent compound and will not produce adverse or harmful effects in their use for treating diseases.

[0599] Prepare pharmaceutically acceptable salts using 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.

[0600] The peptide compounds disclosed herein can be administered in an effective amount, in the form of their alkali metal or alkaline earth metal salts, in parallel, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, particularly and preferably in the form of a pharmaceutical composition, whether by oral, rectal, or parenteral (including subcutaneous) route.

[0601] For example, pharmaceutically acceptable examples of acid addition salts used in the pharmaceutical compositions of the present invention include acid addition salts derived from inorganic 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 acid).

[0602] In certain embodiments, the peptides according to this disclosure are formulated as acetate, Cl- (hydrochloric acid) salts, or Na+ (sodium) salts.

[0603] In some embodiments of the invention (e.g., liquid compositions), the composition is stable during at least 4 days of use, for example, it is physically stable. In a further embodiment, the composition is stable during at least 2 weeks of use and at least 6 months of storage. In still a further embodiment, the composition is stable during at least 2 weeks of use and at least 1 year of storage. In even further embodiments, the composition is stable during at least 4 weeks of use and at least 2 years of storage.

[0604] In this regard, the term "use" for the purposes of this paragraph means removing the pharmaceutical composition from storage and subjecting it to environmental conditions (light, darkness, temperature, agitation, etc.) for the purpose of using the pharmaceutical composition for therapeutic purposes, while the term "storage" for the purposes of this paragraph means storing the pharmaceutical composition in a refrigerator or freezer under non-agitation conditions at a temperature not exceeding about 5 degrees Celsius. Those skilled in the art will understand the typical range of use and storage conditions that these pharmaceutical compositions may experience.

[0605] Application and dosage

[0606] According to this disclosure, peptides as defined herein, or compositions containing peptides as defined herein, are administered to individuals in need of treatment at pharmaceutically effective or therapeutically effective doses. Dosage requirements will vary depending on the specific pharmaceutical 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 also recognize that the optimal amount and interval of individual doses of the peptide compound will depend on 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 conditions can be determined using conventional techniques. Those skilled in the art should also understand that the optimal duration of treatment, i.e., the number of doses of the compound administered daily over a defined number of days, can be determined using conventional treatment duration determination tests.

[0607] In one implementation, the bioactive agent is applied at least once a day, such as once a day, twice a day, three times a day, four times a day, or five times a day.

[0608] Dosage can also be administered at intermittent intervals or intervals where the dose is not administered daily. Instead, one or more doses can be administered every other day, every two days, every three days, every four days, every five days, weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, or at intervals within those ranges (such as every 2 to 4 weeks or every 4 to 6 weeks).

[0609] In one implementation, the dose is administered once a week, such as once a week or one dose per week.

[0610] Application route

[0611] It should be understood that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body, and the active ingredient selected, but may be, for example, subcutaneous.

[0612] Systemic treatment

[0613] For systemic treatments according to this disclosure, the route of administration enables the bioactive agent to be introduced into the bloodstream to ultimately target the desired site of action.

[0614] These routes of administration are any suitable routes, such as enteric routes (including oral, rectal, nasal, pulmonary, buccal, sublingual, percutaneous, intracisional, and intraperitoneal administration), and / or parenteral routes (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal administration).

[0615] External application

[0616] Parenteral administration refers to any route of administration other than oral / enteral, thereby avoiding first-pass degradation in the liver. Therefore, parenteral administration includes any injection and infusion, such as bolus injections or continuous infusions, such as intravenous, intramuscular, or subcutaneous administration. Furthermore, parenteral administration includes inhalation and topical application.

[0617] Therefore, bioactive agents can be applied topically to penetrate any mucous membrane of the animal to which the bioactive substance is to be administered, such as the mucous membranes of the nose, vagina, eyes, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucous membranes of the nose or mouth. Thus, parenteral administration may also include administration via the buccal, sublingual, nasal, rectal, vaginal, and peritoneal routes, as well as administration via inhalation or via the lungs and bronchi through an implant. Furthermore, the agent can also be applied topically to penetrate the skin.

[0618] According to an advantageous embodiment of the invention, the GIP analogue is administered subcutaneously.

[0619] Local treatment

[0620] In one embodiment, the bioactive agent according to the invention can be used for local treatment, i.e., directly introduced into one or more sites of action. Therefore, the bioactive agent can be applied directly to the skin or mucous membranes, or it can be injected into the site of action, such as into diseased tissue or directly into the terminal artery leading to the diseased tissue. These forms of application preferably bypass the blood-brain barrier.

[0621] Set Box

[0622] This disclosure also relates to a kit containing one or more of the above-mentioned bioactive agents and at least one additional or other component, such as one or more second active ingredients.

[0623] References

[0624] 1.Baggio LL,Drucker DJ.Biology of Incretins:GLP-1andGIP.Gastroenterology2007;132(6):2131-2157.

[0625] 2.Holst JJ.On the Physiology of GIP and GLP-1.Horm Metab Res2004;36(11 / 12):747-754.

[0626] 3.Heer J,Rasmussen C,Coy DH,Holst JJ.Glucagon-like peptide-1,but notglucose-dependent insulinotropic peptide,inhibits glucagon secretion viasomatostatin(receptor subtype 2)in the perfused rat pancreas.Diabetologia2008;51(12):2263-2270.

[0627] 4.Gutniak M,Orskov C,Holst JJ,Ahrén B,Efendic S.AntidiabetogenicEffect of Glucagon-like Peptide-1(7-36)amide in Normal Subjects and Patientswith Diabetes Mellitus.N Engl J Med 1992;326(20):1316-1322.

[0628] 5.Christensen M,Vedtofte L,Holst JJ,Vilsboell T,Knop FK.Glucose-Dependent Insulinotropic Polypeptide:A Bifunctional Glucose-DependentRegulator of Glucagon and Insulin Secretion in Humans.Diabetes 2011;60(12):3103-3109.

[0629] 6.Pederson R,Brown J.Interaction of Gastric Inhibitory Polypeptide,Glucose,and Arginine on Insulin and Glucagon Secretion from the Perfused RatPancreas.Endocrinology1978;103(2):610-615.

[0630] 7.Adrian TE,Bloom SR,Hermansen K,Iversen J.Pancreatic polypeptide,glucagon and insulin secretion from the isolated perfused caninepancreas.Diabetologia 1978;14(6):413-417.

[0631] 8.Brunicardi FC,Druck P,Seymour NE,Sun YS,Elahi D,AndersenDK.Selective neurohormonal interactions in islet cell secretion in theisolated perfused human pancreas.Journal of Surgical Research 1990;48(4):273-278.

[0632] 9.Dupre J,Caussignac Y,McDonald TJ,Van Vliet S.Stimulation ofGlucagon Secretion by Gastric Inhibitory Polypeptide in Patients with HepaticCirrhosis and Hyperglucagonemia.The Journal of Clinical Endocrinology&Metabolism 1991;72(1):125-129.

[0633] 10.Ding WG,Renstrom E,Rorsman P,Buschard K,Gromada J.Glucagon-likepeptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediatedmechanism.Diabetes 1997;46(5):792-800.

[0634] 11.Meier JJ,Gallwitz B,Siepmann N et al.Gastric inhibitorypolypeptide(GIP)dose-dependently stimulates glucagon secretion in healthyhuman subjects at euglycaemia.Diabetologia 2003;46(6):798-801.

[0635] 12.Christensen MB,Calanna S,Holst JJ,Vilsboell T,Knop FK.Glucose-dependent Insulinotropic Polypeptide:Blood Glucose Stabilizing Effects inPatients With Type 2Diabetes.The Journal of Clinical Endocrinology&Metabolism2013;99(3):E418-E426.

[0636] 13.Christensen M,Calanna S,Sparre-Ulrich AH et al.Glucose-DependentInsulinotropic Polypeptide Augments Glucagon Responses to Hypoglycemia inType 1Diabetes.Diabetes2014.

[0637] 14.Song DH,Getty-Kaushik L,Tseng E,Simon J,Corkey BE,WolfeMM.Glucose-Dependent Insulinotropic Polypeptide Enhances AdipocyteDevelopment and Glucose Uptake in Part Through AktActivation.Gastroenterology 2007;133(6):1796-1805.

[0638] 15.Miyawaki K,Yamada Y,Ban N et al.Inhibition of gastric inhibitorypolypeptide signaling prevents obesity.Nat Med 2002;8(7):738-742.

[0639] 16.Starich GH,Bar RS,Mazzaferri EL.GIP increases insulin receptoraffinity and cellular sensitivity in adipocytes.Am J Physiol 1985;249(6 Pt1):E603-E607.

[0640] 17.Getty-Kaushik L,Song DH,Boylan MO,Corkey BE,Wolfe MM.Glucose-Dependent Insulinotropic Polypeptide Modulates Adipocyte Lipolysis andReesterification.Obesity2006;14(7):1124-1131.

[0641] 18.Hauner H,Glatting G,Kaminska D,Pfeiffer EF.Effects of gastricinhibitory polypeptide on glucose and lipid metabolism of isolated ratadipocytes.Ann Nutr Metab 1988;32(5-6):282-288.

[0642] 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.

[0643] 20.Nasteska D,Harada N,Suzuki K et al.Chronic Reduction of GIPSecretion Alleviates Obesity and Insulin Resistance Under High-Fat DietConditions.Diabetes2014;63(7):2332-2343.

[0644] 21.Miyawaki K,Yamada Y,Yano H et al.Glucose intolerance caused by adefect in the entero-insular axis:A study in gastric inhibitory polypeptidereceptor knockout mice.Proceedings of the National Academy of Sciences 1999;96(26):14843-14847.

[0645] 22.Ahlqvist E,Osmark P,Kuulasmaa T et al.Link Between GIP andOsteopontin in Adipose Tissue and Insulin Resistance.Diabetes 2013;62(6):2088-2094.

[0646] 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.

[0647] 24.Asmar M,Simonsen L,Madsbad S,Stallknecht B,Holst JJ,BülowJ.Glucose-Dependent Insulinotropic Polypeptide May Enhance Fatty Acid Re-esterification in Subcutaneous Abdominal Adipose Tissue in LeanHumans.Diabetes 2010;59(9):2160-2163.

[0648] 25.Deschamps I,Heptner W,Desjeux JF,Baltakse V,Machinot S,LestradetH.Effects of diet on insulin and gastric inhibitory polypeptide levels inobese children.Pediatr Res1980;14(4 Pt 1):300-303.

[0649] 26. C,Jensen CB,Storgaard H et al.Impact of short-term high-fatfeeding on glucose and insulin metabolism in young healthy men.The Journal ofPhysiology2009;587(10):2387-2397.

[0650] 27.Raufman JP,Singh L,Eng J.Exendin-3,a novel peptide from Helodermahorridum venom,interacts with vasoactive intestinal peptide receptors and anewly described receptor on dispersed acini from guinea pigpancreas.Description of exendin-3(9-39)amide,a specific exendin receptorantagonist.Journal of Biological Chemistry 1991;266(5):2897-2902.

[0651] 28. NB,Dirksen C, KN et al.Exaggerated Glucagon-Like Peptide1 Response Is Important for Improvedβ-Cell Function and GlucoseTolerance After Roux-en-Y Gastric Bypass in Patients With Type 2Diabetes.Diabetes 2013;62(9):3044-3052.

[0652] 29.Nakamura T,Tanimoto H,Mizuno Y,Tsubamoto Y,Noda H.Biological andfunctional characteristics of a novel weight antagonist ofglucose-dependent insulinotropic polypeptide receptor,SKL-14959,in vitro andin vivo.Diabetes,Obesity and Metabolism 2012;14(6):511-517.

[0653] 30.Ebert R,Illmer K,Creutzfeldt W.Release of gastric inhibitorypolypeptide(GIP)by intraduodenal acidification in rats and humans andabolishment of the incretin effect of acid by GIP-antiserum inrats.Gastroenterology 1979;76(3):515-523.

[0654] 31.Fulurija A,Lutz TA,Sladko K et al.Vaccination against GIP for theTreatment of Obesity.PLoS ONE 2008;3(9):e3163.

[0655] 32.Irwin N,McClean PL,Patterson S,Hunter K,Flatt PR.Activeimmunisation against gastric inhibitory polypeptide(GIP)improves bloodglucose control in an animal model of obesity-diabetes.BiologicalChemistry.bchm 390,75.2009.16-7-2014.

[0656] 33.Hinke SA,Manhart S,Pamir N et al.Identification of a bioactivedomain in the amino-terminus of glucose-dependent insulinotropic polypeptide(GIP).Biochimica et Biophysica Acta(BBA)-Protein Structure and MolecularEnzymology 2001;1547(1):143-155.

[0657] 34.Tseng CC,Kieffer TJ,Jarboe LA,Usdin TB,Wolfe MM.Postprandialstimulation of insulin release by glucose-dependent insulinotropicpolypeptide(GIP).Effect of a specific glucose-dependent insulinotropicpolypeptide receptor antagonist in the rat.J Clin Invest1996;98(11):2440-2445.

[0658] 35.Irwin N,Green BD,Parker JC,Gault VA,O'Harte FPM,FlattPR.Biological activity and antidiabetic potential of synthetic fragmentpeptides of glucose-dependent insulinotropic polypeptide,GIP(1-16)and(Pro3)GIP(1-16).Regulatory Peptides 2006;135 :45-53.

[0659] 36.Kerr BD,Flatt AJS,Flatt PR,Gault VA.Characterization andbiological actions of N-terminal truncated forms of glucose-dependentinsulinotropic polypeptide.Biochemical and Biophysical ResearchCommunications 2011;404(3):870-876.

[0660] 37.Gelling RW,Coy DH,Pederson RA et al.GIP(6-30amide)contains thehigh affinity binding region of GIP and is a potent inhibitor of GIP1-42action in vitro.Regulatory Peptides1997;69(3):151-154.

[0661] 38.Deacon CFP.GIP-(3-42)does not antagonize insulinotropic effects ofGIP at physiological concentrations.American Journal of Physiology-Endocrinology and Metabolism 2006;291(3):E468-E475.

[0662] 39.Gault VA,O'Harte FPM,Harriott P,Flatt PR.Characterization of theCellular and Metabolic Effects of a Novel Enzyme-Resistant Antagonist ofGlucose-Dependent Insulinotropic Polypeptide.Biochemical and BiophysicalResearch Communications2002;290(5):1420-1426.

[0663] 40.Ravn P,Madhurantakam C,Kunze S et al.Structural andPharmacological Characterization of Novel Potent and Selective MonoclonalAntibody Antagonists of Glucose-dependent Insulinotropic PolypeptideReceptor.Journal of Biological Chemistry2013;288(27):19760-19772.

[0664] 41.Deacon CF,Plamboeck A,Rosenkilde MM,de Heer J,Holst JJ.GIP-(3-42)does not antagonize insulinotropic effects of GIP at physiologicalconcentrations.American Journal of Physiology-Endocrinology and Metabolism2006;291(3):E468-E475.

[0665] 42.Goetze JP,Hunter I,Lippert SK,Bardram L,Rehfeld JF.Processing-independent analysis of peptide hormones and prohormones in plasma.FrontBiosci 2012;17:1804-1815.

[0666] 43.Goetze JP,Rehfeld JF.Peptide hormones and their prohormones asbiomarkers.Biomarkers Med 2009;3(4):335-338.

[0667] 44.Fujita Y,Asadi A,Yang GK,Kwok YN,Kieffer TJ.Differentialprocessing of pro-glucose-dependent insulinotropic polypeptide ingut.American Journal of Physiology-Gastrointestinal and Liver Physiology2010;298(5):G608-G614.

[0668] 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.

[0669] 46.Graham FL, van der Eb AJ.A new technique for the assay ofinfectivity of human adenovirus 5DNA.Virology 1973;52(2):456-467.

[0670] 47. Kissow H, Hartmann B, Holst JJ et al. Glucagon-like peptide-1(GLP-1)receptor agonism or DPP-4inhibition does not accelerate neoplasia incarcinogen treated mice. Regulatory Peptides 2012;179(1-3):91-100.

[0671] Example

[0672] The embodiments of the present invention support the following conclusions:

[0673] 1) GIP peptide analogs containing substituted A13Aib and / or N24E according to embodiments of this disclosure have increased solubility and / or stability, such as physical stability.

[0674] 2) Individual amino acid substitutions at certain sites, such as Aib at position 13, lead to antagonistic effects on the improvement of GIP receptors.

[0675] 3) Several acylation sites show great potential for GIP(3-30)+Z with substitutions of A13Aib and / or N24E, such as position 18.

[0676] Materials and methods

[0677] WO 2016 / 034186 discloses the generation and function of the GIP(3-30) peptide itself.

[0678] Material

[0679] Human GIP (1-42) was purchased from Phoenix Pharmaceuticals Inc., while the remaining GIP peptide analogs were synthesized from the following: Caslo TM (Lingbi, Denmark) and Almac Group (Craigavian, UK), Peptides & Elephants GmbH (Hanningsdorf, Germany) and WuXi AppTec, China. The cDNA of the human GIP receptor was purchased from Origene (Rockville, MD, USA (SC110906)) and cloned into the pCMV-Script vector.

[0680] Transfection and cell culture

[0681] COS-7 cells were cultured in Durbeco Modified Iger 1885 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 180 IU / ml penicillin, and 45 g / ml streptomycin at 10% CO2 and 37°C. Transient transfection of COS-7 cells with the addition of chloroquine was performed using the calcium phosphate precipitation method to facilitate cAMP accumulation. 46-47 .

[0682] HEK293 cells were cultured in Durbeko modified Iger medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 180 units / ml penicillin, and 45 g / ml streptomycin at 10% CO2 and 37°C. Transient transfection using hGIPR for CisBio assays (Table 2C) was performed using Lipofectamine 2000.

[0683] cAMP assay

[0684] Alternative 1 (also known as CisBio assay):

[0685] The in vitro functional activity of the compound against the receptor can also be determined in HEK-293 cells transiently expressing the human GIP receptor. On the day of 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 plated in 384-well plates at a density of 5000 cells / well. The GIP peptide analog of this disclosure was diluted in HBSS buffer supplemented with 20 mM HEPES, 0.1% pluronic, 0.1% casein, and 500 μM IBMX. To test antagonistic properties, the GIP peptide analogs to be tested were independently added to cells and incubated at 37°C for 20 min, followed by the addition of an agonist (GIP1-42) at an EC50 concentration and incubation at 37°C for 30 min. The resulting reduction in intracellular cAMP was quantitatively determined using the CisBio cAMP Dynamic 2HTRF assay kit. This assay is based on the competition between naturally produced cAMP and cAMP labeled with dye d2 for binding to an antibody labeled with a cryptate complex. A specific signal (i.e., an energy transfer signal) is inversely proportional to the concentration of cAMP in the sample.

[0686] Following the manufacturer's instructions, cAMP-d2 conjugate and antibody-anti-cAMP-cryptoether complex (both diluted in the lysis buffer provided in the kit) were added to the cells. The resulting competitive assay was incubated at room temperature for 60 minutes and analyzed using PerkinElmer. The instrument uses excitation at 320 nm and emission at 665 nm and 620 nm to detect the signal. The HTRF ratio (emission at 665 nm / 620 nm * 10,000) is inversely proportional to the amount of cAMP present, and the HTRF ratio is converted to nM cAMP / well using a cAMP standard curve. The pIC50 value is estimated by fitting a dose-response curve using nonlinear regression analysis (four-logistic equations) in the GraphPad Prism.

[0687] To test the agonistic properties to the GIP receptor, the compound was diluted and added to cells as described above, and incubated at 37°C for 30 min. The resulting increase in intracellular cAMP was determined using the CisBio cAMP Dynamic 2HTRF assay kit, as described above.

[0688] The dose-response curve was fitted using nonlinear regression analysis (four-logistic equation) in GraphPad Prism to estimate the pIC50 value.

[0689] Alternative method 2 (also known as Gaddum assay):

[0690] The antagonist potency (pKb) is estimated in the function settings. The estimated pKb value is calculated from the change in agonist-concentration-response curve in the presence of a single dose of GIP peptide antagonist using the Gaddum 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 the antagonist (EC50), and B is the concentration of the antagonist used.

[0691] In vitro functional assessment of the compound for the receptor was determined in HEK-293 cells transiently expressing the human GIP receptor. On the day of 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 plated in 384-well plates at a density of 3500 cells / well. The GIP peptide analog of this disclosure was 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 independently added to cells at concentrations 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, escalating doses of the agonist (GIP1-42) were added to the cells, and incubation was continued at 37°C for another 30 min. Intracellular cAMP was quantified using the CisBio cAMP Dynamic 2HTRF assay kit. The assay was based on the competition between naturally produced cAMP and cAMP labeled with dye d2 for binding to antibodies labeled with cryptane complexes. A specific signal (i.e., an energy transfer signal) was inversely proportional to the concentration of cAMP in the sample.

[0692] Following the manufacturer's instructions, cAMP-d2 conjugate and antibody-anti-cAMP-cryptoether complex (both diluted in the lysis buffer provided in the kit) were added to the cells. The resulting competitive assay was incubated at room temperature for 60 minutes and analyzed using PerkinElmer. The instrument uses excitation at 320 nm and emission at 665 nm and 620 nm to detect the signal. The HTRF ratio (emission at 665 nm / 620 nm * 10,000) is inversely proportional to the amount of cAMP present, and the HTRF ratio is converted to nM cAMP / well using a cAMP standard curve.

[0693] Alternative Option 3 (also known as Schild analysis):

[0694] The antagonist efficacy was also determined using Schild analysis. GIP1-42EC50 was measured in the absence of an antagonist, and EC50' was measured in the presence of progressively increasing concentrations of the antagonist. These values ​​were used to calculate the dose ratio for each antagonist concentration (DR = EC50' / EC50), 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 on the x-axis.

[0695] In vitro functional assessment of the compound for the receptor was determined in HEK-293 cells transiently expressing the human GIP receptor. On the day of 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 plated in 384-well plates at a density of 3500 cells / well. The GIP peptide analog of this disclosure was diluted in HBSS buffer supplemented with 20 mM HEPES, 0.1% pluronic, 0.1% casein, and 500 μM IBMX. After incubation at 37°C for 20 min, the GIP peptide analogs to be tested were independently added to cells at concentrations of 10, 100, and 1000 nM (for GIP3-30, AT759); 3.16, 31.6, and 316 nM (for AT158, AT364, AT760, and AT761); 1, 10, and 100 nM (for compounds AT762 and AT763); and 31.6, 316, and 3160 nM (for AT758). Subsequently, escalating doses of the agonist (GIP1-42) were added to the cells, and incubation was continued at 37°C for another 30 min. Intracellular cAMP was quantified using the CisBio cAMP Dynamic 2HTRF assay kit. The assay was based on the competition between naturally produced cAMP and cAMP labeled with dye d2 for binding to antibodies labeled with cryptane complexes. The specific signal (i.e. the energy transfer signal) is inversely proportional to the concentration of cAMP in the sample.

[0696] Following the manufacturer's instructions, cAMP-d2 conjugate and antibody-anti-cAMP-cryptoether complex (both diluted in the lysis buffer provided in the kit) were added to the cells. The resulting competitive assay was incubated at room temperature for 60 minutes and analyzed using PerkinElmer. The instrument uses excitation at 320 nm and emission at 665 nm and 620 nm to detect the signal. The HTRF ratio (emission at 665 nm / 620 nm * 10,000) is inversely proportional to the amount of cAMP present, and the HTRF ratio is converted to nM cAMP / well using a cAMP standard curve. The pIC50 value is estimated by fitting a dose-response curve using nonlinear regression analysis (four-logistic equations) in the GraphPad Prism.

[0697] The GIP peptide analogs were also functionally evaluated, with potency determined using the same assays described above (but with some modifications). Functional assessment was performed in CHO cells stably transfected with GIPR. Cells were resuspended in HBSS buffer containing 5 mM HEPES, 0.1% casein, and 500 μM IBMX. Incremental doses of the GIP analogs to be tested were added independently to the cells and incubated at 37°C for 20 min, followed by the addition of agonists (GIP1-42) at EC50–EC80 concentrations, and then incubated at 37°C for 30 min. The resulting reduction in cAMP was quantified as described in the previous section.

[0698] Table 1: Names and structures of GIP peptide analogs, including some reference peptides for comparison. When the linker consists of more than one unit, the intention is to link the first named unit to the peptide and the last named unit to the fatty acid:

[0699]

[0700]

[0701]

[0702]

[0703] Table 2A: Antagonistic effects of reference GIP peptides. CisBio assays (Alternative Method 1 above) were used to determine the antagonistic effects of the GIP peptide analogs listed in Table 2A:

[0704]

[0705] Table 2B: Antagonistic effects of GIP peptide analogs, including some reference peptides. CisBio assays (Alternative Method 1 above) were used to determine the antagonistic effects of the GIP peptide analogs listed in Table 2B:

[0706]

[0707]

[0708] Table 2C: Antagonistic effects of GIP peptide analogs and some reference peptides used for comparison. The Gaddum assay (alternative to method 2 above) was used to determine the antagonistic effects of the GIP peptide analogs listed in Table 2C:

[0709]

[0710]

[0711] Table 2D

[0712]

[0713] result

[0714] As can be seen from Tables 2B and 2C, the substitution of A13Aib in GIP peptide analogs may increase the antagonistic effect on the GIP receptor.

[0715] Solubility and physical stability

[0716] physical stability assessment

[0717] Aggregates in the form of fibrils were detected using the amyloid-specific dye thiosulfate T (ThT), which is often used to demonstrate the presence of fibrils in solution.

[0718] ThT emits weak fluorescence at approximately 527 nm, but exhibits a redshift in its emission spectrum to approximately 486 nm, along with an increase in emission intensity, upon binding to β-sheet-rich structures. Continuous measurements of fluorescence emission at 486 nm can be used as a measure of the fibrillation behavior of peptides and proteins. Here, the time until fibrillation begins, or fibrillation delay time, is estimated by defining the delay time (T-delay) as the point at which the signal reaches 10% of the post-transition baseline relative to the pre-transition baseline.

[0719] Chemicals:

[0720] Sodium dihydrogen phosphate (Na2HPO4, anhydrous, Sigma, batch number: SLBL9126V) (NaH2PO4, anhydrous, Sigma, batch number: SLBP1516V)

[0721] 50 mM sodium phosphate buffer was used to dissolve some samples in ultrapure water with a resistivity of 18.2 MΩ·cm. Prepared according to Reference A+System (Merck). Adjust the buffer to pH 7.4 and filter.

[0722] Before sample preparation, the ultrapure water (MilliQ) used to dissolve some samples was adjusted to pH 7.4 with NaOH and then filtered.

[0723] Sample preparation

[0724] Dissolve the peptides in sodium phosphate buffer (50 mM, pH 7.4, filtered) or in ultrapure (MilliQ) water (adjusted to pH 7.4 with NaOH, filtered) (see Table 3). Prepare all peptide samples at concentrations of 1, 5, 7.5, or 15 mg / ml. All samples (except reference peptides GIP(3-30), AT158, and AT482) dissolve readily and produce clear, colorless solutions with gentle mixing. The peptide samples were then passed through a 0.22 μm nylon filter (…). Filtering was performed using an RR injection filter (13 mm, Frisenette, Denmark) to produce a particle-free solution. At t=0, all samples were added to a 96-well plate reader for ThT determination.

[0725] For each sample, add 22 μL of ThT (1 mM) to 1.2 mL of peptide solution. From this sample mixture, pipette 200 μL / well into four or five wells (n = 4 or 5). A blank sample (buffer + ThT) is also included. Add a 3 mm silica bead to each well containing the sample, and subject the plate to orbital rotation at 300 rpm to agitate and apply pressure to the sample. Maintain the temperature at 25 °C throughout the measurement.

[0726] Board reader settings:

[0727] Excitation wavelength: 450nm

[0728] Dichroic filter: 465nm

[0729] Emission wavelength: 486nm

[0730] Focal length: 3.5mm

[0731] Gain: 1000

[0732] Number of loops: 960

[0733] Loop time: 360s

[0734] Flashes per hole: 20

[0735] result

[0736] A summary of the results can be found in Table 3. Furthermore, Figure 1 This demonstrates a highly stable GIP peptide analog that does not form fibrils (AT763- in phosphate buffer). Figure 1 B) A reference GIP analog with lower physical stability in forming fibrils (AT364- in phosphate buffer). Figure 1 A) Comparison between them.

[0737] As can be seen from Table 3, the substitution of A13Aib and / or N24E in the GIP peptide analogs increases the physical stability of the GIP peptide analogs, as measured by the reduced fibrillation tendency in the ThT assay. See, for example, AT760 compared to AT364, AT762 compared to AT677, and AT763 compared to AT677. The GIP peptide analogs according to embodiments of the invention showed no increase in absorbance intensity during the 96-hour measurement period, indicating that the sample did not fibrillate and the peptides are physically stable in aqueous solution. See, for example, AT673, AT695, and AT696 compared to AT364, and for example, AT749 compared to AT158 and AT719. Also see AT677 compared to AT717 and AT755.

[0738] Table 3 also shows that fatty acids can attach to different positions (e.g., 12, 13, 16, 17, 18, 34, and 40) and retain increased physical stability. See, for example, AT739, AT740, AT741, AT742, AT743, AT744, and AT668, which did not fibrillate within 96 hours.

[0739] Solubility assessment

[0740] A clear visual appearance can serve as an indicator of the immediate solubility of a peptide. Therefore, it can be seen that all peptides according to embodiments of the present invention exhibit increased solubility compared to GIP(3-30), AT158, and AT482. Thus, substitution with A13Aib and / or N24E appears to increase solubility.

[0741] Table 3: Solubility (e.g., measured by visual inspection) and physical stability (e.g., measured by estimated delay time in ThT assay) of the tested GIP peptide analogs.

[0742]

[0743]

[0744]

[0745] Fnd = No fibrils were detected during the experimental period (96 hours) under agitation.

[0746] Most of the peptides precipitate and are captured in the filter during filtration. sequence list <110> Antaibo Pharmaceutical <120> Optimized GIP peptide analogs <130> P5469PC00 <150> PCT / EP2019 / 083506 <151> 2019-12-03 <150> EP20179259.5 <151> 2020-06-10 <160> 102 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X is any amino acid or is omitted; <400> 1 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 2 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X is any amino acid or is omitted. <400> 2 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 3 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X is any amino acid or is omitted. <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 3 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 4 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X is any amino acid or is omitted. <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 4 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 5 <211> 28 <212> PRT <213> Artificial sequence <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 5 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 6 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 6 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 7 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 7 Ser Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 8 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 8 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Gln Trp Leu Leu Ala Gln Lys 20 25 <210> 9 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 9 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 10 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 10 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 11 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 11 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Ala Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 12 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 12 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 13 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 13 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 14 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 14 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 15 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 15 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 16 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 16 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 17 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 17 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Lys Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 18 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 18 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 19 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 19 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 20 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <400> 20 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> twenty one <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> twenty one Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> twenty two <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> twenty two Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> twenty three <400> twenty three 000 <210> twenty four <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> twenty four Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 25 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 25 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 26 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 26 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 27 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <400> 27 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 28 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 28 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 29 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 29 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 30 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 30 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 31 <400> 31 000 <210> 32 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 32 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 33 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 33 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 34 <400> 34 000 <210> 35 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 35 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 36 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 36 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Lys Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 37 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 37 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Lys Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 38 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 38 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 39 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 39 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Lys His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 40 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 40 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Lys 20 25 30 Ala Pro Pro Pro Ser 35 <210> 41 <211> 38 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(38) <223> GIP analogues <400> 41 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser Lys 35 <210> 42 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 42 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Ala Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 43 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 43 Glu Gly Thr Phe Ile Ser Asp Tyr Ala Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 44 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 44 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Glu Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 45 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 45 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Ala Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 46 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 46 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 47 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <400> 47 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 48 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = succinic acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 48 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 49 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = adipic acid <400> 49 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 50 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 50 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 51 <400> 51 000 <210> 52 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 52 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 53 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = Glutaric acid <400> 53 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 54 <211> 37 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues X = Glutaric acid <400> 54 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 55 <211> 4 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(4) <223> connector <400> 55 Gly Pro Ser Ser 1 <210> 56 <211> 5 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(5) <223> connector <400> 56 Gly Pro Ser Ser Gly 1 5 <210> 57 <211> 6 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(6) <223> connector <400> 57 Gly Pro Ser Ser Gly Ala 1 5 <210> 58 <211> 7 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(7) <223> connector <400> 58 Gly Pro Ser Ser Gly Ala Pro 1 5 <210> 59 <211> 8 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(8) <223> connector <400> 59 Gly Pro Ser Ser Gly Ala Pro Pro 1 5 <210> 60 <211> 9 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(9) <223> connector <400> 60 Gly Pro Ser Ser Gly Ala Pro Pro Pro 1 5 <210> 61 <211> 10 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(10) <223> connector <400> 61 Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 10 <210> 62 <211> 4 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(4) <223> connector <400> 62 Pro Ser Ser Gly 1 <210> 63 <211> 5 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(5) <223> connector <400> 63 Pro Ser Ser Gly Ala 1 5 <210> 64 <211> 6 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(6) <223> connector <400> 64 Pro Ser Ser Gly Ala Pro 1 5 <210> 65 <211> 7 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(7) <223> connector <400> 65 Pro Ser Ser Gly Ala Pro Pro 1 5 <210> 66 <211> 8 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(8) <223> connector <400> 66 Pro Ser Ser Gly Ala Pro Pro Pro 1 5 <210> 67 <211> 9 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(9) <223> connector <400> 67 Pro Ser Ser Gly Ala Pro Pro Pro Ser 1 5 <210> 68 <211> 28 <212> PRT <213> Homo sapiens <400> 68 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 69 <211> 39 <212> PRT <213> Homo sapiens <400> 69 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 <210> 70 <211> 28 <212> PRT <213> Artificial sequence <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (10)..(10) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 70 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ala Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 71 <211> 37 <212> PRT <213> Artificial sequence <220> <223> peptide fragments <220> <221> peptides <222> (1)..(37) <223> GIP analogues <220> <221> MOD_RES <222> (10)..(10) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 71 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ala Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys Pro Ser Ser Gly 20 25 30 Ala Pro Pro Pro Ser 35 <210> 72 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 72 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 73 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 73 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 74 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 74 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 75 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 75 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 76 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 76 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 77 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 77 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 78 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 78 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Lys Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 79 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 79 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 80 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 80 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 81 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 81 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 82 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 82 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 83 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 83 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 84 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 84 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 85 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 85 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 86 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <220> <221> MOD_RES <222> (12)..(12) <223> Nle <400> 86 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 87 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <400> 87 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 88 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 88 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 89 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 89 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 90 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 90 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Met Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 91 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 91 Glu Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 92 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 92 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 93 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 93 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Lys Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 94 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 94 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Lys Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 95 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 95 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 96 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 96 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Lys His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 97 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues <400> 97 Glu Gly Thr Phe Ile Ser Asp Tyr Ala Ile Ala Met Asp Lys Lys His 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 98 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = succinic acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 98 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 99 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = adipic acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 99 Xaa Gly Thr Phe Ile Ser Glu Tyr Ser Ile Ala Leu Glu Lys Ile Lys 1 5 10 15 Gln Gln Glu Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 100 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(11) <223> GIP analogues <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 100 Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 <210> 101 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <400> 101 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Glu Trp Leu Leu Ala Gln Lys 20 25 <210> 102 <211> 28 <212> PRT <213> artificial <220> <223> peptide fragments <220> <221> peptides <222> (1)..(28) <223> GIP analogues X = Glutaric acid <220> <221> MOD_RES <222> (11)..(11) <223> Aib <400> 102 Xaa Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys 1 5 10 15 Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25

Claims

1. A glucose-dependent insulinotropic peptide (GIP) analogue or a pharmaceutically acceptable salt thereof, It consists of the following amino acid sequences selected from GIP(3-30) + Cex(31-39): EGTFISEYSIAiBLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 18; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (C16-diacid); D21E; N24E], EGTFISEYSIAibMEKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 33; GIP(3-30)+Cex(31-39) [D9E; A13Aib; D15E; H18K(C16-diacid); N24E], EGTFISDYSIAibMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 12; GIP(3-30)+Cex(31-39) [A13Aib; H18K(C16-diacid); N24E], EGTFISDYSIAibLDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 13; [A13Aib; M14L; H18K (C16-diacid); N24E], EGTFISDYSIAibNleDKIKQQDFVEWLLAQKPSSGAPPPS-C16-diacid / 18K; SEQ ID NO: 14; GIP(3-30)+Cex(31-39) [A13Aib; M14Nle; H18K (C16-diacid); N24E], EGTFISEYSIAiBLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; SEQ IDNO: 18; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (2xAEEAc+yGlu-C18-diacid); D21E; N24E], EGTFISEYSIAiBLEKIKQQEFVEWLLAQKPSSGAPPPS-Aib-C16-diacid / H18K; SEQ ID NO: 18; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (Aib-C16-diacid); D21E; N24E], XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-Diacid / H18K; where X is glutaric acid; SEQ ID NO: 25; GIP(3-30)Cex(31-39) [E3 glutaric acid; D9E; A13Aib; M14L; D15E; H18K(C16-Diacid); D21E; N24E], XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-C16-Diacid / H18K; where X is glutaric acid; SEQ ID NO: 26; GIP(3-30)Cex(31-39) [E3 glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K(C16-Diacid); D21E; N24E], EGTFISEYSIAibMEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / 18K; SEQ IDNO: 29; GIP(3-30)-Cex(31-39) [D9E; A13Aib; D15E; H18K (2xAEEAc+yGlu-C18-diacid); D21E; N24E], EGTFISEYSIAibLDKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; SEQ IDNO: 30; GIP(3-30)Cex(31-39) [D9E; A13Aib; M14L; H18K (2xAEEAc+yGlu-C18-diacid); N24E], XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is succinic acid; SEQ ID NO: 48; GIP(3-30)Cex(31-39) [E3 succinic acid; D9E; A13Aib; M14L; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is adipic acid; SEQ ID NO: 49; GIP(3-30)Cex(31-39) [E3 adipic acid; D9E; A13Aib; M14L; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], EGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 19; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14Nle; D15E; H18K (C16-diacid); D21E; N24E], XGTFISEYSIAibMEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is glutaric acid; SEQ ID NO: 28; GIP(3-30)-Cex(31-39) [E3glutaric acid; D9E; A13Aib; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is glutaric acid; SEQ ID NO: 25; GIP(3-30)Cex(31-39) [E3glutaric acid; D9E; A13Aib; M14L; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], XGTFISDYSIAibMDKIKQQDFVEWLLAQKPSSGAPPPS-C16-Diacid / H18K; where X is glutaric acid; SEQ ID NO: 32; GIP(3-30)Cex(31-39) [E3glutaric acid; A13Aib; H18K(C16-Diacid); N24E], EGTFISEYSIAiBLEKIKQQDFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; SEQ IDNO: 50; GIP(3-30)Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (2xAEEAc+yGlu-C18-diacid); N24E], EGTFISEYSIAibleKIKQQEFVEWLLAQKPSSGAPPPS-(KAAAEKAAAEKAAAE-C16-diacid / H18K); SEQ ID NO: 18; GIP(3-30)Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (KAAAEKAAAEKAAAE-C16-diacid); D21E; N24E], and XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is glutaric acid; SEQ ID NO: 26; GIP(3-30)-Cex(31-39) [E3 glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], Cex(31-39) is a peptide composed of PSSGAPPPS (SEQ ID NO: 67), and The fatty acid molecule is attached to the ε-amino group of the K residue ("H18K") at position 18 of the GIP (3-30) of the amino acid sequence.

2. The GIP analogue or a pharmaceutically acceptable salt thereof according to claim 1, wherein the GIP analogue has improved solubility at pH 7 to 9, such as at pH 7 to 8.5, such as at pH 7.0 to 8.0 or pH 7.5 to 8.5, compared with GIP (3-30) and / or compared with AT364 (SEQ ID NO: 6).

3. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP 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.

4. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP 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 at a pH between 7 and 9, such as about pH 7.5 or about 8.

5. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue has improved physical stability, such as as measured by fibrillation delay time in a ThT assay of more than about 24 hours, such as a fibrillation delay time in a ThT assay of more than about 50 hours, such as a fibrillation delay time in a ThT assay of more than about 96 hours, such as a fibrillation delay time in a ThT assay of more than about 168 hours.

6. The GIP analogue or a pharmaceutically acceptable salt thereof according to claim 1, wherein the GIP analogue has improved physical stability at pH 7 to 9, such as at pH 7 to 8.5, such as at pH 7.0 to 8.0 or pH 7.5 to 8.5, compared with GIP (3-30) and / or compared with AT364 (SEQ ID NO: 6).

7. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue inhibits at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as about 100% of GIPR activity.

8. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue inhibits at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as about 100% of GIPR activity, wherein the inhibition of GIPR activity is determined to be a decrease in intracellular cAMP.

9. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue has GIPR antagonistic potency corresponding to an IC50 value of less than 50 nM, such as an IC50 value of less than 10 nM, such as an IC50 value of less than 5 nM, such as an IC50 value of less than 1 nM, such as an IC50 value of less than 0.001 nM to 1 nM.

10. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue has a free N-terminus.

11. The GIP analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the amino acid sequence is C-terminally amidated (-NH2).

12. The GIP analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the amino acid sequence has a free C-terminal carboxylic acid.

13. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue is selected from: EGTFISEYSIAiBLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 18; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K (C16-diacid); D21E; N24E], XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; where X is glutaric acid; SEQ ID NO: 26; GIP(3-30)-Cex(31-39) [E3glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], and XGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-Diacid / H18K; where X is glutaric acid; SEQ ID NO: 25; GIP(3-30)Cex(31-39) [E3 glutaric acid; D9E; A13Aib; M14L; D15E; H18K(C16-Diacid); D21E; N24E].

14. The GIP analogue of claim 1 or a pharmaceutically acceptable salt thereof, wherein the GIP analogue is EGTFISEYSIAibLEKIKQQEFVEWLLAQKPSSGAPPPS-C16-diacid / H18K; SEQ ID NO: 18; GIP(3-30)+Cex(31-39) [D9E; A13Aib; M14L; D15E; H18K(C16-diacid); D21E; N24E], The amino acid sequence described therein has a free C-terminal carboxylic acid.

15. The GIP analogue or a pharmaceutically acceptable salt thereof according to claim 1, wherein the GIP analogue is XGTFISEYSIAibNleEKIKQQEFVEWLLAQKPSSGAPPPS-2xAEEAc+yGlu-C18-diacid / H18K; wherein X is glutaric acid; SEQ ID NO: 26; GIP(3-30)-Cex(31-39) [E3 glutaric acid; D9E; A13Aib; M14Nle; D15E; H18K(2xAEEAc+yGlu-C18-diacid); D21E; N24E], The amino acid sequence described therein has a free C-terminal carboxylic acid.

16. Use of a GIP analogue or a pharmaceutically acceptable salt thereof according to any one of the preceding claims in the preparation of a medicament for treating obesity.

17. Use of a GIP analogue or a pharmaceutically acceptable salt thereof according to any one of the preceding claims in the preparation of a medicament for treating type 2 diabetes.

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