GIP derivatives and their uses

Abstract

The present invention relates to novel peptides, which are derivatives of glucose-dependent insulinotropic polypeptide (GIP) analogs, having improved physical stability in solution and prolonged action curves. More specifically, the present invention relates to such peptides that are agonists for GIP receptors, and to their use in weight management or treatment of diseases such as obesity, diabetes or non-alcoholic steatohepatitis (NASH).

Description

Technical Field

[0001] The present application relates to novel peptides, which are derivatives of glucose-dependent insulinotropic polypeptide (GIP) analogs, having improved physical stability in solution and prolonged action curve, and to pharmaceutical uses of said GIP derivatives.

[0002] Incorporation by Reference of Sequence Listing

[0003] The sequence listing entitled "180016sequence listing_ST25" is 58,669 bytes, was created on April 9, 2019, and is incorporated herein by reference.

[0004] background

[0005] Glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory peptide) is one of two endogenous incretins, a 42-amino acid peptide hormone released from intestinal K cells after food ingestion. GIP and another incretin, glucagon-like peptide 1 (GLP-1), are enteroendocrine cell-derived gut hormones responsible for the incretin effect, which is estimated to account for more than 70% of the total insulin response to an oral glucose challenge.

[0006] Due to its incretin action, the GIP receptor has become an attractive drug target in the treatment of metabolic diseases such as obesity and diabetes, and GIP receptor agonists can be used alone or in combination with GLP-1 receptor agonists or GLP-1 / glucagon receptor co-agonists. GIP itself has a short plasma half-life due to dipeptidyl peptidase-4 (DPP-IV)-mediated inactivation and has poor physical stability due to its high tendency to form fibrils in solution.

[0007] Patent applications disclosing different GIP receptor agonists and / or their potential medical uses are described, for example, as disclosed in WO 2016 / 066744, WO 2016 / 034186, WO 2012 / 055770 and WO 2012 / 167744. GIP / GLP-1 receptor co-agonists and their medical uses have also been studied, for example, in WO 2013 / 164483 and WO 2014 / 192284.

[0008] The derivatives of the present invention provide novel modified GIP analogs with a prolonged action profile in addition to providing improved stability. Summary of the invention

[0009] The present invention relates to derivatives of GIP analogs having lysine at a position corresponding to position 24 of hGIP (1-31) (SEQ ID NO: 3).

[0010] In some embodiments, the derivative comprises a GIP analog having a lysine at a position corresponding to position 24 of hGIP(1-31) (SEQ ID NO: 3) and having a negatively charged modifying group attached to the epsilon amino group of the lysine.

[0011] In some embodiments, the derivative comprises a GIP analog having a lysine at a position corresponding to position 24 of hGIP (1-31) (SEQ ID NO: 3), having a negatively charged modifying group attached to the epsilon amino group of the lysine, and may contain up to 7 additional amino acid substitutions, i.e., also described as a maximum of 8 substitutions compared to hGIP (1-31).

[0012] In some embodiments, the derivative comprises a GIP analog having a lysine at a position corresponding to position 24 of hGIP (1-31) (SEQ ID NO: 3), having a negatively charged modifying group attached to the epsilon amino group of the lysine, and may comprise up to 7 additional amino acid substitutions, wherein the substitutions are located at one or more positions corresponding to positions 1, 2, 14, 16, 18, 20 and / or 31 of Formula I.

[0013] Furthermore, the present invention relates to pharmaceutical compositions comprising such derivatives of GIP analogs and pharmaceutically acceptable excipients, as well as the medical use of said derivatives.

[0014] In a first aspect, the present invention relates to derivatives of GIP analogues capable of activating the GIP receptor. In a further aspect, said derivatives of GIP analogues are selective in activating the human GIP receptor over the human GLP-1 receptor and the human glucagon receptor.

[0015] Additionally or alternatively, in a second aspect, the invention relates to derivatives of GIP analogues that are active in vivo either alone or in combination with a GLP-1 receptor agonist.

[0016] Additionally or alternatively, in a third aspect, the invention relates to derivatives of GIP analogues having improved pharmacokinetic properties.

[0017] Additionally or alternatively, in a fourth aspect, the invention relates to derivatives of GIP analogues having improved physical stability.

[0018] Additionally or alternatively, in a fifth aspect, the present invention relates to derivatives of GIP analogues having improved chemical stability.

[0019] describe

[0020] The present invention relates to derivatives of GIP analogs having a lysine at a position corresponding to position 24 of hGIP (1-31) (SEQ ID NO: 3). In one aspect, the derivatives of the present invention comprise a lysine at a position corresponding to position 24 of hGIP (1-31) (Formula I; SEQ ID NO: 3) and comprise a negatively charged modifying group attached to the epsilon amino group of the lysine.

[0021] In another aspect, the derivatives of the invention comprise a lysine at a position corresponding to position 24 of hGIP (1-31) (Formula I; SEQ ID NO: 3), comprising a negatively charged modifying group attached to the epsilon amino group of said lysine, and wherein Formula I may comprise up to 7 additional amino acid substitutions, also described as a maximum of 8 substitutions compared to hGIP (1-31).

[0022] In another aspect, the derivatives of the invention comprise a lysine at a position corresponding to position 24 of hGIP (1-31) (Formula I; SEQ ID NO: 3), comprising a negatively charged modifying group attached to the epsilon amino group of said lysine, and wherein Formula I may comprise up to 7 additional amino acid substitutions, wherein said substitutions are located at one or more positions corresponding to positions 1, 2, 14, 16, 18, 20 and / or 31 of Formula I.

[0023] In addition, the present invention relates to pharmaceutical compositions comprising such derivatives of GIP analogs and pharmaceutically acceptable excipients, as well as medical uses of said derivatives.

[0024] In one aspect, the present invention relates to derivatives of GIP analogs capable of activating the GIP receptor. In a further aspect, the derivatives of the GIP analogs are selective in activating the human GIP receptor over the human GLP-1 receptor and the human glucagon receptor. The term "selective" for the GIP receptor over the GLP-1 receptor and the glucagon receptor refers to such derivatives as measured in vitro in a potency assay for receptor function, such as a CRE luciferase functional potency assay, and by EC 50 The glucagon receptor agonist may be a agonist that exhibits at least 10-fold, such as at least 50-fold, at least 500-fold, or at least 1000-fold greater potency at the GIP receptor than at the GLP-1 receptor and the glucagon receptor, as compared to the agonist.

[0025] Additionally or alternatively, the present invention relates to derivatives of GIP analogues that are active in vivo either alone or in combination with a GLP-1 receptor agonist.

[0026] Additionally or alternatively, in one aspect, the invention relates to derivatives of GIP analogs having improved pharmacokinetic properties.

[0027] Additionally or alternatively, in one aspect, the invention relates to derivatives of GIP analogs having improved physical stability.

[0028] Additionally or alternatively, in one aspect, the invention relates to derivatives of GIP analogs having improved chemical stability.

[0029] In the following, Greek letters may be represented by their symbols or corresponding written names, for example: α = alpha; = beta; ε = epsilon; γ = gamma; ω = omega; etc. In addition, the Greek letter μ can also be represented by "u", for example μl = ul or μM = uM.

[0030] Unless otherwise specified in this specification, terms presented in the singular form generally include the plural case as well.

[0031] Also described herein are derivatives, derivatives of GIP analogs, pharmaceutical compositions and uses thereof, wherein open-ended terms such as "comprising" and "including" may be replaced with closed terms such as "consisting of" and the like.

[0032] Compound / Product

[0033] GIP receptor agonists

[0034] Receptor agonists can be defined as compounds that bind to a receptor and elicit a response characteristic of the natural ligand (see, for example, "Principles of Biochemistry", AL Lehninger, DL Nelson, MM Cox, 2nd edition, Worth Publishers, 1993, p. 763).

[0035] As used herein, a "GIP receptor agonist" may be defined as a compound that is capable of activating the GIP receptor.

[0036] GIP analogs

[0037] As used herein, the term "hGIP (1-42)" refers to human glucose-dependent insulinotropic polypeptide, the sequence of which is contained in the sequence listing as SEQ ID NO: 1. The peptide having the sequence of SEQ ID NO: 1 may also be referred to as native hGIP or hGIP.

[0038] As used herein, the term "hGIP(1-31)" refers to a truncated form of hGIP(1-42) comprising amino acids 1-31 of hGIP(1-42), the sequence of which is contained in the sequence listing as SEQ ID NO:2.

[0039] As used herein, the term "GIP analog" refers to a peptide or compound that is a variant of hGIP (1-31). The term "variant" is used to refer to a peptide that contains at least one amino acid substitution compared to hGIP (1-31) and is capable of activating the GIP receptor.

[0040] As used herein, the term "substitution" refers to the replacement of one amino acid in the peptide backbone by another amino acid. In one aspect, amino acids can be replaced by conservative substitutions. As used herein, the term "conservative substitution" means that one or more amino acids are replaced by another residue that is biologically similar. Examples include the replacement of amino acid residues with similar properties, such as small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. In one aspect, the GIP analogs of the derivatives of the present invention may comprise the replacement of one or more non-natural and / or non-amino acids, such as amino acid mimetics, into the GIP analog sequence.

[0041] The GIP analogs of the derivatives of the invention can be described with reference to i) the number of the amino acid residue in hGIP(1-31) or hGIP(1-42) corresponding to the amino acid residue that is altered (i.e., the corresponding position in hGIP(1-31) or hGIP(1-42)), and ii) the actual alteration. For example, [Lys24]-hGIP(1-31) refers to a GIP analog in which position 24 of hGIP(1-31) has been replaced by lysine.

[0042] In one aspect, the GIP analog of the derivative of the invention comprises a lysine residue at a position corresponding to position 24 of hGIP (1-31), as described in Formula I: Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Lys-Trp-Leu-Leu-Ala-Gln-Lys-Gly. Formula I is contained in the sequence listing as SEQ ID NO: 3 and may also be designated as [Lys24]-hGIP (1-31).

[0043] Additionally or alternatively, in one aspect, the GIP analog of the derivative of the invention comprises up to eight amino acid substitutions compared to hGIP (1-31), wherein position 24 is always occupied by a lysine substitution, and up to seven additional substitutions are at positions other than position 24. In a further aspect, the GIP analog of the derivative of the invention comprises up to seven, six, five, four, three, two or one amino acid substitutions compared to hGIP (1-31). In one aspect, the substitutions are present at one or more positions corresponding to positions 1, 2, 14, 16, 18, 20 and 31 of formula I, as described herein in formula II: X1-X2-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-X 14 -Asp-X 16 -Ile-X 18 -Gln-X 20 -Asp-Phe-Val-Lys-Trp-Leu-Leu-Ala-Gln-Lys-X 31 Formula II is contained in the sequence listing as SEQ ID NO: 48. In one aspect, the GIP analog of the derivative of the present invention may be in the form of a C-terminal carboxylic acid or amide.

[0044] Additionally or alternatively, in one aspect, the GIP analog of the derivative of the invention comprises a C-terminal extension of Formula I or Formula II. In a further aspect, the GIP analog of the derivative of the invention comprises a C-terminal extension, as described in Formula III: Lys-X 33 -X 34 -Asp-Trp-Lys-His-Asn-Ile-Thr-Gln, where X 33 is Lys or Glu; X 34 The C-terminal extension is connected to Formula I or Formula II via an amide bond from the C-terminal carboxylic acid of Formula I or Formula II to the N-terminal amino group of Formula III. Formula III is included in the sequence listing as SEQ ID NO: 51.

[0045] The following are non-limiting examples of appropriate analog nomenclature.

[0046] As an example, [Aib2, Nle14, Arg18, Lys24]-hGIP(1-31) contains 4 substitutions compared to hGIP(1-31). As another example, [D-Tyr1, Aib2, Nle14, Arg18, Lys24, Pro31]-hGIP(1-31) contains 6 substitutions compared to hGIP(1-31). Similarly, [D-Tyr1, Aib2, Nle14, Arg18, Lys24, Pro31]-hGIP(1-31) amide contains 6 substitutions compared to hGIP(1-31), because the number of substitutions refers to the backbone.

[0047] An analog that "comprising" certain specified changes may contain further changes compared to the respective general formula. In certain embodiments, an analog "has" the specified changes.

[0048] It is obvious from the above examples that amino acid residues can be represented by their full name, their one-letter code and / or their three-letter code. These three methods are completely equivalent.

[0049] The expression "a position equivalent to" or "a position corresponding to" can be used to characterize the altered site in a variant GIP sequence with reference to a given sequence such as hGIP (1-31) or hGIP (1-42).

[0050] The term "peptide", for example as used in the context of the GIP analogs of the derivatives of the present invention, refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds.

[0051] Amino acids are molecules containing an amino group and a carboxylic acid group and optionally one or more additional groups generally called side chains.

[0052] The term "amino acid" includes proteinogenic (or coded or natural) amino acids (of which there are 20 standard amino acids) and non-proteinogenic (or non-coded or non-natural) amino acids. Proteinogenic amino acids are amino acids that are naturally incorporated into proteins. Standard amino acids are amino acids encoded by the genetic code. Non-proteinogenic amino acids are either not present in proteins or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modifications). Non-limiting examples of non-proteinogenic amino acids are Aib (α-aminoisobutyric acid, Nle (norleucine), and D-isomers of proteinogenic amino acids. Non-limiting examples of D-isomers of proteinogenic amino acids are D-isomers of tyrosine or alanine, which can be written as D-Tyr or D-Ala, respectively.

[0053] Hereinafter, all amino acids of the GIP analogs of the derivatives of the present invention whose optical isomers are not specified should be understood to mean the L-isomer (unless otherwise specified).

[0054] GIP derivatives

[0055] The term "derivative" as used herein in the context of a GIP analog refers to a chemically modified GIP analog in which one or more substituents have been covalently attached to the peptide backbone.

[0056] In one aspect of the invention, the substituent may be an N-terminal substituent.

[0057] Additionally or alternatively, in one aspect, the substituent may be a modifying group, or alternatively referred to as a protracting moiety or an albumin binding moiety.

[0058] As used herein, the term "N-terminal substituent" or "modifying group" refers to a chemical moiety or group that replaces a hydrogen atom.

[0059] In one aspect, the derivative of the GIP analog comprises a substituent covalently linked to the α-amino group of the amino acid residue at the N-terminus of the analog. In one aspect, the N-terminal substituent is an alkanoyl or acyl group. In a specific aspect, the N-terminal substituent is an acetyl group. An example of an amino acid substituted at the N-terminus is Ac-Tyr at position 1. This acetylation will not be considered as a displacement in the peptide backbone compared to hGIP (1-31) because the amino acid in the GIP analog is a natural Tyr, for example, N{1}-acetyl-[Aib2, Nle14, Arg18, Lys24]-hGIP (1-31) comprises 4 displacements compared to hGIP (1-31).

[0060] Additionally or alternatively, in one aspect, the GIP analog comprises a modifying group covalently linked to the amino acid residue corresponding to position 24 of hGIP (1-31) or hGIP (1-42). In a further aspect, the modifying group is capable of forming a non-covalent conjugate with a protein such as albumin, thereby promoting the circulation of the derivative in the blood stream and also having the effect of prolonging the duration of action of the derivative, since the conjugate of the GIP derivative with albumin only slowly decomposes to release the active pharmaceutical ingredient.

[0061] The modifying group can be covalently linked to the lysine residue of the GIP analog by acylation, i.e., via an amide bond formed between the carboxylic acid group of the modifying group and the epsilon amino group of the lysine group. The amino group of lysine can also be coupled to the aldehyde of the modifying group by reductive amination.

[0062] In one aspect, the modifying group is covalently linked to a lysine residue at a position corresponding to position 24 of hGIP(1-31) or hGIP(1-42) by acylation, i.e., via an amide bond formed between the carboxylic acid group of the modifying group and the epsilon amino group of the lysine residue.

[0063] In one embodiment, the modifying group is defined by ABC-, wherein A- is a lipophilic moiety with a negatively charged moiety at the distal end, and BC- is a linker. In one embodiment, the modifying group is defined by ABC-, wherein A- is a lipophilic moiety with a negatively charged moiety at the distal end, and BC- is a linker comprising at least one negatively charged moiety.

[0064] The term "lipophilic moiety" as used herein refers to an aliphatic hydrocarbon chain having 8 to 30 carbon atoms, preferably 10 to 28 carbon atoms, more preferably 12 to 24 carbon atoms, even more preferably 14 to 20 carbon atoms, most preferably 16 or 18 carbon atoms, wherein the hydrocarbon may contain additional substituents.

[0065] As used herein, the term "negatively charged moiety" refers to a negatively charged chemical moiety that is negatively charged at physiological pH (7.4). Examples of negatively charged moieties are carboxylic acids or their isosteres, such as sulfonic acid or tetrazole. In preferred embodiments, the negatively charged moiety is a carboxylic acid moiety.

[0066] The term "distal" as used herein refers to the farthest point (the end) from the connection point of A- and B-.

[0067] The derivatives of the present invention may exist in different stereoisomeric forms with the same molecular formula and bonded atomic sequence but only with different three-dimensional orientations of their atoms in space. In the experimental section, standard nomenclature is used to illustrate the stereoisomerism of the exemplary derivatives of the present invention in terms of name and structure. Unless otherwise indicated, the present invention relates to all stereoisomeric forms of the derivatives claimed.

[0068] The derivatives of the present invention have GIP activity. The term refers to the ability to bind to the GIP receptor and initiate a signal transduction pathway, thereby leading to an insulinotropic effect or other physiological effects known in the art. For example, the GIP activity or stability of the derivatives of the present invention can be determined using the assays described in Examples 1-6 herein.

[0069] Pharmaceutically acceptable salts, amides or esters

[0070] The derivatives of the present invention may be in the form of pharmaceutically acceptable salts or amides.

[0071] For example, salts are formed by chemical reactions between bases and acids, such as: 2NH3+H2SO4→(NH4)2SO4.

[0072] The salt may be a basic salt, an acidic salt, or it may be neither (ie, a neutral salt). In water, basic salts produce hydroxide ions and acidic salts produce hydronium ions.

[0073] Salts of the derivatives of the invention can be formed by adding cations or anions between the anionic or cationic groups, respectively. These groups can be located in the peptide portion and / or in the modifying groups of the derivatives of the invention.

[0074] The non-limiting examples of the anionic groups of derivatives of the present invention are included in the side chain (if any) and in the free carboxyl groups in the peptide portion. The peptide portion usually comprises a free carboxyl group at the C-end, and it can also comprise a free carboxyl group at internal acidic amino acid residues such as Asp and Glu.

[0075] Non-limiting examples of cationic groups in the peptide moiety include the free amino group at the N-terminus (if present), and any free amino groups of internal basic amino acid residues such as His, Arg and Lys.

[0076] Amides of the derivatives according to the invention can be formed, for example, by reaction of a free carboxylic acid group with an amine or a substituted amine, or by reaction of a free or substituted amino group with a carboxylic acid.

[0077] Amide formation may involve the free carboxyl group at the C-terminus of the peptide, any free carboxyl group in a side chain, the free amino group at the N-terminus of the peptide, and / or any free or substituted amino group of the peptide in the peptide and / or side chains.

[0078] In one aspect, the derivatives of the invention are in the form of a pharmaceutically acceptable salt, preferably a sodium salt. Additionally or alternatively, in one aspect, the derivatives of the invention are in the form of a pharmaceutically acceptable amide, preferably having an amide group at the C-terminus of the peptide.

[0079] Functional properties

[0080] In terms of the first function, the derivatives of the present invention have good efficacy on the GIP receptor. Preferably, they are effective GIP receptor agonists, as reflected by their ability to activate the GIP receptor. Additionally or alternatively, in terms of the second function, they have in vivo effects on body weight, food intake and glucose tolerance alone or in combination with a GLP-1 receptor agonist. Additionally or alternatively, in terms of the third function, they have improved pharmacokinetic properties. Additionally or alternatively, in terms of the fourth function, the derivatives of the present invention are physically stable. Additionally or alternatively, in terms of the fifth function, the derivatives of the present invention are chemically stable.

[0081] Biological Activity - In Vitro Potency

[0082] According to a first functional aspect, the derivatives of the present invention and constituent GIP analogs such as [Lys24]-hGIP(1-31) or its analogs are biologically active or potent at the human GIP receptor.

[0083] In one embodiment, potency and / or activity refers to in vitro potency, ie, performance in a functional GIP receptor assay, and more specifically to the ability to activate the human GIP receptor.

[0084] For example, in vitro potency can be determined in a medium containing membranes expressing the human GIP receptor, and / or in an assay using whole cells expressing the human GIP receptor.

[0085] For example, the response of the human GIP receptor can be measured in a reporter gene assay, for example in a stably transfected BHK cell line, which expresses the human GIP receptor and contains a cAMP response element (CRE) DNA and a firefly luciferase (CRE luciferase) gene coupled to a promoter. When cAMP is produced due to the activation of the GIP receptor, this in turn causes luciferase to be expressed. Luciferase can be measured by adding luciferin, which is converted into oxyluciferin by the enzyme and produces bioluminescence, which is measured and used as a measure of in vitro effectiveness. A non-limiting example of this type of test is described in Example 2 described herein.

[0086] The term half maximal effective concentration (EC 50 ) is usually the concentration that induces a response that is halfway between baseline and maximum, referring to a dose-response curve. EC 50 It is used as a measure of compound potency and is the concentration at which 50% of its maximal effect is observed.

[0087] The in vitro potency of the derivatives of the invention can be determined as described above and the EC of the derivative in question is determined. 50 .EC 50 The lower the value, the better the efficacy.

[0088] In a further specific embodiment, the in vitro potency of the derivatives of the invention determined using the method of Example 2 corresponds to an EC of equal to or lower than 5000 pM, more preferably lower than 900 pM, even more preferably lower than 500 pM, or most preferably lower than 200 pM. 50 .

[0089] In a further specific embodiment, the derivatives of the invention are capable of selectively activating the GIP receptor over the human GLP-1 receptor and the human glucagon receptor. When used for activation of the GIP receptor over the GLP-1 receptor and the glucagon receptor, the term "selectively" refers to such derivatives as measured in vitro in potency assays for receptor function such as the CRE luciferase functional potency assay and by EC 50 The term "better potency" of the derivatives of the invention at the GIP receptor as compared to the GLP-1 receptor and the glucagon receptor is defined by the EC values ​​for the GLP-1 receptor and the GIP receptor, respectively. 50 EC ratio for glucagon receptor to GIP receptor 50 The ratio of the values ​​is determined.

[0090] Biological Activity - In Vivo Pharmacology

[0091] According to the second functional aspect, the GIP derivatives and constituent GIP analogs of the present invention, such as [Lys24]-hGIP(1-31) or its analogs, are effective in vivo, which can be determined in any suitable animal model and clinical trials as known in the art.

[0092] Diet-induced obesity (DIO) mice are an example of a suitable animal model, and the effects on body weight, food intake and glucose tolerance can be assessed during subchronic administration in this model. The effects of the GIP derivatives of the present invention on body weight, food intake and glucose tolerance can be determined in such mice, for example, as described in Example 6 herein. Food intake can be assessed by housing individual animals and weighing the food consumed each day. The model can also be used to evaluate the effects on glucose tolerance by performing an oral or intraperitoneal glucose tolerance test (OGTT or IPGTT). These tests are performed by administering a glucose load orally or intraperitoneally to semi-fasted animals, followed by measuring blood glucose for up to three hours.

[0093] Pharmacokinetic profile

[0094] According to the third functional aspect, the derivatives of the invention have improved pharmacokinetic properties, such as a prolonged terminal half-life.

[0095] Prolongation of the terminal half-life means that the compound in question is cleared from the body more slowly. For the derivatives of the invention this entails a prolonged duration of the pharmacological action.

[0096] The pharmacokinetic properties of the derivatives of the invention can be suitably determined in vivo in pharmacokinetic (PK) studies. Such studies are performed to evaluate how a drug compound is absorbed, distributed and eliminated from the body over time, and how these processes affect the concentration of the compound in the body.

[0097] In the discovery and preclinical stages of drug development, animal models such as mice, rats, monkeys, dogs or pigs can be used for such characterization. Any of these models can be used to test the pharmacokinetic properties of the derivatives of the invention.

[0098] In such studies, a single dose of the drug is usually administered intravenously (iv), subcutaneously (sc) or orally (po) to animals in the form of a relevant formulation. Blood samples are drawn at predetermined time points after administration and analyzed for drug concentration using a relevant quantitative test. Based on these measurements, a time-plasma concentration curve for the compound under investigation is plotted and the data are subjected to a so-called non-compartmental pharmacokinetic analysis.

[0099] For most compounds, the terminal portion of the plasma-concentration curve will be linear when plotted on a semi-log plot, indicating that after initial absorption and distribution, the drug is removed from the body at a constant fractional rate. This rate (lambda Z or λ z ) is equal to the negative of the slope of the end of the graph. From this rate, we can also calculate t 1 / 2=ln(2) / λ z The terminal half-life was calculated (see, e.g., Johan Gabrielsson and Daniel Weiner: Pharmacokinetics and Pharmacodynamic Data Analysis. Concepts & Applications, 3rd ed., Swedish Pharmaceutical Press, Stockholm (2000)).

[0100] Clearance can be determined after intravenous administration and is defined as the dose (D) divided by the area under the curve (AUC) on the plasma concentration-time curve (Rowland, M and Tozer TN: Clinical Pharmacokinetics: Concepts and Applications, 3rd edition, 1995 Williams Wilkins).

[0101] In the evaluation of new drug compounds, estimates of terminal half-life and / or clearance are relevant to the evaluation of dosing regimens and are important parameters in drug development.

[0102] Pharmacokinetic profile - in vivo half-life in minipigs

[0103] According to a third functional aspect, the derivatives of the invention have improved pharmacokinetic properties.

[0104] In certain embodiments, the pharmacokinetic properties can be measured as the terminal half-life (t 1 / 2 ) is determined, for example, as described in Example 3 herein.

[0105] In certain embodiments, the terminal half-life in minipigs is at least 24 hours, preferably at least 40 hours, even more preferably at least 60 hours.

[0106] Physical properties

[0107] According to a fourth functional aspect, the derivatives of the invention have improved physical stability in solution.The term "physical stability" refers to the tendency of a polypeptide to form biologically inactive and / or insoluble aggregates such as amyloid fibrils or gels.

[0108] In certain embodiments, improved physical stability can be determined by measuring lag time and / or recovery rate in a thioflavin T (ThT) fibril formation assay, for example, as described in Example 4 herein.

[0109] In further specific embodiments, the derivatives of the present invention have a recovery rate of greater than 70%, preferably greater than 90%, even more preferably greater than 95%, or most preferably greater than 98% in the ThT fibril formation assay, as shown in Example 4 described herein.

[0110] In a further specific embodiment, the derivative of the invention has a lag time in the ThT fibril formation assay of more than 10 hours, preferably more than 20 hours, even more preferably more than 45 hours, as shown in Example 4 described herein.

[0111] In certain embodiments, improved physical stability can be determined by a dynamic light scattering stability index (DLS-SI) assay, e.g., as described in Example 4 herein.

[0112] In a further specific embodiment, the derivatives of the invention have a low DLS-SI value in a DLS-SI assay, preferably less than 7, more preferably less than 2, as shown in Example 4 herein.

[0113] In a further specific embodiment, the derivative of the invention shows no or little precipitation, preferably no precipitation, in a DLS-SI assay, as shown in Example 4 herein.

[0114] Chemical properties

[0115] According to the fifth functional aspect, the derivatives of the present invention have improved chemical stability. The term "chemical stability" refers to chemical (particularly covalent) changes in the polypeptide structure that lead to the formation of chemical degradation products such as high molecular weight proteins (HMWP), deamidation, isomerization and hydrolysis products, which may have reduced biological efficacy and / or increased immunogenic effects compared to the intact polypeptide.

[0116] In certain embodiments, improved chemical stability can be determined by measuring the content and / or purity loss of HMWP, by measuring the amount of chemical degradation products at different time points after exposure to different environmental conditions, such as by SEC-HPLC and / or LCMS, for example, as described in Example 5 herein.

[0117] In further specific embodiments, the monthly purity loss of the derivative of the present invention is less than 35%, preferably less than 15%, more preferably less than 7%, as shown in Example 5 described herein.

[0118] In further specific embodiments, the monthly HMWP formation of the derivatives of the invention is less than 4%, preferably less than 2%, more preferably less than 1%, as shown in Example 5 described herein.

[0119] Additional specific embodiments of the derivatives of the present invention are described in the section entitled "Specific Embodiments".

[0120] Production process

[0121] The production of peptides such as hGIP(1-31) and hGIP analogs is well known in the art.

[0122] For example, GIP analogs (or fragments thereof) of the derivatives of the invention, such as [Lys24]-hGIP (1-31) or analogs or fragments thereof, can be produced by classical peptide synthesis, e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well-established techniques, see, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999, Florencio Zaragoza “Organic Synthesis on solidPhase”, Wiley-VCH Verlag GmbH, 2000, and “Fmoc SolidPhase Peptide Synthesis”, edited by WC Chan and PD White, Oxford University Press, 2000.

[0123] In addition, or alternatively, it can be produced by recombinant methods, i.e., by culturing a host cell containing a DNA sequence encoding the analog and capable of expressing the peptide in a suitable nutrient medium under conditions that allow the peptide to be expressed. Non-limiting examples of host cells suitable for expressing these peptides are: Escherichia coli, Saccharomyces cerevisiae, and mammalian BHK or CHO cell lines.

[0124] Those derivatives of the invention comprising non-natural amino acids and / or covalently linked N-terminal mono- or dipeptide mimetics can be produced, for example, as described in the experimental section. Alternatively, see, for example, Hodgson et al.: "The synthesis of peptides and proteins containing non-natural amino acids", Chemical Society Reviews, vol. 33, no. 7 (2004), p. 422-430.

[0125] Specific examples of methods for preparing various derivatives of the invention are included in the experimental section.

[0126] Pharmaceutical composition

[0127] The injectable compositions comprising the derivatives of the present invention can be prepared using conventional techniques of the pharmaceutical industry, which include dissolving and mixing the components appropriately to obtain the desired end product. Thus, according to one procedure, the derivatives of the present invention are dissolved in a suitable buffer at an appropriate pH to minimize or avoid precipitation. For example, the injectable compositions are made sterile by filtration sterilization.

[0128] The pharmaceutical composition comprising the derivative of the present invention or a pharmaceutically acceptable salt or amide thereof and a pharmaceutically acceptable excipient can be prepared as known in the art.

[0129] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. An excipient can be an inert substance, an inactive substance, and / or a non-pharmaceutically active substance.

[0130] The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995) and any subsequent editions).

[0131] The composition can be a stabilized formulation. The term "stabilized formulation" refers to a formulation with improved physical and / or chemical stability (preferably both). Typically, the formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration time is reached.

[0132] Treatment with the derivatives according to the invention can also be combined with one or more further pharmacologically active substances, for example selected from the group consisting of GLP-1 receptor agonists or GLP-1 / glucagon receptor co-agonists.

[0133] In one aspect of the invention, the derivative of the invention is combined with a GLP-1 receptor agonist. The compound can be provided in a single dose form, wherein the single dose form comprises two compounds, or in the form of a kit comprising a preparation of the derivative of the invention as a first unit dosage form and a preparation of the GLP-1 receptor agonist as a second unit dosage form.

[0134] Non-limiting examples of GLP-1 receptor agonists to be combined with the derivatives of the present invention are liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, tapoglutide and albiglutide. Semaglutide is a GLP-1 receptor agonist which can be prepared as described in Example 4 of WO2006 / 097537 and is also known as N 6,26 -{18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propionic acid), 3,4-L-arginine] human glucagon-like peptide 1 (7-37), see WHO Drug Information Vol. 24, No. 1, 2010 (SEQ ID NO: 57).

[0135] Non-limiting examples of GLP-1 / glucagon receptor co-agonists are described in WO 2014 / 170496, for example, see SEQ ID NO: 52, 53, 54, 55 or 56 therein.

[0136] Drug Indications

[0137] The present invention also relates to derivatives of GIP analogs for use as medicaments.

[0138] In particular embodiments, the derivatives of the invention are useful in the following medical treatments:

[0139] (i) prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity-onset diabetes of the young), gestational diabetes, and / or for the reduction of HbA1C;

[0140] (ii) delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delaying or preventing insulin resistance, and / or delaying the progression of insulin-not-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0141] (iii) preventing and / or treating eating disorders, such as obesity, for example by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence;

[0142] (iv) Weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced)—that is, preventing weight gain after successful weight loss.

[0143] (v) preventing and / or treating liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

[0144] In certain embodiments, the indication is type 2 diabetes and / or obesity.

[0145] In some embodiments, the present invention relates to methods of weight management. In some embodiments, the present invention relates to methods of reducing appetite. In some embodiments, the present invention relates to methods of reducing food intake.

[0146] Generally, all subjects suffering from obesity are also considered to be overweight. In some embodiments, the present invention relates to methods for treating or preventing obesity. In some embodiments, the present invention relates to the use of the derivatives of the present invention for treating or preventing obesity. In some embodiments, the subject suffering from obesity is a human, such as an adult or a pediatric patient (including infants, children and adolescents). Body mass index (BMI) is a measure of body fat based on height and weight. The calculation formula is BMI = weight in kilograms / height in meters 2 A human subject suffering from obesity may have a BMI of ≥30; the subject may also be referred to as obese. In some embodiments, a human subject suffering from obesity may have a BMI of ≥35 or a BMI in the range of ≥30 to <40. In some embodiments, the obesity is severe obesity or morbid obesity, wherein the human subject may have a BMI of ≥40.

[0147] In some embodiments, the present invention relates to a method for treating or preventing overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments, the present invention relates to the use of derivatives of GIP analogs, optionally in the presence of at least one weight-related comorbidity, for treating or preventing overweight. In some embodiments, the subject suffering from overweight is a human, such as an adult or a pediatric patient (including infants, children, and adolescents). In some embodiments, a human subject suffering from overweight may have a BMI of ≥25, such as a BMI of ≥27. In some embodiments, a human subject suffering from overweight has a BMI in the range of 25 to <30 or a BMI in the range of 27 to <30. In some embodiments, the weight-related comorbidity is selected from hypertension, diabetes (such as type 2 diabetes), dyslipidemia, high cholesterol, and obstructive sleep apnea.

[0148] In some embodiments, the present invention relates to methods of reducing weight. In some embodiments, the present invention relates to the use of derivatives of GIP analogs in reducing weight. A person experiencing weight loss according to the present invention may have a BMI of ≥25, such as a BMI of ≥27 or a BMI of ≥30. In some embodiments, a person experiencing weight loss according to the present invention may have a BMI of ≥35 or a BMI of ≥40. The term "weight loss" may include the treatment or prevention of obesity and / or overweight. Specific implementation plan

[0149] The present invention can be further described by the following non-limiting embodiments.

[0150] 1. A derivative of a GIP analog comprising formula I (SEQ ID NO: 3):

[0151] Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Lys-Trp-Leu-Leu-Ala-Gln-Lys-Gly (I),

[0152] wherein a modifying group is covalently attached to the side chain of the epsilon amino group of lysine at position 24, the modifying group being defined by ABC-, wherein A- is a lipophilic moiety having a negatively charged portion at the distal end, and BC- is a linker; and

[0153] wherein the GIP analog has a maximum of 8 amino acid substitutions compared to hGIP (1-31) (SEQ ID NO: 2),

[0154] or a pharmaceutically acceptable salt or amide thereof.

[0155] 2. The derivative according to embodiment 1, comprising formula II (SEQ ID NO: 48): X1-X2-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-X 14 -Asp-X 16 -Ile-X 18 -Gln-X 20 -Asp-Phe-Val-Lys-Trp-Leu-Leu-Ala-Gln-Lys-X 31 (II)

[0156] Wherein Formula II is at positions X1, X2, X 14 , X 16 , X 18 , X 20 and / or X 31 contains any amino acid; and

[0157] The GIP analog has a maximum of 8 amino acid substitutions compared to hGIP (1-31) (SEQ ID NO: 2).

[0158] 3. A derivative according to any one of embodiments 1-2, comprising formula II, wherein positions X1, X2, X 14 , X 16 , X 18 , X 20 and / or X 31 The amino acid at is selected from (SEQ ID NO: 49):

[0159] X1 is Tyr or D-Tyr;

[0160] X2 is Aib, Ala or D-Ala;

[0161] X 14 is Leu, Nle, Asp or Met;

[0162] X 16 is Lys or Ala;

[0163] X 18 is Arg or His;

[0164] X 20 is Gln, Glu or Aib;

[0165] X 31 It is Gly or Pro.

[0166] 4. A derivative according to any one of the preceding embodiments, comprising formula II, wherein positions X1, X2, X 14 , X16 , X 18 , X 20 and / or X 31 The amino acid at is selected from (SEQ ID NO: 64):

[0167] X1 is Tyr or D-Tyr;

[0168] X2 is Aib, Ala or D-Ala;

[0169] X 14 is Leu, Nle or Met;

[0170] X 16 is Lys or Ala;

[0171] X 18 is Arg or His;

[0172] X 20 is Gln, Glu or Aib;

[0173] X 31 It is Gly or Pro.

[0174] 5. A derivative according to any one of the preceding embodiments, comprising formula II, wherein positions X1, X2, X 14 , X 16 , X 18 , X 20 and / or X 31 The amino acid at is selected from (SEQ ID NO: 63):

[0175] X1 is Tyr or D-Tyr;

[0176] X2 is Aib or Ala;

[0177] X 14 is Nle, Asp or Leu;

[0178] X 16 is Lys or Ala;

[0179] X 18 is Arg or His;

[0180] X 20 is Gln, Glu or Aib;

[0181] X 31 It is Gly or Pro.

[0182] 6. A derivative according to any one of the preceding embodiments, comprising formula II, wherein positions X1, X2, X 14 , X 16 , X18 , X 20 and / or X 31 The amino acid at is selected from (SEQ ID NO: 50):

[0183] X1 is Tyr or D-Tyr;

[0184] X2 is Aib or Ala;

[0185] X 14 for Nle;

[0186] X 16 is Lys or Ala;

[0187] X 18 is Arg or His;

[0188] X 20 is Gln, Glu or Aib;

[0189] X 31 It is Gly or Pro.

[0190] 7. A derivative according to any one of the preceding embodiments, wherein the derivative is represented by formula II, wherein positions X1, X2, X 14 , X 16 , X 18 , X 20 and / or X 31 The amino acid at is selected from (SEQ ID NO: 65):

[0191] X1 is Tyr, Ac-Tyr, D-Tyr or Ac-D-Tyr;

[0192] X2 is Aib, Ala or D-Ala;

[0193] X 14 is Leu, Nle, Asp or Met;

[0194] X 16 is Lys or Ala;

[0195] X 18 is Arg or His;

[0196] X 20 is Gln, Glu or Aib;

[0197] X 31 Gly or Pro

[0198] or a pharmaceutically acceptable salt or amide thereof.

[0199] 8. The derivative according to any of the preceding embodiments, wherein the GIP analogue has a maximum of 7 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0200] 9. The derivative according to any of the preceding embodiments, wherein the GIP analogue has a maximum of 6 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0201] 10. The derivative according to any of the preceding embodiments, wherein said GIP analogue has a maximum of 5 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0202] 11. The derivative according to any of the preceding embodiments, wherein the GIP analogue has a maximum of 4 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0203] 12. The derivative according to any of the preceding embodiments, wherein said GIP analogue has a maximum of 3 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0204] 13. The derivative according to any of the preceding embodiments, wherein said GIP analogue has a maximum of 2 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0205] 14. The derivative according to any of the preceding embodiments, wherein the GIP analogue has at most 1 amino acid substitution compared to hGIP(1-31) (SEQ ID NO: 2).

[0206] 15. The derivative according to any one of embodiments 1-12, wherein the GIP analogue has 3 to 6 amino acid substitutions compared to hGIP(1-31) (SEQ ID NO: 2).

[0207] 16. The derivative according to any one of the preceding embodiments, wherein the modifying group is ABC-, wherein A- is a lipophilic moiety having a negatively charged moiety at the distal end, and BC- is a linker comprising at least one negatively charged moiety.

[0208] 17. The derivative according to any of the preceding embodiments, wherein the linker BC- comprises 1 to 6 negatively charged moieties.

[0209] 18. The derivative according to any of the preceding embodiments, wherein the linker BC- comprises 1 to 4 negatively charged moieties.

[0210] 19. The derivative according to any of the preceding embodiments, wherein said at least one negatively charged moiety in said linker BC- is selected from gamma-Glu, Glu and / or Asp.

[0211] 20. The derivative according to any of the preceding embodiments, wherein said at least one negatively charged moiety in said linker BC- is gamma-Glu.

[0212] 21. A derivative according to any one of the preceding embodiments, wherein

[0213] A- is the chemical formula 1

[0214] Chemical formula 1:

[0215] p is an integer in the range of 14-20;

[0216] Wherein * indicates the position of the amide bond connecting A- and B-.

[0217] 22. A derivative according to any one of the preceding embodiments, wherein

[0218] B- is the chemical formula 2

[0219] Chemical formula 2:

[0220] q is an integer in the range of 0-1;

[0221] r is an integer in the range of 1-3;

[0222] Wherein * indicates the position of the amide bond connecting A- and B-, and ** indicates the position of the amide bond connecting B- and C-.

[0223] 23. A derivative according to any one of the preceding embodiments, wherein

[0224] C- is selected from Chemical Formula 3 and Chemical Formula 4

[0225] Chemical formula 3:

[0226] Chemical formula 4:

[0227] s is an integer in the range of 1-3;

[0228] t is an integer in the range of 1-4;

[0229] u is an integer in the range of 1-3;

[0230] wherein ** indicates the position of the amide bond connecting B- and C-, and *** indicates the position of the amide bond connecting C- and the epsilon amino group of lysine at position 24.

[0231] 24. A derivative according to any one of the preceding embodiments, wherein the modifying group is defined by ABC-, wherein

[0232] A- is the chemical formula 1

[0233] Chemical formula 1:

[0234] p is an integer in the range of 14-20;

[0235] Where * indicates the position of the amide bond connecting A- and B-;

[0236] B- is the chemical formula 2

[0237] Chemical formula 2:

[0238] q is an integer in the range of 0-1;

[0239] r is an integer in the range of 1-3;

[0240] Where * indicates the position of the amide bond connecting A- and B-, and ** indicates the position of the amide bond connecting B- and C-

[0241] The position of the amide bond;

[0242] C- is selected from Chemical Formula 3 and Chemical Formula 4

[0243] Chemical formula 3:

[0244] Chemical formula 4:

[0245] s is an integer in the range of 1-3;

[0246] t is an integer in the range of 1-4;

[0247] u is an integer in the range of 1-3;

[0248] Where ** indicates the position of the amide bond connecting B- and C-, and *** indicates the position of the amide bond connecting C- and 24

[0249] The position of the amide bond to the epsilon amino group of lysine.

[0250] 25. The derivative according to any one of embodiments 21 or 24, wherein A- is Formula 1 and p is 16-18.

[0251] 26. The derivative according to any one of embodiments 22 or 24, wherein B- is Formula 2, q is 0-1, and r is 2.

[0252] 27. The derivative according to any one of embodiments 23-24, wherein C- is of Formula 3.

[0253] 28. The derivative according to embodiment 27, wherein C- is of formula 3 and s is 2.

[0254] 29. The derivative according to any one of embodiments 27-28, wherein C- is of formula 3 and t is 2-3.

[0255] 30. The derivative according to any one of embodiments 23-24, wherein C- is of formula 4.

[0256] 31. The derivative according to embodiment 30, wherein C- is of formula 4 and u is 2.

[0257] 32. The derivative according to any one of embodiments 1 to 24, wherein the modifying group ABC- is selected from:

[0258] Chemical formula 5:

[0259]

[0260] Chemical formula 6:

[0261]

[0262] Chemical formula 7:

[0263]

[0264] Chemical formula 8:

[0265]

[0266] Chemical formula 9:

[0267]

[0268] Chemical formula 10:

[0269]

[0270] Chemical formula 11:

[0271]

[0272] Chemical formula 12:

[0273]

[0274] Chemical formula 13:

[0275]

[0276] Chemical formula 14:

[0277]

[0278] Chemical formula 15:

[0279]

[0280] Chemical formula 16:

[0281]

[0282] Chemical formula 17:

[0283] And chemical formula 18:

[0284]

[0285] 33. The derivative according to any one of embodiments 2-32, wherein X1 is Tyr.

[0286] 34. The derivative according to any one of embodiments 2-32, wherein X1 is D-Tyr.

[0287] 35. The derivative according to any one of embodiments 2-34, wherein X2 is Aib.

[0288] 36. The derivative according to any one of embodiments 2-34, wherein X2 is Ala.

[0289] 37. The derivative according to any one of embodiments 2-34, wherein X2 is D-Ala.

[0290] 38. A derivative according to any one of embodiments 2-37, wherein X 14 For Leu.

[0291] 39. A derivative according to any one of embodiments 2-37, wherein X 14 For Nle.

[0292] 40. A derivative according to any one of embodiments 2-37, wherein X 14 For Asp.

[0293] 41. A derivative according to any one of embodiments 2-37, wherein X 14 For Met.

[0294] 42. A derivative according to any one of embodiments 2-41, wherein X 16 For Lys.

[0295] 43. A derivative according to any one of embodiments 2-41, wherein X 16 For Ala.

[0296] 44. A derivative according to any one of embodiments 2-43, wherein X 18 is Arg.

[0297] 45. A derivative according to any one of embodiments 2-43, wherein X 18 For His.

[0298] 46. ​​A derivative according to any one of embodiments 2-45, wherein X 20 For Gln.

[0299] 47. A derivative according to any one of embodiments 2-45, wherein X 20 Glu.

[0300] 48. A derivative according to any one of embodiments 2-45, wherein X 20 For Aib.

[0301] 49. A derivative according to any one of embodiments 2-48, wherein X 31 For Gly.

[0302] 50. A derivative according to any one of embodiments 2-48, wherein X 31 For Pro.

[0303] 51. A derivative according to any one of the preceding embodiments, wherein the peptide defined by Formula III (SEQ ID NO: 51) is linked to the C-terminus of Formula I or Formula II via an amide bond from the C-terminal carboxylic acid group of Formula I or Formula II to the N-terminal amino group of Formula III:

[0304] Lys-X 33 -X 34 -Asp-Trp-Lys-His-Asn-Ile-Thr-Gln (III),

[0305] in

[0306] X 33 Lys, Glu;

[0307] X 34 It is Asn, Glu or Asp.

[0308] 52. The derivative according to embodiment 51, wherein X 33 For Lys.

[0309] 53. The derivative according to embodiment 51, wherein X 33 Glu.

[0310] 54. A derivative according to any one of embodiments 51-53, wherein X 34 Asn.

[0311] 55. A derivative according to any one of embodiments 51-53, wherein X 34 Glu.

[0312] 56. A derivative according to any one of embodiments 51-53, wherein X 34 For Asp.

[0313] 57. The derivative according to any one of the preceding embodiments, wherein the N-terminal amino acid is acylated.

[0314] 58. The derivative according to any one of the preceding embodiments, wherein the N-terminal amino acid is acetylated.

[0315] 59. The derivative according to any one of embodiments 2-33, 35-58, wherein X1 is Ac-Tyr.

[0316] 60. The derivative according to any one of embodiments 2-32, 34-58, wherein X1 is Ac-D-Tyr.

[0317] 61. The derivative according to any one of the preceding embodiments, wherein the GIP analog is a C-terminal carboxylic acid or a C-terminal amide.

[0318] 62. The derivative according to any one of the preceding embodiments, wherein the GIP analog is a C-terminal carboxylic acid.

[0319] 63. A derivative according to any one of embodiments 2-49, 57-62, wherein X 31 It is Gly-OH.

[0320] 64. A derivative according to any one of embodiments 2-48, 50, 57-62, wherein X 31 It is Pro-OH.

[0321] 65. The derivative according to any one of embodiments 1-61, wherein the GIP analog is a C-terminal amide.

[0322] 66. A derivative according to any one of embodiments 2-49, 57-61, 65, wherein X 31 It is Gly-NH2.

[0323] 67. A derivative according to any one of embodiments 2-48, 50, 57-61, 65, wherein X 31 It is Pro-NH2.

[0324] 68. A derivative according to any one of the preceding embodiments, selected from: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 1, SEQ ID NO:6)

[0325]

[0326] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24]-hGIP(1-31) (Compound 2; SEQ ID NO: 7)

[0327]

[0328] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 3; SEQ ID NO: 8)

[0329]

[0330] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 4; SEQ ID NO: 9)

[0331]

[0332] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 5; SEQ ID NO: 10)

[0333]

[0334] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 6; SEQ ID NO: 11)

[0335]

[0336] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 7; SEQ ID NO: 12)

[0337]

[0338] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]-butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 8; SEQ ID NO: 13)

[0339]

[0340] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31) (Compound 9; SEQ ID NO: 14)

[0341]

[0342] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31) (Compound 10; SEQ ID NO: 15)

[0343]

[0344] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31) (Compound 11; SEQ ID NO: 16)

[0345]

[0346] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31) (Compound 12; SEQ ID NO: 17)

[0347]

[0348] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Ala16,Arg18,Lys24]-hGIP(1-31) (Compound 13; SEQ ID NO: 18)

[0349]

[0350] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Ala16,Arg18,Lys24]-hGIP(1-31) (Compound 14; SEQ ID NO: 19)

[0351]

[0352] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 15; SEQ ID NO: 20)

[0353]

[0354] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31) (Compound 16; SEQ ID NO: 21)

[0355]

[0356] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]-butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 17; SEQ ID NO: 22)

[0357]

[0358] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Nle14,Arg18,Lys24]-hGIP(1-31) (Compound 18; SEQ ID NO: 23)

[0359]

[0360] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1, Asp14, Arg18, Glu20, Lys24]-hGIP (1-31) (Compound 19; SEQ ID NO: 24)

[0361]

[0362] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-amino]butyryl]-[Aib2,Nle14,Glu20,Lys24]-hGIP(1-31) (Compound 20; SEQ ID NO: 25)

[0363]

[0364] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]-amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Glu20,Lys24]-hGIP(1-31) (Compound 21; SEQ ID NO: 26)

[0365]

[0366] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Glu20,Lys24]-hGIP(1-31) (Compound 22; SEQ ID NO: 27)

[0367]

[0368] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Glu20,Lys24]-hGIP (1-31) (Compound 23; SEQ ID NO: 28)

[0369]

[0370] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Aib20,Lys24]-hGIP(1-31) (Compound 24; SEQ ID NO: 29)

[0371]

[0372] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP (1-31) (Compound 25; SEQ ID NO: 30)

[0373]

[0374] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide (Compound 26; SEQ ID NO: 31)

[0375]

[0376] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide (Compound 27; SEQ ID NO: 32)

[0377]

[0378] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide (Compound 28; SEQ ID NO: 33)

[0379]

[0380] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Lys24,Pro31]-hGIP(1-31)amide (Compound 29; SEQ ID NO: 34)

[0381]

[0382] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP (1-31) (Compound 30; SEQ ID NO: 35)

[0383]

[0384] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide (Compound 31; SEQ ID NO: 36)

[0385]

[0386] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide (Compound 32; SEQ ID NO: 37)

[0387]

[0388] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33]-hGIP (Compound 33; SEQ ID NO: 38)

[0389]

[0390] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33]-hGIP (Compound 34; SEQ ID NO: 39)

[0391]

[0392] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Lys24,Glu33,Glu34]-hGIP (Compound 35; SEQ ID NO: 40)

[0393]

[0394] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Lys24,Glu33,Asp34]-hGIP (Compound 36, SEQ ID NO:41)

[0395]

[0396] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP (Compound 37; SEQ ID NO: 42)

[0397]

[0398] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33,Glu34]-hGIP (Compound 38; SEQ ID NO: 43)

[0399]

[0400] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33,Asp34]-hGIP (Compound 39; SEQ ID NO: 44)

[0401]

[0402] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP (Compound 40; SEQ ID NO: 45)

[0403]

[0404] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoyl-amino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP (Compound 41; SEQ ID NO: 46)

[0405]

[0406] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP (Compound 42; SEQ ID NO: 47)

[0407]

[0408] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP (1-31) (Compound 43; SEQ ID NO: 59)

[0409]

[0410] N{1}-acetyl, N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide (Compound 44; SEQ ID NO: 60)

[0411]

[0412] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu14,Lys24]-hGIP (Compound 45; SEQ ID NO: 61)

[0413]

[0414] N{1}-acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide (Compound 46; SEQ ID NO: 62)

[0415]

[0416] 69. The derivative according to embodiment 68, which is selected from compound numbers 1-32, 43-44, 46.

[0417] 70. The derivative according to any one of embodiments 68-69, which is selected from compound numbers 1-25, 30, 43.

[0418] 71. The derivative according to any one of embodiments 68-70, which is compound No. 5.

[0419] 72. The derivative according to any one of embodiments 68-70, which is compound No. 25.

[0420] 73. The derivative according to any one of embodiments 68-69, which is selected from compound numbers 26-29, 31-32, 44, 46.

[0421] 74. The derivative according to any one of embodiments 68-69 and 73, which is compound No. 31.

[0422] 75. The derivative according to embodiment 68, which is selected from compound numbers 33-42, 45.

[0423] 76. The derivative according to any one of the preceding embodiments, which is an agonist of the human GIP receptor.

[0424] 77. The derivative according to any one of the preceding embodiments, which is capable of activating the human GIP receptor.

[0425] 78. A derivative according to any of the preceding embodiments, which is capable of activating the human GIP receptor in an assay using whole cells expressing the human GIP receptor.

[0426] 79. A derivative according to any one of the preceding embodiments, which is capable of activating the human GIP receptor in a CRE luciferase assay as described herein in Example 2.

[0427] 80. A derivative according to any one of the preceding embodiments, which is an agonist of the human GIP receptor, as determined in Example 2 described herein, EC 50 Not more than 900pM.

[0428] 81. A derivative according to any one of the preceding embodiments, which is an agonist of the human GIP receptor, as determined in Example 2 described herein, EC 50 Not more than 500pM.

[0429] 82. A derivative according to any one of the preceding embodiments, which is an agonist of the human GIP receptor, as determined in Example 2 described herein, EC 50 Not more than 200pM.

[0430] 83. The derivative according to any of the preceding embodiments, which is selective for the human GIP receptor over the human GLP-1 receptor and the human glucagon receptor.

[0431] 84. A derivative according to any one of the preceding embodiments, which has an EC of 1.004 for human GIP receptor. 50 Lower than the EC at human GLP-1 receptor and human glucagon receptor 50 .

[0432] 85. A derivative according to any one of the preceding embodiments, which has an EC of 1.0 for the human GLP-1 receptor in the CRE luciferase assay. 50 Greater than 100000 pM as determined in Example 2 described herein.

[0433] 86. A derivative according to any one of the preceding embodiments, which has an EC of 1.0 for the human GLP-1 receptor in the CRE luciferase assay. 50 Greater than 10000 pM as determined in Example 2 described herein.

[0434] 87. A derivative according to any one of the preceding embodiments, which has an EC of 1.1 for the human glucagon receptor in a CRE luciferase assay as described in Example 2 herein. 50 Greater than 100000pM.

[0435] 88. The derivative according to any of the preceding embodiments, which has improved pharmacokinetic properties.

[0436] 89. The derivative according to any of the preceding embodiments, which has a prolonged half-life.

[0437] 90. The derivative according to any of the preceding embodiments, which has a prolonged half-life when assayed in minipigs.

[0438] 91. The derivative according to any of the preceding embodiments, which has improved physical stability.

[0439] 92. The derivative according to any one of the preceding embodiments, having a recovery of greater than 95% in a ThT fibrillogenesis assay as determined in Example 4 described herein.

[0440] 93. The derivative according to any of the preceding embodiments, having a lag time of more than 45 hours in a ThT fibrillogenesis assay as determined in Example 4 described herein.

[0441] 94. The derivative according to any one of the preceding embodiments, which has a low DLS-SI value.

[0442] 95. The derivative according to any of the preceding embodiments, having a DLS-SI value of less than 7, preferably less than 2, as determined in Example 4 described herein.

[0443] 96. The derivative according to any one of the preceding embodiments, which has no precipitate in the DLS-SI assay as determined in Example 4 described herein.

[0444] 97. The derivative according to any one of the preceding embodiments, which has improved chemical stability.

[0445] 98. The derivative according to any of the preceding embodiments, having a monthly HMWP formation of no more than 2% as determined in Example 5 described herein.

[0446] 99. The derivative according to any one of the preceding embodiments, having a purity loss of no more than 35% per month as determined in Example 5 as described herein.

[0447] 100. The derivative according to any one of the preceding embodiments, having a purity loss of no more than 6% per month as determined in Example 5 described herein.

[0448] 101. The derivative according to any of the preceding embodiments, which has the in vivo effect of reducing food intake as determined in a subchronic study in DIO mice as described in Example 6 herein.

[0449] 102. The derivative according to any one of the preceding embodiments, which has the in vivo effect of inducing weight loss as determined in a subchronic study in DIO mice as described in Example 6 herein.

[0450] 103. The derivative according to any one of the preceding embodiments, which has the effect of improving glucose tolerance in vivo as determined in a subchronic study in DIO mice as described in Example 6 herein.

[0451] 104. A pharmaceutical composition comprising a derivative according to any one of the preceding embodiments and at least one pharmaceutically acceptable excipient.

[0452] 105. A pharmaceutical composition comprising a derivative according to any one of embodiments 1-103, a GLP-1 receptor agonist and at least one pharmaceutically acceptable excipient.

[0453] 106. A pharmaceutical composition comprising a derivative according to any one of embodiments 1-103, a GLP-1 / glucagon receptor co-agonist and at least one pharmaceutically acceptable excipient.

[0454] 107. The pharmaceutical composition according to embodiment 105, wherein the GLP-1 receptor agonist is liraglutide.

[0455] 108. A pharmaceutical composition according to embodiment 105, wherein the GLP-1 receptor agonist is semaglutide (SEQ ID NO: 57).

[0456] 109. The pharmaceutical composition of embodiment 106, wherein the GLP-1 / glucagon receptor co-agonist is N{Epsilon-28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butyryl]amino]butyryl]-[Acb2,Leu10,Leu16,Arg20,Leu27,Lys28]-glucagon amide (SEQ ID NO: 52):

[0457]

[0458] 110. The pharmaceutical composition of embodiment 106, wherein the GLP-1 / glucagon receptor co-agonist is N{Epsilon-28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(2S)-2-[[(2S)-4-carboxy-2-[[(2S)-2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]-3-hydroxypropanoyl]amino]butyryl]amino]-3-hydroxypropanoyl]amino]butyryl]amino]butyryl]-[Aib2,Leu10,Glu15,Lys17,Arg20,Glu21,Leu27,Lys28]-glucagon amide (SEQ ID NO: 53):

[0459]

[0460] 111. The pharmaceutical composition according to embodiment 106, wherein the GLP-1 / glucagon receptor co-agonist is N{Epsilon-28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butyryl]amino]butyryl]-[Acb2,Leu10,Glu15,Arg20,Glu21,Leu27,Lys28]-glucagon amide (SEQ ID NO: 54):

[0461]

[0462] 112. The pharmaceutical composition of embodiment 106, wherein the GLP-1 / glucagon receptor co-agonist is N{Epsilon-28}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butyryl]amino]butyryl]-[Acb2,Leu10,Leu16,Lys17,Arg20,Glu21,Leu27,Lys28]-glucagon amide (SEQ ID NO: 55):

[0463]

[0464] 113. A pharmaceutical composition according to embodiment 106, wherein the GLP-1 / glucagon receptor co-agonist is N{Epsilon-28}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu10,Ala16,Arg20,Leu27,Lys28]-glucagon amide (SEQ ID NO: 56):

[0465]

[0466] 114. A pharmaceutical composition according to any one of embodiments 104-113, wherein the derivative is SEQ ID NO:10.

[0467] 115. A pharmaceutical composition according to any one of embodiments 104-113, wherein the derivative is SEQ ID NO: 30.

[0468] 116. A pharmaceutical composition according to any one of embodiments 104-113, wherein the derivative is SEQ ID NO:36.

[0469] 117. The pharmaceutical composition according to any one of embodiments 104-116 for use as a medicament.

[0470] 118. The derivative according to any of embodiments 1-103 for use as a medicament.

[0471] 119. The derivative according to any one of embodiments 1-103 for use in the treatment of:

[0472] (i) prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity-onset diabetes of the young), gestational diabetes, and / or for the reduction of HbA1C;

[0473] (ii) delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delaying or preventing insulin resistance, and / or delaying the progression of insulin-not-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0474] (iii) preventing and / or treating eating disorders, such as obesity, for example by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence;

[0475] (iv) weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced)—that is, preventing weight gain after successful weight loss;

[0476] (v) preventing and / or treating liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

[0477] 120. The derivative according to any of embodiments 1-103, for use in the prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and / or for reducing HbA1C.

[0478] 121. A derivative according to any one of embodiments 1-103, for use in delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delaying or preventing insulin resistance, and / or delaying the progression of insulin-free type 2 diabetes to insulin-requiring type 2 diabetes.

[0479] 122. A derivative according to any one of embodiments 1-103, for use in the prevention and / or treatment of eating disorders such as obesity, e.g. by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence.

[0480] 123. A derivative according to any of embodiments 1-103, for use in weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced) - ie preventing weight gain after successful weight loss.

[0481] 124. The derivative according to any one of embodiments 1-103, for use in the prevention and / or treatment of a liver disorder, such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

[0482] 125. The derivative according to any of embodiments 1-103 for use in the treatment and / or prevention of weight management, obesity and obesity-related disorders.

[0483] 126. The derivative according to any of embodiments 1-103, for use in the treatment and / or prevention of all forms of diabetes, such as type 2 diabetes, and conditions associated with diabetes.

[0484] 127. Use of a derivative according to any one of embodiments 1 to 103 in the preparation of a medicament for use

[0485] (i) prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity-onset diabetes of the young), gestational diabetes, and / or for the reduction of HbA1C;

[0486] (ii) delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to insulin-requiring type 2 diabetes, delaying or preventing insulin resistance, and / or delaying the progression of insulin-not-requiring type 2 diabetes to insulin-requiring type 2 diabetes;

[0487] (iii) preventing and / or treating eating disorders, such as obesity, for example by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence;

[0488] (iv) weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced)—that is, preventing weight gain after successful weight loss;

[0489] (v) preventing and / or treating liver disorders such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

[0490] 128. Use of a derivative according to any one of embodiments 1 to 103 for the preparation of a medicament for the prevention and / or treatment of all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity onset diabetes of the young), gestational diabetes, and / or for reducing HbA1C.

[0491] 129. Use of a derivative according to any one of embodiments 1-103 in the preparation of a medicament for delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and / or delaying the progression of type 2 diabetes not requiring insulin to type 2 diabetes requiring insulin.

[0492] 130. Use of a derivative according to any one of embodiments 1-103 for the preparation of a medicament for the prevention and / or treatment of eating disorders such as obesity, e.g. by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence.

[0493] 131. Use of a derivative according to any one of embodiments 1-103 for the preparation of a medicament for weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced) - ie preventing weight gain after successful weight loss.

[0494] 132. Use of a derivative according to any one of embodiments 1-103 for the preparation of a medicament for the prevention and / or treatment of liver disorders, such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver.

[0495] 133. Use of a derivative according to any of embodiments 1-103 for the preparation of a medicament for the treatment and / or prevention of weight management, obesity and obesity-related disorders.

[0496] 134. Use of a derivative according to any of embodiments 1-103 for the preparation of a medicament for the treatment and / or prevention of all forms of diabetes, such as type 2 diabetes, and conditions associated with diabetes.

[0497] 135. A method for preventing and / or treating all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin-dependent diabetes mellitus, MODY (maturity-onset diabetes of the young), gestational diabetes, and / or for reducing HbA1C, the method comprising administering a pharmaceutically active amount of a derivative according to any one of embodiments 1 to 103.

[0498] 136. A method for delaying or preventing the progression of diabetes, such as the progression of type 2 diabetes, delaying the progression of impaired glucose tolerance (IGT) to type 2 diabetes requiring insulin, delaying or preventing insulin resistance, and / or delaying the progression of type 2 diabetes not requiring insulin to type 2 diabetes requiring insulin, the method comprising administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0499] 137. A method for preventing and / or treating eating disorders such as obesity, e.g. by reducing food intake, reducing body weight, suppressing appetite, inducing satiety; treating or preventing binge eating disorder, bulimia nervosa and / or obesity induced by antipsychotic or steroid administration; reducing gastric motility; delaying gastric emptying; increasing physical activity; and / or preventing and / or treating comorbidities of obesity, such as osteoarthritis and / or urinary incontinence, the method comprising administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0500] 138. Weight maintenance after successful weight loss (whether drug-induced or diet and exercise-induced) - i.e. a method for preventing weight gain after successful weight loss, the method comprising administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0501] 139. A method for preventing and / or treating a liver disorder, such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation or fatty liver, the method comprising administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0502] 140. Method for the treatment and / or prevention of weight management, obesity and obesity-related disorders, which method comprises administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0503] 141. Method for the treatment and / or prevention of all forms of diabetes, such as type 2 diabetes, and conditions associated with diabetes, which method comprises administering a pharmaceutically active amount of a derivative according to any one of embodiments 1-103.

[0504] Example

[0505] This experimental section begins with a list of abbreviations, followed by a section containing general methods for synthesizing and characterizing analogs and derivatives of the present invention. This is followed by multiple examples relating to the preparation of specific derivatives of the present invention, and finally includes multiple examples relating to the activities and properties of these analogs and derivatives (a section entitled Pharmacological Methods).

[0506] The examples serve to illustrate the invention.

[0507] Materials and Methods

[0508] List of abbreviations

[0509] The following abbreviations are used below in alphabetical order:

[0510] Ac:Acetyl

[0511] Ado: 8-amino-3,6-dioxaoctanoic acid

[0512] Aib: alpha-aminoisobutyric acid

[0513] AUC: Area Under the Curve

[0514] BHK: Baby Hamster Kidney

[0515] Boc: tert-butyloxycarbonyl

[0516] BW: body weight

[0517] DCC: dicyclohexylcarbodiimide

[0518] DCM: dichloromethane

[0519] DIC: Diisopropylcarbodiimide

[0520] DIO: Diet-Induced Obesity

[0521] DIPEA: N,N--diisopropylethylamine or Hünig's base

[0522] DLS-SI: Dynamic Light Scattering Stability Index

[0523] DMEM: Dulbecco's modified Eagle's medium

[0524] DMF: dimethylformamide

[0525] DODT: 3,6-dioxa-1,8-octanedithiol

[0526] EDTA: Ethylenediaminetetraacetic acid

[0527] ELISA: Enzyme-linked immunosorbent assay

[0528] FBS: Fetal bovine serum

[0529] Fmoc: 9-fluorenylmethoxycarbonyl

[0530] GIP: Glucose-dependent insulinotropic polypeptide

[0531] GLP-1: Glucagon-like peptide 1

[0532] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol

[0533] HMWP: High Molecular Weight Protein

[0534] HOBt: 1-Hydroxybenzotriazole

[0535] HPLC: High Performance Liquid Chromatography

[0536] HSA: Human serum albumin

[0537] ip: intraperitoneal

[0538] IPGTT: intraperitoneal glucose tolerance testiv: intravenous

[0539] kcal: kilocalories

[0540] kg: kilogram

[0541] LCMS: Liquid chromatography mass spectrometry

[0542] MeCN: Acetonitrile

[0543] mM: millimole

[0544] Mtt: 4-methyltrityl

[0545] Nle: Norleucine

[0546] NMP: N-methylpyrrolidone

[0547] Oxyma Ethyl cyano-hydroxyimino-acetate

[0548] PBS: Phosphate buffered saline

[0549] PK: Pharmacokinetics

[0550] pM: picomolar

[0551] QTof: quantitative time of flight

[0552] R h : Stokes radius

[0553] sc: subcutaneous

[0554] SD: Standard deviation

[0555] SEC-HPLC: Size Exclusion High Performance Liquid Chromatography

[0556] SEC-MS: Size Exclusion Chromatography Mass Spectrometry

[0557] SEM: Standard error of the mean

[0558] tBu: tert-butyl

[0559] TFA: trifluoroacetic acid

[0560] ThT: Thioflavin T

[0561] TIS: Triisopropylsilane

[0562] Trt: triphenylmethyl (trityl)

[0563] Trx: tranexamic acid

[0564] UPLC: Ultra-Performance Liquid Chromatography

[0565] General preparation method

[0566] This part involves a solid phase peptide synthesis method (SPPS method, including amino acid deprotection method, method for cleaving peptides from resin, and purification method thereof), and a method for detecting and characterizing the obtained peptides (LCMS method).

[0567] The resin used to prepare the C-terminal peptide amide is PAL Amide AM resin (e.g., loading 0.6 mmol / g) or H-RinkAmide-ChemMatrix resin (e.g., loading 0.5 mmol / g) or Rink Amide Am polystyrene resin (e.g., loading 0.3-0.7 mmol / g). The resin used to prepare the C-terminal peptide glycyl-acid is Fmoc-Gly-Wang polystyrene resin (e.g., loading 0.3-0.7 mmol / g).

[0568] Unless otherwise specified, the Fmoc-protected amino acid derivatives used were recommended standards: for example, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-O-H, provided by AAPPTEC, Anaspec, Bachem, ChemImpex, Iris Biotech, Midwest Biotech, GyrosProtein Technologies, or Novabiochem. H, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH (in the case of methods SPPS_A and SPPS_D) or Fmoc-Trp-OH (in the case of methods SPPS_B and SPPS_C), Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH, Fmoc-Nle-OH, Fmoc-D-Tyr-(tBu)-OH, etc. Unless otherwise specified, the natural L-form of amino acids was used. When the N-terminal amino acid is not acetylated, the N-terminal amino acid is Boc-protected on the α-amino group by using a reagent pre-installed with a Boc group (e.g., Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus) or by exchanging the N-terminal Fmoc protecting group with a Boc protecting group after installation of the amino acid at the N-terminus of the peptide.

[0569] In the case of modular albumin binding moiety attachment using SPPS, appropriately protected building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Fmoc-tranexamic acid (Fmoc-Trx-OH), Fmoc-Glu-OtBu, octadecane dioic acid mono-tert-butyl ester, nonadecanedioic acid mono-tert-butyl ester, eicosanedioic acid mono-tert-butyl ester, tetradecanedioic acid mono-tert-butyl ester or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were used. All operations described below were performed in the synthesis scale range of 100-450 μmol.

[0570] Synthesis of resin-bound protected backbone

[0571] Method: SPPS_A

[0572] SPPS was performed on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ85714U.S.A.) using Fmoc-based chemistry. Fmoc deprotection was achieved with 20% piperidine in DMF. Peptide coupling. The solution (0.3M / 0.3M in DMF, 4-8 times molar excess) was added to the resin, followed by the addition of equal molar equivalents of DIC (1.5M in DMF) and collidine (1.5M in DMF). The stepwise assembly was accomplished using the following steps: 1) pre-swelling the resin with DCM and DMF; 2) using a solution containing 0.1M Oxyma 20% piperidine in DMF for Fmoc-deprotection, two treatments of 10 min each; 3) washing with DMF to remove piperidine; 4) removing the piperidine by adding 4-8 equivalents of Fmoc-amino acid as 0.3 M Oxyma 5) Wash with DMF to remove excess reagents; 6) Perform a final wash with DCM when the assembly is complete. For peptides carrying N-terminal acetylation, the Fmoc-deprotected peptidyl resin is treated with 1 M acetic anhydride in DMF for 30-60 min, followed by washing with DMF and DCM.

[0573] Method: SPPS_B

[0574] Protected peptidyl resins were synthesized on an Applied Biosystems 431A solid phase peptide synthesizer according to the Fmoc strategy using a general Fmoc protocol provided by the manufacturer. Mixing was performed by vortexing and occasional bubbling with nitrogen. The stepwise assembly was completed using the following steps: 1) Fmoc-deprotection was performed by treating with 20% piperidine in NMP for 3 min, followed by a 15 min treatment; 2) washing with NMP to remove piperidine; 3) coupling of Fmoc-amino acids using 5-10 equivalents of Fmoc-amino acids, DIC, and HOBt in NMP for 45-90 min; 4) washing with NMP to remove excess reagents; 5) final washing with DCM when the assembly was complete. The standard protected amino acid derivatives listed above are provided in pre-weighed cartridges (e.g., from Midwest Biotech), while non-standard derivatives are weighed manually. Some amino acids, such as but not limited to sterically hindered amino acids (e.g., Aib), were "double coupled" to ensure the reaction was complete, which means that after the first coupling (e.g., 45 min), the resin was drained, more reagents (Fmoc-amino acid, DIC, HOBt) were added, and the mixture was reacted again (e.g., 45 min). For peptides carrying N-terminal acetylation, the Fmoc-deprotected peptidyl resin was removed from the synthesizer and manually treated with 10% (v / v) acetic anhydride / 10% (v / v) pyridine in DMF for 30-60 min, followed by washing with DMF and DCM.

[0575] Method: SPPS_C

[0576] The protected peptidyl resin was synthesized according to the Fmoc strategy on a Protein Technologies SymphonyX solid phase peptide synthesizer using the manufacturer's protocol with minor modifications. The stepwise assembly was accomplished on a 0.2 mmol basis using the following steps: 1) pre-swelling of the resin in DMF (3 x 8 mL, 15 min each); 2) Fmoc-deprotection by using 20% ​​(v / v) piperidine in DMF (2 x 8 mL, 10 min each); 3) washing with DMF to remove piperidine (5 x 6 mL); 4) addition of Fmoc-amino acid (12 equiv., 2.4 mmol) and Oxyma (12 eq., 2.4 mmol), followed by DIC (12 eq., 2.4 mmol) as a 1.2 M solution in DMF (2 mL), followed by mixing for 0.5-4 h for coupling of Fmoc-amino acids; 4) washing with DMF to remove excess reagent (3×6 mL); 5) final washing with DCM upon completion of assembly. Some amino acids, such as but not limited to sterically hindered amino acids (e.g., Aib) are coupled for extended reaction times (e.g., 4 h) to ensure completion of the reaction. For peptides carrying N-terminal acetylation, or peptides that require installation of an N-terminal protecting group (e.g., exchanging Fmoc for a Boc protecting group) prior to side chain assembly, the N-terminal Fmoc group is removed by treatment with 20% (v / v) piperidine in DMF as described in step 2 above. The peptidyl resin was then removed from the synthesizer and manually treated with 10% (v / v) acetic anhydride / 10% (v / v) DIPEA in DMF for 30-60 min, followed by washing with DMF and DCM.

[0577] Method: SPPS_D

[0578] The protected peptidyl resin was synthesized according to the Fmoc strategy on a Prelude solid phase peptide synthesizer (Protein Technologies, Tucson, USA) using the machine protocol provided by the manufacturer. (Merck, Novabiochem, Switzerland) mediated coupling was used for coupling. As described above, the coupling of Fmoc-amino acid was performed using a 4-8 fold excess of amino acid (4-8 equivalents) relative to the resin substitution. The coupling time was 1-6h. Fmoc-Arg(pbf)-OH was coupled using a double coupling procedure (1h+1h). The step-by-step solid phase assembly was completed on Prelude using the following steps: 1) deprotection (removal of Fmoc) by using 25% piperidine in NMP for 2x4min, step 2) washing with NMP and DCM (removal of piperidine), step 3) 4-8 equivalents excess of Fmoc-amino acid (0.3M Oxyma The coupling was carried out for 1-4 h by adding 1 / 10 volume of 3M DCC in NMP and 1 / 10 volume of collidine in NMP. Mixing was performed by bubbling with nitrogen from time to time, and step 4) washing (using NMP and DCM to remove excess amino acids and reagents). The last step includes washing with DCM, which prepares the resin for attachment of modification groups on lysine side chains.

[0579] Attachment of side chains to the resin-bound protected peptide backbone

[0580] Method: SC_A

[0581] The N-ε-lysine Mtt protecting group was removed by washing the resin with HFIP / TIS / DCM (75:2.5:22.5, v / v / v) (1 x 5 min and 2 x 20 min) followed by washing with piperidine, DMF and DCM.

[0582] Acylation was performed on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 USA) as described in Method SPPS_A, using stepwise addition of building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Fmoc-Glu-OtBu, Fmoc-tranexamic acid (Fmoc-Trx-OH). Introduction of the fatty acid moiety was achieved using Method SPPS_A and appropriate building blocks such as, but not limited to, octadecanedioic acid mono-tert-butyl ester or eicosanedioic acid mono-tert-butyl ester.

[0583] Method: SC_B

[0584] The N-ε-lysine protecting Mtt protecting group was removed by washing the resin twice with 30% HFIP in DCM for 45 min each, followed by washing with DCM and DMF.

[0585] Acylation was performed on an Applied Biosystems 431A solid phase peptide synthesizer using the protocol described in Method SPPS_B with stepwise addition of building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid, Fmoc-tranexamic acid, Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, eicosanedioic acid mono-tert-butyl ester.

[0586] Method: SC_C

[0587] The N-ε-lysine Mtt protecting group was removed by washing the resin twice with 30% (v / v) HFIP in DCM for 1 h each, followed by washing with DCM and DMF.

[0588] Acylation was performed on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 USA) as described in method SPPS_C, with a coupling time of 4 h, using stepwise addition of building blocks, such as, but not limited to, Fmoc-8-amino-3,6-dioxa-octanoic acid (Fmoc-Ado-OH), Fmoc-Glu-OtBu, Fmoc-tranexamic acid (Fmoc-Trx-OH). Introduction of the fatty acid moiety was achieved using method SPPS_C, with a coupling time of 4 h, and using suitable building blocks, such as, but not limited to, tert-butyl octadecanoate or tert-butyl eicosanedioate. In some cases where the building block was insoluble at a stock concentration of 0.6 M in DMF (e.g., Fmoc-Trx-OH and tert-butyl eicosanedioate), a stock concentration of 0.3 M was prepared, and the addition was doubled.

[0589] Method: SC_D

[0590] The N-ε-lysine protecting Mtt protecting group was removed by washing the resin twice with 70% HFIP + 3% TIS in DCM for 15 min each, followed by washing with DCM and NMP.

[0591] Acylation was performed on a Prelude solid phase synthesizer using the protocol described in Method SPPS_D with stepwise addition of building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxoctaoctanedioic acid, Fmoc-tranexamic acid, Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester, docosenedioic acid mono-tert-butyl ester.

[0592] Method: SC_E

[0593] The N-ε-lysine Mtt protecting group was removed by washing the resin with HFIP / TIS / DCM (75:5:20, v / v / v) (2x5 min and 2x30 min) followed by washing with DMF and DCM.

[0594] Acylation was performed on a SymphonyX Solid Phase Peptide Synthesizer from Protein Technologies (Tucson, AZ 85714 USA) as described in Method SPPS_A, using stepwise addition of building blocks such as, but not limited to, Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Fmoc-Glu-OtBu, Fmoc-tranexamic acid (Fmoc-Trx-OH). Introduction of the fatty acid moiety was achieved using Method SPPS_A and appropriate building blocks such as, but not limited to, octadecanedioic acid mono-tert-butyl ester or eicosanedioic acid mono-tert-butyl ester.

[0595] Cleavage and purification of resin-bound peptides

[0596] Method: CP_A

[0597] After synthesis, the resin was washed with DCM, and the peptidyl resin was treated with TFA / TIS / water (95:2.5:2.5, v / v / v) for 1.5-3h, followed by diethyl ether precipitation. The precipitate was washed with diethyl ether and dissolved in a suitable mixture of water, acetic acid and / or MeCN. The crude peptide solution was purified by reverse phase preparative HPLC (Waters Deltaprep 4000) on a column containing C18 silica gel. Elution was performed with a gradient of increasing MeCN in water containing 0.1% TFA. The relevant fractions were checked by analytical UPLC. The fractions containing the pure target peptide were combined and freeze dried.

[0598] When further purification is required, the above-isolated lyophilized peptide TFA salt is dissolved in a neutral aqueous buffer based on common salts (such as but not limited to sodium hydrogen phosphate) and purified by reverse phase preparative HPLC (Waters Deltaprep 4000) on a column containing C18 silica gel. Elution is performed with a gradually increasing gradient of MeCN in an aqueous sodium phosphate solution (90 mM, pH 7.4). The relevant fractions are checked by analytical UPLC. The fractions containing the pure target peptide are combined and, after dilution with water, applied to a second reverse phase preparative HPLC (Waters Deltaprep 4000) on a column containing C18 silica gel. Elution is performed with a gradually increasing gradient of MeCN in water containing 0.1% TFA. The relevant fractions are combined and freeze-dried to obtain the TFA salt of the target peptide.

[0599] Method: CP_B

[0600] After the side chain synthesis is completed, the peptidyl resin is washed with DCM and dried, then treated with TFA / water / TIS (92.5:5:2.5 v / v / v, 10 mL) for 2 h, followed by precipitation with diethyl ether. The precipitate is washed with diethyl ether and dissolved in a suitable solvent (e.g., 2:1 water / MeCN or 4:1 25 mM NH4HCO3 / MeCN aqueous solution). If necessary, the pH of the solution needs to be adjusted to completely dissolve the peptide. The peptide is then purified on a Phenomenex Luna C8 (2) column (10 μM particle size, Purification was performed by reverse phase preparative HPLC (Waters 2545 binary gradient module, Waters 2489 UV / visible detector, Waters fraction collector III) on a 1% HPLC column (100 μm pore size, 250 x 21.2 mm). Separation of impurities and elution of product were accomplished using a gradient of MeCN in water containing 0.1% TFA. Relevant fractions were checked by analytical LCMS. Fractions containing the pure target peptide were combined and freeze-dried to obtain the TFA salt of the target peptide.

[0601] Method: CP_C

[0602] After synthesis, the resin is washed with DCM and the peptidyl resin is treated with TFA / TIS / water / anisole / DODT (90:2.5:2.5:2.5:2.5, v / v / v / v) for 1.5-3h and then precipitated with diethyl ether. The precipitate is washed with diethyl ether and dissolved in aqueous ammonium bicarbonate (concentration, e.g., 50mM) and vortexed. MeCN is added thereto to give a clear yellow solution. The solution is then filtered through a 0.22um Stericup and then purified by reverse phase preparative HPLC (Waters: 2545 pump, 2489 UV-vis, Fr. Collector III) on a column containing C18 silica gel. Elution is performed with an increasing gradient of MeCN in water containing 0.1% TFA. The relevant fractions are checked by analytical UPLC. The fractions containing the pure target peptide are combined and freeze-dried.

[0603] Salt exchange - formation of sodium salt.

[0604] Method: SX_A:

[0605] The lyophilized peptides isolated from methods CP_A, CP_B or CP_C are dissolved in a neutral to weakly alkaline (pH 7-8.5) sodium-containing aqueous buffer, such as 0.1-0.2M sodium acetate or sodium bicarbonate buffer. The peptide-containing buffer solution is salt exchanged using a Sep-Pak C18 column (1-5 g): first equilibrate the column with 4 column volumes of isopropanol, then 4 column volumes of MeCN, and then 8 column volumes of water. The peptide solution is added to the column and the flow-through is reapplied to ensure that the peptide is completely retained. The column is washed with 2-4 column volumes of water and then with 4-15 column volumes of a buffer solution containing a sodium salt (e.g., pH 7.5), such as, but not limited to, NaHCO3, NaOAc, Na2HPO4. The peptide is eluted with 5-10 column volumes of a 50-80% MeCN aqueous solution and lyophilized to obtain a white solid peptide sodium salt, which is used as is.

[0606] Method: SX_B:

[0607] The peptide solution in H2O / MeCN / TFA was adjusted to pH 7-8 with NaOH, with a maximum MeCN content of 20%. The mixture was then subjected to reverse phase preparative HPLC (Waters Deltaprep 4000) on a column containing C18 silica gel. First, equilibrate with approximately 4 column volumes of 0.1M NaOAc, followed by rinsing with 2 column volumes of water. Elution was performed with a gradually increasing gradient of MeCN in water. The relevant fractions were checked by analytical UPLC. The fractions containing the pure target peptide sodium salt were combined and freeze dried.

[0608] General methods for detection and characterization

[0609] LCMS method:

[0610] Method: LCMS_34:

[0611] LCMS_34 was performed on a setup consisting of a Waters Acquity UPLC H Class system and a Waters Xevo G2-XS QTof. Eluents: A: 0.1% formic acid in MQ water; B: 0.1% formic acid in MeCN.

[0612] The analysis was performed at RT (column temperature 40°C) by injecting an appropriate volume of sample onto the column and eluting with a gradient of A and B. UPLC conditions, detector settings and mass spectrometer settings were: Column: Waters Acquity BEH, C-18, 1.7 μm, 2.1 mm x 50 mm. Gradient: Linear 5%-95% B in 4.0 min at 0.4 ml / min. Detection: MS analytical mode, ionization method: ES. Scan: 50-4000 amu.

[0613] Method: LCMS_27:

[0614] LCMS_27 was performed on a setup consisting of an Agilent 1290 infinity series UPLC system and an Agilent Technologies LC / MSD TOF 6230 (G6230A). Eluents: A: 0.02% TFA in water: B: 0.02% TFA in MeCN.

[0615] The analysis was performed at RT (column temperature 40°C) by injecting an appropriate volume of sample onto the column and eluting with a gradient of A and B. Column: Eclipse C18+, 1.8 μm, 2.1 mm x 50 mm. Gradient run time: linear 5-95% B in 4.5 min, then 95% B for 0.5 min, 95-5% B for 0.5 min, 5% B for 0.5 min at a flow rate of 0.40 ml / min. Detection: linear reflector mode (positive); Ionization method: Agilent Jet Stream source. Scan: 100-3200 (m / z)

[0616] Method: LCMS_01

[0617] LCMS_01 is performed on a device consisting of a Waters Acquity UPLC system and a LCT Premier XE mass spectrometer from Micromass. Eluent: A: 0.1% formic acid in MQ water; B: 0.1% formic acid in MeCN. The analysis was performed at RT (column temperature 40°C) by injecting an appropriate volume of sample onto the column and eluting with a gradient of A and B. UPLC conditions, detector settings, and mass spectrometer settings were: Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm x50 mm. Gradient: 5%-95% B linearly in 4.0 min at 0.4 ml / min. Detection: 214 nm (analog output from TUV (tunable UV detector)) MS ionization mode: API-ES. Scan: 500-2000 amu.

[0618] Embodiment 1:

[0619] Synthesis of GIP derivatives

[0620] The derivatives of the present invention are synthesized according to the general preparation methods as described above.

[0621] hGIP(1-42);(SEQ ID NO:1):

[0622] HY AEGTFISDYSIAMDKIHQQDF VNWLLAQKGKK ND WKHNIT Q-OH

[0623] General methods used: SPPS_D, CP_A

[0624] Calculated molecular weight (average): 4983.53 g / mol

[0625] LCMS01: Found (M+4H) 4+1246.17

[0626] hGIP(1-31); (SEQ ID NO:2):

[0627] HY AEGTFISDYSIAMDKIHQQDF VNWLLAQK G-OH

[0628] Common methods used: SPPS_A, CP_A

[0629] Calculated molecular weight (average): 3589.98 g / mol

[0630] LCMS01: Found (M+3H)3+1197.61

[0631] Compound 1 (SEQ ID NO: 6):

[0632] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31)

[0633]

[0634] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0635] Calculated molecular weight (average): 4302.87 g / mol

[0636] LCMS_34: Found (M+4H) 4+1076.82

[0637] Compound 2 (SEQ ID NO: 7):

[0638] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24]-hGIP(1-31)

[0639]

[0640] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0641] Calculated molecular weight (average): 4283.82 g / mol

[0642] LCMS_34: Found (M+4H) 4+1071.82

[0643] Compound 3 (SEQ ID NO: 8):

[0644] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31)

[0645]

[0646] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0647] Calculated molecular weight (average): 4502.20 g / mol

[0648] LCMS_27: Found (M+3H) 3+1501.50

[0649] Compound 4 (SEQ ID NO: 9):

[0650] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31)

[0651]

[0652] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0653] Calculated molecular weight (average): 4470.12 g / mol LCMS_27: Found (M+4H) 4+1118.11

[0654] Compound 5 (SEQ ID NO: 10):

[0655] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31)

[0656]

[0657] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0658] Calculated molecular weight (average): 4344.91 g / mol

[0659] LCMS_27: Found (M+3H) 3+1449.10

[0660] Compound 6 (SEQ ID NO: 11):

[0661] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31)

[0662]

[0663] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0664] Calculated molecular weight (average): 4512.16 g / mol

[0665] LCMS_27: Found (M+3H)3+1504.79

[0666] Compound 7 (SEQ ID NO: 12):

[0667] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31)

[0668]

[0669] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0670] Calculated molecular weight (average): 4330.88 g / mol

[0671] LCMS_27: Found (M+3H) 3+1444.41

[0672] Compound 8 (SEQ ID NO: 13):

[0673] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]-butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31)

[0674]

[0675] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0676] Calculated molecular weight (average): 4498.13 g / mol

[0677] LCMS_27: Found (M+3H) 3+1500.12

[0678] Compound 9 (SEQ ID NO: 14):

[0679] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31)

[0680]

[0681] Common methods used: SPPS_B, SC_B, CP_B

[0682] Calculated molecular weight (average): 4303.86 g / mol

[0683] LCMS_27: Found (M+3H) 3+1435.08

[0684] Compound 10 (SEQ ID NO: 15):

[0685] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31)

[0686]

[0687] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0688] Calculated molecular weight (average): 4471.10 g / mol

[0689] LCMS_27: Found (M+3H)3+1490.79

[0690] Compound 11 (SEQ ID NO: 16):

[0691] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31)

[0692]

[0693] Common methods used: SPPS_C, SC_C, CP_C, SX_A

[0694] Calculated molecular weight (average): 4259.85 g / mol

[0695] LCMS_27: Found (M+3H) 3+1420.74

[0696] Compound 12 (SEQ ID NO: 17):

[0697] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31)

[0698]

[0699] Common methods used: SPPS_C, SC_C, CP_C, SX_A

[0700] Calculated molecular weight (average): 4427.09 g / mol

[0701] LCMS_27: Found (M+3H)3+1476.45

[0702] Compound 13 (SEQ ID NO: 18):

[0703] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Ala16,Arg18,Lys24]-hGIP(1-31)

[0704]

[0705] Common methods used: SPPS_C, SC_C, CP_C

[0706] Calculated molecular weight (average): 4245.78 g / mol

[0707] LCMS_27: Found (M+3H) 3+1416.06

[0708] Compound 14 (SEQ ID NO: 19):

[0709] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Ala16,Arg18,Lys24]-hGIP(1-31)

[0710]

[0711] Common methods used: SPPS_C, SC_C, CP_C

[0712] Calculated molecular weight (average): 4413.02 g / mol

[0713] LCMS_27: Found (M+3H)3+1471.77

[0714] Compound 15 (SEQ ID NO: 20):

[0715] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31)

[0716]

[0717] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0718] Calculated molecular weight (average): 4362.97 g / mol

[0719] LCMS_27: Found (M+3H)3+1455.09

[0720] Compound 16 (SEQ ID NO: 21):

[0721] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31)

[0722]

[0723] Common methods used: SPPS_C, SC_C, CP_C

[0724] Calculated molecular weight (average): 4363.95 g / mol

[0725] LCMS_27: Found (M+3H)3+1455.42

[0726] Compound 17 (SEQ ID NO: 22):

[0727] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]-butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31)

[0728]

[0729] Common methods used: SPPS_B, SC_B, CP_B, SX_A

[0730] Calculated molecular weight (average): 4621.19 g / mol

[0731] LCMS_27: Found (M+3H)3+1541.12

[0732] Compound 18 (SEQ ID NO: 23):

[0733] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Nle14,Arg18,Lys24]-hGIP(1-31)

[0734]

[0735] Common methods used: SPPS_C, SC_C, CP_C

[0736] Calculated molecular weight (average): 4330.88 g / mol

[0737] LCMS_27: Found (M+3H) 3+1444.40

[0738] Compound 19 (SEQ ID NO: 24):

[0739] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Asp14,Arg18,Glu20,Lys24]-hGIP(1-31)

[0740]

[0741] Common methods used: SPPS_C, SC_C, CP_C

[0742] Calculated molecular weight (average): 4365.88 g / mol

[0743] LCMS_27: Found (M+3H)3+1456.07

[0744] Compound 20 (SEQ ID NO: 25):

[0745] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-amino]butyryl]-[Aib2,Nle14,Glu20,Lys24]-hGIP(1-31)

[0746]

[0747] Common methods used: SPPS_A, SC_A, CP_A

[0748] Calculated molecular weight (average): 4413.92 g / mol

[0749] LCMS_27: Found (M+3H)3+1472.07

[0750] Compound 21 (SEQ ID NO: 26):

[0751] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]-amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Glu20,Lys24]-hGIP(1-31)

[0752]

[0753] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0754] Calculated molecular weight (average): 4581.17 g / mol

[0755] LCMS_27: Found (M+3H) 3+1527.78

[0756] Compound 22 (SEQ ID NO: 27):

[0757] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Glu20,Lys24]-hGIP(1-31)

[0758]

[0759] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0760] Calculated molecular weight (average): 4480.07 g / mol

[0761] LCMS_27: Found (M+3H)3+1494.13

[0762] Compound 23 (SEQ ID NO: 28):

[0763] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31)

[0764]

[0765] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0766] Calculated molecular weight (average): 4499.11 g / mol

[0767] LCMS_27: Found (M+3H) 3+1500.47

[0768] Compound 24 (SEQ ID NO: 29):

[0769] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Aib20,Lys24]-hGIP(1-31)

[0770]

[0771] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0772] Calculated molecular weight (average): 4436.06 g / mol

[0773] LCMS_27: Found (M+3H) 3+1479.46

[0774] Compound 25 (SEQ ID NO: 30):

[0775] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31)

[0776]

[0777] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0778] Calculated molecular weight (average): 4455.10 g / mol

[0779] LCMS_27: Found (M+3H)3+1485.81

[0780] Compound 26 (SEQ ID NO: 31):

[0781] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide

[0782]

[0783] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0784] Calculated molecular weight (average): 4509.20 g / mol

[0785] LCMS_27: Found (M+3H)3+1503.84

[0786] Compound 27 (SEQ ID NO: 32):

[0787] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide

[0788]

[0789] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0790] Calculated molecular weight (average): 4490.15 g / mol

[0791] LCMS_27: Found (M+3H)3+1497.49

[0792] Compound 28 (SEQ ID NO: 33):

[0793] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide

[0794]

[0795] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0796] Calculated molecular weight (average): 4537.21 g / mol

[0797] LCMS_34: Found (M+4H) 4+1135.13

[0798] Compound 29 (SEQ ID NO: 34):

[0799] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Lys24,Pro31]-hGIP(1-31)amide

[0800]

[0801] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0802] Calculated molecular weight (average): 4518.16 g / mol

[0803] LCMS_34: Found (M+4H) 4+1130.37

[0804] Compound 30 (SEQ ID NO: 35):

[0805] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31)

[0806]

[0807] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0808] Calculated molecular weight (average): 4627.24 g / mol

[0809] LCMS_34: Found (M+4H) 4+1130.37

[0810] Compound 31 (SEQ ID NO: 36):

[0811] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide

[0812] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0813] Calculated molecular weight (average): 4551.23 g / mol

[0814] LCMS_27: Found (M+3H)3+1517.81

[0815] Compound 32 (SEQ ID NO: 37):

[0816] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide

[0817] Common methods used: SPPS_C, SC_C, CP_C, SX_A

[0818] Calculated molecular weight (average): 4532.19 g / mol

[0819] LCMS_27: Found (M+3H) 3+1511.44

[0820] Compound 33 (SEQ ID NO: 38):

[0821] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33]-hGIP

[0822]

[0823] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0824] Calculated molecular weight (average): 5678.32 g / mol

[0825] LCMS_27: Found (M+4H) 4+1420.54

[0826] Compound 34 (SEQ ID NO: 39):

[0827] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33]-hGIP

[0828]

[0829] Common methods used: SPPS_A, SC_A, CP_A, SX_A

[0830] Calculated molecular weight (average): 5697.36 g / mol

[0831] LCMS_27: Found (M+4H) 4+1425.29

[0832] Compound 35 (SEQ ID NO: 40):

[0833] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Lys24,Glu33,Glu34]-hGIP

[0834]

[0835] Common methods used: SPPS_A, SC_A, CP_A

[0836] Calculated molecular weight (average): 5725.41 g / mol

[0837] LCMS_27: Found (M+4H) 4+1432.25

[0838] Compound 36 (SEQ ID NO: 41):

[0839] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Lys24,Glu33,Asp34]-hGIP

[0840]

[0841] Common methods used: SPPS_A, SC_A, CP_A

[0842] Calculated molecular weight (average): 5711.39 g / mol

[0843] LCMS_27: Found (M+4H) 4+1428.75

[0844] Compound 37 (SEQ ID NO: 42):

[0845] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP

[0846]

[0847] Common methods used: SPPS_A, SC_A, CP_A

[0848] Calculated molecular weight (average): 5744.46 g / mol

[0849] LCMS_27: Found (M+4H) 4+1437.03

[0850] Compound 38 (SEQ ID NO: 43):

[0851] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33,Glu34]-hGIP

[0852]

[0853] Common methods used: SPPS_A, SC_A, CP_A

[0854] Calculated molecular weight (average): 5693.33 g / mol

[0855] LCMS_27: Found (M+4H) 4+1424.24

[0856] Compound 39 (SEQ ID NO: 44):

[0857] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Glu33,Asp34]-hGIP

[0858]

[0859] Common methods used: SPPS_A, SC_A, CP_A

[0860] Calculated molecular weight (average): 5679.30 g / mol

[0861] LCMS_27: Found (M+4H) 4+1420.79

[0862] Compound 40 (SEQ ID NO: 45):

[0863] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP

[0864]

[0865] Common methods used: SPPS_A, SC_A, CP_A

[0866] Calculated molecular weight (average): 5712.37 g / mol

[0867] LCMS_27: Found (M+4H) 4+1429.00

[0868] Compound 41 (SEQ ID NO: 46):

[0869] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoyl-amino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP

[0870]

[0871] Common methods used: SPPS_A, SC_A, CP_A

[0872] Calculated molecular weight (average): 5911.71 g / mol

[0873] LCMS_27: Found (M+4H) 4+1478.81

[0874] Compound 42 (SEQ ID NO: 47):

[0875] N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Glu33,Glu34]-hGIP

[0876]

[0877] Common methods used: SPPS_A, SC_A, CP_A

[0878] Calculated molecular weight (average): 5879.62 g / mol

[0879] LCMS_27: Found (M+4H) 4+1470.81

[0880] Compound 43 (SEQ ID NO: 59):

[0881] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31)

[0882]

[0883] Common methods used: SPPS_A, SC_E, CP_A, SX_A

[0884] Calculated molecular weight (average): 4358.08 g / mol

[0885] LCMS_34: Found (M+3H)3+1452.82

[0886] Compound 44 (SEQ ID NO: 60):

[0887] N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide

[0888] Common methods used: SPPS_A, SC_E, CP_A, SX_A

[0889] Calculated molecular weight (average): 4454.21 g / mol

[0890] LCMS_34: Found (M+3H)3+1484.83

[0891] Compound 45 (SEQ ID NO: 61)

[0892] N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu14,Lys24]-hGIP

[0893]

[0894] Common methods used: SPPS_D, SC_D, CP_A

[0895] Calculated molecular weight (average): 5709.46 g / mol

[0896] LCMS_34: Found (M+4H) 4+1428.36

[0897] Compound 46 (SEQ ID NO: 62)

[0898] N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide

[0899]

[0900] Common methods used: SPPS_A, SC_A, CP_A, SX_B

[0901] Calculated molecular weight (average): 4383.99 g / mol

[0902] LCMS_34: Found (M+3H)3+1461.99

[0903] Pharmacological methods

[0904] The use of the derivatives of the invention as pharmacologically active agents in reducing weight gain and treating obesity and in treating diabetes and NASH in mammals such as humans can be demonstrated by agonist activity in conventional assays as well as in vitro and in vivo tests as described below.

[0905] Such assays also provide a means by which the activity of the derivatives of the invention can be compared with the activity of known compounds.

[0906] Embodiment 2:

[0907] In vitro potency (CRE luciferase; whole cells)

[0908] The purpose of this example is to test the activity or potency of the derivatives in vitro at the human GIP receptor as well as at the human GLP-1 receptor and the human glucagon receptor. In vitro potency is a measure of activation of the human GIP, GLP-1 or glucagon receptor, respectively, in a whole cell assay.

[0909] The potency of the derivatives of Example 1 was determined as follows. hGIP and C-terminally truncated hGIP (1-31) as well as hGLP-1 (7-37) (SEQ ID NO: 4) and human glucagon (hGcg) (SEQ ID NO: 5) were included for comparison.

[0910] principle:

[0911] In vitro efficacy is determined by measuring the response of human GIP, GLP-1 or glucagon receptor in a reporter gene assay in a separate cell line. The test is carried out in a BHK cell line expressing human GIP receptor, human GLP-1 receptor or human glucagon receptor and containing cAMP response element (CRE) DNA and firefly luciferase (CRE luciferase) gene coupled to a promoter. When each receptor is activated, it causes the generation of cAMP, which in turn causes luciferase protein to be expressed. When the test is incubated, luciferase substrate (luciferin) is added, and the enzyme converts luciferin into oxyluciferin to produce bioluminescence. Measure the luminescence as the readout of the test.

[0912] Cell culture and preparation

[0913] The cells used in this experiment (BHK CRE luc2P hGIPR clone #5, BHK CRE luc2P hGLP-1R clone #6, BHK CRE luc2P hGCGR clone #18) are BHK cells with BHKTS13 as the parental cell line. The cells were derived from clones containing the CRE luciferase element and were established by further transfection with the respective receptors to obtain the present clones.

[0914] The cells were cultured in cell culture medium under 5% CO2. The cells were aliquoted and stored in liquid nitrogen. The cells were maintained at passages and seeded the day before each assay.

[0915] Material

[0916] The following chemicals were used in this assay: Pluronic F-68 (10%) (Gibco 2404), human serum albumin (HSA) (Sigma A9511), 10% FBS (fetal bovine serum; Invitrogen 16140-071), fetal ovalbumin (Sigma A5503), DMEM without phenol red (Gibco 11880-028), 1 M Hepes (Gibco 15630), Glutamax 100x (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin (Invitrogen 10687-010), 1% pen / strep (penicillin / streptomycin; Invitrogen 15140-122), and steadylite plus (PerkinElmer 6016757).

[0917] Buffer

[0918] The cell culture medium consisted of DMEM medium containing 10% FBS, 1 mg / ml G418, 1 mg / ml hygromycin and 1% pen / strep (penicillin / streptomycin; Invitrogen 15140-122).

[0919] The assay medium consisted of DMEM without phenol red, 10 mM Hepes, and 1x Glutamax. The assay buffer consisted of 1% ovalbumin and 0.1% Pluronic F-68 in the assay medium supplemented with twice the indicated concentration of human serum albumin. The assay medium was mixed 1:1 with an equal volume of the test compound in assay buffer to give a final assay concentration of serum albumin.

[0920] program

[0921] 1) Cells were plated at 5000 cells / well and incubated overnight.

[0922] 2) Wash cells three times in PBS.

[0923] 3) Add assay medium (50 μl aliquots) with or without serum albumin to each well of the assay plate.

[0924] 4) Dilute the stock solutions of the test compound and reference compound to a concentration of 0.2 μM in assay buffer. Dilute the compound 10-fold to obtain the following concentrations: 2x10 -7 M, 2x10 -8 M, 2x10 -9 M, 2x10 -10M, 2x10 -11 M, 2x10 -12 M, 2x10 -13 M and 2x10 -14 M.

[0925] 5) Transfer 50 μl aliquots of compound or blank from the dilution plate to the assay plate. Test compounds at the following final concentrations: 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M and 1x10 -14 M.

[0926] 6) Incubate the assay plate at 37°C in a 5% CO2 incubator for 3 h.

[0927] 7) Take the assay plate out of the incubator and allow it to stand at room temperature for 15 minutes.

[0928] 8) Wash the cells 3 times with PBS plus some liquid in each well.

[0929] 9) Add 100 μl aliquots of steadylite plus reagent (the reagent is light sensitive) to each well of the assay plate.

[0930] 10) Cover each assay plate with aluminum foil to avoid light and shake at room temperature for 30 min.

[0931] 11) Read each assay plate in a microtiter plate reader.

[0932] Calculations and Results

[0933] Data from the microtiter plate reader were transferred to GraphPad Prism software. The software ran a nonlinear regression (log(agonist) vs. response). EC was calculated using the software and reported in pM. 50 The values ​​are shown in Table 1 below.

[0934] A minimum of two replicates were measured for each sample. The reported values ​​are the mean of the replicate measurements.

[0935] Table 1: Potency, EC 50

[0936]

[0937]

[0938] *Assayed in the presence of 1% HSA; nd = not determined.

[0939] Under the given conditions, the derivatives of the present invention all show good GIP potency and have essentially no activity or no measurable activity at the human GLP-1 receptor and the human glucagon receptor.

[0940] Embodiment 3:

[0941] Pharmacokinetic studies in miniature pigs

[0942] The aim of this study was to determine the in vivo half-life of the derivatives of the invention after intravenous administration to minipigs, i.e. the extension of their time in the body and thus the extension of their duration of action. This was done in a pharmacokinetic (PK) study to determine the terminal half-life of the derivative in question. The terminal half-life generally refers to the length of time it takes for a certain plasma concentration to be halved, measured after the initial distribution phase.

[0943] Research:

[0944] female Miniature Pigs from Ellegaard Minipigs (Dalmose, Denmark) were obtained and used in the study at approximately 7-14 months of age and weighing approximately 16-35 kg. Minipigs were housed individually and restricted fed once daily with a SDS minipig diet (Special Diets Services, Essex, UK).

[0945] After 3 weeks of acclimatization, two permanent central venous catheters were implanted in the caudal vena cava of each animal. Animals were allowed to recover for 1 week after surgery and then used for repeated pharmacokinetic studies with appropriate washout periods between consecutive derivative administrations.

[0946] Animals were fasted for approximately 18 hours prior to dosing and from 0 to 4 hours after dosing but had free access to water during the entire period.

[0947] The GIP derivative of Example 1 is dissolved in 8 mM sodium phosphate buffer at pH 7.8 containing 236 mM propylene glycol to a concentration of 50 nmol / ml. The compound is injected intravenously through a catheter (a volume corresponding to typically 5 nmol / kg, e.g. 0.1 ml / kg), and blood samples are collected at predetermined time points, continuing up to 13 days after administration (preferably through another catheter). Blood samples (e.g. 0.8 ml) are collected in EDTA buffer (8 mM) and then centrifuged at 4°C and 1942G for 10 minutes.

[0948] Sampling and analysis:

[0949] The plasma was pipetted into Micronic tubes on dry ice and kept at -20°C until the plasma concentration of the corresponding GIP peptide derivative was analyzed using ELISA or similar antibody-based assays or LCMS. Each plasma concentration-time curve was analyzed by a non-compartmental model in Phoenix WinNonLinver.6.4 (Pharsight Inc., Mountain View, CA, USA), and the resulting terminal half-life (harmonic mean) was determined.

[0950] result:

[0951] Table 2: Half-life (t 1 / 2 )

[0952]

[0953] Compared to the half-life of hGIP(1-42) measured in humans of about 5 min [Meier et al., Diabetes, Vol. 59, 2004, 654-662] or 7 min [Deacon et al., J. Clin. Endocrinol. & Metab., Vol. 85, No. 10, 2000, 3575-3581], the half-life of the tested GIP derivatives is very long.

[0954] Embodiment 4:

[0955] Physical stability of peptide compositions (ThT fibril formation assay and DLS_SI)

[0956] The aim of this study was to evaluate the physical stability of the derivatives of the invention in aqueous solution in the ThT test and the DLS-SI test as described below.

[0957] ThT test:

[0958] The thioflavin T assay was performed as outlined by Schlein (2017), AAPS J, 19(2), 397-408.

[0959] The low physical stability of peptides can lead to amyloid fibril formation, which is observed in the sample as an ordered linear macromolecular structure, which may eventually lead to gel formation. Traditionally, this is measured by visual inspection of the sample. However, this measurement is very cumbersome and depends on the observer. Therefore, the application of small molecule indicator probes is more advantageous. Thioflavin T (ThT) is such a probe, which has a unique fluorescence characteristic when combined with fibrils [Naiki et al. (1989) Anal. Biochem. 177, 244-249; LeVine (1999) Methods. Enzymol. 309, 274-284].

[0960] The time course of fibril formation can be described by a sigmoidal curve with the following expression [Nielsen et al. (2001) Biochemistry 40, 6036-6046]:

[0961]

[0962] Where F is the ThT fluorescence at time t. The constant t0 is the time required to reach 50% of the maximum fluorescence. Two important parameters describing fibril formation are the lag time calculated from t0–2τ and the apparent rate constant kapp 1 / τ.

[0963] It has been proposed that the formation of partially folded intermediates of the peptide is the general initiation mechanism of fibril formation. A small amount of these intermediates nucleate to form a template onto which other intermediates can assemble and fibril formation occurs. The lag time corresponds to the time interval for the critical mass of nuclei to build up, while the apparent rate constant is the rate at which the fibrils themselves form.

[0964] Samples were prepared freshly before each assay. The bulk drug substance was dissolved in 8 mM phosphate, 14 mg / mL propylene glycol, 58 mM phenol (pH 7.4) at 250 μM GIP derivative. The pH of the samples was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCl. Thioflavin T was added to the samples from a stock solution in H2O to a final concentration of 1 μM.

[0965] 200 μl sample aliquots were placed in 96-well microtiter plates (Packard OptiPlate TM -96, white polystyrene). Four replicates of each sample (corresponding to one test condition) were placed in a column of wells. The plate was sealed with Scotch Pad (Qiagen).

[0966] Incubate, shake and measure ThT fluorescence emission in Fluoroskan Ascent FL fluorescence plate reader (Thermo Labsystems). Orbital shaking is adjusted to 960rpm, 1mm amplitude, and the plate is incubated at 37°C. Fluorescence measurement is performed using excitation through a 444nm filter and emission through a 485nm filter. The assay is completed after incubation for 45 hours.

[0967] Each run was initiated by incubating the plate at the assay temperature for 10 min. The plate was measured every 20 minutes for the desired period of time. Between each measurement, the plate was shaken and heated as described.

[0968] After the ThT assay was completed, duplicates of each sample were combined and centrifuged at 20,000 rpm for 30 minutes at 18°C. The supernatant was filtered through a 0.22 μm filter and aliquots were transferred to HPLC vials. The concentration (percentage) of the filtered sample relative to the initial sample was reported as the recovery.

[0969] The measurement points are saved in Microsoft Excel format for further processing and curve drawing and fitting are performed using GraphPad Prism. In the absence of protofibrils, the background emission from ThT can be ignored. The data points are the average of four replicates and are shown with standard deviation error bars. Only data obtained in the same experiment (i.e., samples on the same plate) are shown in the same figure to ensure relative measurement of protofibril formation between experiments.

[0970] The data set can be fitted to equation (1). However, the lag time before fibril formation reported herein was determined by visual inspection of the curves, determining the time point at which ThT fluorescence increased significantly above background levels. No increase in ThT fluorescence during the duration of the assay was reported as a lag time of 45 hours.

[0971] Dynamic Light Scattering Stability Index (DLS-SI) for evaluating the physical stability of peptides in solution:

[0972] The hydrodynamic radius of the peptide in solution (R h , synonym: Stokes radius) is an indicator of the size and oligomeric state of a biomolecule in solution and can be measured by dynamic light scattering (DLS). h ) over time can be an indicator of changes in size and oligomeric state, and therefore an indicator of the physical instability of the peptide in solution.

[0973] Samples were prepared freshly before each assay. The bulk drug was dissolved at 250 μM GIP derivative in 8 mM phosphate, 14 mg / mL propylene glycol, 58 mM phenol (pH 7.4). The pH of the samples was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCl.

[0974] Non-sterile via pore size 0.02 μm Filter 400 μL of each freshly prepared sample through a 10 syringe filter, discarding the first two drops. Place 25 μL of filtered sample per well in a 384-well microtiter plate ( 3540 Polystyrene, black, flat bottom, transparent), each sample was analyzed in triplicate. The sample in each well was covered with 15 μL of silicone oil (Sigma-Aldrich, viscosity 20 cSt at 25°C). The plate was centrifuged at 1200 rpm for 5 min ( Centrifuge 5430, rotor A 2MTP) and placed in Wyatt DynaPro Plate Reader II for 30min before starting the measurement to reach the temperature equilibrium of the sample to 25°C. Wyatt DynaPro Plate Reader II is equipped with 830nm laser and Dynamics v7.5 software. Each sample in each well is analyzed with a data acquisition interval of 5 seconds and 40 acquisitions per sample. After the measurement, the plate is covered with an adhesive film for microplates (VWR polypropylene heat-resistant film) and incubated at 25°C for 4 weeks. The samples are re-measured by DLS with the same parameters after 1, 2 and 4 weeks.

[0975] After measurement, the data of each sample were first filtered by Dynamics software v7.5, minimum amplitude: 0.03, maximum amplitude: 1, baseline limit (1+ / -): 0.005, maximum SOS: 100. Afterwards, the temporal autocorrelation function of each measured sample was peer reviewed before calculating the DLS-SI according to equation (2).

[0976] In DLS-SI analysis, the cumulative R h The change in is defined as the stability index as a function of time:

[0977]

[0978] where DLS-SI is the stability index that depends on time (t), and the cumulative hydrodynamic radius R at the beginning of the stability study (t0) is h and R at the end (t) h The differences between the two were normalized by the statistically significant variability 2σ. hThe analytical variability of the measurements yielded σ = 0.3 nm. A DLS-SI value greater than 1 indicates that R h The larger the DLS-SI value, the higher the R h The greater the change over time.

[0979] result:

[0980] Table 3: ThT assay lag time and recovery at pH 7.4, and DLS-SI

[0981]

[0982]

[0983] nd = not determined; # The concentration of hGIP(1-42) in DLS-SI was only 156 μM instead of 250 μM as stated in the sample preparation. ## The concentration of hGIP(1-31) in DLS-SI was only 5 μM instead of 250 μM as stated in sample preparation. *The concentration was only 162 μM instead of 250 μM as stated in sample preparation. **Compound was not soluble at the given pH.

[0984] The GIP derivatives tested in the ThT assay showed no fibril formation after 45 hours (lag time) and a very high recovery, i.e., the concentration of the GIP derivatives recovered after the assay relative to the initial concentration, compared to hGIP (1-42) and hGIP (1-31). In addition, a tendency of hGIP (1-42) and hGIP (1-31) to form fibrils in solution was shown.

[0985] DLS-SI data showed that for hGIP (1-42) and the GIP derivatives of the present invention, there was no increase or only a slight increase in the hydrodynamic radius, and no precipitation was visible. h The changes over time were dramatic and in the DLS instrument the formation of large aggregates became visible along with the precipitation.

[0986] Therefore, the GIP derivative of the present invention has high physical stability compared to hGIP (1-42) and hGIP (1-31).

[0987] Embodiment 5:

[0988] Chemical stability of GIP derivative compositions

[0989] The purpose of this study was to determine the chemical stability of the GIP derivative compositions. As a measure of the stability of the GIP derivative compositions, the high molecular weight peptide formation (% HMWP) over time was analyzed by size exclusion chromatography (SEC-MS). In addition, the purity loss of the GIP derivative compositions was measured by LCMS.

[0990] preparation:

[0991] Samples for chemical stability assays were prepared freshly before each assay. The drug substance was dissolved at 250 μM GIP derivative in 8 mM phosphate, 14 mg / mL propylene glycol, 58 mM phenol (pH 7.4). The pH of the samples was adjusted to the desired value using appropriate amounts of concentrated NaOH and HCl.

[0992] Incubation:

[0993] Preparations of the corresponding GIP derivatives were stored in an incubator at 37°C for 4 weeks before being tested for HMWP formation and purity loss as described below.

[0994] HMWP formation analysis:

[0995] The formation of covalent oligomers (HMWP) was analyzed and identified by SEC-MS method. Waters Acquity i-class UPLC equipped with Acquity BEH200SEC column, 4.6mmx150mm, 1.7μm particle size and The pore size was 0.05%. At a column temperature of 45°C, isocratic elution was performed with 0.05% TFA in 55% MeCN at a flow rate of 0.2 ml / min. The LC system was coupled to a TUV detector running at 215 nm and 10 Hz and to a high-resolution QToF mass spectrometer (Synapt G2S) from Waters running in positive ion mode with an m / z range of 100 to 4000 and with normal resolution settings. Lock mass correction was performed every 31 seconds relative to Leu-enkephalin. The spectrum was processed in MassLynx version 4.1 and MaxEnt deconvolution was performed. The results are shown in Table 5.

[0996] Purity analysis by LCMS:

[0997] To speed up the chemical stability testing, the relevant samples were analyzed on a Waters Acquity I-stage UPLC system coupled to a Waters Synapt G2S high-resolution QToF mass spectrometry system for purity and impurity identification and characterization. The UPLC system was equipped with a Waters Acquity CSH C18 column with a particle size of 1.7 μm, an inner diameter of 1 mm, and a length of 150 mm. The column oven was maintained at 55°C. The solvents used were 0.1% formic acid in water (eluent A), premixed by Thermo (Optima LS118-1), while for the B eluent, a 0.1% formic acid in MeCN solution was also premixed by Thermo (Optima LS120-1). The solvents were pumped out from the binary solvent manager system and mixed in 50 μl volumes on the high pressure side of the mixer. The gradients and flow rates can be seen in Table 4:

[0998] Table 4: LC gradient table

[0999]

[1000] 1 μl of each sample was injected using a flow-through needle autosampler at 8°C. The effluent passed through an adjustable UV detector (TUV) adjustable to 215nm. The outlet of the UV detector was connected to the electrospray source of the mass spectrometer. The capillary was kept at 3kV and dry nitrogen was purged at a flow rate of 750l / h and a temperature of 250°C. The source block was kept at 120°C and the large-bore cone was flushed with nitrogen at 50l / h, and the rest of the instrument had a standard electrode potential. The MS was operated in high-resolution mode with a nominal resolution of 35,000. The ToF analyzer was operated in ADC mode with an m / z window of 100-2000. A second function with higher collision energy in the trap T-wave region was used for MSE-type experiments, in which the voltage of the high-energy ramp was between 32 and 52V.

[1001] result:

[1002] Table 5: HMWP formation and purity loss at 37°C

[1003]

[1004]

[1005] As can be seen from Table 5, the GIP derivatives of the present invention show low HMWP formation and have low monthly purity loss. Therefore, they are considered to be chemically stable in solution.

[1006] Embodiment 6:

[1007] Subchronic in vivo studies in obese mice

[1008] The purpose of this example is to evaluate the in vivo effects of the GIP derivatives of the present invention alone and in combination with a GLP-1 receptor agonist on food intake, body weight and glucose tolerance in diet-induced obese (DIO) mice. The GLP-1 receptor agonist used in this example is a semaglutide-like molecule, which has the same pharmacological properties as semaglutide but slightly different structures. The compound can be synthesized using methods known in the art, such as described in Example 1 herein or as described in Example 4 of WO 2006 / 097537.

[1009] Semaglutide-like molecule (compound No. 47; SEQ ID NO: 58): N 6,26 -{18-[N-(17-carboxyheptadecanyl)-D-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propionic acid), 34-L-arginine] human glucagon-like peptide 1 (7-37)

[1010]

[1011] Animals were treated once daily with a GLP-1 receptor agonist (Compound No. 47) and / or a GIP derivative of the invention (exemplified by Compound Nos. 5, 25, and 31) to assess weight loss, efficacy, and glucose tolerance.

[1012] Animals and diet

[1013] All animal protocols were approved by the Institutional Animal Care and Use Committee and the Ethical Review Committee of Novo Nordisk. Animals were housed according to Novo Nordisk rodent housing standards and received food and water ad libitum under controlled lighting (12h:12h light / dark cycle; lights off from 18:00-06:00), temperature (23±2°C) and relative humidity (50±20%). DIO male C57BL / 6J mice fed a high-fat diet (45% kcal fat, RD12451, Research Diets, New Brunswick, NJ, USA) for 22 weeks were obtained from Charles River (France). Upon arrival, mice were housed in single cages (one mouse per cage) and adapted to the new environment for 2 weeks before treatment was started.

[1014] Grouping and drug administration

[1015] Animals were single caged and acclimated to the treatment for 7 days before the start of the study. DIO mice were grouped (n=8 / group) to minimize statistical differences in the means and standard deviations of fat mass and body weight between groups. Animals were administered vehicle or test compound subcutaneously once daily.

[1016] Preparation of buffer

[1017] All compounds in this study were prepared in the following buffer: 50 mM phosphate; 70 mM sodium chloride; 0.05% polysorbate 80, pH 7.4. Dosing solutions were prepared in glass vials and stored at 2-8°C. Dosing solutions were warmed to room temperature prior to dosing and returned to 2-8°C after dosing.

[1018] Body weight and food intake

[1019] Body weight (BW) and food intake were measured every day before administration. Before treatment began, the average starting weight of mice was 45.2 ± 0.2 grams. The results are shown in Tables 6-8.

[1020] IPGTT (Intraperitoneal Glucose Tolerance Test)

[1021] On the day of the glucose tolerance test (day 15), the animals were fasted for 4 h. The food was removed and the animals were transferred to new cages. The animals could drink water, but could not eat food. The tail blood glucose level was measured, and the mice were injected (t=0) with an intraperitoneal (ip) glucose load of 2 g / kg (200 mg / ml glucose solution, dose volume 10 ml / kg). The tail blood glucose level was measured at 0, 15, 30, 60, 90, 120 minutes after the intraperitoneal injection of the glucose load. The animal stratification during the IPGTT was such that, for example, two mice from group 1 were dosed, followed by two mice from groups 2, 3, and 4, followed by the following two mice from groups 1, 2, 3, etc. This allowed the "time of day" to be evenly distributed among all groups.

[1022] result:

[1023] Table 6: Study 1, Effects on food intake, body weight and glucose tolerance in DIO mice treated with GLP-1 receptor agonist (Compound No. 47, 2 nmol / kg) and / or GIP Compound No. 5 (30 nmol / kg).

[1024]

[1025] a-dp<0.05, one-way ANOVA and Tukey's multiple comparison test for each day; groups not connected by the same letter (in each column) are significantly different from each other. Results are expressed as mean±SEM, n=6-8. iAUC=area under the curve minus baseline.

[1026] Table 7: Study 2, GLP-1 receptor agonist (Compound No. 47, 2 nmol / kg) and / or GIP compound No. Effects of 25 (30 nmol / kg) on ​​food intake, body weight and glucose tolerance in DIO mice treated with 25.

[1027]

[1028] a-d p<0.05, one-way ANOVA and Tukey's multiple comparison test for each day; groups not connected by the same letter (in each column) are significantly different from each other. Results are expressed as mean±SEM, n=8. iAUC=area under the curve minus baseline.

[1029] Table 8: Study 3, effects on food intake, body weight and glucose tolerance in DIO mice treated with GLP-1 receptor agonist (Compound No. 47, 2 nmol / kg) and GIP Compound No. 31 (30 nmol / kg).

[1030]

[1031] a-d p<0.05, one-way ANOVA and Tukey's multiple comparison test for each day; groups not connected by the same letter (in each column) are significantly different from each other. Results are expressed as mean±SEM, n=8. iAUC=area under the curve minus baseline.

[1032] As can be seen from Tables 6-8, monotherapy with the GLP-1 receptor agonist compound No. 47 (2nmol / kg) resulted in a reduction in food intake, leading to weight loss and improved glucose tolerance. Compared with compound 47, monotherapy with the GIP derivatives of the present invention (compounds 5, 25, 31; 30nmol / kg) had less effect on food intake, resulting in less weight loss. Monotherapy with GIP derivatives 5, 25, and 31 all improved glucose tolerance. Compared with monotherapy, combination therapy with compound 47 (2nmol / kg) and each of the GIP derivatives 5, 25, and 31 (30nmol / kg) enhanced the reduction in food intake and weight loss, and the effect was better than the cumulative effect. Glucose tolerance with combination therapy did not exceed the improvement achieved with monotherapy with the GIP derivatives (Tables 6-8).

[1033] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and changes that fall within the true scope of the invention.

Claims

1. A derivative of a GIP analog, or a pharmaceutically acceptable salt or amide thereof, wherein the derivative is a GIP analog covalently linked to a modifying group, wherein the GIP analog is represented by Formula II (SEQ ID NO: 48): X1-X2-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-X 14 -Asp-X 16 -Ile-X 18 -Gln-X 20 -Asp-Phe-Val-Lys-Trp-Leu-Leu-Ala-Gln-Lys-X 31 (II), wherein, X 16 for Lys, and X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 for Gly; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 For His, X 20 Gln, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 for Gly; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Glu, X 31 for Gly; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 For Aib, X 31 for Gly; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 For His, X 20 Glu, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 For His, X 20 Glu, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 Arg, X 20 Glu, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 For His, X 20 For Aib, X 31 for Gly; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 Arg, X 20 For Aib, X 31 for Gly; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 For Pro; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 For His, X 20 Gln, X 31 For Pro; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 For Pro; X1 is Ac-D-Tyr, X2 is Ala, X 14 For Nle, X 18 For His, X 20 Gln, X 31 For Pro; X1 is Ac-D-Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 is a Pro; or X1 is Ac-D-Tyr, X2 is Aib, X 14 For Nle, X 18 For His, X 20 Gln, X 31 For Pro, or The GIP analog further comprises a C-terminal extension of Formula II, wherein the C-terminal extension is shown in Formula III (SEQ ID NO: 51), which is connected to Formula II via an amide bond from the C-terminal carboxylic acid of Formula II to the N-terminal amino group of Formula III, Lys-X 33 -X 34 -Asp-Trp-Lys-His-Asn-Ile-Thr-Gln(III) Among them, X 16 for Lys, and X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 For His, X 20 Gln, X 31 Gly, X 33 Glu, X 34 Asn; X1 is Tyr, X2 is Aib, X 14 For Nle, X 18 Arg, X 20 Gln, X 31 Gly, X 33 Glu, X 34 is Asn; or X1 is Tyr, X2 is Aib, X 14 Leu, X 18 For His, X 20 Gln, X 31 Gly, X 33 Lys, X 34 Asn, wherein the modifying group is covalently linked to the side chain of the epsilon amino group of lysine at position 24, and the modifying group is defined by ABC-, Where A- is the chemical formula 1 Chemical formula 1: p is an integer in the range of 14-20; Where * indicates the position of the amide bond connecting A- and B-, And BC- is the connector; B- is the chemical formula 2 Chemical formula 2: q is an integer in the range of 0-1; r is an integer in the range of 1-3; wherein * indicates the position of the amide bond connecting A- and B-, and ** indicates the position of the amide bond connecting B- and C-, and C- is selected from Chemical Formula 3 and Chemical Formula 4 Chemical formula 3: Chemical formula 4: s is an integer in the range of 1-3; t is an integer in the range of 1-4; u is an integer in the range of 1-3; wherein ** indicates the position of the amide bond connecting B- and C-, and *** indicates the position of the amide bond connecting C- and the epsilon amino group of lysine at position 24.

2. The derivative according to claim 1, or a pharmaceutically acceptable salt or amide thereof, wherein the modifying group ABC- is selected from: Chemical formula 5: Chemical formula 6: Chemical formula 7: Chemical formula 8: Chemical formula 9: Chemical formula 10: Chemical formula 11: Chemical formula 12: Chemical formula 13: Chemical formula 14: Chemical formula 15: Chemical formula 16:

3. A derivative, or a pharmaceutically acceptable salt or amide thereof, selected from the following: Compound 1, as shown in SEQ ID NO: 6: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl [Aib2, Nle14, Arg18, Lys24]-hGIP(1-31) Compound 2, as shown in SEQ ID NO:7: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl [Aib2,Nle14,Lys24]-hGIP(1-31) Compound 3, as shown in SEQ ID NO: 8: N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 4, as shown in SEQ ID NO:9: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 5, as shown in SEQ ID NO: 10: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 6, as shown in SEQ ID NO: 11: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 7, as shown in SEQ ID NO: 12: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 8, as shown in SEQ ID NO: 13: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]-butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 10, as shown in SEQ ID NO: 15: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31) Compound 11, as shown in SEQ ID NO: 16: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl [Aib2, Nle14, Arg18, Aib20, Lys24]-hGIP(1-31) Compound 12, as shown in SEQ ID NO: 17: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31) Compound 15, as shown in SEQ ID NO: 20: N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 17, as shown in SEQ ID NO: 22: N{1}-Acetyl,N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]-butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[D-Tyr1,Nle14,Arg18,Lys24]-hGIP(1-31) Compound 21, as shown in SEQ ID NO: 26: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]-amino]butyryl]amino]butyryl]amino]butyryl [Aib2, Nle14, Glu20, Lys24]-hGIP(1-31) Compound 22, as shown in SEQ ID NO: 27: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Glu20,Lys24]-hGIP(1-31) Compound 23, as shown in SEQ ID NO: 28: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Glu20,Lys24]-hGIP(1-31) Compound 24, as shown in SEQ ID NO: 29: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Aib20,Lys24]-hGIP(1-31) Compound 25, as shown in SEQ ID NO: 30: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31) Compound 26, as shown in SEQ ID NO:31: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide Compound 27, as shown in SEQ ID NO: 32: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]amino]-butyryl]amino]butyryl]-[Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide Compound 28, as shown in SEQ ID NO: 33: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide Compound 29, as shown in SEQ ID NO: 34: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Lys24,Pro31]-hGIP(1-31)amide Compound 30, as shown in SEQ ID NO:35: N{1}-Acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl [D-Tyr1, Nle14, Arg18, Lys24]-hGIP(1-31) Compound 31, as shown in SEQ ID NO: 36: N{1}-Acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl [D-Tyr1, Aib2, Nle14, Arg18, Lys24, Pro31]-hGIP(1-31)amide Compound 32, as shown in SEQ ID NO: 37: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Lys24,Pro31]-hGIP(1-31)amide Compound 33, as shown in SEQ ID NO: 38: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl -[Aib2,Nle14,Lys24,Glu33]-hGIP Compound 34, as shown in SEQ ID NO: 39: N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl -[Aib2,Nle14,Arg18,Lys24,Glu33]-hGIP Compound 45, as shown in SEQ ID NO:61: N{Epsilon-24}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib2,Leu14,Lys24]-hGIP Compound 46, as shown in SEQ ID NO:62: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24,Pro31]-hGIP(1-31)amide 4. The derivative according to claim 3, or a pharmaceutically acceptable salt or amide thereof, wherein the derivative is: Compound 5, as shown in SEQ ID NO: 10: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butyryl]amino]butyryl]amino]butyryl]-[D-Tyr1,Aib2,Nle14,Arg18,Lys24]-hGIP(1-31) 5. The derivative according to claim 3, or a pharmaceutically acceptable salt or amide thereof, wherein the derivative is: Compound 25, as shown in SEQ ID NO: 30: N{1}-Acetyl,N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]amino]butyryl]-amino]butyryl]amino]butyryl]-[D-Tyr1,Nle14,Arg18,Aib20,Lys24]-hGIP(1-31) 6. The derivative according to claim 3, or a pharmaceutically acceptable salt or amide thereof, wherein the derivative is: Compound 31, as shown in SEQ ID NO: 36: N{1}-Acetyl, N{Epsilon-24}-[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[[4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl]-amino]butyryl]amino]butyryl]amino]butyryl]amino]butyryl [D-Tyr1, Aib2, Nle14, Arg18, Lys24, Pro31]-hGIP(1-31)amide 7. A pharmaceutical composition comprising a derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or amide thereof, and at least one pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, further comprising a GLP-1 receptor agonist or a GLP-1 / glucagon receptor co-agonist.

9. The pharmaceutical composition according to claim 8, wherein the GLP-1 receptor agonist is semaglutide as shown in SEQ ID NO:

57.

10. Use of the derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or amide thereof in the preparation of a medicament for treating and / or preventing obesity.

11. Use of a derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or amide thereof in the preparation of a medicament for treating and / or preventing all forms of diabetes.

12. Use of the derivative according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or amide thereof in the preparation of a medicament for treating and / or preventing type 2 diabetes.

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