WO2023139106A2

WO2023139106A2 - Long-acting gipr antagonists

Info

Publication number: WO2023139106A2
Application number: PCT/EP2023/051098
Authority: WO
Inventors: Brian Patrick Finan; Bin Shubin YANG
Original assignee: Novo Nordisk A/S
Priority date: 2022-01-18
Filing date: 2023-01-18
Publication date: 2023-07-27
Also published as: WO2023139106A3

Abstract

The present application relates to novel peptides that are derivatives of glucose-dependent insulinotropic peptide (GIP) analogues with improved GIP antagonistic activity, and to the pharmaceutical use of the GIP derivatives. The GIP derivatives of the invention are modified by including a lipophilic moiety.

Description

LONG-ACTING GIPR ANTAGONISTS

TECHNICAL FIELD

The present application relates to novel antagonists that are derivatives of glucosedependent insulinotropic peptide (GIP) analogues with improved GIP antagonistic activity, and to the pharmaceutical use of the GIP derivatives.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference.

BACKGROUND

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

Due to the incretin effect, the GIP receptor has become an attractive drug target in the treatment of metabolic diseases such as obesity and diabetes, with GIP receptor agonists either as a standalone, in combination with GLP-1 receptor agonists, or in combination with GLP-1/glucagon receptor co-agonists. GIP itself has a short plasma half-life due to dipeptidyl peptidase-4 (DPP-IV) mediated inactivation, and poor physical stability due to high tendency to form fibrils in solution. If GIP(1-42) or GIP(1-30) are secreted into the circulation in humans, the cleavages catalyzed by DPP-4 would result in GIP(3-42) or GIP(3- 30) which both are inactive GIP hormones.

WO 2020/115048 and US2020/087373 discloses GIP analogues that act as hGIP receptor antagonists. These GIP analogues are modified by introducing a modified lysine at one or more of positions 3 to 29, wherein the modification is primarily a fatty acid in the form of a diacid attached to the epsilon-amino group of the modified lysine and the primary site for attachment of said modification is in position 18.

It is, however, desirable achieving hGIP receptor antagonists with increased activity. For the purpose of developing a GIP based drug, it is also important to have good correlation of the activity from in vitro assays to animal models. Accordingly, it is also desirable to obtain GIP receptor antagonists having antagonist activity at the human GIP receptor and at the same time on a model GIP receptor e.g. the mGIP receptor. These objectives have been achieved with the GIP derivatives according to the present invention.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention concerns a glucose-dependent insulinotropic peptide (GIP) derivative consisting of a modifying group and a GIP analogue wherein the GIP analogue is defined by Formula la, Ila, or Illa:

Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-Gln-Xxxso-Xxxsi (la), (SEQ ID NO: 46);

Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn (Ila), (SEQ ID NO: 47);

Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (Illa), (SEQ ID NO: 48); wherein Xxxs is Thr or absent, Xxxe is Phe or L-3-phenyllactic acid (Pla), Xxx is Tyr or Lys, Xxxn is Ser or Lys, Xxxi4 is Met, Leu, lie or Nle, Xxxis is His, Arg, Orn, homoArg, XXX21 is Asp, Glu or homoGlu, Xxxso is Lys or absent, and Xxxsi is Gly or absent; provided that when Xxx₆ is Pla, then Xxx₅ is absent; wherein said GIP analogue of Formula la or Ila comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula Illa comprises a modified lysine in position 43, said modified lysine comprises the modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine in one of positions 11 , 10 or 43, the modifying group being defined by A-B-C-, wherein A- is a mono fatty acid and B-C- is a linker, which may be absent or present; or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof. In another aspect, the invention concerns the GIP derivative according to the invention for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

It has been found that the compounds of the invention provide for an even better effect on weight loss in combination with a GLP-1 receptor agonist. Accordingly, in still another aspect, the invention concerns a dosage form comprising a combination of a GIP derivative according to the invention and a GLP-1 receptor agonist.

In yet another aspect, the invention concerns a kit-of-parts comprising a first dosage form comprising a GIP derivative according to the invention and a second dosage form comprising a GLP-1 receptor agonist.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows percent change in body weight over 26 days of treatment of DIO mice when administering vehicle (open circles), semaglutide 2 nmol/kg (closed squares), compound 7 500 nmol/kg (open triangles-up), compound 7 1500 nmol/kg (open triangles-down), semaglutide 2 nmol/kg + compound 7 500 nmol/kg (closed triangles-up), and semaglutide 2 nmol/kg + compound 7 1500 nmol/kg (closed triangles-down).

Fig. 2 shows cumulative food intake over 26 days of treatment of DIO mice when administering vehicle (open circles), semaglutide 2 nmol/kg (closed squares), compound 7 500 nmol/kg (open triangles-up), compound 7 1500 nmol/kg (open triangles-down), semaglutide 2 nmol/kg + compound 7 500 nmol/kg (closed triangles-up), and semaglutide 2 nmol/kg + compound 7 1500 nmol/kg (closed triangles-down).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In what follows, Greek letters may be represented by their symbol or the corresponding written name, for example: a = alpha; p = beta; s = epsilon; y = gamma; co = omega; etc. Also, the Greek letter of p may be represented by "u", e.g. in pl=ul, or in pM=uM.

Unless otherwise indicated in the specification, terms presented in singular form generally also include the plural situation.

Also described herein are derivatives of GIP analogues, pharmaceutical compositions and uses thereof in which open ended terms like “comprises” and ’’comprising” are replaced with closed terms such as “consists of”, “consisting of”, and the like. GIP receptor antagonists

A receptor antagonist may be defined as a compound that binds to a receptor and is capable of inhibiting or reducing a response typical of the natural ligand. As described herein, a “GIP antagonist” may be described as a compound capable of binding to the GIP receptor without activating the GIP receptor or reducing the activation compared to the native ligand.

GIP analogues

The term “hGIP(1-42)” as used herein refers to the human glucose-dependent insulinotropic polypeptide, the sequence of which is included in the sequence listing as SEQ ID NO: 5. The peptide having the sequence of SEQ ID NO: 5 may also be designated native hGIP or hGIP.

The term “hGIP(1-31)” as used herein refers to a truncated version of hGIP(1-42), comprising amino acids 1-31 of hGIP(1-42), the sequence of hGIP(1-31) is included in the sequence listings as SEQ ID NO: 4.

The term “hGIP(3-31)” as used herein refers to a truncated version of hGIP(1-42) comprising amino acids 3-31 of hGIP(1-42), and is a GIP antagonist. The sequence of hGIP(3-31) is included in the sequence listings a SEQ ID NO: 6.

The term “hGIP(3-42)” as used herein refers to truncated version of hGIP(1-42) comprising amino acids 3-42 of hGIP(1-42), and is a GIP antagonist. The sequence of hGIP(3-42) is included in the sequence listings as SEQ ID NO: 7.

The term “GIP analogue” as used herein refers to a peptide, or a compound, which is a variant of hGIP(1-31) or hGIP(1-42). The term “variant” is used for peptides comprising at least one amino acid substitution as compared to hGIP(1-31) or hGIP(1-42) and is capable of binding to the GIP receptor.

In the context of the present invention, the term “amino acid modification” refers to the substitution of an amino acid for another, the deletion of an amino acid, or the addition of an amino acid.

The term “substitution” as used herein refers to one amino acid being replaced by another in the backbone of the peptide. The GIP derivatives of the invention may comprise substitutions of one or more unnatural and/or non-amino acids, e.g., amino acid mimetics, into the sequence of the GIP derivative. As an example, replacing the serine in position 11 of hGIP(1-31) with lysine is considered a “substitution”.

The term “deletion” as used herein refers to one amino acid in the backbone of the peptide being removed without replacing it with another moiety. As an example, deleting the first four amino acids of hGIP(1-31) to prepare hGIP(5-31) (SEQ ID NO: 8) is considered a “deletion” of each of the four amino acids.

The term “addition” as used herein refers to adding one amino acid, or another moiety that may form part of the peptide backbone, into the backbone of the peptide. As an example, adding an amino acid at the C-terminal end of hGIP(1-31) to prepare (a modified) hGIP(1-32) is considered an “addition”.

GIP analogues of the derivatives of the invention may be described by reference to i) the number of the amino acid residue in hGIP(1-31) or hGIP(1-42) which corresponds to the amino acid residue which is changed (i.e. the corresponding position in hGIP(1-31) or hGIP(1-42), and to ii) the actual change. For example, [M14L]-hGIP(1-31) refers to a GIP analogue in which the methionine of position 14 of hGIP(1-31) has been replaced by a leucine.

The numbering of positions in the GIP analogues of the present invention is made with reference to hGIP(1-31) or hGIP(1-42), even if some of the amino acids in the N- terminal end have been deleted. As an example, the first position of hGIP(5-31) is referred to as position 5, even though it is the first amino acid of the sequence.

In one aspect, the GIP analogues of the derivatives of the invention comprises Formula la, Ila, or Illa (SEQ ID NOs: 46, 47, 48):

Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Xxx2o-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (Illa), (SEQ ID NO: 48); wherein Xxxs is Thr or absent, Xxxe is Phe or L-3-phenyllactic acid (Pla), Xxx is Tyr or Lys, Xxxn is Ser or Lys, Xxxi4 is Met, Leu, lie or Nle, Xxxis is His, Arg, Orn, homoArg, XXX21 is Asp, Glu or homoGlu, Xxxso is Lys or absent, and Xxxsi is Gly or absent; provided that when Xxxe is Pla, then Xxxs is absent, wherein said GIP analogue of Formula la or Ila comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula Illa comprises a modified lysine in position 43, or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

In an alternative aspect, the GIP analogues of the derivatives of the invention comprises Formula I, II, or III (SEQ ID NOs: 1, 2, 3):

Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- As n-Trp- Le u- Le u- A I a- G I n-Xxxso-Xxxsi ( I ) , Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn (II), Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (III), wherein Xxxs is Glu or absent, Xxx4 is Gly or absent, Xxxs is Thr or absent, Xxxso is Lys or absent, and Xxxsi is Gly or absent; wherein said GIP analogue of Formula I or II comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula III comprises a modified lysine in position 43; wherein the GIP analogue has a maximum of 10 amino acid modifications as compared to positions 3 to 31 of hGIP(1-31) (SEQ ID NO: 4), or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

The position of the modified lysine of the GIP analogue of the derivative of the invention depends on whether the GIP derivative of the invention comprises a GIP analogue of Formula I, II, III, la, Ila, or Illa. If the GIP derivative of the invention comprises a GIP analogue of Formula I, II, la or Ila, the modified lysine is in one of positions 11 or 10. If the GIP derivative of the invention comprises a GIP analogue of Formula III or Illa, the modified lysine is in position 43.

In one embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula la or Ila. In a further embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula la. In another embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula Illa.

In one embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula I or II. In a further embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula I. In another embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula III.

In still a further embodiment, the GIP derivative of the invention comprises a GIP analogue of Formula I, II, la or Ila, wherein the modified lysine is in one of positions 11 and 10. In yet a further embodiment, the modified lysine is in position 11 .

The GIP analogues of the derivatives of the present invention do not have any amino acids in positions 1 and 2 corresponding to positions in hGIP(1-31). In one embodiment, the derivatives do not have any amino acids in position 1 , 2, 3 and 4 corresponding to hGIP(1-31). In one embodiment, Xxx₅ is Thr and Xxx₆ is Phe. In a further embodiment, an additional deletion may be present in position 5. Accordingly, in one embodiment, Xxxs is absent. In another embodiment, Xxxs is absent and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla).

The GIP analogue of the derivative of the invention may have a maximum of 10 amino acid modifications as compared to positions 3 to 31 of hGIP(1-31) (i.e. compared to hGIP(3-31)), some of which may be substitutions. Accordingly, in one embodiment, Met in position 14 is substituted by Leu. In a further embodiment, His in position 18 is substituted by Arg. In still a further embodiment, Asp in position 21 is substituted by Glu. In yet a further embodiment, the GIP analogue of the derivative of the invention has a maximum of 8 amino acid modifications as compared to hGIP(3-31). In another embodiment, the GIP analogue of the derivative of the invention has a maximum of 7 amino acid modifications as compared to hGIP(3-31). In still another embodiment, the GIP analogue of the derivative of the invention has a maximum of 6 amino acid modifications as compared to hGIP(3-31). In one embodiment, Xxxi4 is Leu, Xxxis is Arg and XXX21 is Glu.

The GIP analogue of the derivatives of the present invention may have a combination of the features defined above. Accordingly, in one embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula la, wherein the modified lysine is in position 11 (Xxxn is Lys), Xxx₅ is Thr, Xxx₆ is Phe, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a-acetylate. In another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula la or Ila, wherein the modified lysine is in position 10 (Xxx is Lys), Xxxs is Thr, Xxxe is Phe, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a-acetylate.

In an alternative embodiment, the GIP analogue of the derivatives of the present invention is a GIP analogue of Formula I, wherein the modified lysine is in position 11 , Xxx₃ and Xxx4 are absent, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a-acetylate. In another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula I or II, wherein the modified lysine is in position 10, Xxx₃ and Xxx4 are absent, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a- acetylate.

In still another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula Illa, wherein the modified lysine is in position 43 and the N-terminal end is substituted with an a-acetylate. In yet another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula la, wherein the modified lysine is in position 11 (Xxxn is Lys), Xxxs is absent, and Xxx₃ is L-3-phenyllactic acid (Pla). In yet another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula la, wherein the modified lysine is in position 11 (Xxxn is Lys), Xxxs is absent, Xxx₃o is Lys or absent, Xxx_3i is Gly or absent, and Xxx₃ is L-3-phenyllactic acid (Pla). In a further embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula la or Ila, wherein the modified lysine is in position 10 (Xxx is Lys), Xxxs is absent, Xxx₃o is Lys, Xxx_3i is Gly, and Xxx₃ is L-3-phenyllactic acid (Pla).

In still another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula III, wherein the modified lysine is in position 43, Xxx₃ and Xxx4 are absent, and the N-terminal end is substituted with an a-acetylate. In yet another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula I, wherein the modified lysine is in position 11 , Xxx₃, Xxx4 and Xxxs are absent, and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla). In yet another embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula I, wherein the modified lysine is in position 11 , Xxx₃, Xxx4, and Xxxs are absent, Xxx₃o is Lys or absent, Xxx_3i is Gly or absent, and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla). In a further embodiment, the GIP analogue of the derivative of the invention is a GIP analogue of Formula I or II, wherein the modified lysine is in position 10, Xxx₃, Xxx4, and Xxxs are absent, Xxx₃o is Lys, Xxx_3i is Gly, and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla).

In some embodiment, the GIP analogues of the derivatives of the invention may comprise a substitution selected from the group consisting of: M14L, H18R, D21 E as compared to hGIP(1-31). In some embodiment, the GIP analogue of the derivative of the invention comprises all of substitutions M14L, H18R and D21 E as compared to hGIP(1-31). In one embodiment, the GIP analogue of the derivatives of the invention comprises the backbone of [S11 K, M14L, H18R, D21 E]-hGIP(6-29) (SEQ ID NO: 9). Also or alternatively, in one embodiment, the GIP analogue of the derivatives of the invention comprises the backbone of [F6Pla, S11 K, M14L, H18R, D21 E]-hGIP(6-29) (SEQ ID NO: 10). Also or alternatively, in one embodiment, the GIP analogue of the derivatives of the invention comprises the backbone of [Y10K, M14L, H18R, D21 E]-hGIP(6-29) (SEQ ID NO: 11).

In some embodiment, further substitutions may be present in additional positions. In one embodiment, the amino acid in position 13 is selected from Ala or Lys. In one embodiment, position 16 is selected from Lys or Aib. In one embodiment, the amino acid in position 17 is selected from lie or Lys. In some embodiment, position 20 is selected from Gin or Aib. In one embodiment, the amino acid in position 24 is selected from Asn, Gin or Lys. In one embodiment, the amino acid in position 30 is selected from Lys, Arg or is absent. In one embodiment, the amino acid in position 31 is selected from Gly, Pro or is absent.

Backbone of the GIP analogues of the derivative of the invention are included in the sequence listing. In some embodiment, the GIP analogue of the derivative of the invention is selected from SEQ ID NO: 12-45, such as 17-45. In some embodiment, the GIP analogue of the derivative of the invention is be selected from any one of SEQ ID NO: 17-31 and 43-45, such as SEQ ID NO: 17-26, 28-31 and 43-44. In one embodiment, the GIP analogue of the derivative of the invention is selected from the group consisting of SEQ ID NO: 17-23, 31, 43- 44 such as 17-18, 21-23 or such as 19-20. In some embodiment, the GIP analogue of the derivative of the invention is selected from the group consisting of SEQ ID NO: 24-25, 28-30, such as 24-25, 30 or such as 28-30. In some embodiment the GIP analogue of the derivative of the invention is SEQ ID NO: 26.

Analogues “comprising” certain specified changes may comprise further changes, when compared to the respective formula. In a particular embodiment, the analogue "has" the specified changes.

As is apparent from the above examples, amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code. These three ways are fully equivalent.

GIP derivatives

The term “GIP derivative” as used herein refers to a chemically modified GIP analogue, in which one or more substituents have been covalently attached to the peptide backbone.

In one aspect of the invention, the substituent may be an N-terminal substituent. Also or alternatively, in one aspect, the substituent may be a modifying group or, alternatively, referred to as a protracting moiety or albumin binding moiety.

The term “N-terminal substituent” or “modifying group” as used herein, means a chemical moiety or group replacing a hydrogen atom. In one aspect, the derivative of a GIP analogue comprises a substituent covalently attached to the alpha-amino group of the amino acid residue in the N-terminus of the analogue. In one aspect, the N-terminal substituent is an alkanoyl or acyl group. In a particular aspect, the N-terminal substituent is an acetyl group or glutaric acid. As an example of an N-terminal substituted amino acid is Ac-Thr at position 5. Such N-acetylation would not count as a substitution in the peptide backbone compared with hGI P(1-31 ), because the amino acid in the GIP analogue is the native Thr, e.g. N“-Ac- [Lys11 ,Met14,Arg18,Glu21]-hGIP(5-31) comprises 4 substitutions as compared to hGIP(5- 31).

Also or alternatively, in one aspect, the GIP analogue comprises a modifying group covalently attached to a lysine residue at one of positions 11, 10, 21 or 43. In a further aspect, the modifying group is capable of forming non-covalent conjugates with proteins, e.g. albumin, thereby promoting the circulation of the derivative with the blood stream, and also having the effect of protracting the time of action of the derivative, due to the fact that the conjugate of the GIP derivative and albumin is only slowly disintegrated to release the active pharmaceutical ingredient.

The modifying group may be covalently attached to a lysine residue of the GIP analogue by acylation, i.e. via an amide bond formed between a carboxylic acid group of the modifying group and the epsilon amino group of said lysine group.

In one aspect, the modifying group is covalently attached to a lysine residue at one of positions 11 , 10, 21 or 43 by acylation, i.e. via an amide bond formed between a carboxylic acid group of the modifying group and the epsilon amino group of the lysine residue.

In one aspect, the modifying group is defined by A-B-C-, wherein A- is a mono fatty acid and B-C- is a linker, which may be absent or present.

It has been found that A- provides increased hGIP receptor antagonistic activity. It has in particular been found that antagonistic activity of comparative compounds, wherein an additional carboxylic acid group, has been added to A- to prepare a diacid derivative instead of a mono fatty acid derivative, is lower. Accordingly, in one embodiment, A- is a mono fatty acid. In a further embodiment, the mono fatty acid is a straight-chain mono fatty acid. In still a further embodiment, the straight-chain mono fatty acid is a C14 to C18 fatty acid, such as a C16 fatty acid. In yet a further embodiment, the fatty acid is a saturated fatty acid or an unsaturated fatty acid, such as a mono-unsaturated acid. In another embodiment, the fatty acid is a saturated fatty acid. In a more particular embodiment, the fatty acid is palmitic acid (C16). In the context of the present invention, the term mono fatty acid refers to a monoacid having one carboxylic acid group attached to the lysine in the GIP analogue. A mono fatty acid may be straight or branched and it may be saturated or unsaturated. A mono fatty acid typically has an even number of carbon atoms from 4 to 28, such as from 14 to 18 carbon atoms. A straight chain mono fatty acid may also be described by formula -C=(O)- (CH₂)_n-CH₃, where n is an integer between 2-26, preferably n is 12, 14 of 16. The monoacids could use abbreviation such as C14, C16 and C18, herein C16 refer to palmitic acid.

The type of linker represented by B-C- in the GIP derivatives of the present invention is known in the art and may vary. In a typical variation, B is an amino acid. Accordingly, in one embodiment, B is an amino acid linker. In a further embodiment, B is selected from y-Glu-y-Glu, y-Glu, Glu, Lys, and Asp, wherein y-Glu is gamma-glutamic acid represented by Chem. 1 :

Chem. 1 :

In still a further embodiment, B is selected from y-Glu and y-Glu-y-Glu. In yet a further embodiment, B is y-Glu. In another embodiment, B is absent.

B may be attached directly to the peptide backbone of the GIP derivatives of the invention, or the spacer C may be inserted in-between. Accordingly, in one embodiment, C is absent or selected from AEEA2 and AEEA, wherein AEEA is 8-amino-3,6-dioxaoctanoic acid represented by Chem. 2:

Chem. 2:

In another embodiment, C is absent or AEEA2. In still another embodiment, C is absent. In yet another embodiment, B-C is y-Glu. In a further embodiment, B-C is y-Glu and A is a C16 fatty acid, such as palmitic acid and as represented by Chem. 3:

Chem.

A number of GIP derivatives according to the present invention has been tested for its hGIP and mGIP receptors antagonistic activity. Accordingly, in one embodiment, the GIP derivative of the invention is selected from the group consisting of: compound No. 7, compound No. 8, compound No. 9, compound No. 19, compound No. 17, compound No. 21, compound No. 22, compound No. 49, compound No. 50, compound No. 52 and compound No. 53 of example 1 herein.

Pharmaceutical indications

Inhibitors of the human GIP receptor are known as useful candidates for treating or preventing obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome. Accordingly, one aspect of the invention concerns the GIP derivative according to the invention for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

Other compounds known for their usefulness in these indications are GLP-1 receptor agonists. Hence, in a further aspect of the invention it concerns the GIP derivative according to the invention in combination with a GLP-1 receptor agonist for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome. In one embodiment, the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide. In a further embodiment, the GLP-1 receptor agonist is semaglutide.

Pharmaceutical compositions

Pharmaceutical compositions comprising a derivative of the invention or a pharmaceutically acceptable salt, or amide thereof, and a pharmaceutically acceptable excipient may be prepared as is known in the art. The term "excipient" broadly refers to any component other than the active therapeutic ingredient(s). The excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance.

The formulation of pharmaceutically active ingredients with various excipients is known in the art, see e.g. Remington: The Science and Practice of Pharmacy (e.g. 19th edition (1995), and any later editions).

The pharmaceutical composition comprising the derivative of the invention may be of several dosage forms, e.g. a solution, a suspension, a tablet, and a capsule. The pharmaceutical composition comprising the derivative of the invention may be administered to a patient in need thereof at several sites, e.g. at topical sites such as skin or mucosal sites; at sites which bypass absorption such as in an artery, in a vein, or in the heart; and at sites which involve absorption, such as in the skin, under the skin, in a muscle, orally, or in the abdomen.

In one embodiment, the composition comprising the derivative of the invention is an injectable composition comprising GIP derivatives of the present invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, a GIP derivative of this invention is dissolved in a suitable buffer at a suitable pH so precipitation is minimised or avoided. The injectable composition is made sterile, for example, by sterile filtration.

In one embodiment, the composition comprising the derivative of the invention is a solid formulation, e.g. a freeze-dried or spray-dried composition, which may be used as is, or whereto the physician or the patient adds solvents, and/or diluents prior to use. In one embodiment, the pharmaceutical composition is in the form of a tablet.

A composition may be a stabilised formulation. The term “stabilised formulation” refers to a formulation with increased physical and/or chemical stability, preferably both. In general, a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.

In view of the combination use discussed above, a pharmaceutical composition may also be a dosage form combining a GIP derivative of the invention and a GLP-1 receptor agonist. Accordingly, in one aspect the present invention concerns a dosage form comprising a combination of a GIP derivative according to the invention and a GLP-1 receptor agonist, in terms of loose-dose combination and fixed-dose combination therapy of the GIP derivatives and the GLP-1 receptor agonists. In one embodiment, the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide. In a further embodiment, the GLP-1 receptor agonist is semaglutide.

The two active components may also be in separate dosage forms. Accordingly, in another aspect, the invention concerns a kit-of-parts comprising a first dosage form comprising a GIP derivative according to the invention and a second dosage form comprising a GLP-1 receptor agonist. In one embodiment, the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide. In a further embodiment, the GLP-1 receptor agonist is semaglutide. Pharmaceutically acceptable salts

In some embodiments, the derivatives as described herein are in the form of a pharmaceutically acceptable salt. Salts are e.g. formed by a chemical reaction between a base and an acid, e.g.: 2NH3 + H2SO4 — > (NH^SC . The salt may be a basic salt, an acid salt, or it may be neither (i.e. a neutral salt). Basic salts produce hydroxide ions and acid salts hydronium ions in water. The salts of the derivatives may be formed with added cations or anions between anionic or cationic groups, respectively. These groups may be situated in the peptide and/or in the substituent of the derivatives. Non-limiting examples of anionic groups include any free carboxylic acid groups in the substituent, if any, as well as in the peptide. The peptide may include a free carboxylic acid group at the C-terminus, if present, as well as any free carboxylic acid group of internal acidic amino acid residues such as aspartic acid and glutamic acid.

Non-limiting examples of cationic groups include any free amino groups in the substituent, if any, as well as in the peptide. The peptide may include a free amino group at the N-terminus, if present, as well as any free imidazole or amino group of internal basic amino acid residues such as histidine, arginine, and lysine.

Production processes

The production of peptides like hGIP(1-31) and hGIP(1-42) analogues is well known in the art.

The GIP derivatives of the invention (or fragments thereof) may for instance 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 Dorwald, “Organic Synthesis on solid Phase”, Wiley-VCH Verlag GmbH, 2000, and moc Solid Phase Peptide Synthesis”, Edited by W.C. Chan and P.D. White, Oxford University Press, 2000.

Also, or alternatively, they may be produced by recombinant methods, viz. by culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the peptide in a suitable nutrient medium under conditions permitting the expression of the peptide. Non-limiting examples of host cells suitable for expression of these peptides are: Escherichia coli, Saccharomyces cerevisiae, as well as mammalian BHK or CHO cell lines.

Those derivatives of the invention which include non-natural amino acids and/or a covalently attached N-terminal mono- or dipeptide mimetic may e.g. be produced as described in the experimental part. Or see e.g., 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.

Specific examples of methods of preparing a number of the derivatives of the invention are included in the experimental part.

PARTICULAR EMBODIMENTS

The invention may be further described by the following non-limiting embodiments:

Embodiment 1:

A glucose-dependent insulinotropic peptide (GIP) derivative comprising a GIP analogue comprising Formula I, II, or III:

Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-Gln-Xxxso-Xxxsi (I), (SEQ ID NO: 1) Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn (II), (SEQ ID NO: 2)

Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (III), (SEQ ID NO: 3) wherein Xxxs is Glu or absent, Xxx4 is Gly or absent, Xxxs is Thr or absent, Xxxso is Lys or absent, and Xxxsi is Gly or absent; wherein said GIP analogue of Formula I or II comprises a modified lysine in one of positions 10-32 and wherein said GIP analogue of Formula III comprises a modified lysine in position 43, said modified lysine comprising a modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine, the modifying group being defined by A-B-C-, wherein A- is a fatty acid and B-C- is a linker, which may be absent or present; and wherein the GIP analogue has a maximum of 10 amino acid modifications as compared to positions 3 to 31 of hGI P(1 -31 ) , or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

Embodiment 2: A glucose-dependent insulinotropic peptide (GIP) derivative comprising a GIP analogue comprising Formula I, II, or III:

Xxx3-Xxx4-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-Gln-Xxxso-Xxxsi (I), (SEQ ID NO: 1) Xxx3-Xxx₄-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn (II), (SEQ ID NO: 2) Xxx3-Xxx₄-Xxx5-Phe-lle-Ser-Asp-Tyr-Ser-lle-Ala-Met-Asp-Lys-lle-His-Gln-Gln-Asp-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (III), (SEQ ID NO: 3) wherein Xxxs is Glu or absent, Xxx₄ is Gly or absent, Xxxs is Thr or absent, Xxxso is Lys or absent, and Xxxsi is Gly or absent; wherein said GIP analogue of Formula I or II comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula III comprises a modified lysine in position 43, said modified lysine comprising a modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine, the modifying group being defined by A-B-C-, wherein A- is a fatty acid and B-C- is a linker, which may be absent or present; and wherein the GIP analogue has a maximum of 10 amino acid modifications as compared to positions 3 to 31 of hGI P(1 -31 ) , or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

Embodiment 3:

The GIP derivative according to Embodiment 2, wherein the modified lysine of the GIP analogue is in one of positions 11 and 10.

Embodiment 4:

The GIP derivative according to any one of Embodiments 2-3, wherein the modified lysine is in position 11.

Embodiment 5:

The GIP derivative according to any one of Embodiments 2-3, wherein the modified lysine is in position 10. Embodiment 6:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula I.

Embodiment 7:

The GIP derivative according to Embodiment 2, wherein the modified lysine of the GIP analogue is in position 43.

Embodiment 8:

The GIP derivative according to Embodiments 1-2 or 7, wherein the GIP analogue is of Formula III.

Embodiment 9:

The GIP derivative according to any one of the preceding Embodiments, wherein Xxxs is absent.

Embodiment 10:

The GIP derivative according to any one of the preceding Embodiments, wherein Xxxs and Xxx4 are absent, such as wherein Xxxs, Xxx4, and Xxxs are absent.

Embodiment 11 :

The GIP derivative according to any one of the preceding Embodiments, wherein Met in position 14 is substituted by Leu, lie or Nle.

Embodiment 12:

The GIP derivative according to any one of the preceding Embodiments, wherein Met in position 14 is substituted by Leu.

Embodiment 13:

The GIP derivative according to any one of the preceding Embodiments, wherein His in position 18 is substituted by Arg, Orn or homoArg.

Embodiment 14:

The GIP derivative according to any one of the preceding Embodiments, wherein His in position 18 is substituted by Arg. Embodiment 15:

The GIP derivative according to any one of the preceding Embodiments, wherein Asp in position 21 is substituted by Glu or homoGlu.

Embodiment 16:

The GIP derivative according to any one of the preceding Embodiments, wherein Asp in position 21 is substituted by Glu.

Embodiment 17:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of formula I, wherein the modified lysine is in position 11 , Xxx₃ and Xxx4 are absent, Xxx₃o is Lys, Xxxsi is Gly, and the N-terminal end is substituted with an a-acetylate.

Embodiment 18:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula I or II, wherein the modified lysine is in position 10, Xxx₃ and Xxx4 are absent, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a-acetylate.

Embodiment 19:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula III, wherein the modified lysine is in position 43, Xxx₃ and Xxx4 are absent, and the N-terminal end is substituted with an a-acetylate.

Embodiment 20:

The GIP derivative according to any one of the preceding Embodiments, wherein Xxx₃, Xxx4, and Xxxs are absent and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla).

Embodiment 21 :

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula I, wherein the modified lysine is in position 11 , Xxx₃, Xxx4, and Xxxs are absent, Xxx₃o is Lys or absent, Xxx_3i is Gly or absent, and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla).

Embodiment 22: The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula I or II, wherein the modified lysine is in position 10, Xxxs, Xxx4, and Xxxs are absent, Xxxso is Lys or absent, Xxxsi is Gly or absent, and Phe in position 6 is substituted by L-3-phenyllactic acid (Pla).

Embodiment 23:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue has a maximum of 8 amino acid modifications as compared to positions 3 to 31 of hGIP(1-31).

Embodiment 24:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue has a maximum of 7 amino acid modifications as compared to positions 3 to 31 of hGIP(1-31).

Embodiment 25:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue has a maximum of 6 amino acid modifications as compared to positions 3 to 31 of hGIP(1-31).

Embodiment 26:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a straight-chain fatty acid.

Embodiment 27:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a C14 to C18 fatty acid, such as a C16 fatty acid.

Embodiment 28:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a saturated fatty acid.

Embodiment 29:

The GIP derivative according to any one of the preceding Embodiments, wherein A is an unsaturated fatty acid. Embodiment 30:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a mono-unsaturated fatty acid.

Embodiment 31 :

The GIP derivative according to any one of the preceding Embodiments, wherein B is an amino acid linker.

Embodiment 32:

The GIP derivative according to any one of the preceding Embodiments, wherein B is selected from y-Glu-y-Glu, y-Glu, Glu, Lys, and Asp.

Embodiment 33:

The GIP derivative according to any one of the preceding Embodiments, wherein B is selected from y-Glu and y-Glu-y-Glu.

Embodiment 34:

The GIP derivative according to any one of the preceding Embodiments, wherein B is y-Glu.

Embodiment 35:

The GIP derivative according to any one of the preceding Embodiments, wherein B is absent.

Embodiment 36:

The GIP derivative according to any one of the preceding Embodiments, wherein C is absent or selected from AEEA2 and AEEA.

Embodiment 37:

The GIP derivative according to any one of the preceding Embodiments, wherein C is absent or AEEA₂.

Embodiment 38:

The GIP derivative according to any one of the preceding Embodiments, wherein C is AEEA₂. Embodiment 39:

The GIP derivative according to any one of the preceding Embodiments, wherein C is absent.

Embodiment 40:

The GIP derivative according to any one of the preceding Embodiments, wherein B-C is y- Glu and A is a C16 fatty acid.

Embodiment 41 :

The GIP derivative according to any one of the preceding Embodiments, wherein A is palmitic acid.

Embodiment 42:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 6, compound No. 7, compound No. 8, compound No. 9, compound No. 17, compound No. 19, compound No. 21 , compound No. 22, compound No. 49 and compound No. 50 as shown in example 1 herein.

Embodiment 43:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7, compound No. 8, compound No. 9, compound No. 17, compound No. 19, compound No. 21 , and compound No. 22 as shown in example 1 herein.

Embodiment 44:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7, compound No. 8, compound No. 9, compound No. 17, compound No. 19, and compound No. 21 as shown in example 1 herein.

Embodiment 45:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7, compound No. 8, compound No. 9, compound No. 17 and compound No. 19 as shown in example 1 herein.

Embodiment 46: The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7, compound No. 8, compound No. 9, and compound No. 19 as shown in example 1 herein.

Embodiment 47:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7, compound No. 8, and compound No. 9 as shown in example 1 herein.

Embodiment 48:

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 7 and compound No. 8 as shown in example 1 herein.

Embodiment 49:

The GIP derivative according to any one of the preceding Embodiments which is compound No. 7 as shown in example 1 herein.

Embodiment 50:

The GIP derivative according to any one of the preceding Embodiments which is an antagonist at the human GIP receptor.

Embodiment 51 :

The GIP derivative according to any one of the preceding Embodiments which binds to the human GIP receptor.

Embodiment 52:

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

Embodiment 53:

A pharmaceutical composition comprising a derivative according to any one of embodiments 1-51 , a GLP-1 receptor agonist, and at least one pharmaceutically acceptable excipient.

Embodiment 54: The pharmaceutical composition according to embodiment 53, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 55:

The pharmaceutical composition according to embodiments 53-54, wherein the GLP-1 receptor agonist is semaglutide.

Embodiment 56:

The pharmaceutical composition according to any one of embodiments 52-55 for use as a medicament.

Embodiment 57:

The GIP derivative according to any one of Embodiments 1-51 for use as a medicament.

Embodiment 58:

The GIP derivative according to any one of Embodiments 1-51 for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

Embodiment 59:

The GIP derivative for use according to Embodiment 58 in combination with a GLP-1 receptor agonist.

Embodiment 60:

The GIP derivative for use according to Embodiment 59, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 61 :

The GIP derivative for use according to Embodiment 60, wherein the GLP-1 receptor agonist is semaglutide.

Embodiment 62: A dosage form comprising a combination of a GIP derivative according to any one of Embodiments 1 to 51 and a GLP-1 receptor agonist.

Embodiment 63:

The dosage form according to Embodiment 62, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 64:

The dosage form according to Embodiment 63, wherein the GLP-1 receptor agonist is semaglutide.

Embodiment 65:

A kit-of-parts comprising a first dosage form comprising a GIP derivative according to any one of Embodiments 1 to 51 and a second dosage form comprising a GLP-1 receptor agonist.

Embodiment 66:

The kits-of-parts according to Embodiment 67, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 67:

The kits-of-parts according to Embodiment 66, wherein the GLP-1 receptor agonist is semaglutide.

Embodiment 68:

Use of the GIP derivative according to any one of Embodiments 1-51 for the manufacture of a medicament for the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

Embodiment 69:

A method of prevention and/or treatment of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome comprising administering a pharmaceutically active amount of the derivative according to any one of embodiments 1-51.

Embodiment 70:

The method according to embodiment 69 administering the derivative according to any one of embodiments in combination with a pharmaceutically active amount of a GLP-1 receptor agonist.

Embodiment 71 :

The method according to embodiment 70, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 72:

The method according to embodiment 71, wherein the GLP-1 receptor agonist is semaglutide.

ADDITIONAL PARTICULAR EMBODIMENTS:

Embodiment 1:

A glucose-dependent insulinotropic peptide (GIP) derivative comprising of a GIP analogue comprising Formula la, Ila, or Illa and a modifying group, wherein Formula la, Ila, and Illa are defined by:

Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-Gln-Xxx₃o-Xxx_3i (la), (SEQ ID NO: 46) Xxx5-Xxx6-lle-Ser-Asp-Xxxio-Xxxii-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn (Ila), (SEQ ID NO: 47)

Xxx5-Xxx6-lle-Ser-Asp-Tyr-Ser-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val-Asn- Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (Illa), (SEQ ID NO: 48) wherein Xxxs is Thr or absent; Xxxe is Phe or L-3-phenyllactic acid (Pla); Xxx is Tyr or Lys; Xxxn is Ser or Lys; Xxxi4 is Met, Leu, lie or Nle; Xxxis is His, Arg, Orn, homoArg; XXX21 is Asp, Glu, or homoGlu; Xxx₃o is Lys or absent; and Xxx_3i is Gly or absent; provided that when Xxxe is Pla, then Xxxs is absent; wherein said GIP analogue of Formula la or Ila comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula Illa comprises a modified lysine in position 43, said modified lysine comprises the modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine, the modifying group being defined by A-B-C-, wherein A- is a mono fatty acid and B-C- is a linker, which may be absent or present; and wherein the GIP analogue has a maximum of 10 amino acid modifications as compared to positions 3 to 31 of hGI P(1 -31 ) , such as maximum 8, 7 or 6 modifications compared to positions 3 to 31 of hGIP(1-31); or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

Embodiment 2:

A glucose-dependent insulinotropic peptide (GIP) derivative consisting of a GIP analogue of Formula la, Ila, or Illa and a modifying group, wherein Formula la, Ila, and Illa are defined by:

Xxx5-Xxx6-lle-Ser-Asp-Tyr-Ser-lle-Ala-Xxxi4-Asp-Lys-lle-Xxxi8-Gln-Gln-Xxx2i-Phe-Val-Asn- Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-lle-Thr-GIn-Lys (Illa), (SEQ ID NO: 48) wherein Xxxs is Thr or absent, Xxxe is Phe or L-3-phenyllactic acid (Pla), Xxx is Tyr or Lys; Xxxn is Ser or Lys; Xxxi4 is Met, Leu, lie or Nle; Xxxis is His, Arg, Orn, homoArg; XXX21 is Asp, Glu, or homoGlu; Xxx₃o is Lys or absent, and Xxx_3i is Gly or absent; provided that when Xxxe is Pla, then Xxxs is absent; wherein said GIP analogue of Formula la or Ila comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula Illa comprises a modified lysine in position 43, said modified lysine comprises the modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine, the modifying group being defined by A-B-C-, wherein A- is a mono fatty acid and B-C- is a linker, which may be absent or present; or a pharmaceutically acceptable salt, amide, or a-N acetylate thereof.

Embodiment 3:

The glucose-dependent insulinotropic peptide (GIP) derivative according to embodiments 1-2 wherein wherein Xxxs is Thr or absent, Xxxe is Phe or L-3-phenyllactic acid (Pla), Xxx is Tyr or Lys, Xxxn is Ser or Lys, Xxxi4 is Leu, lie or Nle, Xxxis is Arg, Orn, homoArg, XXX21 is Glu or homoGlu, Xxxso is Lys or absent, and Xxxsi is Gly or absent; provided that when Xxxe is Pla, then Xxxs is absent.

Embodiment 4:

The GIP derivative according to any one of the preceding embodiments wherein Xxxi4 is Leu.

Embodiment 5:

The GIP derivative according to any one of the preceding embodiments wherein Xxxis is His or Arg.

Embodiment 6:

The GIP derivative according to any one of the preceding embodiments wherein Xxxi₈ is Arg.

Embodiment 7:

The GIP derivative according to any one of the preceding embodiments wherein Xxx_2i is Glu.

Embodiment 8:

The GIP derivative according to any one of the preceding embodiments wherein Xxxi4 is Leu, Xxxis is Arg and Xxx_2i is Glu.

Embodiment 9:

The GIP derivative according to any one of the preceding embodiments, wherein the modified lysine of the GIP analogue is in one of positions 11 and 10.

Embodiment 10: The GIP derivative according to any one of the preceding embodiments, wherein the GIP analogue is of Formula la or Ila.

Embodiment 11 :

The GIP derivative according to any one of the preceding Embodiment, wherein the modified lysine is in position 11.

Embodiment 12:

The GIP derivative according to any one of the preceding embodiments, wherein the GIP analogue is of Formula la or Ila, wherein Xxx is Tyr and Xxxn is Lys.

Embodiment 13:

The GIP derivative according to any one of Embodiments 1-10, wherein the modified lysine is in position 10.

Embodiment 14:

The GIP derivative according to any one of embodiments 1-10 or 13, wherein the GIP analogue is of Formula la or Ila, wherein Xxx is Lys and Xxxn is Ser.

Embodiment 15:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula la.

Embodiment 16:

The GIP derivative according to any one of Embodiments 1-8, wherein the modified lysine of the GIP analogue is in position 43.

Embodiment 17:

The GIP derivative according to any one of Embodiments 1-8 or 16, wherein the GIP analogue is of Formula Illa.

Embodiment 18:

The GIP derivative according to any one of the preceding Embodiments, wherein Xxxs is absent. Embodiment 19:

The GIP derivative according to any one of Embodiments 1-17, wherein Xxxs is Thr.

Embodiment 20:

The GIP derivative according to any one of the preceding Embodiments, wherein Xxxe is Phe.

Embodiment 21 :

The GIP derivative according to any one of Embodiments 1-18, wherein Xxx₆ is L-3- phenyllactic acid (Pla).

Embodiment 22:

The GIP derivative according to any one of Embodiments 1-12, 15, 18-21 wherein Xxx is Tyr.

Embodiment 23:

The GIP derivative according to any one of Embodiments 1-10, 13-15, 18-21 , wherein Xxx is Lys.

Embodiment 24:

The GIP derivative according to any one of Embodiments 1-12, 15, 18-22, wherein Xxxn is Lys.

Embodiment 25:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of formula la, wherein the modified lysine is in position 11 , Xxx is Tyr, Xxxn is Lys, Xxxso is Lys, Xxxsi is Gly, and the N-terminal end is substituted with an a-acetylate.

Embodiment 26:

The GIP derivative according to embodiment 25, wherein Xxxi4 is Leu, Xxxis is Arg, and XXX21 is Glu.

Embodiment 27:

The GIP derivative according to any one of the Embodiments 1-24, wherein the GIP analogue is of Formula la or Ila, wherein the modified lysine is in position 10, Xxx is Lys, Xxxn is Ser, Xxx₃o is Lys, Xxx_3i is Gly, and the N-terminal end is substituted with an a- acetylate.

Embodiment 28:

The GIP derivative according to embodiment 27, wherein Xxxi₄ is Leu, Xxxi₈ is Arg, and Xxx_2i is Glu.

Embodiment 29:

The GIP derivative according to any one of Embodiments 1-24, wherein the GIP analogue is of Formula Illa, wherein the modified lysine is in position 43, and the N-terminal end is substituted with an a-acetylate.

Embodiment 30:

The GIP derivative according to Embodiment 29, wherein Xxxi₄ is Leu, Xxxis is Arg, and XXX21 is Glu.

Embodiment 31 :

The GIP derivative according to any one of the preceding Embodiments, wherein Xxx₅ are absent and Xxx₆ is L-3-phenyllactic acid (Pla).

Embodiment 32:

The GIP derivative according to any one of the preceding Embodiments, wherein the GIP analogue is of Formula la, wherein the modified lysine is in position 11 , Xxxs is absent, Xxx₈ is L-3-phenyllactic acid (Pla), Xxx is Tyr, Xxxn is Lys, Xxx₃o is Lys or absent, Xxx_3i is Gly or absent.

Embodiment 33:

The GIP derivative according to any one of Embodiments 31-32, wherein Xxxi₄ is Leu, Xxxis is Arg, and Xxx_2i is Glu.

Embodiment 34:

The GIP derivative according to any one of Embodiments 1-31 , wherein the GIP analogue is of Formula la or Ila, wherein the modified lysine is in position 10, Xxxs is absent, Xxx₈ is L-3- phenyllactic acid (Pla), Xxx is Lys, Xxxn is Ser, Xxx₃o is Lys or absent, Xxx_3i is Gly or absent. Embodiment 35:

The GIP derivative according to Embodiment 34, wherein Xxxi4 is Leu, Xxxis is Arg, and XXX21 is Glu.

Embodiment 36:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a straight-chain fatty acid of formula -C=(O)-(CH2)n-CH₃, where n is an integer between 2-26, such as 8-20, preferably n is 12, 14 or 16.

Embodiment 37:

The GIP derivative according to embodiment 36, wherein n is 14.

Embodiment 38:

The GIP derivative according to any one of the preceding Embodiments, wherein A is a C14 to C18 mono fatty acid, such as a C16 mono fatty acid.

Embodiment 39:

Embodiment 40:

Embodiment 41 :

Embodiment 42:

Embodiment 43:

The GIP derivative according to any one of the preceding Embodiments, wherein B is y-Glu. Embodiment 44:

Embodiment 45:

The GIP derivative according to any one of the preceding Embodiments, wherein C is absent or selected from AEEA₂ and AEEA.

Embodiment 46:

Embodiment 47:

The GIP derivative according to any one of the preceding Embodiments, wherein C is AEEA₂.

Embodiment 48:

Embodiment 49:

The GIP derivative according to any one of the preceding Embodiments, wherein B-C is y- Glu and A is a C16 mono fatty acid.

Embodiment 50:

Embodiment 51 :

The GIP derivative according to any one of the preceding Embodiments selected from the group consisting of: compound No. 6, compound No. 7, compound No. 8, compound No. 9, compound No. 17, compound No. 19, compound No. 21 , compound No. 22, compound No. 23, compound No. 25, compound No. 28, compound No. 49 and compound No. 50, compound No. 52, compound No. 53 as shown in example 1 herein. Embodiment 52:

Embodiment 53:

Embodiment 54:

Embodiment 55:

Embodiment 56:

Embodiment 57:

The GIP derivative according to any one of the preceding Embodiments which is an antagonist at the human GIP receptor and an antagonist at the mouse GIP receptor.

Embodiment 58:

Embodiment 59:

A pharmaceutical composition comprising a derivative according to any one of embodiments 1-57, a GLP-1 receptor agonist, and at least one pharmaceutically acceptable excipient. Embodiment 60:

The pharmaceutical composition according to embodiment 59, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 61 :

The pharmaceutical composition according to embodiments 59-60, wherein the GLP-1 receptor agonist is semaglutide.

Embodiment 62:

The pharmaceutical composition according to any one of embodiments 58-61 for use as a medicament.

Embodiment 63:

The GIP derivative according to any one of Embodiments 1-57 for use as a medicament.

Embodiment 64:

The GIP derivative according to any one of Embodiments 1-57 for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

Embodiment 65:

The GIP derivative for use according to Embodiment 64 in combination with a GLP-1 receptor agonist.

Embodiment 66:

The GIP derivative for use according to Embodiment 65, wherein the GLP-1 receptor agonist is selected from the group consisting of liraglutide, semaglutide, exenatide, dulaglutide, lixisenatide, taspoglutide, and albiglutide.

Embodiment 67:

The GIP derivative for use according to Embodiment 65-66, wherein the GLP-1 receptor agonist is semaglutide. Embodiment 68:

Use of the GIP derivative according to any one of Embodiments 1-574 for the manufacture of a medicament for the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome.

Embodiment 69:

A method of prevention and/or treatment of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome comprising administering a pharmaceutically active amount of the derivative according to any one of embodiments 1-57.

Embodiment 70:

Embodiment 71 :

Embodiment 72:

The method according to embodiment 70-71, wherein the GLP-1 receptor agonist is semaglutide.

EXAMPLES

This experimental part starts with a list of abbreviations and is followed by a section including general methods for synthesizing and characterising the GIP antagonists as described herein. Then follows a number of examples which relate to the preparation of the specific GIP antagonists of the invention and selected comparative compounds, and at the end a number of examples relating to the activity and properties of the GIP antagonists have been included.

Examples serve to illustrate the invention. List of Abbreviations yE: y-glutamic acid

Aib: a-aminoisobutyric acid

6-CI-HOBt: 6-chloro-1 -hydroxybenzotriazole

ACN: Acetonitrile

AEEA: 8-amino-3,6-dioxaoctanoic acid

BW: Body weight

C16: Palmitic acid

C18-OH: Octadecanedioic acid

DCM: Dichloromethane

DEPBT: Diethyl 3,4-dihydro-4-oxo-1 ,2,3-benzotriazin-3-yl phosphate

DIG: N, N-Diisopropylcarbodiimide

DIEA/DIPEA: N,N-Diisopropylethylamine

DIO: Diet-induced obese

DMF: Dimethylformamide

DODT : 3,6-dioxa-1 ,8-octanedithiol

Fmoc: Fluorenylmethyloxycarbonyl

GIP: Glucose-dependent insulinotropic peptide

GIPR: Glucose-dependent insulinotropic peptide receptor

GLP-1: Glucagon-like peptide 1

HFD: High fat diet

HFIP: Hexafluoroisopropanol hGIP: Human glucose-dependent insulinotropic peptide hGIPR: Human glucose-dependent insulinotropic peptide receptor

HPLC: High-performance liquid chromatography

IPGTT: Intra-peritoneal glucose tolerance test mGIP: Mouse glucose-dependent insulinotropic peptide mGIPR: Mouse glucose-dependent insulinotropic peptide receptor

Mtt: 4-Methyltrityl

OGTT: Oral glucose tolerance test

OxymaPure: ethyl 2-cyano-2-(hydroxyimino)acetate

Pla: L-(-)-3-phenyllactic acid I L-3-phenyllactic acid I S-3-phenyllactic acid tBu: tert-butyl

TFA: Trifluoroacetic acid

Tis: Triisopropylsilane General Methods of Peptide Synthesis

Generally, peptides were synthesized by automated Fmoc/tBu solid-phase methodology employing a Symphony peptide synthesizer (Gyros Protein Technologies, Tucson, AZ) and Applied Biosystems ABI 433A peptide synthesizer, starting with preloaded Wang resin (AAPPtec, Louisville, KY; Novabiochem, San Diego, CA) for acid C-terminal peptides, and H-Rink Amide ChemMatrix® (PCAS BioMatrix Inc, Saint-Jean-sur-Richelieu, Quebec, Canada J3B 8J8) for C-terminal amide peptides. All Fmoc-amino acids were coupling with 6-CI-HOBt/DIC or OxymaPure/DIC activations in DMF. All common Fmoc- amino acids, 6-CI-HOBt, OxymaPure and DIC were purchased from Midwest Biotech (Fisher, IN), AAPPtec and Gyros Protein Technologies. The Fmoc were removed by 20% piperidine in DMF. The N-terminal acetylation was performed on resin in the presence of tenfold excess of acetic anhydride/DIEA in DCM for 1 h. The N-terminal L-3-Phenyllactic acid (Pla) was coupled manually in 5-fold excess by DEPBT in DMF/DIEA. For the peptide with fatty acids, the residues were pre-incorporated with Fmoc-L-Lys(Mtt) - OH, after peptide assembling done and the N-terminal acetylation, the Mtt was deprotected with 1 - 2%TFA/5%Tis in DCM or with 30% HFIP/5% Tis in DCM.

The linkage amino acid of y-glutamic acid was coupled by Fmoc-Glu-OtBu; AEEA was coupled by Fmoc-8-amino-3,6-dioxaoctanoic acid/Fmoc-AEEA-OH (AAPPTec, Louisville, KY). Palmitic acid was coupled by 5-fold excess with DEPBT/DIEA in DMF for about 4h; the octadecanedioic acid acylation was done by coupling octadecanedioic acid mono-tert-butyl ester (Novo Nordisk, Malov, DK) with 5-fold excess DEPBT/DIEA in DMF for about 4h. Completed peptidyl resins were cleaved by standard TFA cleavage containing 5% Tis and 5% H₂O, added 2.5% 2-Mercaptoethanol or the odourless thiol scavenger DODT (Sigma-Aldrich, St. Louis, MS) for the cysteine and methionine residue containing peptides for 2 h with agitation.

The cleaved peptides in TFA were precipitated with ether, centrifuged and washed twice with ether, the crude peptides were dissolved in 20% ACN containing 2% acetic acid and injected to a Luna 19 x 250nm/10 pm C8 column (Phenomenex, Torrance, CA) to purify with 0.1% TFA/ACN eluent solvents on the Waters 2545 preparative HPLC system.

General LC-MS Methods of Peptide Detection and Characterisation

Peptide molecular weights characterization were measured by liquid chromatography-mass spectrometry on an Agilent 1260 lnfinity/6120 Quadrupole instrument with a Kinetex C8 column. Eluent A is water with 0.05% TFA and eluent B is 10% water, 90% ACN and 0.05% TFA. Eluent gradient methods are Method A: a gradient of 10%-80% eluent B in 10 min with 1 ml/min flow rate; Method B: a gradient of 20%-100% eluent B in 10 min with 1 ml/min flow rate; Method C: a gradient of 30%-80% eluent B in 3.5 min with 4 ml/min flow rate; Method D: a gradient of 40%-100% eluent B in 3.5 min with 4 ml/min flow rate.

Pharmacological Methods

The utility of the derivatives of the present invention as pharmacologically active agents e.g. in the reduction of weight gain and treatment of obesity in mammals, such as humans may be demonstrated by the activity of the antagonists in conventional assays and in the in vitro and in vivo assays described below.

Such assays also provide a means whereby the activities of the antagonists of the invention can be compared with activities of known compounds

General methods of measuring in vitro receptor binding

The receptor binding assay may be performed at the desired receptors, herein the human GIP receptors (“hGIPR”) and mouse GIP receptors (“mGIPR”) are tested.

GIP receptors were inserted in pcDNA3 vectors under the control of a CMV promotor with Geneticin as the selectable marker. The pGL4.29[luc2P/CRE/Hygro] vector from Promega (USA, E8471) contains a cAMP response element (CRE), that drives the transcription of the luciferase (luc) reporter gene luc2P. Luc2P is a synthetically-derived luciferase sequence optimized for expression in mammalian cells. Upon transfection of Baby Hamster Kidney (BHK) cells with these plasmids stable clones expressing receptor as well as reporter gene were selected in culture media containing hygromycin and geneticin (G418). The clones used were: Human (hGIPR/BHK CRELuc2p clone#5), mouse (mGIPR/BHK CRELuc2p clone#3).

Binding of peptide antagonists to the full length GIP receptor was assessed in whole cells with competitive binding using radiolabelled human GIP (¹²⁵l-NNC0090-0554, produced at Novo Nordisk A/S). BHK cells stably expressing the GIP receptor were seeded in poly-D- lysine coated white 96-well plates with clear bottom (Corning, cat. no. 354651). 5000-10000 cells are seeded pr. well in DMEM medium (cat. no 61965-026) supplemented with Geneticin (Gibco, cat. no 10131-027), Hygromycin B (Gibco cat no 10687-010) and 10% fetal bovine serum (FBS) (Gibco, cat. no 16140-071). The following day the medium was removed, and cells were rinsed twice with HBSS (Gibco, cat. no 14025), and the binding assay was performed. The assay buffer consists of HBSS (Gibco, cat no 14025), 10mM HEPES (Gibco, cat. no 15630), 0.1 % pluronic F-68 (Gibco, cat. no 2404,) and 0.1 % ovalbumin (SigmaAldrich, cat. no A5503), and pH is adjusted to 7.4. GIP receptor peptide antagonists were diluted in assay buffer and tested in 10-fold dilutions from 10 pM downwards. To each well was added 50pl assay buffer, 25 pl peptide (or assay buffer) and 25 pl radiolabelled GIP (equivalent of ~60pM). Compounds were tested in duplicates. The plate was sealed and incubated at 4°C overnight. The supernatant was discarded, and the plates were washed twice with ice cold PBS (Gibco, cat. no 14040-091). The cells were lysed with addition of 50pl 0.1M NaOH followed by 5 minutes shaking. 100 pl microscint-40 (Perkin Elmer, cat. no 6013641) was added to each well, and after incubation at room temperature for 30 minutes the plates were counted in a scintillation counter (Topcount® NXT™ HTS from Packard). Nonlinear regression analysis on the output files was performed in the Windows program GraphPad Prism 7 (GraphPad software, USA) using the equation “log(inhibitor) vs response (three parameters)”. Data were reported as geometric mean of IC50 values with 95% Cl.

General methods of measuring in vitro functional potency

The ability of peptides to activate or antagonize incretin receptors was studied in vitro using firefly luciferase reporter gene assay designed to indirectly measure cAMP production. BHK-21 cells (ATCC CCL-10) were transfected with human or mouse GLP-1 or GIP receptor and firefly luciferase under control of cAMP-response element. The cells were grown in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher 10566-016) supplemented with 10% Fetal Bovine Serum (Thermo Fisher 10082147), 100 lU/ml penicillin, 100 pg/mL streptomycin, and 10 mM HEPES at 37°C, 5% CO2 and 90% humidity for 2-3 days. Sixteen to twenty hours prior to the experiment the cells were plated at 5x104 cells per well in 96 well Isoplate (Perkin-Elmer 6005040, Waltham, MA). The plate was gently washed with warm Hank’s Balanced Salt Solution pH 7.4 (14025092, Invitrogen, Carlsbad, CA) and filled with 0.1 ml/well of serial dilutions of peptides prepared in sterile DMEM containing 1% Ovalbumin (A5503, Sigma-Aldrich). For the antagonism experiments the peptides were premixed with EC90 concentration- (75 - 95% maximal signal) of native ligand. After 3h incubation at 37°C and 5% CO2 in humidified atmosphere, the plates were washed with warm Hank’s Balanced Salt Solution pH 7.4 and 0.1 ml/well of Steady Lite HTS luminescence substrate reagent (Perkin-Elmer, Waltham, MA) was added to each well. The plate was sealed and shaken 10 min at 800 rpm. Luminescence signal was measured on Perkin-Elmer Envision plate reader. The luminescence data were plotted against peptide concentrations and EC50 or IC50 values were calculated by using logistic nonlinear 3 parameter regression analysis in GraphPad Prism 7 (GraphPad Software, La Jolla, CA). General methods for pharmacokinetic study in mice

C57BL/6J mice (58% HFD diet, male, body weight 61-74 g), were given subcutaneous injections of compounds 7, 9 and 22 at a single dose of 500 nmol/kg; each time point dosed 4 mice (n = 4). Plasma was collected at time points of 5min, 30min, 1 h, 2h, 6h, 8h, 24h, 48h, 72h, 96h, and 120h. The last time point of compound 9 is 48h. To measure plasma concentration, standards of each peptide were prepared in mouse plasma ranging from 0.5 to 4000 nM. A protein precipitation was performed by organic solvent extraction with a 14 - fold dilution with methanol. The samples were centrifuged for 20 min at 4°C at 13000g- force. Supernatants of samples were collected and diluted 3-fold with 0.1% formic acid in water. The diluted samples were then subjected to LC - MS analysis.

LC-MS analyses were carried out on a Thermo Q Exactive HF mass spectrometer interfaced with a Vanquish LIPLC. LC separations were performed on an Acquity LIPLC BEH C18 1.7 pm, 1.0 x 50 mm column. Mobile phase A was composed of 0.1% formic acid in water and mobile phase B was composed of 0.1% formic acid in ACN. The LC flow rate was set to 0.4 pL/min using a gradient elution from 10 to 95% B over the course of 4.0 min. Selected ion monitoring was used for all molecules, where the 3+ ion 1212.3491 m/z were chosen for compound 7, the 3+ ion 1102.9574 m/z were chosen for compound 9, and the 5+ ion 1017.1634 m/z for compound 22, respectively. Plasma data were analyzed by noncompartmental methods with sparse sampling using Phoenix WinNonlinTM 8.3 (Certara USA, Inc., Princeton, NJ).

General methods for pharmacodynamic study in mice

All mice studies were performed in accordance with Institutional Animal Care and Use Committee guidelines at University of Cincinnati. Male C57BL/6J mice (Jackson Laboratories) were housed 4 per cage under 12 h/12 h light-dark cycle at 22°C with ad libitum access to water and 58% fat, high-sugar diet (D12331 , Research Diets) for ~12 weeks. Mice achieved body weight (BW) greater than 55 g on average and were subsequently assigned to treatment groups (n = 8 per group) which were normalized for body weight. Treatment groups received either vehicle, semaglutide (2nmol/kg/d), GIPR antagonist (500 or 1500nmol/kg/d), semaglutide + GIPR antagonist (2 + 500 or 1500nmol/kg/d; co-formulated single injection), or semaglutide + GIPR antibody (2nmol/kg/d + 30mg/kg/w; separate injections). Test compounds (200pM) were dissolved in a vehicle (pH 7.4) containing 0.05% polysorbate-80, 50 mM sodium phosphate, and 70 mM sodium chloride; test compounds were administered once daily (semaglutide and GIPR antagonist) for 27d, subcutaneously during the light cycle at a volume of 3.9|jL/gram BW. Body weight and food intake were measured prior to dosing every other day; the percent change in body was calculated for each mouse based on its initial body weight. Food intake was measured on a per cage basis (n = 2 cages/group). Oral glucose tolerance tests (OGTT) were performed on day 0 (1h post-compound injection) and 22 (24h post-compound injection) and an intraperitoneal (i.p.) glucose tolerance test (IPGTT) was performed on d 27 (24h post-compound injection). For IPGTT and OGTT tests, animals were fasted for 4 hours prior to the test with access to water. Baseline tail-vein blood glucose was measured, then mice were injected i.p. or gavaged orally with 2.5 g/kg of 20% glucose solution (12uL/g). Tail blood glucose levels were measured 0, 15, 30, 60, 90, and 120 minutes following the glucose load. An additional measure of 6h fasting insulin (Crystal Chem 90080), resistin (R&D Systems MRSN00), CTX (Novus Biologicals NBP2-69074) and total GIP (Crystal Chem 81527) levels were taken at day 27.

Example 1 : Synthesis of compounds

The peptide analogues and derivative of the present invention were synthesized according to the General Methods of Peptide Synthesis as describe above. Name, structure, and properties are shown below for each compound. SEQ ID NO’s are included for the peptide backbone.

Compound No. 1 (reference cpd): hGIP(3-30) amide (SEQ ID NO: 12)

H— E G T F I S D Y S I A M D K I H Q Q D F V N W L L A Q K-N H₂

Formula Weight: 3297.7; Exact Mass: 3295.63; Formula: C_d„H,,_RN,_sO..S LCMS: [M+3H]3+: 1100.0; [M+4H]4+: 825.2; RT = 6.5min (Method A)

Compound No. 2 (reference cpd):

[M14L, H18R, D21E]-hGIP(3-30) amide (SEQ ID NO: 13)

H— E G T F I S D Y S I A L D K I R Q Q E F V N W L L A Q K-NH₂

Formula Weight: 3312.7; Exact Mass: 3310.74; Formula: C_d„H„_RN,_QO..

LCMS: [M+3H]3+: 1105.0; [M+4H]4+: 828.7; RT = 1.68min (Method C)

Compound No. 3 (reference cpd):

[M14L, H18R, D21E]-hGIP(5-31) (SEQ ID NO: 14) 210081WO01 42 Formula Weight: 3184.6; Exact Mass: 3182.68; Formula: C₁₄₇H₂₂₇N₃₇O₄₂ LCMS: [M+2H]2+: 1593.7; [M+3H]3+: 1062.2; [M+4H]4+: 797.0; RT = 1.97min (Method C) 5 Compound No.4 (reference cpd): [M14L, H18R, D21E]-hGIP(6-31) (SEQ ID NO: 15) Formula Weight: 3083.5; Exact Mass: 3081.63; Formula: C₁₄₃H₂₂₀N₃₆O₄₀ LCMS: [M+2H]2+: 1542.6; [M+3H]3+: 1028.6; [M+4H]4+: 771.7; RT = 1.54min (Method C) 10 Compound No.5 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) (SEQ ID NO: 16)

Formula Weight: 3084.5; Exact Mass: 3082.61; Formula: C₁₄₃H₂₁₉N₃₅O₄₁ 15 LCMS: [M+2H]2+: 1542.9; [M+3H]3+: 1029.0; RT = 1.96min (Method C) Compound No.6: [M14L, H18R, D21E]-hGIP(3-30) - K11( ^E-C16) amide (SEQ ID NO: 17)

20 Formula Weight: 3721.3; Exact Mass: 3719.07; Formula: C₁₇₆H₂₇₉N₄₁O₄₇ LCMS: [M+2H]2+: 1861.5; [M+3H]3+: 1241.1; [M+4H]4+: 931.2; RT = 6.37min (Method B) Compound No.7: [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 18) 210081WO01 43

Formula Weight: 3635.2; Exact Mass: 3633.02; Formula: C₁₇₃H₂₇₃N₃₉O₄₆ LCMS: [M+2H]2+: 1817.9; [M+3H]3+: 1212.6; [M+4H]4+: 909.8; RT = 6.88min (Method B) 5 Compound No.8: [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K11( ^E-C16) (SEQ ID NO: 19)

Formula Weight: 3493.1; Exact Mass: 3490.95; Formula: C₁₆₇H₂₆₃N₃₇O₄₄ LCMS: [M+2H]2+: 1746.9; [M+3H]3+: 1165.1; RT = 7.19min (Method B) 10 Compound No.9: [F6Pla, M14L, H18R, D21E]-hGIP(6-29) - K11( ^E-C16) amide (SEQ ID NO: 20)

Formula Weight: 3306.9; Exact Mass: 3304.85; Formula: C₁₅₉H₂₄₉N₃₅O₄₁ 15 LCMS: [M+2H]2+: 1653.9; [M+3H]3+: 1102.8; RT = 2.51min (Method D) Compound No.10 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-29) - K11(AEEA₂- ^E-C18-OH) amide (SEQ ID NO: 20) 210081WO01 44

Formula Weight: 3655.2; Exact Mass: 3653.00; Formula: C₁₇₃H₂₇₃N₃₇O₄₉ LCMS: [M+2H]2+: 1828.5; [M+3H]3+: 1219.3; RT = 6.55min (Method B) 5 Compound No.11 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K11(AEEA₂- ^E-C18-OH) (SEQ ID NO: 19)

Formula Weight: 3841.4; Exact Mass: 3839.10; Formula: C₁₈₁H₂₈₇N₃₉O₅₂ LCMS: [M+2H]2+: 1921.3; [M+3H]3+: 1281.2; [M+4H]4+: 961.3; RT = 2.78min (Method C) 10 Compound No.12 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K11(Lys₂- ^E-C18-OH) (SEQ ID NO: 19)

Formula Weight: 3807.5; Exact Mass: 3805.14; Formula: C₁₈₁H₂₈₉N₄₁O₄₈ 15 LCMS: [M+2H]2+: 1904.2; [M+3H]3+: 1270.1; [M+4H]4+: 952.7; RT = 2.28min (Method C) 210081WO01 45 Compound No.13 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(6-31) - K11(AEEA₂- ^E-C18-OH) (SEQ ID NO: 21)

Formula Weight: 3882.5; Exact Mass: 3880.13; Formula: C₁₈₃H₂₉₀N₄₀O₅₂ 5 LCMS: [M+2H]2+: 1942.1; [M+3H]3+: 1294.9; [M+4H]4+: 971.5; RT = 6.12min (Method B) Compound No.14 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K11(AEEA₂- ^E-C18-OH) (SEQ ID NO: 18)

10 Formula Weight: 3983.6; Exact Mass: 3981.18; Formula: C₁₈₇H₂₉₇N₄₁O₅₄ LCMS: [M+2H]2+: 1992.6; [M+3H]3+: 1328.6; [M+4H]4+: 996.8; RT = 6.05min (Method B) Compound No.15 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K11(AEEA₂-C18-OH) (SEQ ID NO: 18) 15

Formula Weight: 3854.5; Exact Mass: 3852.13; Formula: C₁₈₂H₂₉₀N₄₀O₅₁ LCMS: [M+2H]2+: 1927.8; [M+3H]3+: 1285.6; [M+4H]4+: 964.5; RT = 2.67min (Method C) 210081WO01 46 Compound No.16 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K11(-C18-OH) (SEQ ID NO: 18)

5 Formula Weight: 3564.2; Exact Mass: 3561.99; Formula: C₁₇₀H₂₆₈N₃₈O₄₅ LCMS: [M+2H]2+: 1782.7; [M+3H]3+: 1188.8; RT = 2.86min (Method C) Compound No.17: N^α -Ac - hGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 22) 10

Formula Weight: 3620.2; Exact Mass: 3617.92; Formula: C₁₇₁H₂₆₄N₃₈O₄₆S LCMS: [M+2H]2+: 1810.5; [M+3H]3+: 1207.4; RT = 2.22 min (Method C) Compound No.18 (reference cpd): 15 N^α -Ac - hGIP(5-31) - K11(AEEA₂- ^E-C18-OH) (SEQ ID NO: 22)

Formula Weight: 3968.6; Exact Mass: 3966.07; Formula: C₁₈₅H₂₈₈N₄₀O₅₄S LCMS: [M+2H]2+: 1984.9; [M+3H]3+: 1323.4; [M+4H]4+: 992.7; RT = 1.74 min (Method C) 210081WO01 47 Compound No.19: N^α -Ac - mGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 23)

5 Formula Weight: 3667.3; Exact Mass: 3664.70; Formula: C₁₇₁H₂₆₉N₄₁O₄₆S LCMS: [M+2H]2+: 1834.8; [M+3H]3+: 1223.2; RT = 6.87 min (Method B) Compound No.20 (reference cpd): N^α -Ac - mGIP(5-31) - K11(AEEA₂- ^E-C18-OH) (SEQ ID NO: 23) 10

Formula Weight: 4015.6; Exact Mass: 4013.12; Formula: C₁₈₅H₂₉₃N₄₃O₅₄S LCMS: [M+3H]3+: 1339.2; [M+4H]4+: 1004.7; RT = 6.83 min (Method B) 15 Compound No.21: [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K10( ^E-C16) (SEQ ID NO: 24)

Formula Weight: 3559.2; Exact Mass: 3557.00; Formula: C₁₆₇H₂₆₉N₃₉O₄₆ 210081WO01 48 LCMS: [M+2H]2+: 1780.2; [M+3H]3+: 1187.2; [M+4H]4+: 890.7; RT = 2.12min (Method D) Compound No.22: [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K10( ^E ^E-C16) amide (SEQ ID NO: 25) 5

Formula Weight: 5080.8; Exact Mass: 5077.78; Formula: C₂₃₄H₃₇₃N₆₁O₆₅ LCMS: [M+3H]3+: 1694.2; [M+4H]4+: 1271.0; [M+5H]5+: 1017.0; RT = 2.37 min (Method C) Compound No.23: 10 [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43( ^E ^E-C16) amide (SEQ ID NO: 26)

Formula Weight: 5244.0; Exact Mass: 5240.84; Formula: C₂₄₃H₃₈₂N₆₂O₆₇ LCMS: [M+3H]3+: 1748.6; [M+4H]4+: 1311.8; [M+5H]5+: 1049.8; RT = 1.59 min (Method D) 15 Compound No.24 [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) amide (SEQ ID NO: 27)

Formula Weight: 4619.2; Exact Mass: 4616.43; Formula: C₂₁₁H₃₂₆N₅₈O₅₉ LCMS: [M+3H]3+: 1540.4; [M+4H]4+: 1155.6; RT = 1.55 min (Method C) 20 Compound No.25: 210081WO01 49 [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K10( ^E ^E-C16) (SEQ ID NO: 28)

Formula Weight: 3546.1; Exact Mass: 3543.96; Formula: C₁₆₆H₂₆₆N₃₈O₄₇ LCMS: [M+2H]2+: 1774.0; [M+3H]3+: 1182.8; [M+4H]4+: 887.4; RT = 2.86 min (Method C) 5 Compound No.26 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K10( ^E ^E-C18-OH) (SEQ ID NO: 28)

Formula Weight: 3604.2; Exact Mass: 3601.96; Formula: C₁₆₈H₂₆₈N₃₈O₄₉ 10 LCMS: [M+2H]2+: 1802.0; [M+3H]3+: 1202.2; RT = 2.51 min (Method C) Compound No.27 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31)-K10(AEEA₂- ^E-C18-OH) (SEQ ID NO: 28)

15 Formula Weight: 3765.3; Exact Mass: 3763.07; Formula: C₁₇₅H₂₈₃N₃₉O₅₂ LCMS: [M+2H]2+: 1883.4; [M+3H]3+: 1256.0; [M+4H]4+: 942.0; RT = 2.59 min (Method C) Compound No.28: 210081WO01 50 [F6Pla6, M14L, H18R, D21E]-hGIP(6-42) - K10( ^E ^E-C16) amide (SEQ ID NO: 29) Formula Weight: 4938.7; Exact Mass: 4935.70; Formula: C₂₂₈H₃₆₃N₅₉O₆₃ LCMS: [M+3H]3+: 1647.2; [M+4H]4+: 1235.5; [M+5H]5+: 988.9; RT = 2.51 min (Method C) 5 Compound No.29 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K10(AEEA₂- ^E-C18-OH) amide (SEQ ID NO: 30) Formula Weight: 5300.1; Exact Mass: 5296.89; Formula: C₂₄₃H₃₉₀N₆₂O₇₀ 10 LCMS: [M+3H]3+: 1767.8; [M+4H]4+: 1325.8; [M+5H]5+: 1060.9; RT = 2.13 min (Method C) Compound No.30 (reference cpd): [N^α-Ac, M14L, H18R, D21E]-hGIP(5-42) - K11(AEEA₂- ^E-C18-OH) amide (SEQ ID NO: 31) 15 Formula Weight: 5376.2; Exact Mass: 5372.92; Formula: C₂₄₉H₃₉₄N₆₂O₇₀ LCMS: [M+3H]3+ : 1792.6; [M+4H]4+: 1345.1; RT = 2.29 min (Method C) Compound No.31 (reference cpd): 210081WO01 51 [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K13(AEEA₂- ^E-C18-OH) (SEQ ID NO: 32)

Formula Weight: 3857.4; Exact Mass: 3855.10; Formula: C₁₈₁H₂₈₇N₃₉O₅₃ LCMS: [M+2H]2+: 1928.2; [M+3H]3+: 1286.8; [M+4H]4+: 965.3; RT = 2.55 min (Method C) 5 Compound No.32 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K13(AEEA₂- ^E-C18-OH) amide (SEQ ID NO: 33)

Formula Weight: 5392.2; Exact Mass: 5388.91; Formula: C₂₄₉H₃₉₄N₆₂O₇₁ 10 LCMS: [M+3H]3+: 1798.3; [M+4H]4+: 1348.8; [M+5H]5+: 1079.2; RT = 2.18 min (Method C) Compound No.33: [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K16( ^E ^E-C16) (SEQ ID NO: 16)

15 Formula Weight: 3581.1; Exact Mass: 3578.93; Formula: C₁₆₉H₂₆₃N₃₇O₄₈ LCMS: [M+2H]2+: 1791.5; [M+3H]3+: 1194.5; RT = 2.42 min (Method D) Compound No.34 (reference cpd): 210081WO01 52 [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K17(AEEA₂- ^E-C18-OH) amide (SEQ ID NO: 34)

Formula Weight: 5350.1; Exact Mass: 5346.87; Formula: C₂₄₆H₃₈₈N₆₂O₇₁ LCMS: [M+3H]3+: 1784.1; [M+4H]4+: 13336.3; [M+5H]5+: 1070.8; RT = 2.1 min (Method C) 5 Compound No.35 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K17(AEEA₂- ^E-C18-OH) (SEQ ID NO: 35)

Formula Weight: 3815.4; Exact Mass: 3813.05; Formula: C₁₇₈H₂₈₁N₃₉O₅₃ 10 LCMS: [M+2H]2+: 1908.5; [M+3H]3+: 1272.5; [M+4H]4+: 954.7; RT = 2.48 min (Method C) Compound No.36 (reference cpd): [F6Pla, M14L, H18R]-hGIP(6-31) - K21( ^E ^E-C16) (SEQ ID NO: 36)

15 Formula Weight: 3580.2; Exact Mass: 3577.98; Formula: C₁₇₀H₂₆₈N₃₈O₄₆ LCMS: [M+2H]2+: 1790.2; [M+3H]3+: 1194.2; RT = 2.90 min (Method C) Compound No.37 (reference cpd): 210081WO01 53 [F6Pla, M14L, H18R]-hGIP(6-31) - K21( ^E ^E-C18-OH) (SEQ ID NO: 36) Formula Weight: 3638.2; Exact Mass: 3635.99; Formula: C₁₇₂H₂₇₀N₃₈O₄₈ LCMS: [M+2H]2+: 1820.3; [M+3H]3+: 1213.4; RT = 2.53 min (Method C) 5 Compound No.38 (reference cpd): [N^α -Ac, M14L, H18R]-hGIP(5-31) - K21( ^E ^E-C18-OH) (SEQ ID NO: 37) Formula Weight: 3780.4; Exact Mass: 3778.06; Formula: C₁₇₈H₂₈₀N₄₀O₅₀ 10 LCMS: [M+2H]2+: 1891.0; [M+3H]3+: 1260.8; [M+4H]4+: 945.9; RT = 2.53 min (Method C) Compound No.39 (reference cpd): [N^α -Ac, M14L, H18R, G31P]-hGIP(5-31) - K21( ^E ^E ^E-C18-OH) amide (SEQ ID NO: 38) 15 Formula Weight: 3948.6; Exact Mass: 3946.15; Formula: C₁₈₆H₂₉₂N₄₂O₅₂ LCMS: [M+2H]2+: 1974.7; [M+3H]3+: 1317.1; [M+4H]4+: 988.2; RT = 2.45 min (Method C) Compound No.40 (reference cpd): [N^α -Ac, M14L, H18R]-hGIP(5-42) - K21( ^E ^E ^E-C18-OH) amide (SEQ ID NO: 39) 210081WO01 54 Formula Weight: 5303.0; Exact Mass: 5299.83; Formula: C₂₄₅H₃₈₄N₆₂O₆₉ LCMS: [M+3H]3+: 1768.7; [M+4H]4+: 1326.5; [M+5H]5+: 1061.4; RT = 2.08 min (Method C) 5 Compound No.41 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K24( ^E ^E-C16) (SEQ ID NO: 40) Formula Weight: 3595.2; Exact Mass: 3593.0; Formula: C₁₇₁H₂₆₉N₃₇O₄₇ 10 LCMS: [M+2H]2+: 1798.5; [M+3H]3+: 1199.2; RT = 2.21 min (Method D) Compound No.42 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K24( ^E ^E-C16) amide (SEQ ID NO: 41) 15 Formula Weight: 5129.9; Exact Mass: 5126.80; Formula: C₂₃₉H₃₇₆N₆₀O₆₅ LCMS: [M+3H]3+: 1710.7; [M+4H]4+: 1283.3; [M+5H]5+: 1026.7; RT = 2.38 min (Method C) Compound No.43 (reference cpd): [F6Pla, M14L, H18R, D21E]-hGIP(6-31) - K30( ^E ^E-C16) (SEQ ID NO: 16)

Formula Weight: 3581.1; Exact Mass: 3578.93; Formula: C₁₆₉H₂₆₃N₃₇O₄₈ LCMS: [M+2H]2+: 1790.6; [M+3H]3+: 1194.1; RT = 2.34 min (Method D) 5 Compound No.44 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-42) - K30( ^E ^E-C16) amide (SEQ ID NO: 27)

Formula Weight: 5115.8; Exact Mass: 5112.74; Formula: C₂₃₇H₃₇₀N₆₀O₆₆ 10 LCMS: [M+3H]3+: 1705.6; [M+4H]4+: 1279.7; [M+5H]5+: 1024.1; RT = 2.37 min (Method C) Compound No.45 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43( ^E ^E ^E-C18-OH) amide (SEQ ID NO: 26)

15 Formula Weight: 5431.2; Exact Mass: 5427.89; Formula: C₂₅₀H₃₉₁N₆₃O₇₂ LCMS: [M+3H]3+: 1811.1; [M+4H]4+: 1358.7; [M+5H]5+: 1087.2; RT = 1.29 min (Method D) Compound No.46 (reference cpd): 210081WO01 56 [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43( ^E ^E-C18-OH) amide (SEQ ID NO: 26)

Formula Weight: 5302.0; Exact Mass: 5298.84; Formula: C₂₄₅H₃₈₄N₆₂O₆₉ LCMS: [M+3H]3+: 1768.4; [M+4H]4+: 1326.3; [M+5H]5+: 1061.4; RT = 1.29 min (Method D) 5 Compound No.47 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43( ^E-C18-OH) amide (SEQ ID NO: 26)

Formula Weight: 5172.9; Exact Mass: 5169.80; Formula: C₂₄₀H₃₇₇N₆₁O₆₆ 10 LCMS: [M+3H]3+: 1725.1; [M+4H]4+: 1294.0; [M+5H]5+: 1035.3; RT = 1.34 min (Method D) Compound No.48 (reference cpd): [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43(-C18-OH) amide (SEQ ID NO: 26)

15 Formula Weight: 5043.8; Exact Mass: 5040.76; Formula: C₂₃₅H₃₇₀N₆₀O₆₃ LCMS: [M+3H]3+: 1681.9; [M+4H]4+: 1261.8; [M+5H]5+: 1009.5; RT = 1.39 min (Method D) Compound No.49: 210081WO01 57 [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43(-C16) amide (SEQ ID NO: 26)

Formula Weight: 4985.8; Exact Mass: 4982.75; Formula: C₂₃₃H₃₆₈N₆₀O₆₁ LCMS: [M+3H]3+: 1662.6; [M+4H]4+: 1247.2; [M+5H]5+: 998.0; RT = 1.66 min (Method D) 5 Compound No.50: [N^α -Ac, M14L, H18R, D21E]-hGIP(5-43) - K43( ^E-C16) amide (SEQ ID NO: 26)

Formula Weight: 5114.9; Exact Mass: 5111.80; Formula: C₂₃₈H₃₇₅N₆₁O₆₄ 10 LCMS: [M+3H]3+: 1705.8; [M+4H]4+: 1279.5; [M+5H]5+: 1023.8; RT = 1.61 min (Method D) Compound No.51 (reference cpd): [N^α -Ac, M14L, D21E] -hGIP(5-31) – K18( ^E-C16) (SEQ ID NO: 42)

15 Formula Weight : 3566.1 ; Exact Mass : 3564.0 ; Formula : C₁₇₀H₂₆₆N₃₆O₄₇ LCMS: [M+3H]3+ : 1189.6 ; [M+2H]2+ : 1783.5 ; RT = 7.18 min (Method B) Compound No.52: [N^α -Ac, M14L, Aib16, H18R, D21E] - hGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 43) 210081WO01 58

Formula Weight : 3592.2 ; Exact Mass : 3590.0 ; Formula : C₁₇₁H₂₆₈N₃₈O₄₆ LCMS: [M+2H]2+ : 1796.6 ; [M+3H]3+ : 1198.1 ; RT = 7.48min (Method B) 5 Compound No.53: [N^α -Ac, M14L, H18R, Aib20, D21E] - hGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 44)

Formula Weight : 3592.2 ; Exact Mass : 3590.0 ; Formula : C₁₇₂H₂₇₂N₃₈O₄₅ LCMS: [M+2H]2+ : 1796.6 ; [M+3H]3+ : 1198.1 ; RT = 7.15min (Method B) 10 Compound No.54 (reference cpd): [N^α -glutaric, M14L, H18R, D21E] - hGIP(5-31) - K11(OEG2- ^E-C18-OH) (SEQ ID NO: 45)

Formula Weight : 4055.7 ; Exact Mass : 4053.2 ; Formula : C₁₉₀H₃₀₁N₄₁O₅₆ 15 LCMS: [M+3H]3+ : 1352.6 ; [M+4H]4+ : 1014.6 ; RT = 6.07min (Method B) Example 2: In vitro functional potency (CRE luciferase; whole cells) and binding

The purpose of this example is to test the activity, or potency, of the derivatives in vitro at the human or mouse GIP receptor.

The potencies and binding of the analogues and derivatives of Example 1 were determined as described under General methods of measuring in vitro receptor binding and General methods of measuring in vitro functional potency above at the human and mouse GIP receptor. All IC50 data are average ± SD of at least two independent experiments or average from one duplicate experiment. Percentage indicating the maximal inhibition, the inhibition is 95 ~ 100% for those without notice. Results are shown in Table 1.

Table 1 : Potency and binding of test compounds at hGIPR and mGIPR

inhibitions are above 95%. **: compound has di-fatty acid in side chain; nd: not determined or not available; Partial weak antagonism: with below 60% inhibition. All IC50 data are average ± SD of at least two independent experiments or average from one duplicate experiment.

From the compounds without an acyl group (1-5, 24) it is seen that going from hGIP (cpd 1) to 2-5, 24 all having M14L, H18R, D21 E substitutions especially antagonism at the mouse receptor is improved. When attaching a side chain with a mono fatty acid at Lys in position 11 (compounds 6-9, 17, 19, 52-53), potency is significantly improved on both receptors, especially on the truncated versions (compounds 7-9, 17, 19, 52-53), compared to attaching a di-fatty acid as present in all of compounds 10-16, 18, 20, 30, 54.

Generally, the above data shows that acylation with mono fatty acid acylation at one of positions 10, 11 , or 43, in particular 11 or 10, compared to fatty acid acylation at positions 13, 16, 17, 18, 21 , 24, or 30, result in very potent antagonists on both hGIPR and mGIPR, with mono fatty acid acylation at position 11 being the best antagonists on mGIPR. 210081WO01 62 Comparing the mono fatty acid acylation with diacid acylation, mono fatty acid acylated derivatives show better antagonism than comparative derivatives with diacid acylation (see e.g. compound 7 and 16). Example 3: In vivo pharmacokinetic study 5 The purpose of this study is to determine the half-life in vivo of the derivatives of the present invention after a single s.c. administration to mice, i.e. the prolongation of their time in the body and thereby their time of action. This is done in a pharmacokinetic (PK) study, as described under General methods for pharmacokinetic study in mice where the terminal half- life of the derivative in question is determined. By terminal half-life is generally meant the 10 period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase. Results are shown in Table 2: Table 2: Maximal conc (C_max), time taken to reach C_max (T_max), and half-life (t_½)

15 Data shows that compounds 7 and 9 have long half-lives while compound 22 has shorter half-life. Example 4: In vivo pharmacodynamic study The purpose of this example is to assess the in vivo effect of the derivatives of the 20 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 for this example was semaglutide. The experiment was performed as described under General methods for pharmacodynamic study in mice. The results are shown below in Tables 3-6: 25 Table 3. Average (n=8) body weight changes in grams and percentage of body weight compared to day 0.

The data of Table 3 is also shown in Figure 1. It is seen that administration of compound No. 7 (500 nmol/kg) results in reduced body weight gain compared to vehicle and administration of compound No. 7 (1500 nmol/kg) results in a slight body weight reduction compared to day 0. Furthermore, combination of compound No. 7 with semaglutide significantly improves body weight lowering beyond that of semaglutide alone at both doses.

Table 4. Cumulative food intake in grams (average, n=8).

The data in Table 4 is also shown in Figure 2. It is seen that administration of the compound No. 7 at both 500 nmol/kg and 1500 nmol/kg results in reduced food intake compared to vehicle. Furthermore, when combined with semaglutide, food intake is lowered beyond that of semaglutide alone.

Table 5. Blood glucose levels glucose tolerance tests (AUC, average ±SEM, n=8)

On day 0, the OGTT shows that dosing compound No. 7 at 500 nmol/kg (‘low dose’) did not affect glucose level compared to vehicle. However, dosing compound No. 7 at 1500 nmol/kg (‘high dose’) increased glucose levels on day 0. This effect was overcome when combining both the low dose and high dose of compound No. 7 with GLP-1R agonist semaglutide 2 nmol/kg.

Results from day 22 OGTT and day 27 IPGTT shows that after chronical administration of compound No. 7 both alone and in combination with GLP-1R agonist semaglutide, the GIPR antagonist improves glucose control. Both high and low dose of compound No. 7 on day 22 (OGTT) and day 27 (IPGTT) improves glucose level compared to vehicle, and in combination with semaglutide showed significantly improved glucose levels compared to semaglutide alone on day 27 IPGTT.

Table 6. Insulin, GIP, resistin, and C-terminal telopeptide (CTX) levels on day 27 after 6h fasting (average ±SEM, n=8)

The data in Table 6 shows that compared to vehicle compound No. 7 and the combination of semaglutide and compound No. 7 improves insulin sensitivity and resistance (insulin), reduce the GIP response (GIP), does not significantly affect resistin level (resistin), and does not affect bone health (CTX).

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS Claim 1: A glucose-dependent insulinotropic peptide (GIP) derivative consisting of a GIP analogue of 5 Formula Ia, IIa, or IIIa and a modifying group, wherein Formulae Ia, IIa, and IIIa are defined by: Xxx₅-Xxx₆-lle-Ser-Asp-Xxx₁₀-Xxx₁₁-lle-Ala-Xxx₁₄-Asp-Lys-lle-Xxx₁₈-Gln-Gln-Xxx₂₁-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Xxx₃₀-Xxx₃₁ (Ia), (SEQ ID NO: 46) 10 Xxx₅-Xxx₆-lle-Ser-Asp-Xxx₁₀-Xxx₁₁-lle-Ala-Xxx₁₄-Asp-Lys-lle-Xxx₁₈-Gln-Gln-Xxx₂₁-Phe-Val- Asn-Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln (IIa), (SEQ ID NO: 47) Xxx₅-Xxx₆-lle-Ser-Asp-Tyr-Ser-lle-Ala-Xxx₁₄-Asp-Lys-lle-Xxx₁₈-Gln-Gln-Xxx₂₁-Phe-Val-Asn- Trp-Leu-Leu-Ala-GIn-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln-Lys (IIIa), (SEQ 15 ID NO: 48) wherein Xxx₅ is Thr or absent, Xxx₆ is Phe or L-3-phenyllactic acid (Pla), Xxx₁₀ is Tyr or Lys; Xxx₁₁ is Ser or Lys; Xxx₁₄ is Met, Leu, Ile or Nle; Xxx₁₈ is His, Arg, Orn, homoArg; Xxx₂₁ is Asp, Glu, or homoGlu; Xxx₃₀ is Lys or absent, and Xxx₃₁ is Gly or absent; provided that when 20 Xxx₆ is Pla, then Xxx₅ is absent; wherein said GIP analogue of Formula Ia or IIa comprises a modified lysine in one of positions 11 or 10 and wherein said GIP analogue of Formula IIIa comprises a modified lysine in position 43, said modified lysine comprises the modifying group that is covalently attached to the side chain of the epsilon amino group of the lysine, the modifying group being 25 defined by A-B-C-, wherein A- is a mono fatty acid and B-C- is a linker, which may be absent or present; or a pharmaceutically acceptable salt, amide, or ^-N acetylate thereof. Claim 2: The GIP derivative according to claim 1, wherein 30 Xxx₅ is Thr or absent, Xxx₆ is Phe or L-3-phenyllactic acid (Pla), Xxx₁₀ is Tyr or Lys, Xxx₁₁ is Ser or Lys, 35 Xxx₁₄ is Leu, 210081WO01 67 Xxx₁₈ is His or Arg, Xxx₂₁ is Glu, Xxx₃₀ is Lys or absent, and Xxx₃₁ is Gly or absent; 5 provided that when Xxx₆ is Pla, then Xxx₅ is absent. Claim 3: The GIP derivative according to any of claims 1-2, wherein the Xxx₁₈ is Arg. 10 Claim 4: The GIP derivative according to any one of claims 1-3, wherein the modified lysine is in position 11. Claim 5: 15 The GIP derivative according to any one of the preceding claims, wherein the GIP analogue is of Formula Ia. Claim 6: The GIP derivative according to any one of the preceding claims, wherein the GIP analogue 20 is of formula Ia, wherein the modified lysine is in position 11, Xxx₁₀ is Tyr, Xxx₁₁ is Lys, Xxx₃₀ is Lys, Xxx₃₁ is Gly, and the N-terminal end optionally substituted with an ^-acetylate. Claim 7: The GIP derivative according to any one of the preceding claims, wherein Xxx₅ is Thr and 25 Xxx₆ is Phe. Claim 8: The GIP derivative according to any one of claims 1-6, wherein Xxx₅ is absent and Xxx₆ is L- 3-phenyllactic acid (Pla). 30 Claim 9: The GIP derivative according to any one of the preceding claims, wherein B is selected from ^-Glu and ^-Glu- ^-Glu. 35 Claim 10: 210081WO01 68 The GIP derivative according to any one of the preceding claims, wherein B-C is ^-Glu and A is a C14 to C18 mono fatty acid, such as palmitic acid. Claim 11: 5 The GIP derivative according to any one of the preceding claims selected from the group consisting of: compound No.7, compound No.8, compound No.9, compound No.17, compound No.19, compound No.21, compound No.22, compound No.23, compound No. 25, compound No.28, compound No.49 and compound No.50 as shown in example 1 herein. 10 Claim 12: The GIP derivative according to any one of the preceding claims which is compound No.7: [N^α -Ac, M14L, H18R, D21E]-hGIP(5-31) - K11( ^E-C16) (SEQ ID NO: 18)

15 . Claim 13: A pharmaceutical composition comprising a derivative according to any one of the preceding claims, and at least one pharmaceutically acceptable excipient. 20 Claim 14: The GIP derivative according to any one of claims 1-13 for use in the treatment or prevention of obesity, diabetes, such as type II diabetes, hyperinsulinemia, such as congenital hyperinsulinemia, and Cushing’s syndrome. 25 Claim 15: The GIP derivative for use according to claim 14 in combination with a GLP-1 receptor agonist.